Application of KDM5B in the treatment of renal fibrosis

Abstract

The present invention belongs to the field of biomedicine technology and specifically relates to the use of KDM5B in the treatment of renal fibrosis. The present invention has found that KDM5B expression is upregulated in fibrotic human renal tubular epithelial cells and in fibrotic mouse kidney tissue. Furthermore, inhibition of KDM5B expression or activity by KDM5B-specific small interfering RNA and its specific inhibitor, TK-129, can significantly reduce or alleviate renal fibrosis. Therefore, KDM5B can be used as a drug, drug target, or target gene in gene therapy for the prevention, alleviation, and / or treatment of renal fibrosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to application of KDM5B in the treatment of renal fibrosis. Background Art

[0002] Renal fibrosis is a chronic and progressive pathophysiological process characterized by damage to renal intrinsic cells caused by multiple pathogenic factors, regardless of the underlying etiology. Renal fibrosis, characterized by abnormal deposition of extracellular matrix (ECM), is a key pathological stage in the progression of renal function from normal to chronic kidney disease (CKD) and then to end-stage renal disease (ESRD). It is considered the best histological predictor of renal function decline and one of the major factors affecting the prognosis of renal transplant patients. Renal fibrosis affects half of adults over 70 years old and 10% of the world's population, placing a significant economic burden on society. However, there are currently no targeted drugs to treat renal fibrosis. Therefore, in-depth exploration of the pathogenesis and new targets of renal fibrosis, and the development of new drugs based on these targets, are of great research significance and clinical value.

[0003] Renal fibrosis is a complex pathophysiological process involving multiple factors and signaling pathways, and its pathogenesis remains largely unexplained. Studies have shown that epigenetic histone modifications significantly regulate TGFβ1-induced renal fibrosis and ECM gene expression, as well as downstream pro-fibrotic genes. Histone lysine demethylase 5B (KDM5B) specifically removes dimethyl and trimethyl groups (H3K4me2 / me3) from lysine 4 (K4) of histone 3 (H3), thereby inhibiting target gene expression. Aberrant expression of KDM5B is closely associated with the development and progression of various diseases. Evidence suggests that KDM5B is a key regulator of the H3K4 methylome during early mouse preimplantation embryonic development. KDM5B is also overexpressed, amplified, or mutated in many cancer types. In cancer cells, KDM5B regulates the expression of oncogenes and tumor suppressors by mediating H3K4 methylation levels. Furthermore, KDM5B has been found to bind to the promoter of activating transcription factor 3 (Atf3) and inhibit ATF3 expression by demethylating activated H3K4me2 / 3 modifications, thereby promoting heart failure. However, as a key molecule in epigenetic histone modification, the role of KDM5B in the progression of renal fibrosis has not been reported. Because histone modification is closely related to the pathogenesis of renal fibrosis, elucidating the key epigenetic regulatory role of KDM5B in the pathogenesis of abnormal renal fibrosis is expected to make it an important candidate target for intervention in renal fibrosis. Summary of the Invention

[0004] In view of the problems and shortcomings in the prior art, the present invention aims to provide an application of KDM5B in the treatment of renal fibrosis.

[0005] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a biomarker that can be used for auxiliary diagnosis of renal fibrosis. The biomarker is a KDM5B gene, KDM5B mRNA, KDM5B protein, or a peptide segment of a KDM5B protein.

[0007] Western Blot was used to detect the expression of KDM5B in human renal tubular epithelial cells (HK-2) that underwent fibrotic changes induced by TGFβ1. It was found that the expression level of KDM5B in HK-2 cells that underwent fibrotic changes induced by TGFβ1 was significantly higher than that in normal HK-2 cells, and the difference was statistically significant. In addition, Western Blot and immunohistochemistry staining detected that the expression level of KDM5B in the fibrotic kidney tissue of UUO (unilateral ureteral obstruction) mice was significantly higher than that in sham-operated mice, and the difference was statistically significant.

[0008] The second aspect of the present invention provides the use of KDM5B gene, KDM5B mRNA, KDM5B protein, or peptide segments of KDM5B protein as drug targets for screening drugs for preventing, alleviating, or / and treating renal fibrosis.

[0009] According to the above application, preferably, the renal fibrosis is renal fibrosis caused by various kidney diseases.

[0010] A third aspect of the present invention provides the use of a substance that inhibits the expression and / or function of the KDM5B gene, KDM5B mRNA, KDM5B protein, or a peptide fragment of the KDM5B protein in the preparation of a drug for preventing, alleviating, or / and treating renal fibrosis.

[0011] According to the above application, preferably, the substance that inhibits the expression and / or function of the KDM5B gene or KDM5B mRNA or KDM5B protein or a peptide segment of the KDM5B protein is an siRNA that specifically targets the KDM5B gene, an shRNA that specifically targets the KDM5B gene, an inhibitor of the KDM5B protein or an inhibitor of a peptide segment of the KDM5B protein.

[0012] According to the above application, preferably, the siRNA specifically targeting the KDM5B gene is siRNA 1, siRNA 2 or siRNA 3; wherein,

[0013] The nucleotide sequence of siRNA 1 is:

[0014] siRNA 1forward:5'-GGACUUAUUUCAGCUUAAUTT-3'(SEQ ID NO.1),

[0015] siRNA 1reverse: 5'-AUUAAGCCUGAAAUAAGUCCTT-3' (SEQ ID NO. 2);

[0016] The nucleotide sequence of siRNA 2 is:

[0017] siRNA 2forward: 5'-GACCUGGAGAGCUUUAATT-3' (SEQ ID NO.3),

[0018] siRNA 2reverse: 5'-UUAAAGCUCUCUCCAGGUCTT-3' (SEQ ID NO.4);

[0019] The nucleotide sequence of siRNA 3 is:

[0020] siRNA 3forward: 5'-GCCCAAGAGUCGAUCUAAATT-3' (SEQ ID NO.5),

[0021] siRNA 3reverse: 5'-UUUAGAUCGACUCUUGGGCTT-3' (SEQ ID NO. 6).

