Biomarker of GRAMD1B in renal tubular epithelial cell injury and diagnostic application

Abstract

According to the biomarker of GRAMD1B in renal tubular epithelial cell injury and the diagnostic application, the mRNA expression level and / or protein expression level of GRAMD1B in a detection sample are / is detected, and the expression level in the detection sample is compared with the corresponding expression level in a reference sample; based on joint expression information of mRNA (messenger ribonucleic acid) expression level and protein expression level, the GRAMD1B is used for carrying out auxiliary diagnosis on whether the renal tubular epithelial cells are injured or not and the injury-related state of the renal tubular epithelial cells, so that the GRAMD1B is used as a biological marker related to the injury of the renal tubular epithelial cells; the renal tubular epithelial cell injury state is evaluated on the basis of changes of mRNA expression level and / or protein expression level, and the method has the advantages of being clear in diagnosis directivity, high in early and local injury sensitivity, suitable for multiple sample types, flexible in detection mode and convenient to achieve accurate evaluation and dynamic monitoring.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and molecular diagnostics, and in particular to GRAMD1B as a biomarker and diagnostic application in renal tubular epithelial cell injury. Background Technology

[0002] Chronic kidney disease (CKD) is a group of diseases characterized by progressive damage to the structure and function of the kidneys. It is characterized by insidious onset, prolonged course, and high rates of disability and mortality, and has become a major public health problem worldwide. Among them, renal tubular epithelial cell damage plays a key role in the occurrence and progression of CKD. Especially under the influence of factors such as increased metabolic load, ischemia and hypoxia, or drug toxicity, renal tubular epithelial cells are prone to structural and functional damage, which can subsequently lead to inflammatory responses, interstitial fibrosis, and a continuous decline in renal function. Therefore, early identification and effective assessment of renal tubular epithelial cell damage are of great significance for the prevention and treatment of chronic kidney disease.

[0003] Currently, the main indicators used in clinical practice to assess the degree of kidney damage include serum creatinine, blood urea nitrogen, and estimated glomerular filtration rate. However, these indicators mostly reflect the overall renal function status and are difficult to reflect the early renal tubular epithelial cell damage in a timely and specific manner. In recent years, although some molecules have been proposed to assist in assessing the state of kidney damage, their stability and specificity in renal tubular damage of different etiologies and at different stages are still limited, and they are still difficult to meet the clinical needs for accurate diagnosis and dynamic monitoring.

[0004] There is an urgent need to provide a new biomarker and its diagnostic application scheme to solve the problems that still exist in the current technology, such as the lack of specific biomarkers for renal tubular epithelial cell injury, insufficient sensitivity of diagnostic methods to early and local injuries, and difficulty in achieving accurate assessment and dynamic monitoring of renal tubular epithelial cell injury. Summary of the Invention

[0005] In view of this, the present invention proposes GRAMD1B as a biomarker and diagnostic application in renal tubular epithelial cell injury, which involves the fields of biomedical and molecular diagnostic technology, and solves the existing technical problems of lack of specific biomarkers, insufficient sensitivity of diagnostic methods, inability to perform accurate assessment and dynamic monitoring.

[0006] To achieve the above-mentioned technical objectives, the specific technical solution adopted by the present invention is as follows: The study investigates the biomarker and diagnostic applications of GRAMD1B in renal tubular epithelial cell injury. This involves detecting the mRNA and / or protein expression levels of GRAMD1B in test samples and comparing these levels with those in reference samples. Based on the combined expression information of mRNA and protein levels, the study aims to assist in the diagnosis of whether renal tubular epithelial cells are damaged and their related states.

[0007] Furthermore, the test samples include kidney tissue samples, serum samples, blood samples, and urine samples. The test samples for GRAMD1B as a biomarker and diagnostic application in renal tubular epithelial cell injury were obtained from individuals with different etiologies of chronic kidney disease, individuals from animal models of renal tubular injury, and in vitro models of renal tubular epithelial cell injury. By detecting the expression level of GRAMD1B in the test samples, reference information can be provided for early diagnosis, risk assessment, disease progression analysis, and / or efficacy monitoring of renal tubular injury.

