A medicament for treating renal fibrosis and use thereof

Abstract

The application discloses a medicine for treating renal fibrosis and application thereof, and relates to a medicine for treating renal fibrosis, which comprises an inhibitor capable of inhibiting epithelial mesenchymal transition of tubular epithelial cells HK-2 stimulated by TGF-beta 1; the application proves that a Kisspeptin analogue TAK-448 significantly improves epithelial mesenchymal transition of tubular epithelial cells HK-2 stimulated by TGF-beta 1, improves renal fibrosis of UUO and FA mice, so that the application has a wide clinical application prospect, has great potential in preparation of medicines for treating and / or improving renal fibrosis diseases, and has high industrial utilization value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a drug for treating renal fibrosis and its application. Background Technology

[0002] Chronic kidney disease (CHD) is a condition caused by various etiologies, resulting in persistent kidney damage or a decline in kidney filtration function, affecting 8%–16% of the global population. Renal fibrosis is considered the common pathway and primary pathological basis for the progression of almost all types of chronic kidney disease to end-stage renal disease, and its occurrence and development stem from multiple pathological processes. Because renal fibrosis is a complex process involving multiple factors and pathways, and there is a lack of specific and highly effective treatments in clinical practice, further research and development of effective drugs for treating renal fibrosis is of paramount clinical importance. Summary of the Invention

[0003] In view of the shortcomings of the prior art, the present invention proposes a drug for treating renal fibrosis and its application.

[0004] One drug for treating kidney fibrosis includes an inhibitor that can suppress the epithelial-mesenchymal transition of TGF-β1-stimulated renal tubular epithelial cells HK-2.

[0005] Preferably, the inhibitor includes the Kisspeptins analog TAK-448.

[0006] Preferably, the sequence of TAK-448 is WYPDTFGLQ.

[0007] Preferably, the chemical structural formula of TAK-448 is as follows:

[0008]

[0009] The above-mentioned inhibitors are used in the preparation of drugs for treating TGF-β1-induced epithelial-mesenchymal transition of renal tubular epithelial cells.

[0010] The above-mentioned inhibitors are used in the preparation of drugs for treating renal pathological damage and renal interstitial collagen fiber deposition caused by ureteral obstruction.

[0011] The above-mentioned inhibitors are used in the preparation of drugs for treating kidney pathological damage and renal interstitial collagen fiber deposition caused by folic acid injection.

[0012] The above-mentioned inhibitors are used in the preparation of drugs for treating renal fibrosis caused by ureteral obstruction.

[0013] The above-mentioned inhibitors are used in the preparation of drugs for treating folic acid injection-induced renal fibrosis.

[0014] This invention demonstrates that the Kisspeptin analog TAK-448 significantly improves the epithelial-mesenchymal transition of TGF-β1-stimulated renal tubular epithelial cells HK-2 and significantly improves renal fibrosis in UUO and FA mice. This gives the invention broad prospects for clinical application and great potential in the preparation of drugs for treating and / or improving renal fibrosis, and it has high industrial utilization value. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0016] Figure 1 Table A shows the results of Western blotting experiments on KISS1 and epithelial-mesenchymal transition markers (Fibronectin, α-SMA) in HK-2 cells after stimulation with different concentrations of TGF-β1 (0, 2.5, 5, 10, and 20 ng / μL) for 48 h. Table B shows the results of Western blotting experiments on KISS1 and epithelial-mesenchymal transition markers (Fibronectin, Collagen-I) in UUO mouse kidney tissue. Table C shows the statistical analysis of KISS1 protein expression results in Figure B. n=4, data are expressed as Mean±SDs, and Student's t-tests were used for analysis. ***: P<0.001. TGF-β1, transforming growth factor β1; UUO, unilateral ureteral obstruction; α-SMA, smooth muscle actin.