[0022] According to the above application, preferably, the inhibitor is TK-129.

[0023] According to the above application, preferably, the renal fibrosis is renal fibrosis caused by various kidney diseases.

[0024] A fourth aspect of the present invention provides a drug for preventing, alleviating or / and treating renal fibrosis, wherein the drug contains a substance that inhibits the expression and / or function of the KDM5B gene, KDM5B mRNA, KDM5B protein or a peptide segment of the KDM5B protein.

[0025] According to the above-mentioned drug, preferably, the substance that inhibits the expression and / or function of the KDM5B gene or KDM5B mRNA or KDM5B protein or a peptide segment of the KDM5B protein is an siRNA that specifically targets the KDM5B gene, an shRNA that specifically targets the KDM5B gene, an inhibitor of the KDM5B protein or an inhibitor of a peptide segment of the KDM5B protein.

[0026] According to the above-mentioned drug, preferably, the siRNA specifically targeting the KDM5B gene is siRNA 1, siRNA 2 or siRNA 3; wherein,

[0027] The nucleotide sequence of siRNA 1 is:

[0028] siRNA 1forward:5'-GGACUUAUUUCAGCUUAAUTT-3'(SEQ ID NO.1),

[0029] siRNA 1reverse: 5'-AUUAAGCCUGAAAUAAGUCCTT-3' (SEQ ID NO. 2);

[0030] The nucleotide sequence of siRNA 2 is:

[0031] siRNA 2forward: 5'-GACCUGGAGAGCUUUAATT-3' (SEQ ID NO.3),

[0032] siRNA 2reverse: 5'-UUAAAGCUCUCUCCAGGUCTT-3' (SEQ ID NO.4);

[0033] The nucleotide sequence of siRNA 3 is:

[0034] siRNA 3forward: 5'-GCCCAAGAGUCGAUCUAAATT-3' (SEQ ID NO.5),

[0035] siRNA 3reverse: 5'-UUUAGAUCGACUCUUGGGCTT-3' (SEQ ID NO. 6).

[0036] According to the above-mentioned drug, preferably, the inhibitor is TK-129.

[0037] According to the above-mentioned medicine, preferably, the medicine further contains a pharmaceutically acceptable carrier / excipient.

[0038] According to the above-mentioned medicine, preferably, the renal fibrosis is renal fibrosis caused by various kidney diseases.

[0039] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0040] (1) The present invention first discovered that KDM5B is upregulated in fibrotic human renal tubular epithelial cells and that KDM5B expression is upregulated in fibrotic mouse kidney tissue. Therefore, KDM5B can be used as an auxiliary diagnostic indicator for renal fibrosis. Furthermore, the receiver operating characteristic curve (AUC) for distinguishing mice with renal fibrosis from normal control mice using KDM5B reached 0.882, indicating that KDM5B has a high diagnostic value for the auxiliary diagnosis of renal fibrosis.

[0041] (2) The present invention uses KDM5B-specific small interfering RNA (si-KDM5B) and its specific inhibitor TK-129 to inhibit KDM5B expression or activity, which can significantly reduce and alleviate renal fibrosis. Therefore, KDM5B can be used as a drug, drug target or target gene in gene therapy for the prevention, alleviation and / or treatment of renal fibrosis, which can provide a new strategy for the prevention and treatment of renal fibrosis. At the same time, it will also provide a new direction for further research on the causes of renal fibrosis and corresponding prevention and treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Figure 2 is the Western Blot analysis result of FN1, α-SMA and KDM5B protein expression levels in human renal tubular epithelial cells HK-2 induced by TGFβ1; A is a representative Western Blot band image; B and D are the results of Western Blot quantitative analysis; GAPDH was used as the internal control, n=3, **p<0.01;

[0043] Figure 2 Figure 3 shows the results of Masson staining to detect renal fibrosis in UUO and Sham mice; A is a representative Masson staining image, scale bar is 100 μm; B is the statistical results of renal fibrosis area in UUO and Sham mice; n=6, ***p<0.001;

[0044] Figure 3 Figure 3 Western Blot analysis of the expression of fibrosis markers FN1 and α-SMA in kidney tissues of UUO and Sham mice; A is a representative Western Blot image; B and C are the results of Western Blot quantitative analysis; GAPDH was used as an internal control, n = 3, **p < 0.01, ***p < 0.001;

[0045] Figure 4 Figure 1: Immunohistochemical staining and Western blot analysis of KDM5B expression in renal tissues of UUO-induced fibrotic mice. A: Representative immunohistochemical staining images. B: Quantitative analysis of immunohistochemical staining, scale bar: 20 μm. C: Representative Western blot images. D: Quantitative Western blot analysis, using GAPDH as an internal control. n = 3, **p < 0.01, ***p < 0.001.

