Use of CF48 kidney injury biomarker and kidney injury therapeutic agent

Abstract

Provided are a Cf48 small molecule peptide and a diagnostic reagent kit as markers for the condition of kidney injury, the use of the Cf48 small molecule peptide in the preparation of a therapeutic agent for kidney injury as a drug intervention target, and a system for evaluating the condition of kidney injury in a measurement target subject based on the Cf48 biomarker. The Cf48 small molecule peptide has the sequence shown in SEQ ID NO.1. The Cf48 small molecule peptide may be used as a marker for the diagnosis of kidney disease, or the Cf48 small molecule peptide may be used as a marker for determining the degree of kidney injury, or the Cf48 small molecule peptide may be used as a marker for predicting the prognosis of a target subject, or the Cf48 small molecule peptide may be used as a marker for the degree of renal fibrosis, or the Cf48 small molecule peptide may be used as a marker for the activity of renal fibrosis, or the Cf48 small molecule peptide may be used as one or more markers for monitoring the therapeutic effect of kidney disease patients. As a new marker for the diagnosis of kidney disease and its diagnostic reagent kit, kidney injury can be determined by detecting the Cf48 content in kidney tissue and blood. It has strong specificity, solves the defect of the prior art that basically depends on the combination of biopsy and pathology to determine kidney injury, the detection is simple, and the result is accurate.

Description

Technical Field

[0001] The present invention relates to the medical field of kidney diseases, and particularly to the use of C4orf48 (abbreviated as Cf48) low molecular weight peptide as a marker for the status of kidney injury in a measurement target subject, as a diagnostic marker for the status of kidney injury, and in the aspect of an intervention target drug and an evaluation system for the status of kidney injury.

Background Art

[0002] Kidney diseases, particularly chronic kidney disease (CKD), are increasingly affecting the adult population worldwide. In China, the prevalence of CKD is 10.8%, and a significant number of patients among them develop end-stage renal disease (ESRD) and require renal replacement therapy.

[0003] Chronic kidney disease (CKD) refers to structural and functional disorders of the kidneys due to various causes, with progressive decline in renal function, mainly leading to progressive and irreversible loss of nephrons, reduced renal regenerative capacity, metabolic changes, oxidative stress and inflammation, and ultimately resulting in renal fibrosis. Renal fibrosis is the final outcome of various types of CKD and is also one of the main causes of end-stage renal disease (ESRD). Fibrosis is part of the normal repair process of the kidney. Under injury conditions, fibroblasts produce an appropriate amount of extracellular matrix (ECM) to maintain the structural and functional integrity of the tissue. When the cause of injury persists and the repair process becomes dysregulated, renal fibroblasts are activated or epithelial cells undergo transdifferentiation into myofibroblasts, proliferate massively, produce excessive ECM, replace the original renal parenchymal tissue, and cause renal fibrosis. When podocytes are lost and replaced by ECM, it is called glomerulosclerosis, and damage to renal tubular cells and subsequent pathological deposition of ECM are called renal interstitial fibrosis (RIF). Compared with glomerulosclerosis, renal interstitial fibrosis is more strongly correlated with proteinuria in patients and the severity of the disease.

[0004] Acute Kidney Injury (AKI) refers to a rapid decline in renal function over a short period, characterized by programmed cell death of endogenous cells in the kidney and an excessive inflammatory response. Epidemiological studies have revealed that patients with CKD have a high risk of developing AKI, and aging and CKD significantly increase the incidence and severity of AKI. Similarly, severe or recurrent AKI may progress to CKD or ESRD (known as the transition from AKI to CKD). One study reported that there are more than 5,000 cases per year per million AKI patients who do not require dialysis, and a significant proportion of them progress to CKD or ESRD. The severity, incidence, and duration of AKI are related to the subsequent incidence of CKD. Even mild AKI may progress to renal fibrosis. As a result, AKI is considered an important risk factor for CKD and ESRD. However, except for supportive therapy, effective and specific treatment methods to delay the progression of AKI are still lacking.

[0005] The disease processes of kidney diseases, especially chronic kidney disease (CKD), are complex and heterogeneous. Current clinical evaluations and diagnoses of kidney diseases largely depend on glomerular function. That is, renal function is reflected by the evaluation of glomerular filtration function. Over the past 100 years, the diagnosis of kidney diseases has mainly relied on the detection of serum creatinine levels. Serum creatinine is a metabolite of creatine / phosphocreatine and is freely filtered by the glomerulus in the presence of normal glomerular filtration. However, as the demand for sensitivity in clinical diagnosis increases, serum creatinine and other molecules for evaluating glomerular filtration function have limitations as biomarkers for kidney injury. These limitations include: (1) insufficient diagnostic sensitivity: Serum creatinine is an indirect marker of kidney injury and may delay diagnosis; (2) insufficient diagnostic specificity: Serum creatinine may increase even in the absence of injury to the glomeruli or tubules; (3) insufficient diagnostic directionality: It is impossible to distinguish whether the increase in serum creatinine is due to tubular injury or glomerular injury; (4) in patients with good pre-renal function, serum creatinine may not increase even in the presence of severe tubular injury.

[0006] To address these limitations, it is necessary to provide biomarkers in blood or urine that may be associated with kidney injury, particularly biomarkers directly produced by the kidney or biomarkers that increase in blood or urine due to abnormal kidney function after kidney injury. Finding biomarkers with specificity in the onset and progression of kidney injury and drugs that act on potential therapeutic targets is highly important for the treatment of kidney injury.

Summary of the Invention

[0007] An object of the present invention is to provide a Cf48 low-molecular peptide as a marker for the condition of kidney injury and a diagnostic reagent kit, its use in the preparation of a therapeutic agent for kidney injury as a drug intervention target, and a system for evaluating the condition of kidney injury in a measurement target subject based on the Cf48 biomarker.

[0008] The above object of the present invention is achieved by the following technical means.

[0009] The first technical solution of the present invention provides the use of a Cf48 low-molecular peptide biomarker having the sequence shown in SEQ ID NO.1 as a marker for determining the condition of kidney injury in a measurement target subject.

[0010] Furthermore, the Cf48 low-molecular peptide is used as a marker for the diagnosis of kidney disease, or the Cf48 low-molecular peptide is used as a marker for determining the degree of kidney injury, or the Cf48 low-molecular peptide is used as a marker for predicting the prognosis of the target subject, or the Cf48 low-molecular peptide is used as a marker for the degree of renal fibrosis, or the Cf48 low-molecular peptide is used as a marker for the activity of renal fibrosis, or the Cf48 low-molecular peptide serves as one or more markers for monitoring the treatment effect of kidney disease patients.

[0011] The first technical solution of the present invention provides the use of CF48 low-molecular-weight peptide as a biomarker in a diagnostic reagent or reagent kit for determining the status of kidney injury in a measurement target subject. The reagent or reagent kit may be a reagent or kit using a mass spectrometry method or an immunoassay method, and the immunoassay method includes, but is not limited to, enzyme-linked immunosorbent assay, chemiluminescence immunoassay, immunochip method, fluorescence immunoassay, immunohistochemistry, immunochromatography, stress immunity, immunoturbidimetry, etc.

[0012] Furthermore, detecting the expression level (i.e., content) of Cf48 low-molecular-weight peptide in a biological sample, and a higher expression level than the reference level indicates the presence of kidney injury, and the sample is kidney tissue or serum or plasma or whole blood.

[0013] Furthermore, by detecting the expression level of Cf48 low-molecular-weight peptide in a biological sample, it is determined whether the target subject has kidney disease, or

[0014] By detecting the expression level of Cf48 low-molecular-weight peptide in a biological sample, the degree of kidney injury is determined, or By detecting the expression level of Cf48 low-molecular-weight peptide in a biological sample, the prognosis of the patient is predicted, or By detecting the expression level of Cf48 low-molecular-weight peptide in a biological sample, the degree of renal fibrosis is determined, or By detecting the expression level of Cf48 low-molecular-weight peptide in a biological sample, the activity of renal fibrosis is determined, or By detecting the expression level of Cf48 low-molecular-weight peptide in a biological sample, at least one of the treatment effect monitoring of kidney disease patients is performed.

[0015] Furthermore, according to the content of Cf48 low-molecular-weight peptide detected in the biological sample, it is determined that there is damage to the renal tubules.

[0016] Furthermore, the higher the content of Cf48 low-molecular-weight peptide detected in the biological sample, the higher the degree of kidney injury, The higher the content of Cf48 peptide in the detected biological sample, the higher the prognosis prediction of the target subject, that is, it indicates that the ability of the kidney disease to progress in a more serious direction is stronger. Use (application) according to claim 5, characterized in that the higher the content of Cf48 low molecular weight peptide in the detected biological sample, the higher the activity of renal fibrosis, that is, the higher the degree of progressive renal fibrosis in the subject to be measured.

[0017] Furthermore, the content of Cf48 low molecular weight peptide in the detected biological sample is compared with a first threshold value. When the content of Cf48 low molecular weight peptide in the detected biological sample is equal to or higher than the first threshold value, it indicates that the activity of renal fibrosis is strong, indicating that the activity of progressive fibrosis in the kidney is high and intervention is necessary. When the content of Cf48 low molecular weight peptide in the detected biological sample is smaller than the first threshold value and larger than the second threshold value, then it is detected whether TGF-β1 is present in the biological sample. When TGF-β1 is present in the measured biological sample, it indicates that the activity of renal fibrosis is strong. When the content of Cf48 low molecular weight peptide in the detected biological sample is smaller than the second threshold value, it indicates that the activity of renal fibrosis is weak, indicating that the activity of progressive fibrosis in the kidney is low and the ability of renal fibrosis to progress is absent. Use (application) according to claim 7, characterized in that the first threshold value is larger than the second threshold value.

[0018] The third technical solution of the present invention provides the use (application) of a Cf48 low molecular weight peptide marker as an intervention target in the preparation of a therapeutic agent for kidney injury.

[0019] Preferably, the therapeutic agent for kidney injury treats kidney injury or delays the progression of kidney injury by targeting and inhibiting or removing the expression of Cf48 low molecular weight peptide.

[0020] Furthermore, the therapeutic agent for kidney injury avoids the phenomenon of fibroblast activation caused by Cf48 low-molecular-weight peptide inhibiting the amino acid channel SLC3A in fibroblasts by targeting and inhibiting the expression of Cf48 low-molecular-weight peptide.

[0021] Furthermore, the therapeutic agent for kidney injury avoids the occurrence of induction of EMT and the promotion of the formation of renal fibrosis caused by Cf48 low-molecular-weight peptide inhibiting the amino acid channel SLC3A2 in tubular cells and inhibiting the energy metabolism of tubular cells by targeting and inhibiting the expression of Cf48 low-molecular-weight peptide.

[0022] Furthermore, the therapeutic agent for kidney injury avoids the occurrence of induction of EMT and the promotion of the formation of renal fibrosis caused by Cf48 activating the signal pathway downstream of TFRC by inhibiting the binding of Cf48 low-molecular-weight peptide to transferrin receptor protein 1 (TFRC).

[0023] Furthermore, the therapeutic agent for kidney injury avoids the phenomenon of Cf48 acting synergistically with other factors that promote the formation of renal fibrosis and promoting the formation of renal fibrosis by targeting and inhibiting the expression of Cf48 low-molecular-weight peptide.

[0024] The fourth technical solution of the present invention provides a system for evaluating the status of kidney injury in a measurement target subject based on the Cf48 biomarker. In this system, a data collection module that collects at least the amount of Cf48 in a biological sample of the measurement target subject, an evaluation module that compares the content of Cf48 in the biological sample with a collation parameter and obtains a level result corresponding to the kidney injury of the measurement target subject according to the comparison result, are provided.

[0025] Preferably, the evaluation module is provided with a parameter storage unit and an analysis unit, the parameter storage unit stores a first reference value and a second reference value, The analysis unit compares the content of Cf48 in the detected biological sample transmitted by the data collection module with a first reference value and a second reference value. When the content of Cf48 low-molecular-weight peptide in the detected biological sample is equal to or greater than the first reference value, the analysis unit gives a first-level result, and the first level indicates that the condition of kidney injury is severe and intervention is necessary. When the content of Cf48 low-molecular-weight peptide in the detected biological sample is less than the second reference value, the analysis unit gives a low-level result, and the low level indicates that the condition of kidney injury is mild and intervention is not necessary. Here, the first reference value is greater than the second reference value.

[0026] Preferably, the data collection module further collects whether TGF-β1 exists in the measured biological sample. The analysis unit compares the content of Cf48 in the detected biological sample transmitted by the data collection module with a first reference value and a second reference value. When the content of Cf48 low-molecular-weight peptide in the detected biological sample is less than the first reference value and greater than the second reference value, the analysis unit obtains the result of whether TGF-β1 exists in the measured biological sample collected by the data collection module. When TGF-β1 exists in the measured biological sample, the analysis unit gives a second-level result, and the second level indicates that the condition of kidney injury is severe and intervention is necessary.

[0027] The present invention provides a Cf48 low-molecular-weight peptide, which is a marker capable of identifying kidney injury, and a diagnostic reagent kit thereof. By detecting the Cf48 content in kidney tissue and blood, kidney injury can be determined. It has strong specificity and solves the defect of the prior art that basically depends on the combination of biopsy and pathology to determine kidney injury. At the same time, the marker of the present invention can also identify the degree of change of kidney injury, and can make up for the technical defect that the prior art could not determine the change ability of the situation of kidney injury. By using the Cf48 low-molecular-weight peptide as a marker to conduct characteristic evaluation on the situation of kidney injury, the situation of kidney injury can be determined without biopsy symptoms, overcoming the limitations of various requirements in clinical diagnosis in the prior art, and it may also be detected as an index for health diagnosis.

[0028] The use (application) of the Cf48 low-molecular-weight peptide marker as an intervention target provided by the present invention in a therapeutic agent for kidney injury can inhibit or treat the situation of kidney injury by targeting and inhibiting the expression of the Cf48 low-molecular-weight peptide.

[0029] The system for evaluating the situation of kidney injury in a measurement target subject based on the Cf48 biomarker provided by the present invention has the characteristics that it is easy to detect and the results are accurate by collecting the amount of Cf48 in the biological sample of the measurement target subject and evaluating and obtaining the level of the situation of kidney injury.

Brief Description of the Drawings

[0030] A further description of the present invention is made with reference to the drawings, but the content of the drawings does not constitute any limitation to the present invention.

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Mode for Carrying Out the Invention

[0034] The present invention will be described in more detail with reference to the following examples. <Example 1>

[0035] This example provides the use (application) of the Cf48 small molecule peptide biomarker having the sequence shown in SEQ ID NO.1 as a biomarker for judging the status of kidney injury of a measurement target subject.

[0036] The Cf48 low-molecular-weight peptide is the abbreviation of the C4orf48 low-molecular-weight peptide. The human gene C4orf48 (NCBI Gene ID: 401115) is located in the open reading frame at position 48 on chromosome 4 (Chr4: 2,035,610 - 2,043,970), consists of 9 exons, can translate a low-molecular-weight peptide with an amino acid length of 90aa, and the amino acid sequence is as follows.

[0037] The amino acid sequence of human C4orf48 is: MAPPPACRSPMSPPPPPLLLLLLSLALLGARARA EPAGSAVPAQSRPCVDCHAFEFMQRALQDLRKTACSLDARTETLLLQAERRALCACWPAGH (95aa). The cDNA sequence of human C4orf48 is: ATGGCCCCCCCGCCCGCGTGCCGGTCCCCGATGTCACCGCCGCCGCCGCCGCTGCTGCTGCTGCTGCTGAGTCTGGCGCTGCTGGGCGCC CGGGCCCGCGCCGAGCCCGCCGGGAGTGCCGTCCCCGCGCAGAGCCGCCCATGCGTGGACTGCCACGCCTTCGAGTTCATGCAGCGCGCCCTGCAGGACCTGCGGAAGACAGCCTGCAGCCTGGACGCGCGGACGGAGACCCTACTGCTGCAGGCAGAGCGCCGTGCCCTGTGTGCCTGCTGGCCAGCGGGGCACTGAG. (Note: Underlined part: signal peptide, other sequences: secreted peptide).

[0038] According to the experimental results, it was found that the Cf48 low-molecular-weight peptide can be used as a diagnostic marker for kidney diseases. By measuring the content of the Cf48 low-molecular-weight peptide in the target sample, it is possible to determine whether the target has kidney diseases. Specifically, by detecting the expression of the Cf48 low-molecular-weight peptide in the tissues and blood of the target subject, it can be determined whether the target subject has kidney diseases.

[0039] The Cf48 low molecular weight peptide can also be used as a marker for judging the degree of kidney damage. By detecting the expression of the Cf48 low molecular weight peptide in the target subject's tissue or blood, according to the content of the detected Cf48 low molecular weight peptide, the damage status of the kidney structure (such as structural changes), the damage status of kidney function (such as filtration function damage), and the degree of kidney damage including the degree of kidney inflammation are judged.

[0040] The CF48 low molecular weight peptide can also be used as a marker for predicting the prognosis of the target subject. The prognosis prediction of the target subject represents the current kidney damage progression ability of the target subject. When the current kidney damage progression ability is high, it indicates a high ability to change in a serious direction. When the current kidney damage progression ability is low, it indicates that when the current kidney damage progression ability is low, the current kidney damage of the kidney disease does not have the ability to progress in a serious direction, and the current kidney damage situation basically maintains the current status or is alleviated. By detecting the expression of the Cf48 low molecular weight peptide in the target subject's tissue or blood, the progression of kidney damage can be judged, and the prognosis prediction result of the target subject can be obtained.

[0041] The Cf48 low molecular weight peptide can also be used as a marker for the degree of renal fibrosis, which is an important feature of kidney damage, especially kidney disease.

[0042] The Cf48 low molecular weight peptide may also be a marker for the activity of renal fibrosis. The activity of renal fibrosis is a characteristic of the current degree of renal fibrosis. A high current fibrosis activity indicates the degree to which the kidney is currently fibrotic, and the target subject is rapidly fibrotic, indicating the need for timely intervention. A low current fibrosis activity indicates a low current fibrotic ability of the kidney. The kidney either maintains the current status or the fibrotic ability decreases, making it difficult to fibrotic, suggesting that in most cases, intervention is not necessary.

[0043] The Cf48 low molecular weight peptide can also be used as a marker for monitoring the treatment effect of kidney disease patients, and the treatment effect of the patient can be monitored through the Cf48 low molecular weight peptide.

[0044] In this embodiment, the Cf48 low-molecular-weight peptide is used as a marker for the kidney, providing a new marker. By detecting the Cf48 content in kidney tissue and blood, kidney damage can be determined. It solves the defect of the prior art that basically depends on the combination of biopsy and pathology to determine kidney damage. At the same time, the marker of the present invention can also specify the degree of change in kidney damage, and can make up for the technical defect that the prior art could not determine the change ability of the situation of kidney damage. By performing characteristic evaluation on the situation of kidney damage using the Cf48 low-molecular-weight peptide as a marker, the situation of kidney damage can be determined without biopsy symptoms, overcoming the limitations of various requirements of clinical diagnosis in the prior art, and it may also be detected as one of the indicators for health diagnosis.

[0045] <Example 2> Provide the use (application) of CF48 low-molecular-weight peptide as a biomarker in a diagnostic reagent or reagent kit for determining the situation of kidney damage in a measurement target subject. Detect the expression level of the Cf48 low-molecular-weight peptide in a biological sample, and a higher expression level than the reference level indicates the risk of the presence of kidney damage, and the sample may be kidney tissue or serum or plasma or whole blood.

[0046] Specifically, by using the Cf48 low-molecular-weight peptide as a marker for the diagnosis of kidney disease and detecting the expression level of the Cf48 low-molecular-weight peptide in a biological sample, it can be determined whether the target subject has kidney disease. Or, by using the Cf48 low-molecular-weight peptide as a marker for the degree of kidney damage and detecting the expression level of the Cf48 low-molecular-weight peptide in a biological sample, the degree of kidney damage can be determined. Or, by using the Cf48 low-molecular-weight peptide as a marker for predicting the prognosis of a target subject with kidney damage and detecting the expression level of the Cf48 low-molecular-weight peptide in a biological sample, the prognosis of the patient can be predicted. Or, By detecting the expression level of Cf48 low-molecular-weight peptide in a biological sample, the degree of renal fibrosis is determined using Cf48 low-molecular-weight peptide as a marker for the degree of renal fibrosis. Or, by detecting the expression level of Cf48 low-molecular-weight peptide in a biological sample, the activity of renal fibrosis is determined using Cf48 low-molecular-weight peptide as a marker for the activity of renal fibrosis. Or, by detecting the expression level of Cf48 low-molecular-weight peptide in a biological sample, the therapeutic effect monitoring of patients with kidney disease is performed using Cf48 low-molecular-weight peptide as a marker for the therapeutic effect monitoring of patients with kidney disease.

[0047] Specifically, detecting the expression level of Cf48 low-molecular-weight peptide in a biological sample can be determined according to the content of Cf48 low-molecular-weight peptide in the detected biological sample.

