Marker for determining severity of igan renal tissue lesions and use thereof

Abstract

The application discloses a marker for determining the severity of IgAN kidney tissue lesions and application thereof, and inventors find that ACTN4, ACADS and COL1A1 are significantly differentially expressed proteins in kidney tissues. The significant down-regulation of ACTN4 may cause the change of actin cytoskeleton of IgAN glomerular podocytes; the significant down-regulation of ACAD may indirectly participate in the occurrence process of abnormal structure and function of IgAN renal tubular epithelial cells by affecting fatty acid metabolism in the cells; and the significant up-regulation of COL1A1 may participate in the accumulation of extracellular matrix in the renal interstitium of IgAN, and play a certain role in promoting the renal interstitial fibrosis. The combination of ACTN4, ACADS and COL1A1 has good prediction efficiency for the diagnosis of the severity of IgAN kidney tissue lesions, and the area under the ROC curve is 0.815, P 0.043. In addition, the immunohistochemical results also confirm the expression trend of the three proteins ACTN4, ACADS and COL1A1 in the corresponding kidney tissue substructures in proteomics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a marker for determining the severity of IgAN renal tissue lesions and application thereof. BACKGROUND

[0002] IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide and also an important cause of end-stage renal disease (ESRD) [1-5] . The prevalence of IgAN varies significantly by geographic location worldwide, with the highest incidence in East Asia and the lowest in Central Africa [6] . The occurrence of IgAN may be related to upper respiratory tract infection [7] , with a peak incidence at 20 and 30 years of age [8] , and more common in male patients [9,10] . The susceptibility of IgAN and the risk of disease progression are affected by the combined effects of genetic and environmental factors

[11] . Literature reports that the incidence of IgAN in adults is at least 2.5 / 100,000 per year

[12] . The clinical manifestations of IgAN patients are relatively wide-ranging, from asymptomatic microscopic hematuria to more severe courses characterized by persistent proteinuria and rapid deterioration of renal function [2] . The most common symptoms are hematuria and proteinuria of varying degrees

[13] . The current diagnosis of the disease still relies on pathological examination of renal tissue by kidney biopsy [1,5,9] , mainly IgA deposition in the mesangial area [5] , which is characterized by coarse granular, dispersed distribution, and complement C3 deposition in the mesangial area is also common, and can be accompanied by deposition of other immunoglobulins such as IgM and IgG [1] . The glomerular IgA eluted from the renal tissue samples of IgAN patients belongs to the IgA1 subclass and mainly exists in the form of polymers, and the most important is abnormal glycosylation

[14] . There is no disease-specific treatment at present, and the treatment is based on the severity of the disease to control blood pressure and maintain renal function in patients, and patients with mild urinary abnormalities, normal blood pressure and normal glomerular filtration rate usually show good performance and only need regular monitoring; for other patients, the treatment options are limited, including non-specific treatment by RAS blockers to reduce blood pressure and proteinuria, and other general treatments such as lipid-lowering, sodium intake control, smoking cessation, avoidance of non-steroidal anti-inflammatory drugs and other nephrotoxins; for patients with a large number of crescent formation, immunosuppressive agents combined with steroids can be used for treatment [5]Early intervention and subsequent treatment can help to prevent or delay the progression of kidney failure and reduce the number of patients requiring kidney replacement therapy

[15] IgAN patients have a variable clinical presentation and a highly variable prognosis [4] Some clinical parameters have been identified as strong indicators of progression of the disease, such as: elevated serum creatinine concentration (reduced glomerular filtration rate), hypertension, severe proteinuria

[16] , severe histopathological lesions [1] 30-40% of patients eventually progress to ESRD stage within 20-30 years of diagnosis [5] There are also studies that show a good long-term prognosis in patients with mild urinary abnormalities and normal renal function

[17] However, crescent IgAN has a poor prognosis

[18] Although there has been progress in understanding the pathogenesis of IgAN [5] It has not been fully elucidated and understanding the abnormal deposition of pathogenic IgA at the molecular level is promising for the treatment of IgAN

[16] There is an urgent need to improve treatment strategies, possibly based on further disease subclassification based on specific genetic and biological markers in the underlying pathogenic processes [2] .