[0046] Figure 5The results of Masson staining to detect renal fibrosis in UUO and Sham mice; A is a representative image of Masson staining; B is the quantitative analysis result of Masson staining, scale bar is 100 μm, n=12, ***p<0.001;

[0047] Figure 6 Figure 2 shows the results of immunohistochemical staining to detect the expression of KDM5B in kidney tissues of UUO and Sham mice; A is a representative image of immunohistochemical staining; B is the quantitative analysis result of immunohistochemical staining, scale bar is 50 μm, n=12, ***p<0.001;

[0048] Figure 7 ROC curve for KDM5B diagnosis to distinguish UUO mice from Sham mice;

[0049] Figure 8 Figure 2 shows the results of Western Blot detection of KDM5B knockdown in HK-2 cells; A is a representative Western Blot image, and B is the quantitative analysis result of Western Blot; GAPDH was used as an internal control, n=3, ***p<0.001;

[0050] Figure 9 Figure 3: Western Blot analysis of the effect of KDM5B knockdown on TGFβ1-induced FN1 and α-SMA protein expression in HK-2 cells. A is a representative Western Blot band. B is the quantitative Western Blot analysis of KDM5B protein. C is the quantitative Western Blot analysis of FN1 protein. D is the quantitative Western Blot analysis of α-SMA protein. GAPDH was used as an internal control. n = 3, **p < 0.01, ***p < 0.001.

[0051] Figure 10 The figure shows the results of the CCK-8 experiment to detect the survival rate of HK-2 cells treated with TK-129 solutions at concentrations of 2.5μM, 5μM and 10μM for 24 hours;

[0052] Figure 11Figure 3: Western Blot analysis of the effect of TK-129 on TGFβ1-induced FN1 and α-SMA protein expression in HK-2 cells by inhibiting KDM5B activity. A is a representative Western Blot band. B is the quantitative Western Blot analysis of FN1 protein. C is the quantitative Western Blot analysis of α-SMA protein. GAPDH was used as an internal control. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001.

[0053] Figure 12 Figure 3 shows the results of RT-PCR and Western Blot detection of KDM5B overexpression in HK-2 cells; A is a schematic diagram of the KDM5B overexpression plasmid structure; B is the RT-PCR detection of KDM5B mRNA expression; C is a representative Western Blot band; D is the Western Blot quantitative analysis of KDM5B protein; GAPDH was used as an internal control, n = 3, **p < 0.01, ***p < 0.001;

[0054] Figure 13 Figure 2 shows the effect of KDM5B overexpression on the expression levels of FN1 and α-SMA proteins in HK-2 cells; A is a representative Western Blot band; B is the quantitative Western Blot analysis result of FN1 protein; C is the quantitative Western Blot analysis result of α-SMA protein; GAPDH was used as an internal control, n=3, *p<0.05, **p<0.01;

[0055] Figure 14 Figure 3: Western Blot analysis of KDM5B expression in renal tissues of renal tubular epithelial cell-specific KDM5B knockout mice. A is a schematic diagram of the structure of the KDM5B knockout adeno-associated virus AAV2 / 9-KDM5B-shRNA. B is a schematic diagram of in situ injection into mouse kidneys. C is a representative Western Blot band. D is the quantitative Western Blot analysis of KDM5B protein, using GAPDH as an internal control. n = 3, ***p < 0.001.

[0056] Figure 15 The results of Masson staining to detect the effect of tubular epithelial cell-specific KDM5B knockout on UUO-induced renal fibrosis in mice; A is a representative Masson staining image, scale bar is 100 μm; B is the statistical results of renal fibrosis area in mice in each experimental group; n = 3, ***p < 0.001;

[0057] Figure 16Figure 3: Immunofluorescence staining results of the effect of tubular epithelial cell-specific KDM5B knockout on the expression of FN1 and α-SMA in the renal tissue of mice induced by UUO; A is a representative FN1 immunofluorescence staining image, with a scale bar of 100 μm; B is the statistical results of FN1 protein immunofluorescence staining in the renal tissue of mice in each experimental group; C is a representative α-SMA immunofluorescence staining image, with a scale bar of 100 μm; D is the statistical results of α-SMA protein immunofluorescence staining in the renal tissue of mice in each experimental group; n=3, *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0058] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0059] The following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0060] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, components and / or combinations thereof.

[0061] The experimental methods in the following examples without specifying specific conditions were all based on conventional techniques in the art or the conditions recommended by the manufacturers; the reagents or instruments used without specifying the manufacturers were all conventional products that can be purchased commercially.

[0062] Example 1: Study on the expression level of KDM5B in TGFβ1-induced human renal tubular epithelial cells (HK-2)

[0063] Western Blot was used to detect the expression level of KDM5B in TGFβ1-induced HK-2 cells and normal HK-2 cells.

[0064] 1. Experimental cells and culture methods:

[0065] Human renal tubular epithelial cells (HK-2) were obtained from the Shanghai ATCC Cell Bank. Discard the old culture medium in the culture flask, add 2 mL of sterile PBS, shake gently, and discard. Add 2 mL of 0.25% trypsin preheated at 37°C and digest for 2-3 minutes. Gently shake the culture dish to detach the cells, and add 2 mL of fresh DMEM / F12 complete medium (10% FBS + 1% double antibody) to terminate the digestion. Collect the cells, centrifuge at 1000 rpm for 5 minutes at room temperature, discard the supernatant, resuspend the cells in fresh complete medium, add to the culture dish, and continue culturing in a 37°C, 5% CO2 incubator.

[0066] 2. Experimental methods:

[0067] HK-2 cells were collected, counted using a cell counting plate, and plated in 6-well plates at 1×10 5 The cells were divided into a control group and a TGFβ1 group. The next day, after the cell morphology had fully recovered, the TGFβ1 group was treated with a TGFβ1 solution (the solvent was citric acid, the concentration of the TGFβ1 solution was 10 μg / mL, and it needed to be diluted 1000-fold, with a final active concentration of 10 ng / mL). The control group was treated with the same dose of the control solvent (citric acid). After 24 hours, the cells were collected. Western blotting was used to detect changes in the protein expression of fibrosis markers fibronectin (FN1), α-SMA, and KDM5B in the control and TGFβ1 groups, respectively.

