Use of an srx2 inhibitor in the manufacture of a medicament for the treatment of renal fibrosis

Abstract

This invention relates to the field of pharmaceutical technology, specifically disclosing the application of SRPX2 inhibitors in the preparation of drugs for treating renal fibrosis, including SRPX2 inhibitors. SRPX2 inhibitors regulate the expression of iron metabolism-related proteins by inhibiting the binding of SRPX2 to IRP1, a key regulatory protein in iron metabolism, thereby restoring iron homeostasis and alleviating renal fibrosis. In patients with chronic kidney disease and animal models of renal fibrosis, SRPX2 is highly expressed in renal tubular epithelial cells and is positively correlated with the degree of fibrosis. The use of SRPX2 inhibitors can significantly reduce the pathological changes of renal fibrosis and improve renal function. This invention provides a new treatment strategy and drug candidate for renal fibrosis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more particularly to the use of SRPX2 inhibitors in the preparation of medicaments for the treatment of renal fibrosis. Background Technology

[0002] Renal fibrosis is a common pathological process in the progression of various chronic kidney diseases (CKD) to end-stage renal disease, characterized by glomerular sclerosis and tubulointerstitial fibrosis. The pathogenesis of renal fibrosis is not yet fully understood, and effective treatments are lacking clinically. Sushi repeat-containing protein X-linked2 (SRPX2) is a secreted extracellular matrix protein belonging to the chondroitin sulfate proteoglycan family. It is expressed in various tissues, including neurons, the heart, and the lungs, and can promote angiogenesis, cell proliferation, and adhesion. It is also associated with the development of the nervous system and the occurrence and development of tumors. Studies suggest that SRPX2 can participate in bleomycin-induced pulmonary fibrosis in mice by activating the TGF-β1 / Smad3 signaling pathway in lung fibroblasts, and that reducing SRPX2 expression can inhibit pulmonary fibrosis. However, the expression sites and functions of SRPX2 in chronic kidney disease remain unclear. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and to propose the application of SRPX2 inhibitors in the preparation of drugs for the treatment of renal fibrosis.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: One of the drugs used to treat kidney fibrosis includes SRPX2 inhibitors.

[0005] Preferably, the SRPX2 inhibitor is a nucleic acid inhibitor that targets SRPX2.

[0006] Preferably, the nucleic acid inhibitor targeting SRPX2 is siRNA, shRNA, or antisense oligonucleotide.

[0007] The use of one of the above-mentioned SRPX2 inhibitors in the preparation of a drug for treating renal fibrosis.

[0008] Preferably, the treatment of renal fibrosis includes inhibiting the binding of SRPX2 to IRP1, a key regulatory protein of iron metabolism, to restore iron homeostasis.

[0009] Preferably, the renal fibrosis includes chronic kidney disease, unilateral ureteral obstruction, or folic acid-induced renal fibrosis.

[0010] A kit for diagnosing kidney fibrosis.

[0011] Preferably, the kit contains an antibody for detecting SRPX2 protein expression or primers for detecting SRPX2 gene expression.

[0012] A method for constructing a mouse model of renal fibrosis includes the following steps: A: Provide mice with specific knockout of the Srpx2 gene in renal tubular epithelial cells; B: The mice were subjected to unilateral ureteral obstruction treatment or folic acid induction treatment; C: Obtain a mouse model of renal fibrosis.

[0013] Preferably, the mice with Srpx2 gene knocked out specifically in renal tubular epithelial cells are obtained by the following method: Srpx2 flox / flox Mice were mated with Cdh16-cre mice, and the genotype Srpx2 was obtained through screening. flox / Y ;Cdh 16+ / Cre Male mice.

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention breaks through the limitation of existing technologies that only associate SRPX2 with pulmonary fibrosis. It reveals for the first time that SRPX2 is highly expressed in renal tubular epithelial cells and participates in the process of renal fibrosis by regulating iron metabolism, providing a novel molecular target for the treatment of renal fibrosis.

[0015] 2: This invention has demonstrated through various animal models and cell experiments that inhibiting SRPX2 expression can significantly reduce the degree of renal fibrosis and decrease the expression of fibrosis-related proteins (α-SMA, Collagen I, fibronectin), providing experimental evidence for the development of drugs to treat renal fibrosis.

[0016] 3: This invention is the first to discover that SRPX2 directly interacts with IRP1, a key protein in iron metabolism, and confirms that SRPX2 mediates iron homeostasis imbalance by regulating the expression of IRP1 and its downstream iron metabolism-related proteins (TFR1, FTH1, FPN), thereby promoting renal fibrosis, revealing a new mechanism by which SRPX2 promotes renal fibrosis.

[0017] 4. This invention constructs a mouse model of renal tubular epithelial cell-specific Srpx2 gene knockout, providing an important tool for the study of renal fibrosis mechanisms and drug screening. This model has higher cell specificity and can be used to evaluate therapeutic strategies targeting SRPX2 in renal tubular epithelial cells. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 A graph showing the expression analysis data of SRPX2 in kidney samples; Figure 2 The graph shows the analytical data showing that SRPX2 was significantly elevated in CKD patients compared to healthy subjects, and was negatively correlated with estimated glomerular filtration rate (eGFR). Figure 3 This is a graph showing the analytical data of upregulated SRPX2 expression in mouse fibrotic kidney tissue induced by a unilateral ureteral obstruction (UUO) model; Figure 4 A graph showing the analytical data on the upregulation of SRPX2 expression in folic acid-induced fibrotic kidney tissue in mice; Figure 5 This is a graph showing the analysis data of SRPX2 expression in proximal renal tubular cells, distal renal tubular cells, and collecting duct cells of mice. Figure 6 Graph of analytical data showing how knocking out SRPX2 can alleviate UUO-induced renal fibrosis; Figure 7 The graph shows the analytical data of how SRPX2 overexpression exacerbates UUO-mediated kidney injury and fibrosis. Figure 8 Graph of analytical data showing that SRPX2 knockdown improves the fibrotic phenotype of HK-2 cells; Figure 9 A graph showing the analytical data of protein-protein interaction between SRPX2 and iron regulatory protein 1 (IRP1); Figure 10 Analytical data plots to verify the interaction between SRPX2 and IRP1; Figure 11 Comparative proteomics analysis of renal biopsy tissue from human CKD patients; Figure 12 Graphs showing the data analysis of how SRPX2 gene knockdown altered the inflammatory and metabolic pathways in UUO mice; Figure 13 SRPX2 inhibits IRP1-mediated iron homeostasis regulation in renal tubular epithelial cells (TECs); Figure 14 A schematic diagram of the breeding model of Srpx2 gene knockout mice in renal tubular epithelial cells; Figure 15 for Srpx2 Image of gel electrophoresis results for gene knockout mice; Figure 16 Image of gel electrophoresis results for Srpx2-FLAG gene knock-in mice; Figure 17 Renal tubular epithelial cells Srpx2 Image of gel electrophoresis results for conditional gene knockout mice. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0021] Unless otherwise specified, the reagents or instruments used in this invention are commercially available.

