Biomarker of CTSA in kidney podocyte injury and podocytosis and diagnostic application of CTSA in kidney podocyte injury and podocytosis

Abstract

The invention relates to a biomarker and diagnostic application of CTSA in kidney podocyte injury and podocytosis, the diagnostic application takes uninjured in-vitro cultured podocyte as a control, and when the in-vitro cultured podocyte is induced by adriamycin, angiotensin II or palmitic acid to form a podocyte injury state, the podocyte injury state is changed into the podocyte injury state. CTSA shows stable and detectable expression up-regulation relative to control podocyte under the condition of podocyte injury, the mRNA expression level change of the CTSA is detected, and the CTSA is used as a biomarker for reflecting the state of the podocyte injury of the kidney and the podocyte injury related to the podocyte disease and is used for in-vitro diagnosis of the podocyte injury of the kidney and the podocyte disease, and in a word, the CTSA can be used for in-vitro diagnosis of the podocyte injury of the kidney and the podocyte disease. Under the condition of an in-vitro podocyte injury model, the expression change of the CTSA can stably and detectably reflect the podocyte injury related state, thereby supporting the technical scheme of using the CTSA as a biomarker of kidney podocyte injury and podocytosis and diagnosis application.

Description

Technical Field

[0001] This invention relates to the field of molecular biological detection technology for kidney diseases, and in particular to the biomarker and diagnostic application of CTSA in renal podocyte injury and podocyte disease. Background Technology

[0002] Podocytes are key cells in maintaining the integrity of the glomerular filtration barrier. Their structural and functional stability is crucial for normal kidney function. In various kidney diseases, podocytes are susceptible to damage, leading to foot process fusion, cytoskeleton rearrangement, and abnormal cell function, which in turn triggers or exacerbates clinical manifestations such as proteinuria. Podocyte injury is considered a significant pathological basis for the occurrence and development of various podocyte diseases and related kidney diseases. Currently, the diagnosis of renal podocyte injury and podocyte diseases mainly relies on clinical indicators, imaging examinations, and histopathological analysis. However, these methods still have limitations in terms of sensitivity, specificity, and the ability to reflect early podocyte injury. Furthermore, some detection methods are invasive, hindering dynamic monitoring of podocyte injury. Therefore, exploring molecular biological indicators that can objectively reflect the degree of podocyte injury and possess good stability and reproducibility remains of significant research importance and application value.

[0003] Existing studies suggest that various molecules related to cell metabolism, lysosomal function, and proteolysis may undergo expression changes in podocyte injury and related pathological states. However, the expression of different molecules varies under different injury models and pathological conditions, and their applicability and stability as diagnostic markers still need further clarification. Therefore, it is still necessary to screen and validate biomarkers that can reflect the state of renal podocyte injury based on the molecular changes associated with podocyte injury, in order to meet the practical needs of diagnosing renal podocyte injury and podocyte disease.

[0004] Based on the above situation, there is an urgent need to provide a new technical solution to address the problems of insufficient sensitivity, limited stability and reproducibility of existing diagnostic methods in reflecting the state of podocyte injury, thereby providing more reliable technical support for the diagnosis of renal podocyte injury and podocyte disease. Summary of the Invention

[0005] In view of this, the present invention proposes CTSA as a biomarker and diagnostic application in renal podocyte injury and podocyte disease, which belongs to the field of molecular biology detection of kidney diseases and solves the technical problems of insufficient sensitivity, limited stability and reproducibility of current diagnostic methods in reflecting the state of podocyte injury.

[0006] To achieve the above-mentioned technical objectives, the specific technical solution adopted by the present invention is as follows: This invention mentions the biomarker and diagnostic application of CTSA in renal podocyte injury and podocyte disease. The diagnostic application uses undamaged in vitro cultured podocytes as a control. When in vitro cultured podocytes are induced to form a podocyte injury state by doxorubicin, angiotensin II, or palmitic acid, CTSA shows a stable and detectable upregulation of expression under podocyte injury conditions compared to control podocytes. By detecting changes in CTSA mRNA expression levels, it serves as a biomarker reflecting the podocyte injury state associated with renal podocyte injury and podocyte disease, and is used for the in vitro diagnosis of renal podocyte injury and podocyte disease.

[0007] Furthermore, in diagnostic applications, the expression level of CTSA mRNA in cultured podocytes was detected by real-time quantitative polymerase chain reaction, and the results were compared with the expression level of CTSA mRNA in control podocytes to determine the upregulation of CTSA expression under podocyte injury conditions for in vitro diagnostic purposes.