[0068] 3. Experimental results:

[0069] Western Blot was used to detect the expression changes of fibrosis markers FN1, α-SMA and KDM5B in human renal tubular epithelial cells HK-2 induced by TGFβ1. Figure 1 shown.

[0070] Depend on Figure 1 As shown, compared with the control group, the expression of fibrosis markers FN1 and α-SMA proteins in HK-2 cells treated with TGFβ1 was significantly increased, and the difference was statistically significant, indicating that TGFβ1 induces fibrotic changes in HK-2 cells. In addition, the expression of KDM5B was significantly increased in fibrotic HK-2 cells, and the difference was statistically significant.

[0071] Example 2: Study on the expression level of KDM5B in fibrotic kidney tissue of mice

[0072] Western Blot was used to detect the expression level of KDM5B in the kidney tissue of mice with renal fibrosis.

[0073] 1. Selection of experimental animals and feeding conditions:

[0074] Eight-week-old SPF-grade wild (WT) male C57BL / 6 mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and raised in an SPF environment with 12 h light / 12 h dark, temperature controlled at 18-23°C, and humidity maintained at 40%-60%, and were fed normal mouse chow.

[0075] 2. Experimental methods:

[0076] After one week of adaptive feeding, the mice were randomly divided into sham operation (Sham) group and unilateral ureteral obstruction (UUO) group, with 6 mice in each group.

[0077] The UUO model was established as follows: under isoflurane anesthesia, mice were placed in the lateral decubitus position on an operating table. After disinfecting the skin with alcohol, a 1-cm long abdominal incision was made on the left side of the abdomen to expose and free the left ureter. The ureter was ligated with 6-0 surgical sutures at the upper and middle thirds of the ureter, and the ureter was transected between the two ligatures. Finally, the abdominal cavity and skin were sutured layer by layer. In the sham group, only the abdominal cavity was opened and the left ureter was freed, without ligation.

[0078] Seven days after the model was established, mice in each group were killed and their kidney tissues were collected. Half of the kidney tissues were fixed with 4% paraformaldehyde for 48 hours, embedded in paraffin, and cut into 4-μM thick sections. Masson staining was performed to detect the fibrosis of the kidney tissues in UUO mice to evaluate whether the UUO-induced renal fibrosis model in mice was successfully established. The remaining kidney tissues were quickly frozen in liquid nitrogen and stored at -80°C. Western blot was then used to detect the expression of fibrosis markers FN1 and α-SMA in the kidney tissues of UUO mice. Immunohistochemistry staining and Western blot experiments were also used to detect the expression changes of KDM5B in the fibrotic kidney tissues of UUO mice.

[0079] 3. Experimental results

[0080] (1) Masson staining:

[0081] Masson staining results Figure 2 shown.

[0082] Depend on Figure 2 It can be seen that compared with the mice in the Sham group, the collagen deposition in the renal tissue of the mice in the UUO group was obvious, and the statistical fibrosis area was significantly increased, indicating that the renal fibrosis model of UUO mice was successfully established.

[0083] (2) Western Blot analysis of the expression of fibrosis markers FN1 and α-SMA in the kidney tissues of UUO and Sham mice:

[0084] Western Blot analysis of fibrosis markers FN1 and α-SMA in kidney tissue of UUO mice Figure 3 shown.

[0085] Depend on Figure 3 It can be seen that compared with the sham group mice, the expression of fibrosis markers FN1 and α-SMA proteins in the kidney tissue of UUO mice was significantly increased, and the difference was statistically significant, further indicating that the renal fibrosis model of UUO mice was successfully established.

[0086] (3) Immunohistochemical staining and Western Blot analysis of KDM5B expression in renal fibrosis tissues of UUO and Sham mice:

[0087] The results of KDM5B immunohistochemical staining and Western Blot detection in fibrotic renal tissues of UUO and Sham mice are shown in Figure 2. Figure 4 As shown. Figure 4 It can be seen that the expression of KDM5B in the fibrotic renal tissue of UUO mice was significantly higher than that in Sham mice, and the difference was statistically significant.

[0088] Example 3: Evaluation of the value of KDM5B for the diagnosis of renal fibrosis

[0089] 1. Selection of experimental animals and feeding conditions:

[0090] SPF-grade 7-week-old C57 BL / 6 male mice, weighing 16-18 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and housed in an SPF environment with 12 h light / 12 h dark, a temperature controlled at 18-23°C, and a humidity maintained at 40%-60%. They were given normal drinking water and fed with ordinary mouse chow.

[0091] 2. Experimental methods:

[0092] After one week of adaptive feeding, the mice were randomly divided into a sham operation (Sham) group and a unilateral ureteral obstruction (UUO) group, with 12 mice in each group. The method for constructing the UUO model was the same as in Example 2 and will not be described in detail here.

[0093] Seven days after the model was established, mice in each group were sacrificed, and kidney tissue was collected. The tissues were fixed with 4% paraformaldehyde for 48 hours, embedded in paraffin, and cut into 4-μM-thick sections. Masson staining was used to examine renal fibrosis in UUO-induced mice. Immunohistochemistry was used to examine the expression of KDM5B in the kidneys of UUO and sham mice. The results were statistically analyzed, and receiver operating characteristic (ROC) curves were plotted and area under the curves (AUCs) were calculated to assess the diagnostic value of KDM5B for renal fibrosis.