[0017] Figure 2 To detect the protein expression of epithelial-mesenchymal transition-related markers (fibronectin, E-cadherin, α-SMA) in HK-2 cells after treatment with different concentrations of KPA (0, 5, 10, and 20 nM) for 48 h using Western blotting assays. TGF-β1 (transforming growth factor β-1); α-SMA (α-smooth muscle actin); KPA (kisspeptin analogue, KP analogue, TAK-448).

[0018] Figure 3Image A shows the left kidney tissue photographed on day 7 after euthanasia of mice following UUO modeling; Image B shows the serum creatinine (Scr) level measured from blood collected from the orbital vein; Image C shows the serum blood urea nitrogen (BUN) level measured from blood collected from the orbital vein. n=3. Data are expressed as mean ± SDs. One-way ANOVA was used for comparisons between groups. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ns: not significant. UUO: unilateral ureteral obstruction; Scr: creatinine; BUN: blood urea nitrogen; KPA: Kisspeptin analog (KP analog, KPA) TAK-448.

[0019] Figure 4 Image A shows a representative image of Masson staining, scale bar = 100 μm or 50 μm; Image B shows a quantitative analysis of collagen area fraction in Image A, n = 4, data are expressed as Mean ± SDs, one-way ANOVA analysis was used for intergroup comparisons, ****: P < 0.0001, ns: not significant; Image C shows a representative image of HE staining showing pathological changes in the kidney tissue of mice in each group, scale bar = 100 μm or 50 μm; Image D shows a quantitative analysis of renal tubular injury score based on HE staining in Image C, n = 4, data are expressed as Mean ± SDs, one-way ANOVA analysis was used for intergroup comparisons, **: P < 0.01, ****: P < 0.0001, ns: not significant. UUO, unilateral ureteral obstruction; Masson staining; HE, hematoxylin-eosin; KPA, Kisspeptin analog (KP analog, KPA) TAK-448.

[0020] Figure 5Figure A shows representative immunohistochemical staining images of Collagen-I, Fibronectin, and α-SMA in the renal tubules and interstitium of mice in each group, scale bar = 100 μm; Figures B and D show quantitative analysis of protein levels of Collagen-I, Fibronectin, and α-SMA in the renal tubules and interstitium based on Figure A, n = 3, data are expressed as Mean ± SDs, one-way ANOVA analysis was used for comparisons between groups, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant; Figure E shows the levels of fibrotic proteins (Fibronectin, Vimentin, and α-SMA) in the kidney tissue of mice in each group as detected by Western blotting; Figure FH shows the statistical analysis of the results of Figure E, n = 4, data are expressed as Mean ± SDs, one-way ANOVA analysis was used for comparisons between groups. ANOVA analysis, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001, ns: not significant; IK is the expression level map of Fn1, Vim, and Acta2 mRNA in the kidney tissue of mice in each group detected by RT-qPCR. The results are counted as 4 technical replicates. Data are expressed as Mean ± SDs. One-way ANOVA analysis was used for comparison between groups, **: P<0.01, ***: P<0.001, ****: P<0.0001, ns: not significant. UUO, unilateral ureteral obstruction; α-SMA, α-smooth muscle actin; RT-qPCR, reverse transcription real-time quantitative polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KPA, Kisspeptin analog (KPanalog, KPA) TAK-448.

[0021] Figure 6 Image A shows the weight gain curve of mice after FA modeling, obtained by regular weighing (n=3). Data are expressed as Mean ± SDs. One-way ANOVA analysis was used for comparisons between groups. *: P < 0.05, ns: not significant. Image B shows the kidneys of mice euthanized 28 days after modeling. Image C shows the serum creatinine (Scr) measured from the orbital vein of mice. Image D shows the serum urea nitrogen (BUN) measured from the orbital vein of mice (n=3). Data are expressed as Mean ± SDs. One-way ANOVA analysis was used for comparisons between groups. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ns: not significant. FA: folic acid; BUN: blood urea nitrogen; Scr: serum creatinine; KPA: Kisspeptin analog (KP analog, KPA) TAK-448.

