Application of ubiquitin specific peptidase 13 inhibitor in preparation of medicine for treating chronic kidney diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a ubiquitin specific peptidase 13 inhibitor in preparation of a medicine for treating chronic kidney diseases. According to the present invention, as the acknowledged ubiquitin specific peptidase 13 inhibitor, the results of the in vivo unilateral ureteral ligation animal experiment and the in vitro TGF-beta 1 induced cell experiment show that the Spautin-1 can be used as the ubiquitin specific peptidase 13 inhibitor, the Spautin-1 can be used for alleviating UUO induced renal interstitial fibrosis, alleviating in-vitro TGF-beta1 induced renal fibroblast activation and regulating and controlling the cell cycle process of the TGF-beta1 induced renal fibroblast. The Spautin-1 has the advantages that the UUO induced renal interstitial fibrosis can be alleviated, and the in-vitro TGF-beta1 induced renal fibroblast activation can be alleviated; therefore, the Spautin-1 can relieve the CKD-related diseases and can be used for preparing the medicine for treating the CKD and the related diseases. The invention provides a new medicine source for relieving and treating CKD, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to the application of a ubiquitin-specific peptidase 13 inhibitor in the preparation of a drug for treating chronic kidney disease. Background Technology

[0002] Chronic kidney disease (CKD) is a serious global public health problem, with its incidence and mortality rates rising year by year. Multiple epidemiological studies show that although the incidence of CKD varies across different regions and countries, the global overall incidence is approximately 10%, and about 2.6 million people die annually from CKD and its cardiovascular complications. Currently, the pathogenesis of CKD is not fully understood, and effective clinical interventions are still lacking. This leads some CKD patients to eventually develop end-stage renal disease (ESRD), requiring expensive renal replacement therapy or kidney transplantation, placing a heavy burden on families and society. In the development and progression of CKD, damage to renal tubular epithelial cells and activation of renal interstitial fibroblasts are important pathological factors. Fibroblasts are mesenchymal cells normally present in the renal interstitium, maintaining the integrity of kidney structure by producing basal levels of extracellular matrix (ECM). However, after damage to renal tubular epithelial cells, inflammatory cell infiltration and the release of inflammatory factors activate fibroblasts, leading to fibroblast proliferation, transformation into myofibroblasts, and ECM production, resulting in the continuous progression of renal interstitial fibrosis. Therefore, effectively inhibiting the proliferation and activation of renal interstitial fibroblasts and finding effective intervention targets may be key to slowing the progression of CKD and blocking renal interstitial fibrosis.

[0003] Ubiquitin-specific proteases (USPs) are the largest class of deubiquitinating enzymes, mainly composed of a core catalytic domain and other functional domains such as the ubiquitin-associated domain (UBA), ubiquitin-associated motif (UIM), and zinc finger domain (ZNF). USP13 is a member of the USP family; its UBA domain can bind ubiquitin molecules and catalyze the hydrolysis of polyubiquitinated chains. Studies have shown that USP13 participates in various physiological processes, including cell cycle regulation, DNA damage repair, myoblast differentiation, autophagy, and endoplasmic reticulum quality control. Research indicates that USP13 plays a crucial role in neurodegenerative diseases by regulating autophagy, and inhibiting USP13 can alleviate the progression of neurodegenerative diseases. In viral infections, USP13 inhibits DNA virus-triggered signal transduction by deubiquitinizing STING. Furthermore, USP13 can promote the development of spontaneous pulmonary fibrosis by stabilizing Smad4 through deubiquitination. Current research on USP13 mainly focuses on the field of oncology. USP13 can promote the proliferation of tumor cells, and it plays the role of a tumor suppressor gene in bladder cancer and oral squamous cell carcinoma.

[0004] Spautin-1 directly binds to and inhibits the enzymatic activity of USP13, preventing it from performing deubiquitination. This ultimately leads to the target protein being over-ubiquitinated and subsequently recognized and degraded by the cell's proteasome system. USP13, as the primary target of Spautin-1, is a core step in achieving its function. Currently, Spautin-1 is primarily used as a tool compound in basic scientific research to explore the function of autophagy and cancer treatment strategies in cell and animal models; it has not yet become an approved clinical drug. The discovery of Spautin-1 in basic research on chronic kidney disease will drive its development in the field of clinical treatment, laying the foundation for the development of subsequent drugs. Summary of the Invention

[0005] The purpose of this invention is to provide the application of a ubiquitin-specific peptidase 13 inhibitor in the preparation of a drug for treating chronic kidney disease, so as to achieve effective treatment of chronic kidney disease.