[0094] 3. Experimental results:

[0095] Masson staining results Figure 5 As shown. Figure 5 It can be seen that compared with Sham mice, the renal tissues of UUO mice showed obvious fibrosis, indicating that the UUO-induced mouse renal fibrosis model was successfully established.

[0096] The immunohistochemical staining results of KDM5B in renal tissues of UUO mice with fibrosis are shown in Figure 2. Figure 6 As shown. Figure 6 It can be seen that the expression of KDM5B in the fibrotic kidney tissue of UUO mice was significantly higher than that in Sham mice, and the difference was statistically significant.

[0097] The immunohistochemical staining results of KDM5B were statistically analyzed, and the ROC curve of KDM5B diagnosis and differentiation between UUO group mice and Sham mice was drawn. Figure 7 shown.

[0098] Depend on Figure 7 The AUC of the ROC curve for KDM5B in differentiating UUO mice from sham mice reached 0.882, effectively distinguishing mice with renal fibrosis from normal controls. This suggests that KDM5B can be used as an auxiliary diagnosis for renal fibrosis and has high diagnostic value.

[0099] Example 4: Effects of KDM5B knockdown or activity inhibition on TGFβ1-induced fibrosis in human renal tubular epithelial cells (HK-2) in vitro

[0100] 1. KDM5B knockdown in HK-2 cells:

[0101] (1) Experimental cells and culture methods

[0102] Human renal tubular epithelial cells (HK-2) were obtained from the Shanghai ATCC Cell Bank. Discard the old culture medium in the culture flask, add 2 mL of sterile PBS, wash gently, and then discard. Add 2 mL of 0.25% trypsin preheated at 37°C and digest for 2-3 minutes. Gently shake the culture dish to detach the cells, and add 2 mL of fresh DMEM / F12 complete medium (10% FBS + 1% double antibody) to terminate the digestion. Collect the cells, centrifuge at 1000 rpm for 5 minutes at room temperature, discard the supernatant, resuspend the cells in fresh complete medium, add to the culture dish, and continue culturing in a 37°C, 5% CO2 incubator.

[0103] (2) Experimental methods

[0104] KDM5B-specific small interfering RNAs (si-KDM5B-1, si-KDM5B-2, si-KDM5B-3) and control siRNA (si-Ctrol) were designed and synthesized by Genema. Their nucleotide sequences are:

[0105] si-KDM5B-1forward 5'-GGACUUAUUUCAGCUUAAUTT-3',

[0106] si-KDM5B-1reverse 5'-AUUAAGCUGAAAAUAAGUCCT-3';

[0107] si-KDM5B-2forward 5'-GACCUGGAGAGAGCUUUAATT-3',

[0108] si-KDM5B-2reverse 5'-UUAAAGCUCUCUCCAGGUCTT-3';

[0109] si-KDM5B-3forward 5'-GCCCAAGAGUCGAUCUAAATT-3',

[0110] si-KDM5B-3reverse 5'-UUUAGAUCGACUCUUGGGCTT-3';

[0111] si-Ctrol forward 5'-UUCUCCGAACGUGUCACGUTT-3',

[0112] si-Ctrol reverse 5'-ACGUGACACGUUCGGAGAATT-3'.

[0113] Human renal tubular epithelial cells HK-2 were cultured in DMEM / F12 (10% FBS, 100 U / ml penicillin, 100 μg / mL streptomycin) medium at 37°C in a 5% CO2 incubator. When the cell density reached 80%, the cells were trypsinized, resuspended in fresh medium, counted, and plated in 6-well plates at a density of 1 × 10 cells per well. 5 The cells were cultured overnight in a cell culture incubator. The next day, KDM5B-specific small interfering RNAs (si-KDM5B-1, si-KDM5B-2, and si-KDM5B-3) and a control RNA (si-Ctrol) were transfected using Lipofectamine RNAiMAX Transfection Reagent. After 48 hours, cells were harvested and KDM5B knockdown in HK-2 cells was assessed by western blot.

[0114] (3) Experimental results

[0115] The experimental results of Western Blot detection of KDM5B knockdown are as follows Figure 8 shown.

[0116] Depend on Figure 8 It can be seen that compared with si-Ctrol, the expression of KDM5B protein in the si-KDM5B-1, si-KDM5B-2, and si-KDM5B-3 treatment groups was significantly decreased, indicating that KDM5B was successfully knocked down in HK-2, and the si-KDM5B-1 sequence had the best knockdown effect. This sequence was used in subsequent experiments.

[0117] 2. Western Blot analysis of the effect of KDM5B knockdown on TGFβ1-induced FN1 and α-SMA protein expression in HK-2 cells:

[0118] (1) Experimental cells and culture methods

[0119] Human renal tubular epithelial cells (HK-2) were obtained from the Shanghai ATCC Cell Bank. Discard the old culture medium from the culture flask, gently wash with 2 mL of sterile PBS, and then discard. Add 2 mL of 0.25% trypsin, preheated at 37°C, and digest for 2-3 minutes. Gently shake the culture dish to dislodge the cells, and add 2 mL of fresh DMEM / F12 complete medium (10% FBS, 100 U / ml penicillin, 100 μg / mL streptomycin) to terminate the digestion. Collect the cells, centrifuge at 1000 rpm for 5 minutes at room temperature, discard the supernatant, resuspend the cells in fresh complete medium, add the culture dish, and continue culturing in a 37°C, 5% CO2 incubator.

