Application of WIF1 in preparation of medicine for preventing and treating focal segmental glomerulosclerosis

Abstract

The invention belongs to the technical field of biological medicine, discloses application of WIF1 in preparation of a medicine for preventing and treating focal segmental glomerulosclerosis, and researches the relationship between the WIF1 and the occurrence and development of the focal segmental glomerulosclerosis by designing and constructing a mouse model in which a WIF1 gene is knocked out and a mouse model in which the WIF1 is overexpressed. Experimental results show that: knockout of the Wif1 gene can significantly aggravate damage of podocyte in focal segmental glomerulosclerosis and increase generation of proteinuria in focal segmental glomerulosclerosis; the overexpression of the Wif1 gene can significantly reduce the level of proteinuria in focal segmental glomerulosclerosis, significantly reduce the glomerular injury index in focal segmental glomerulosclerosis, and significantly improve podocyte injury. Therefore, the WIF1 has protection and improvement effects on the occurrence and development of the focal segmental glomerulosclerosis, and the WIF1 has a new application of preventing and treating the focal segmental glomerulosclerosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of WIF1 in the preparation of drugs for preventing and treating focal segmental glomerulosclerosis. Background Art

[0002] Focal segmental glomerular sclerosis (FSGS) is a pathological phenomenon in common clinical kidney diseases and a common cause of nephrotic syndrome and end-stage renal disease (ESRD) in adults and children. Currently, FSGS has been regarded as one of the common causes of nephrotic syndrome in adults and children, accounting for about 20% of nephrotic syndrome in children and 40% of nephrotic syndrome in adults, with an estimated incidence of seven per million. FSGS mainly occurs in glomeruli. The disease has a relatively insidious onset and complex etiology, which brings difficulties to early clinical intervention. Currently, there is a lack of particularly effective treatment means. Research shows that podocyte injury is the core link of FSGS, and the dysfunction and reduction of podocytes are related to the development of proteinuria and the progression of various diseases. The main features of FSGS are glomerular capillary loop occlusion, podocyte degeneration, increased extracellular matrix, renal interstitial fibrosis, segmental hyperplasia of endothelial cells and mesangial cells, etc., and the main clinical manifestations are hypertension, proteinuria, microscopic hematuria, and nephrotic syndrome, and finally develop into end-stage renal disease.

[0003] Combined with clinical history, laboratory results, renal biopsy, and gene detection results, etc., clinically, FSGS cases are divided into 4 categories, namely primary, secondary, hereditary, and idiopathic FSGS. Primary FSGS is generally considered to be induced by circulating permeability factors (cardiotrophin-like cytokine 1, soluble urokinase-type plasminogen activator receptor, anti-CD40 antibody, apolipoprotein A1, etc.), and these factors can cause sudden and extensive damage to podocytes. Secondary FSGS is a general term for FSGS lesions caused by various pathogenic factors, and can be subdivided into maladaptive FSGS, drug-induced FSGS, virus-induced FSGS, and FSGS damage superimposed on other glomerular diseases. Defects in podocyte and GBM proteins are considered to be the causes of hereditary FSGS. The main factors for the onset of FSGS are podocyte apoptosis, foot process fusion, and shedding. Podocytes have extremely poor proliferative ability. Research shows that the loss and destruction of podocytes in glomerular mesangium will show moderate hyperplasia of mesangial cells accompanied by persistent microproteinuria and glomerular capsule fibrous adhesion, and finally form focal segmental glomerulosclerosis. Therefore, podocyte dysfunction is the core factor in the occurrence of FSGS. Therefore, preventing and treating podocyte dysfunction is one of the treatment strategies for FSGS.

[0004] WNT inhibitory factor 1 (WIF1) belongs to the Wnt antagonist family. It directly binds to Wnt proteins, preventing Wnt from binding to the receptor protein complex, so that β-catenin in the cytoplasm cannot accumulate due to phosphorylation, thereby blocking both the canonical Wnt signaling pathway and the non-canonical Wnt signaling pathway. Multiple studies have shown that the activation of the Wnt / β-catenin signaling pathway can promote podocyte dysfunction in patients with FSGS, and upregulation of Wnt1 and active β-catenin expression has also been observed in the podocytes of FSGS patients. WIF1 is highly expressed in podocytes. Therefore, whether WIF1 is involved in the occurrence and development of FSGS and whether it can improve the disease process of FSGS by inhibiting the Wnt signaling pathway remain unclear.

[0005] Through the above analysis, the problems and defects existing in the prior art are as follows:

[0006] Whether WIF1 is involved in the occurrence and development of FSGS and whether it can improve the disease process of FSGS by inhibiting the Wnt signaling pathway remain unclear. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides the application of WIF1 in the preparation of drugs for preventing and treating FSGS.

[0008] The present invention is realized in the following way. A method for studying the relationship between WIF1 and the occurrence and development of focal segmental glomerulosclerosis (FSGS) includes the following steps:

[0009] 1. Construct a mouse model with the Wif1 gene knocked out to simulate the situation of WIF1 deficiency;

[0010] 2. Construct a mouse model with overexpression of the Wif1 gene to simulate the situation of WIF1 overexpression;

[0011] 3. Induce focal segmental glomerulosclerosis (FSGS) in the above two mouse models and monitor the development of the disease course;

[0012] 4. Compare and analyze the performance of the mouse models with the Wif-1 gene knocked out and overexpressed during the occurrence and development of FSGS to study the relationship between WIF1 and FSGS.

[0013] The present invention also provides a drug for preventing and treating focal segmental glomerulosclerosis (FSGS). The drug contains the WIF1 overexpression viral particle AAV-Wif1, and its working principle is as follows:

[0014] 1. Introduce the AAV-Wif1 viral particle containing the Wif1 gene into the patient's body by injection or other administration methods;

[0015] 2. The AAV-Wif1 virus particles selectively infect and integrate into kidney cells in patients;

[0016] 3. In kidney cells, AAV-Wif1 drives the expression of the Wif1 gene, thereby increasing the level of WIF1 protein in kidney cells;

[0017] 4. Through the biological effects of the WIF1 protein, such as inhibiting the Wnt signaling pathway, reducing kidney inflammation, promoting kidney cell repair and regeneration, etc., focal segmental glomerulosclerosis (FSGS) is prevented and treated.

[0018] The present invention also provides a method for using WIF1 in the preparation of a drug for preventing and treating FSGS, which includes the following steps:

[0019] By designing and constructing a mouse model with the Wif1 gene knocked out and a mouse model with overexpression of Wif1, the relationship between WIF1 and the occurrence and development of FSGS is studied.

[0020] Furthermore, the drug is the WIF1 overexpression virus particle AAV-Wif1.