[0048] Experimental results showed that the higher the content of Cf48 peptide in the detected biological sample, the higher the severity of kidney damage. The higher the content of Cf48 low-molecular-weight peptide in the detected biological sample indicates a higher degree of kidney damage. The higher the content of Cf48 low-molecular-weight peptide in the detected biological sample indicates a higher prognosis prediction for the target subject, that is, a stronger ability for the kidney disease to progress in a more severe direction. The higher the content of Cf48 low-molecular-weight peptide in the detected biological sample, the higher the activity of renal fibrosis, that is, the degree of progressive renal fibrosis in the measured subject is high, as described in claim 5. When the content of Cf48 low-molecular-weight peptide in the detected biological sample is high, there is damage to the renal tubules.

[0049] In actual use, a corresponding threshold range can be set according to specific subjects and detection requirements, and kidney damage can be determined through the threshold range. For example, the content of Cf48 low-molecular-weight peptide in the detected biological sample is compared with a first threshold. When the content of Cf48 low-molecular-weight peptide in the detected biological sample is equal to or higher than the first threshold, it indicates that the activity of renal fibrosis is strong, the activity of progressive fibrosis in the kidney is high, and intervention is required.

[0050] When the content of the Cf48 low-molecular peptide in the detected biological sample is less than the first threshold and greater than the second threshold, next, it is detected whether TGF-β1 is present in the biological sample. When TGF-β1 is present in the measured biological sample, it indicates that the activity of renal fibrosis is strong.

[0051] When the content of the Cf48 low-molecular peptide in the detected biological sample is less than the second threshold, it indicates that the activity of renal fibrosis is weak, indicating that the activity of the progressing fibrosis in the kidney is low and the ability of renal fibrosis to progress is absent.

[0052] Here, the first threshold is greater than the second threshold. Note that the first threshold and the second threshold can be set according to the needs for various purposes. For example, the value range of the first threshold is 40 ng / mL or more, while the value range of the second threshold is from 0 ng / mL to 6 ng / mL.

[0053] The present invention can identify a diagnostic reagent kit for kidney injury by Cf48 low-molecular peptide, and can judge the situation of kidney injury by detecting the Cf48 content in kidney tissue and blood. It solves the defect of the prior art that depends basically on the combination of biopsy and pathology to judge kidney injury. At the same time, the degree of change of kidney injury can also be identified, and it can make up for the technical defect that the prior art could not judge the change ability of the situation of kidney injury. By using the Cf48 low-molecular peptide as a marker to conduct characteristic evaluation on the situation of kidney injury, the situation of kidney injury can be judged without biopsy symptoms, overcoming the limitations of various requirements of clinical diagnosis in the prior art, and it may be detected as one of the indicators for health diagnosis. The reagent kit of the present invention can be various immunological diagnostic kits such as immunohistochemistry, chemiluminescence, ELISA, Western blot, etc.

[0054] <Example 3> Hereinafter, the present invention will be described in more detail in conjunction with a specific experimental process.

[0055] Part 1, Acquisition of Cf48 1.1 Research purpose: Utilize the means of translatom sequencing to screen for new small peptides encoded by sORFs specific to the occurrence and progression of CKD.

[0056] 1.2 Experimental materials (1) The NRK-49F cell line and the human embryonic kidney cell (293T) cell line were purchased from ATCC (American Type Culture Collection, USA); (2) Streptozotocin, Sigma, USA; (3) Recombinant TGF-β1 stimulator, Peprotech, USA; (4) Serum-free expression medium, Thermo, USA; (5) RIPA cell lysate, Milipore, USA; (6) BCA reagent kit, Thermo, USA; (7) Antibodies: The α-SMA antibody was purchased from Sigma (USA), the GPADH antibody was purchased from SAB (China), and the goat anti-rabbit antibody was purchased from Cell Signaling Technology (USA).

[0057] 1.3 Research methods 1.3.1. Experimental animals eNOS- / - mice with a C57BL / 6 background were purchased from GemPharmatech Co., Ltd. The modeling of the diabetic nephropathy model was carried out at the Experimental Animal Center of Sun Yat-sen University (North Campus).

[0058] 1.3.2 Preparation of animal models Mouse model of induced progressive diabetes: Streptozotocin (STZ)-induced diabetic nephropathy mice of eNOS- / - mice. The method of continuous intraperitoneal injection for 5 days with a low dose was used to cause partial damage to the pancreatic islet cells of the mice. After 12 weeks (w refers to weeks) after the successful modeling, the mice that successfully induced DN (DN group) were sacrificed for examination, and the eNOS- / - mice were sacrificed for examination with the same dose of solvent as the normal control group (NC group) at the same time.

[0059] 1.3.3 Target of sequencing Translatome sequencing was performed on kidney tissues from 5 eNOS- / - mice with STZ-induced progressive diabetic nephropathy and 4 age-matched eNOS- / - normal control mice.

[0060] 1.3.4 Translatome sequencing Translatome sequencing and analysis were completed by entrusting to Guangzhou Genedenovo Biological Technology Co., Ltd. The main processes include translation inhibition fixation (addition of cycloheximide), RNase digestion, ribosome removal, ribosome footprint extraction, reverse transcription, database construction, quality control, and sequencing.

[0061] 1.3.5 Analysis steps (1) Calculate the translational abundance of ribosome footprints a) Alignment and identification of ribosome footprints (RF): Ribosome footprints are the locations where ribosome complexes slide and stay on RNA during translation. When translation is terminated, treating the ribosome-nascent peptide chain complex with low-concentration RNase retains only the fragments of mRNA covered by ribosomes. After ribosome removal, these small RNA fragments of about 30 bp protected by ribosomes are called ribosome footprints. Each RF represents the ribosome position of the transcript to be translated.

[0062] RFs were directly sequenced by next-generation sequencing to obtain RF reads. The RF reads were aligned to the mouse genome via the software bowtie2, and only the RF reads that could be uniquely aligned to genes were retained for subsequent quantitative analysis.

[0063] b) Calculation of the translational expression level of open reading frames (ORFs): Based on the RF alignment results, the abundance of RF reads in each ORF (the number of RF reads overlapping with the ORF) is further counted, and the expression level of the ORF is represented by RPKM (Reads Per Kilobase per Million mapped reads).

[0064] (2) Analysis of differences between ORF groups After calculating the abundance (RPKM) of RF reads in various ORFs, edgeR software is used to compare the ORFs with differential expression between the DN and NC groups, and the fold change (FC) is calculated. Genes with differential translation (differentially translated gene, DTG) are selected using FDR and log2FC, and the selection criteria are |log2FC| > 1 and FDR < 0.05.

[0065] (3) Evaluation of sORF encoding potential The following three criteria are used to comprehensively judge the potential of sORF encoding: a) ORFscore: Since RF follows a three-base rhythm and ribosomes pause temporarily at the three-base codons of RNA during translation, RF signals with the potential for translation concentrate in the main reading frame (also called Frame1) of the ORF sequence. The score of ORF score is based on the fact that RF has the potential for translation.

[0066] b) RRSscore: Since ribosomes are released from RNA when they encounter a stop codon, the abundance of RF with translation potential is significantly less downstream of the stop codon than in the upstream ORF region. The score of RRS score is based on the attributes of RF with potential.

[0067] c) Fickett score: Since various coding sequences and non-coding sequences have different sequence composition characteristics, the known coding sequences of this type can be used as a reference database to evaluate whether an unannotated sequence has coding ability. By using this method to calculate the Fickett score, generally, a Fickett score ≧ 0.74 is used as the criterion for the coding potential of RF.

[0068] Based on the criteria that the ORFscore is greater than the threshold, the RRSscore is greater than the threshold, and the Fickett score ≧ 0.74 are simultaneously satisfied, the potentially translatable sORFs are selected.

[0069] (4) ORF annotation Annotate ORFs with a translation potential with a translation difference multiple greater than 2 between two groups, align the potential protein sequences encoded by sORFs to the pfam database (version 26.0), and try to discover the protein domains potentially included in sORFs.

[0070] 1.3.6 Stimulation of fibroblasts with a medium overexpressing secreted small peptides The vector used for overexpression of the selected secreted small peptides and the control plasmid was pMSCV-IRES-GFP, and the plasmid synthesis was entrusted to GenScript Biotech Corporation. The synthesized plasmids for overexpressing secreted small peptides are Cf48-pMSCV-IRES-GFP, Hilpda-pMSCV-IRES-GFP, Apoc1-pMSCV-IRES-GFP. The synthesized control plasmid is pMSCV-IRES-GFP.

[0071] (1) Transfect the plasmids for overexpressing secreted small peptides and the control plasmid according to the operating procedure of Lipofectamine 3000. The specific procedure is as follows. a) The 293T tool cells were uniformly seeded in a 6-well cell plate with cell growth medium (DMEM / F12 + 10% FBS). b) After the cells adhered to the wall and grew uniformly and stably, the cell medium was replaced with serum-free medium, and 0.5 mL per well was used. c) Prepare the reaction solution in an amount of 500 μL of Opti-MEM + 2.5 μg of DNA + 3.75 μL of Lipofetamine 3000 + 5 μL of P3000 per well, mix well at room temperature for 15 minutes, and add 500 μL of the reaction solution containing the overexpression plasmid or the control plasmid to each well. d) 12 hours after the reaction, replace it with cell growth medium (DMEM / F12 + 10% FBS), and continue culturing in a cell culture incubator.

[0072] (2) The overexpression of the secreted small molecule peptide was collected. After 24 hours of transfection of the control: plasmid, the cells were observed with an upright fluorescence microscope. Bright green fluorescence of the cells was visible, and the density of the cells expressing green fluorescence was about 70%. The cell growth medium was replaced with serum-free expression medium. After 36 hours of reaction, the overexpression of the secreted small molecule peptide and the blank control medium were collected respectively, and the cell debris was discarded after centrifugation.

[0073] (3) The NRK-49F cells were uniformly seeded in a 6-well cell plate using cell growth medium (DMEM / F12 + 10% FBS).

[0074] (4) The overexpression of the collected secreted small molecule peptide and the control medium were added to NRK-49F cells with or without the addition of 1 ng / mL TGF-β1. After 48 hours of reaction, the cell proteins were collected, and the expression of α-SMA was observed by Western blotting.

[0075] 1.3.7 Cell Protein Extraction (1) Lysing proteins: Place a 6-well cell plate on ice, aspirate the culture medium supernatant, wash off cell debris with pre-chilled clean PBS, add 60 μL of cell lysate to each well, and incubate on ice for 15 min. (2) Using a cell scraper, scrape the protein in a specific direction in each well and transfer the cell lysate into a 1.5 mL EP tube. (3) Centrifuge at 15,000 rpm, 4°C for 30 minutes and aspirate the lysate supernatant for later use.

[0076] 1.3.8 Measuring protein concentration by the BCA method According to the operating instructions of the BCA reagent kit (Thermo), the specific steps are as follows: (1) After diluting the protein stock solution to the corresponding ratio, add 5 μL of protein and 5 μL of protein standard into each well of the corresponding 96-well plate. (2) Prepare working solution (solution A:solution B = 50:1) and add 200 μL of working solution to each well of protein and standard solution according to the formula. (3) Incubate at 37°C for 30 minutes. (4) The absorbance at a wavelength of 562 nm is measured using a multi-function microplate reader, and a standard curve is plotted according to the standard solution to calculate the protein concentration.

[0077] 1.3.9 Western blot detection of proteins (1) PAGE gel stripping According to the operating instructions of the PAGE gel rapid preparation kit (雅▲bai▼, the kanji for ▲bai▼ are the characters for "酉" and "和" combined on the left and right), a 10% separating gel and a laminating gel were prepared. The specific steps are as follows: a) Clean a 1.5 mm glass plate, fix it on the adhesive rack, fix it on the adhesive filling strip, then add ddH20 to check whether the glass plate is leaking and whether the liquid level is level. b) Prepare the lower gel buffer according to the ratio of the operation process: 4 mL of the lower gel solution + 4 mL of the lower gel buffer. After complete mixing, add 80 μL of the coagulant, mix uniformly, add a glass spray plate, and then slowly add anhydrous ethanol to flatten the lower gel. Leave it for about 15 minutes, and a clear boundary between the lower gel and anhydrous ethanol can be seen, which means the lower gel has condensed. Pour out the anhydrous ethanol and let it volatilize and dry at room temperature. c) Prepare the upper gel buffer according to the ratio of the operation process: 2 mL of the upper gel solution + 2 mL of the upper gel buffer. After complete mixing, add 40 μL of the coagulation accelerator, mix uniformly, add a glass plate, slowly insert a 1.5 mm 15-well comb obliquely, and wait for about 15 minutes until the upper gel condenses. The prepared PAGE gel is stored at 4°C in a wet state.

[0078] (2) SDS-PAGE electrophoresis Clamp the prepared PAGE gel into the vertical electrophoresis tank with electrophoresis clips, take out the denatured protein after trimming, heat it in a 100°C air bath for 5 minutes to completely melt the protein, then take it out and use it later. Pour the prepared 1x solution into the electrophoresis tank, slowly and gently remove the comb, add 30 μL of protein to each well, and add 5 μL of protein molecular weight indicator to both ends of the PAGE gel. Start electrophoresis. First, run the gel at a constant voltage of 90 V and wait until all protein samples enter the separation gel. After the protein bands become narrow horizontal lines, increase the voltage and adjust it to a constant voltage of 120 V, and stop electrophoresis until all protein indicators enter the electrophoresis solution.

[0079] (3) Membrane transfer Immersion in methanol activates the PVDF membrane. Prepare the membrane transfer "sandwich" device: from the negative electrode to the positive electrode, use thick filter paper, separation gel, PVDF membrane, thick filter paper, and a roller to drive out the bubbles, cover the electrode cover, and it is an ice bath device. Transfer at a constant voltage of 100 V for 120 minutes.

[0080] (4) Sealing: Prepare 5% milk (non-fat dry milk in TBST). After the transfer is completed, the PVDF membrane is washed with ddH2O and sealed with 5% milk at room temperature for 1 hour.

[0081] (5) Incubation with primary antibody: Dilute the primary antibody with 5% milk (1:1000) and incubate overnight on a shaker at 4°C.

[0082] (6) Membrane washing: After the primary antibody is recovered, wash the membrane 3 times with TBST shaker for 10 minutes each.

[0083] (7) Incubation with secondary antibody: Dilute the secondary antibody with 5% milk (1:5000) and incubate at room temperature for 1 hour.

[0084] (8) Membrane washing: After the secondary antibody is recovered, wash the membrane 3 times with TBST shaker for 10 minutes each.

[0085] (9) Development: Under light-avoiding conditions, uniformly drip the chemiluminescent solution onto the PVDF membrane, react at room temperature for 3 minutes, place the PVDF membrane in an automatic chemiluminescence imager, develop the image in the automatic exposure mode. If the signal is too strong or too weak, change to the manual exposure mode for development.

[0086] (10) Protein stripping: Add protein stripping buffer to the PVDF membrane, wash on a shaker for 15 minutes, then wash with TBST for 5 minutes, seal with 5% milk at room temperature for 1 hour, and continue to incubate with the next antibody.

[0087] (11) Result analysis: Quantitatively analyze the gray value of the target protein band by an automatic chemiluminescence imaging system, calculate the relative content of the target protein using the internal reference protein as an internal control, and perform statistical analysis.

[0088] 1.4 Experimental results 1.4.1 Differentially expressed genes between progressive diabetic nephropathy mice and normal control mice in the NC group In ORFs with encoding potential, differentially expressed genes between progressive diabetic nephropathy mice (DN) and normal control mice (NC) were selected based on the selection criteria (FDR < 0.05, |log2FC| > 1). As a result, 1981 significantly differentially expressed genes were found between the two groups, of which 1622 were significantly up-regulated genes and 359 were significantly down-regulated genes. There were significant differences in the overall gene expression patterns between the DN group and the NC group. As shown in Figure 1-1, most genes in the DN group showed an up-regulated expression pattern, while the NC group showed the opposite result. The expression patterns of the samples belonging to the NC group (NC1-NC4) and the samples belonging to the DN group (DN1-DN5) were similar, indicating that the samples of NC mice and DN mice had good reproducibility.

[0089] 1.4.2 Selection criteria for target genes Target genes are selected based on the following selection criteria. (1) Target genes need to have good conservation: Since the experiment was sequenced in mouse kidney tissue, in order to enhance the clinical application value, the research object needs to have good conservation and be able to be expressed in both humans and mice. According to the database, 39 out of 1981 differentially expressed genes have good conservation and can be expressed in both humans and mice (39 / 1981). (2) Target genes can be translated into small peptides: Due to the advantages of translatome sequencing, it is expected that new small peptides related to diseases can be selected. Among the 39 genes with good conservation, 11 genes can be translated into small peptides with amino acid length < 100 AA (11 / 39). (3) Target genes must be secreted small peptides: Further, using software (SignalP-5.0), 3 out of 11 small peptides are predicted to be secreted small peptides (3 / 11). (4) The target gene has potential biological functions: Among the three differentially expressed secreted peptides, in vitro experiments are conducted to pre-confirm whether the secreted peptide has biological functions.

[0090] 1.4.3 Verification of the fibrotic promotion effect of the secreted small peptide The expression of the Cf48 secreted small peptide obtained by the screening conditions in 1.4.2 was increased in the kidney tissues of mice with diabetic nephropathy: (1) Cf48 (MGI:2686519, DN / NC: Log2(FC)=2.96, FDR=0.046, 90aa). Considering the secretion characteristics of the small peptide, the following external experiments were designed. An overexpression plasmid of the secreted small peptide with increased Cf48 expression in control and diabetic kidney mice was transfected into the tool cells. After complete expression, the supernatants of the blank vector and the small peptide overexpression medium were collected. Fibroblasts (NRK-49F) were stimulated under the conditions with or without TGF-β1 to observe the expression of α-SMA in the fibroblasts, and the results are shown in Figure 1-2 and Figure 1-3. According to Figure 1-2 and Figure 1-3, it can be seen that the supernatant of the overexpression medium containing Cf48 enhanced the activation response of TGF-β1-induced fibroblasts and increased the expression of α-SMA compared with the conditioned medium of the blank control vector.

[0091] 1.4.4 The small peptide CF48 has high conservation The mouse gene Cf48 (gene name: mouse Gm1673, MGI: 2686519, NCBI gene ID: 381633) is located on chromosome 5 (Chr5: 34,140,777 - 34,142,353), consists of 4 exons, can translate a low-molecular-weight peptide with an amino acid length of 90aa, and the amino acid sequence is SEQ:2. The human gene Cf48 (NCBI gene ID: 401115) is located in the open reading frame at the 48th position on chromosome 4 (Chr4: 2,035,610 - 2,043,970), consists of 9 exons, can be translated into a low-molecular-weight peptide with an amino acid length of 90aa, and the amino acid sequence is SEQ:1. Comparing the gene structures of Cf48 in mouse and human, the gene structures of Cf48 in human and mouse are similar and have excellent gene homology. From the results of multiple sequence alignment of the Cf48 protein sequence among multiple species, it was concluded that the low-molecular-weight peptide Cf48 has a high degree of conservation among species.

[0092] The amino acid sequence of mouse Cf48 is: MAPALRSLLSPRTLLLLLLSLALLGARA EPATGSAVPAQSRPCVDCHAFEFMQRALQDLRKTAYSLDARTETLLLQAERRALCACWPAGR (90aa). The cDNA sequence of mouse Cf48 is: ATGGCCCCCGCGCTCAGGTCCTTGCTGTCGCCACGGACTTTACTGCTGCTGCTGCTGAGCCTGGCGCTGCTGGGCGCCCGCGCC GAGCCCGCCACCGGGAGCGCTGTCCCCGCTCAGAGCCGCCCGTGCGTGGATTGCCACGCATTTGAATTCATGCAGCGCGCCCTTCAGGACCTACGGAAAACCGCCTACAGCCTGGACGCACGGACGGAGACCCTCCTGCTGCAGGCTGAGCGCCGGGCTCTGTGTGCCTGCTGGCCAGCTGGACGCTGA. (Note: Underlined part: signal peptide, other sequences: secreted peptide).

[0093] 1.4.5 The low-molecular-weight peptide Cf48 is a secreted low-molecular-weight peptide The secretion potential of the small peptide Cf48 was predicted by SignalP-5.0 (http: / / www.cbs.dtu.dk / services / SignalP-5.0 / ), and the result showed that the small peptide Cf48 is a secreted small peptide in both mice and humans, and the cleavage sites of the signal terminus and the secretion terminus are the same. The cleavage position is between amino acids 28 and 29 in mice and between amino acids 34 and 35 in humans, and it can be cleaved into a signal peptide located intracellularly and a secreted peptide secreted extracellularly.

[0094] 1.4.6 The Cf48 overexpression medium enhances the TGF-β1-induced fibroblast activation response As described above, according to the results in Figures 1-3, the Cf48 overexpression conditioned medium enhanced the expression of TGF-β1-induced α-SMA and promoted the activation of fibroblasts compared with the blank control. Secreted Cf48 has a biological function of promoting fibrosis.