[0008] Furthermore, the reference samples are healthy control samples and / or preset reference samples. The expression level of GRAMD1B in the test samples is compared with the corresponding expression level in the reference samples to obtain expression difference information for characterizing the state related to renal tubular epithelial cell damage.

[0009] Furthermore, diagnostic applications are achieved through kits. The GRAMD1B biomarker and diagnostic application kit for renal tubular epithelial cell injury includes a detection reagent for detecting GRAMD1B expression levels, and a nucleic acid detection reagent for detecting GRAMD1B mRNA expression levels and / or an immunoassay reagent for detecting GRAMD1B protein expression levels.

[0010] Furthermore, the detection reagents include nucleotide primers and / or probes for detecting GRAMD1B mRNA expression levels, and / or antibodies and their functional fragments for detecting GRAMD1B protein expression levels. By detecting GRAMD1B mRNA expression levels and / or protein expression levels, molecular-level expression information for subsequent analysis can be obtained.

[0011] Furthermore, the detection of GRAMD1B mRNA expression level and / or protein expression level is achieved by one or more of the following methods: real-time quantitative polymerase chain reaction, Western blotting, enzyme-linked immunosorbent assay, in situ hybridization, nucleic acid microarray, or protein microarray.

[0012] Furthermore, in diagnostic applications, the mRNA and protein expression levels of GRAMD1B were obtained by separate or simultaneous detection.

[0013] Furthermore, in diagnostic applications, simultaneous detection is used to establish the correspondence between GRAMD1B mRNA expression levels and protein expression levels.

[0014] This invention is based on research into the mechanism of renal tubular epithelial cell damage during the development of chronic kidney disease. It has been found that when renal tubular epithelial cells undergo structural and functional abnormalities under the influence of various pathogenic factors, their intracellular molecular expression profiles will change accordingly. Among these changes, some molecular changes can serve as important indicators reflecting the state of renal tubular epithelial cell damage. GRAMD1B, as a molecule with expression characteristics in kidney tissue, has an expression level that is closely related to the physiological state of renal tubular epithelial cells. When renal tubular epithelial cells are damaged by factors such as increased metabolic load, ischemia-hypoxia, or toxic stimulation, the intracellular homeostasis regulation process is disrupted, and the expression level of GRAMD1B changes accordingly. This expression change is correlated with the state of renal tubular epithelial cell damage and can serve as an important molecular signal reflecting renal tubular epithelial cell damage.

[0015] Based on the above understanding, this invention can detect the mRNA and / or protein expression levels of GRAMD1B in the test sample, and combine this with a comparative analysis of the corresponding expression levels in the reference sample to obtain molecular-level expression information related to renal tubular epithelial cell damage. Furthermore, by comprehensively analyzing the expression information, it can provide a reliable basis for assessing the state of renal tubular epithelial cell damage, thereby realizing the application of GRAMD1B in the diagnosis of renal tubular epithelial cell damage.

[0016] By adopting the above technical solution, the present invention can also bring the following beneficial effects: 1. This invention relates to GRAMD1B as a biomarker and its diagnostic application in renal tubular epithelial cell injury. By using GRAMD1B as a biomarker related to renal tubular epithelial cell injury, the status of renal tubular epithelial cell injury can be assessed based on changes in its mRNA expression level and / or protein expression level. This overcomes the problem that existing diagnostic indicators mainly reflect overall renal function and are difficult to reflect renal tubular epithelial cell injury in a timely and specific manner, thereby improving the specificity and reliability of renal tubular epithelial cell injury identification and having the advantage of clear diagnostic direction.

[0017] 2. This invention relates to GRAMD1B as a biomarker and diagnostic application in renal tubular epithelial cell injury. By comparing and analyzing the expression level of GRAMD1B in the test sample with the corresponding expression level in the reference sample, molecular-level expression information related to renal tubular epithelial cell injury is obtained. This information is used to assist in assessing the occurrence and trend of renal tubular epithelial cell injury, which is beneficial for achieving accurate assessment and dynamic monitoring of the state of renal tubular epithelial cell injury. It has the advantages of rich assessment dimensions and high information sensitivity.