[0021] Furthermore, the administration method of the WIF1 overexpression virus particle AAV-Wif1 is intravascular injection, intramuscular injection, in-situ tissue injection, and oral administration.

[0022] Furthermore, the drug is a drug that enhances the expression of the Wif1 gene and / or the WIF1 protein.

[0023] Furthermore, the drug is a drug that enhances the activity of the WIF1 protein.

[0024] Combined with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by the present invention are as follows:

[0025] First, the present invention adopts the following technical solutions:

[0026] By designing and constructing a mouse model with the Wif1 gene knocked out and a mouse model with overexpression of Wif1, the relationship between WIF1 and the occurrence and development of FSGS is studied. The experimental results show that:

[0027] (1) Knocking out the Wif1 gene: can significantly exacerbate the damage of podocytes in FSGS and increase the production of proteinuria in FSGS;

[0028] (2) Overexpressing the Wif1 gene: can significantly reduce the level of proteinuria in FSGS, significantly reduce the glomerular injury index in FSGS, and significantly improve podocyte injury.

[0029] It can be seen that WIF1 itself has a protective and improving effect on the occurrence and development of FSGS, and WIF1 has a new use in preventing and treating FSGS.

[0030] Through research, it is found that WIF1 can significantly improve the clinicopathological features of FSGS, without side effects, with remarkable therapeutic effects and no occurrence of complications, and can fundamentally improve the clinical symptoms of FSGS and prevent the occurrence of FSGS.

[0031] Second, the current treatments for FSGS mainly include:

[0032] 1. General treatment: Pay attention to rest, prevent infection, control diet and weight, and reduce protein intake;

[0033] 2. RAAS blockers: ACEI, ARB, mineralocorticoid receptor antagonists (MRA);

[0034] 3. Immunosuppressive drugs: Glucocorticoids and calcineurin inhibitors (CNIs);

[0035] 4. RAS inhibitors: Prostaglandin E

[0036] 4. Plasma exchange or immunoadsorption.

[0037] Different from traditional treatment methods, this scheme directly targets podocytes, uses adeno-associated virus as a vector, directly delivers the Wif1 gene to target cells, realizes the expression of WIF1 protein, inhibits the overactivation of the Wnt signaling pathway in podocytes of the kidneys in FSGS, and achieves the effects of treating and preventing FSGS.

[0038] Third, as the creative auxiliary evidence of the claims of the present invention, it is also reflected in the following important aspects:

[0039] (1) The expected benefits and commercial value after the transformation of the technical scheme of the present invention are:

[0040] After the clinical transformation of this scheme, it can provide a new treatment method for FSGS patients, provide precise targeted treatment for glomerular damage in the FSGS patient group, reduce the risk of kidney damage, save medical resources, and reduce medical expenses.

[0041] (2) The technical scheme of the present invention fills the technical gaps in the domestic and international industries:

[0042] There has been no report on the therapeutic effect of WIF1 in focal segmental glomerulosclerosis. Therefore, this technical scheme clarifies the role of WIF1 in focal segmental glomerulosclerosis; there is currently no research on the therapeutic effect of WIF1 in focal segmental glomerulosclerosis, and this scheme fills this gap.

[0043] (3)Whether the technical solution of the present invention solves the technical problems that people have been eager to solve but have never been successful in solving:

[0044] The application of WIF1 in improving focal segmental glomerulosclerosis has the following significant technological advancements:

[0045] 1. Targeted delivery: This solution uses adeno-associated virus as a vector to directly target and deliver the WIF1 plasmid to podocytes.

[0046] 2. Action target of WIF1: WIF1 is an effective action target for improving focal segmental glomerulosclerosis. By using the adeno-associated virus-targeted delivery strategy to preferentially deliver the WIF1 plasmid DNA to renal podocytes, the high-efficiency expression of WIF1 can be achieved, thereby exerting its therapeutic effect on focal segmental glomerulosclerosis.

[0047] 3. Gene expression regulation: By delivering the WIF1 plasmid DNA to renal podocytes through adeno-associated virus, the expression of WIF1 is achieved, regulating the downstream signaling pathway, thereby changing the pathological process of focal segmental glomerulosclerosis and achieving the therapeutic effect.

[0048] Generally speaking, through strategies such as the targeted delivery and gene expression regulation of WIF1, the precise treatment of focal segmental glomerulosclerosis is achieved, with good therapeutic effects and biological safety, bringing significant technological advancements to the field of focal segmental glomerulosclerosis treatment.

[0049] Fourth, the existing technical problems solved by the present invention and the significant technological advancements obtained

[0050] In the field of prevention and treatment of focal segmental glomerulosclerosis (FSGS), the existing treatment methods mainly focus on hormone and immunosuppressive drug treatments. However, these methods have limitations in delaying the progression of FSGS and improving renal function, and the treatment effects are not ideal enough. In addition, the research on the molecular mechanism of the occurrence and development of FSGS, especially the role of WIF1 (Wnt inhibitory factor 1) in FSGS and its regulatory mechanism, is not deep enough, which restricts the research and clinical application of FSGS treatment drugs targeting the WIF1 target.

[0051] The present invention deeply studied the role and regulatory mechanism of WIF1 in the occurrence and development of FSGS by constructing mouse models with Wif1 gene knockout and overexpression of Wif1 gene. Further, the present invention proposed to use WIF1 overexpression virus particles (AAV-Wif1) and WIF1 enhancers as drugs for the prevention and treatment of FSGS, and explored different administration methods and treatment effects. These innovative methods not only provide new ideas for the prevention and treatment of FSGS, but also lay a foundation for the research and clinical application of drugs targeting the WIF1 target.

[0052] 1) Deeply revealing the mechanism of action of WIF1 in FSGS: Through studies on mouse models, the present invention deeply reveals the role and regulatory mechanism of WIF1 in the occurrence and development of FSGS, providing a new theoretical basis for the prevention and treatment of FSGS.

[0053] 2) Innovative drug application methods: The present invention proposes the use of WIF1 overexpressing viral particles (AAV-Wif1) and WIF1 enhancers as drugs for the prevention and treatment of FSGS, and explores different administration methods. These innovative methods not only improve the therapeutic effect of the drugs, but also reduce side effects and safety issues.

[0054] 3) Providing a new target for drug development for FSGS: The present invention discovers that WIF1 is a potential therapeutic target for FSGS. Drug development targeting the WIF1 target will provide new strategies and methods for the prevention and treatment of FSGS.

[0055] 4) Promoting personalized treatment of FSGS: Based on the individual differences in WIF1 expression levels, the method of the present invention is expected to promote personalized treatment of FSGS, improving the therapeutic effect and the quality of life of patients.