[0022] Figure 7 Figure A shows representative images of Masson staining, scale bar = 100 μm or 50 μm; Figure B shows quantitative analysis of collagen area fraction in Figure A, n = 3, data are expressed as Mean ± SDs, one-way ANOVA analysis was used for intergroup comparisons, *: P < 0.05, **: P < 0.01, ****: P < 0.0001, ns: not significant; Figure C shows representative images of renal tissue pathological damage in each group showing HE staining, scale bar = 100 μm or 50 μm; Figure D shows statistical analysis of renal tubular pathological damage scores based on Figure C, n = 3, data are expressed as Mean ± SDs, one-way ANOVA analysis was used for intergroup comparisons, *: P < 0.05, **: P < 0.01, ****: P < 0.0001, ns: not significant. FA, folic acid; Masson, Masson stain; HE, hematoxylin-eosin; KPA, Kisspeptin analog (KP analog, KPA) TAK-448.

[0023] Figure 8Figure A shows representative immunohistochemical staining images of Fibronectin, α-SMA, and Vimentin in the renal tubule interstitium of mice in each group, scale bar = 100 μm; Figures B and D show quantitative analysis of protein levels of Fibronectin, α-SMA, and Vimentin in the renal tubule interstitium based on Figure A, n = 3. Data are expressed as Mean ± SDs. One-way ANOVA analysis was used for comparisons between groups. *: P < 0.05, ***: P < 0.001, ****: P < 0.0001, ns: not The graph E represents the levels of fibrotic proteins (Fibronectin, E-cadherin, Vimentin, and α-SMA) in the kidney tissue of mice in each group as detected by Western blotting. FI represents the statistical analysis of the results of graph E, n=3. Data are expressed as Mean ± SD. One-way ANOVA analysis was used for comparisons between groups. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001, ns: not significant. JM represents the expression levels of Fn1, Col1a1, Vim, and Acta2 mRNA in the kidneys of FA mice detected by RT-qPCR, n=3. Data are expressed as Mean ± SD. One-way ANOVA analysis was used for comparisons between groups. ****: P < 0.0001, ns: not significant. FA, folic acid; α-SMA, α-smooth muscle actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KPA, Kisspeptin analog (KP analog, KPA) TAK-448. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] The modification process of TAK-448 is as follows: ① Amide reaction occurs on L-tryptophan, ② Acetylation occurs on the amino group of tyrosine, ③ Hydroxylation occurs on the 4R configuration of proline, ④ 1-azaglycosyl modification occurs on glycine, which usually means replacing a carbon atom with a nitrogen atom in the glycosyl structure and undergoing an acylation reaction, ⑤ Imino(methylamino)methylation occurs at the N5 position of ornithine, and the remaining amino acids are not specifically modified.

[0026] The sequence of TAK-448 is WYPDTFGLQ.

[0027] Example 1: KISS1's role in renal fibrosis

[0028] This embodiment verifies that KISS1 is highly expressed in renal tubular epithelial cells undergoing epithelial-mesenchymal transition and in fibrotic kidney tissue. Specifically, KISS1 expression is upregulated in TGF-β1-stimulated human proximal renal tubular epithelial cells (HK-2) and in the UUO renal fibrosis mouse model.

[0029] In this embodiment, an epithelial-mesenchymal transition (EMT) cell model was established in HK-2 cells by stimulating the cells with different concentrations of TGF-β1 (0, 2.5, 5, 10, and 20 ng / μL). The protein expression of EMT-related markers (Fibronectin, Collagen-I) and KISS1 before and after stimulation was detected by Western blotting. The experimental results are as follows: Figure 1 As shown in Figure A, the results showed that Fibronectin and Collagen-I were upregulated in TGF-β1-stimulated HK-2 cells, indicating that the cell model of epithelial-mesenchymal transition was successfully constructed. At the same time, as the concentration of TGF-β1 increased, the protein expression of KISS1 also gradually increased, suggesting that KISS1 protein may be involved in the epithelial-mesenchymal transition process of HK-2 cells.