[0022] Example 1 To investigate the potential role of SRPX2 in renal fibrosis, we first analyzed the expression of SRPX2 in renal tissues from patients with different types of kidney disease using the publicly available renal transcriptome database Nephroseq (https: / / nephroseq.org / resource / main.html). Specifically... Figure 1 As shown in figure a, compared with the healthy control group (n=31), the expression level of SRPX2 was significantly increased in renal biopsy tissues of diabetic nephropathy (n=18), focal segmental glomerulosclerosis (n=28), lupus nephritis (n=47), and vasculitis (n=21).

[0023] To verify the elevated SRPX2 expression levels in patients with different types of renal fibrosis, adjacent kidney tissue from patients with renal tumors (healthy controls) and kidney biopsy tissue from patients with different degrees of renal fibrosis confirmed by renal biopsy were collected. All kidney tissues were obtained from the Department of Nephrology and the Urology Center of the Second Affiliated Hospital of Soochow University between 2022 and 2023, and were approved by the Research Ethics Committee of the Second Affiliated Hospital of Soochow University (Approval No.: JD-HG-2023-67). All participants signed informed consent forms. Immunohistochemical staining was performed on the collected kidney tissues. (Specific details are as follows...) Figure 1As shown in b, renal fibrosis biopsy specimens were classified into three categories—mild (n=20), moderate (n=20), and severe (n=20)—based on the degree of kidney damage indicated by hematoxylin and eosin (H&E) staining and the area of ​​collagen deposition shown by Masson trichrome (Masson) staining. Twenty adjacent kidney tissue samples served as healthy controls. Immunohistochemical results showed that, compared with healthy controls, SRPX2 expression was increased in the kidney tissues of patients with renal fibrosis, and the expression was most significant in the kidney tissues of patients with severe renal fibrosis.

[0024] Furthermore, immunohistochemical staining of renal biopsy tissue samples from patients with different types of CKD showed that SRPX2 was expressed to varying degrees in renal tissues of different types of CKD, specifically as follows: Figure 1 As shown in c, the diseases include diabetic nephropathy (n=10), IgA nephropathy (n=10), focal segmental glomerulosclerosis (n=10), thrombotic microangiopathy (n=5), and vasculitis (n=5).

[0025] The relationship between serum SRPX2 levels and clinicopathological features in CKD patients was further evaluated. Serum samples were collected from 30 healthy controls and 146 CKD patients diagnosed by renal biopsy between 2022 and 2023 from the Department of Nephrology and the Physical Examination Center of the Second Affiliated Hospital of Soochow University. Each serum sample consisted of 2 mL. The sample collection process followed the principles of the Declaration of Helsinki and was approved by the Research Ethics Committee of the Second Affiliated Hospital of Soochow University (Approval No.: JD-HG-2023-67). All participants signed informed consent forms. Serum SRPX2 concentration was measured using ELISA, and relevant data including gender, age, creatinine, and estimated glomerular filtration rate (eGFR) were recorded. The results showed that serum SRPX2 concentration was significantly higher in CKD patients compared to healthy controls. Figure 2 a), and it gradually increases with the increase of CKD stage ( Figure 2 b). Furthermore, Pearson correlation analysis showed a significant negative correlation between SRPX2 levels and eGFR ( Figure 2 c). These findings suggest that SRPX2 may be a potential serum biomarker for CKD progression and renal fibrosis.

[0026] Example 2 First, such as Figure 3As shown in Figure a, male mice underwent left ureteral double ligation after anesthesia to establish a mouse model of unilateral ureteral obstruction (UUO) and renal fibrosis. In the sham surgery group, only laparotomy to expose the ureter was performed, without ligation. Mice were sacrificed on postoperative days 3, 7, and 14, and the obstructed kidney was collected for further analysis. Type I collagen, as a major component of the extracellular matrix of interstitial cells, affects the typical structure and function of the kidney. α-SMA (α-smooth muscle actin) is a marker of myofibroblasts and participates in the renal fibrosis process. The expression levels of these two renal fibrosis markers and SPRX2 in the renal tissues of the UUO and Sham mouse models were detected by Western blot and immunofluorescence methods. (Details are omitted as they are not relevant to the main text.) Figure 3 As shown in b and 3c, Western blot results indicated that the expression levels of Collagen I and α-SMA in the kidney tissue of Sham group mice were low, while the expression levels of Collagen I and α-SMA in the kidney tissue of UUO group mice increased on day 3 (D3), increased significantly on day 7 (D7), and peaked on day 14 (D14), suggesting successful model establishment. The expression level of SPRX2 protein in the kidney tissue of Sham group mice was low, while the expression level of SPRX2 protein in the kidney tissue of UUO group mice was increased, and gradually increased over time, consistent with the expression trends of Collagen I and α-SMA.

[0027] Fibronectin is also a marker of kidney fibrosis. For example... Figure 3 As shown in d, immunofluorescence staining results showed that the expression levels of SPRX2, α-SMA and Fibronectin in the kidney tissue of Sham group mice were low, while the expression levels of SPRX2, α-SMA and Fibronectin in the kidney tissue of UUOD14 mice were significantly increased.