[0095] 1.4.7 From the above experiments, the following conclusions can be drawn: Translatome sequencing of diabetic nephropathy mice showed that the expression of the small peptide Cf48 was significantly increased in the kidney tissues of progressive diabetic nephropathy mice. The overexpression medium containing secreted Cf48 promotes the activation of TGF-β1-induced fibroblasts and promotes the activation of fibroblasts

[0096] Part 2. Expression status of the small peptide Cf48 in patients with chronic kidney disease 2.1 Research purpose: Analyze the expression of Cf48 in the renal tissues and sera of chronic kidney disease (CKD) patients with different etiologies, and explore the correlation between Cf48 and the disease activity and the severity of renal injury.

[0097] 2.2 Experimental materials 2.2.1 Main instruments and equipment for the experiment (1) Multifunctional microplate reader, Molecular Devices, USA; (2) Automatic plate washer, Biorad, USA; (11) Vortex mixer XW-80A, Shanghai Medical Instrument Factory, China. 2.2.2 Main consumables for the experiment (1) Immunohistochemical pen, Zhongshan Golden Bridge, China; (2) Positively charged glass slides, Thermo, USA; (3) Cover glass, Guangzhou Weijia Technology Co., Ltd., China. 2.2.3 Main reagents for the experiment (1) PBS powder, Guangzhou Weijia Technology Co., Ltd.; (2) BSA powder, Guangzhou Weijia Technology Co., Ltd.; (3) 30% hydrogen peroxide, Guangzhou Weijia Technology Co., Ltd.; (4) Immunohistochemistry kit, Dako, Denmark; (5) Mayer hematoxylin, Abcam, USA; (6) Neutral resin, Hesi Biology, China; (7) DAPI, Thermo, USA; (8) Fluorescent mounting medium, Dako, Denmark; (9) 50X antigen retrieval solution, Guangzhou Weijia Technology Co., Ltd.; (10) Cf48 ELISA kit, C USA BIO, China; (11) Antibodies: The anti-Cf48 antibody was purchased from Abcam (USA), the directly labeled α-SMA-cy3 antibody, and the anti-α-SMA antibody was purchased from Sigma (USA), and the Alexa Fluro 488-labeled goat anti-rabbit-IgG was purchased from life technology (USA).

[0098] 2.3 Research methods 2.3.1 Research subjects 2.3.1.1 Clinical research ethics This study was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University, and all patients, diseases, and healthy controls signed the informed consent form, and the ethics approval number was "Sun Yat-sen University First Affiliated Hospital Ethics Review (2016) No. 215".

[0099] 2.3.1.2 Selection criteria and exclusion criteria for CKD patients Cases confirmed by renal biopsy in the Department of Nephrology, the First Affiliated Hospital of Sun Yat-sen University are included, and the expression of Cf48 in renal tissue and the content of free Cf48 in serum are detected. CKD diseases include diabetic nephropathy (DN), lupus nephritis (LN), IgA nephropathy (IgAN), etc. Patient selection criteria: Patients who underwent renal needle biopsy in the Department of Nephrology, the First Affiliated Hospital of Sun Yat-sen University from September 2018 to September 2021 were included; the results of renal puncture were consistent with any of DN, LN, or IgAN; aged 18 - 65 years, male or female, CKD1 - 5, and with the intention to sign an informed consent. Exclusion criteria for patients: Severe complications such as malignant tumors, heart failure, malignant hypertension, cerebrovascular disorders, etc.

[0100] 2.3.1.3 Selection criteria for disease control group and healthy control group Selection criteria for diabetic nephropathy disease control group (DM): Aged 18 - 65 years, diagnosed with diabetes revised by the American Diabetes Association (ADA) in 2020, and without accompanying symptoms of kidney damage. With the intention to sign an informed consent form. Selection criteria for healthy control group: Aged 18 to 65 years, normal renal function, without microscopic hematuria and proteinuria, without recent infections, and without a history of serious diseases. With the intention to sign an informed consent form.

[0101] 2.3.1.4 Sample collection All CKD patients and control groups had 5 ml of peripheral venous blood collected, centrifuged, and the separated serum was frozen at -80 °C for later use. Renal tissue sections of CKD patients were obtained from paraffin sections of renal biopsies, and normal control sections were obtained from paraffin sections of adjacent normal renal tissues of kidney cancer patients.

[0102] 2.3.1.5 Collection of clinical information (1) Demographic data: Collected the gender, age, and other demographic data of the selected CKD patients, DM disease controls, and healthy controls. (2) Disease severity indicators: The main course of the disease, history of hypertension, diabetes, and cardiovascular disease of the selected CKD patients were collected; serum urea nitrogen (BUN), serum creatinine, blood albumin, blood uric acid, fasting blood glucose, glycosylated hemoglobin, and 24-hour urinary protein indicators were collected; eGFR was calculated using the simplified MRDR formula. (3) Pathological indicators: a) Diabetic nephropathy patients: According to the pathological histological diagnosis of renal biopsy, using the pathological classification proposed by Tervaet et al., DN patients were classified into grade 2, grade 3, and grade 4. b) Lupus nephritis patients: Based on the pathological histological diagnosis of renal biopsy, the pathological classification of ISN / RPS was used. c) IgA nephropathy patients: According to the pathological histological diagnosis of renal biopsy, using Lee’s Grading System, IgA nephropathy patients were divided into two groups according to Lee's grade ≤ 3 and Lee's grade > 3. The Lee grading criteria are based on the Lee grading criteria in 1982.

[0103] 2.3.2 Fluorescent in situ hybridization staining (Cf48) Fluorescent in situ hybridization staining was performed on paraffin sections (5 μm) of DN patients and normal renal tissues adjacent to renal cancer, and probe synthesis and in situ hybridization staining were completed by entrusting Guangzhou Saiwei'er Biotechnology Co., Ltd.

[0104] 2.3.3 Immunohistochemical staining (Cf48, α-SMA) (1) Bake the slices: Paraffin slices with a thickness of 5 μm were baked in an oven at 60 °C for 1 hour until the wax of the slices was completely removed. (2) Deparaffinize and hydrate in the following order: Xylene (20 minutes, 2 times), absolute ethanol (10 minutes, 2 times), 95% ethanol (10 minutes, 2 times), 75% ethanol (10 minutes, 2 times), PBS buffer (5 minutes, 1 time). (3) Antigen activation: Prepare 1X antigen activation solution (sodium citrate salt, 0.01 mol / L, pH = 6), activate it in an autoclave, boil it to the maximum boiling point of the autoclave, and then let it cool naturally to room temperature. The whole process takes about 3 hours. Use PBS buffer for 5 minutes to wash away the antigen activation solution. (4) Draw a circle with a histochemical pen: Wipe the water around the tissue, gently draw a circle with an immunohistochemical pen to completely surround the tissue, and place it in a wet box. During this period, the drying of the tissue slice should be avoided. (5) Catalase inactivation: Drop 50 μL of 3% hydrogen peroxide solution into each histochemical circle, incubate at room temperature in the dark for 15 minutes, place it in PBS buffer on a shaker for 5 minutes, and wash 3 times. (6) Blocking: Drop 50 μL of 5% BSA (prepared with PBS) solution into each histochemical circle and block at room temperature for 10 minutes. (7) Primary antibody incubation: Dilute the primary antibody with PBS buffer at the corresponding concentration (Cf48, 1:100, α-SMA, 1:1000) and incubate at room temperature for 1 hour. (8) Secondary antibody incubation: Place it in PBS buffer on a shaker for 10 minutes and wash 3 times. Drop 50 μL of solution A (a combination of pica and secondary antibody) of the immunohistochemistry kit into each histochemical circle and incubate at room temperature in the dark for 1 hour. (9) DAB color development: Place it in PBS buffer on a shaker for 10 minutes and wash 3 times. Prepare the DAB reaction working solution by mixing solutions B and C of the immunohistochemistry kit at a ratio of 50:1, drop it into the histochemical circle, observe the reaction rate under a microscope, and wash it away with ddH20 after the reaction is completed. (10) Hematoxylin counterstaining: Immerse the section in Mayer's hematoxylin for 3 minutes and then wash it away with ddH20. (11) Conventional dehydration and mounting in the following order: 75% ethanol (5 minutes, 2 times), 95% ethanol (5 minutes, 2 times), absolute ethanol (5 minutes, 2 times), xylene (5 minutes, 2 times), neutral resin mounting. (12) Observe with an upright microscope and take pictures.

[0105] 2.3.4 Immunofluorescence staining (Cf48, α-SMA) (1) Paraffin sections with a thickness of 5 μm are baked, dewaxed, hydrated, and the antigen is activated in the usual way. The step of drawing a circle with a histochemical pen is the same as 2.3.3(1)-(4). (2) Blocking: 50 μL of 1% BSA (prepared with PBS) solution is dropped into each histochemical circle and blocked at room temperature for 1 hour. (3) Incubation with primary antibody: The primary antibody is diluted with 1% BSA solution at the corresponding concentration (Cf48, 1:50, α-SMA, 1:5000) and incubated overnight at 4°C in the dark. (4) Incubation with secondary antibody: Place it on a shaker in PBS buffer for 10 minutes, wash 3 times, dilute the corresponding secondary antibody with 1% BSA solution at 1:1000, and incubate at room temperature for 1 hour. (5) DAPI counterstaining: Place it on a shaker in PBS buffer for 10 minutes, wash 3 times, and incubate DAPI at a concentration of 1:100 at room temperature for 5 minutes. (6) Mounting: Place it on a shaker in PBS buffer for 10 minutes, wash 3 times, rotate the water, then drop the mounting medium, cover with a coverslip, and dry overnight at room temperature. (7) Observe with a laser confocal microscope and take pictures.

[0106] 2.3.5 Measurement of the immunostaining area Glass slides that require quantification are scanned with a fully automatic scanner. By the single-blind method, 10 renal cortex regions are randomly selected under a high magnification (×200) field of view. The expression intensity of the positive signal is quantified by Image j (http: / / www.imagej.nih.gov / ij / ). The ratio of the positive expression intensity in each visual field was used as the area of one visual field and taken as the expression level of the target molecule, and the average expression level of 10 visual fields was taken as the expression level of the target molecule (Cf48, α-SMA).

[0107] 2.3.6 Detection of serum Cf48 content Follow the procedure of the Cf48 ELISA kit, and the specific steps are as follows. (1) All reagents are returned to room temperature 30 minutes in advance. (2) Prepare the standard: Through sample dilution, dissolve the standard to a stock solution concentration of 40 ng / mL, and perform serial dilutions to obtain standards with concentrations of 40, 20, 10, 5, 2.5, 0.625, and 0 ng / mL respectively. (3) Plate incubation: Add 100 μL of the standard solution and serum for each experimental group to each well, attach a film, gently shake, and incubate in an incubator at 37 °C for 2 hours. (4) Discard the supernatant directly, gently tap the liquid remaining on the paper, add 100 μL of biotinylated biotin antibody per well, attach a film, gently shake, and incubate in an incubator at 37 °C for 1 hour. (5) Plate washing: Using an automatic plate washer, add 300 μL of washing solution to each well, aspirate the washing solution, repeat the residual liquid 3 times, and then gently tap on the paper to dry. (6) Add 100 μL of horseradish peroxidase - biotin complex (HRP - avidin) to each well, attach a film, gently shake, and incubate in an incubator at 37 °C for 1 hour. (7) Wash the plate: Repeat the same procedure (6) 5 times, and gently tap the liquid remaining on the paper to dry. (8) Add 90 μL of TMB substrate chromogenic solution to each well, and incubate in the dark in an incubator at 37 °C for 30 minutes until the liquid in the sample and standard wells gradually turns blue. (9) Add 50 μL of stop solution to each well, and gently mix until the liquid changes from blue to yellow. (10) The microplate reader reads the absorbance (OD value) at a wavelength of 450 nm. (11) Draw a standard curve and calculate the sample concentration.

[0108] 2.3.7 Statistical analysis Data are presented as mean ± standard deviation, and data analysis was performed using SPSS 26.0 software and GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA). Student’s t test was used for comparison between two groups, analysis of variance (ANOVA) was used for comparison among multiple groups, and then multiple comparisons among three or more groups were performed by Tukey’s Post-test. Correlation analysis was performed using Spearman correlation analysis. A P value < 0.05 was defined as statistically significant.

[0109] 2.4 Experimental results 2.4.1 Upregulation of Cf48 mRNA expression in the kidney tissue of DN patients To clarify the expression of Cf48 in clinical patients, fluorescence in situ hybridization (FISH) staining was performed on renal biopsy tissues and adjacent normal tissues of renal cancer in DN patients to clarify the localization and expression of Cf48 mRNA in DN patients. As shown in Figure 2-1, compared with normal renal tissues, the expression of Cf48 mRNA in DN patients mainly increased significantly in renal tubules, and the expression was high in significantly damaged and dilated tubules, but was high in glomeruli. The expression of Cf48 mRNA increased significantly in damaged tubules of diabetic kidney patients, and the expression of Cf48 mRNA was not seen in glomeruli and normal renal tissues of diabetic nephrosis patients, confirming that this low molecular weight peptide Cf48 is produced by damaged tubular cells.

[0110] 2.4.2 The expression of Cf48 increases in the kidney tissue of DN patients Immunohistochemical staining was used to detect the expression of Cf48 in DN patients and normal kidney tissues. As shown in Figure 2-2, Cf48 was significantly increased in the kidney tissues of DN patients, and the positive signals were concentrated in the tubulointerstitium. Furthermore, when the expression intensity of Cf48 in the tubulointerstitium was statistically analyzed, it was shown that the expression intensity of Cf48 in the tubulointerstitial region of DN patients was significantly higher than that in normal kidney tissues, and the difference was statistically significant (P<0.001).

[0111] 2.4.3 The expression of Cf48 is increased in the kidney tissues of CKD patients The expression of Cf48 in the kidney tissues of CKD patients with other etiologies was further detected to determine whether the increased expression of the low-molecular-weight peptide Cf48 in the kidney tissues is specific or non-specific to diabetic nephropathy. The expression of Cf48 was detected by immunofluorescence in the adjacent normal kidney tissues of renal cancer adjacent to normal kidney cancer, the renal biopsy tissues of patients with minimal change nephropathy (MCD) as a disease control, and the renal biopsy tissues of CKD patients (including LN, IgAN, DN). The results are shown in Figure 2-3. According to the results of Figure 2-3, the expression levels of Cf48 in the kidney tissues of normal controls and MCD patients were low, there was no significant expression, and the α-SMA staining was not increased. However, the expression of Cf48 in the kidney tissues of CKD patients including LN, IgAN, and DN patients was significantly increased, the signals were mainly located in the tubular epithelial cells, and the expression of α-SMA was more significant. It can be seen that the expression of the low-molecular-weight peptide Cf48 is increased in the kidney tissues of CKD patients with different etiologies and is correlated with the expression of the fibrosis index α-SMA. The low-molecular-weight peptide Cf48 can be used as a marker for nephropathy and characterize the degree of nephropathy and fibrosis.

[0112] 2.4.4 Secreted Cf48 is increased in the sera of CKD patients Since Cf48 is a small secreted peptide, its serum content in healthy volunteers (CTL), disease management without diabetic nephropathy (DM), and patients with diabetic nephropathy (DN) was detected using an ELISA kit. As shown in Figures 2-4, the serum Cf48 content in DN patients was significantly higher than that in the normal control group (9.678±4.661 vs 1.243±1.566, P<0.0001) and also increased significantly compared with DM patients (9.678±4.661 vs 1.626±1.150, P<0.0001). However, there was no significant difference in serum Cf48 content between the normal control group and DM patients. These results suggest that the secreted small peptide Cf48 is a key factor promoting the progression of kidney disease and its level is independent of the patient's glucose tolerance level. Therefore, the serum Cf48 content in CKD patients with other non-DN etiologies was further detected. As a result, the serum Cf48 content in IgAN and LN patients was 7.131 ng / mL and 8.752 ng / mL respectively, which were significantly higher than those in the normal controls (P<0.0001). These results suggest that the content of the small peptide Cf48 in serum increases in CKD patients with different etiologies, and its upregulation is related to the progression of kidney disease but not to its etiology, indicating that it may be involved in the general mechanism of CKD progression.

[0113] 2.4.5 Serum secreted Cf48 content in CKD patients is significantly negatively correlated with eGFR The correlation between the serum-secreted Cf48 content and eGFR in CKD patients was analyzed, and the results are shown in Figure 2-5A. According to Figure 2-5A, the serum-secreted Cf48 content in CKD patients showed a significant negative correlation with eGFR (correlation coefficient r = -0.496, P < 0.0001, n = 73). Figure 2-5B shows the relationship between the serum-secreted Cf48 content and the CKD grade in CKD patients. The serum Cf48 content in CKD patients reflects the CKD stage of the patients and shows a significant positive correlation (correlation coefficient r = 0.625, P < 0.0001, b = 73). That is, the higher the Cf48 content, the higher the CKD stage. It can be seen that the serum-secreted Cf48 content can characterize the degree of nephropathy and the CKD stage of the patients.

[0114] 2.4.6 There is a significant positive correlation between the serum-secreted CF48 content and the degree of renal fibrosis To explore the relationship between the serum Cf48 content and the degree of renal fibrosis in patients, pathological slides of DN patients who underwent renal biopsy in the Department of Nephrology, the First Affiliated Hospital of Sun Yat-sen University were taken out, and the degree of renal fibrosis in the patients was reflected by α-SMA immunohistochemical staining, and a correlation analysis with the serum Cf48 content was performed. As shown in Figure 2-6, the serum Cf48 content in DN patients showed a significant positive correlation with the expression intensity of α-SMA in kidney tissues (correlation coefficient r = 0.815, P < 0.0001, n = 16). Among the representative 2 cases, the serum Cf48 content of patient 1 was 15.128 ng / mL, and the positive area of α-SMA staining was 14.94%, while the Cf48 content and the positive area of α-SMA staining of patient 2 were 3.718 ng / mL, and the positive area of -SMA staining was 3.78%. It can be seen that the Cf48 content can characterize the degree of fibrosis of kidney tissues and can be used as a marker for characterizing the degree of fibrosis of kidney tissues.

[0115] 2.47 Correlation analysis of serum-secreted Cf48 content and clinical indicators in DN patients Next, the CKD patients included in this study were stratified and analyzed according to the type of disease. The correlation between the serum-secreted Cf48 content and clinical indicators in DN patients was analyzed, and the results are shown in Table 2-1. According to Table 2-1, the serum-secreted Cf48 content in DN patients was significantly positively correlated with blood urea nitrogen and serum creatinine (correlation coefficient r = 0.514, P = 0.002; correlation coefficient r = 0.446, P = 0.009), significantly negatively correlated with the patients' eGFR (correlation coefficient r = -0.446, P = 0.009), and also positively correlated with the CKD stage (correlation coefficient r = 0.669, P < 0.0001). Figure 2-7 shows that the higher the serum-secreted Cf48 content, the higher the CKD stage in DN patients. Furthermore, in the correlation analysis between the serum Cf48 content and the pathological grade of DN patients, the higher the serum Cf48 content in DN patients, the more severe the pathological grade of the renal tissue, and the two were significantly positively correlated (correlation coefficient r = 0.385, P = 0.027).

[0116]

Table 2-1

[0117] 2.4.8 Correlation analysis between serum-secreted Cf48 content and clinical indicators in LN patients The correlation between the serum-secreted Cf48 content and clinical indicators in LN patients was analyzed, and the results are shown in Table 2-2 and Figure 2-8. As shown in Figure 2-8, the serum-secreted Cf48 content in LN patients was significantly positively correlated with blood urea nitrogen and serum creatinine (correlation coefficient r = 0.610, P = 0.004; correlation coefficient r = 0.669, P = 0.002), negatively correlated with eGFR (correlation coefficient r = -0.609, P = 0.004), and positively correlated with the CKD stage (correlation coefficient r = 0.693, P < 0.0001). In the correlation analysis between the serum Cf48 content and the ISN / RPS pathological grade in LN patients, no significant correlation was found (correlation coefficient r = -0.097, P = 0.731).

[0118]

Table 2-2

[0119] 2.4.9 Correlation analysis between serum secreted Cf48 content and clinical indicators in IgAN patients The correlation between serum secreted Cf48 content and clinical indicators in IgAN patients was analyzed, and the results are shown in Table 2-3. The results showed that the serum secreted Cf48 content in IgAN patients was significantly positively correlated with the Lee's grade in renal tissue biopsy results (correlation coefficient r = 0.564, P = 0.023). It has been shown that the higher the serum Cf48 content of the patients, the higher the Lee's grade in renal puncture results (see Figure 2-9).