[0056] By deeply studying the mechanism of action of WIF1 in FSGS, the present invention proposes innovative drug application methods, providing new strategies and methods for the prevention and treatment of FSGS and achieving significant technological progress. Description of the Drawings

[0057] Figure 1 is the expression change diagram provided by the embodiment of the present invention; wherein: (a) is the expression change diagram of Wif1 mRNA detected by Quantitative Real-time PCR (qRT-PCR) in the renal tissue of FSGS mice induced by Adriamycin (ADR); (b) is the expression change diagram of WIF1 protein detected by Immunofluorescence (IF) in the kidney tissue of FSGS mice induced by ADR.

[0058] Figure 2It is a pathological change diagram of the kidney tissue of FSGS mice after WIF1 knockout analyzed at the morphological level provided by the embodiments of the present invention; wherein: (a) Transmission electron microscopy was used to observe the changes in the ultrastructure of glomeruli in mouse kidney tissue; (b) Statistical chart of the number of podocytes in glomeruli in the transmission electron microscopy results; (c) Statistical chart of the width of the glomerular basement membrane in the transmission electron microscopy results; (d) PAS staining was used to analyze the morphological changes of glomeruli in mouse tissue; (e) Statistical chart of the glomerulosclerosis index according to the PAS staining pathological score; (f) Image J software was used for relative quantitative analysis of the percentage of mesangial matrix area in the glomerular area.

[0059] Figure 3 It is a diagram showing the change of UACR in FSGS mice after WIF1 knockout provided by the embodiments of the present invention.

[0060] Figure 4 It is a diagram showing the change of the expression of podocyte-related molecules in FSGS mice after WIF1 knockout provided by the embodiments of the present invention; wherein: (a) Diagram of the change in the transcriptional levels of Nphs1, Nphs2, and Podxl genes in isolated glomeruli detected by qRT-PCR; (b) Diagram of the change in the protein expression of NPHS1, NPHS2, PODXL, and Desmin in glomeruli detected by immunofluorescence.

[0061] Figure 5 It is a diagram showing the change of UACR in FSGS mice after overexpression of WIF1 provided by the embodiments of the present invention.

[0062] Figure 6 It is a pathological change diagram of the kidney tissue of FSGS mice after overexpression of WIF1 analyzed at the morphological level provided by the embodiments of the present invention: Statistical chart of the glomerulosclerosis index according to the PAS pathological score.

[0063] Figure 7 It is a diagram showing the change of the expression of podocyte-related molecules in FSGS mice after overexpression of WIF1 provided by the embodiments of the present invention. Among them: (a) Diagram of the change in the transcriptional levels of Wt1, Nphs1, Nphs2, and Podxl genes in isolated glomeruli detected by qRT-PCR; (b) Diagram of the change in the protein expression of NPHS1, NPHS2, and PODXL in glomeruli detected by immunofluorescence. Detailed implementation manners

[0064] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0065] Focal segmental glomerulosclerosis (FSGS) is a pathological phenomenon in common clinical kidney diseases, seriously endangering the health of patients. In recent years, studies have found that WIF1 (Wnt inhibitory factor 1) plays an important role in the occurrence and development of FSGS. Therefore, based on the research of WIF1, it provides new ideas for the prevention and treatment of FSGS.

[0066] 1. Model construction:

[0067] Mouse model with Wif1 gene knockout: To study the impact of WIF1 deficiency on FSGS, scientists designed and constructed a mouse model with Wif1 gene knockout. This model simulates the situation of WIF1 deficiency, which helps to observe and analyze the function of WIF1 in FSGS.

[0068] Mouse model with overexpression of Wif1: At the same time, to study the impact of WIF1 overexpression on FSGS, a mouse model with overexpression of Wif1 gene was also constructed. This model simulates the situation of WIF1 overexpression, which helps to evaluate the preventive and therapeutic effects of increasing the expression level of WIF1 on FSGS.

[0069] 2. Studying the relationship between WIF1 and FSGS:

[0070] By comparing the performance of mouse models with Wif1 gene knockout and overexpression during the occurrence and development of FSGS, scientists can deeply study the relationship between WIF1 and FSGS. This includes analyzing the role of WIF1 in the pathogenesis of FSGS, the impact of WIF1 expression on the course of FSGS, etc.

[0071] The drug provided in the embodiment of the present invention is the WIF1 overexpression viral particle AAV-Wif1. The working principle of this drug is as follows:

[0072] 1. Gene delivery: The AAV-Wif1 viral particle has the ability to deliver the Wif1 gene to target cells. When AAV-Wif1 is injected into the body, it can selectively infect and integrate into kidney cells.

[0073] 2. Gene expression: Once AAV-Wif1 integrates into the genome of kidney cells, it can drive the expression of the Wif-1 gene. This results in an increase in the level of WIF1 protein in kidney cells.

[0074] 3. Biological effects: The increased WIF1 protein can exert biological effects through various pathways, thereby preventing and treating FSGS. For example, WIF1 alleviates kidney inflammation, promotes the repair and regeneration of kidney cells, and improves the structure and function of the kidney by inhibiting the Wnt signaling pathway.

[0075] 4. Long-term treatment: Since AAV-Wif1 can stably integrate into the genome of kidney cells, it can achieve long-term and stable expression of WIF1 protein. This makes AAV-Wif1 a therapeutic drug for FSGS with long-term therapeutic effects.

[0076] In this invention, mouse models with knocked-out and overexpressed Wif1 genes were designed and constructed to deeply study the relationship between WIF1 and FSGS. Based on these studies, it was proposed to use the WIF1 overexpressing viral particle AAV-Wif1 as a drug for preventing and treating FSGS. This drug exerts biological effects by increasing the expression level of WIF1 protein in kidney cells, has long-term therapeutic effects and good application prospects.

[0077] The application of WIF1 in the preparation of drugs for preventing and treating FSGS provided by the embodiments of this invention includes the following steps:

[0078] By designing and constructing a mouse model with a knocked-out Wif1 gene and a mouse model with overexpressed Wif1, study the relationship between the occurrence and development of WIF1 and FSGS.

[0079] The drug provided by the embodiments of this invention is the WIF1 overexpressing viral particle AAV-Wif1.

[0080] The administration methods of the WIF1 overexpressing viral particle AAV-Wif1 provided by the embodiments of this invention are intravascular injection, intramuscular injection, in-situ tissue injection and oral administration.

[0081] The drug provided by the embodiments of this invention is a drug that enhances the expression of the Wif1 gene and / or WIF1 protein.

[0082] The drug provided by the embodiments of this invention is a drug that enhances the activity of WIF1 protein.