[0028] Furthermore, CD68 is a marker of macrophages, and kidney injury molecule-1 (TIM1) is a marker of renal tubular injury. The expression levels of these two proteins, along with SPRX2, in the kidney tissues of UUO and Sham mice were detected using immunofluorescence staining. Specifically... Figure 3 As shown in e (CD68) and 3f (TIM1), the expression levels of SPRX2, CD68 and TIM1 in the kidney tissue of Sham group mice were low, while the expression levels of SPRX2, CD68 and TIM1 in the kidney tissue of UUO D14 mice were increased, suggesting the activation of inflammatory response during renal fibrosis.

[0029] Another animal model of kidney fibrosis—the folic acid (FA) nephropathy mouse model—was used to observe the kidney fibrosis process over 28 days. The modeling process is as follows: Figure 4 As shown in figure a. Western blotting and immunofluorescence were used in a FA mouse model to verify the presence of renal fibrosis and changes in SPRX2 protein expression. Specifically, as shown in figure a. Figure 4 As shown in b and 4c, Western blot results indicated that the expression levels of α-SMA and Fibronectin proteins were low in the kidney tissue of control mice, while their expression was increased in the kidney tissue of FA group mice. Specifically, the expression level increased on day 7 (D7), significantly increased on day 14 (D14), and peaked on day 28 (D28), indicating successful model establishment. The expression level of SPRX2 protein was low in the kidney tissue of control mice, compared to which the expression level was increased in the kidney tissue of FA group mice, and gradually increased over time, consistent with the expression trends of α-SMA and Fibronectin.

[0030] like Figure 4 As shown in d, immunofluorescence staining results showed that the expression levels of SPRX2, α-SMA and Fibronectin in the kidney tissue of control mice were low, while the expression levels of SPRX2, α-SMA and Fibronectin in the kidney tissue of FAD28 mice were significantly increased.

[0031] In addition, the expression levels of CD68, TIM1, and SPRX2 in the kidney tissues of FA D28 and control mice were detected by immunofluorescence staining. Specifically... Figure 4 As shown in e(CD68) and 4f(TIM1), the expression levels of SPRX2, CD68, and TIM1 were low in the kidney tissue of control mice, while the expression levels of SPRX2, CD68, and TIM1 were increased in the kidney tissue of FA D28 mice. In conclusion, SRPX2 expression is upregulated in the fibrotic kidney tissue of UUO and FA nephropathy mice.

[0032] Example 3 To clarify the specific expression site of SRPX2 in mouse kidneys, a customized... Srpx2 mRNA probes were used to detect mRNA in the kidney tissue of healthy mice using RNAscope technology. Srpx2 mRNA expression was measured and co-stained with cellular markers from different segments of four renal units. RNAscope technology is a probe hybridization-based signal amplification method used for in situ detection of mRNA expression. Specifically... Figure 5 As shown in a, podocyte spike protein (NPHS2) is a marker for podocytes, solute carrier family 5 member 2 (SLC5A2) is a marker for proximal tubular epithelial cells, urokinase (UMOD) is a marker for distal tubular epithelial cells, and aquaporin 2 (AQP2) is a marker for collecting duct epithelial cells.

[0033] The test results are as follows: Figure 5 As shown in b. Srpx2 It mainly co-stained with SLC5A2, UMOD, and AQP2, and co-stained with NPHS2 to a small extent, indicating that Srpx2 It is mainly expressed in the proximal renal tubular cells, distal renal tubular cells, and collecting duct cells of mice, with a small amount expressed in the glomeruli.

[0034] Example 4 Using the UUO 14D model in Srpx2 knockout mice ( Srpx2 - / Y ) and wild-type mice ( Srpx2 + / Y The effects of SRPX2 deficiency on renal fibrosis were studied in a study published in WT. Specific modeling methods are as follows: Figure 6 As shown in a.

[0035] and Srpx2 + / Y Compared to mice, Srpx2 - / Y The mice exhibited normal body weight and kidney function. Further analysis was performed using H&E and Masson staining. Srpx2 - / Y and Srpx2 + / Y Kidney tissue sections after UUO 14D and Sham mouse models. Specific results are as follows: Figure 6 As shown in b, compared to the Sham treatment, Srpx2 - / Y and Srpx2 + / Y Mice treated with UUO 14D all exhibited pathological morphological features of renal fibrosis, such as luminal dilation, epithelial cell atrophy, and interstitial inflammation. However, after treatment with UUO 14D, compared with... Srpx2 + / Y Compared to mice, the pathological changes of fibrosis are... Srpx2 - / Y The effect was even milder in mouse kidney tissue, suggesting that SRPX2 deficiency significantly reduced the pathological changes of renal fibrosis after UUO 14D treatment.

[0036] In addition, Western blot was used to detect... Srpx2 - / Y and Srpx2 + / Y The expression levels of fibrosis markers in kidney tissue after UUO 14D and Sham mouse models were shown in the following figures. Figure 6 c and Figure 6 As shown in d, compared to the Sham treatment, Srpx2 - / Y and Srpx2 + / Y In mice treated with UUO 14D, the expression levels of Collagen I, fibronectin, and α-SMA proteins in kidney tissue were all increased. However, after UUO 14D treatment, the expression levels of these proteins were significantly reduced compared to... Srpx2 + / Y Compared with mice, the expression levels of Collagen I, fibronectin, and α-SMA proteins were... Srpx2 - / Y The levels of SRPX2 were significantly reduced in mouse kidney tissue, further indicating that SRPX2 deficiency significantly alleviated UUO-induced renal fibrosis in mice.

[0037] Next, we will explore the functional role of adeno-associated virus 9 (AAV9)-mediated SRPX2 overexpression in UUO-induced renal fibrosis. Specific modeling methods are as follows: Figure 7 As shown in a, directly... AAV9-Vector (p) cAAV-CMV-GdGreen )and AAV9- Srpx2 Virus( pcAAV-CMV-Srpx2-GdGreen It was injected into the renal cortex of mice.

[0038] E-Cadherin is a marker of epithelial cells. Four weeks after injection into the renal cortex, some mice were sacrificed to verify the success of SRPX2 overexpression. Specifically... Figure 7 As shown in b, the kidney tissue section was subjected to... AAV9-Srpx2 co-staining with E-Cadherin using immunofluorescence confirmed that... AAV9-Srpx2 It has successfully entered the renal tubular epithelial cells (TECs).