[0003] The pathogenesis of IgAN is not fully elucidated [13,16,19] It has been reported that IgA1 in the circulation of IgAN patients is abnormally glycosylated [9,20] and that IgA1 eluted from the glomeruli is also abnormally glycosylated, and that these abnormally glycosylated IgA1 selectively deposits in the mesangial area of the glomerulus

[21] Earlier studies have also found that galactosylation and sialylation of IgA1 isolated from kidney biopsy specimens is reduced, and that this reduction in galactosylation and sialylation can play a key role in the deposition of IgA1 in the glomerulus

[22] Hematuria is typical in IgAN patients, with macrohematuria often occurring simultaneously with upper respiratory tract infections [23-25] There are also articles suggesting that IgAN can be linked to changes in the function of the intestinal mucosal immune system [25,26] Disorders of the mucosal immune system, lack of immune tolerance to commensal or common pathogens, can be a key factor in triggering the disease

[23] An animal experiment study found that mice with impaired mucosal immune tolerance mechanisms developed hematuria, persistent proteinuria and nephritis after intranasal administration of an infectious Sendai virus, leading to the conclusion that repeated heavy exposure to infectious viruses in the respiratory tract can disrupt the normally functioning mucosal immune tolerance mechanisms

[27] Abnormally glycosylated IgA1 can be produced after mucosal antigen stimulation[25,28] and then autoantibodies IgG and IgA against the abnormal glycosylated IgA1 are produced in vivo

[13] The antigen binds to the antibody to form an immune complex, which is finally deposited in the kidney [5] In vitro experiments have confirmed that circulating immune complexes rich in galactose-deficient IgA1 can stimulate mesangial cell proliferation

[29] The immune complex deposited in the mesangial area of the glomerulus can also activate mesangial cells [5] and activate the complement system (as complement C3 can usually be detected in kidney tissue specimens from kidney biopsy), ultimately leading to damage to the kidney tissue

[14] The current mainstream explanation for the immunopathogenesis of IgAN is the "multiple hit" theory [5,14,23] Both genetic and environmental factors may play a role

[23] Specifically, the production of abnormally glycosylated IgA1 is the first hit; the production of antibodies in circulation that recognize the self-antigen is the second hit; the formation of pathogenic IgA1-containing immune complexes through immune recognition is the third hit; the fourth hit is the deposition of immune complexes in the mesangial area of the glomerulus and the activation of mesangial cells, stimulation of cytokine secretion, activation of the complement system, etc., leading to kidney damage [5,14]

[0004] Although the pathogenesis of IgAN is not fully understood, there has been significant progress in the study of the disease. A whole genome association analysis study of IgAN found that ITGAM-ITGAX, VAV3, CARD9, HLA-DQB1 and DEFA are genetic loci associated with IgAN

[30] In addition, the susceptibility loci associated with IgAN also include TNFSF13, MHC

[31] Studies have also shown that the gene risk score prediction model composed of rs11150612, rs7634389, rs2412971 and rs2856717 single nucleotide polymorphisms is independently associated with the progression of IgAN and can improve the predictive efficiency of clinical or clinical-pathological risk models for the progression of IgAN [4] Among them, rs2856717 may affect the clinical features and poor prognosis of IgAN, but further studies are needed to explore the association mechanism between this genotype-disease-phenotype

[32] ​A study on immunology found that the length of complementarity-determining region 3 (CDR3) of T cell receptor beta chain and immunoglobulin heavy chain in peripheral blood mononuclear cells of IgAN patients was significantly shorter than that of healthy controls, and the study suggested that the disease-related clonality of T cell receptor beta chain and immunoglobulin heavy chain could be used as a potential biomarker to help screen and diagnose IgAN

[33] . Another study found that whole-genome DNA methylation screening of CD4 +T T cells from IgAN patients found that some specific DNA regions were abnormally methylated, which could lead to changes in the expression of T cell receptor signaling-related genes and a decrease in the intensity of T cell receptor signaling in CD4+ T cells. Abnormal responses and activation of CD4+ T cells may explain the imbalance of helper T cells. The increased expression of transforming growth factor-beta (TGF-β) genes in CD4 +T T cells in the circulation of IgAN patients may promote glomerulosclerosis in IgAN, as TGF-β can enhance extracellular matrix accumulation

[35] . Another study showed that the percentage of regulatory T cells in peripheral blood mononuclear cells of IgAN patients was significantly lower than that of healthy controls, however, the expression levels of miR-133a and miR-133b in IgAN patients were significantly higher than those of controls, and the percentage of regulatory T cells was negatively correlated with the expression of miR-133a and miR-133b, while the expression level of transcription factor FOXP3 (forkhead box P3) mRNA in IgAN patients was significantly lower than that of controls and was also negatively correlated with the expression of miR-133a and miR-133b. In addition, the study confirmed that miR-133a and miR-133b mimics could significantly reduce the expression of FOXP3, while inhibitors of miR-133a and miR-133b could have the opposite effect

[36] , as FOXP3 is crucial for the differentiation, development, and function of regulatory T cells [37,38] , the study suggested that the inhibition of miR-133a and miR-133b on the differentiation of regulatory T cells was caused by the action on FOXP3

[36] In renal tissue affected by IgAN, diffuse IgA deposition in the mesangial area is a major characteristic. A single-cell transcriptomics study found that the gene JCHAIN ​​was significantly upregulated in glomerular mesangial cells of IgAN, and this was confirmed by in situ immunofluorescence staining on frozen kidney tissue sections. Since JCHAIN ​​is essential for immunoglobulin polymerization and IgA transmucosal epithelial transport, researchers hypothesize that glomerular mesangial cells in IgAN may recognize and transport IgA by upregulating JCHAIN ​​expression, potentially guiding specific IgA deposition in the glomerular mesangium. Furthermore, this study also found that the interaction between mesangial cells and endothelial cells was most increased in IgAN. This study systematically analyzed the molecular events related to the onset and progression of IgAN, providing a promising prospect for the treatment of the disease.