[0006] Therefore, the present invention provides the following technical solution.

[0007] The first aspect of the present invention provides the use of a ubiquitin-specific peptidase 13 inhibitor as a target for the treatment of chronic kidney disease in the preparation of a medicament.

[0008] In a preferred embodiment of the present invention, the ubiquitin-specific peptidase 13 inhibitor is Spautin-1.

[0009] In a preferred embodiment of the present invention, the pathological features of the chronic kidney disease include renal interstitial fibrosis, renal tubular dilatation, and renal tubular epithelial cell phenotypic transformation.

[0010] A second aspect of the present invention provides the use of a ubiquitin-specific peptidase 13 inhibitor in the preparation of a medicament for treating chronic kidney disease.

[0011] In a preferred embodiment of the present invention, the ubiquitin-specific peptidase 13 inhibitor is Spautin-1.

[0012] In a preferred embodiment of the present invention, the pathological features of the chronic kidney disease include renal interstitial fibrosis, renal tubular dilatation, and renal tubular epithelial cell phenotypic transformation.

[0013] In a preferred embodiment of the invention, the drug further includes a pharmaceutically acceptable carrier or excipient.

[0014] In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier or excipient includes at least one of solvents, polymers, liposomes, recombinant viral vectors, and eukaryotic recombinant expression vectors.

[0015] In a preferred embodiment of the present invention, the route of administration of the drug includes intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, or transdermal administration.

[0016] In a preferred embodiment of the present invention, the dosage form of the drug is tablets, granules, pills, powders, capsules, or liquids.

[0017] By employing the above technical solution, the present invention has at least the following advantages: Spautin-1, a recognized ubiquitin-specific peptidase 13 inhibitor, has been shown in in vivo unilateral ureteral ligation animal experiments and in vitro TGF-β1-induced cell experiments to alleviate UUO-induced renal interstitial fibrosis, reduce in vitro TGF-β1-induced renal fibroblast activation, and regulate the cell cycle progression of TGF-β1-induced renal fibroblasts. This indicates that Spautin-1 can alleviate CKD-related symptoms and can be used in the preparation of drugs for the treatment of CKD and related conditions. This invention provides a new drug source for the relief and treatment of CKD and has promising clinical application prospects.

[0018] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0019] Figure 1 To detect changes in fibrosis markers after Spautin-1 intervention in a UUO mouse model; where A: Western blot results; B: RT-qPCR results; C: Masson staining results.

[0020] Figure 2 To detect changes in fibrosis markers in renal fibroblasts stimulated by TGF-β1 using Western blot.

[0021] Figure 3 Western blot analysis was performed to detect changes in USP13 expression in UUO and 5 / 6Nx-induced mouse CKD models: A: Western blot results; B: Western blot results.

[0022] Figure 4 To detect changes in fibrosis markers in UUO and 5 / 6Nx-induced mouse CKD models; where A: Western blot results; B: Masson staining results; C: Immunohistochemical staining results.

[0023] Figure 5 To detect changes in activation markers of TGF-β1-stimulated renal fibroblasts; where A: Western blot results; B: RT-qPCR results. Detailed Implementation

[0024] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0025] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0026] In this document, the terms “contains,” “includes,” “containing,” and similar terms encompass the meanings of “basically composed of” and “composed of.” For example, when this document discloses “A contains B and C,” “A is basically composed of B and C” and “A is composed of B and C” should be considered as having been disclosed in this document.

[0027] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0028] In this document, when describing embodiments or examples, it should be understood that it is not intended to limit the invention to those embodiments or examples. Rather, all alternatives, modifications, and equivalents of the methods and materials described herein are covered within the scope defined by the claims.

[0029] For the sake of brevity, not all possible combinations of the technical features in each embodiment or example are described herein. Therefore, as long as the combinations of these technical features are not contradictory, the technical features in each embodiment or example can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification. In this invention, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components comprising the formulation and / or the mammals treated with it. Preferably, "pharmaceutically acceptable" as used herein means approved by federal regulatory agencies or national governments, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, particularly humans.