[0039] Then on AAV9-Vector and AAV9-Srpx2 Mice were treated with UUO or Sham, and sacrificed 10 days after the onset of renal fibrosis. Indicators related to renal fibrosis were then measured. Figure 7 As shown in c, H&E and Masson staining indicate that, with AAV9- Vector Compared to the group, AAV9-Srpx2 The renal tubular dilation and fibrosis were more severe in the group. Figure 7 c). Western blot results showed that SRPX2 overexpression exacerbated UUO-induced fibrosis, and Collagen I and Fibronectin protein levels were elevated ( Figure 7 The above results indicate that SRPX2 overexpression exacerbates UUO-induced renal tubular dilatation and collagen deposition.

[0040] To investigate the effects of SRPX2 on renal fibrosis in vitro, three SRPX2 gene interference vectors—SRPX2 siRNA, siRNA-2, and siRNA-3—were designed and synthesized. Using negative siRNA as a control, these vectors were transfected into proximal tubular epithelial cells (HK-2). Western blotting was performed to assess silencing efficiency after 48 hours. The results showed that SRPX2 protein expression was decreased in all three interference groups compared to the negative control group, with the SRPX2 siRNA-2 group exhibiting the highest silencing efficiency at approximately 76%. (Details are as follows...) Figure 8 As shown in a and 8b. siRNA-2 was subsequently selected as the interfering siRNA for the experiment. Its gene sequence is: upstream (5-3): GUGCUCCUAUGAAGAUUAATT, downstream (5-3'): UUAAUCUUCAUAGGAGCACTT.

[0041] HK-2 cells were then pretreated with this siRNA, with negative siRNA as a control. An epithelial-mesenchymal transition (EMT) model was induced by stimulating HK-2 cells with 5 ng / mL TGF-β1 for 24 h, dividing them into four groups. Western blotting was used to detect the expression of fibrosis-related proteins. The results showed that TGF-β increased the expression of Collagen I and α-SMA, but this effect was significantly weakened in SRPX2 knockdown cells, specifically as follows: Figure 8 As shown in c and 8d. These results suggest that knocking down SRPX2 expression can suppress the TGF-β-induced profibrotic effect.

[0042] Example 5 To explore the mechanism by which SRPX2 exacerbates renal fibrosis, extract... Srpx2 Flag / Y and Srpx2 + / Y SRPX2 and its interacting proteins were enriched by immunoprecipitation of mouse kidney tissue proteins. The eluted interacting proteome samples were then digested with various proteases to generate peptides. High-performance liquid chromatography (HPLC) was used to separate and purify the peptides, followed by LC-MS / MS analysis to obtain mass spectrometry data. Finally, database searches and bioinformatics analyses were performed on the mass spectrometry data to identify potential binding partners for SRPX2. The specific procedure is as follows: Figure 9 As shown in figure a. Through protein recognition and functional classification, the Venn diagram displays 37 specific proteins expressed in SRPX2-tagged protein precipitates (…). Figure 9(b and 9c), among which iron regulatory protein 1 (IRP1), NADPH, and SlC25A5 are the top three proteins that may bind to SRPX2. KEGG analysis showed that these proteins are closely related to several key metabolic pathways, including the citric acid cycle, 2-oxoacetic acid homeostasis, the cGMP-PKG signaling pathway, and fatty acid metabolism (b and 9c). Figure 9 d). Special attention was paid to IRP1, as it was found to potentially bind to SRPX2 in multiple samples. IRP1 is associated with cis-regulatory iron response elements (IREs) in the untranslated regions of mRNA, which play a crucial role in regulating iron metabolism.

[0043] Subsequently, in vivo and in vitro co-immunoprecipitation (Co-IP) experiments confirmed the binding of SRPX2 to IRP1, while NADPH and SlC25A5 did not bind to SRPX2. Srpx2 FlAG / Y and littermates Srpx2 + / Y SRPX2-FLAG was immunoprecipitated in the renal tissue lysate, and the presence of IRP1 in the FLAG-labeled SRPX2 protein precipitate was identified by Western blot assay. Specifically... Figure 10 As shown in a. Next, co-transfected... IRP1 and SRPX2-FLAG HEK293T cells containing the plasmid underwent Co-IP experiments to explore their interactions, and IRP1 and SRPX2-FLAG proteins were identified in the elution buffer. The results showed a significant interaction between exogenously expressed SRPX2 and IRP1, specifically as follows: Figure 10 As shown in b. Furthermore, immunofluorescence staining of wild-type mouse kidney tissue further revealed that SRPX2 and IRP1 directly co-localize in renal tubular cells (as shown in b). Figure 10 c).

[0044] IRP1 exhibits a dual function through conformational changes: in its open conformation, it inhibits target mRNA translation or promotes degradation by binding to IREs, specifically as follows: Figure 10As shown in d. Upon binding to the iron-sulfur cluster, IRP1 transforms into a closed conformation, functioning as aconitase. To investigate the theoretical interaction mechanism between IRP1 and the target molecule SRPX2, this study used the ClusPro protein-protein docking platform for molecular docking simulation and screened interaction models with significant binding characteristics through structural clustering analysis. Predicted complex structures showed that SRPX2 protein binds with high affinity to the central domain of the open conformation IRP1 through its C-terminal domain. Molecular interaction analysis revealed that the conformational stability of the SRPX2-IRP1 binding relationship is primarily maintained by polar interactions. Specifically, the negatively charged residues D354, D375, and E436 of SRPX2 form salt bridges with the positively charged residues K615, R541, and H207 of IRP1, respectively. Simultaneously, residues Q368, R394, Y360, S365, T439, R358, R416, R413, and D430 of SRPX2 form a network of hydrogen bonds with the side chains S436, S441, T295, E302, Q143, N707, and N685 of IRP1, as well as the main chain atoms G474 and P703. (Specific details are omitted as they are not translated.) Figure 10 As shown in e, these interaction sites have spatial consistency with previously reported key regions of iron metabolism-related protein interactions. Notably, this study suggests that SRPX2 may affect iron homeostasis by regulating the mRNA binding activity of IRP1, thereby interfering with the expression of downstream targets related to iron storage, transport, and utilization. This provides new theoretical evidence for elucidating the role of the SRPX2-IRP1 regulatory axis in iron metabolism. In this invention, iron metabolism-related proteins refer to proteins involved in iron ion uptake, storage, efflux, and regulation, including but not limited to IRP1, TFR1, FTH1, and FPN. In summary, SRPX2 directly binds to the key iron metabolism regulator IRP1, regulating the expression of downstream iron metabolism-related proteins, thereby affecting iron homeostasis and the progression of renal fibrosis.