[0120]

Table 2-3

[0121] 2.4.10 Expression of Cf48 in mouse lung and heart fibrous tissues To further clarify the expression of Cf48 in other fibrotic organs, the expression of Cf48 in mouse lung fibrous tissue was detected 4 weeks after bleomycin induction. Masson Chrome staining shows normal lung (Figure 2-10A) and fibrotic lung (Figure 2-10B). Immunohistochemistry shows that, as shown in Figure 2-10C, the expression of Cf48 is not detected in the lungs of normal mice. The expression of Cf48 at a few scattered dots could only be detected in a part of the lung tissue with obvious fibrosis, as shown in Figure 2-10D. As a result of immunohistochemistry, as shown in Figure 2-10E, the expression of Cf48 is not detected in the hearts of normal mice, and the expression of Cf48 in fibrotic hearts is also low (Figure 2-10F), indicating that only a few scattered dots are expressed.

[0122] 2.4.11 Expression level of CF48 in peripheral blood of patients with lung diseases To further determine whether Cf48 is elevated in the peripheral blood of patients with pulmonary fibrosis, samples with KL-6 test values exceeding 800 were examined using a Cf48 Elisa reagent kit (KL-6 is an indicator of interstitial lung disease, representing hyperplasia of type II alveolar epithelial cells, and is usually less than 500 in normal individuals). Elisa showed that the level of Cf48 in the peripheral blood of patients with KL-6 values exceeding 800 was 1.07 ± 0.64 ng / ml (mean ± SD, n = 57), while the level of Cf48 in the control of other lung diseases (including pneumonia, pulmonary embolism, lung cancer, etc.) was 0.72 ± 0.52 ng / ml (mean ± SD, n = 32). This strongly suggests that the level of Cf48 in the peripheral blood of patients with renal fibrosis is significantly higher than that of patients with lung diseases including pulmonary fibrosis and non-pulmonary fibrosis, suggesting that the elevation of Cf48 can be used as a specific indicator of renal fibrosis.

[0123] 2.5 Summary In conclusion, the expression of the low-molecular-weight peptide Cf48 was significantly increased in the renal tissues of CKD patients with different etiologies. The content of the secreted low-molecular-weight peptide Cf48 was significantly increased in the sera of CKD patients, showing a negative correlation with the patients' eGFR and a positive correlation with the degree of renal fibrosis and the severity of renal pathology. In the lungs and hearts of normal mice, the expression of Cf48 was hardly detected by immunohistochemistry, the expression of Cf48 in bleomycin-induced pulmonary fibrosis tissue could be detected in trace amounts at almost negligible levels, and the expression of Cf48 in angiotensin II-induced cardiac fibrosis tissue was only slightly visible. The level of Cf48 in the peripheral blood of patients with lung diseases was not elevated, and the elevated Cf48 can be used as a specific indicator of renal fibrosis.

[0124] Part 3, Role and Mechanism of Low-Molecular-Weight Peptide Cf48 in Renal Fibrosis and Inflammation in Mice 3.1.1 Research Objective: To investigate the effect of overexpression of Cf48 on renal fibrosis and inflammation in the disease progression of mice. 3.1.2 Experimental Materials 3.1.2.1 Experimental animals: C57BL / 6 was purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd., and the Cf48 transgenic parent mice were entrusted to Guangzhou Saiye Biotechnology Co., Ltd. for breeding.

[0125] 3.1.2.2 Main experimental reagents (1) Streptomycin, Sigma, USA; (2) Folic acid, Sigma, USA; (3) Mouse genotype rapid identification kit, Guangzhou Novizan Biotechnology Co., Ltd.; (4) 50X TAE solution, Guangzhou Jingxin Biotechnology Co., Ltd.; (5) Agarose, Sigma, USA; (6) Nucleic acid stain, Guangzhou Novizan Biotechnology Co., Ltd.; (7) PAGE gel rapid preparation kit, Ya ▼ bai ▼ (the Chinese characters of ▼ bai ▼ are the combination of "you" and "mei" on the left and right), China; (8) 10X electrophoresis solution: 144 g Glycine + 30.2 g Tris base + 10 g SDS in 1000 mL ddH20; (9) 10X transfer solution: 144 g Glycine + 30.2 g Tris base in 1000 mL ddH20; (10) 1X TBST solution: TBST powder (Guangzhou Weijia Biotechnology Co., Ltd.) + 2 mL Tween-20 in 2000 mL ddH20; (11) Tween-20, Sigma, USA; (12) Protein stripping buffer, Thermo, USA; (13) Non-fat dry milk, BD, USA; (14) BSA powder, Guangzhou Weijia Biotechnology Co., Ltd.; (15) 2×SDS loading buffer, Sigma, USA; (16) Molecular weight marker of stained protein, Thermo, USA; (17) RIPA protein lysate, Millipore, USA; (18) Protease inhibitor, Cell Signaling Technology, USA; (19) ECL luminescent solution, Millipore, USA; (20) Horseradish peroxidase (HRP)-labeled donkey anti-mouse IgG, Cell Signaling Technology, USA; (21) Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG, Cell Signaling Technology, USA; (22) PVDF membrane, Millipore, USA; (23) Trizol Reagent, Invitrogen, USA; (24) DEPC water, Guangzhou Weijia Biotechnology Co., Ltd.; (25) Chemical reagents: chloroform, isopropanol, absolute ethanol, 95% ethanol, 75% ethanol, methanol, xylene, etc. are all pure reagents for analysis and were purchased from the Second Factory of Guangzhou Chemical Reagents;(26) Transcriptor First Strand cDNA Synthesis Kit, Roche, Switzerland; (27) Sybrgreen real-time PCR kit, Roche, Switzerland; (28) Alexa Fluro 488-labeled goat anti-mouse IgG, Life Technology, USA (29) Alexa Fluro 488-labeled goat anti-rabbit IgG, Life Technology, USA (30) Alexa Fluro 488-labeled donkey anti-goat IgG, Life Technology, USA; (31) Alexa Fluro 555-labeled goat anti-rabbit IgG, Life Technology, USA (32) Alexa Fluro 647-labeled goat anti-rabbit IgG, Life Technology, USA; (33) DAPI, Thermo, USA; (34) Fluorescent mounting medium, Dako, Denmark; (35) Immunohistochemistry kit, Dako, Denmark; (36) Neutral resin, Hesi Biology, China; (37) Cystatin-C ELISA kit, Abcam, USA; (38) Albumin ELISA kit, Abcam, USA; (39) Cretanine ELISA kit, R&D, USA, (40) Masson staining kit, Sigma, USA; (41) PAS staining kit, Wuhan Bolf Biotechnology Co., Ltd.; (42) Antibodies: Anti-ColIV antibody was purchased from Southern Biotech (USA); Anti-ColI antibody was purchased from Boster (China); Anti-Fn antibody was purchased from Abcam (USA); Anti-F4 / 80 antibody and anti-α-Tubulin antibody were purchased from Cell Signaling Technology (USA); Directly labeled anti-α-SMA-Cy3 antibody and anti-α-SMA antibody were purchased from Sigma (USA); (43) The rabbit anti-mouse Cf48 antibody used in the Western blotting experiment was customized by entrusting Hangzhou Mingyan Biotechnology Co., Ltd. The recognized antigen sequence is RALQDLRKTAYSLDARTETLLLQAE.;

[0126] 3.1.3 Research methods 3.1.3.1 Breeding of genetically modified mice A pair of male and female Cf48 transgenic mice (Cf48-Tg) with C57BL / 6 background was prepared by Guangzhou Saiye Biotechnology Co., Ltd. Through vector construction, purification, and infection reagents, they were injected into fertilized eggs, and then transplanted into the oviducts of surrogate mother mice. After PCR identification, the first positive rats were obtained. Subsequently, they were bred at the Experimental Animal Center of Sun Yat-sen University, and Cf48-Tg was mated with Cf48-WT mice. The obtained offspring were identified by PCR to obtain Cf48-Tg and Cf48-WT mice suitable for the experiment.

[0127] The Tg and WT mice used in the experiment were 8-10-week-old male mice with a body weight of 25-30 g, and were bred at the Experimental Animal Center of Sun Yat-sen University. The environment was specific pathogen-free animal grade (SPF), provided with standard feed and clean water, given a 12-hour / 12-hour day-night cycle, and the environment was maintained at 20-25 °C. During the experiment, the mice could eat and drink freely. This experiment was reviewed and approved by the Experimental Animal Ethics Committee of Sun Yat-sen University, and the experiment approval number was "SYSU-IACUC-2022-000134".

[0128] 3.1.3.2 Genotype Identification of Transgenic Mice Cut the tail tissue of 4-week-old weaned mice and operate according to the instructions of the Mouse Genotype Rapid Identification Kit (Guangzhou Novizan Biotechnology Co., Ltd.). The procedure is as follows: (1) Tissue DNA extraction: Add 100 μL of lysis solution and 2 μL of proteinase K to the tail tissue of each mouse, heat in a 56 °C water bath for 30 minutes, and then heat in a 98 °C water bath for 5 minutes to inactivate proteinase K. Absorb the supernatant and store it at -20 °C for later use; (2) Preparation of PCR System According to the following system, the tail tissue of each mouse is required: The PCR primer sequences for the identification of Tg mice in JPEG2025520135000005.jpg69154 are as follows: The size of the expected transgenic mouse PCR product: 232 bp; Primer F: GGCAACGTGCTGGTTATTGTG; Primer R: GTATTGCCTCTGAAAGCATGTTC.

[0129] (3) PCR procedure The reaction was carried out according to the following PCR procedure: JPEG2025520135000006.jpg82126

[0130] (4) Preparation of agarose horizontal electrophoresis gel Place the mold on the surface of the horizontal platform and insert a 25-well comb. Prepare 1XTAE solution, 200 mL of the solution is required for each culture mold, add 2% agarose and mix well. Heat the solution in a microwave oven until it boils, take it out and shake it vigorously, then put it back into the microwave oven. After boiling, take it out again and shake it vigorously until the solution becomes transparent and the agarose is completely dissolved. Add 6 μL of nucleic acid stain, mix well and pour it into the mold. Wait for the solution to cool and solidify into a gel for later use.

[0131] (5) Horizontal electrophoresis Put the agarose gum into the horizontal electrophoresis tank and add 1XTAE solution. After mixing the PCR products, add 10 μL per well to the agarose gum and run it at a constant voltage of 120 V for 20 minutes, then observe and photograph it under ultraviolet light. 3.1.3.3 Preparation and grouping of animal models 3.1.3.3.1 Establishment and grouping of a mouse model of streptozotocin-induced diabetic nephropathy (DN) The DN induction method of streptozotocin (STZ) was the same as 1.3.2. Cf48-Tg mice and WT mice with a C57BL / 6 background were fasted for 4 hours, then dissolved in a 0.1 mmol / L sodium citrate solution at pH 4.5 together with 55 mg / kg of STZ and injected intraperitoneally for 5 consecutive days. Two weeks after STZ induction, the fasting blood glucose level was measured. Mice with a fasting blood glucose level exceeding 16.7 mmol / L were successfully modeled, and the induction of a diabetes (DM) model was successful. Mice that could not achieve the target fasting blood glucose level were sacrificed. Glycated hemoglobin was measured two weeks after DM modeling. Mice with glycated hemoglobin > 7% were successfully modeled, and mice that could not achieve the target glycated hemoglobin were sacrificed. The remaining mice were successfully modeled and sacrificed at 12 w after DM modeling (DN group). The control mice were administered the same dose of sodium citrate solution as the solvent control group (NC group). After DM modeling, random urine was collected through a metabolic cage for 4 hours, and urinary albumin was detected at the 3rd, 6th, 8th, 10th, and 12th weeks respectively.

[0132] Tg mice and WT mice were randomly divided into 4 groups with 8 - 15 mice in each group by the random number table method: (1) Cf48 wild type, solvent group (WT-NC group); (2) Cf48 wild type, diabetic nephropathy group (WT-DN group); (3) Cf48 gene recombination, solvent group (Tg-NC group); (4) Cf48 gene recombination, diabetic nephropathy (Tg-DN group).

[0133] 3.1.3.2.2 Establishment and grouping of a mouse model of folic acid-induced folic acid nephropathy (FAN) Folic acid (FA) induces folic acid nephropathy in mice (FAN) by folic acid, and the specific process is as follows: The toxic tubular necrotic folic acid necrotic folic acid nephropathy model is induced by folic acid by a single injection of a large dose. FA was dissolved in 0.3 mol / L sodium bicarbonate solution at 0.25 mg / g and intraperitoneally injected once. Two days after the folic acid introduction, the body weight was measured, and the blood sampling method from the orbital ophthalmic vein after anesthesia was used for a small amount of blood sampling two days after the folic acid introduction, and the blood CysC was measured using the serum obtained by centrifugation. Mice with a serum CysC content exceeding 1500 μg / L two days after folic acid induction and those that failed in modeling were euthanized. Tg mice and WT mice were randomly divided into four groups of 8 - 15 mice each by the random number table method. (1) Cf48 wild type, solvent group (WT - NC group); Cf48 wild type folic acid nephropathy group (WT - FAN group); Cf48 transgenic, solvent group (Tg - NC group); Cf48 transgenic folic acid nephropathy group (Tg - FAN group).

[0134] 3.1.3.4 Extraction of total protein from kidney tissue (1) Approximately 3 mg of mouse tissue from each group was taken on a cold table and placed in a 1.5 mL EP tube, and 200 μL of protein lysate (1x RIPA + protease inhibitor) was added to each EP tube. (2) One grinding bead with a diameter of 3 mm was added to each EP tube, and it was ground twice at 0 °C, 60 HZ, with a parameter of 15 seconds at 8 - second intervals using a grinder. (3) After 30 minutes on ice, it was centrifuged at 15,000 rpm at 4 °C for 30 minutes. (4) The protein supernatant was aspirated, diluted 20 - fold with ddH20, and the protein concentration was measured by the BCA method. (5) When trimming the protein in the kidney tissue of each group of mice, each electrophoresis system has 50 μg of protein and the total volume is 20 μL. (6) 2x loading buffer was added, and the protein was decomposed at 98 °C in an air bath for 10 minutes. After denaturation, the protein was stored at -20 °C for later use.

[0135] 3.1.3.5 Measuring protein concentration by the BCA method: The procedure is the same as 1.3.8. 3.1.3.6 Protein Western blot detection For low molecular weight indicators such as Cf48, SDS-PAGE electrophoresis is performed using a 15% PAGE gel with a separation gel concentration, and semi-dry transfer is used for transfer. The low molecular weight target protein (Cf48) is semi-dry transferred. According to the instructions, prepare a semi-dry transfer solution, load 100 mL onto each gel, and the formula is 20 mL of 5-fold semi-dry transfer solution, 20 mL of absolute ethanol, and 60 mL of ddH20. Soak thick filter paper in the semi-dry transfer dedicated transfer solution 30 minutes in advance, soak the filter paper in the transfer solution, and soak in methanol to activate the PVDF membrane. From the negative electrode to the positive electrode, there is thick filter paper, PVDF membrane, separation adhesive, and thick filter paper. Use a roller to drive out the bubbles, cover the electrode cover, the constant current is limited to 2.5 A, the voltage is limited to 25 V, and the membrane is transferred for 7 minutes. The remaining procedure is the same as 1.3.9.

[0136] 3.1.3.7 Extraction of total RNA from kidney tissue (1) Cut about 3 mg of kidney tissue from each group of mice on a cold table, put it into a 1.5 mL EP tube, and add 500 μL of Trizol solution to each EP tube. (2) Add one grinding bead with a diameter of 3 mm to each EP tube, and grind it 4 times at intervals of 8 seconds at a parameter of 4 °C, 60 HZ, and 15 seconds using a grinder. (3) After adding 500 μL of Krizol to each EP tube, add 200 μL of chloroform, mix vigorously up and down for 30 seconds, and let it stand for 10 minutes until the mixture is clearly separated into an aqueous phase (RNA, DNA) and a chloroform phase (protein). (4) Centrifuge at 15,000 rpm, 4°C for 30 minutes. (5) Add 500 μL of isopropanol to each EP tube, absorb the uppermost aqueous phase of the centrifuged mixture, add it to the isopropanol, mix it upside down, and leave it standing on ice for 2 hours. (6) Centrifuge at 15,000 rpm, 4°C for 30 minutes. (7) After centrifugation, a precipitate is visible and the supernatant is discarded. (8) Prepare 70% ethanol with anhydrous ethanol and enzyme-free water (DEPC water), and add 1 mL of enzyme-free 70% ethanol to each tube and pipette. (9) Centrifuge at 15,000 rpm, 4°C for 5 minutes. (10) Repeat (7)-(9). (11) Discard the supernatant, confirm that there is no ethanol residue, and dry it on ice. (12) When the precipitate becomes transparent, add 20 μL of enzyme-free water to each tube to dissolve the RNA. (13) Measure the RNA concentration with a spectrophotometer and freeze it at -80°C for later use or reverse transcribe it into cDNA.

[0137] 3.1.3.8 Reverse transcription reaction Follow the instructions of the reverse transcription kit (Roche) and use the following two-step method. (1) Prepare the reagents for the first step of the reaction. The following dosages are required for each serving of RNA. JPEG2025520135000007.jpg61144(2) Reaction in the PCR instrument: Heat reaction at 65°C for 10 minutes. (3) Set the reagents for the second-step reaction to 7 μL per well, add them to the reacted system, and mix well. JPEG2025520135000008.jpg56146(4) Reaction in the PCR device: The reaction is carried out according to the following procedure. JPEG2025520135000009.jpg49170 (5) After the reverse transcription is measured, the concentration of cDNA is measured with a spectrophotometer and then frozen at -20°C for later use.

[0138] 3.1.3.9 Real-time PCR (qRT-PCR) Follow the instructions of the real-time PCR kit from (Roche) and perform the following steps: (1) Dilute the cDNA: Dilute the cDNA 20-fold with enzyme-free water to make the total amount of cDNA in the final system 20 ng; (2) Prepare the reaction mixture: The dosage of the mixture for each well is as follows. JPEG2025520135000010.jpg52155 (3) Add to the plate: Add 4 μL of cDNA and 6 μL of the reaction mixture to each well of a 96-well plate or a 384-well plate; (4) Real-time PCR reaction: The reaction system is as follows: JPEG2025520135000011.jpg59126 The synthesis of qRT-PCR primers was entrusted to DeepSense BGI Tech Co., Ltd., and the specific sequences are as follows: JPEG2025520135000012.jpg71128

[0139] 3.1.3.10 Detection of blood glucose level and glycosylated hemoglobin in mice After 6 hours of fasting, a small part of the tail tissue of the rat was cut off in a quiet environment. Fasting blood glucose measurement: Insert a blood glucose test strip into the blood glucose meter. After the device displays the prompt for testing, squeeze a drop of blood at the tip of the tail, drop it on the blood glucose test strip, wait for 3 seconds, and the recorded result will be displayed. The measurement of glycosylated hemoglobin is carried out as follows according to the operating procedure of the glycosylated hemoglobin analyzer (Sannuo). (1) Re-warm the reagent for 30 minutes. (2) Power on: Insert the code card attached to the kit into the glycosylated hemoglobin meter and check the information of the code card. (3) Sample collection: Using a sampler, squeeze blood from the tip of the mouse tail until the blood reaches the upper part of the capillary, insert the sampler into the R1 reagent container, force the blood sample in the capillary into the R1 reagent, shake well, and let it stand for 2 minutes. (4) Loading: To avoid air bubbles, drop the R1 mixture onto the glycated hemoglobin test strip, then add the R2 reagent vertically to the test strip, and let it stand for 15 seconds to confirm that the R2 reagent has completely penetrated the test strip membrane. (5) Test reading result: Insert the test strip into the glycated hemoglobin meter, wait for 30 seconds, and display the recorded result.

[0140] 3.1.3.11 Mouse renal function test Two days after FA introduction, 200 μL of the mouse's whole blood was collected from the medial orbital vein, left to stand at room temperature for 1 hour, then centrifuged at 3000 rpm for 10 minutes, and the upper serum was collected to detect CysC in the mouse blood. When sacrificing the mouse, after anesthesia, collect the mouse's whole blood by abdominal aorta blood collection method, let it stand at room temperature for 1 hour, then centrifuge at 3000 rpm for 10 minutes, and then collect the upper serum to detect serum creatinine, blood urea nitrogen, and blood CysC at the end point of the disease. The serum creatinine and blood urea nitrogen in the mouse were detected by an automatic biochemical analyzer, and the blood CysC in the mouse was detected by ELISA (Abcam, USA). The serum was diluted 2000-fold according to the operation instructions, and the specific procedure was the same as 2.3.6.

[0141] 3.1.3.12 Detection of mouse urinary albumin DN and control mice were collected through metabolic cages at the 3rd, 6th, 8th, 10th, and 12th weeks of 4-hour random urine of the disease. ELISA detection of urinary albumin (Albumin, Abcam, USA) and urinary creatinine (Cretanine, R&D, USA) was performed according to the operating procedure. For the detection of urinary albumin, the urine was diluted 2000-fold, and for the detection of urinary creatinine, a stock solution was used. The specific procedure was the same as 2.3.6. The urinary albumin / creatinine ratio was used to reflect the urinary albumin of the mice.