[39] .

[0005] Currently, finding reliable biomarkers in IgAN is more challenging.

[40] However, some studies have explored the biological markers of this disease. Most IgAN patients have elevated serum galactose-deficient IgA1 levels. [41,42] Furthermore, elevated serum galactose-deficient IgA1 levels are associated with poor prognosis in IgAN.

[42] Studies have shown that serum galactose-deficient IgA1 in IgAN patients is a useful diagnostic and prognostic biomarker.

[43] Studies have also shown that serum standardized IgG autoantibody levels and total IgA autoantibody levels are significantly elevated in IgAN patients, and that serum standardized IgG and total IgA autoantibody levels are closely related to the progression of IgAN.

[44] Zhang et al.

[45] Serum IgA / C3 levels have been found to be a useful predictor of poor prognosis in IgAN. Other studies have shown that serum C3 levels and their fluctuations may reflect disease activity in IgAN patients.

[46] Furthermore, elevated levels of circulating advanced oxidation protein products (AOPPs) are also a biomarker reflecting poor renal function prognosis in IgAN patients. [47,48] Elevated plasma mannose-binding lectin levels may also indicate poor renal prognosis in IgAN patients.

[49] Studies have also found that plasma and peptide levels are associated with the severity and progression of IgAN disease.

[50] The level of circulating complement factor H-related protein 5 (FHR-5) was significantly elevated in IgAN patients and was an independent risk factor for disease progression in IgAN patients.

[51] In addition, IgG in kidney tissue

[52] C3a and C5a

[53] C4d

[54] C3 and C4d deposition is associated with poor renal outcomes in IgAN patients, and deposition of C3 and C4d in the glomerular mesangial area is also an independent risk factor for disease progression.

[55] Regarding the exploration of biological markers in urine, studies have found that the level of IgA-IgG immune complexes in the urine of IgAN patients is significantly higher compared to non-IgAN patients and healthy controls.

[19] Ding et al. discovered that neutrophil gelatinase-associated lipocalin (NGAL) in urine holds promise as an early biomarker for tubulointerstitial damage in IgAN patients.

[56] Studies have also found that mannose-binding lectin levels are significantly elevated in the urine of IgAN patients, and urinary mannose-binding lectin is significantly correlated with known clinical predictive parameters for IgAN prognosis. Furthermore, patients with non-remission had significantly higher urinary mannose-binding lectin levels at the end of follow-up than those with remission. This study suggests that urinary mannose-binding lectin can serve as a reliable and non-invasive biomarker for assessing disease severity and predicting prognosis.

[57] Furthermore, studies have shown that the level of matrix metalloproteinase 7 (MMP-7) in the urine of IgAN patients is an independent predictor of disease progression.

[58] Furthermore, measuring soluble transferrin receptors in the urine of IgAN patients is a novel and sensitive method for detecting potential biomarkers of the disease.

[59] .

[0006] To better assess the severity of IgAN renal tissue lesions, it is necessary to find new biomarker groups. Summary of the Invention

[0007] The purpose of this invention is to overcome at least one deficiency of the prior art and provide...

[0008] The technical solution adopted in this invention is:

[0009] The first aspect of the present invention provides:

[0010] A marker for determining the severity of renal tissue lesions in IgAN, consisting of ACTN4, ACADS and COL1A1 in combination.

[0011] In a second aspect of the present application, there is provided:

[0012] Use of a quantitative reagent for quantifying the amount of ACTN4, ACADS and COL1A1 protein in a sample in the preparation of a diagnostic reagent for the severity of renal tissue lesions in IgAN.

[0013] In some examples of use, the sample is a kidney biopsy tissue section.

[0014] In some examples of use, the quantitative reagent is selected from the group consisting of an ELISA reagent, an immunofluorescence reagent.

[0015] In some examples of use, the detection threshold is determined by plotting a ROC curve.

[0016] In a third aspect of the present application, there is provided:

[0017] A system for determining the severity of renal tissue lesions in IgAN, comprising:

[0018] a quantitative device for quantifying the amount of ACTN4, ACADS and COL1A1 protein in a sample;

[0019] an analysis device for determining the severity of renal tissue lesions in IgAN based on the amount of ACTN4, ACADS and COL1A1 protein in the sample;

[0020] a result output device for outputting the analysis result of the analysis device.

[0021] In some examples of the system, the quantitative device is selected from the group consisting of an ELISA quantitative device, an immunofluorescence quantitative device, a data-independent acquisition mass spectrometry quantitative device.