[0083] Example 1

[0084] 1. Construction of AAV-Wif1: First, construct a Wif1 overexpressing plasmid, and then package and concentrate the Wif1 plasmid, AAV vector, and helper plasmid to obtain AAV-Wif1;

[0085] 2. Targeted delivery: Inject AAV-Wif1 through the tail vein to target the delivery of Wif1;

[0086] 3. Gene expression regulation: The WIF1 plasmid DNA is released in kidney podocytes, expresses WIF1 protein, and regulates the downstream signaling pathway, thereby improving the pathological process of focal segmental glomerulosclerosis.

[0087] III. Some positive effects have been achieved during the R & D or use of the embodiments of the present invention, and there are indeed great advantages compared with the prior art. The following content will be described in combination with the data, charts, etc. of the experimental process.

[0088] 1. Experimental methods

[0089] 1.1 Experimental animals

[0090] (1) C57BL / 6J mice

[0091] The C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0092] (2) Wif1 loxP / loxP mice

[0093] Wif1 loxP / loxP mice were obtained by the inventor using the CRISPR Cas9 technology. The construction process is as follows:

[0094] On both sides of the 3rd exon of the Wif1 gene in vitro, a gene sequence containing loxP sites was inserted respectively. Subsequently, the constructed recombinant gene sequence was transferred into the embryonic stem cells of C57BL / 6J mice by electroporation technology. Then, the embryonic stem cells were re-implanted into the uterus of pseudopregnant mice to develop into a complete embryo and produce a transgenic mouse. Subsequently, the transgenic mouse was crossed with C57BL / 6J mice (the purpose of crossing is to remove the Neo transgene) to obtain Wif1 loxP / - mice. After that, the Wif1 loxP / - mice were backcrossed, and finally Wif1 loxP / loxP mice were produced.

[0095] (3) Nphs2-Cre mice

[0096] The Nphs2-Cre mice were purchased from Jackson Laboratory.

[0097] (4) Nphs2-Cre + / - / Wif1 loxP / loxP : Wif1 podocyte-specific knockout mice (KO), Nphs2-Cre - / - / Wif1 loxP / loxP : Littermate control mice (CTL), both were obtained by the inventor by crossing Wif1 loxP / loxP mice and Nphs2-Cre + / - mice. The obtaining process is as follows:

[0098] Let the Nphs2-Cre + / - mice be crossed with Wif1 loxP / loxPMice were crossed to obtain Nphs2-Cre + / - / Wif1 loxP / - mice. Subsequently, Nphs2-Cre + / - / Wif1 loxP / - was crossed with Wif1 loxP / loxP mice to obtain Nphs2-Cre + / - / Wif1 loxP / loxP : Wif1 podocyte-specific knockout mice (KO);

[0099] Nphs2-Cre + / - / Wif1 loxP / loxP : Wif1 podocyte-specific knockout mice (KO) were crossed with Wif1 loxP / loxP mice to obtain Nphs2-Cre + / - / Wif1 loxP / loxP : Wif1 podocyte-specific knockout mice (KO) and Nphs2-Cre - / - / Wif1 loxP / loxP : littermate control mice (CTL).

[0100] All mice were housed in a specific pathogen-free animal facility, maintained at an environmental temperature of 22 - 25°C and an environmental humidity of 40 - 60%, and subjected to a 12-hour light-dark cycle. All experiments and procedures were approved by the Animal Ethics Committee of Binzhou Medical University.

[0101] 1.2 Adriamycin (ADR)-induced FSGS

[0102] Twenty-four 8-week-old male BALB / c mice with similar body weights were selected and divided into 2 groups of 12 mice each. Urine was collected before dosing, and body weights were measured. One group was given ADR (10.2 mg / kg BW), and the other group was given an equal volume of normal saline. Before dosing, the mice were anesthetized with isoflurane gas using an anesthesia machine, and the drug was administered by retro-orbital venous injection. Urine was collected weekly after dosing, and urinary albumin was monitored by Coomassie Brilliant Blue staining. At 5 weeks, 24-hour urine was collected using a metabolic cage, and urinary albumin in each week was detected by ELISA. Samples were collected at 5 weeks, and transmission electron microscopy, histological staining, immunofluorescence staining, and qRT-PCR were performed.

[0103] 1.3 The overexpressing viral particle of WIF1, AAV-Wif1, was constructed using the following method

[0104] (I) Construction of plasmids

[0105] (1) Amplification of the full-length target gene of Wif1

[0106] Obtain the full-length coding sequence of the murine Wif1 gene from the NCBI database, with the gene number: NM_011915.2, and design the primer sequences for the target gene, where:

[0107] AAV-m-Wif1-B / K-F: gacctccatagaagacaccgggatccgccaccATGGCTCGGAGAAG; AAV-m-Wif1-B / K-R: ccttgtagtcgttaattaaggtaccCCAGATGTAATTGGATTCAG.

[0108] The design of this primer includes swapped pairing bases, BamHI restriction site, KpnI restriction site, expression enhancement sequence, and a partial sequence of the 5' end of the target gene.

[0109] Extract total RNA from mouse tissues and reverse transcribe it into cDNA. Using this cDNA as a template, after PCR amplification (PCR cycling parameters: 95°C, 5 min for 1 cycle; then 95°C, 30 s, 55°C, 30 s, 72°C, 60 s for 30 cycles, and again 72°C, 10 min for 1 cycle; finally 4°C, ∞ for 1 cycle), electrophorese and excise the gel to recover the murine Wif1 gene fragment.

[0110] (2) Construction and extraction of recombinant plasmids

[0111] First, use the restriction endonucleases BamHI and KpnI to perform double digestion on the vector pHBAAV-CMV-MCS-3flag-T2A-ZsGreen and the recovered murine Wif1 gene fragment. The final digestion system is a total of 50 μL, where: 10×CutSmart Buffer 5 μL, vector 10 μL, BamHI 1 μL, KpnI 1 μL, and the remaining system is made up with double-distilled water.

[0112] Next, mix the linearized vector DNA and the purified PCR product at a ratio of 1:2 (molar ratio), and use the HB infusion TM one-step cloning ligation system. Under the action of the HB infusion TM Master mix, react at 50°C for 30 min, then cool in an ice-water bath for 5 min, and then perform transformation. Add 10 μL of the above reaction product to 100 μL of DH5α competent cells, mix well, place on ice for 30 min, then heat shock at 42°C for 90 s, and then incubate on ice for 3 min and add 200 μL of LB medium. Shake and culture in a 37°C shaker for 1 h.

[0113] Finally, spread all the above bacterial solutions evenly on the plate containing ampicillin antibiotic and incubate in an incubator for 12 h.