[0008] Furthermore, in diagnostic applications, the mRNA expression level of CTSA in an in vitro podocyte injury model is compared with the mRNA expression level of CTSA in undamaged in vitro cultured podocytes. When the mRNA expression level of CTSA is higher than that in undamaged podocytes, the kidney podocytes corresponding to the in vitro cultured podocytes are determined to be in a damaged state, which is used for in vitro diagnosis.

[0009] Furthermore, in in vitro podocyte injury models constructed under different podocyte injury induction conditions, the mRNA expression level of CTSA was upregulated compared with that of undamaged in vitro cultured podocytes, thus making CTSA a suitable biomarker for the diagnosis of different types of podocyte injury states as a renal podocyte injury and podocyte disease.

[0010] Furthermore, the in vitro podocyte injury models include podocyte injury models induced by doxorubicin, angiotensin II, and palmitic acid, respectively. Thus, in different podocyte injury models, the mRNA expression level of CTSA can be used to distinguish between podocyte injury and uninjured states.

[0011] Furthermore, diagnostic applications are achieved through any of the reagents or kits used for in vitro testing.

[0012] Furthermore, the kit includes a detection reagent for detecting the mRNA expression level of CTSA, and based on the detection results of the CTSA mRNA expression level, it provides a diagnostic basis for determining whether podocytes are in a damaged state.

[0013] Because CTSA (cathepsin A) is a carboxypeptidase located within lysosomes, it participates in lysosomal enzyme activation, extracellular matrix-related protein processing, and the maturation of various glycoproteins, playing a crucial role in maintaining intracellular metabolic homeostasis and protein degradation balance. CTSA not only has fundamental functions in lysosomal physiological processes, but its expression and activity changes are also related to cellular stress responses and signal regulation in various disease states. Furthermore, in disease-related studies such as tumors, CTSA has been found to be associated with the regulation of multiple cellular signaling pathways, and its expression or activity changes are related to biological behaviors such as cell proliferation, survival, and microenvironmental adaptation. In various disease models and clinical studies, changes in CTSA expression levels or circulating levels have been used to reflect disease progression. The development and progression of disease-related states, such as in cardiovascular studies, have shown that CTSA can be detected in circulation early after an acute injury event. Its level changes are correlated with the degree of tissue damage and subsequent structural remodeling. This indicates that CTSA, as a molecule closely related to lysosomal function, cellular metabolic homeostasis, and stress response, can reflect changes in cellular or tissue states under different disease backgrounds, providing a biological basis for its application in other disease-related states. Based on the above biological characteristics of CTSA participating in cellular homeostasis regulation and reflecting changes in cellular stress states in various disease-related states, its expression changes in renal podocyte injury and podocyte disease-related states are worthy of further research.

[0014] From the perspective of cell biology mechanisms, podocytes may experience changes in intracellular homeostasis, lysosomal function, and protein metabolism under conditions of injury and podocyte disease. Lysosomal-associated proteins participate in intracellular degradation and metabolic regulation during these processes, and changes in their expression levels can reflect the functional state of podocytes to some extent. Based on this principle, when studying the state of podocytes under conditions of renal podocyte injury and podocyte disease, it can be observed that the expression level of CTSA in podocytes shows an increasing trend. This expression change is correlated with the state of podocyte injury and can reflect the changes in the state of podocytes related to renal podocyte injury and podocyte disease at the molecular level.

[0015] Meanwhile, changes in CTSA expression levels can also reflect podocyte injury-related risk status. By detecting and analyzing the levels of CTSA in test samples, the podocyte injury-related status can be assessed and analyzed. Furthermore, as the podocyte injury-related status changes at different stages, the expression level of CTSA may exhibit dynamic changes at different time points. By comparing the changes in CTSA expression levels in test samples at different time points, it is helpful to analyze the development trend of podocyte injury status, thereby providing a molecular biological reference for the in vitro analysis of renal podocyte injury and podocyte disease-related status.

[0016] By adopting the above technical solution, the present invention can also bring the following beneficial effects: 1. The present invention relates to the biomarker and diagnostic application of CTSA in renal podocyte injury and podocyte disease. By introducing CTSA as a detection indicator, the changes in CTSA mRNA expression level are analyzed in an in vitro podocyte injury model, so that the podocyte injury status can be reflected at the molecular level. Compared with detection methods that rely on histopathology or indirect clinical indicators, the present invention starts from the molecular expression changes of podocytes themselves, which can more directly reflect the podocyte injury-related status, thus having the advantages of clear diagnostic direction and accurate detection basis.