[0142] 3.1.3.13 Histopathological Staining of Mouse Kidneys (1) PAS Staining: According to the instruction manual, the specific procedure is as follows. A) Normal deparaffinization and hydration of paraffin slides with a thickness of 5 μm in the same steps as 2.3.3(1)-(2); b) Wash with distilled water for 1-2 minutes; c) Oxidize with periodic acid solution for 10 minutes. d) Distilled water to wash away the floating color. e) Stain with Schiff reagent for 30 minutes. f) Distilled water to wash away the floating color. g) Stain with Mayer hematoxylin for 3 minutes. h) Distilled water to wash away the floating color. i) Conventional dehydration, clearing, and mounting with neutral resin are the same procedures as 2.3.3(11). k) Observe with an upright microscope and take pictures, or scan the film with a fully automatic scanner. (2) Masson Staining: According to the instruction manual (Sigma), the specific procedure is as follows. a) Conventional deparaffinization and hydration of 5-μm-thick paraffin slides, the steps are the same as in the case of 2.3.3(1)-(2); b) 1% acetic acid solution (200 mL in ddH20), iron-containing hematoxylin solution (100 mL solution A + 100 mL solution B), phosphate solution (50 mL solution C + 50 mL solution D + 100 mL ddH20); e) Stain with iron-containing hematoxylin solution for 5 minutes. f) Rinse with running water for 5 minutes to remove the floating color and wash with ddH20. g) Stain with magenta solution for 5 minutes. h) Wash with ddH20 to wash away the floating color. i) Stain with phosphate solution for 5 minutes. j) Wash with ddH20 to remove the floating color. k) Stain with Anline Blue solution for 20 minutes. l) Remove the floating color by washing with ddH20. m) Rinse up and down twice with 1% acetic acid solution. Wash away the floating color. n) The conventional dehydration procedure for mounting with transparent, neutral resin is the same as in 2.3.3(11). o) Observe and photograph with an upright microscope or scan the film with a fully automatic scanner.

[0143] 3.1.3.14 Immunohistochemistry / Immunofluorescence Staining of Mouse Kidney Tissue Prepare paraffin slides with a thickness of 5 μm and follow procedures 2.3.3 and 2.3.4.

[0144] 3.1.3.15 Immunohistochemistry F4 / 80 + Macrophage Measurement The films scanned with a fully automatic scanner were randomly selected for 10 renal cortex fields by high magnification (×400) method and single - blind method. The selected fields need to avoid glomeruli and blood vessels, and the number of F4 / 80+ cells in each field is quantified, The average number of cells in 10 visual fields was in the and obtained as the number of F4 / 80+ macrophages.

[0145] 3.1.3.16 Statistical Method: The statistical analysis method is the same as in 2.3.7. 3.1.4 Experimental Results 3.1.4.1 Identification of Cf48 Transgenic Mice According to the genotype identification results of the mice, from the PCR results, it was shown that Cf48Tg mice expressing a product with a size of 232 bp were neonatal mice. From the results of Western blotting, it was shown that the expression of Cf48 protein in the kidney tissue of Cf48Tg mice was significantly increased compared with the WT group.

[0146] 3.1.4.2 Increase in Cf48 Expression in the Kidney Tissue of DN Mice The expression of Cf48 in the kidney tissues of DN mice was detected by immunoblotting, qRT-PCR, and immunofluorescence. As a result, it was shown that the mRNA expression level and protein expression level of Cf48 in DN mice were significantly higher than those in the kidney tissues of NC mice. Immunofluorescence revealed that Cf48 was highly expressed in the kidney tissues of DN mice, mainly in renal tubules and renal interstitium. As shown in Figure 3-1, Cf48 was shown to co-localize with α-SMA positive myofibroblasts by co-staining.

[0147] 3.1.4.3 Deterioration of renal function progression in diabetic nephropathy mice with overexpression of Cf48 As a result of blood glucose measurement, the blood glucose level and glycosylated hemoglobin of DN mice were significantly higher than those of NC mice that met the diabetes criteria. There was no significant difference in fasting blood glucose level and glycosylated hemoglobin between WT mice and Tg2 mice. From the above results, it was shown that overexpression of Cf48 could not change the glucose tolerance of mice (Figure 3-2A, B). As shown in Figure 3-2C, the kidney weight / body weight ratio of Tg-DN mice was significantly higher than that of WT-DN mice, indicating that the renal ultrafiltration capacity of diabetic nephropathy mice with Cf48 overexpression was high. The urinary albumin status of mice in the WT-DN group and Tg-DN group during the progression of diabetic nephropathy was dynamically observed. As a result, it was shown that the urinary albumin excretion amount of DN mice gradually increased with the progression of diabetic nephropathy, indicating that the diabetic nephropathy model was successfully established. As shown in Figure 3-2D, E, there was no significant difference in the urinary albumin / creatinine ratio between the two groups at the initial stage (3w) of diabetic nephropathy. At the mid-stage (6w) of the progression of diabetic nephropathy, the urinary albumin of Tg mice was significantly higher than that of WT mice, and the more severe urinary albumin in Tg mice continued until the late stage (12w) of diabetic nephropathy at death.

[0148] The renal function of mice is reflected by measuring the status of cystatin C (CysC) in the blood of mice. As shown in Figure 3-2F, since the serum CysC of DN mice was significantly higher than that of normal control mice at the time of killing, it was shown that the renal function of the model mice had severely progressed. There was no significant difference in the serum CysC values between Tg mice and WT mice in the NC group, and the blood CysC value of Tg mice in the DN group was significantly higher than that of WT mice. These results indicated that in diabetic nephropathy mice, overexpression of Cf48 may worsen the progression of renal function without changing glucose tolerance.

[0149] Regarding the results of PAS for the worsening of renal pathological damage in diabetic nephropathy mice with Cf48 overexpression, compared with NC, it was shown that the glomerular basement membrane of DN mice was thickened, mesangial cells proliferated, and the mesangial area was thickened, which was consistent with the changes in diabetic nephropathy. As shown in Figure 3-3, the diabetic nephropathy lesions of Tg mice were more prominent compared with WT mice, and significant thickening of the tubular basement membrane and significant increase in interstitial interstitial lesions outside the tubules were observed.

[0150] 3.1.4.5 Overexpression of Cf48 in diabetic nephropathy mice exacerbates renal fibrosis The results of immunofluorescence showed that extracellular matrix collagen (Collagen IV, Col), fibronectin (Fibronectin, Fn), and α-smooth muscle actin (α-SMA), a myofibroblast marker, in DN mice were significantly increased compared with NC mice. In the DN model, the increase in Col, Fn, and α-SMA in Tg mice was more prominent than that in WT mice, indicating that the interstitial fibrosis of the kidneys in Tg-DN mice had worsened. Compared with WT mice, the deposition of Col in the glomeruli of Tg-DN mice was significantly increased, suggesting that overexpression of Cf48 may also worsen glomerulosclerosis in DN mice. The results of Western blot showed a significant increase in α Tg compared with WT mice, which was consistent with the results of immunofluorescence as shown in Figure 3-4.

[0151] 3.1.4.6 Worsening of Cf48 overexpression in diabetic nephropathy mice F4 / 80 is a marker for macrophages. The number of F4 / 80+ macrophages in the renal cortex of mice was counted by immunohistochemistry to observe the inflammation of the mouse kidneys. As a result, it was shown that the number of unmodeled macrophages in normal mice was very small and there was no significant difference in the number of macrophages between the WT group and the Tg group. As shown in Figure 3-5A, the number of F4 / 80+ macrophages in the renal cortex region of DN mice was significantly increased compared with that of NC mice, and the number of F4 / 80+ macrophages in Tg-DN mice was significantly increased compared with that of WT-DN mice.

[0152] To further observe the inflammatory response in the kidneys of mice in each group, qRT-PCR was used to detect the mRNA levels of the inflammatory factors MCP-1 and TNF-α in kidney tissues. As a result, as shown in Figure 3-5B, the mRNA expression levels of MCP-1 and TNF-α were significantly higher in DN mice compared with the NC group, and the expression levels of the inflammatory factors were further increased in Tg-DN mice.

[0153] 23.1.4.7 Upregulation of Cf48 expression in the kidney tissues of FAN mice The mRNA expression of Cf48 in kidney tissues 2 days, 7 days, 14 days, and 28 days after folic acid injection was detected by qRT-PCR. As a result, it was shown that the mRNA level of Cf48 was significantly increased on the 2nd day after folic acid induction, and the mRNA level of Cf48 gradually increased with the progression of the disease, and the expression level of Cf48 was the highest on the 28th day of FAN. The FAN 28d model was selected as the research object. Western blot showed that the protein expression level of Cf48 in FAN-28d kidney tissues was significantly higher than that in normal kidney tissues. The results of immunofluorescence showed that, as shown in Figure 3-6, the expression of Cf48 was significantly increased mainly in renal tubules in the FAN model and increased in more significantly expanded and severely damaged tubules.

[0154] 3.1.4.8 Cf48 Overexpression in Folic Acid Nephropathy Mice with Deteriorated Renal Function Progression Two days after folic acid modeling, the blood CysC in FA mice increased significantly, which was consistent with the changes in acute kidney injury. Twenty-eight days after folic acid modeling, the blood CysC, serum creatinine (Cr), and blood urea nitrogen (BUN) in FA mice increased significantly compared with those in NC mice, indicating that the model of folic acid nephropathy was successfully modeled. In the FA group, compared with WT mice, the blood CysC and blood Cr in Tg mice increased significantly, and the difference was statistically different. The blood BUN tended to increase. As shown in Figure 3-7, the above results indicated that the overexpression of Cf48 28 days after FA introduction might deteriorate the renal function progression in mice.

[0155] 3.1.4.9 Overexpression of Folic Acid Nephropathy by Cf48 Worsens Nephropathy Twenty-eight days after FA introduction, the kidneys of the mice were significantly shrunk, and the surface was granular and uneven. As a result of Masson staining, the renal tubules of folic acid nephropathy mice were significantly dilated, the arrangement of the tubules was disrupted, the tubules were necrotic, and partially necrotic tubules fell off into the lumen to form cell cylinders. Interstitial edema was obvious, accompanied by inflammatory cell infiltration. Masson staining showed a significant increase in collagen deposition and obvious fibrosis in the renal tissue of FA mice. In the FA group, the damage of Tg was stronger than that of WT mice. A large number of tubular cells were necrotic, the cell cylinders increased, and the collagen deposition increased significantly. From the calculation results of the collagen deposition ratio under Masson staining, the collagen deposition ratio in the kidneys of FA mice was significantly higher than that in NC mice, and the collagen deposition ratio in Tg mice was significantly higher than that in WT mice, indicating that the degree of renal fibrosis was severe.

[0156] 3.1.4.10 Worsening of Renal Fibrosis in Folic Acid Nephropathy Mice with Cf48 Overexpression As a result of immunofluorescence, it was shown that Col and Fn were mainly expressed in the basement membrane and morphological rules, and α-SMA was mainly expressed in blood vessels. Col, Fn, and α-SMA staining in the kidney tissues of FA mice were mainly distributed in the renal interstitium, with significant increases, thickening, and disordered alignment. In the FA model, the increase in renal fibrosis-related factors in Tg mice was more significant than that in WT mice. The results of Western blot showed that, as shown in Figure 3-8, the expression of Col and α-SMA in Tg mice of the FA group was significantly increased, which was consistent with the results of immunofluorescence.

[0157] 3.1.4.11 Deterioration of renal inflammation in folic acid nephropathy kidneys with Cf48 overexpression As a result of immunohistochemistry, it was shown that the number of F4 / 80+ macrophages in FA mice was significantly increased compared with that in NC mice, and the inflammatory reaction in the kidney tissues of folic acid nephropathy mice was significantly worsened. In the FA mouse model, the number of F4 / 80+ macrophages in Tg mice was significantly higher than that in WT mice, and the difference was statistically significant. As a result of qRT-PCR, as shown in Figure 3-9, the mRNA expression levels of MCP-1, TNF-α, and IL-1 in FA mice were significantly higher than those in NC mice, and the expression levels of inflammation-related factors in Tg mice were further increased compared with those in WT mice.

[0158] 3.2 Role of CF48 gene knockout in mouse models of folic acid nephropathy and obstructive nephropathy 3.2.1 Objective: To verify the effect of Cf48 gene knockout on renal inflammation and fibrosis in the progression of mouse diseases. 3.2.2 Experimental materials: C57BL / 6 was purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd., and Cf48 gene knockout parental mice were entrusted to Saiye Biotechnology Co., Ltd. for cultivation.

[0159] 3.2.3 Research methods 3.2.3.1 Breeding of Cf48 knockout mice Pairs of male and female C57BL / 6 background Cf48+ / - mice (HZ) were prepared by Saiye Biotech Co., Ltd. Using CRISPR / Cas9 technology, sgRNA was designed, and Cf48+ / - was obtained by PCR identification through high-throughput electroporation of fertilized eggs. Subsequently, they were bred at the Experimental Animal Center of Sun Yat-sen University (North Campus), and male and female Cf48-HZ mice were mated. The offspring obtained were identified by PCR, and Cf48- / -(KO) mice and Cf48+ / +(WT) mice suitable for the experiment were obtained. The KO mice and WT mice used in the experiment were male mice aged 8-10 weeks with a body weight of 25-30 g, and they were bred at the Experimental Animal Center of Sun Yat-sen University. This experiment was reviewed and approved by the Experimental Animal Ethics Committee of Sun Yat-sen University, and the experiment approval number is "SYSU-IACUC-2022-000134".

[0160] 3.2.3.2 Genotyping of Genetically Modified Mice The procedures for rat tail DNA extraction, PCR reaction, gel preparation, and horizontal electrophoresis are the same as those in 3.1.3.2. The PCR primer sequences for the identification of KO mice are as follows: The expected size of the PCR product of KO mice: 246 bp; Primer F1: TTGGTCTCCCACACGAATGG; Primer R1: CCAGAGCTCAGCTTCCACAA. The PCR primer sequences for the identification of WT mice are as follows: The expected size of the PCR product of WT mice: 265 bp; Primer F2: GAGCAGGCATCCACTCCTTT; Primer R2: AGAGCTCTCCTCCTTCCCAG. The PCR procedure is as follows. JPEG2025520135000013.jpg54138

[0161] 3.2.3.3 Preparation, Grouping, and Research Plan of Animal Models 3.2.3.3.1 Establishment and Grouping of a Folic Acid-Induced Mouse Model of Folic Acid Nephropathy (FAN) The modeling method of folic acid nephropathy is the same as 3.1.3.2.2. KO mice and WT mice were randomly divided into 4 groups of 8 - 15 mice each using the random number table method: (1) Cf48 wild type, solvent group (WT-NC group). (2) Cf48 wild type folic acid nephropathy group (WT-FAN group); (3) Cf48 gene knockout, solvent group (KO-NC group); (4) Cf48 gene knockout, folic acid nephropathy group (KO-FAN group).

[0162] 3.2.3.3.2 Establishment and grouping of the unilateral ureteral obstruction mouse model (UUO) The specific process of modeling the unilateral ureteral obstruction mouse model (UUO) is as follows: Mice in the surgery group (UUO) were anesthetized with pentobarbital (45 mg / kg), the skin was routinely sterilized and prepared, the skin was incised near the midline of the abdomen at the base of the right thigh of the mouse, the skin and muscle tissues were separated in sequence, the abdominal cavity was exposed, the oblique mouse ureter was found along the posterior abdominal wall, and the ureter was cut after being ligated at the proximal and distal ends. The abdominal organs were retracted, and the muscle layer and skin layer were sutured in sequence. In the sham operation group (Sham), ligation was not performed after the ureter was freed, and the remaining operations were the same as those in the UUO group. On the 7th day after surgery, the mice were sacrificed for examination.

[0163] KO mice and WT mice were randomly divided into 4 groups of 8 - 15 mice each using the random number table method: (1) Cf48 wild type, sham group (WT-Sham group); (2) Cf48 wild type, UUO group (WT-UUO group); (3) Cf48 gene knockout, sham group (KO-Sham group); (4) Cf48 gene knockout, UUO group (KO-UUO group). 3.2.3.4 The remaining investigation methods are the same as 3.1.3

[0164] 3.2.4 Experimental results 3.2.4.1 Identification of Cf48 knockout mice According to the results of the genotype gel electrophoresis of neonatal mice, the target product with a length of 246 bp was amplified by the P1F1 (KO) primer of Cf48 - / - mice, but not by P2F2 (WT). Cf48 + / + mice could not amplify the product with the P1F1 (KO) primer, but P2F2 (WT) could amplify the target product with a length of 265 bp. Cf48 + / - mice were amplified by the P1F1 (KO) primer and P2F2 (WT) to identify the genotype of the mice.

[0165] 3.2.4.2 Reduction of kidney injury in CF48 knockout mice with folic acid nephropathy Two days after folic acid modeling, the blood CysC in FA mice increased significantly, which was consistent with the changes in acute kidney injury and the above results. Twenty-eight days after folic acid modeling, the blood CysC, blood Cr, and blood BUN in FA mice increased significantly compared with those in NC mice. In the FA group, compared with WT mice, the increase in blood CysC, blood Cr, and blood BUN in KO mice decreased significantly, and the difference was statistically different. However, as shown in Figure 3 - 10, the decrease could not return to the normal level of renal function.

[0166] 3.2.4.3 Reduction of renal pathological injury in CF48 knockout mice with folic acid nephropathy By Masson staining, it was observed that the renal morphology of NC mice was basically normal, and the tubular cells were arranged regularly and neatly. The morphological changes of Masson staining in mice 28 days after folic acid modeling were consistent with the above, with obvious interstitial fibrosis, infiltration of a large number of inflammatory cells, necrosis of tubular epithelial cells, collapse of tubules, and visible cell casts. Compared with WT - FA mice, as shown in Figure 3 - 11, the severity of tubular injury was reduced, and the area of interstitial fibrosis decreased significantly.

[0167] 3.2.4.4 Reduction of renal fibrosis in CF48 knockout mice with folic acid nephropathy Immunofluorescence staining was consistent with the above results. The extracellular matrix of NC mice was mainly expressed in the basement membrane with a regular morphology, and α-SMA was mainly expressed in blood vessels. Col, Fn, and α-SMA staining in the kidney tissues of FA mice were mainly distributed in the renal interstitium, showing significant increases, thickening, and disordered alignment. Compared with WT mice, the staining intensity and the area of fibrosis-related indicators decreased. The results of Western blot showed that the expression of ColI and α-SMA in KO was significantly decreased compared with that in WT mice in the FA model, which was consistent with the immunofluorescence results as shown in Figure 3-12.

[0168] 3.2.4.5 Reduction of renal inflammation in CF48 knockout mice with folic acid nephropathy The results of immunohistochemistry showed that the number of F4 / 80+ macrophages in FA mice was significantly increased, indicating significant inflammatory infiltration in the renal cortex of mice with folic acid nephropathy. There was no significant difference in the proportion of F4 / 80+ cells between KO mice and WT mice in the NC group, but the proportion of F4 / 80 macrophages in KO mice was lower than that in WT mice in the FA model, and the difference was statistically significant. The results of qRT-PCR showed that, as shown in Figure 3-13, the expression levels of inflammation-related factors MCP-1 and TNF-α in FA mice were significantly higher than those in NC mice, and the mRNA expression of inflammation-related factors in KO mice was significantly lower than that in WT mice. + The results of qRT-PCR showed that, as shown in Figure 3-13, the expression levels of inflammation-related factors MCP-1 and TNF-α in FA mice were significantly higher than those in NC mice, and the mRNA expression of inflammation-related factors in KO mice was significantly lower than that in WT mice.

[0169] 3.2.4.6 Increased Cf48 expression in the kidney tissues of UUO mice The expression of Cf48 in the kidney tissues of UUO mice was detected by qRT-PCR. The results showed that, as shown in Figure 3-14, the mRNA expression level of Cf48 in UUO mice was significantly higher than that in the kidney tissues of sham mice.

[0170] 3.2.4.7 Reduction of renal pathological damage in Cf48 knockout UUO mice By Masson staining, it was found that the tubular cells in the sham group were neatly arranged, the basement membrane was intact, and there was no edema or extracellular matrix deposition in the renal interstitium. The renal tubules of UUO mice were highly dilated, some cells were necrotic and shed, the extracellular matrix increased significantly, a large amount of collagen was deposited, and renal tissue fibrosis was obvious. As shown in Figure 3-15, there was no significant difference in the morphology between WT mice and KO mice in the sham mice. In the UUO group, compared with WT mice, it was found that the interstitial collagen deposition in KO mice was significantly reduced.

[0171] 3.2.4.8 Reduction of renal fibrosis in Cf48 knockout UUO mice By immunofluorescence staining, it was shown that the level of extracellular matrix in the Sham group was normal and mainly distributed in the basement membrane and blood vessels. The expressions of Col, Fn, and α-SMA in the renal interstitium of UUO mice were significantly increased, the basement membrane was significantly thickened, and interstitial fibrosis was obvious. Compared with WT mice, the expression of extracellular matrix was significantly reduced, and the degree of fibrosis was alleviated. 3.2.4.9 Reduction of renal inflammation in Cf48 knockout UUO mice As a result of immunohistochemistry, the renal interstitial morphology of Sham mice was normal, and a small number of F4 / 80 + macrophage infiltration was observed around the blood vessels. The tubules of UUO mice were significantly dilated, the inflammatory cells were significantly increased, showing sheet-like aggregation. The inflammatory cells in KO mice were significantly reduced compared with WT mice, with a statistical difference. From the results of qRT-PCR, the expression levels of the inflammation-related factors MCP-1 and TNF-α in UUO mice were significantly higher than those in NC mice. In the UUO model group, as shown in Figure 3-16, the expression of inflammation-related factors in KO mice was significantly lower than that in WT mice.