[0120] (2) Experimental methods

[0121] HK-2 cells were cultured in vitro, digested with trypsin, resuspended in fresh DMEM / F12 complete medium, and plated in 6-well plates at 1×10 cells per well. 5The experimental groups were as follows: control (si-Ctrol), KDM5B knockdown (si-KDM5B), TGFβ1-treated (si-Ctrol+TGFβ1), and KDM5B knockdown + TGFβ1-treated (si-KDM5B+TGFβ1). The next day, si-KDM5B and si-Ctrol were transfected using Lipofectamine RNAiMAX Transfection Reagent. Twenty-four hours later, TGFβ1 (10 μg / mL in citric acid, diluted 1000-fold to a final concentration of 10 ng / mL) was added and incubated for an additional 24 hours. Cells were harvested, lysed using RIPA, and total protein concentration was determined using a BCA protein quantification kit. Western blot analysis was performed to examine the effect of KDM5B knockdown on TGFβ1-induced protein expression of KDM5B, FN1, and α-SMA in HK-2 cells.

[0122] (3) Experimental results:

[0123] Western Blot analysis of the effects of KDM5B knockdown on TGFβ1-induced FN1 and α-SMA protein expression in HK-2 cells. Figure 9 shown.

[0124] Depend on Figure 9 Compared with the control group, the expression levels of KDM5B, as well as the fibrosis markers FN1 and α-SMA, were significantly increased in the TGFβ1-treated group, indicating that KDM5B is upregulated in TGFβ1-induced fibrosis in HK-2 cells. Furthermore, compared with the TGFβ1-treated group, KDM5B knockdown significantly reduced TGFβ1-induced FN1 and α-SMA expression. These results demonstrate that KDM5B knockdown in vitro significantly ameliorates TGFβ1-induced fibrosis in HK-2 cells.

[0125] 3. Western Blot analysis of the effect of KDM5B activity inhibition on TGFβ1-induced FN1 and α-SMA protein expression in HK-2 cells:

[0126] (1) Experimental cells and culture methods

[0127] Human renal tubular epithelial cells (HK-2) were obtained from the Shanghai ATCC Cell Bank. Discard the old culture medium from the culture flask, gently wash with 2 mL of sterile PBS, and then discard. Add 2 mL of 0.25% trypsin, preheated at 37°C, and digest for 2-3 minutes. Gently shake the culture dish to dislodge the cells, and add 2 mL of fresh DMEM / F12 complete medium (10% FBS, 100 U / ml penicillin, 100 μg / mL streptomycin) to terminate the digestion. Collect the cells, centrifuge at 1000 rpm for 5 minutes at room temperature, discard the supernatant, resuspend the cells in fresh complete medium, add the culture dish, and continue culturing in a 37°C, 5% CO2 incubator.

[0128] (2) Screening of the effective concentration of the KDM5B activity inhibitor TK-129 ((R)-1-Cyclopropyl-3-(1-(3-isopropyl-1H-pyrazole-5-carbonyl)-pyrrolidin-3-yl)urea)

[0129] HK-2 cells were cultured in vitro, digested with trypsin, resuspended and plated in 6-well plates, with 1×10 5 cells; a series of concentrations of KDM5B-specific small molecule inhibitor TK-129 solution (TK-129 solution concentrations of 2.5μM, 5μM, 10μM; TK-129 solution solvent is DMSO) were added to 96-well plates and allowed to act for 24 hours. The cell viability was detected using the CCK-8 assay to screen the effective concentration of TK-129. The results are shown in Figure 2. Figure 10 shown.

[0130] Depend on Figure 10 It can be seen that compared with the control (Control), there was no statistical difference in the survival rate of HK-2 cells after 24 hours of treatment with 2.5μM, 5μM, and 10μM TK-129 solutions, indicating that 2.5μM, 5μM, and 10μM TK-129 did not show cytotoxicity. Subsequently, related experiments were carried out using 5μM and 10μM solutions, respectively.

[0131] (3) Effect of KDM5B activity inhibitor TK-129 on TGFβ1-induced FN1 and α-SMA protein expression in HK-2 cells

[0132] HK-2 cells were cultured in vitro, digested with trypsin, resuspended and plated in 6-well plates, with 1×10 5cells. The experimental groupings were as follows: control group (Ctrl), TK-129 treatment group (TK-129 5μM, TK-129 10μM), TGFβ1 treatment group (TGFβ1), TGFβ1 + TK-129 administration group (TGFβ1 + TK-129 5μM, TGFβ1 + TK-129 10μM). The next day, control reagent (DMSO) and 5μM and 10μM TK-129 solutions (the solvent of TK-129 solution is DMSO) were added to HK-2 cells respectively. After two hours of action, TGFβ1 was added (TGFβ1 solution was citric acid, the concentration was 10ug / mL, it needed to be diluted 1000 times, and the final action concentration was 10ng / mL) and incubated for another 24 hours. The cells were collected, lysed using RIPA, and the total protein concentration was determined using a BCA protein quantification kit. Western Blot was used to detect the effect of the KDM5B activity inhibitor TK-129 on the expression of fibrosis markers FN1 and α-SMA proteins in HK-2 cells induced by TGFβ1. Figure 11 shown.

[0133] Depend on Figure 11 As shown, compared with the control group, the expression levels of fibrosis markers FN1 and α-SMA in the TGFβ1-treated group were significantly increased. Furthermore, compared with the TGFβ1-treated group, high-dose TK-129 (10 μM) significantly reduced TGFβ1-induced FN1 and α-SMA expression. These results indicate that inhibiting KDM5B activity with TK-129 in vitro can significantly mitigate TGFβ1-induced fibrosis in HK-2 cells.