[0018] 3. This invention relates to GRAMD1B as a biomarker and diagnostic application in renal tubular epithelial cell injury. It is applicable to various test samples such as renal tissue samples, blood samples, serum samples and / or urine samples, and can be combined with various detection methods such as real-time quantitative polymerase chain reaction, immunoblotting, enzyme-linked immunosorbent assay, in situ hybridization, nucleic acid microarray or protein microarray. The detection method is flexible, has a wide range of applications, and is easy to implement under different detection conditions and application scenarios. It has the advantages of strong method versatility and good prospects for promotion and application. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The graph shows the expression of GRAMD1B in the kidney tissues of CKD patients with different etiologies and the relationship between the expression level of GRAMD1B and the glomerular filtration rate of the patients. Figure 2 qPCR results of NGAL markers for the reversibility of palmitic acid and carotenoid-induced damage in HK-2 cells by overexpression of GRAMD1B; Figure 3 WB and qPCR images of decreased GRAMD1B expression in the renal cortex of folic acid and cisplatin model mice; Figure 4 A graph showing the level of renal cortical cholesterol in a mouse model of folic acid and cisplatin. Detailed Implementation

[0021] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0022] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0024] Furthermore, specific details are provided in the following description to facilitate a thorough understanding of the examples. However, those skilled in the art will understand that the described aspects can be practiced without these specific details.

[0025] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0026] To achieve the above-mentioned technical objectives, the specific technical solution adopted by the present invention is as follows: Example 1: Test samples from different subjects were used as test subjects. The subjects included healthy controls and individuals at risk of kidney dysfunction. The test samples included one or more of the following: kidney tissue samples, serum samples, and urine samples.

[0027] After routine pretreatment of the samples, the expression level of GRAMD1B in the samples was detected using molecular biology and immunological methods, respectively. Molecular biological detection methods were used to obtain the mRNA expression level of GRAMD1B; Immunological assays were used to obtain the protein expression level of GRAMD1B.

[0028] The test results showed that GRAMD1B expression signals could be detected in samples from different sources, and its expression levels exhibited distinguishable variations among different subjects. These results demonstrate that detecting GRAMD1B expression levels based on different types of samples is technically feasible, can stably acquire detection signals, and provides a foundation for subsequent analysis.

[0029] Example 2: Using test samples from the same subject as the test subjects, the mRNA and protein expression levels of GRAMD1B in the biomarker of renal tubular epithelial cell injury and in the diagnostic application test samples were detected and analyzed.

[0030] The mRNA expression level of GRAMD1B in renal tubular epithelial cell injury was obtained by real-time quantitative polymerase chain reaction, and the protein expression level of GRAMD1B in renal tubular epithelial cell injury was obtained by Western blotting or enzyme-linked immunosorbent assay.

[0031] In the same test sample: Simultaneous detection was used to obtain the mRNA and protein expression information of GRAMD1B; Alternatively, asynchronous detection methods can be used to obtain the above-mentioned expression information separately.

[0032] The obtained mRNA expression information and protein expression information were correlated and analyzed.

[0033] The test results showed that the mRNA expression level and protein expression level of GRAMD1B in the same test sample were consistent in their changing trends. These results indicate that by jointly obtaining the expression information of GRAMD1B at different molecular levels, it is possible to conduct a consistent analysis of the renal tubular epithelial cell injury-related status from multiple levels.

[0034] Example 3: Healthy control samples and / or preset reference samples were selected as reference samples, and test samples from subjects suspected of having renal tubular epithelial cell damage were selected as test subjects.

[0035] The expression level of GRAMD1B in the test samples was obtained, and the expression level of GRAMD1B as a biomarker and diagnostic application in renal tubular epithelial cell injury was compared and analyzed with the corresponding expression level in the reference sample.

[0036] A comparative analysis of GRAMD1B as a biomarker and diagnostic application in renal tubular epithelial cell injury was conducted to develop diagnostic information for assessing the status of renal tubular epithelial cell injury.

[0037] Comparative analysis showed that the expression level of GRAMD1B in the tested samples exhibited a stable decreasing trend compared to the reference samples. These results indicate that by comparing the tested samples with the reference samples, stable and reproducible expression differences can be obtained, providing reliable molecular-level reference information for assessing renal tubular epithelial cell injury-related status.

[0038] Example 4: Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression level of GRAMD1B in the test sample.