[0022] In some examples of the system, the analysis device determines the detection threshold by plotting a ROC curve.

[0023] In some examples of the system, the sample is a kidney biopsy tissue section.

[0024] The beneficial effects of the present application are:

[0025] The inventors found that, through ROC curve analysis of joint prediction of ACTN4, ACADS and COL1A1, it is concluded that the combination of the three protein indicators helps to evaluate the severity of IgAN renal tissue lesions, has the potential to distinguish the severity of IgAN renal histopathology, and may be a biological marker reflecting the degree of IgAN renal tissue lesions. It provides a more explicit basis for the diagnosis and treatment of IgA nephropathy. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a representative picture of kidney biopsy renal tissue pathology. A IgAN group PAS staining, PASM staining, Masson staining, IgA immunofluorescence picture (from left to right); B control group HE staining, PAS staining, PASM staining, Masson staining picture (from left to right).

[0027] Figure 2 is a representative picture of the substructure of the kidney glomerulus (A), the renal tubule (B), and the renal interstitium (C) before (left picture in each group of pictures) and after (right picture in each group of pictures) laser capture microdissection. The arrow in the picture indicates the target substructure, i.e., the glomerulus, the renal tubule, and the renal interstitium before and after microdissection.

[0028] Figure 3 is a representative picture of the cap of the PCR tube before laser capture microdissection (A) and the representative picture of the target tissue falling into the cap of the PCR tube after the tissue sample is cut (B). The arrow in the picture indicates the target tissue falling into the cap of the PCR tube after being cut.

[0029] Figure 4 is the expression amount of ACTN4 (A), ACADS (B), and COL1A1 (C) in the IgAN group and the control group.

[0030] Figure 5 is the ROC curve of the joint diagnosis of ACTN4, ACADS, and COL1A1 for the severity (mild, moderate and severe) of IgAN renal tissue lesions.

[0031] Figure 6 is the expression of ACTN4 protein on the representative renal tissue sections of the control group (A) and the IgAN group (B) (the pictures in each group come from three different research objects). The brown color in the picture indicates the positive expression of the protein, and it can be seen that the positive staining is mainly concentrated in the glomerular part.

[0032] Figure 7 is the statistical result of the expression of ACTN4 protein on the renal tissue sections of the IgAN group and the control group.

[0033] Figure 8are representative pictures of ACADS protein expression on kidney tissue sections from control group (A) and IgAN group (B) (the pictures from each group are from three different subjects, respectively). The brown color indicates the positive expression of the protein, and it can be seen that the positive staining is mainly concentrated in the renal tubular part.

[0034] Figure 9 are statistical results of COL1A1 protein expression on kidney tissue sections from IgAN group and control group.

[0035] Figure 10 are representative pictures of COL1A1 protein expression on kidney tissue sections from control group (A) and IgAN group (B) (the pictures from each group are from three different subjects, respectively). The brown color indicates the positive expression of the protein, and it can be seen that the positive staining is mainly concentrated in the renal interstitial part.

[0036] Figure 11 are statistical results of COL1A1 protein expression on kidney tissue sections from IgAN group and control group. DETAILED DESCRIPTION

[0037] The technical solutions of the present application are further illustrated below in combination with experiments.

[0038] MATERIALS AND METHODS

[0039] Sample source

[0040] The kidney biopsy tissue samples of the IgAN group and the control group were obtained from the Second Clinical Medical College of Jinan University, and were the formalin-fixed paraffin-embedded tissues remaining after routine kidney pathological diagnosis [60,69,70,86] . The clinical and relevant laboratory test index information of the IgAN patients in this batch, such as age, blood pressure, serum albumin, serum creatinine, blood urea, blood uric acid, blood total cholesterol, blood triglyceride, cystatin C, and 24-hour urine protein quantification, were also collected. The inclusion criteria for the IgAN patients were: the kidney biopsy tissue pathological examination was diagnosed as IgAN, and Lee pathological grading was performed [87,88] (Lee pathological grading is shown in Table 2.1); the patient was > 18 years old, and had not used glucocorticoid, immunosuppressant, etc. drug treatment before the puncture operation; no evidence of allergic purpura, systemic lupus erythematosus, rheumatoid arthritis, malignant tumor, hepatitis B virus related nephritis, cirrhosis, etc. was found; the exclusion criteria: patients with diabetic nephropathy, obesity related nephropathy, interstitial nephritis and other kidney diseases. The control group was > 18 years old and the kidney biopsy tissue pathology was roughly normal (had hematuria and microalbuminuria when the kidney biopsy was performed)

[69] The IgAN group included 18 cases, of which 5 cases were Lee grade II, 8 cases were Lee grade III, and 5 cases were Lee grade IV; the control group included 11 cases. The present application research was approved by the Medical Ethics Committee of the Second Clinical Medical College of Jinan University. All the included research objects have signed the informed consent form.