[0017] 2. The biomarker and diagnostic application of CTSA mentioned in this invention in renal podocyte injury and podocyte disease, through research on in vitro podocyte injury models under different podocyte injury induction conditions, found that CTSA showed an upregulated expression trend relative to undamaged podocytes under various podocyte injury conditions, making this biomarker usable for analyzing podocyte injury status without relying on a single injury induction method; therefore, CTSA as a biomarker has good applicability in different podocyte injury models, which is beneficial to improving the stability and consistency of in vitro podocyte injury analysis results, thus having the advantages of wide applicability and good reproducibility.

[0018] 3. The biomarker and diagnostic application of CTSA in renal podocyte injury and podocyte disease mentioned in this invention is achieved through real-time quantitative polymerase chain reaction and corresponding reagents or kits. By detecting the expression level of CTSA mRNA and comparing it with control podocytes, analytical basis for determining whether podocytes are in a state of injury can be obtained, making the detection process more standardized and the operation method more standardized. This method is easy to implement under routine laboratory conditions, which is conducive to promoting the application of in vitro podocyte injury-related analysis methods, thus having the advantages of simple operation and easy standardization. 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 To identify the populations of healthy and damaged podocytes in podocyte patients during single-nuclear transcriptome sequencing, and the proportion of different types of podocyte diseases; Figure 2 Venn diagram for differential gene analysis of healthy and damaged podocytes in humans and mice in single-nuclear transcriptome sequencing; Figure 3 This figure shows the expression of CTSA in healthy and damaged mouse podocytes during single-nuclear transcriptome sequencing. Figure 4 This figure shows the expression of CTSA in healthy and damaged human podocytes during single-nuclear transcriptome sequencing. Figure 5 The bar chart shows the changes in CTSA mRNA expression levels in in vitro podocyte injury models induced by doxorubicin, angiotensin II, and palmitic acid, respectively. Figure 6 A bar chart showing the effect of CCK8 assay on the increased podocyte viability after transfection with CTSA-overexpressing lentivirus following doxorubicin-induced damage. 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: This example uses single-nucleus transcriptome sequencing technology to analyze the expression distribution of CTSA in kidney cells and the changes in expression under conditions of podocyte injury and podocyte disease.

[0027] Renal biopsy samples were collected from patients with clinical podocyte injury and podocyte disease (samples were obtained from Sir Run Run Shaw Hospital, affiliated with Zhejiang University School of Medicine, ethics approval number: 20220606-016), as well as renal cortex samples from podocyte disease mice and healthy mice. Single-nuclear transcriptome sequencing was used to detect CTSA expression. Single-nucleus sequencing specifically includes the following steps: (1) Collect kidney tissues from mice and humans and prepare them into single-cell nuclei suspensions. The specific steps include tissue homogenization, filtration and nuclear extraction. (2) The samples were subjected to single-nucleus sequencing (snRNA-seq) using the commercial 10X Genomics sequencing platform to obtain high-throughput transcriptome data. (3) The raw data obtained from sequencing are subjected to quality control and low-quality cell nuclei are removed, and preliminary data processing is performed, including filtering, removal of double cell nuclei and batch effect correction. (4) The Seurat package in R language was used to analyze the processed single-cell nucleus data.

[0028] Figure 1 and Figure 2 This indicates that CTSA expression differs significantly between injured podocytes in humans and mice. Figure 3 and Figure 4 This indicates that CTSA expression was significantly upregulated in both podocyte disease patients and mice, through... Figures 1 to 4The results confirmed that CTSA was mainly expressed in podocytes in the kidney tissues of humans and mice, while the expression of CTSA was significantly increased in podocytes of patients with podocyte injury and podocyte disease and mice.

[0029] In summary, CTSA is upregulated in the kidney tissues of patients with podocyte injury and podocyte disease, as well as mice, and is mainly expressed in podocytes.

[0030] Example 2: This example demonstrates that CTSA expression is significantly increased in an in vitro podocyte injury model.

[0031] Human podocytes were selected as experimental subjects and cultured under constant temperature and suitable gas composition conditions. After the cells were stable, doxorubicin, angiotensin II or palmitic acid were used to induce in vitro podocyte injury models, and undamaged in vitro cultured podocytes were used as controls.

[0032] In this embodiment, human podocytes (from Wuhan Pronosai Biotechnology Co., Ltd.) were cultured and treated with doxorubicin (ADR, 10 μM per ml of culture medium), angiotensin II (AngII, 1 μM), or palmitic acid (PA, 200 μol / L). After these treatments, the degree of podocyte damage and changes in CTSA expression were observed and evaluated. At appropriate time points after damage, the expression level of CTSA was detected by methods such as real-time quantitative PCR.