[0030] In this embodiment, a mouse model of renal fibrosis (UUO: Unilateral Uretera Obstruction, renal interstitial fibrosis model) was further constructed through unilateral ureteral obstruction surgery. On the 7th day post-surgery, the protein expression of fibrosis-related markers (fibronectin, α-SMA) and KISS1 in the experimental group (UUO) and the control group (Sham) was detected by immunoblotting. The experimental results are as follows: Figure 1 As shown in BC. The results showed that Fibronectin and α-SMA were upregulated in the UUO animal model, indicating that the fibrosis animal model was successfully established. At the same time, the protein expression of KISS1 was also upregulated in UUO mice, suggesting that KISS1 protein may be involved in renal fibrosis in UUO mice.

[0031] These experimental results confirmed, both in vitro and in vivo, that KISS1 protein expression was increased in fibrotic kidney tissue, suggesting that KISS1 is involved in kidney fibrosis.

[0032] Example 2: Kisspeptin analog (KP analog, KPA) TAK-448 significantly improved the epithelial-mesenchymal transition of TGF-β1-stimulated renal tubular epithelial cells.

[0033] Example 1 has demonstrated that KISS1 participates in renal fibrosis at both the cellular and animal levels. This example further explores the alleviating effect of KPA on epithelial-mesenchymal transition (EMT) at the cellular level. TGF-β1-stimulated HK-2 cells were treated with different concentrations of KPA (0, 5, 10, and 20 nM) for 48 h. Western blotting was used to detect the protein expression of EMT-related markers (fibronectin, E-cadherin, α-SMA) in HK-2 cells after KPA treatment. The results showed (…). Figure 2 After TGF-β1 stimulation, the expression of Fibronectin, E-cadherin, and α-SMA proteins in HK-2 cells increased significantly, indicating that the fibrotic cell model was successfully constructed by TGF-β1 stimulation of HK-2 cells. After administration of different concentrations of KPA, the expression of proteins related to epithelial-mesenchymal transition (EMT) was significantly reduced in a concentration-dependent manner, suggesting that KPA can significantly inhibit TGF-β1-induced EMT in HK-2 renal tubular epithelial cells.

[0034] Example 3: Kisspeptin analog (KP analog, KPA) TAK-448 improves kidney morphology and renal function in UUO mice.

[0035] This embodiment further explores the effect of KPA on renal fibrosis. In Example 3, the effects of KPA on renal morphology and renal function were verified in a fibrotic UUO mouse model.

[0036] In this embodiment, in the UUO mouse model experiment, mice were randomly divided into 7 groups of 4 mice each: sham operation group, Sham+KPA group (Sham-KPA, KPA dosage of 0.6 mg / (kg·day)), model group (UUO), UUO+KPA low-dose group (UUO-KPA, KPA dosage of 0.15 mg / (kg·day)), UUO+KPA medium-dose group (UUO-KPA, KPA dosage of 0.3 mg / (kg·day)), UUO+KPA high-dose group (UUO-KPA, KPA dosage of 0.6 mg / (kg·day)), and positive control group, UUO+enalapril (Enalapril dosage of 10 mg / (kg·day)). KPA was dissolved in physiological saline and administered subcutaneously. Enalapril was dissolved in sodium carboxymethyl cellulose (CMC-Na) and administered by gavage. Administration began 24 hours after UUO surgery, once daily until 7 days post-surgery. Kidney tissue was harvested from mice 7 days later. After administration, mice were anesthetized, and blood was collected from the orbital rim for serum creatinine (Scr) and blood urea nitrogen (BUN) testing. Simultaneously, the left (obstructed) kidney was removed and photographed.