[0114] (3) Identification of recombinant plasmid

[0115] Pick 5 single colonies from the plate and place them in 5 LB media containing ampicillin antibiotic respectively. Shake and culture the bacterial solutions on a shaker at 37 °C. After 12 h, take 1 mL of bacterial solution from each medium and send it to Sangon Biotech Co., Ltd. for sequencing to identify the accuracy of the recombinant plasmid. Preserve the remaining bacterial solutions with 30% glycerol and store them in a -80 °C refrigerator for future use.

[0116] (II) AAV-293 cell culture

[0117] (1) Resuscitation of AAV-293 cells:

[0118] ① Set the water bath temperature to 37 °C;

[0119] ② Take out the cryopreserved AAV-293 cells from the liquid nitrogen tank, quickly drop them into the water bath and shake rapidly to completely dissolve the cell solution within 1 min as much as possible;

[0120] ③ Transfer the cell solution to a 15 mL centrifuge tube, add 5 mL of fresh complete medium to the centrifuge tube, mix well and centrifuge at 1000 rpm for 5 min;

[0121] ④ Discard the supernatant, add 5 mL of fresh complete medium, mix the precipitate well and transfer it into a T225 culture flask. Add DMEM complete medium (DMEM + penicillin + streptomycin + 10% fetal bovine serum), and place the culture flask smoothly in an incubator at 37 °C and 5% CO2 for culture.

[0122] (2) Digest the above cells with 0.25% trypsin for 5 min, then remove the trypsin, add fresh complete medium and pipette to mix well. Then transfer them into a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min. Discard the supernatant and add 5 mL of complete medium to resuspend the precipitate.

[0123] (3) Take 50 μL of the mixed cells and place them in a 1.5 mL EP tube, add 450 μL of DMEM complete medium and mix well. Take 10 μL of the cells and count them on a counting plate. Passage the AAV-293 cells into a 100 mm culture dish (for transfection), and record the day of plating as the first day.

[0124] (4) Place the above culture dish in an incubator at 37 °C and 5% CO2 for culture. Observe the cell density after culturing for two days. When the culture dish is 80% - 90% full, transfection can be carried out.

[0125] (3) AAV Packaging and Concentration

[0126] (1) Plasmid Amplification

[0127] The constructed AAV vector, packaging plasmid, and helper plasmid (the vector plasmid carrying the target gene, pAAV-RC, and pHelper respectively) are extracted in large quantities using the Qiagen large-scale extraction kit. The concentration should be greater than 1 μg / μL, and the A260 / 280 should be between 1.7 and 1.8, which can be used for virus packaging.

[0128] (2) Replace the medium in the culture dish containing AAV-293 cells (when the cell density reaches 80% - 90% confluence in monolayer morphology), and then use LipofiterTM transfection reagent (purchased from Hanheng Biotechnology, and the usage instructions refer to the LipofiterTM instruction manual) for cell transfection.

[0129] (3) Mix LipofiterTM transfection reagent with DMEM medium without antibiotics and serum at a volume ratio of 1:25 (to obtain mixture 1). Mix the above AAV vector, packaging plasmid, and helper plasmid at a volume ratio of 1:2:1 (to obtain mixture 2). Mix mixture 1 and mixture 2 at a volume ratio of 1:1, and add the mixture dropwise to the pre-plated AAV-293 cells. Incubate at 37°C and 5% CO2. After 6 h, replace the medium with complete DMEM medium. Place the culture dish with the transfected plasmid in the incubator and continue to culture for 72 h.

[0130] (4) Harvesting AAV Virus: After 72 h of transfection, use a cell scraper to harvest the cells from a 150 mm culture dish, centrifuge at 150 g for 3 min to collect the cell pellet. Then resuspend the cell pellet in 300 μL of PBS. Freeze and thaw the centrifuge tube containing the cells three times in liquid nitrogen and 37°C water bath. Then centrifuge at 4°C at 2000 g for 5 min to remove cell debris, and collect the lysate supernatant containing AAV particles (crude virus extract).

[0131] (5) Purification of AAV:

[0132] ① Treatment with Benzonase Nuclease: Add 0.1 μL of Benonase enzyme to every 1 mL of crude virus extract, incubate in a 37°C water bath for 1 h to remove the cellular genome and residual plasmid DNA in the crude virus extract. Then centrifuge at 4°C at 600 g for 10 min and take the supernatant;

[0133] ② Column Purification: Purify the AAV virus using the Biomiga Adeno-Associated Virus Purification Kit V1469-01 according to the operation manual.

[0134] ③ Centrifugation: Add the AAV virus sample liquid (4 mL) obtained by column purification into an ultrafiltration tube, centrifuge at 1400 g for 30 min to obtain AAV virus (about 1 mL), collect the finally purified AAV virus, and store it at -80 °C.

[0135] (6) Determination of AAV virus packaging titer (using qRT-PCR method):

[0136] Take 3 μL of AAV virus, add 10 μL of DNAse, 41 μL of high-pressure water, and 6 μL of Dilution Buffer, mix well, incubate in a water bath at 37 °C for 1 h, then heat-treat in a metal bath at 100 °C for 10 min. After naturally cooling to room temperature, add 2 μL of proteinase K, incubate in a water bath at 55 °C for 1 h, then quickly heat-treat in a metal bath at 100 °C for 10 min. After naturally cooling to room temperature again, centrifuge to make the liquid on the tube wall converge at the bottom of the tube. After treatment, dilute 10-fold and use it for qRT-PCR to detect the titer.

[0137] 1.4 Systemic delivery of AAV-Wif1

[0138] Inject AAV-Wif1 via the tail vein 2 weeks after ADR injection, and collect samples 3 weeks after injection.

[0139] 1.5 UACR determination

[0140] Detect urinary albumin according to the instructions of the enzyme-linked immunosorbent assay (ELISA) kit, detect urinary creatinine with a creatinine kit, and calculate the urinary albumin / creatinine ratio (UACR).

[0141] 1.6 Histological staining

[0142] (I) Preparation of paraffin sections

[0143] (1) Fix the kidney tissue with PFA for 24 hours and rinse with tap water overnight.

[0144] (2) Tissue dehydration, clearing, and infiltration with wax:

[0145] Go through the cylinders in the following steps in sequence: 1) 75% ethanol, overnight treatment; 2) 85% ethanol, 3 h; 3) 95% ethanol I, 2 h; 4) 95% ethanol I, 1 h; 5) absolute ethanol I, 1 h; 6) absolute ethanol II, 1 h; 7) xylene I, 30 min; 8) xylene II, 30 min; 9) soft wax, 2 h; 10) hard wax, 1 h.