[0134] Example 5: Effect of KDM5B overexpression on human renal tubular epithelial cells (HK-2) in vitro

[0135] 1. Experimental cells and culture methods

[0136] Human renal tubular epithelial cells (HK-2) were obtained from the Shanghai ATCC Cell Bank. Discard the old culture medium from the culture flask, gently wash with 2 mL of sterile PBS, and then discard. Add 2 mL of 0.25% trypsin, preheated at 37°C, and digest for 2-3 minutes. Gently shake the culture dish to dislodge the cells, and add 2 mL of fresh DMEM / F12 complete medium (10% FBS, 100 U / ml penicillin, 100 μg / mL streptomycin) to terminate the digestion. Collect the cells, centrifuge at 1000 rpm for 5 minutes at room temperature, discard the supernatant, resuspend the cells in fresh complete medium, add the culture dish, and continue culturing in a 37°C, 5% CO2 incubator.

[0137] 2. Experimental methods

[0138] The KDM5B overexpression plasmid (pc-KDM5B) and control plasmid (pc-DNA3.1) were designed and synthesized by Hanbio. The structural map of pc-KDM5B is shown in Figure 12 As shown in A. HK-2 cells were plated in 6-well plates, with 1×10 cells per well. 5 The next day, Lipofectamine TM Plasmids were transfected with 3000 transfection reagent. After 48 hours, cells were harvested and KDM5B expression in HK-2 cells was detected by RT-PCR and Western blotting. Western blotting was also used to examine the effect of KDM5B overexpression on the expression of fibrosis markers FN1 and α-SMA in HK-2 cells.

[0139] 3. Experimental results

[0140] (1) RT-PCR and Western Blot detection of KDM5B overexpression in HK-2

[0141] The results of RT-PCR and Western Blot detection of KDM5B overexpression in HK-2 are shown in Figure 2. Figure 12 As shown. Figure 12 It can be seen that compared with the pc-cDNA3.1 control group, the KDM5B mRNA and protein levels in the pc-KDM5B group were significantly increased, indicating that KDM5B was successfully overexpressed in HK-2.

[0142] (2) Western Blot analysis of the effect of KDM5B overexpression on the expression levels of FN1 and α-SMA proteins in HK-2 cells

[0143] The results of Western Blot analysis of the effect of KDM5B overexpression on the expression levels of FN1 and α-SMA proteins in HK-2 cells are shown in Figure 2. Figure 13 As shown. Figure 13 It can be seen that compared with the pc-cDNA3.1 control group, the expression of fibrosis markers FN1 and α-SMA proteins in HK-2 in the pc-KDM5B group was significantly increased, indicating that KDM5B overexpression can induce HK-2 fibrosis response.

[0144] Example 6: Effect of tubular epithelial cell-specific KDM5B knockout on renal fibrosis in UUO model mice

[0145] 1. Selection of experimental animals and feeding conditions

[0146] SPF-grade 8-week-old C57 BL / 6 male mice, weighing 16-18 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and housed in an SPF environment with 12 h light / 12 h dark, a temperature controlled at 18-23°C, and a humidity maintained at 40%-60%. They were fed normal mouse chow.

[0147] 2. Experimental methods

[0148] Hanheng Bio used the renal tubular epithelial cell-specific promoter Ksp-cadherin to construct a KDM5B knockout adeno-associated virus (AAV2 / 9-KDM5B-shRNA, the structure of AAV2 / 9-KDM5B-shRNA is shown in the figure). Figure 14 The nucleotide sequence of KDM5B shRNA is: 5'-TTCGCTTGTGATGTCGATAAA-3', and the control nucleotide sequence is: 5'-TTCTCCGAACGTGTCACGT-3'. The experimental mice were randomly divided into AAV9-Ctrol Sham group, AAV9-KDM5B-shRNA Sham group, AAV9-Ctrol UUO group and AAV9-KDM5B-shRNA UUO group. The kidney was injected in situ with an injection volume of 60 μL and the AAV9-KDM5B-shRNA virus titer was 1.3×10 12 vg / mL, and the titer of AAV9-Ctrol virus was 1.4×10 12 vg / mL. The specific procedure was as follows: Under isoflurane anesthesia, mice were secured in a recumbent position on an operating table. After skin disinfection, a 1-cm long incision was made on the left side of the abdomen to expose the left kidney. The kidney was carefully secured with the fingers, and the virus was injected using a microsyringe (100 μL range) at six locations, one above the kidney and one below the kidney, along with the lateral margin. After the injection, the kidney was replaced, the muscles and skin were sutured, and the mouse was returned to its cage for normal care after it fully recovered.

[0149] After 3 weeks, each group of mice was subjected to UUO or Sham model. For specific operations, see Example 2. One week after the UUO model, the mice were killed, and the left kidney tissue was taken. Half of the kidney tissue was fixed with 4% paraformaldehyde for 48 hours and then embedded in paraffin. The kidney paraffin tissue was prepared into 4 μm thick sections using a microtome for standby use. The other half of the kidney tissue was quick-frozen in liquid nitrogen for 5 minutes and then stored at -80°C for standby use. Kidney tissue was taken and the expression of KDM5B was detected by western blot. Masson staining was further used to detect the effect of tubular epithelial cell-specific KDM5B knockout on renal fibrosis in mice. Immunofluorescence staining was used to detect the effect of tubular epithelial cell-specific KDM5B knockout on the expression of fibrosis markers FN1 and α-SMA in the kidney tissue of UUO-induced mice.

