A medicament for treating renal fibrosis

Abstract

The application discloses a medicine for treating renal fibrosis. It is found that kemajorone can inhibit the activation of renal fibroblasts and the production of extracellular matrix (ECM) by inhibiting the activity of KLF4 in the kidney, thereby slowing the renal fibrosis induced by unilateral ureteral ligation, and can be used as an effective clinical medicine for preventing and treating renal fibrosis.

Description

[0001] This application is a divisional application. The original application was entitled "Use of keparonone in the preparation of a drug for alleviating renal fibrosis induced by unilateral ureteral ligation"; application number 202510289128.2, application date March 12, 2025. Technical Field

[0002] This invention relates to a novel pharmaceutical use of Kenpaullone, specifically its use in the preparation of a drug for alleviating renal fibrosis caused by unilateral ureteral ligation. Background Art

[0003] Renal fibrosis is a major cause of progression in CKD (Chronic Kidney Disease) and a common outcome of various chronic kidney diseases. Its main characteristics include inflammatory cell infiltration, myofibroblast activation, and massive extracellular matrix deposition, ultimately leading to the destruction of normal kidney structure and the formation of scar tissue. The mechanisms of renal fibrosis development have long remained unclear. Recent studies have shown that the main source of extracellular matrix is ​​myofibroblasts in fibrotic kidneys. Further lineage tracing and single-cell sequencing technologies have demonstrated that activated fibroblasts are the primary source of myofibroblasts. Therefore, blocking fibroblast activation is key to preventing and treating renal fibrosis. Current clinical treatments for renal fibrosis are limited, primarily targeting the primary disease or symptoms, and kidney transplantation. However, problems remain, including a lack of specific targeted drugs, significant drug side effects, and uncertain efficacy. Therefore, developing new and effective drugs to improve renal fibrosis is of significant clinical importance.

[0004] KLF4 is a transcription factor with a CYS2-His2 zinc finger structure. As a major regulator of chromatin remodeling, it plays a crucial role in the reprogramming mixture of fibroblasts into induced pluripotent stem cells (iPSCs). KLF4 involvement can be observed in many diseases, particularly cancer. However, its role in the progression of chronic kidney disease remains unclear.

[0005] Kenpaullone, a KLF4 inhibitor, is a small molecule compound with the following structural formula:

[0006]

[0007] Currently, there are no publicly available publications reporting that Kenpaullone can inhibit the expression of KLF4 in renal fibroblasts under pathological conditions. Summary of the Invention

[0008] Purpose of the invention: This invention provides the use of Kenpaullone in the preparation of a medicament for alleviating renal fibrosis induced by unilateral ureteral ligation.

[0009] This application unexpectedly discovered a significant increase in KLF4 protein levels in the kidney tissue of mice with unilateral ureteral ligation-induced renal fibrosis, suggesting that intervention with KLF4 function using related small molecule compounds may improve unilateral ureteral ligation-induced renal fibrosis, and further experimentally verified the above hypothesis.

[0010] Specifically, this invention provides the use of the KLF4 protein inhibitor Kenpaullone in the preparation of drugs for renal fibrosis, solving the technical problem that there are no suitable drugs in the prior art for treating renal structural and functional damage caused by unilateral ureteral ligation.

[0011] Specifically, this application proposes that Kenpaullone inhibits the activity of transcription factor KLF4, thereby suppressing fibroblast activation and thus alleviating renal fibrosis induced by unilateral ureteral ligation.

[0012] This invention explores the potential use of Kenpaullone as a KLF4 inhibitor in alleviating symptoms associated with unilateral ureteral ligation-induced renal fibrosis in mouse models and in vitro cell models. Results showed that Kenpaullone intervention in both the unilateral ureteral ligation mouse model and in vitro cell models significantly improved renal structure and function in fibrotic kidneys and inhibited renal fibroblast activation. Our findings may provide an effective clinical drug for the prevention and treatment of renal fibrosis.