[0146] (3) Paraffin embedding of tissues and sectioning: Place solid paraffin in an embedding machine to fully dissolve it. Put the tissue in a metal embedding cassette, and pour the dissolved paraffin into the cassette after it is dissolved. Transfer the embedding cassette to a cooling table (pre-cooled by turning on the machine 2 hours in advance) and let it solidify overnight. Take out the solidified wax block, section it with a microtome, with a section thickness of 5 μm. Spread the sections in warm water, and use a soft brush to pick up the sections and place them on glass slides.

[0147] (4) Baking the slides: Incubate the tissues on the glass slides in an oven at 60 °C for 2 h.

[0148] (II) PAS staining

[0149] Go through the cylinders in the following steps in sequence: 1) Xylene I, 15 minutes; 2) Xylene II, 15 minutes; 3) Absolute ethanol I, 5 minutes; 4) Absolute ethanol II, 5 minutes; 5) 95% ethanol I, 5 minutes; 6) 95% ethanol II, 5 minutes; 7) 85% ethanol, 5 minutes; 8) 75% ethanol, 5 minutes; 9) Distilled water, 5 minutes; 10) Alcian blue, 10 - 20 seconds; 11) Distilled water, 2 minutes; 12) Oxidizing agent, 5 minutes; 13) Tap water, 1 minute; 14) Schiff Reagent, 10 - 20 minutes; 15) Running water, 10 minutes; 16) Hematoxylin, 1 - 2 minutes; 17) Tap water, 2 minutes; 18) Acidic differentiating solution, 2 - 5 seconds; 19) Tap water, 1 minute; 20) Scott bluing and counterstaining solution, 10 minutes; 21) Tap water, 3 minutes; 22) 85% ethanol, 5 minutes; 23) 95% ethanol I, 5 minutes; 24) 95% ethanol II, 5 minutes; 25) Absolute ethanol I, 5 minutes; 26) Absolute ethanol II, 5 minutes; 27) Xylene I, 5 minutes; 28) Xylene II, 5 minutes. Mount the sections with neutral balsam.

[0150] (III) After photographing the PAS - stained sections with an optical microscope, the photos are analyzed using Image J software to statistically calculate the percentage of the mesangial matrix area in the glomerular area (n = 4 for each group, and 5 fields of view are taken from each sample). At the same time, calculate the glomerulosclerosis index (GSI): The degree of glomerulosclerosis is divided into 4 grades: Grade 0: Basically normal or the glomerular sclerosis area < 25%; Grade 1: ~ 50%; Grade 2: ~ 75%; Grade 3: > 75%. Calculate the GSI to represent the degree of glomerulosclerosis based on the integral of the average sclerosis area of glomeruli per section. The formula is: GSI = (1×n1 + 2×n2 + 3×n3) / total number of glomeruli per section]×100% (n represents the number of diseased glomeruli).

[0151] 1.7 Immunofluorescence (IF)

[0152] (1) Kidney frozen sections: Take out the OCT-embedded kidney tissue from the -80°C refrigerator, place it in the -20°C refrigerator to equilibrate for 30 min - 1 h. Pre-cool the temperature of the cryostat chamber to -20°C in advance, and lower the temperature of the cryostat blade to -20°C before use. After trimming the tissue properly, adjust the section thickness to 5 μm, and use a glass slide to traction and adhere the frozen sections.

[0153] (2) Fixation: Place the sections in ice-cold methanol and fix them in the -20°C refrigerator for 20 min.

[0154] (3) Blocking: Wash the fixed sections with PBS to remove the surrounding OCT, draw a circle around the tissue with an immunohistochemistry pen to lock in the water, place them in a light-tight box, and add 60 μL of blocking solution (9 mL PBS + 1 mL FBS + 20 μL Tween-20). Block at room temperature for 1 h, taking care not to let the sections dry out.

[0155] (4) Antibody incubation: Dilute the primary antibody with the blocking solution according to the antibody instruction manual. Add no less than 50 μL of antibody dilution to each section and incubate overnight at 4°C. After incubation, discard the primary antibody, and wash the sections three times with 1×PBS for 10 min each time. Dilute the secondary antibody with the blocking solution at a certain ratio and incubate at room temperature for 1.5 - 2 h. After incubation, wash the sections three times with 1×PBS for 10 min each time. After washing, add 50 μL of DAPI to stain the nuclei, incubate at room temperature for 5 min, and wash the sections three times with 1×PBS for 10 min each time. Pay attention to avoiding light after incubating the secondary antibody.

[0156] (5) Mounting with mounting medium and photographing: Mount the sections with pre-prepared Mowiol, image with a laser confocal microscope, and analyze the results.

[0157] 1.8 Isolation of glomeruli (magnetic bead method)

[0158] (1) Pre-cool HBSS (calcium and magnesium containing) buffer in the 4°C refrigerator. Add 200 μL of mixed magnetic beads (Dynabeads TTM M-450 Tosylactivate-d) to every 20 mL of HBSS buffer, invert and mix well, place on ice for later use, and mix well again before use.

[0159] (2) Intraperitoneal injection of anesthetic drug in mice: 0.5% pentobarbital (9 μL / g). After complete anesthesia, disinfect the ventral skin with iodophor, open the thoracic and abdominal cavities with ophthalmic scissors, expose the heart, pull open the pericardium, and fully expose the right atrium.

[0160] (3) Withdraw the pre-mixed magnetic beads with a 20 mL syringe, connect a disposable intravenous infusion needle (0.55 mm), empty the air in the infusion needle, and place it on a semi-automatic intravenous infusion pump.

[0161] (4) Insert the tip of the infusion needle into the left ventricle of the mouse heart, pay attention to the depth and direction, cut open the right auricle with an auricle scissors to establish the systemic circulation, and turn on the infusion pump to start perfusion.

[0162] (5) During the perfusion process, it can be seen that the liquid flowing out of the right auricle gradually turns clear from red, the liver gradually changes from pink to yellowish-brown, and there is no edema in the lungs, indicating successful perfusion. After waiting for the end of the 20 mL magnetic bead perfusion, take the kidney.

[0163] (6) Rinse the kidney with sterile HBSS, remove the outer capsule of the kidney, and chop the kidney with a surgical blade.

[0164] (7) Place a disposable sieve with a 100 μm pore size on a 50 mL centrifuge tube, transfer the chopped kidney tissue into the sieve, pull out the syringe plunger of a 5 mL syringe to replace the grinding rod, fully grind it and then add 20 mL of pre-cooled HBSS buffer, and collect the filtrate in the centrifuge tube.

[0165] (8) Filter the above filtrate again through a sieve with a 100 μm pore size, and gently rinse with 15 mL of HBSS. Place the obtained filtrate in a centrifuge and centrifuge: 4 °C, 1770 rpm, 5 min. Retain the precipitate.