[0150] 3. Experimental results

[0151] (1) Western Blot was used to detect the expression of KDM5B in the kidney tissue of renal tubular epithelial cell-specific KDM5B knockout mice:

[0152] Western Blot analysis of KDM5B expression in renal tissues of renal tubular epithelial cell-specific KDM5B knockout mice. Figure 14 As shown. Figure 14 The results showed that KDM5B expression in the kidney tissues of AA2 / 9-Ctrol UUO mice was significantly increased compared with AA2 / 9-Ctrol Sham mice. In contrast, KDM5B expression in the kidney tissues of AA2 / 9-KDM5B-shRNA UUO mice was significantly decreased compared with AA2 / 9-Ctrol UUO mice, indicating that renal tubular epithelial cell-specific KDM5B knockout mice were successfully constructed.

[0153] (2) Masson staining was used to examine the effect of tubular epithelial cell-specific KDM5B knockout on UUO-induced renal fibrosis in mice.

[0154] Masson staining test results Figure 15 As shown. Figure 15It can be seen that compared with the AAV9-Ctrol Sham group, no obvious changes were observed in the renal tissue of mice in the AAV9-KDM5B-shRNA Sham group, indicating that tubular epithelial cell-specific KDM5B knockout had no effect on the mouse kidneys under the basal state, while the renal tubules of mice in the AAV9-Ctrol UUO model group were dilated and the area of ​​renal fibrosis was significantly increased, indicating that the renal fibrosis model was successfully constructed; compared with the AAV9-Ctrol UUO model group, the degree of renal tubular dilation and the area of ​​renal fibrosis in mice in the AAV9-KDM5B-shRNA UUO group were reduced, indicating that tubular epithelial cell-specific KDM5B knockout can significantly alleviate UUO-induced renal fibrosis in mice.

[0155] (3) Immunofluorescence staining was used to detect the effect of tubular epithelial cell-specific KDM5B knockout on the expression of fibrosis markers FN1 and α-SMA in the kidney tissue of UUO-induced mice:

[0156] The results of immunofluorescence staining to detect the effect of tubular epithelial cell-specific KDM5B knockout on the expression of FN1 and α-SMA in the renal tissue of UUO-induced mice are as follows: Figure 16 shown.

[0157] Depend on Figure 16 It can be seen that compared with the AAV9-Ctrol Sham group, the expression of fibrosis markers FN1 and α-SMA in the renal tissue of mice in the AAV9-Ctrol UUO model group was also significantly increased, while compared with the AAV9-Ctrol UUO model group, the expression of FN1 and α-SMA in the renal tissue of mice in the AAV9-KDM5B-shRNA UUO group was significantly decreased, further indicating that tubular epithelial cell-specific knockout of KDM5B can significantly improve UUO-induced renal fibrosis in mice.

[0158] In summary, in vitro KDM5B knockdown or activity inhibition significantly alleviated TGFβ1-induced fibrosis in human renal tubular epithelial HK-2 cells. Specific KDM5B ablation in renal tubular epithelial cells in vivo also significantly improved renal fibrosis in mice.

[0159] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of KDM5B gene or KDM5B protein as a drug target for screening drugs for preventing, alleviating and / or treating renal fibrosis.

2. Use of a substance that inhibits the expression and / or function of the KDM5B gene or KDM5B protein in the preparation of a medicament for preventing, alleviating, or / and treating renal fibrosis, wherein the substance that inhibits the expression and / or function of the KDM5B gene or KDM5B protein is an siRNA that specifically targets the KDM5B gene or an inhibitor of the KDM5B protein, wherein the siRNA that specifically targets the KDM5B gene is siRNA 1, siRNA 2, or siRNA 3; wherein, The nucleotide sequence of siRNA 1 is: siRNA 1 forward: 5'-GGACUUAUUUCAGCUUAAUTT-3', siRNA 1 reverse: 5'-AUUAAGCCUGAAAUAAGUCCTT-3'; The nucleotide sequence of siRNA 2 is: siRNA 2 forward: 5'-GACCUGGAGAGAGCUUUAATT-3', siRNA 2 reverse: 5'-UUAAAGCUCUCUCCAGGUCTT-3'; The nucleotide sequence of siRNA 3 is: siRNA 3 forward: 5'-GCCCAAGAGUCGAUCUAAATT-3', siRNA 3 reverse: 5'-UUUAGAUCGACUCUUGGGCTT-3'; The inhibitor is TK-129.

3. A drug, characterized in that The drug is used to prevent, alleviate or / and treat renal fibrosis, and contains a substance that inhibits the expression and / or function of the KDM5B gene; the substance that inhibits the expression and / or function of the KDM5B gene is an siRNA that specifically targets the KDM5B gene, and the siRNA that specifically targets the KDM5B gene is siRNA 1, siRNA 2 or siRNA 3; wherein, The nucleotide sequence of siRNA 1 is: siRNA 1 forward: 5'-GGACUUAUUUCAGCUUAAUTT-3', siRNA 1 reverse: 5'-AUUAAGCCUGAAAUAAGUCCTT-3'; The nucleotide sequence of siRNA 2 is: siRNA 2 forward: 5'-GACCUGGAGAGAGCUUUAATT-3', siRNA 2 reverse: 5'-UUAAAGCUCUCUCCAGGUCTT-3'; The nucleotide sequence of siRNA 3 is: siRNA 3 forward: 5'-GCCCAAGAGUCGAUCUAAATT-3', siRNA 3 reverse: 5'-UUUAGAUCGACUCUUGGGCTT-3'.

4. The drug according to claim 3, characterized in that The medicine also contains pharmaceutically acceptable carriers / excipients.

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