[0033] The specific steps of real-time quantitative PCR are as follows: (1) Total RNA was extracted from cells using RNA isoPlus reagent; (2) Reverse transcription of cDNA was performed using PrimeScript™ RT Master Mix; (3) Real-time PCR amplification was performed using SYBR Green PCR Master Mix and ABI 7-hour PCR detection system; The temperature cycling conditions were: 10 min at 95℃, 15 s at 95℃, and 1 min at 60℃. In this study, the relative expression level of mRNA was normalized relative to GAPDH, and 2 was used. -ΔΔCT Method calculation; (4) The required primers were designed and synthesized by Sangon Biotech Co., Ltd., and their primer sequences are shown in Table 1 below: Table 1. Primer sequences used in the study Statistical analysis includes the following: (1) All quantitative values ​​are expressed as mean ± standard error of mean SEM; (2) The statistical differences between the two groups were analyzed using the two-tailed Student t test with GraphPad Prism. (3) One-way ANOVA is used for comparisons between multiple groups; P < 0.05 is considered statistically significant.

[0034] like Figure 5 As shown, real-time quantitative PCR results indicate that after podocyte injury induced by doxorubicin, angiotensin II, and palmitic acid, the mRNA level of CTSA in podocytes significantly increased. The above experiments were performed under independent replication conditions, and the results showed a consistent trend.

[0035] Example 3 demonstrates that increasing CTSA expression can improve doxorubicin-induced podocyte injury and enhance the correlation between CTSA expression and changes in podocyte state.

[0036] Based on the construction of doxorubicin-induced podocyte injury models under different time gradients, human podocytes were seeded in culture plates and cultured to an appropriate density. CTSA was then overexpressed in podocytes via lentivirus-mediated expression. Subsequently, the survival ability of podocytes in different treatment groups was evaluated using the CCK-8 (Cell Counting Kit-8) cell viability assay, thereby analyzing the ameliorative effect of increased CTSA expression on podocyte injury.

[0037] The specific steps for CCK-8 testing are as follows: Cell seeding and treatment: Human podocytes were seeded in 96-well plates. After the cells adhered and reached a suitable density, doxorubicin solution was added to induce cell damage. At the same time, lentivirus was added to the experimental group to increase CTSA expression, and empty vector virus was added to the control group. The treatment time was, for example, 24 hours.

[0038] Add CCK-8 reagent: After treatment, add 10 μL of CCK-8 reagent to each well, gently shake to mix, and continue to incubate at 37 ℃ and 5% CO2 for 1-2 hours.

[0039] Absorbance detection: The absorbance (OD450) of each well was measured at a wavelength of 450 nm using an ELISA reader, and the background value of the blank wells was subtracted.

[0040] Data collection: Using the control group as a baseline, the relative cell viability of each treatment group was calculated to evaluate the effects of podocyte injury and CTSA overexpression on cell viability.

[0041] Data Analysis: GraphPad Prsm was used to perform statistical processing on the experimental data. A two-tailed Student's t-test was used for comparisons between the two groups, and a p-value < 0.05 was considered statistically significant.

[0042] like Figure 6 As shown, the CCK8 assay results indicate that doxorubicin-induced podocyte injury significantly reduced podocyte viability, while treatment with CTSA transcription factors to enhance CTSA expression significantly improved podocyte viability. These results suggest that in the in vitro podocyte injury model, changes in CTSA expression levels are correlated with changes in podocyte status, providing experimental evidence for understanding the role of CTSA in renal podocyte injury and podocyte disease-related states.

[0043] Example 4: This example demonstrates the detection and comparative analysis of CTSA mRNA expression levels using an in vitro podocyte injury model and a kit for in vitro detection.

[0044] In an in vitro podocyte injury model, the mRNA expression level of CTSA was detected using any one of the following methods: real-time quantitative reverse transcription polymerase chain reaction, nuclease protection assay, in situ hybridization, nucleic acid microarray, or RNA blotting. The kit used included one or more of the following: pre-coated plate, standards, coating buffer, sample dilution buffer, blocking buffer, enzyme label, elution buffer, and chromogenic agent.