[0013] Specifically, kenparone improves kidney damage in renal fibrosis induced by unilateral ureteral ligation, and can effectively improve renal pathological damage such as renal tubular dilatation, renal tubular epithelial cell shedding, brush border atrophy, inflammatory cell infiltration, and renal tubular casts.

[0014] Meanwhile, Kenpaullone improves collagen fiber deposition in renal fibrosis induced by unilateral ureteral ligation. Specifically, Kenpaullone can significantly reduce the expression of FN, α-SMA, and Col-I in the renal interstitium of fibrotic kidneys.

[0015] Kemparone reduces TGFβ1-induced fibroblast activation. Attached Figure Description

[0016] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0017] Figure 1 The study showed that in a renal fibrosis model induced by unilateral ureteral ligation, the level of KLF4 protein in mouse kidney tissue was significantly increased.

[0018] Figure 2 Kenpaullone showed that it significantly improved renal damage in renal fibrosis induced by unilateral ureteral ligation;

[0019] Figure 3 Kenpaullone showed that in a renal fibrosis model induced by unilateral ureteral ligation, Kenpaullone downregulated the levels of FN, Col-I, and α-SMA proteins in renal tissue.

[0020] Figure 4 In a unilateral ureteral ligation-induced renal fibrosis model, Kenpaullone significantly inhibited the level of KLF4 protein in α-SMA+ myofibroblasts and the mRNA expression level of KLF4 in the kidney.

[0021] Figure 5 In an in vitro renal fibroblast model, Kenpaullone significantly reduced TGFβ1-induced fibroblast activation. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments. The embodiments will help to understand the present invention, but the scope of protection of the present invention is not limited to the following embodiments.

[0023] The materials and methods used in the following embodiments are as follows:

[0024] (I) Materials and reagents:

[0025] Normal rat renal fibroblasts (NRK-49F) were purchased from American Type Culture Collection; Kenpaullone was purchased from MCE (USA); TGFβ1 was purchased from R&D Systems; KLF4 antibody was purchased from Santa Cruz; α-SMA and Fibronectin antibodies were purchased from Sigma-Aldrich; Col-I antibody was purchased from Southern Biotech; GAPDH antibody was purchased from Proteintech; and fluorescent secondary antibody was purchased from Invitrogen.

[0026] (II) Cell Culture and Treatment:

[0027] Normal rat renal fibroblasts (NRK-49F) were cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin at 37°C, 5% carbon dioxide, and 95% air. To investigate the role of Kenpaullone in TGFβ1-induced activation of renal fibroblasts, when NRK-49F cells reached approximately 70% confluence, they were switched to DMEM medium without fetal bovine serum and starved for 10-12 hours. Then, Kenpaullone (dissolved in DMSO) was added for pretreatment for 30 minutes, or the same treatment with DMSO was performed, followed by the addition of TGFβ1. The cells were then incubated for another 24 hours. Finally, the cells were collected for Western blotting and immunofluorescence analysis.

[0028] (III) Mouse model of renal fibrosis induced by unilateral ureteral ligation and experimental grouping:

[0029] In a unilateral ureteral ligation-induced renal fibrosis model, 8-12 week old CD-1 mice were used and randomly divided into four groups of seven mice each: a unilateral ureteral ligation model group (UUO) and a unilateral ureteral ligation + Kenpaullone group (UUO + Kenpaullone). Kenpaullone was administered intraperitoneally at a dose of 5 mg / kg. Mice in the control group or Kenpaullone treatment group were given intraperitoneal injections of either the solvent or Kenpaullone every other day, starting one day before the UUO procedure. Seven days later, all mice in all groups were euthanized, and kidney specimens were collected. All experimental animals were managed and used in accordance with Chinese regulations on laboratory animal management.

[0030] (iv) Renal tubular injury and collagen fiber deposition score:

[0031] The degree of tubular damage was observed in the PAS pathological staining of the kidney, and the degree of collagen fiber deposition was observed in the Masson and Sirius red pathological staining. The ImageJ software was used to perform semi-quantitative scoring of collagen fiber deposition.