[0172] 3.3.1 Objective: To investigate whether inhibiting the expression of renal C4orf48 by injecting LNA C4orf48 in vitro in the urinary tract and STZ-DKD (Nos3- / - mice) model can alleviate renal fibrosis and delay the progression of DKD. In this study, a mouse fibrosis model consisting of UUO and a progressive STZ-DKD model (Nos3- / - mice) was established. Subsequently, mice were injected with locked nucleic acid (LNA) antisense oligonucleotide C4orf48 (locked nuclei acid (LNA) anti-sense oligonucleotide C4orf48, LNA C4orf48) to reduce endogenous C4orf48 in the kidneys. It was observed whether the fibrosis of UUO and STZ-DKD could be alleviated and the progression of STZ-DKD could be delayed by intervening in the expression of C4orf48.

[0173] 3.3.2 Experimental materials Control LNA (LNA CTL): 5'-AACACGTCTATACGC-3', the first "A" and the last "C" are LNAs, and the entire strand contains a phosphorothioate backbone. LNA1 C4orf48 type: 5'-CGCTGCATGAATTCAA-3', the first "A" and the last "C" are LNAs, and the entire strand contains a phosphorothioate backbone. LNA2 C4orf48 type: 5'-CCGTAGGTCCTGAAGG-3', the first "A" and the last "C" are LNAs, and the sequence of the entire strand contains a phosphorothioate backbone. LNA was synthesized by Shanghai GeneBiogist.

[0174] 3.3.3 Method: C57bl6 / J male mice at 8 - 12 weeks of age were subjected to sham surgery (sham) or UUO surgery. The UUO group was further randomly divided into an intraperitoneal injection of LNA CTL (10 mg / kg) group, LNA1 C4orf48 10 mg / kg group, LNA2 C4orf48 10 mg / kg group, LNA1 C4orf48 10 mg / kg group, and LNA1 C4orf48 10 mg / kg group + LNA2 C4orf48 10 mg / kg group. 8 mice per group. The same dose was intraperitoneally injected once on the 6th day after surgery, and the mice were sacrificed on the 7th day, and kidney tissues were collected for various analyses. WB showed that the expression of C4orf48 in UUO kidneys was significantly inhibited in the LNA1 C4orf48 10 mg / kg + LNA2 C4orf48 10 mg / kg group. LNA1 C4orf48 10 mg / kg + LNA2 C4orf48 10 mg / kg was intraperitoneally administered once a week as a therapeutic agent for STZ - DKD. STZ 55 mg / kg was intraperitoneally administered to Nos3 - / - male mice at 10 - 12 weeks of age for 5 consecutive days. Two weeks later, the fasting blood glucose level of the mice was measured and found to be 250 mg / dl or higher, and they were diagnosed with diabetes. The diabetic mice were randomly divided into two groups, and one group was intraperitoneally injected with LNA CTL (20 mg / kg) once a week. One group was intraperitoneally injected with LNA1 C4orf48 10 mg / kg + LNA2 C4orf48 10 mg / kg once a week for 6 weeks, and the mice were sacrificed at the 7th week. Furthermore, Nos3 - / - male mice at 10 - 12 weeks of age were intraperitoneally injected with the same amount of solvent as the control group for 5 consecutive days. 12 - 14 mice / group. Collection of materials such as blood, urine, kidneys, liver, spleen, heart, and other tissues such as those in 3.1.3.

[0175] 3.3.4 Experimental Results 3.3.4.1 LNA Cf48 injection reduces Cf48 expression in UUO kidneys Western blotting showed that the expression of Cf48 in the sham group was very low, the expression of Cf48 in the UUO group increased significantly after 7 days, and the expression of Cf48 could be decreased by injecting LNA1 Cf48 or LNA2 Cf48. Co-injection of LNA1 Cf48 and LNA2 Cf48 further suppressed the expression of Cf48, and the inhibition rate reached more than 90%. As a result, as shown in Figure 3-17, it was found that co-administration of LNA1 Cf48 and LNA2 Cf48 once a week could inhibit the expression of endogenous Cf48 in the kidney.

[0176] 3.3.4.2 LNA Cf48 injection reduces UUO renal fibrosis Western blotting showed that co-injection of LNA1 Cf48 and LNA2 Cf48 decreased the production of extracellular matrix α-SMA and collagen I in UUO, injection of LNA1 Cf48 and LNA2 Cf48 decreased renal fibrosis, and as shown in Figure 3-18, it was suggested that interfering with the expression of Cf48 could inhibit renal fibrosis.

[0177] 3.3.4.3 LNA Cf48 injection reduces proteinuria production, reduces renal fibrosis, and delays the decline of renal function in STZ-DKD There was no difference in fasting blood glucose and glycosylated hemoglobin between the STZ-DKD+LNA CTL group and the STZ-DKD+LNA Cf48 group. Collagen IV immunohistochemistry showed that LNA Cf48 treatment reduced renal fibrosis in STZ-DKD. Compared with the STZ-DKD+LNA CTL group, the STZ-DKD+LNA Cf48 group decreased proteinuria production. Also, as shown in Figure 3-19, LNA Cf48 treatment decreased the levels of Cr and Cystatin-c in STZ-DKD, indicating that LNA Cf48 treatment could delay the decline of renal function in STZ-DKD. In conclusion, the injection of LNA Cf48 can reduce renal fibrosis and delay the occurrence and progression of STZ-DKD. Cf48 has been proven to be usable as a target substance, and by regulating the content of Cf48, it can be used as a drug to control kidney diseases and nephrotic fibrosis.

[0178] Part 4, Molecular mechanism of low molecular weight peptide Cf48 in the promotion of renal fibrosis 4.1 Objective: To investigate the direct effects of low molecular weight peptide Cf48 on fibroblasts and tubular cells in vitro, screen the downstream molecules of Cf48, and explore the molecular mechanism of Cf48 in the activation of fibroblasts and the promotion of EMT in tubular cells. 4.2 Experimental materials 4.2.1 Experimental cells Rat kidney fibroblasts (NRK-49F), rat kidney tubular cells (NRK-52E), and human embryonic kidney cells (293T) were purchased from ATCC (American Type Culture Collection, USA). 4.2.2 Main experimental instruments and equipment (1) Clean bench, Esco, Singapore. (2) 5% CO2 incubator, Thermo Eclectron Corporation, USA. (3) Cell counter, Thermo, USA. (4) Autoclave, Hirayama, Japan. (5) Electric thermostatic drying oven, Guangzhou Dongfang Electric Heating Drying Equipment Factory. (6) Inverted mixer reactor, Thermo, USA. (7) Ultrasonic crusher, Helser UP200S, Germany; (8) SeahorseXFe / XF analyzer, Agilent, USA; (9) Others: same as 3.1.2.2. 4.2.3. Main experimental consumables (1) Cell culture flask, 75 cm 2, Corning, USA; (2) Cell culture dish, Corning, USA. (3) 96-well cell plate, 6-well cell plate, Corning, USA; (4) Cell counting plate, Thermo, USA. (5) Cell sieve (22μm), Corning, USA. (6) 2mL vial, Corning, USA; (7) Chamber Slide, Thermo, USA; (8) Others: same as 3.1.2.3.

[0179] 4.2.4 Main experimental reagents (1) Fetal bovine serum, GIBCO, USA; (2) DMEM / F12 medium, GIBCO, USA. (3) 0.25% Trypsin, GIBCO, USA; (4) Penicillin / Streptomycin sulfate, GIBCO, USA; (5) TGF-β1 recombinant protein, R&D, USA; (6) Lipofectamine3000, Thermo, USA. (7) Opti-MEM medium, GIBCO, USA; (8) Protein A / G Agarose, Santa Cruz, USA; (9) Blasticidin S, Solarbio, China; (10)Puromycin, Sigma, USA. (11)Polybrene transfection reagent, Kuman Biotechnology, China; (12)Seahorse-related reagents: RPMI medium, pyruvic acid, glutamine, glucose, oligomycin, FCCP, rotenone / antimycin A purchased from Agilent (USA). (13)Antibodies: Directly labeled anti-α-SMA-Cy3 antibody, anti-α-SMA antibody purchased from Sigma (USA). Anti-His antibody and anti-Pax8 antibody were purchased from Abcam (USA). Anti-α-Tubulin antibody purchased from Cell Signaling Technology, Inc. (USA). Anti-SLC3A2 antibody was purchased from Thermo Corporation (USA) and Cell Signaling Technology (USA). (14)SLC3A2-sgRNA-CRISPR / Cas9 virus and control virus were commissioned to be synthesized by Shanghai Kuman Biotechnology Co., Ltd. (15)SLC3A2-siRNA and CTL-siRNA were commissioned to be synthesized by Shanghai Jiying Biotechnology Co., Ltd. (16)Cf48 recombinant protein was completed by commissioning Wuhan Pujian Biotechnology Co., Ltd. (17)The synthesis of hCf48-his-GFP plasmid was commissioned to Genewiz Biotechnology Co., Ltd., (18)Others: The same as 3.1.2.4.

[0180] 4.3 Research methods 4.3.1 Synthesis of Cf48 recombinant protein The CF48 recombinant protein has been commissioned to Pujian Biotechnology (Wuhan) Co., Ltd., and the specific process is as follows: (1) Gene synthesis and expression vector cloning a) Synthesize the cDNA sequence of the Fc segment with an Fc-Cf48 tag and add it to the expression vector pATX1 (cloning site: EcoRI / NotI); b) The expression vector Fc-Cf48-pATX1 was introduced into CHO cells (tool cells: Chinese hamster ovary cells), and the CHO cells were proliferated on a large scale to obtain as many expressed amino acids as possible. The expressed amino acid sequence is expected to be as follows. MKHLWFFLLLVAAPRWVLSCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKDDDDKSR EPATGSAVPAQSRPCVDCHAFEFMQRALQDLRKTAYSLDARTETLLLQAERRALCACWPAGR *. Note: Cf48 signal peptide [1:19], Fc tag [20:238], enzyme cleavage site [239:245], Cf48 secretion peptide [246:307].

[0181] (2) Affinity purification of the expressed amino acids Cf48 containing the Fc tag was purified by protein resin A affinity. Since the synthesized Cf48 is the secretion terminus, cell lysate (CLB) and cell culture supernatant (Medium) were collected for affinity purification, and intracellular Fc-Cf48 (isolated from the cell lysate) and extracellular secreted Cf48 (sFc-Cf48, isolated from the cell culture medium) were obtained respectively. The obtained proteins were verified by SDS-PAGE gel and Kaoma staining. (3) Protease digestion to remove the Fc segment C-Cf48 and s-Cf48 without the Fc tag were obtained by digestion of the lyase cleavage site by enterokinase digestion, and it was confirmed by SDS-PAGE gel and Kaoma staining that the free Fc tag was removed by the adhesion of protein resin A.

[0182] 4.3.2 Cell culture The NRK-49F and NRK-52E cell lines were cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37°C with 95% air and 5% CO2.

[0183] 4.3.3 The Cf48 recombinant protein stimulates fibroblasts (1) NRK-49F cells were evenly seeded into 6-well cell plates together with the growth medium (DMEM / F12 + 10% FBS). (2) When the cells adhered uniformly and stably and grew to a density of 70%, different types of Cf48 recombinant proteins (s-Cf48, c-Cf48) were diluted in 0.5% FBS medium (DMEM / F12), or Cf48 was diluted to different concentrations and added to NRK-49F cells. TGF-β1 was not added to the NC group, and TGF-β1 was added to the TGF-β1 stimulation group at a total concentration of 1 ng / mL, and the cells were co-stimulated for 48 hours. 4.3.4 Cell protein extraction: The same as 1.3.7. 4.3.5 Western blot: The same as 1.3.9.

[0184] 4.3.6 Cell immunofluorescence (1) Seed NRK-52E cells in a chamber slide at a density of 5000 cells / well with growth medium (DMEM / F12 + 10% FBS); (2) Cell stimulation: After the cells adhere, stimulate with s-Cf48 in 0.5% FBS medium (DMEM / F12) at concentrations of 0 ng / mL, 20 ng / mL, 40 ng / mL, 80 ng / mL with or without TGF-β1 (1 ng / mL) for 48 hours; (3) Aspirate the medium in the chamber slide and wash 3 times with pre-chilled PBS; (4) Fixation: Fix the cells with 4% paraformaldehyde at room temperature for 5 minutes; (5) Place in PBS buffer, shake on a shaker for 10 minutes, and wash 3 times; (6) Tissue pen circle: Remove the cell separation plate of the chamber slide, wipe the water around the stem cells, gently draw a circle with an immunohistochemistry pen to completely enclose the cells, and place in a humid box. During this time, prevent the tissue from drying the film; (7) Blocking: Drop 50 μL of 1% BSA (in PBS) solution onto each histochemical circle and block at room temperature for 1 hour; (8) Incubation with primary antibody: Dilute the primary antibody in 1% BSA solution at the corresponding concentration (α-SMA-Cy3, 1:5000) and incubate overnight in the dark; (9) Place in PBS buffer, shake on a shaker for 10 minutes, and wash 3 times; (10) DAPI double staining: Incubate DAPI at a concentration of 1:100 at room temperature for 5 minutes; (11) Cover: Place in PBS buffer, shake on a shaker for 10 minutes, wash 3 times, then centrifuge to remove water, drop mount medium, cover with a coverslip, and dry overnight at room temperature; (12) Observe with a laser confocal microscope and take pictures.

[0185] 4.3.7 Co-Immunoprecipitation and Identification of Protein Complexes (1) Transfection of hCf48-his-IRES-GFP plasmid and protein expression The synthesis of the hCf48-his-IRES-GFP plasmid was entrusted to Genewiz Biotechnology Co., Ltd., and the specific synthesis method was as follows. a) 293T cells were evenly seeded into three 10-cm cell culture dishes. When the cell density reached 80%, the medium was replaced with serum-free medium. b) The transfection mixture was prepared at a ratio of 67.5 μg of DNA, 33.75 μL of Lipofectamine 3000, and 45 μL of P3000 per dish, and mixed well at room temperature for 15 minutes. Then, the cells were added. After 12 hours of transfection, the cell culture medium containing 10% FBS was replaced, and the culture was continued for 36 hours. Under an upright fluorescence microscope, the cells had a normal morphology, and approximately 70% of the cells expressed green fluorescence. (2) Protein degradation After each large dish was washed three times with clean pre-cooled PBS, 300 μL of RIPA protein lysate containing protease inhibitor was added, and the reaction was carried out on ice for 15 minutes. The cells were scraped with a cell scraper, and the protein lysate was pipetted into a 1.5-mL EP tube. After the protein was disrupted with an ultrasonic breaker, it was centrifuged at high speed (15,000 rpm, 4°C) for 30 minutes, and the protein supernatant was aspirated to remove the precipitate. (3) Reaction of specific primary antibody with agarose When 400 μL of PBS was added per 100 μL of protein lysate, the total reaction volume became 500 μL. Anti-His antibody was added to each mixture at a ratio of 1:100, and the reaction was mixed by inverting at 4°C for 1 hour. Subsequently, 50 μL of protein A / G agarose was added to each mixture, and the reaction was mixed by inverting at 4°C overnight. (4) Agarose precipitation The mixture was centrifuged at high speed (10,000 rpm, 4°C) for 1 minute overnight until the agarose precipitate was visible. The supernatant was discarded, washed three times with clean PBS, and centrifuged at high speed (10,000 rpm, 4°C) for 1 minute to remove the supernatant. (5) Protein denaturation After removing the supernatant, 2X Loading Buffer was directly added to the precipitate, boiled at 100°C for 15 minutes, centrifuged at high speed (15,000 rpm, 4°C) for 30 minutes, and the supernatant protein lysate was loaded onto the gel. (6) Protein running adhesive, Coomassie staining Take 100 μL of the protein lysate after boiling and denaturation, run the gel on an SDS-PAGE gel, wait for the protein to flow into the separation gel for about 1.5 cm in length, stop the electrophoresis, perform Coomassie staining, and look at the protein bands. The cleavage area is about 1 cm 2 is. (7) Identification of the protein mixture The excised gel flow sample was sent to Jingjie Biotech Co., Ltd., and the mass spectrometry identification of the protein mixture was entrusted to Jingjie Biotech Co., Ltd.

[0186] 4.3.8 Lentiviral transfection for constructing SLC3A2 knockdown cell line (1) Knock out the SLC3A2 target gene using the CRISPR / Cas9 system. CAS9 selects the resistance vector by Blasticidin S (methicillin S) and the gRNA by the Puromycin (puromycin) resistance vector (PGMLV-GM1:U6-gRNA-EF1a-Puro). The synthesis of two lentiviruses and the control lentivirus was entrusted to Shanghai GenePharma Co., Ltd. The designed and synthesized SLC3A2 sgRNA oligomer single-stranded oligonucleotide sequences are as follows (rat). Primer-T2 CACCGAGCAGCAGCAGCGCCCAGC; Primer-B2 AAACGCTGGGCGCTGCTGCTGCTC 。 (2) Infect NRK-49F cells with Cas9-Blasticidin S virus a) NRK-49F cells were uniformly seeded into a 6-well plate at a density of 105 cells / well with cell growth medium (DMEM / F12 + 10% FBS). b) After the cells adhered, the cells were changed to serum-free medium containing polybrene transfection auxiliary reagent (working concentration: 6 μg / mL), and Cas9-blastocidin S virus solution (MOI 1:100) was added to infect the cells. After 24 hours, the cells were changed to cell growth medium (DMEM / F12 + 10% FBS), and blank control cells were added only to serum-free medium containing the same concentration of polybrene. c) Blasticidin S screening of positive transfection cells: 48 hours after transfection, the cells were passaged with fresh medium containing a working concentration of 25 μg / mL Blasticidin S, and then the medium was taken out every 3 - 4 days, and fresh medium containing the antibiotic was added until all the cells in the blank control wells died. (3) NRK-49F and the Cas9 working system were infected with SLC3A2-sgRNA and CTL-sgRNA a) NRK-49F cells expressing the Cas9 working system after screening were uniformly seeded into a 6-well cell plate at a density of 105 cells / well with cell growth medium (DMEM / F12 + 10% FBS). b) Infection with SLC3A2-sgRNA and CTL-sgRNA viruses by the same method as in (2b); c) Puromycin screening of positive cells: 48 hours after transfection, the cells were passaged with fresh medium containing a working concentration of 8 μg / mL puromycin, and then the medium was taken out every 3 - 4 days, and fresh medium containing the antibiotic was added until all the cells in the blank control wells died. d) Verify the knockdown efficiency of SLC3A2 in fibroblasts by Western blot.

[0187] 4.3.9 Transfection of SLC3A2-siRNA and CTL-siRNA SLC3A2-siRNA and CTL-siRNA were synthesized by commissioning Shanghai Jiying Biotechnology Co., Ltd. The SLC3A2-siRNA (rat) sequences used are as follows. Primer-F: GAGGCAUAGCUGGUCUGAA; Primer-R: UUCAGACCAGCUAUGCCUC; Transfect SLC3A2-siRNA and CTL-siRNA as follows using the Lipofectamine 3000 procedure. (1) Seed NRK-49F cells evenly in a 6-well cell plate at a density of 105 cells / well with cell growth medium (DMEM / F12 + 10% FBS). (2) After the cell adherents have grown uniformly and stably, replace the cell growth medium with serum-free medium (1.5 mL per well). (3) Dissolve the siRNA in enzyme-free water and prepare to store the stock solution at a concentration of 20 μM. (4) According to the amount of 500 μL Opti-MEM + 10 μL siRNA stock solution + 6 μL of Lipofectamine 3000 per well, configure the siRNA working solution so that the working concentration of the siRNA is 100 nM, and add 500 μL of the working solution containing 100 nM siRNA to each well. (5) After a 48-hour reaction, replace the liquid for the next step.