[0166] (9) Resuspend with 4 mL of HBSS buffer and transfer it to a sterilized 5 mL centrifuge tube. Place the centrifuge tube on a magnetic stand, carefully tilt the magnetic stand about 30°, let it stand for 2 min, and then carefully suck out the liquid from the opposite side of the magnetic pole using a Pasteur pipette.

[0167] (10) Take out the centrifuge tube from the magnetic stand, add 4 mL of pre-cooled HBSS and resuspend it again, and wash it repeatedly 3 times. Pay attention to operating on ice for the above steps. Finally, collect the glomeruli adsorbed on the magnetic stand for the next experiment (extracting glomerular RNA, extracting glomerular proteins).

[0168] 1.9 Real-time fluorescence quantitative PCR (qRT-PCR)

[0169] Place the glomeruli in Trizol (Ambion, USA) for homogenization to obtain total RNA. According to the instructions provided by the manufacturer (Takara, Japan), use a reverse transcription system kit to reverse transcribe the RNA, and then use a ThermoFisher QS3 PCR instrument to perform fluorescence quantitative PCR according to the instructions in the SYBR Green PCR Master Mix (ABI, USA) kit. The internal reference gene is β-actin, based on ΔCT = Ct gene -Ct β-actin Calculate the relative expression levels of the mRNA of each target gene, and the results are expressed as 2 -ΔCt values. The specific primer sequences are shown in the following table:

[0170]

[0171] 1.10 Data analysis

[0172] The experimental data were statistically analyzed using Prism 8 software. Unpaired t-tests were used for comparisons between two groups, and one-way analysis of variance (one-way ANOVA) was used for comparisons among multiple groups. The results were expressed as the mean ± standard error of the mean (Mean ± SEM). A P value < 0.05 was considered statistically significant.

[0173] 2. Experimental results

[0174] 2.1 Downregulation of WIF1 expression in glomeruli of FSGS mice

[0175] First, by searching the results of single-cell sequencing and transcriptome analysis in the database, it was found that WIF1 was highly expressed in glomerular podocytes. To explore the role of WIF1 in the development of FSGS, a mouse model of FSGS was constructed on BALB / c mice. After 5 weeks of modeling, the kidneys were collected, and glomeruli were isolated using magnetic beads to extract RNA. The qRT-PCR results showed that the gene transcription level of WIF1 in glomeruli of ADR-induced FSGS was significantly downregulated compared with the control group (P < 0.0001, n = 5)( Figure 1 a). The protein level of WIF1 was detected by IF, and the results showed that the protein expression of WIF1 in glomeruli of ADR-induced FSGS decreased compared with the control group( Figure 1 b).

[0176] 2.2 Podocyte-specific knockout of Wif1 exacerbates ADR-induced focal segmental glomerulosclerosis

[0177] To further explore the effect of WIF1 deficiency on focal segmental glomerulosclerosis, podocyte-specific knockout of Wif1 mice (Nphs2-Cre + / - / Wif1 loxP / loxP : podocyte-specific knockout mice of Wif-1 (KO) and Nphs2-Cre - / - / Wif1 loxP / loxP : littermate control mice (CTL)) were constructed using the Cre-Loxp system. FSGS mice (n = 10) were sampled after 5 weeks of modeling. The changes in glomerular ultrastructure between the two groups were analyzed by transmission electron microscopy. The results showed that compared with the CTL group, the foot processes of glomerular podocytes in the KO group were widened, fused, and missing, and the basement membrane was significantly thickened( Figure 2 a-c). The results of PAS staining showed that compared with the CTL group, the mesangial matrix in the KO group was significantly widened, and the degree of glomerular injury was obvious( Figure 2(d - e). Consistent with the histological results, the proteinuria level in the KO group of mice was significantly increased ( Figure 3 ). The podocyte - related molecules NPHS1, NPHS2, and PODXL were significantly decreased at both the mRNA and protein levels, and the expression level of Desmin protein was increased ( Figure 4 ). The above results indicate that knocking out Wif1 in podocytes exacerbates kidney injury in mice with focal segmental glomerulosclerosis.

[0178] 2.3 Systemic delivery of WIF1 alleviates ADR - induced focal segmental glomerulosclerosis

[0179] To verify that exogenous administration of WIF1 can be used as a treatment for ADR - induced kidney injury, AAV - Wif1 was designed and assembled and administered via the tail vein system to C57BL / 6J mice 2 weeks after ADR induction. At 5 weeks after ADR induction, the urinary albumin / creatinine ratio was measured and it was found that compared with the AAV - CTL group, the UACR in the AAV - Wif1 treatment group was significantly decreased (P < 0.01, n = 10)( Figure 5 ); The results of PAS staining showed less glomerular injury in the AAV - Wif1 treatment group ( Figure 6 ); Compared with the AAV - CTL group, the transcriptional levels of podocyte - related molecules Wt1, Nphs1, Nphs2, and Podxl were up - regulated in the AAV - Wif1 treatment group, and the protein levels of NPHS1, NPHS2, and PODXL were increased ( Figure 7 ).

[0180] In summary, overexpression of WIF1 can significantly reduce the proteinuria level in focal segmental glomerulosclerosis, improve the glomerular morphological damage in FSGS, and improve the podocyte dysfunction in FSGS. Therefore, formulating WIF1 into a medicament (such as: the WIF1 - overexpressing viral particle AAV - Wif1) and administering it in a suitable way (such as: intravascular injection, intramuscular injection, in - situ tissue injection, and oral administration) can significantly improve the pathological characteristics of FSGS, without side effects, with a significant therapeutic effect, no complications occurring, and can fundamentally improve FSGS and prevent the occurrence of FSGS.

[0181] The following are two specific application examples:

[0182] Example 1: Study on the preventive effect of WIF1 overexpression on FSGS

[0183] Step 1: Construction of mouse model

[0184] 1. Design and construct a mouse model with Wif1 gene knockout to simulate the situation of WIF1 deficiency.

[0185] 2. Design and construct a mouse model overexpressing the Wif1 gene to simulate the situation of WIF1 overexpression.

[0186] Step 2: Induce focal segmental glomerulosclerosis

[0187] 1. Use adriamycin (ADR) or other appropriate methods to induce focal segmental glomerulosclerosis in two groups of mice.

[0188] Step 3: Administer drugs

[0189] 1. For the mouse model overexpressing the Wif1 gene, no additional drug administration is required because they already overexpress WIF1.

[0190] 2. For the mouse model with the Wif1 gene knocked out, the WIF1 overexpression viral particle AAV-Wif1 can be selectively used for drug administration to study the preventive effect of exogenous WIF1 on FSGS.