[0041] Table 2.1 IgAN kidney biopsy renal tissue pathological Lee grading

[0042]

[0043] Experimental method

[0044] The collected formalin-fixed paraffin-embedded kidney tissue samples of each case were cut respectively using a conventional pathological sectioning machine, and the thickness of the cut tissue slices was 10 μm [60,86] , then the cut tissue slices were transferred to the frame film slide (PET FrameSlides, No. 11505190, Leica Microsystems, Germany) prepared in advance for laser capture microdissection, and then deparaffinization, drying and laser capture microdissection steps were performed

[86] . The operation steps of laser capture microdissection of kidney tissue substructure: place the frame film slide loaded with kidney tissue slices on the stage of the laser capture microdissection instrument (Leica LMD7000, Germany), and place the 0.2ml standard PCR tube (special tube for laser capture microdissection instrument, used to collect samples) in the sample collection position, find the kidney tissue using a low-power objective, then adjust to a high-power objective and find the kidney tissue again on the computer screen connected to the instrument, get a clear tissue image by fine adjustment, then use the mouse to select the glomerulus on the computer screen, set appropriate laser beam, laser energy and cutting speed, click the start cutting button, and you can see the cutting track of the laser according to the previously selected tissue shape on the computer screen, after completion of the cutting, the previously selected tissue (i.e. the tissue that has been completely cut) will be separated from the surrounding tissue, under the action of gravity, the cut tissue will fall into the PCR tube cap for sample collection, then take out the PCR tube and carefully cap it, the cut tissue is collected in the PCR tube and labeled, thus completing the laser capture microdissection and collection of the glomerular part of the kidney tissue. The microdissection and collection of the tubular and interstitial parts of the kidney tissue are performed according to the above method respectively. Finally, the laser capture microdissection and collection of each substructure of the kidney tissue of each research object are completed.

[0045] Statistical analysis

[0046] The collected clinical data were statistically analyzed by SPSS 22.0 statistical software, and the data were expressed as mean ± standard deviation.

[0047] Results

[0048] Clinical information

[0049] The clinical and related laboratory test index information of the included IgAN patients is shown in Table 2.2, and the representative kidney biopsy renal tissue pathology pictures of the IgAN group are shown in Figure 1 A; and the representative kidney biopsy renal tissue pathology pictures of the control group are shown in Figure 1 B.

[0050] Table 2.2 Clinical and related laboratory test index of IgAN patients

[0051]

[0052] Results of laser capture microdissection of renal tissue

[0053] After the frame membrane slide loaded with renal tissue slices was prepared, the inventors used a laser capture microdissection instrument (Leica LMD7000, Germany) to perform microdissection and collection of the glomerulus, renal tubule, and renal interstitium of the renal tissue on the frame membrane slide of each research subject. Representative pictures of the renal tissue substructures glomerulus, renal tubule, and renal interstitium before and after microdissection are shown in Figure 2 , and representative pictures of the PCR tube cap used for tissue sample collection before laser capture microdissection and the tissue sample falling into the PCR tube cap after being cut are shown in Figure 3 .

[0054] Proteomic analysis of IgA nephropathy renal tissue substructures

[0055] The laser capture microdissection technique was used to separate the glomerulus, renal tubule, and renal interstitium of the kidney biopsy renal tissue of the IgAN group and the control group, and then DIA proteomic analysis was performed on the tissues in these parts. By comparing the protein expression of the glomerulus, renal tubule, and renal interstitium of the IgAN group and the control group, respectively, the differentially expressed proteins of each comparison group (i.e., the glomerular protein comparison group of the IgAN group and the control group, the renal tubular protein comparison group of the IgAN group and the control group, and the renal interstitial protein comparison group of the IgAN group and the control group) were obtained. Then, the differentially expressed proteins of each comparison group were analyzed by bioinformatics, and the differentially expressed proteins specific to the glomerulus, renal tubule, and renal interstitium of the IgAN were also analyzed by bioinformatics.

[0056] According to the KEGG pathway enrichment results of differentially expressed proteins in each comparison group and existing relevant literature knowledge, the important proteins with significant differences on the pathways related to the pathogenesis of IgAN were discussed. In the KEGG pathway significantly enriched results of differentially expressed proteins in glomerulus, the actin cytoskeleton regulation pathway may be related to the podocyte injury of IgAN glomerulus, and the significant down-regulation of ACTN4 protein on the pathway may lead to the changes of podocyte actin cytoskeleton, and then promote the abnormalities of podocyte morphology and function. In the KEGG pathway significantly enriched results of differentially expressed proteins in renal tubule, the fatty acid degradation pathway may be related to the fatty acid metabolic process of IgAN renal tubule, and the ACADS protein on the pathway is the starting enzyme of fatty acid mitochondrial beta oxidation, and the down-regulated ACADS protein may indirectly participate in the occurrence process of IgAN renal tubular structural and functional abnormalities by limiting the fatty acid metabolism in renal tubular epithelial cells. In the KEGG pathway significantly enriched results of differentially expressed proteins in renal interstitium, the ECM (extracellular matrix) receptor interaction pathway may be related to the extracellular matrix of IgAN renal interstitium; the significantly up-regulated COL1A1 protein on the pathway indicates that its expression in IgAN renal interstitium significantly increases, which suggests that it may be involved in the accumulation of extracellular matrix in IgAN renal interstitium and promote the fibrosis process of renal interstitium. The above results and conclusions show that the differentially expressed proteins (i.e. the proteins obtained by comparing the proteins in the corresponding parts of IgAN group and control group) in the glomerulus, renal tubule and renal interstitium of IgAN kidney tissue exist different changes and interactions, and the protein signal transduction pathways in each substructure part also exist certain degree of differences.