[0032] (V) Histological and immunofluorescence staining:

[0033] Kidney tissue was fixed with 4% paraformaldehyde, paraffin-embedded, and sectioned for histological and immunofluorescence staining. The specific procedures were as follows: paraffin-embedded sections were dewaxed to water, rinsed with distilled water, and rinsed three times with phosphate-buffered saline (PBS). Tris-EDTA alkaline antigen retrieval solution was used for microwave retrieval for 20 min, followed by natural cooling to room temperature. The sections were rinsed three times with PBS, blocked with Beyotime QuickBlock immunostaining blocking solution for one hour, discarded, and incubated overnight at 4°C with the appropriate primary antibody. After three PBS rinses, the appropriate secondary antibody was added, and the sections were incubated at room temperature for 1 h, followed by three PBS rinses. For immunohistochemistry, the subsequent steps were: adding DAB for staining at room temperature to the appropriate depth, counterstaining the cell nuclei with hematoxylin, rinsing with running water to invert blue, dehydrating and clearing, and finally mounting. For immunofluorescence, the subsequent steps were: mounting with a DAPI + anti-fluorescence quenching mounting medium, observing under a fluorescence microscope, and photographing the slides. For the corresponding primary antibody concentrations, the FN concentration was 1:200, and the Col-I concentration was 1:100. NRK-49F was fixed with paraformaldehyde and then subjected to immunofluorescence staining. The primary antibody concentrations for FN, α-SMA, etc., were all 1:200, and the secondary fluorescent antibody concentration was 1:200. DAPI staining was performed according to the labeling instructions.

[0034] (vi) Western blotting of proteins:

[0035] Total protein was extracted from kidney tissue using RIPA lysis buffer, and protein concentration was determined using the BCA method. 20 μg of protein was loaded onto a PVDF membrane and wet-transferred using 10% polyacrylamide gel electrophoresis (SDS-PAGE) at 400 mA for 2 hours. The membrane was blocked with 5% skim milk at room temperature for 2 hours, eluted with TBST, and then incubated with primary antibody at 4°C overnight. The membrane was washed three times with TBST for 15 minutes each time, incubated with the corresponding secondary antibody at room temperature for 2 hours, and then washed three more times. Antigen-antibody complexes were visualized using enhanced chemiluminescence (ECL) on X-ray film exposed and scanned in a darkroom. Protein quantification was performed by analyzing the grayscale values ​​of the target bands, with the relative expression level of the target protein expressed as the grayscale value of the target band / GAPDH grayscale value. Western blot results were analyzed using ImageJ software.

[0036] (vii) Statistical Analysis:

[0037] Data are expressed as mean ± SEM. A t-test was used to compare data between two groups. A p-value < 0.05 was considered statistically significant.

[0038] Example 1: Kenpaullone significantly improved kidney damage in renal fibrosis induced by unilateral ureteral ligation.

[0039] In a unilateral ureteral ligation-induced renal fibrosis model, the level of KLF4 protein in mouse renal tissue was significantly elevated. Figure 1 (A, 1B). To evaluate the protective effect of Kenpaullone in renal fibrosis, we induced chronic renal fibrosis using unilateral ureteral ligation. Glycogen staining (PAS) showed that 7 days after unilateral ureteral ligation, renal pathological damage, including renal tubular dilatation, tubular epithelial cell shedding, brush border atrophy, inflammatory cell infiltration, and renal tubular casts, was more severe. However, after Kenpaullone treatment, the corresponding renal damage significantly decreased. Figure 2 (A, 2B). These results indicate that Kenpaullone can protect against renal fibrosis induced by unilateral ureteral ligation, and that it itself has no significant toxic side effects.

[0040] Example 2: Kenpaullone significantly improved collagen fiber deposition in renal fibrosis induced by unilateral ureteral ligation.