[0037] Mouse kidney tissue morphology ( Figure 3 A) showed that, compared with the Sham group, the UUO group exhibited ureteral dilation and severe kidney tissue damage, with urine squeezing the kidney tissue down to a thin layer of cortex, indicating successful modeling. Compared with the UUO group, the UUO + different concentrations of KPA groups showed less ureteral dilation and less kidney tissue compression than the corresponding UUO groups, suggesting that KPA can reduce postoperative kidney tissue damage after UUO and exert a renal protective effect. Furthermore, Scr( Figure 3 B) and BUN( Figure 3 C) The test results showed that compared with the Sham group, the Scr level in the UUO group was significantly increased, indicating that UUO modeling damaged kidney function. However, after administration of KPA 0.6 mg / (kg·day), the Scr and BUN levels were significantly reduced, indicating that KPA has a protective effect on kidney function.

[0038] Example 4: Kisspeptin analog (KP analog, KPA) TAK-448 significantly improved renal interstitial collagen fiber deposition and renal pathological damage in UUO mice.

[0039] In this embodiment, the effects of KPA on renal interstitial collagen fiber deposition and renal pathological damage were further examined in the constructed UUO mouse model using Masson staining and HE staining. Masson staining results showed that UUO mice had extensive collagen fiber deposition in the renal interstitium, and administration of different concentrations of KPA significantly improved collagen fiber deposition, suggesting that KPA can effectively alleviate collagen deposition in the renal tissue of UUO mice. Figure 4 (AB) and has a certain concentration dependence. The degree of tubulointerstitial injury stained with HE was judged by inflammation score, renal tubular dilatation score, and renal tubular cast score. The scoring criteria were: 0 points, normal kidney (no damage); 1 point, slight damage (≤10%); 2 points, moderate damage (11%~25%); 3 points, severe damage (26%~45%); 4 points, very severe damage (46%~75%); 5 points, large area damage (>75%). The results showed that KPA effectively reduced renal tubular epithelial cell atrophy, brush border loss, lumen dilatation and lumen collapse in a dose-dependent manner in the UUO mouse model. Figure 4 The above results suggest that KPA can significantly improve kidney pathological damage in UUO mice.

[0040] Example 5: Kisspeptin analog (KP analog, KPA) TAK-448 improves renal fibrosis in UUO mice.

[0041] This embodiment further investigated the ameliorative effect of KPA on renal fibrosis in UUO mice. In the UUO mouse model constructed above, this embodiment first detected the effect of KPA on the expression of fibrosis marker proteins (Collagen-I, Fibronectin, and α-SMA) by immunohistochemistry, then detected the effect of different concentrations of KPA on the expression of fibrosis marker proteins (Fibronectin, Vimentin, and α-SMA) by Western blotting, and detected the changes in the mRNA of fibrosis markers (Fn1, Vim, Acta2) by RT-qPCR. The results showed that the expression of fibrosis marker proteins in the kidney tissue of UUO mice (… Figure 5 AH) and mRNA levels ( Figure 5 IK) levels were significantly increased, while these indicators were significantly reduced after administration of different concentrations of KPA, suggesting that KPA can effectively alleviate renal fibrosis in UUO mice.

[0042] Example 6: Kisspeptin analog (KP analog, KPA) TAK-448 significantly improved renal morphology and renal function in FA mice.

[0043] In this embodiment, in the FA mouse model experiment, mice were randomly divided into 4 groups of 3 mice each: Saline group, Saline+KPA group (Saline-KPA, KPA dosage 0.6 mg / (kg·day)), model group (FA FA: 250 mg / kg), and FA+KPA group (FA-KPA FA: 250 mg / kg, KPA dosage 0.6 mg / (kg·day)). FA was dissolved in 300 mM sodium bicarbonate and administered via intraperitoneal injection, while KPA was dissolved in physiological saline and administered via subcutaneous injection. KPA administration began 18 days after FA model establishment, once daily for 10 days. After administration, the mice were anesthetized, and blood was collected from the orbital rim for serum creatinine (Scr) and blood urea nitrogen (BUN) testing. Simultaneously, the left and right kidneys were harvested, weighed, and photographed.