[0057] In the KEGG pathway significantly enriched results of IgAN glomerular differential expression proteins (obtained by comparing the glomerular proteins of the IgAN group and the control group), and according to the relevant literature knowledge, the ACTN4 protein on the actin cytoskeleton regulation pathway was discussed, and it was considered that the significant down-regulation of the ACTN4 protein may be related to the podocyte injury of the IgAN glomerulus. In the KEGG pathway significantly enriched results of IgAN tubular differential expression proteins (obtained by comparing the tubular proteins of the IgAN group and the control group), according to the relevant literature content, the ACADS protein on the fatty acid degradation pathway was discussed, and it was considered that the significantly down-regulated ACADS protein may be indirectly involved in the occurrence process of the structural and functional abnormalities of the IgAN tubule by affecting the fatty acid metabolism in the renal tubular epithelial cells. In the KEGG pathway significantly enriched results of IgAN interstitial differential expression proteins (obtained by comparing the interstitial proteins of the IgAN group and the control group), according to the relevant literature knowledge, the COL1A1 protein on the ECM (extracellular matrix) receptor interaction pathway was discussed, and it was considered that the significantly up-regulated COL1A1 protein may be involved in the accumulation of extracellular matrix in the IgAN interstitial and promote the process of renal interstitial fibrosis. In summary, the important proteins (i.e. ACTN4, ACADS, COL1A1) of the glomerulus, tubule and interstitial part of the IgAN kidney tissue may be related to the process of tissue lesions, therefore, the severity of the kidney tissue lesions of the IgAN patients included in the present application is divided according to the Lee pathological grading of the kidney biopsy of the kidney tissue pathology, and the ROC (receiver operating characteristic curve) curve analysis of the joint prediction of the three indicators of ACTN4, ACADS and COL1A1 is performed by using SPSS22.0.

[0058] Statistical analysis method

[0059] The statistical analysis was performed by using Graphpad Prism 7.0 and SPSS22.0 statistical software. The expression amounts of the three proteins of ACTN4, ACADS and COL1A1 in the IgAN group and the control group in the present application were represented by mean ± standard deviation. The diagnostic prediction ability of the joint of the three proteins of ACTN4, ACADS and COL1A1 was analyzed by drawing the ROC curve in the SPSS22.0 software, so as to judge the diagnostic prediction value of the joint for the severity of the IgAN kidney tissue lesions. The P value <0.05 was considered to have statistical significance. Among them, the severity of the kidney tissue lesions of the IgAN patients included in the present application was divided according to the Lee pathological grading, and the mild was Lee grading II, and the moderate to severe was Lee grading III-IV.

[0060] Results

[0061] ACTN4, ACADS, COL1A1 three protein expression amount display

[0062] According to the protein quantification results identified by DIA proteomics, the expression amounts of the three proteins ACTN4, ACADS and COL1A1 discussed in the application in the IgAN group and the control group are displayed, see Figure 4 .

[0063] Through the drawn ROC curve (see Figure 5 ), it can be concluded that the combination of ACTN4, ACADS and COL1A1 has good diagnostic prediction performance for the severity of IgAN kidney tissue lesions, with an area under the curve of 0.815 and a P value of 0.043, less than 0.05, which is statistically significant, with a sensitivity of 84.6% and a specificity of 80%.

[0064] Through ROC curve analysis of the combined prediction of ACTN4, ACADS and COL1A1, it is concluded that the combination of the three protein indicators helps to evaluate the severity of IgAN kidney tissue lesions, has the potential to distinguish the severity of IgAN kidney histopathology, and may be a biological marker reflecting the severity of IgAN kidney tissue lesions.

[0065] IgA nephropathy kidney tissue lesion severity marker verification

[0066] In order to verify the reliability of the detection and identification results of proteomics, and also for further analysis, the application adopts the method of immunohistochemistry to verify the important proteins, and carries out immunohistochemical verification of the kidney biopsy tissue sections of the IgAN glomerular differential expression protein ACTN4, the tubular differential expression protein ACADS and the renal interstitial differential expression protein COL1A1. According to the immunohistochemical staining results on the kidney tissue pathological sections, the expression of the above three proteins in the kidney tissues of another selected IgAN patient group and the control group is judged.