[0045] Example 6 To further elucidate the association between abnormal iron metabolism and chronic kidney disease (CKD), this study performed proteomic analysis on renal biopsy tissues from patients with membranous nephropathy and normal adjacent healthy kidney tissues (controls). The specific procedure is as follows: Figure 11 As shown in figure a. A total of 5,355 proteins were identified and quantified using tissue protein extraction, enzymatic digestion, and label-free quantification techniques based on LC-MS / MS. Partial least squares discriminant analysis (PLS-DA) model showed significant separation between the control group and the membranous nephropathy group (…). Figure 11 b). Volcano plots show a significant dysregulation of iron homeostasis-related proteins in patients with membranous nephropathy. Figure 11 c), in which ferritin light chain (FTL) expression was significantly increased, while transferrin (TF) and heme-binding protein (HPX) levels were significantly decreased. Figure 11 d). The increase in FTL and the decrease in TF indicate the pathological regulation of iron homeostasis imbalance in membranous nephropathy. Gene ontology analysis showed that differentially regulated proteins were mainly enriched in biological processes such as Golgi vesicle transport, while downregulated proteins were associated with iron homeostasis; cellular component analysis showed that differentially regulated proteins were mainly located in early endosomes and the major histocompatibility complex (MHC), suggesting that immune system abnormalities and tissue homeostasis imbalance may be involved in the pathological process of CKD. Figure 11 e).

[0046] To further explore the role of SRPX2 in iron metabolism via IRP1 regulation, and to investigate the effects of UUO-induced iron metabolism... Srpx2 + / Y and Srpx2 - / Y Mice underwent transcriptome sequencing (RNA-seq) analysis to identify potential target genes and enriched pathways. Principal component analysis (PCA) revealed significant separation in gene expression profiles across groups. Figure 12 a). With Srpx2 + / Y Compared to the sham group, in Srpx2 + / Y In the UUO group, 3312 genes were significantly upregulated and 2535 were significantly downregulated. Furthermore, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. Srpx2 + / Y Compared to the UUO group Srpx2 - / Y In the UUO group, 701 genes were upregulated and 1559 genes were downregulated. Figure 12 b). KEGG pathway enrichment analysis showed that, compared with... Srpx2 + / Y Compared to the sham group, Srpx 2+ / Y The UUO group upregulated genes were mainly enriched in cytokine-receptor interactions and inflammatory pathways, while downregulated genes were related to metabolic pathways. Figure 12 (c and 12d) It is worth noting that, Srpx2 After gene knockout, the expression patterns of inflammation and metabolism-related pathways were significantly reversed. Figure 12 e and 12f). Further analysis revealed that the expression levels of iron homeostasis-related genes in UUO kidney tissue were at... Srpx2 Significant changes were observed after deletion ( Figure 12 g). The integrated analysis of the above proteomics and transcriptomics data provides important clues for a deeper understanding of the role of iron metabolism pathways in the pathogenesis of CKD.

[0047] Since the research mainly focuses on the expression of SRPX2 in the renal tubules, in order to further explore the effect of renal tubular SRPX2 on iron homeostasis in progressive renal fibrosis, mice with SRPX2 specifically knocked out by renal tubular epithelial cells (TECs) were constructed. Srpx2 flox / Y ; Cdh16 + / Cre (cKO), and a UUO model was established. For details of the modeling process, please refer to Figure 13 As shown in Figure a, iron regulatory proteins (IRPs) can control the expression of specific messenger RNAs (mRNAs) by binding to IREs in their 5' or 3' untranslated regions (UTRs) to regulate intracellular iron metabolism. Specifically, IRPs can inhibit mRNA translation by binding to IREs in the 5' UTRs of genes such as ferritin heavy chain polypeptide 1 (FTH1), ferritin light chain (FTL), and iron transporter 1 (FPN1). Conversely, they can enhance mRNA stability by interacting with IREs in the 3' UTRs of genes such as transferrin receptor 1 (TFR1) and divalent metal ion transporter 1 (DMT1), thereby protecting these mRNAs from nuclease degradation. Therefore, genes related to iron homeostasis in a renal fibrosis model were detected by real-time quantitative PCR (qRT-PCR) and Western blot. Irp1 , Tfrc , Fth1 and Slc40a1 mRNA and protein levels. Compared with the control group, the renal fibrosis mice had... Irp1 mRNA levels were significantly reduced. Conversely, in the WT-UUO group, mRNA levels involved in iron storage and export were significantly decreased. Fth1 and Slc40a1 The mRNA level was significantly increased ( Figure 13 b). Furthermore, Western blot and immunofluorescence assays showed that, compared to the control group, UUO-induced renal IRP1 and TFR1 protein expression was significantly decreased, while FTH1 and FPN protein levels were significantly increased. Notably, SRPX2 knockout in TECs significantly reversed these changes (b). Figure 13 Furthermore, compared to the WT-UUO group, the loss of Srpx2 in renal tubular epithelial cells alleviated UUO-induced renal pathological damage, specifically as follows: Figure 13As shown in f, H&E staining in row 1 indicates reduced renal tubular dilation, reduced casts, and preserved brush borders; Masson staining in row 2 indicates reduced collagen deposition area; and immunohistochemistry in rows 3 and 4 indicates reduced expression of Collagen I and Fibronectin. Therefore, the results indicate that SRPX2 regulates the expression of downstream iron metabolism-related proteins by binding to IRP1, a key regulatory protein in iron metabolism, thereby restoring iron homeostasis and improving renal fibrosis.

[0048] Example 7 Laboratory animal breeding 1. Srpx2 Gene knockout mice ( Srpx2 - / Y , KO) and Srpx2 -FLAG gene knock-in mice ( Srpx2 Flag / Y (KI) was kindly provided by other research groups.