[0044] Mouse weight gain curve results ( Figure 6 A) This shows that after FA modeling, mice experienced significant weight loss, and administration of KPA improved this weight loss. Morphology of mouse kidney tissue ( Figure 6 (B) The results showed that, compared with the Saline group, the kidneys in the FA group were atrophied and scarred on the kidney surface, indicating successful modeling. Compared with the FA group, the kidneys in the FA+KPA group had better size, weight, and surface smoothness, and their morphology was closer to that of the Saline group, suggesting that KPA can reduce kidney tissue damage after FA modeling and exert a nephroprotective effect. Furthermore, Scr( Figure 6 C) and BUNFigure 6 D) The test results showed that, compared with the Saline group, the Scr and BUN levels in the FA group were significantly increased, suggesting that FA modeling impaired kidney function. However, after KPA administration, Scr and BUN levels significantly decreased, indicating that KPA has a protective effect on kidney function. The above experimental results confirm that KPA can significantly improve morphological changes and kidney function in FA mice.

[0045] Example 7: Kisspeptin analog (KP analog, KPA) TAK-448 improves renal interstitial collagen fiber deposition and renal pathological damage in FA mice.

[0046] In this embodiment, the effects of KPA on renal interstitial collagen fiber deposition and renal pathological damage were further examined in the constructed FA mouse model using Masson staining and HE staining. Masson staining ( Figure 7 AB and HE staining results (Figures CD) showed that KPA significantly improved collagen deposition and tubulointerstitial damage induced by high-dose FA. These results are consistent with those in the UUO mouse model, suggesting that KPA can significantly improve renal interstitial collagen fiber deposition and renal pathological damage caused by FA injury.

[0047] Example 8: Kisspeptin analog (KP analog, KPA) TAK-448 significantly improved renal fibrosis in FA mice.

[0048] This embodiment further investigated the ameliorative effect of KPA on renal fibrosis in FA mice. In the FA mouse model constructed above, this embodiment used immunohistochemistry to detect the effect of KPA on the expression of fibrosis marker proteins (Fibronectin, α-SMA, and Vimentin), Western blotting to detect the effect of KPA on the expression of fibrosis marker proteins (Fibronectin, E-cadherin, Vimentin, and α-SMA), and RT-qPCR to detect changes in the mRNA of fibrosis markers (Fn1, Collar, Vim, and Acta2). The results showed that the expression of fibrosis marker proteins in the kidney tissue of FA mice (Fn1, Collar, Vim, and Acta2) was significantly improved. Figure 8 AI) and mRNA Figure 8 The levels of JM were significantly increased, while these indicators were significantly downregulated after KPA treatment, suggesting that KPA significantly reduced the expression of fibrosis markers and alleviated renal fibrosis in FA mice.

[0049] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0050] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. The application of TAK-448 in the preparation of drugs for treating renal fibrosis, characterized in that, Inhibits TGF-β1-induced epithelial-mesenchymal transition in renal tubular epithelial cells; The chemical structural formula of TAK-448 is as follows: 。 2. The use of TAK-448 as described in claim 1 in the preparation of a drug for treating renal fibrosis caused by ureteral obstruction.

3. The application according to claim 2, characterized in that, It improves kidney pathological damage and the deposition of collagen fibers in the renal interstitium.

4. The use of TAK-448 as described in claim 1 in the preparation of a drug for treating renal fibrosis caused by folic acid injection.

5. The application according to claim 4, characterized in that, It improves kidney pathological damage and renal interstitial collagen fiber deposition caused by folic acid injection.

Images