[0030] In this invention, the term "pharmaceutically acceptable carrier" includes any solvent, pharmaceutical stabilizer, or combination thereof, all of which are known to those skilled in the art. It covers the use of any conventional carrier in therapeutic or pharmaceutical compositions, except in cases where any conventional carrier is incompatible with the active ingredient.

[0031] In this invention, the term "pharmaceuticalally acceptable excipient" may include any solvent suitable for a particular target dosage form. The use of any conventional excipients, except those incompatible with the exosomes disclosed herein, for example, for any adverse biological effects or harmful interactions with any other component of a pharmaceutically acceptable composition, is also within the scope of this invention.

[0032] In this invention, the terms "treatment" and "relief" both refer to the attainment of desired pharmacological and / or physiological effects. These effects may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of symptoms in susceptible individuals who have not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need. The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0033] The following embodiments involve and mention: 1. Mouse source and feeding The C57BL / 6 mice used in this embodiment were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and were housed in an SPF-grade barrier environment at the Experimental Animal Center of Nanjing Medical University. They had free access to water, were fed standard rodent food, and maintained a constant temperature and humidity with a 12 / 12h circadian rhythm.

[0034] 2. Cell Culture and Processing Mouse kidney fibroblasts (NRK-49F) were cultured in DMEM medium containing 10% fetal bovine serum, 0.5% penicillin, and streptomycin at 37°C with 5% carbon dioxide and 95% air. When NRK-49F cells reached 60-70% confluency, they were transfected with small interfering RNA and then treated with TGF-β1 (CA59, purchased from Novoprotein) for 24 h. Cells were then collected for subsequent experiments.

[0035] 3. Transient cell transfection Grouping: siNC and siRNA. First, discard the cell supernatant from the plated cells and add 600 mL of basal culture medium to each well. For each group, prepare 200 mL / well basal culture medium + 5 mL / well Lipo2000 and add to a centrifuge tube, gently vortex and incubate for 5 min. Then, prepare 200 mL / well basal culture medium + 3 mL / well siNC (or siRNA) and add to the centrifuge tube, vortex and incubate for 10 min. Add 400 mL of the prepared transfection solution to each group, mix gently, and incubate at 37°C.

[0036] 4. Western blot Cell and tissue samples were added with an appropriate amount of protein lysis buffer, centrifuged at 12,000 rpm for 15 min in a pre-cooled centrifuge at 4°C, and the supernatant was collected. The protein concentration was determined using a BCA kit, and 5×SDS buffer was added in proportion. After mixing, the mixture was boiled in a metal bath at 100°C for 5 min. A 10% polyacrylamide gel was prepared using the YARN PAGE gel rapid preparation kit. After loading the sample, the gel was kept at a constant voltage of 80V for 30 min and then at 120V for 1 h until the bromophenol blue indicator reached the bottom of the separating gel. The gel was wet-transferred at a constant current of 30 mA for 1.5 h to a PVDF membrane. The PVDF membrane was blocked in 5% skim milk for 1 h and then incubated with primary antibody overnight at 4°C on a shaker: Fibronectin (FN1, Abcam, ab268020), USP13 (proteintech, 16840-1-AP), Collagen I (Bioss, bs-10423R), GAPDH (Proteintech, 60004-1-Ig)). Afterwards, the cells were incubated with secondary antibodies (HRP-labeled rabbit secondary antibody (Beyotime, A0208) and HRP-labeled mouse secondary antibody (Beyotime, A0216)) for 1 hour, and then developed using ECL chemiluminescence solution on a gel imaging system. Semi-quantitative analysis of protein bands was performed using ImageJ.