[0067] Sample source

[0068] In addition, the present application selects kidney biopsy kidney tissue samples of IgAN patient group and control group for immunohistochemical verification experiment. Among them, the inclusion criteria of IgAN group and control group are consistent with the foregoing inclusion criteria, 16 cases of IgAN patients (5 cases of Lee grading II, 8 cases of Lee grading III, and 3 cases of Lee grading IV) are included in the verification experiment of this part, 5 cases of control group, and all the research objects included have signed the informed consent form. The obtained kidney tissue samples are also the formalin-fixed paraffin-embedded tissues remaining after routine kidney pathological diagnosis, and part of the clinical data of the IgAN patients included in the verification experiment are collected.

[0069] Immunohistochemical experimental method and picture collection and analysis

[0070] (1) The collected formalin-fixed paraffin-embedded kidney tissue samples of each case were cut by a conventional pathological sectioning machine, the thickness of the cut tissue slices was 4 μm, and tissue sections (triplicate) were prepared. Used for subsequent immunohistochemical experiments.

[0071] (2) Tissue section deparaffinization: sequentially place the tissue sections into dimethylbenzene I for 15 minutes, dimethylbenzene II for 15 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and distilled water.

[0072] (3) Antigen repair: place the tissue sections in a repair box containing EDTA (pH 9.0) antigen repair solution, then perform the antigen repair process in a microwave oven. During this process, prevent the repair buffer from evaporating too much, and avoid dry pieces. After natural cooling, place the tissue sections in PBS (pH 7.4) buffer, and shake and wash on a decolorizing shaker for 3 times, each time for 5 minutes.

[0073] (4) Endogenous peroxidase blocking: place the tissue sections in 3% hydrogen peroxide solution, incubate at room temperature in the dark for 25 minutes, then place the tissue sections in PBS (pH 7.4) buffer, and shake and wash on a decolorizing shaker for 3 times, each time for 5 minutes.

[0074] (5) BSA blocking: after slightly shaking the tissue sections, use a group chemical pen to draw a circle around the tissue to prevent the antibody from flowing away, add 3% BSA evenly on the tissue on the section by dropping, then block at room temperature for 30 minutes.

[0075] (6) Add primary antibody: gently shake off the blocking solution, add the prepared primary antibody on the tissue sections according to a certain proportion, and place the tissue sections flat in a wet box for incubation overnight at 4°C. A small amount of water is added to the wet box to prevent evaporation of the antibody.

[0076] (7) Add secondary antibody: Place the tissue section in PBS (pH 7.4) buffer and shake on a shaking table for 3 times, each time for 5 minutes. After the tissue section is slightly spun dry, add the corresponding secondary antibody (HRP labeled) of the primary antibody in the kit to cover the tissue, and incubate at room temperature for 50 minutes.

[0077] (8) DAB color development: Place the tissue section in PBS (pH 7.4) buffer and shake on a shaking table for 3 times, each time for 5 minutes. After the tissue section is slightly spun dry, add freshly prepared DAB color developing solution in the circle, control the color development time under a microscope, and the brownish yellow color indicates positive, then use tap water to rinse the tissue section to stop the color development.

[0078] (9) Nucleus restaining: Hematoxylin staining solution is restained for about 3 minutes, rinsed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, then rinsed with tap water, ammonia water is returned to blue, and finally rinsed with running water.

[0079] (10) Dehydration and mounting: Place the tissue section in 75% alcohol for 6 minutes, 85% alcohol for 6 minutes, absolute ethanol I for 6 minutes, absolute ethanol II for 6 minutes, and xylene I for 5 minutes for dehydration and transparency, then take the tissue section out of the xylene and slightly dry, and mount it with neutral gum.

[0080] (11) Microscope examination and picture collection and analysis: Image-Pro Plus 6.0 software is used for analysis, 3 fields of view are randomly collected for each tissue section, then each photo is analyzed to obtain the positive optical density value of each photo, and finally the average optical density value is used to represent the expression amount of the protein to be verified on each tissue section.

[0081] Statistical analysis method

[0082] Graphpad Prism 7.0 and SPSS22.0 statistical software are used for statistical analysis of data, SPSS22.0 statistical software is used for statistical analysis of collected clinical data, and data are represented by mean ± standard deviation. Graphpad Prism 7.0 statistical software is used for statistical analysis of the final results of immunohistochemical staining of IgAN group and control group (non-parametric test), and the mean ± standard deviation is used. P<0.05 is considered statistically significant.

[0083] Results

[0084] Clinical information

[0085] The part of the verification experiment of the IgAN patients included in this part is shown in Table 5.3.