[0045] This study analyzed the changes in the state of cultured podocytes under different treatment conditions by comparing the mRNA expression levels of CTSA in an in vitro podocyte injury model with those in undamaged in vitro cultured podocytes. Tools used to detect CTSA expression levels included one or more of the following: nucleotides, antibodies and their functional fragments, ligands, test strips, chips, plates, membranes, nanoparticles, or sequencing libraries. Nucleotides included primer pairs, probes, or antisense nucleotides that specifically bind to the CTSA gene; antibodies and their functional fragments included antibodies capable of specifically binding to CTSA or their antigen-binding fragments; chips included gene chips or microfluidic chips; and nanoparticles included magnetic beads, organic nanoparticles, quantum dot nanoparticles, or rare earth complex nanoparticles. The study ultimately demonstrated the detection methods for CTSA mRNA expression levels under in vitro conditions and their application in the analysis of in vitro podocyte injury states.

[0046] Examples 1 to 4 demonstrate that this invention systematically verifies the feasibility and rationality of CTSA as a biomarker and for diagnostic applications, focusing on the expression changes of CTSA in renal podocyte injury and podocyte disease-related states. Example 1, through mononuclear transcriptome sequencing of human and mouse kidney tissues, shows that CTSA is mainly expressed in podocytes in kidney tissue and exhibits upregulation under podocyte injury and podocyte disease-related conditions, clarifying the podocyte-specific expression basis of CTSA. Example 2 further confirms, in an in vitro cultured podocyte injury model, that the mRNA expression level of CTSA is stably increased relative to the uninjured control under different induction conditions such as doxorubicin, angiotensin II, and palmitic acid, with consistent and reproducible results. Example 3 analyzes the correlation between CTSA expression changes and podocyte state changes in an in vitro podocyte injury model, providing experimental evidence for understanding the biological significance of CTSA in podocyte injury-related states. Example 4 illustrates, from an in vitro detection perspective, the detection of CTSA mRNA using reagents or kits. The method of detecting expression levels and comparing them with controls clarifies its application path in in vitro podocyte injury status analysis. In summary, under in vitro podocyte injury model conditions, changes in CTSA expression can stably and detectably reflect podocyte injury-related status, thus supporting the technical solution of using CTSA as a biomarker and diagnostic tool for renal podocyte injury and podocyte disease.

[0047] 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 application of CTSA as a biomarker and diagnostic tool in renal podocyte injury and podocyte disease, characterized by: The diagnostic application is implemented in an in vitro podocyte injury model, using undamaged in vitro cultured podocytes as a control. When in vitro cultured podocytes are induced to form a podocyte injury state by doxorubicin, angiotensin II, or palmitic acid, CTSA shows a stable and detectable upregulation of expression under podocyte injury conditions compared to control podocytes. By detecting changes in CTSA mRNA expression levels, it serves as a biomarker reflecting renal podocyte injury and podocyte disease-related podocyte injury status, and is used for the in vitro diagnosis of renal podocyte injury and podocyte disease.

2. The application according to claim 1, characterized in that: The diagnostic application uses real-time quantitative polymerase chain reaction to detect the expression level of CTSA mRNA in cultured podocytes in vitro, and compares the detection results with the CTSA mRNA expression level in control podocytes to determine the upregulation of CTSA expression under podocyte injury conditions, for in vitro diagnosis.

3. The application according to claim 2, characterized in that: The diagnostic application compares the mRNA expression level of CTSA in an in vitro podocyte injury model with the mRNA expression level of CTSA in undamaged in vitro cultured podocytes. When the mRNA expression level of CTSA is higher than that in undamaged podocytes, the kidney podocytes corresponding to the in vitro cultured podocytes are determined to be in a damaged state, which is used for in vitro diagnosis.

4. The application according to claim 3, characterized in that: In in vitro podocyte injury models constructed under different podocyte injury induction conditions, the mRNA expression level of CTSA was upregulated compared with that of undamaged in vitro cultured podocytes, thus making CTSA a suitable biomarker for the diagnosis of different types of podocyte injury states as a renal podocyte injury and podocyte disease.

5. The application according to claim 4, characterized in that: The in vitro podocyte injury models include podocyte injury models induced by doxorubicin, angiotensin II, and palmitic acid, respectively. Thus, the mRNA expression level of CTSA in different podocyte injury models can be used to distinguish between podocyte injury and uninjured states.

6. The application according to claim 5, characterized in that: The diagnostic application is achieved through any of the reagents or kits used for in vitro detection.

7. The application according to claim 6, characterized in that: The kit includes a detection reagent for detecting the mRNA expression level of CTSA, and based on the detection results of the CTSA mRNA expression level, it provides a diagnostic basis for determining whether podocytes are in a damaged state.

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