[0041] Masson staining and Sirius red staining showed that 7 days after unilateral ureteral ligation, significant collagen fiber deposition was observed in the renal interstitium, while after Kenpaullone intervention, the deposition of collagen fibers in the renal interstitium was significantly reduced. Figure 2 (A, 2B). To further demonstrate the protective effect of Kenpaullone in renal fibrosis, we examined extracellular matrix-specific markers, FN and Col-I, and the myofibroblast-specific marker α-SMA. Figure 3 Immunofluorescence showed that after unilateral ureteral ligation, FN, α-SMA, and Col-I were highly expressed in the kidneys with renal fibrosis induced by unilateral ureteral ligation. Kenpaullone significantly reduced the expression of FN, α-SMA, and Col-I in the renal interstitium of fibrotic kidneys. Figure 3 A, 3B). Western blot also showed that in kidneys with renal fibrosis induced by unilateral ureteral ligation, the expression levels of FN, α-SMA, and Col-I were significantly decreased after Kenpaullone treatment. Figure 3 C). The above results indicate that Kenpaullone can improve renal fibrosis induced by unilateral ureteral ligation.

[0042] Example 3: In a renal fibrosis model induced by unilateral ureteral ligation, Kenpaullone significantly suppressed KLF4 protein levels.

[0043] We further explored the possible mechanism of action of Kenpaullone. Our literature review revealed that Kenpaullone specifically inhibits the expression of KLF4, a transcription factor with a zinc finger structure and a major regulator of chromatin remodeling, playing a crucial role in renal fibrosis. Our experimental results showed that Kenpaullone treatment significantly downregulated KLF4 protein expression in unilateral ureteral ligation-induced fibrotic kidneys. Figure 4 (A, 4B). Example 4: In an in vitro TGFβ1 model of kidney fibroblasts, Kenpaullone intervention improved TGFβ1-induced fibroblast activation.

[0044] Furthermore, we cultured normal rat kidney fibroblasts (NRK-49F) in vitro, pretreated them with Kenpaullone at concentrations of 0.1, 0.5, and 5 μM for 30 min, and then continued treatment with 2 ng / ml TGFβ1 for 24 h. Cells were then harvested and KLF4 levels and extracellular matrix production were detected by Western blotting. Figure 5 A) After TGFβ1 intervention, the levels of KLF4, the fibroblast activation marker α-SMA, and the production of extracellular matrix FN and Col-1 in fibroblasts were significantly increased. However, after Kenpaullone intervention, the expression levels of these proteins significantly decreased in a dose-dependent manner. Furthermore, using a Kenpaullone concentration of 0.5 μM, immunofluorescence showed that after TGFβ1 intervention, the production of fibroblast activation marker α-SMA and extracellular matrix FN significantly increased, while after Kenpaullone intervention, the expression levels of these proteins significantly decreased. Figure 5 B).

[0045] In summary, this invention provides the use of Kenpaullone, a transcription factor KLF4 inhibitor, in the preparation of a medicament for alleviating symptoms associated with unilateral ureteral ligation-induced renal fibrosis. The medicament is administered via intraperitoneal injection at a mouse dose of 5 mg / kg / day. It improves renal fibrosis by inhibiting the synthesis of KLF4 protein in fibroblasts, inhibiting fibroblast activation, and inhibiting the production of extracellular matrix.

[0046] This invention provides an approach and method for using kenparone in the preparation of a drug to alleviate renal fibrosis induced by unilateral ureteral ligation. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A medicament for treating renal fibrosis, characterized in that, The drug includes tamparone; the renal fibrosis is renal fibrosis induced by unilateral ureteral ligation; the drug is used to improve the deposition of collagen fibers in renal fibrosis induced by unilateral ureteral ligation; reduce the expression of FN, α-SMA, and Col-I in the renal interstitium of fibrotic kidneys; and reduce the activation of fibroblasts induced by TGFβ1.

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