[0049] 2. Renal tubular epithelial cells Srpx2 Gene condition knockout mouse construction and mating strategy To construct renal tubular epithelial cells Srpx2 Gene conditional knockout mice were generated using the Cre / LoxP knockout system. Female mice were... Srpx2 fl / fl mice and Cdh16-cre By mating male mice of the transgenic strain, one can obtain ( Srpx2 flox / Y ; Cdh16 + / Cre cKO mice and littermate controls Srpx2 flox / Y Mouse (WT). Specifically, as follows... Figure 14 As shown.

[0050] 3. Methods for Gene Identification in Laboratory Mice (1) Identification of rat tail lysis Five days after birth, the tails of mice were cut off approximately 0.5 cm from the end using finely shredded shears that had been subjected to high temperature and autoclaving. The tails were then placed into sterile 200 μL centrifuge tubes without enzymes. 1×MousetissueLysisBuffer and proteinase K were added to the centrifuge tubes at a ratio of 50:1. The tails were lysed using a PCR instrument following the reaction program: 55°C, 1 h; 95°C, 10 min; 12°C, 20 min. After the reaction, the tails were stored at 4°C.

[0051] (2) Primer information Srpx2 - / Y The primers for gene identification are as follows: Srpx2 FLAG / Y The primers for gene identification are as follows: Cdh16-Cre The primers for gene identification are as follows: Srpx2 fl / fl The primers for gene identification are as follows: (3) PCR reaction system Srpx2 - / Y PCR reaction system Srpx2 FLAG / Y PCR reaction system Cdh16-Cre PCR reaction system Srpx2 fl / fl PCR reaction system (4) PCR reaction procedure Srpx2 - / Y PCR reaction procedure Srpx2 FLAG / Y PCR reaction procedure Cdh16-Cre PCR reaction procedure Srpx2 fl / fl PCR reaction procedure (5) Gel electrophoresis results Specifically, such as Figure 15 As shown, Srpx2 Gel electrophoresis results of gene knockout mice.

[0052] The PCR products were separated by gel electrophoresis using a 1.5% agarose gel (constant current 100mA, 60min). The results showed that the wild-type band was approximately 500bp and the mutant band was approximately 274bp. Figure 15 The genotypes of the six mice shown are as follows: Srpx2 - / + 、Srpx2 + / + (Female KO) 、Srpx2 - / + 、Srpx2 - / Y (Male WT) 、 Srpx2 + / Y (Male KO) 、Srpx2 - / - (Female WT)

[0053] Specifically, such as Figure 16 As shown, Srpx2 - Gel electrophoresis results of FLAG gene knock-in mice.

[0054] The PCR products were separated by gel electrophoresis using a 2% agarose gel (constant current 100mA, 60min). The results showed that the wild-type band was approximately 200bp and the mutant band was approximately 2204bp. Figure 16 The genotypes of the three mice shown are as follows: Srpx2 - / Y (Male WT) 、Srpx2 FLAG / Y (Male KI) 、Srpx2 - / Y (Male WT)

[0055] specific Figure 17 As shown, renal tubular epithelial cells Srpx2 Gel electrophoresis results of gene conditional knockout mice.

[0056] The PCR products were separated by gel electrophoresis using a 2% agarose gel (constant current 100mA, 60min). The results showed... Cdh16-Cre In gene identification, the wild-type Cre band was 420 bp ( Figure 17 (Left image) Srpx2-flox In gene identification, the wild-type band was 221 bp, and the flux band was 328 bp. Figure 17 (Right image). Figure 5 The genotypes of the four mice shown are as follows: Srpx2 fl / - (Female WT) Srpx2 fl / Y(Male WT) Cdh16 + / Cre (Male WT) Srpx2 flox / Y ; Cdh16 + / Cre (Male cKO).

[0057] Adeno-associated virus (AAV) infection in mice Adeno-associated virus type 9 (AAV9) vector (carrying) provided by Shanghai Aobian Biotechnology Co., Ltd. Srpx2 Gene delivery was achieved via targeted injection into the renal parenchyma using a control virus and a TBE (avodin, 1.25% 2,2,2-tribromoethanol) at a dose of 0.02 mL / g body weight. Five minutes later, the mouse paws were gently clamped with forceps until the mouse became unresponsive, at which point the left kidney was exposed. Five to six sites in the renal cortex were then injected using glass microneedles. AAV9-Srpx2 ( pcAAV-CMV-Srpx2-GdGreen (or control virus) AAV9-vector ( pcAAV-CMV-GdGreen ), inject 10 μL at each site, with a viral titer of 8 × 10¹² vector genome / mL (vg / mL).

[0058] Cell culture and transfection 1. TGF-β-induced epithelial-mesenchymal transition (EMT) model in HK-2 cells This study used human renal proximal tubular epithelial cells (HK-2, Fuheng Bio, FH0228) cultured in DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics.

[0059] (1) Cell resuscitation and passage: Resuscitate HK-2 cells and passage them to the logarithmic growth phase to ensure cell viability >90%.

[0060] (2) Plating: HK-2 cells were plated at an appropriate density (5×10⁻⁶). 4 Cells / wells were seeded in 6-well plates and cultured at 37°C until the confluence reached 60-70%.

[0061] (3) Serum-free starvation: Remove the original culture medium and culture the cells in serum-free culture medium for 12-24 hours to synchronize the cells.

[0062] (4) TGF-β treatment: Serum-free culture medium containing TGF-β1 was added, and a concentration gradient was set (0, 2, 5, 10 ng / mL). The optimal induction concentration was screened after 24 hours of treatment with HK-2 cells, and then the treatment duration was determined by stimulating with 10 ng / mL TGF-β for 0, 6, 12, and 24 hours, respectively. The control group was only treated with serum-free culture.

[0063] 2. SRPX2-siRNA cell transfection experiment: (1) Cell preparation: Seed HK-2 cells into appropriate culture plates (such as 6-well plates), adjust the density to the confluence required for transfection (usually 60-80%), and pre-culture in a medium containing serum but without antibiotics.