[0037] 5. Real-time quantitative PCR (RT-qPCR) An appropriate amount of TRIzol was added to the tissue sample to extract total RNA. After determining the RNA concentration, it was reverse transcribed into cDNA. RT-qPCR was performed using the Roche SYBR Green method. The composition of the 10 μL reaction system and primer sequence information are shown in Tables 1 and 2 below, and the reaction program is shown in Table 3 below. Table 1 Reaction System Table 2 Primer sequences Table 3 Reaction Procedure 6. Masson staining Tissues were fixed in 4% paraformaldehyde and routinely dehydrated and embedded. Sections were dewaxed to 4 μm and rinsed with distilled water. Sections were then mordanted with 1× mordant and dried in a 60°C oven for 1 h, followed by three 3-min rinses with distilled water. Azurite blue staining solution was applied for 2 min, followed by two 10-s rinses with distilled water. Hematoxylin staining solution was applied for 2 min, followed by two 10-s rinses with distilled water. Differentiation was performed with acidic differentiation solution for a few seconds, followed by a rinse with distilled water to terminate differentiation, and then rinsed with tap water for 10 min to restore blue color. Ponceau and fuchsin staining solution were applied for 5 min, followed by two 10-s rinses with distilled water. Differentiation was then performed with phosphomolybdic acid solution for 5 min. The supernatant was discarded, and sections were stained directly with aniline blue staining solution for 5 min without rinsing. After rinsing with weak acid working solution to remove the aniline blue solution, weak acid working solution was applied again to cover the sections for 2 min. Sections were then rapidly dehydrated with 95% ethanol for 3-5 s, followed by two 10-s rinses with anhydrous ethanol. S, xylene clearing twice, 2 min each time; neutral resin mounting.

[0038] 7. Immunohistochemistry (IHC) Paraffin sections were dewaxed to water: xylene dewaxing for 10 min × 2 times, followed by gradient alcohol hydration (anhydrous ethanol 3 min → 95% ethanol 3 min → 85% ethanol 3 min → 75% ethanol 3 min), soaking in double-distilled water, and washing with PBS for 3 min × 3 times. Heat retrieval: sections were immersed in diluted 1× sodium citrate antigen retrieval solution, boiled at medium-high temperature → heated at medium-low temperature for 20 min (do not dry the sections) → cooled to room temperature, and then washed with PBS for 3 min × 3 times. Endogenous peroxidase was added, and incubated at room temperature for 20 min. After incubation, sections were washed with PBS for 3 min × 3 times. After washing, normal goat serum blocking solution was added, and incubation was carried out at room temperature for 60 min. After incubation, the blocking solution was discarded, and no PBS washing was required. Then, diluted primary antibody (Fibronectin (FN1, Abcam, ab268020)) was added, and incubated overnight at 4°C. After rewarming, sections were washed with PBS for 5 min × 3 times, and then enzyme-labeled goat anti-mouse / rabbit IgG polymer was added and incubated at room temperature for 20 min. After incubation, rinse with PBS for 5 min × 3 times, then add reaction enhancement solution (Zhongshan Jinqiao) and incubate at room temperature for 20 min. After incubation, rinse with PBS for 5 min × 3 times, then add diaminobenzidine (DAB) solution (freshly prepared for use) and observe the color development under a microscope (approximately 1 min). Rinse with tap water for 20 min, stain with hematoxylin for 2 min, rinse with tap water, dehydrate with graded alcohols, clear with xylene, and mount with neutral resin.

[0039] Example 1: Effects of Spautin-1 drug intervention on renal interstitial fibrosis in UUO-induced CKD mice Establishment of unilateral ureteral obstruction (UUO) mouse model: After anesthesia with isoflurane, the abdominal cavity of mice was exposed, and the left ureter was ligated before the abdominal cavity was closed to establish a unilateral ureteral obstruction mouse model, namely the UUO model mouse.

[0040] Mice were divided into four groups: Vehicle+Sham group, Vehicle+UUO group, Spautin-1+Sham group, and Spautin-1+UUO group, with 6 mice in each group. The mice in each group were then treated as follows: Spautin-1+UUO group: mice were pretreated with Spautin-1 by intraperitoneal injection at a dose of 10 mg / kg for 1 day. Then, UUO model mice were constructed according to the above method, and Spautin-1 was administered intraperitoneally at a dose of 10 mg / kg for 7 consecutive days. Spautin-1+Sham group: Spautin-1 (CAS No.: 1262888-28-7, purchased from MCE, the same below) was administered to mice via intraperitoneal injection at 10 mg / kg for 1 day as a pretreatment, followed by intraperitoneal injection of Spautin-1 at 10 mg / kg for 7 consecutive days. Vehicle+UUO group: Mice were pretreated with the same dose of physiological saline intraperitoneally for 1 day, and then UUO model mice were constructed according to the above method. Then, the same dose of physiological saline was injected intraperitoneally for 7 consecutive days. Vehicle+Sham group: Mice were pretreated with the same dose of saline intraperitoneally for 1 day, and then administered the same dose of saline intraperitoneally for 7 consecutive days.