[0086] Table 5.3 Partial clinical information of IgAN patients

[0087]

[0088] Results of glomerular ACTN4 protein immunohistochemistry experiment:

[0089] ACTN4 protein immunohistochemistry staining results

[0090] The present application verifies the glomerular differential expression protein ACTN4 on kidney tissue sections by immunohistochemistry method. The ACTN4 protein expression on kidney tissue sections of IgAN group and control group is shown in Table 5.4. Figure 6

[0091] ACTN4 protein expression difference statistics

[0092] The ACTN4 protein expression difference statistics on kidney tissue sections of IgAN group and control group is shown in Table 5.5. Figure 7 The statistical result shows that although the average expression amount of ACTN4 protein in the disease group is lower than that of the control group, the P value is greater than 0.05, which is not statistically significant.

[0093] Renal tubular ACADS protein immunohistochemistry experiment results

[0094] ACADS protein immunohistochemistry staining results

[0095] The present application verifies the renal tubular differential expression protein ACADS on kidney tissue sections by immunohistochemistry method. The ACADS protein expression on kidney tissue sections of IgAN group and control group is shown in Table 5.6. Figure 8

[0096] ACADS protein expression difference statistics

[0097] The ACADS protein expression difference statistics on kidney tissue sections of IgAN group and control group is shown in Table 5.7. Figure 9 The statistical result shows that although the average expression amount of ACADS protein in the disease group is lower than that of the control group, the P value is greater than 0.05, which is not statistically significant.

[0098] Renal interstitial COL1A1 protein immunohistochemistry experiment results

[0099] COL1A1 protein immunohistochemistry staining results

[0100] ​​The present application verifies the kidney interstitial differential expression protein COL1A1 on the kidney tissue section by the method of immunohistochemistry. The COL1A1 protein expression on the kidney tissue section of the IgAN group and the control group is as shown in Figure 10 .

[0101] COL1A1 protein expression difference statistics

[0102] The COL1A1 protein expression difference statistics result on the kidney tissue section of the IgAN group and the control group is as shown in Figure 11 . The statistical result shows that although the average expression amount of the COL1A1 protein in the disease group is higher than that of the control group, the P value is greater than 0.05, which is not statistically significant.

[0103] The present application verifies the three proteins ACTN4, ACADS and COL1A1 on the kidney tissue section by the immunohistochemistry experiment, although the difference of the expression amount of the protein obtained according to the immunohistochemistry staining result between the disease group and the control group is not statistically significant, but from the immunohistochemistry staining result of each substructure part: for the verified glomerular differential expression protein ACTN4, it can be seen from Figure 7 that compared with the control group, its expression amount in the glomerulus of the IgAN group has a decreasing trend; for the verified renal tubular differential expression protein ACADS, it can be seen from Figure 8 that compared with the control group, its expression amount in the renal tubule part of the IgAN group has a decreasing trend; for the verified kidney interstitial differential expression protein COL1A1, it can be seen from Figure 10 that compared with the control group, its expression amount in the kidney interstitial part of the IgAN group has an increasing trend. But the final difference statistics is not statistically significant, and the reason is that the sample capacity selected for the verification experiment is relatively small. In the future, the sample amount needs to be expanded, and more accurate verification needs to be carried out.

[0104] The above is the further detailed description of the present application, which cannot be regarded as the limitation of the specific implementation of the present application. For the ordinary skilled person in the technical field to which the present application belongs, the simple deduction or replacement without departing from the concept of the present application is within the protection scope of the present application.

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Claims

1. A biomarker for determining the severity of renal lesions in IgAN, consisting of ACTN4, ACADS, and COL1A1.

2. The application of quantitative reagents in the preparation of diagnostic reagents for the severity of IgAN renal tissue lesions, characterized in that, The quantitative reagent is used to quantify the amount of ACTN4, ACADS, and COL1A1 proteins in the sample.

3. The application according to claim 2, characterized in that, The sample was a kidney tissue section obtained from a kidney biopsy.

4. The application according to claim 2, characterized in that, The quantitative reagents are selected from ELISA reagents and immunofluorescence reagents.

5. The application according to claim 2, characterized in that, The detection threshold is determined by plotting an ROC curve.

6. A system for determining the severity of IgAN renal tissue lesions, comprising: A quantitative device for quantifying the amounts of ACTN4, ACADS, and COL1A1 proteins in a sample; The analytical device determines the severity of IgAN renal tissue lesions based on the levels of ACTN4, ACADS, and COL1A1 proteins in the sample. The result output device is used to output the analysis results of the analysis device.

7. The system according to claim 6, characterized in that, The quantitative device is selected from ELISA quantitative devices, immunofluorescence quantitative devices, and data-independent acquisition mass spectrometry quantitative devices.

8. The system according to claim 6, characterized in that, The analytical device determines the detection threshold by plotting an ROC curve.

9. The system according to claim 6, characterized in that, The sample was a kidney tissue section obtained from a kidney biopsy.

Images