[0064] (2) Preparation of siRNA-Lipofectamine complex: a. According to the Lipofectamine 2000 instructions, dilute the SRPX2-targeting siRNA (si-SRPX2-1 / 2 / 3) and the negative control siRNA (scramble siRNA) in Opti-MEM medium.

[0065] b. Mix Lipofectamine 2000 with Opti-MEM in the specified ratio and incubate at room temperature for 5 minutes.

[0066] c. Mix the diluted siRNA with Lipofectamine 2000 and let it stand at room temperature for 15-20 minutes to form a transfection complex.

[0067] (3) Transfection procedure: Add the complex evenly to the wells of HK-2 cell culture and gently shake to mix. Incubate at 37°C and 5% CO2 for 4-6 hours, then replace with serum-containing complete culture medium.

[0068] (4) After TGF-β stimulation and grouping, cells were collected after culturing for 24 hours for subsequent detection.

[0069] 3. Procedure for transfection of 293T cells with SRPX2–FLAG-pCAG and pCAG-IRP1 plasmids: (1) Cell preparation: 293T cells were seeded in 6-well plates at a density of about 70-80% confluence (about 2×10⁻⁶ cells per well). 6 Cells / well). Pre-cultured in DMEM medium containing 10% FBS for 24 hours at 37°C in a 5% CO2 incubator.

[0070] (2) Plasmid combination design: Co-transfection group: SRPX2-FLAG (2μg) + pCAG-IRP1 (2μg).

[0071] Control group: SRPX2-FLAG (2 μg) + empty vector pCAG (2 μg).

[0072] (3) Preparation of transfection reagent: Mix DNA, P3000 reagent and liposomes according to the instructions.

[0073] (4) Transfection procedure: a. Remove the old culture medium and add 1 ml of fresh culture medium (containing serum).

[0074] b. Add the DNA-transfection reagent complex dropwise into the wells and gently shake to mix.

[0075] c. Replace with complete culture medium after 6 hours (to reduce toxicity).

[0076] (5) Culture and detection: Collect samples 24-48 hours later for protein expression detection.

[0077] Construction of siRNA and plasmid Human source SRPX2 siRNA (si- SRPX2 -1 / 2 / 3) and scramble siRNA were purchased from Gemma Gene. The siRNA sequences are as follows: Plasmid construction: (1) pCAG-IRP1 was constructed by the following steps: First, the IRP1 gene (NM_001419952.1) was amplified from mouse kidney cDNA using PCR technology. Then, the PCR fragment was subcloned into the pCAG vector between the Sph1 and XhoI restriction sites. (2) The construction process of the SRPX2–FLAG-pCAG vector was as follows: The SRPX2 gene (NM_026838) was amplified by PCR from complementary DNA from mouse kidney. The obtained PCR fragment was fused with the C-terminal FLAG tag and then subcloned into the pCAG vector between the NotI and EcoRV restriction sites.

[0078] In vitro and in vivo co-immunoprecipitation assay (CO-IP) 1. In vivo CO-IP experiment Choose 2-month-old Srpx2 + / Y and Srpx2 FLAG / Y Genotyped mice were anesthetized and their kidneys were perfused with PBS. The renal cortex tissue was rapidly separated and homogenized in NP-40 lysis buffer (Beyotime, P0013F) containing 1 mM PMSF (Beyotime, ST506). After incubation with the lysis buffer at 4°C for 30 minutes, the supernatant was obtained by centrifugation at 12000×g for 10 minutes.

[0079] 2. In vitro CO-IP experiment In vitro experiments were performed using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019) to co-transfect HEK293T cells with pCAG-SRPX2-FLAG and pCAG-IRP1 plasmids. Four hours after transfection, the cells were cultured in Opti-MEM medium for another 48 hours. Cells were collected and centrifuged at 5000×g for 10 minutes to obtain the cell pellet. Lysis was performed using the same NP-40 lysis system, followed by centrifugation at 12000×g for 10 minutes at 4°C to collect the supernatant.

[0080] The protein supernatant prepared above was incubated overnight at 4°C with Anti-FLAG magnetic beads (Beyotime, P2115-2ML). The magnetic beads were then thoroughly washed with lysis buffer and TBS buffer (50mM TrisHCl, 150mM NaCl, pH 7.4), and the protein complex was finally eluted with 5× Loading Buffer.

[0081] Identification of interacting proteins 1. Sample preparation (1) Select 2-month-old infants Srpx2 + / Y and Srpx2 FLAG / Y Genotyped mice were anesthetized and their kidneys were perfused with PBS. The renal cortex tissue was rapidly separated and homogenized in NP-40 lysis buffer (Beyotime, P0013F) containing 1 mM PMSF (Beyotime, ST506). After incubation with the lysis buffer at 4°C for 30 minutes, the supernatant was obtained by centrifugation at 12000×g for 10 minutes.

[0082] (2) The protein supernatant prepared above was incubated overnight at 4°C with Anti-FLAG magnetic beads (Beyotime, P2115-2ML). The magnetic beads were then thoroughly washed with lysis buffer and TBS buffer (50mM TrisHCl, 150mM NaCl, pH 7.4) in sequence, and the protein complex was finally eluted with 5× Loading Buffer.

[0083] (3) The renal cortical immunoprecipitation eluate was separated by SDS-PAGE electrophoresis and then stained with Coomassie brilliant blue. Subsequent sample processing was completed by Putai Biotechnology (Suzhou, China) according to standard procedures: peptides were extracted after in-gel enzymatic digestion and vacuum dried, and then reconstituted in a solution containing 2% acetonitrile, 97.5% water and 0.5% formic acid. After purification by DTT reduction, IAA alkylation and desalting column, the samples were analyzed using a nano-liquid chromatography-electrospray ionization tandem mass spectrometry system (NanoLC-ESI-MS / MS).

[0084] 2. Nano-Liquid Chromatography-Mass Spectrometry Analysis An Agilent high-performance liquid chromatography system equipped with a 75 μm inner diameter, 8 cm long self-packed C18 reversed-phase capillary column was used for online ESI ionization with a Thermo linear ion trap mass spectrometer (LTQ). Mass spectrometry data were retrieved from the UniProt protein database using ProtQuest software, and protein relative abundance was calculated according to standard methods. Venn diagrams were constructed using software developed by Bardou et al. to visualize differentially expressed proteins. Interaction network analysis was performed using the STRING database (https: / / cn.string-db.org / ), and KEGG pathway enrichment analysis was completed based on the clusterProfiler package (v4.10.0) and the org.Mm.eg.db database (v3.18.0).