[0041] Mice in each group were sacrificed on day 8, and the expression levels of relevant proteins and genes in each group were analyzed. The experimental results are shown below. Figure 1 .like Figure 1 As shown: Compared with the Vehicle group, the protein expression level of the fibrosis marker FN was significantly downregulated in the Spautin-1 intervention group, and the mRNA levels of FN, ACTA2, and POSTN were also significantly decreased, with statistically significant differences. Figure 1 AB); Masson staining results also showed that the fibrotic area in the Spautin-1 group was significantly reduced, and the difference was statistically significant. Figure 1 C).

[0042] Example 2: Effects of Spautin-1 drug intervention on TGF-β1-induced renal fibroblasts Kidney fibroblasts were cultured in vitro and divided into four groups: Vehicle+Ctrl group, Vehicle+TGF-β1 group, Spautin-1+Ctrl group, and Spautin-1+TGF-β1 group. The cells in each group were then treated as follows: Spautin-1+TGF-β1 group: Spautin-1 drug was added at a concentration of 5 μM to kidney fibroblasts with a density of 70% for 12 h of pretreatment, followed by treatment with Spautin-1 (5 μM) + TGF-β1 (5 ng / μL) for 24 h. Spautin-1+Ctrl group: Spautin-1 drug was added at a concentration of 5 μM to kidney fibroblasts with a density of 70% for 12 h of pretreatment, followed by treatment with Spautin-1 (5 μM) for 24 h. Vehicle+TGF-β1 group: After the renal fibroblasts grew to 70% density, they were treated with TGF-β1 (5 ng / μL) for 24 h; Vehicle+Ctrl group: After the kidney fibroblasts grew to 70% density, they were starved for 24 hours in basal culture medium.

[0043] After culture, the FN protein level in each group of cells was detected, and the experimental results are shown in [the table below]. Figure 2 .like Figure 2 As shown: Compared with the Ctrl group, the Spautin-1 intervention group showed a significant decrease in the protein level of the fibrosis marker FN, and the difference was statistically significant. Figure 2 A).

[0044] Example 3: Changes in USP13 expression in kidney tissues of UUO and 5 / 6Nx-induced CKD mice Construction of UUO model mice: The construction steps are the same as in Example 1.

[0045] Construction of a 5 / 6 nephrectomy (5 / 6Nx) mouse model: After anesthesia with isoflurane, the abdominal cavity of mice was exposed, and a total right nephrectomy and a 2 / 3 left nephrectomy were performed. The abdominal cavity was then closed to construct a 5 / 6 nephrectomy mouse model.

[0046] The mice to be tested were divided into four groups: WT+Sham group and WT+UUO group; WT+Sham group and WT+5 / 6Nx group; with 6 mice in each group. The mice in each group were then treated as follows: WT+Sham group: After the mice were opened, no other procedures were performed, and the abdominal cavity was closed; WT+UUO group: Mice were used to construct a UUO model according to the method described in Example 1 above; WT+Sham group: After the mice were opened, no other procedures were performed, and the abdominal cavity was closed; WT+5 / 6Nx group: The 5 / 6Nx model was constructed in mice according to the above method.

[0047] The expression level of USP13 protein in each group of mice was then measured, and the results are shown in the table below. Figure 3 .like Figure 3 As shown: Compared with the Sham group, the expression level of USP13 protein in the UUO group and the 5 / 6Nx group was significantly reduced, and the difference was statistically significant. Figure 3 AB).

[0048] Example 4: Effect of USP13 knockdown on renal interstitial fibrosis in UUO-induced CKD mice The experimental mice used in this embodiment include: wild-type mice and systemic knockout USP13 heterozygous mice.