[0191] Drug administration method: Intravascular injection or intramuscular injection.

[0192] Drug administration dose and frequency: Determine according to the experimental design and mouse body weight.

[0193] Step 4: Observation and evaluation

[0194] 1. Regularly detect the renal function indexes of mice, such as blood urea nitrogen (BUN), serum creatinine (Cr), etc.

[0195] 2. Conduct histopathological examinations to evaluate the changes in renal structure and function.

[0196] 3. Analyze the relationship between the WIF1 expression level and the occurrence and development of FSGS.

[0197] Step 5: Data analysis

[0198] 1. Statistically analyze the experimental results and compare the differences between the two groups of mice in terms of renal function, histopathology, etc.

[0199] 2. Evaluate the preventive effect of WIF1 overexpression on FSGS.

[0200] Example 2: Study the therapeutic effect of WIF1 enhancer on FSGS

[0201] Step 1: Mouse model construction

[0202] 1. Use the established mouse model of focal segmental glomerulosclerosis as the experimental group.

[0203] Step 2: Administer drugs

[0204] 1. Administer a WIF1 enhancer to the experimental group of mice, such as a drug that can enhance the expression of the WIF1 gene or the activity of the WIF1 protein.

[0205] Route of administration: Oral administration or in-situ tissue injection.

[0206] Dosage and frequency of administration: Determined according to the experimental design and the body weight of the mice.

[0207] Step 3: Observation and evaluation

[0208] 1. Regularly detect the renal function indexes of the mice.

[0209] 2. Conduct histopathological examinations to evaluate the changes in the structure and function of the kidneys.

[0210] 3. Analyze the therapeutic effect of the WIF1 enhancer on FSGS.

[0211] Step 4: Data analysis

[0212] 1. Statistically analyze the experimental results and compare the differences in renal function, histopathology, etc. of the mice before and after administration.

[0213] 2. Evaluate the therapeutic effect of the WIF1 enhancer on FSGS, as well as side effects and safety issues.

[0214] As described above, it is only a specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be covered by the protection scope of the present invention.

Claims

1. Application of WIF1 in the preparation of drugs for preventing and treating focal segmental glomerulosclerosis (FSGS).

2. The use of WIF according to claim 1 in the preparation of a medicament for preventing and treating focal segmental glomerulosclerosis (FSGS), characterized in that: The following steps are involved: By designing and constructing a mouse model with Wif1 knockout and a mouse model with Wif1 overexpression, the relationship between WIF1 and the occurrence and development of focal segmental glomerulosclerosis (FSGS) was studied.

3. The use of WIF according to claim 1 in the preparation of a medicament for preventing and treating focal segmental glomerulosclerosis (FSGS), characterized in that: The drug is WIF1 overexpression virus particle AAV-Wif1.

4. The use of WIF1 according to claim 3 in the preparation of a medicament for preventing and treating focal segmental glomerulosclerosis (FSGS), characterized in that: The administration method of the WIF1 overexpression virus particle AAV-Wif1 is intravascular injection, intramuscular injection, in situ tissue injection and oral administration.

5. The use of WIF1 according to claim 1 in the preparation of a medicament for preventing and treating focal segmental glomerulosclerosis (FSGS), characterized in that: The drug is a drug that enhances the expression of the Wif1 gene and / or the WIF1 protein.

6. The use of WIF1 according to claim 1 in the preparation of a medicament for preventing and treating focal segmental glomerulosclerosis (FSGS), characterized in that: The drug is one that enhances the activity of the WIF1 protein.

7. A method for studying the relationship between WIF1 and the occurrence and development of focal segmental glomerulosclerosis (FSGS) based on the application of claim 1, the method comprising the following steps: A mouse model with Wif1 gene knockout was constructed as a control group to simulate the condition of WIF1 deficiency; A mouse model overexpressing the Wif1 gene was constructed as an experimental group to simulate the condition of WIF1 overexpression; Focal segmental glomerulosclerosis (FSGS) was induced in the two mouse models and the disease course was monitored; To compare and analyze the performance of mouse models with Wif1 knockout and overexpression in the development of focal segmental glomerulosclerosis (FSGS) and to study the relationship between WIF1 and focal segmental glomerulosclerosis (FSGS).

8. A drug for preventing and treating focal segmental glomerulosclerosis (FSGS) based on the use of claim 1, the drug comprising WIF1 overexpression virus particles AAV-Wif1, and its working principle is: Introducing AAV-Wif1 virus particles containing the Wif1 gene into the patient by injection; AAV-Wif1 viral particles selectively infect and integrate into kidney cells in patients; In kidney cells, AAV-Wif1 drives the expression of the Wif1 gene, thereby increasing the level of WIF1 protein in kidney cells; Through the biological effects of WIF1 protein, including inhibiting the Wnt signaling pathway, reducing renal inflammatory response, and promoting renal cell repair and regeneration, focal segmental glomerulosclerosis (FSGS) can be prevented and treated.

9. A method for using a drug for preventing focal segmental glomerulosclerosis (FSGS), characterized in that: The method comprises the following steps: A mouse model with Wif1 gene knockout was constructed as a control group, and a mouse model with Wif1 gene overexpression was constructed as an experimental group; Focal segmental glomerulosclerosis (FSGS) was induced in two groups of mice using doxorubicin (ADR); For the mouse model with knockout of Wif1 gene, WIF1 overexpression virus particles AAV-Wif1 were used for administration, and the administration method was intravascular injection or intramuscular injection. The dosage and frequency of administration were determined according to the experimental design and mouse weight; The renal function indicators of mice, including blood urea nitrogen (BUN) and serum creatinine (Cr), were regularly tested, and histopathological examinations were performed to evaluate changes in renal structure and function; The experimental results were statistically analyzed, and the differences in renal function and histopathology between the two groups of mice were compared to evaluate the preventive effect of WIF1 overexpression on focal segmental glomerulosclerosis (FSGS).

10. A method for using a drug for treating focal segmental glomerulosclerosis (FSGS), characterized in that: The method comprises the following steps: An established FSGS mouse model was used as the experimental group; The mice in the experimental group were given a WIF1 enhancer, which can enhance the WIF1 gene expression or WIF1 protein activity. The administration method was oral administration or in situ tissue injection. The dosage and frequency of administration were determined according to the experimental design and the weight of the mice. The renal function indexes of mice were tested regularly, and histopathological examinations were performed to evaluate the changes in renal structure and function; The experimental results were statistically analyzed, and the differences in renal function and histopathology of mice before and after administration were compared to evaluate the therapeutic effect of WIF1 enhancer on FSGS, and consider its side effects and safety issues.

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