[0085] Protein-protein docking analysis Based on the 3D structures of IRP1 and SRPX2 predicted by AlphaFold394, the stereochemical rationality and energy stability of the model were verified using Ramachandran plots and ProSA-web. Molecular docking was performed using the ClusPro server: firstly, the protein structure was preprocessed with hydrogenation and energy minimization using the GROMACS software package in conjunction with the OPLS-AA force field to eliminate steric hindrance and optimize the side chain conformation. The IRP1-SRPX2 binary complex was generated using default parameters. Finally, key interaction features such as hydrogen bonds were visualized and analyzed using the PyMOL molecular graphics system (version 2.4.0, Schrödinger).

[0086] The expression sites of SRPX2 in the kidney were investigated using RNAscope technology. (1) Tissue collection: TBE (Avodin, 1.25% 2,2,2-tribromoethanol) was injected intraperitoneally at a dose of 0.02 mL / g body weight. After 5 min, the mouse paws were gently clamped with forceps. After the mouse showed no reaction, it was dissected. The constant flow pump was set to a flow rate of 5 mL / min, and the kidney tissue was perfused with pre-cooled 1×PBS for 4 min.

[0087] (2) Sectioning and preservation: The kidney tissue was placed in a glass dish containing a mixture of isopentane and dry ice, and frozen for 1 min. Then it was transferred to -80°C for OCT embedding. The tissue was then sectioned using a cryostat with a thickness of 20 μm. The sections were stored at -20°C for 1 h and then transferred to -80°C.

[0088] (3) Fresh 20μm frozen sections of mice were fixed in 4% paraformaldehyde (PFA) solution at 4°C for 1h, and then dehydrated in a gradient of 50%, 70%, 100%, and 100% ethanol for 5min each.

[0089] (4) After treating with hydrogen peroxide (H2O2) in a humidified chamber for 10 min, wash the slides twice with at least 200 mL of Milli-Q ultrapure water for 4 min each time, ensuring that all slides are submerged in water. Place the slides in the humidified chamber rack and add 100 µL of primary antibody diluted with Co-Detection Antibody Diluent to each slide (NPHS2-labeled glomeruli, SLC5A2-labeled proximal tubules, UMOD-labeled distal tubules, and Aquaporin-2-labeled collecting ducts), ensuring that there is enough solution to completely cover each slide. Incubate overnight at 4°C.

[0090] (5) After primary antibody incubation, wash the slides twice with PBST for 2 min each time, immerse in 4% PFA at room temperature for 30 min, and wash the slides four times with PBS-T for 2 min each time. Place the slides in a humidified chamber rack, add 2-4 drops of RNAscope protease IV to each slice, and incubate at room temperature for 30 min. Wash the slides twice with PBST for 2 min each time. (6) Remove excess liquid from the slides and add 40 µL of [unspecified solution] to the slides. Srpx2 Prepare the probe mixture to completely cover each slide. Close the tray and incubate in a hybridization oven at 40°C for 2 hours. Wash the slides twice at room temperature for 2 minutes each time with at least 200 mL of 1×WashBuffer.

[0091] (7) Then, in a hybridization oven at 40°C, the following steps were performed: AMP1 incubation for 30 min, AMP2 incubation for 30 min, AMP3 incubation for 30 min, HRP-C2 incubation for 30 min, opal570Reagent incubation for 30 min, and HRP blocking agent incubation for 15 min to amplify the signal. The slide was washed with 200 mL of 1×Wash Buffer between each step.

[0092] (8) Add the fluorescently coupled secondary antibody and DAPI diluted with Co-DetectionAntibodyDiluent to completely cover the slide. Incubate the slide in a dark box at room temperature for 30 min. Wash twice with fresh PBS-T for 2 min each time. Wash the slide twice at room temperature for 2 min each time. Finally, stain with DAPI for 2 min and mount the slide with mounting medium.

[0093] (9) The Zeiss (LSM700) laser confocal microscope was used to take pictures at a high magnification (63×), and the intensity and distribution of RNA probe signals, as well as colocalization with cell marker signals, were analyzed using Fiji software.

[0094] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A drug for treating renal fibrosis, characterized in that, Including SRPX2 inhibitors.

2. The medicament for treating renal fibrosis according to claim 1, characterized in that, The SRPX2 inhibitor is a nucleic acid inhibitor that targets SRPX2.

3. The medicament for treating renal fibrosis according to claim 2, characterized in that, The nucleic acid inhibitors targeting SRPX2 are siRNA, shRNA, or antisense oligonucleotides.

4. The use of an SRPX2 inhibitor as described in any one of claims 1-3 in the preparation of a medicament for treating renal fibrosis.

5. The application according to claim 4, characterized in that, The treatment for renal fibrosis includes inhibiting the binding of SRPX2 to IRP1, a key regulatory protein of iron metabolism, to restore iron homeostasis.

6. The application according to claim 4, characterized in that, The renal fibrosis includes chronic kidney disease, unilateral ureteral obstruction, or folic acid-induced renal fibrosis.

7. A kit for diagnosing renal fibrosis.

8. The reagent kit according to claim 7, characterized in that, The kit contains an antibody for detecting SRPX2 protein expression or primers for detecting SRPX2 gene expression.

9. A method for constructing a mouse model of renal fibrosis, characterized in that, Includes the following steps: A: Provide mice with specific knockout of the Srpx2 gene in renal tubular epithelial cells; B: The mice were subjected to unilateral ureteral obstruction treatment or folic acid induction treatment; C: Obtain a mouse model of renal fibrosis.

10. The model according to claim 9, characterized in that, The mice with Srpx2 gene knocked out specifically in renal tubular epithelial cells were obtained through the following method: Srpx2... flox / flox Mice were mated with Cdh16-cre mice, and the genotype Srpx2 was obtained through screening. flox / Y ;Cdh 16+ / Cre Male mice.

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