[0188] 4.3.10 Cell Mitochondrial Loading Test (Seahorse XF) (1) Cell plating: Seed NRK-52E cells in a Seahorse XF cell culture microplate at a density of 5000 cells / well with cell growth medium (DMEM / F12 + 10% FBS). (2) Cell stimulation: After the cells have adhered (about 6 hours), add the corresponding stimuli (NC, 40 ng / mL Cf48, 1 ng / mL TGF-β1, 1 ng / mL TGF-β1 + 40 ng / mL Cf48) to each experimental well in serum-free medium for a total of 48 hours. The transfection method for CTL-siRNA and SLC3A2-siRNA is the same as in 4.3.9. (3) Follow the experimental procedure of the Agilent Seahorse XF cell mitochondrial stress test. The specific procedure is as follows. a) Sensor hydration: Place the sensor probe plate in ddH20 and hydrate it overnight at 37°C in a CO2-free incubator. b) Sensor calibration: After hydrating the sensor overnight, place the probe in the calibration solution and leave it in a CO2-free incubator at 37°C for 45 - 60 minutes for calibration. c) Prepare the basic detection solution: Add additives to RPMI medium to prepare a basic assay solution containing 1 mmol / L sodium pyruvate, 2 mmol / L glutamine, and 10 mmol / L glucose. d) Configure the working solution: Dissolve in the basic detection solution to configure the working solution: oligomycin, FCCP, and Rotenone / Antimycin A; e) Plate addition: Oligomycin, FCCP, Rotenone / Antimycin A were added to the calibrated sensor probe plate in wells A, B, C in that order, so that the final concentrations in the wells were 2 μM, 1 μM, 0.5 μM respectively. f) Follow the machine's cell mitochondrial stress program.

[0189] 4.4 Experimental results 4.4.1 Secreted Cf48 promotes fibroblast activation During the synthesis of the small molecule peptide Cf48 in vitro, two recombinant peptides Cf48, sCf48 (secreted Cf48) from the culture supernatant and cCf48 (intracellular Cf48) from the cell lysate, were obtained by concentration and purification from the culture supernatant and the cell lysates of the tool cells. To observe the effect of synthetic Cf48 on fibroblasts, two types of recombinant Cf48 were added to rat fibroblasts (NRK-49F) for 48 hours with or without TGF-β1 (1 ng / mL). As shown in Figure 4-1, the expression of α-SMA increased in fibroblasts without TGF-β1 addition, while the expression of α-SMA did not change significantly in fibroblasts with intracellular Cf48 (cCf48) addition. After TGF-β1 addition, the expression of α-SMA in the solvent group increased, indicating that TGF-β1 may promote the activation of fibroblasts. The expression of α-SMA in fibroblasts added with sCf48 under the condition of TGF-β1 further increased, while there was no significant difference in fibroblasts added with cCf48 compared with the solvent group. This result is consistent with the result that the Cf48 overexpression-conditioned medium confirmed in the first part promotes the activation of fibroblasts, and it is further confirmed that secreted Cf48 has the biological effect of promoting the activation of fibroblasts. Therefore, secreted Cf48 (sCf48) purified from the supernatant of cell culture fluid was selected for the next experiment.

[0190] 4.4.2 Secreted Cf48 promotes the activation of fibroblasts in a concentration-dependent manner Furthermore, regardless of the presence or absence of TGF-β1, different concentrations of sCf48 were added to NRK-49F cells, and the expression of α-SMA was observed by Western blot. As shown in Figure 4-2A, in the absence of TGF-β1, even when sCf48 was less than 5 ng / ml, there was no change in the expression of α-SMA. With the increase in sCf48 concentration, the expression of α-SMA gradually increased. High-concentration sCf48 itself has the effect of promoting the activation of fibroblasts, and the effect is more significant at 40 ng / mL. Under the condition of TGF-β1, the effect of sCf48 on fibroblast activation was significantly enhanced. The expression of α-SMA increased significantly at sCf48 concentrations exceeding 5 ng / mL, indicating that the fibrotic promotion effects of sCf48 and TGF-β1 were synergistic. The results of three independent experiments are shown in Figures 4-2 B and C. The expression of α-SMA at 40 ng / mL sC4orf48 can be significantly enhanced regardless of the presence or absence of TGF-β1. This is statistically different. Therefore, for further experiments, a concentration of 40 ng / mL of sCf48 was selected on fibroblasts.

[0191] 4.4.3 Identification of the CF48 co-immunoprecipitation mixture To further investigate the molecular mechanism of Cf48 that promotes the fibrotic reaction, the expression plasmid of Cf48 was transfected into 293T cells, and possible molecules of Cf48 were explored by co-immunoprecipitation and protein spectrometry. According to the SDS-PAGE map of the protein mixture interacting with Cf48, the consistency of the three biological replicates was good, and it was found that the distribution of the protein mixture was more consistent. The target protein Cf48 was detected in the three mixture identifications. It was found that Cf48 was successfully transfected and pulled down during the co-immunoprecipitation process, and the co-immunoprecipitated protein molecules were identified by mass spectrometry. Cf48 binds to cell surface receptors, transmits messages to cells, and causes a series of biological effects. To find the Cf48 receptor, the molecule had a cytoplasmic domain, a transmembrane domain, and an extracellular domain as screening criteria. In the identification of the three mixtures, both TFRC and SLC3A2 could be detected, meeting the screening criteria and having a high protein score. From the concentration gradient binding curves of Cf48 with TFR and SLC3A2 by surface plasmon resonance (SPR), it was shown that the dissociation equilibrium constants of Cf48 from TFRC and SLC3A2d [KD(M)] were 5.71e-7 and 3.52e-8, respectively. TFRC is Transferrin receptor protein 1, which mediates iron absorption and Ferroptosis. Reducing the expression of TFRC can delay the fibrosis of UUO and STZ-induced diabetic nephropathy (Yasumura et al. Hypertension. February 2020; 75(2):413-421). SLC3A2 is the heavy chain of an amino acid transport channel protein located in the cell membrane. It binds to various light chains via disulfide bonds and plays a role in stably fixing the amino acid transport channel protein to the cell membrane. Since the transmembrane transport of amino acids plays an important role in maintaining the energy metabolism of cells and other normal cell functions, SLC3A2 is widely expressed in renal tissue, especially in renal tubular epithelial cells, and is involved in the reabsorption function of renal tubules. The above information suggests that TFRC and SLC3A2 are involved in the mechanism of action of Cf48, and TFRC and SLC3A2 may be receptors and / or targets of Cf48.

[0192] 4.4.4 Secreted Cf48 reduces the expression level of SLC3A2 in fibroblasts To observe the effect of Cf48 on SLC3A2, fibroblasts were stimulated with sCf48 at concentrations of 0, 5 ng / mL, 10 ng / mL, and 20 ng / mL for 48 hours, and its effect on SLC3A2 was observed. As shown in Figure 4-3 by Western blotting, it was found that sCf48 decreased the expression of SLC3A2 in a concentration-dependent manner, and it was able to decrease the expression of SLC3A2 at a low concentration (5 ng / mL).

[0193] 4.4.5 Downregulation of SLC3A2 expression promotes the activation of fibroblasts To investigate the effect of decreased SLC3A2 expression on fibroblasts, the expression of SLC3A2 was suppressed by lentiviral infection and siRNA transfection, and the effect on fibroblasts was observed. As shown in Figure 4-4A, SLC3A2-sgRNA-infected SLC3A2-sgRNA could partially knockdown the expression of SLC3A2 compared with the control virus. The expression of α-SMA in fibroblasts infected with SLC3A2-sgRNA decreased along with the expression of SLC3A2, and the expression of cellular α-SMA increased. Next, as shown in Figure 4-4B, since the expression of SLC3A2 was knocked down by transfection with different concentrations of control siRNA and SLC3A2-siRNA, the expression of SLC3A2 gradually decreased with the increase in the concentration of SLC3A2-siRNA, and it was found that SLC3A2 could achieve a complete knockout effect at a concentration of 100 nmol / L of SLC3A2-siRNA. As the expression of SLC3A2 decreased, the expression of α-SMA in fibroblasts gradually increased. It can be seen that the decrease in the expression of SLC3A2 can increase the expression of α-SMA in fibroblasts and promote the activation of fibroblasts.

[0194] 4.4.6 Downregulation of SLC3A2 enhances the role of secreted Cf48-activated fibroblasts In Figure 4-5, the expression of α-SMA was observed in fibroblasts transfected with or without the combined use of CTL-sgRNA and SLC3A2-sgRNA. As a result, it was shown that in fibroblasts transfected with CTL-sgRNA, the expression of SLC3A2 decreased and the expression of α-SMA increased, which was consistent with the above results. In transfected SLC3A2-sgRNA fibroblasts, the expression of SLC3A2 decreased and the expression of α-SMA increased. Furthermore, when sCf48 was added, it was shown that the expression of SLC3A2 further decreased and the expression of α-SMA further increased, indicating that the downregulation of SLC3A2 expression can further promote the activation of sCf48. From these results, it was shown that the downregulation of SLC3A2 has a synergistic effect with sCf48 in the activation of fibroblasts.

[0195] 4.4.7 Downregulation of SLC3A2 enhances the role of TGF-β1 in fibroblast activation Since TGF-β1 can promote the activation of fibroblasts, to determine whether the decreased expression of SLC3A2 has a synergistic effect with TGF-β1, fibroblasts transfected with CTL-sgRNA and SLC3A2-sgRNA were stimulated with or without TGF-β1, and the effect on fibroblast activation was observed. As shown in Figure 4-6, when TGF-β1 was added to fibroblasts transfected with CTL-sgRNA, the expression of α-SMA increased, indicating that TGF-β1 itself can promote the activation of fibroblasts. Compared with CTL-sgRNA, in fibroblasts transfected with SLC3A2-sgRNA, the expression of SLC3A2 decreased and the expression of α-SMA increased, which was consistent with the above results. The expression of α-SMA further increased after the expression of SLC3A2 was knocked down and stimulated by TGF-β1, indicating that the downregulation of SLC3A2 can further enhance the activation of TGF-β1 in fibroblasts, suggesting that sCf48 may enhance the fibrotic promoting effect of TGF-β1 by decreasing the expression of SLC3A2. 4.4.8 Secreted Cf48 promotes the differentiation and transformation of renal tubular cells To observe the effect of secreted Cf48 on other cells, different concentrations of sCf48 were added to rat renal tubular epithelial cells (NRK-52E) for 48 hours with or without TGF-β1 (1 ng / mL), and the expression of α-SMA in renal tubular cells was observed by immunofluorescence staining of cells. The results showed that renal tubular cells in the solvent group did not express α-SMA under the condition without TGF-β1, indicating that tubular cells did not undergo differentiation and transformation under normal conditions. After stimulation with sCf48 for 48 hours, renal tubular cells expressing α-SMA were observed at a concentration of 20 ng / mL. As the concentration of sCf48 increased, the number of renal tubular cells expressing α-SMA also increased, indicating that sCf48 can promote the differentiation and transformation of renal tubular cells in a concentration-dependent manner. After adding 1 ng / mL of TGF-β1 and stimulating for 48 hours, it was found that renal tubular cells could express α-SMA, indicating that TGF-β1 can promote the differentiation and transformation of renal tubular cells. At the same time, different concentrations (1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL) of sCf48 were added, and the expression of α-SMA in renal tubular cells was further enhanced, indicating that sCf48 can enhance the differentiation and transformation of TGF-β1 on renal tubular cells in a concentration-dependent manner.

[0196] 4.4.9 The number of differentiated and migrated renal tubular cells in CF48 transgenic folic acid nephrosis mice is significantly increased To further clarify the role of Cf48, in an in vitro model of folic acid nephrosis in Cf48 overexpressing and control mice, renal tubular cells were labeled with Pax8 staining and α-SMA staining as mesenchymal cell markers. Since Pax8 is a nuclear marker of renal tubular cells, co-localization of Pax8 and DAPI was used to identify renal tubular cells in kidney tissues. In wild-type folic acid nephropathy mice, a small number of tubular cells expressing Pax8 were found, and α-SMA staining was observed in the cytoplasm, indicating that the tubular cells had undergone differentiation and transformation. However, in mice overexpressing Cf48 with folic acid nephropathy, the number of tubular cells expressing Pax8 and α-SMA increased significantly. These results indicate that upregulation of Cf48 expression in vivo promotes the differentiation and transformation of tubular cells in the diseased state, which is consistent with the results found in in vitro experiments.

[0197] 4.4.10 Secreted Cf48 decreases the expression of SLC3A2 in tubular cells To observe the effect of secreted Cf48 on the expression of SLC3A2 in tubular cells, different concentrations of sCf48 were added to NRK-52E cells and stimulated for 5 days to observe changes in the expression of SLC3A2. Western blot showed that sCf48 could decrease the expression of SLC3A2 in a concentration-dependent manner, and the effect of decreasing the expression of SLC3A2 was more obvious at a concentration of 40 ng / mL. At the same time, with the increase in the concentration of sCf48 and the decrease in the expression of SLC3A2, the expression of α-SMA gradually increased, and as shown in Figure 4-7, it was observed that it was consistent with the previous results. 4.4.11 Downregulation of SLC3A2 promotes the differentiation and transformation of tubular cells To investigate the effect of downregulation of SLC3A2 on the differentiation and transformation of tubular cells, the expression of SLC3A2 was suppressed by transfecting NRK-52E with control siRNA and SLC3A2-siRNA. As shown in Figure 4-8, the expression of SLC3A2 decreased, and the expression of E-cadherin, a marker of tubular cells, and the expression of α-SMA, a marker of mesenchymal cells, decreased after transfection with SLC3A2-siRNA, indicating the possibility that the decrease in the expression of SLC3A2 leads to the differentiation and transformation of tubular cells.

[0198] 4.4.12 Downregulation of SLC3A2 expression results in a decrease in mitochondrial function in tubular cells The expression of SLC3A2 was knocked down in renal tubular epithelial cells, and its mitochondrial function was detected. Specifically, after rat renal tubular cells were treated with CTL-siRNA and SLC3A2-siRNA for 48 hours, the mitochondrial function of the cells was detected by Tatno Otoshi Go, and the results are shown in Figures 4-9. The OCR curve of tubular cells expressing SLC3A2 decreased significantly. The basal respiration, maximum respiration rate, ATP production capacity, and spare respiration capacity indicating mitochondrial function decreased significantly in tubular cells with decreased SLC3A2 expression. Since SLC3A2 is involved in the formation of the amino-terminal transport channel, normal amino acid metabolism process is a prerequisite for energy supply, and the high energy demand and dependence on aerobic metabolism of renal tubular cells lead to abnormal energy metabolism in tubular cells.

[0199] 4.4.13 Secreted Cf48 causes a decrease in the mitochondrial function of tubular cells sCf48 was directly added to tubular cells, and mitochondrial function was detected regardless of the presence or absence of TGF-β1. As a result, compared with the normal control (NC), the OCR curve of tubular cells stimulated by sCf48 decreased significantly, and the basal respiration volume, maximum respiration volume, ATP production capacity, and spare respiration capacity decreased significantly, which was shown to be statistically different. The OCR curve of tubular cells stimulated by TGF-β1 was significantly lower than that of NC and was basically the same as that of tubular cells stimulated by sCf48, indicating that the effects of sCf48 and TGF-β1 on reducing the mitochondrial function of tubular cells are equivalent. The addition of sCf48 under TGF-β1 stimulation showed that the OCR curve of tubular cells further decreased, and the basal respiration capacity, ATP production capacity, and spare respiration capacity of tubular cells decreased significantly. From the above results, as shown in Figure 4-10, it was found that sCf48 causes a decrease in the mitochondrial function of tubular cells and may further exacerbate the mitochondrial dysfunction of tubular cells caused by TGF-β1.

[0200] 4.4.14 Decreased expression of SLC3A2 in folic acid nephrosis mice To further observe the relationship between the low-molecular-weight peptide Cf48 and SLC3A2 in in vivo experiments, immunofluorescence staining of SLC3A2 was performed on kidney tissues of folic acid nephrosis mice and normal control mice, and the in vitro expression of SLC3A2 was observed. As a result, high expression of SLC3A2 was found in the glomeruli and renal tubules of normal mice, and it was shown that the expression of SLC3A2 was significantly decreased in the glomeruli and renal tubules of FAN mice.

[0201] 4.4.15 The expression of SLC3A2 was down-regulated in Cf48 overexpressing mice, and the degree of fibrosis deteriorated To observe the relationship between the expression of Cf48 and SLC3A2 and the degree of fibrosis in vivo, in a folic acid nephrosis model of Cf48 transgenic mice, the expression of SLC3A2 and the fibrosis index α-SMA were detected. As can be seen from the results of Western blotting in Figure 4-11, in normal control mice, compared with WT mice, the expression of Cf48 increased in Tg mice, and at the same time, the expression of SLC3A2 decreased, and the expression of α-SMA increased slightly. In WT mice of the FAN model, the expression of Cf48 increased, the expression of SLC3A2 decreased significantly compared with normal mice, and the expression of α-SMA increased significantly, which was consistent with the above results. Compared with WT-FAN mice, the expression of Cf48 further increased in Tg-FAN mice, and the expressions of SLC3A2 and α-SMA increased significantly. 4.4.16 The expression of SLC3A2 increased in Cf48 knockout mice, and the degree of fibrosis decreased Furthermore, the expressions of SLC3A2 and the fibrosis index α-SMA were detected in the UUO model of Cf48 knockout mice. As shown in Figure 4-12, the expression of SLC3A2 increased slightly in Cf48-KO mice compared with WT mice in the sham group. After UUO surgery, the expression of SLC3A2 decreased significantly in WT mice, but the expression of α-SMA increased significantly. Compared with WT-UUO mice, the expression of SLC3A2 increased in KO-UUO mice, and with the decrease in the expression of α-SMA, the degree of renal fibrosis in KO-UUO mice was reduced.

[0202] 4.5 Mice with streptozotocin-induced advanced diabetic nephropathy obtained in this implementation were treated with Cf48 locked nucleic acid. After administering to diabetic nephropathy mice induced by diabetes for 4 weeks, LNA-CTL or LNA-c4orf48 (20 mg / kg) was intraperitoneally injected into the mice once a week for 6 weeks, and then the mice were sacrificed. Normal mice with matched age were also administered LNA-CTL or LNA-c4orf48 (20 mg / kg) as a control. As a result, it was shown that Cf48 locked nucleic acid treatment knocked down the expression of endogenous Cf48 in mice and delayed the occurrence and onset of advanced diabetic nephropathy mice. It can be seen that kidney damage can be suppressed or treated by inhibiting the expression of Cf48 in the target kidney. It is possible to achieve drug treatment by targeting and inhibiting the production of Cf48 in the target kidney.

[0203] 4.4.17 Effects of Cf48 on reducing SLC3A2 expression and promoting fibrosis via TFRC To further clarify the role of TFRC in the biological effects of Cf48, when TFRC was knocked down using siRNA technology in rat kidney fibroblasts, it was shown that knocking down TFRC significantly decreased the expression of TGF-β1-induced α-SMA by Cf48, and the fibrosis-promoting reaction by Cf48 was significantly weakened. As shown in Figure 4-13, it was suggested that TFRC is a receptor that mediates the fibrosis-promoting reaction of Cf48.

[0204] 4.6 Summary Maintaining normal renal function depends on the complex and precise interactions among a large number of renal parenchymal cells. This delicate balance among cells largely depends on the action of intercellular communication factors. When this balance is disrupted by factors such as metabolic disorders, hemodynamic abnormalities, and toxic substances, renal tubular cells are often the first to be attacked, and as a result, intercellular communication factors are abnormally secreted. If the damaging factors persist, the progressive action of the communication factors will cause irreversible damage to the parenchymal cells of the kidney, ultimately leading to renal fibrosis.

[0205] In this section, in vitro experiments were used to confirm that the small molecule peptide Cf48 secreted from damaged renal tubular cells can be used as an intercellular communication factor. Secreted Cf48 (sCf48) can act on fibroblasts via the paracrine pathway, increase the expression of α-SMA, and activate ECM-producing myofibroblasts. Secreted Cf48 can promote the differentiation and transformation of tubular cells (EMT) via the autocrine pathway, thereby worsening the progression of fibrosis. In in vivo experiments, it has also been observed that upregulation of Cf48 expression leads to a significant increase in the differentiation and transformation of tubular cells in mice under diseased conditions.

[0206] The mechanism of action of the small molecule peptide Cf48 is similar to that of TGF-β1, which has been well studied so far. It is produced by damaged tubular cells and then acts on renal parenchymal cells through humoral communication to promote fibrosis. TGF-β1 can be expressed in many tissues and cells in the body and is involved in many biological signal transduction pathways. In some preclinical trials, it has been shown that therapeutic strategies targeting the TGF-β1 pathway have an antifibrotic effect. However, since many signal transduction pathways are involved, antifibrosis may also have other adverse effects on the kidneys and cardiovascular system. The clinical development of anti-TGF-β1 neutralizing antibodies in kidney diseases has been interrupted due to the lack of beneficial effects of TGF-β1 signal transduction blockers in clinical trials for focal segmental glomerulosclerosis and diabetic nephropathy. Different from TGF-β1, the small molecule peptide Cf48 has a small molecular weight and strong specificity for targeting antibodies. No significant phenotypic abnormalities were observed in Cf48 gene knockout mice. Considering that the small molecule peptide Cf48 and TGF-β1 play similar roles in the activation of fibroblasts and the promotion of tubular cell EMT, targeted therapeutic drugs targeting Cf48 are expected to have great applications.