[0049] The mice were first divided into four groups: WT+Sham group, WT+UUO group, HET+Sham group, and HET+UUO group, with 7 mice in each group. The mice in each group were then treated as follows: WT+Sham group: Wild-type mice underwent abdominal surgery, with no other procedures performed, and the abdominal cavity was closed. WT+UUO group: Wild-type mice were used to construct the UUO model according to the method described in Example 1 above; HET+Sham group: After the laparotomy of the systemically knocked-out USP13 heterozygous mice, no other procedures were performed, and the abdominal cavity was closed. HET+UUO group: UUO model was constructed in systemically knocked-out USP13 heterozygous mice according to the method described in Example 1 above.

[0050] The changes in fibrosis markers in each group of mice were then statistically analyzed, and the experimental results are shown below. Figure 4 .like Figure 4 As shown: In the UUO-induced mouse CKD model, compared with the Sham group, the protein expression levels of fibrosis markers FN and Col-I in the UUO group were significantly different. Figure 4 A); Masson and IHC staining results showed that the degree of fibrosis in the HET+UUO group was significantly reduced compared with the WT+UUO group, and the difference was statistically significant. Figure 4 B).

[0051] Example 5: Effect of USP13 knockdown on TGF-β1-induced renal fibroblasts In this embodiment, USP13 expression in kidney fibroblasts was knocked down by transfecting them with si-USP13. si-USP13 is a small interfering RNA (SRNA) with its target sequence shown in SEQ ID NO. 11: TGACTACTCACCATTAGAT. After transfection, it specifically knocked down USP13 expression and was purchased from Ribobio. si-NC was a control SRNA; transfection with si-NC had no effect on USP13 expression.

[0052] Cells were divided into four groups: si-NC+Ctrl, si-NC+TGF-β1, si-USP13+Ctrl, and si-USP13+TGF-β1. Each group of cells underwent the following treatments: si-USP13+TGF-β1 group: After kidney fibroblasts were transfected with si-USP13 for 24 h, the supernatant was discarded, and the cells were cultured in basic medium containing TGF-β1 (5 ng / μL) for 24 h. si-USP13+Ctrl group: Kidney fibroblasts were transfected with si-USP13 for 24 h, the supernatant was discarded, and the cells were cultured in basal medium for 24 h. si-NC+TGF-β1 group: After kidney fibroblasts were transfected with si-NC for 24 h, the supernatant was discarded, and the cells were cultured in basic medium containing TGF-β1 (5 ng / μL) for 24 h. si-NC+Ctrl group: Kidney fibroblasts were transfected with si-NC for 24 h, the supernatant was discarded, and the cells were cultured in basal medium for 24 h.

[0053] After the culture was completed, the changes in cell fibrosis indicators in each group were statistically analyzed. The experimental results are shown in […]. Figure 5 .like Figure 5 As shown: In the TGF-β1-induced renal fibroblast model, compared with the Ctrl group, the protein expression of fibrosis markers FN and α-SMA in the TGF-β1 group was significantly decreased, and the difference was statistically significant. Figure 5 A); FN, α-SMA, and Col-I expression were also significantly reduced at the mRNA level, and the differences were statistically significant. Figure 5 B).

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. Application of ubiquitin-specific peptidase 13 inhibitors as targets for the treatment of chronic kidney disease in drug preparation.

2. The application according to claim 1, characterized in that, The ubiquitin-specific peptidase 13 inhibitor is Spautin-1.

3. The application according to claim 1, characterized in that, The pathological features of the chronic kidney disease include renal interstitial fibrosis, renal tubular dilatation, and phenotypic transformation of renal tubular epithelial cells.

4. Application of ubiquitin-specific peptidase 13 inhibitors in the preparation of drugs for the treatment of chronic kidney disease.

5. The application according to claim 4, characterized in that, The ubiquitin-specific peptidase 13 inhibitor is Spautin-1.

6. The application according to claim 4, characterized in that, The pathological features of the chronic kidney disease include renal interstitial fibrosis, renal tubular dilatation, and phenotypic transformation of renal tubular epithelial cells.

7. The application according to claim 1 or 2, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

8. The application according to claim 7, characterized in that, The pharmaceutically acceptable carrier or excipient includes at least one of solvents, polymers, liposomes, recombinant viral vectors, and eukaryotic recombinant expression vectors.

9. The application according to claim 1 or 2, characterized in that, The routes of administration of the drug include intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, or transdermal administration.

10. The application according to claim 1 or 2, characterized in that, The dosage form of the drug is tablets, granules, pills, powders, capsules, or liquids.

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