[0039] Kidney tissue sample: Take approximately 10 mg of fresh or frozen kidney tissue; Total RNA was lysed and extracted using RNAiso Plus reagent; RNA purity was controlled at 1.8–2.0 A260 / A280.

[0040] Serum / urine samples: Take 200 μL of sample; Centrifuge at 12,000 rpm for 10 minutes to remove impurities; RNA was extracted using the miRNeasy kit.

[0041] Reverse transcription conditions: Use PrimeScript RT Master Mix; React at 37℃ for 15 minutes, then inactivate at 85℃ for 5 seconds.

[0042] qPCR amplification system and conditions: SYBR Green PCR Master Mix 10μL; 2 μL of cDNA template; Primers, 0.5 μM each; Total volume: 20 μL; Cyclic conditions: 95℃ pre-denaturation for 10 minutes, 95℃ for 15 seconds, 60℃ for 1 minute, for a total of 40 cycles.

[0043] Data were collected using an ABI 7500 system, and relative expression levels were calculated using the 2^(-ΔΔCt) method, with GAPDH as an internal reference.

[0044] Each sample was prepared in triplicate, and water was used instead of template for the negative control.

[0045] The test results showed that the mRNA expression level of GRAMD1B in kidney injury-related samples decreased by about 40%–60% compared with healthy control samples.

[0046] Figure 1This study elucidates the expression differences of GRAMD1B in kidney injury-related samples and healthy control samples, and its relationship with kidney function indicators. It demonstrates that the overall expression level of GRAMD1B in kidney-impaired samples is lower than that in healthy control samples, and its expression trend is consistent with the changes in kidney function indicators. The expression level of GRAMD1B can reflect the state related to renal tubular epithelial cell injury and has the feasibility of being used as a biomarker for evaluation and analysis. Combined with the above results, it is clear that detecting the mRNA expression level of GRAMD1B based on qRT-PCR can stably obtain expression difference information that can be used for the assessment of renal tubular epithelial cell injury.

[0047] Example 5: An in vitro model of renal tubular epithelial cell injury was constructed using human renal tubular epithelial cells HK-2.

[0048] Cell culture conditions: DMEM / F12 medium containing 10% FBS, 37℃, 5% CO2.

[0049] Damage induction conditions: Palmitic acid (PA) treatment group: final concentration 10 μM, treatment for 48 hours; Toxic carotenoid (TG) treatment group: final concentration 1 μM, treatment for 48 hours.

[0050] After damage treatment: The mRNA expression level of GRAMD1B was detected by qRT-PCR; The protein expression level of GRAMD1B was detected by Western blotting; Calibration was performed using β-actin and other internal references.

[0051] The results showed that under PA or TG-induced damage conditions, the mRNA expression level and protein expression level of GRAMD1B both decreased synchronously.

[0052] Figure 2 The changes in GRAMD1B expression level and related damage indicators in the HK-2 cell injury model induced by palmitic acid and carotenoids are shown. It can be demonstrated that the expression level of GRAMD1B is significantly reduced under the injury induction conditions, and is consistent with the changes in cell injury indicators. Combined with the above results, it can be concluded that the expression changes of GRAMD1B can stably reflect the cell injury status in the in vitro renal tubular epithelial cell injury model, verifying its feasibility and consistency at the cellular level.

[0053] Example 6: An in vivo kidney injury model was established using male C57BL / 6 mice, 8 weeks old and weighing 20–25g.

[0054] Keeping conditions: SPF grade environment, 22±2℃, humidity 50%, 12-hour light and dark cycle.

[0055] Modeling method: Folic acid model: Intraperitoneal injection of folic acid 250 mg / kg; Cisplatin model: Intraperitoneal injection of cisplatin 20 mg / kg.

[0056] Urine, blood, and kidney tissue samples were collected on days 7, 14, and 28 after modeling.

[0057] The testing content includes: Urine protein / creatinine ratio; Renal function indicators such as serum creatinine; The mRNA and protein expression levels of GRAMD1B in kidney tissue.

[0058] LRP2 was used as a marker for proximal tubule co-staining in immunofluorescence assay.

[0059] The test results showed that the expression level of GRAMD1B consistently decreased under different model conditions.