[0207] By co-immunoprecipitation protein profiling, the protein molecules that interact with Cf48 include TFRC and SLC3A2. The KDs of Cf48, TFRC, and SLC3A2 are 5.71e-7M and 3.52e-8M, respectively, and TFRC and SLC3A2 were the receptors and / or target targets of Cf48. The small peptide Cf48 can reduce the expression of SLC3A2 in fibroblasts and renal tubular epithelial cells. SLC3A2 belongs to the SLC3 family, which is a family of proteins that make up the heavy chain of amino acid transport channels. Since amino acids are essential raw materials for all living cells and organisms to function properly, there are special transport channels that mediate the transport of amino acids across the plasma membrane. The SLC3 family and the SLC7 family form heteromeric amino acid transporters (HATs). HATs are composed of a light chain and a heavy chain, with SLC3 family members forming the heavy chain of HAT and SLC7 family members forming the light chain of HAT. Among many amino acid substrates, SLC3A2 binds to various light chains via disulfide bonds and plays a role in maintaining the structural stability of the amino acid transport channel by anchoring HAT to the plasma membrane. Since it is an exchange channel, it is essential for the reabsorption function of the kidney and small intestine and the redox reaction of cells. LC3A2 plays an important role in cell energy supply and tissue metabolic homeostasis. In in vivo experiments, it was shown that the expression of SLC3A2 was significantly decreased in the kidney tissue of folic acid nephrosis mice. In vitro experiments revealed the expression of SLC3A2 in silencing fibroblasts, and it was found that fibroblasts may be activated into myofibroblasts expressing α-SMA. Similarly, silencing the expression of SLC3A2 in renal tubular epithelial cells causes EMT in tubular cells. Therefore, although SLC3A2 itself may be involved in the occurrence and development of fibrosis, by what mechanism does SLC3A2 mediate the fibrotic-promoting changes in renal parenchymal cells?

[0208] The kidney is an organ that consumes high energy in normal physiological processes. Its large energy demand is mainly due to the reabsorption function of the proximal renal tubule, which contains Na+, K+, glucose, amino acids, etc. in the original urine. Among them, the transport of sodium, especially sodium, requires tight regulation of energy supply and demand. Some studies have shown that defects in energy metabolism may promote the occurrence and development of renal fibrosis, and the progression of CKD is known to be associated with abnormal fatty acid oxidation (FAO) and glucose metabolism disorders. Many studies have confirmed that when the energy supply of tubular cells is insufficient, it becomes difficult to maintain the morphology and function of the renal tubules, and differentiation and transformation occur. For example, silencing the enzyme AMPK (protein kinase related to AMP activation) related to the energy metabolism of tubular cells inhibits the energy metabolism of tubular cells, promotes EMT of tubular cells, and worsens the fibrosis process. Inhibiting the expression of the amino acid channel SLC3A2 was found to significantly reduce the mitochondrial function of tubular cells, resulting in insufficient cell energy supply and leading to the occurrence of EMT. The small molecule peptide Cf48 inhibits the mitochondrial function of tubular cells and further worsens the decrease in mitochondrial function through TGF-β1, thereby inhibiting the energy metabolism of tubular cells. Since the small molecule peptide Cf48 can reduce the expression of SLC3A2, it can be concluded that the small molecule peptide Cf48 inhibits the energy metabolism of tubular cells, promotes the occurrence of EMT, and can promote the progression of fibrosis by reducing the expression of the amino acid channel SLC3A2.

[0209] It was also found that the small molecule peptide Cf48 may also lead to a decrease in SLC3A2 in fibroblasts. As the expression of SLC3A2 in fibroblasts decreases, the expression of α-SMA gradually increases. Therefore, it was found that the small molecule peptide Cf48 can activate fibroblasts by leading to a decrease in SLC3A2.

[0210] In in-vivo experiments, a significant decrease in SLC3A2 expression was observed in a mouse model of renal fibrosis. When Cf48 expression was upregulated, a decrease in SLC3A2 expression and an increase in the degree of fibrosis were observed in mice. Similarly, when Cf48 expression was downregulated, SLC3A2 expression increased and the degree of fibrosis in mice also decreased. In summary, the research in this section found that the small peptide Cf48 may lead to the activation of fibroblasts, promote EMT of tubular cells, and thereby exacerbate the progression of renal fibrosis. In addition, the small peptide Cf48 can reduce the expression of the amino acid channel SLC3A2 in fibroblasts and promote the activation of fibroblasts. The small peptide Cf48 can reduce the expression of the amino acid channel SLC3A2 in tubular cells, inhibit the energy metabolism of tubular cells, and promote the occurrence of EMT.

[0211] In summary, the following can be obtained. a. Secreted Cf48 promotes the activation of fibroblasts and the differentiation and transformation (EMT) of renal tubular epithelial cells. b. Secreted Cf48 reduces the expression of SLC3A2 in fibroblasts and renal tubular epithelial cells. Secreted Cf48 inhibits the amino acid channel SLC3A2 and promotes the activation of fibroblasts. d. Secreted Cf48 inhibits the amino acid channel SLC3A2, reduces the mitochondrial function of tubular cells, and promotes EMT. In in-vivo experiments, upregulation of Cf48 expression may lead to a decrease in SLC3A2 expression and an increase in fibrosis. When Cf48 expression is downregulated, SLC3A2 expression may increase and fibrosis may decrease.

[0212] <Example 4> Provided is the use of a Cf48 low molecular weight peptide marker as a target for the intervention of a kidney injury drug. The kidney injury drug treats kidney injury or delays the progression of kidney injury by targeting and inhibiting or removing the expression of the Cf48 low molecular weight peptide. Specifically, the kidney injury drug avoids the phenomenon of fibroblast activation caused by the inhibition of the amino acid channel SLC3A in fibroblasts by the Cf48 low molecular weight peptide by targeting and inhibiting the expression of the Cf48 low molecular weight peptide. The therapeutic drug for kidney injury avoids the occurrence of the induction of EMT and the promotion of the formation of renal fibrosis caused by the inhibition of the amino acid channel SLC3A2 in tubular cells and the inhibition of the energy metabolism of tubular cells by the Cf48 low molecular weight peptide by targeting and inhibiting the expression of the Cf48 low molecular weight peptide. Furthermore, as can be seen from the experiments, the therapeutic drug for kidney injury avoids the phenomenon of promoting the formation of renal fibrosis by acting synergistically with other factors by which Cf48 promotes the formation of renal fibrosis by targeting and inhibiting the expression of the Cf48 low molecular weight peptide. Note that the drug that inhibits the expression of the Cf48 low molecular weight peptide may be a locked nucleic acid not described herein or other drugs capable of achieving Cf48 inhibition.

[0213] <Example 5> A system for evaluating the status of kidney injury in a measurement target subject based on the Cf48 biomarker, A data collection module that collects at least the amount of Cf48 in a biological sample of the measurement target subject, An evaluation module is provided that compares the content of Cf48 in the biological sample with a collation parameter and obtains a level result corresponding to the kidney injury of the measurement target subject according to the comparison result. Specifically, the evaluation module is provided with a parameter storage unit and an analysis unit, and the parameter storage unit stores a first reference value and a second reference value.

[0214] The analysis unit compares the content of Cf48 in the detected biological sample transmitted by the data collection module with a first reference value and a second reference value. When the content of Cf48 low-molecular-weight peptides in the detected biological sample is equal to or higher than the first reference value, the analysis unit gives a first-level result, where the first level indicates that the condition of kidney injury is severe and intervention is necessary. When the content of Cf48 low-molecular-weight peptides in the detected biological sample is less than the second reference value, the analysis unit gives a low-level result, where the low level indicates that the condition of kidney injury is mild and intervention is not necessary. Here, the first threshold is greater than the second threshold. It should be noted that the specific values and numerical ranges of the first reference value and the second reference value can be flexibly selected and set according to specific purposes and usage occasions. Furthermore, the data collection module further collects whether TGF-β1 is present in the measured biological sample. The analysis unit compares the content of Cf48 in the detected biological sample transmitted by the data collection module with a first reference value and a second reference value. When the content of Cf48 low-molecular-weight peptides in the detected biological sample is less than the first reference value and greater than the second reference value, the analysis unit obtains the result of whether TGF-β1 is present in the measured biological sample collected by the data collection module. When TGF-β1 is present in the measured biological sample, the analysis unit gives a second-level result, where the second level indicates that the condition of kidney injury is severe and intervention is necessary.

[0215] A system for evaluating the condition of kidney injury of a measurement target subject based on the Cf48 biomarker has the characteristics of being easy to detect and having accurate results by collecting the amount of Cf48 in the biological sample of the measurement target subject and evaluating and obtaining the level of the condition of kidney injury. With this system, it is possible to track and evaluate various situations to be utilized for various purposes, such as the condition of kidney disease, the degree of kidney injury, prognosis prediction of the subject, the degree of renal fibrosis, the activity of renal fibrosis, and monitoring the treatment effect of kidney disease patients. According to various needs, corresponding first reference values and second reference values can be set to achieve evaluations for multiple purposes.

[0216] <Example 6> Based on the Cf48 biomarker of Embodiment 5, the activity of renal fibrosis is determined using a system for evaluating the status of kidney injury in a measurement target subject. The first reference value and the second reference value stored in the parameter storage unit are the first threshold value and the second threshold value, respectively. The analysis unit compares the content of Cf48 in the detected biological sample transmitted by the data collection module with the first reference value and the second reference value. When the content of Cf48 low-molecular peptide in the detected biological sample is greater than or equal to the first reference value, it indicates that the activity of renal fibrosis is strong activity. The analysis unit outputs a result at the first level, and the first level indicates that the status of kidney injury is serious and intervention is necessary. When the content of Cf48 low-molecular peptide in the detected biological sample is less than the second threshold value, it indicates that the activity of renal fibrosis is weak activity. The analysis unit outputs a result at a low level, and the low level indicates that the activity of progressive renal fibrosis is low and the ability of renal fibrosis to progress is absent. Here, the first threshold value is greater than the second threshold value, and the first and second threshold values can be set according to the actual detection purpose and control.

[0217] Based on the Cf48 biomarker, the system for evaluating the status of kidney injury in a measurement target subject further collects whether TGF-β1 is present in the measured biological sample by the data collection module. The analysis unit compares the content of Cf48 in the detected biological sample transmitted by the data collection module with the first threshold value and the second threshold value. When the content of Cf48 low-molecular peptide in the detected biological sample is less than the first threshold value and greater than the second threshold value, the analysis unit obtains the result of whether TGF-β1 is present in the measured biological sample collected by the data collection module. When TGF-β1 is present in the measured biological sample, it indicates that the activity of renal fibrosis is strong activity. The analysis unit outputs a result at the second level, and the second level indicates that the degree of activity of progressive renal fibrosis is high and intervention is necessary. When the system evaluates the level of the kidney damage situation by collecting the amount of Cf48 in the biological sample of the measurement target subject, it has the characteristics that the detection is easy and the result is accurate.

[0218] <Example 7> Some usage scenarios of the markers of the present invention are provided below. Mr. Li (48 years old, male) has no obvious abnormality in the kidneys and does not meet the indications for the examination and treatment of clinical kidney diseases. In a normal health check, the Cf48 content in the blood sample is measured at a level of less than 0.1 ng / mL, indicating that there is no risk of kidney damage. Mr. Wang (53 years old, male) has no obvious abnormality in the kidneys and does not meet the indications for the examination and treatment of clinical kidney diseases. In a normal health check, the Cf48 content in the blood sample is measured to exceed 10 ng / mL and be less than 30 ng / mL, indicating mild kidney damage. By implementing targeted measures to reduce the Cf48 content for a certain period and then reexamining the Cf48 content in the blood sample, it has been shown that the Cf48 content has decreased significantly, indicating that the treatment is effective. Ms. Chen (49 years old, female) has a high urinary albumin / creatinine in the kidneys but does not meet the indications for clinical kidney biopsy. Through examination, the Cf48 content in the blood sample is as high as 32 ng / mL, indicating severe kidney damage, a strong ability to progress in a more severe direction, a high degree of kidney fibrosis, a high activity of kidney fibrosis, and the need to suppress the progression as early as possible. It is suggested that timely treatment is required in the clinical setting, and the treatment effect can be judged by measuring the Cf48 content in the blood sample after the treatment period.

[0219] <Example 8> To confirm the specific expression of C4orf48 in nephrosis and renal fibrosis, in this example, the ELISA method was used to detect C4orf48 in serum samples of healthy people (people who went to Zhongshan Traditional Chinese Medicine Hospital and Yichang Central Hospital for physical examinations and had normal test results). The reagents were purchased from CUSABIO, and the operations were carried out according to the instructions of the manufacturer. A standard curve was established with six standard products at different concentration levels in Table 1, and a conversion formula between the absorbance value (OD) and the concentration was obtained. The OD value measured by the sample was substituted into the formula to calculate the sample concentration value. A total of 178 whole blood samples from healthy individuals were examined, with a mean value of 2.48 ng / ml and a standard deviation of 1.01 ng / ml. Table 2 shows the test results of samples 1 - 10 and samples 90 - 99 among the 178 samples. As can be seen from the test results, the content of Cf48 in the serum of healthy people is very low. By detecting the CF48 level, healthy people can be distinguished from patients with kidney damage.

Table 1

Table 2

[0220] <Example 9> To verify the specificity of serum C4orf48 in the diagnosis of nephrosis and renal fibrosis, this example used the same ELISA method as in Example 8 to detect the serum Cf48 content in patients with lung diseases having a tendency to develop pulmonary fibrosis, including 57 cases of interstitial lung disease, 33 cases of pulmonary infections, severe pneumonia, lung cancer, chronic obstructive pulmonary disease and other lung diseases. The test results of 10 cases of patients with interstitial lung disease and 10 cases of other lung diseases were randomly selected and shown in Table 3. According to the test results, the mean value of Cf48 in the serum samples of patients with lung injury was 0.95 ng / ml, and the standard deviation was 0.62 ng / ml. The experimental results indicated that the content of Cf48 could not characterize lung injury, proving the specific identification effect of Cf48 on kidney diseases.

Table 3

[0221] Finally, it should be noted that the above embodiments are not restrictive and are only used to illustrate the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above relatively preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modifications or equivalent replacements of the solutions that do not deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of a Cf48 low-molecular-weight peptide biomarker having the sequence shown in SEQ ID NO. 1 as a biomarker for determining the status of kidney injury in a subject to be measured.

2. The Cf48 low-molecular-weight peptide is used as a biomarker for the diagnosis of kidney disease, or The Cf48 low-molecular-weight peptide is used as a biomarker for determining the degree of kidney injury, or The Cf48 low-molecular-weight peptide is used as a biomarker for predicting the prognosis of a subject, or The Cf48 low-molecular-weight peptide is used as a biomarker for the degree of renal fibrosis, or The Cf48 low-molecular-weight peptide is used as a biomarker for the activity of renal fibrosis, or The use according to claim 1, wherein the Cf48 low-molecular-weight peptide is used as one or more biomarkers for monitoring the treatment effect of a kidney disease patient.

3. Use of the CF48 low-molecular-weight peptide as a biomarker in a diagnostic reagent kit for determining the status of kidney injury in a subject to be measured.

4. Detecting the expression level of the Cf48 low-molecular-weight peptide in a biological sample, and that the expression level being higher than the reference level indicates the presence of kidney injury, wherein the sample is kidney tissue or serum or plasma or whole blood, the use according to claim 3.

5. By detecting the expression level of the Cf48 low-molecular-weight peptide in a biological sample, determining whether the subject has kidney disease, or By detecting the expression level of the Cf48 low-molecular-weight peptide in a biological sample, determining the degree of kidney injury, or By detecting the expression level of the Cf48 low-molecular-weight peptide in a biological sample, performing a prognosis prediction of the patient, or By detecting the expression level of the Cf48 low-molecular-weight peptide in a biological sample, determining the degree of renal fibrosis, or By detecting the expression level of the Cf48 low-molecular-weight peptide in a biological sample, determining the activity of renal fibrosis, or By detecting the expression level of the Cf48 low-molecular-weight peptide in a biological sample, performing at least one of monitoring the treatment effect of a kidney disease patient, the use according to claim 3 or 4.

6. Determining whether there is damage to renal tubules according to the content of the Cf48 low-molecular-weight peptide in the detected biological sample, the use according to claim 5.

7. The higher the content of the Cf48 low-molecular-weight peptide in the detected biological sample, the higher the degree of kidney injury, The higher the content of Cf48 low-molecular-weight peptide in the detected biological sample, the higher the prognosis prediction of the target subject, that is, it indicates that the ability of the kidney disease to progress in a more severe direction becomes stronger. The use according to claim 5, characterized in that the higher the content of Cf48 low-molecular-weight peptide in the detected biological sample, the higher the activity of renal fibrosis, that is, the higher the degree of progressing renal fibrosis in the subject to be measured.

8. Compare the content of Cf48 low-molecular-weight peptide in the detected biological sample with a first threshold value. When the content of Cf48 low-molecular-weight peptide in the detected biological sample is equal to or higher than the first threshold value, it indicates that the activity of renal fibrosis is strong, the activity of progressing fibrosis in the kidney is high, and intervention is necessary. When the content of Cf48 low-molecular-weight peptide in the detected biological sample is smaller than the first threshold value and larger than the second threshold value, then detect whether TGF-β1 is present in the biological sample. When TGF-β1 is present in the measured biological sample, it indicates that the activity of renal fibrosis is strong. When the content of Cf48 low-molecular-weight peptide in the detected biological sample is smaller than the second threshold value, it indicates that the activity of renal fibrosis is weak, the activity of progressing fibrosis in the kidney is low, and it indicates that there is no ability for renal fibrosis to progress. The use according to claim 7, characterized in that the first threshold value is larger than the second threshold value.

9. Use of the Cf48 low-molecular-weight peptide marker as an intervention target in the preparation of a therapeutic agent for kidney injury.

10. The use according to claim 9, characterized in that the therapeutic agent for kidney injury treats kidney injury or delays the progression of kidney injury by targeting and inhibiting or removing the expression of Cf48 low-molecular-weight peptide.

11. The use according to claim 10, characterized in that the therapeutic agent for kidney injury avoids the phenomenon of fibroblast activation caused by Cf48 low-molecular-weight peptide inhibiting the amino acid channel SLC3A in fibroblasts by targeting and inhibiting the expression of Cf48 low-molecular-weight peptide.

12. The therapeutic agent for kidney injury avoids the occurrence of the induction of EMT and the promotion of the formation of renal fibrosis caused by Cf48 low-molecular-weight peptide inhibiting the amino acid channel SLC3A2 in tubular cells and inhibiting the energy metabolism of tubular cells by targeting and inhibiting the expression of Cf48 low-molecular-weight peptide. The therapeutic agent for kidney injury inhibits the binding of Cf48 low-molecular peptide to transferrin receptor protein 1, thereby avoiding the occurrence of induction of EMT caused by Cf48 activating the signal pathway downstream of TFRC and the occurrence of promotion of formation of renal fibrosis, and is the use according to any one of claims 9 to 11.

13. The therapeutic agent for kidney injury inhibits the expression of Cf48 low-molecular peptide as a target, thereby avoiding the phenomenon that Cf48 acts synergistically with other factors promoting the formation of renal fibrosis and promotes the formation of renal fibrosis, and is the use according to claim 12.

14. A system for evaluating the status of kidney injury in a measurement target subject based on the Cf48 biomarker, comprising: a data collection module that collects at least the amount of Cf48 in a biological sample of the measurement target subject; an evaluation module that compares the content of Cf48 in the biological sample with a collation parameter and obtains a level result corresponding to the kidney injury of the measurement target subject according to the comparison result, and is a system for evaluating the status of kidney injury in a measurement target subject based on the Cf48 biomarker.

15. The evaluation module is provided with a parameter storage unit and an analysis unit, the parameter storage unit stores a first reference value and a second reference value, the analysis unit compares the content of Cf48 in the detected biological sample transmitted by the data collection module with the first reference value and the second reference value, and when the content of Cf48 low-molecular peptide in the detected biological sample is equal to or higher than the first reference value, the analysis unit gives a result of the first level, and the first level indicates that the status of kidney injury is serious and intervention is necessary; when the content of Cf48 low-molecular peptide in the detected biological sample is smaller than the second reference value, the analysis unit gives a result of a low level, and the low level indicates that the status of kidney injury is mild and intervention is not necessary; the first reference value is larger than the second reference value, and is a system for evaluating the status of kidney injury in a measurement target subject based on the Cf48 biomarker according to claim 14.

16. The data collection module further collects whether TGF-β1 is present in the measurement biological sample. The analysis unit compares the content of Cf48 in the detected biological sample transmitted by the data collection module with a first reference value and a second reference value. When the content of Cf48 low molecular peptide in the detected biological sample is less than the first reference value and greater than the second reference value, The analysis unit obtains the result of whether TGF-β1 is present in the measured biological sample collected by the data collection module. When TGF-β1 is present in the measured biological sample, the analysis unit gives a result at the second level, and the second level indicates that the situation of kidney injury is serious and intervention is necessary. A system for evaluating the situation of kidney injury of a measurement target subject based on the Cf48 biomarker according to claim 15, characterized in that.

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