[0060] Figure 3 The changes in GRAMD1B expression in mice with folic acid or cisplatin-induced kidney injury models are shown. It can be demonstrated that under different modeling methods and different time points, the expression level of GRAMD1B shows a consistent decreasing trend. Combined with the above results, GRAMD1B has stable and reproducible expression changes in different in vivo kidney injury models, which can reflect the occurrence of renal tubular epithelial cell injury.

[0061] Example 7: Based on the animal model of Example 6, a correlation analysis was performed on the changes in GRAMD1B expression and metabolic-related states.

[0062] The accumulation of free cholesterol in the cell membrane was observed using Filipin III staining (5 μg / mL).

[0063] The protein expression information and mRNA expression information of GRAMD1B were acquired simultaneously or separately for corresponding analysis.

[0064] The test results showed that cholesterol accumulation was more pronounced in samples with reduced GRAMD1B expression levels.

[0065] Figure 4The study showed changes in renal cortical cholesterol levels in a kidney injury model, demonstrating that cholesterol accumulation was more pronounced in samples with decreased GRAMD1B expression. These results, combined with the findings, indicate a clear association between changes in GRAMD1B expression and kidney injury-related metabolic abnormalities, providing metabolic-level support for its use as a biomarker for renal tubular epithelial cell injury.

[0066] In summary, the above embodiments demonstrate that the expression level of GRAMD1B can be stably detected under different sources of samples, different detection levels, and different kidney injury-related models. Its expression changes show a consistent trend in both in vitro cell models and in vivo animal models, and it has a clear correlation with kidney injury-related metabolic states. In conclusion, this invention has the advantages of a clear detection pathway, sufficient experimental support, and reliable results.

[0067] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The biomarker and diagnostic application of GRAMD1B in renal tubular epithelial cell injury, characterized by: By detecting the mRNA and / or protein expression levels of GRAMD1B in the test samples and comparing the expression levels in the test samples with the corresponding expression levels in the reference samples, the combined expression information of mRNA and protein expression levels is used to assist in the diagnosis of whether renal tubular epithelial cells are damaged and their damage-related status.

2. The diagnostic application according to claim 1, characterized in that: The test samples include kidney tissue samples, serum samples, blood samples, and urine samples. The test samples are derived from individuals with different etiologies of chronic kidney disease, individuals from animal models of renal tubular injury, and in vitro models of renal tubular epithelial cell injury. The expression level of GRAMD1B in the test samples is detected to provide reference information for early diagnosis, risk assessment, disease progression analysis, and / or efficacy monitoring of renal tubular injury.

3. The diagnostic application according to claim 2, characterized in that: The reference sample uses healthy control samples and / or preset reference samples. The expression level of GRAMD1B in the test sample is compared with the corresponding expression level in the reference sample to obtain expression difference information for characterizing the state related to renal tubular epithelial cell damage.

4. The diagnostic application according to claim 3, characterized in that: The diagnostic application is implemented in the form of a kit, which includes a detection reagent for detecting GRAMD1B expression level, and the detection reagent includes a nucleic acid detection reagent for detecting GRAMD1B mRNA expression level and / or an immunoassay reagent for detecting GRAMD1B protein expression level.

5. The diagnostic application according to claim 4, characterized in that: The detection reagent includes nucleotide primers and / or probes for detecting GRAMD1B mRNA expression levels, and / or antibodies and their functional fragments for detecting GRAMD1B protein expression levels. By detecting GRAMD1B mRNA expression levels and / or protein expression levels, molecular-level expression information for subsequent analysis can be obtained.

6. The diagnostic application according to claim 5, characterized in that: The detection of GRAMD1B mRNA expression level and / or protein expression level is achieved by one or more of the following methods: real-time quantitative polymerase chain reaction, Western blotting, enzyme-linked immunosorbent assay, in situ hybridization, nucleic acid microarray, or protein microarray.

7. The diagnostic application according to any one of claims 1–6, characterized in that: In the diagnostic application, the mRNA and protein expression levels of GRAMD1B were obtained by separate or simultaneous detection.

8. The diagnostic application according to claim 7, characterized in that: The diagnostic application simultaneously detects the correlation between the mRNA expression level and the protein expression level of GRAMD1B.

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