Use of substance for inhibiting CAPG activity in preparation of product for treating renal fibrosis caused by kidney injury

Abstract

The present invention pertains to the technical field of biomedicine. Specifically disclosed is use of a substance for inhibiting the activity and / or expression of a CAPG gene or protein in the preparation of a product for treating renal fibrosis caused by kidney injury. First, research has found that CAPG is expressed at low levels in normal kidney tissues, while its expression level is increased in kidney tissues of patients with acute kidney injury, minimal change disease, IgA nephropathy, lupus nephritis, and diabetic kidney disease, indicating that the expression of CAPG may be involved in the occurrence and development of renal fibrosis caused by kidney injury. Then, by means of constructing a CAPG knockout mouse model, it has been further found that knocking out CAPG can effectively alleviate the progression of renal fibrosis; and by means of culturing renal fibroblasts in vitro, it has been found that silencing or knocking down CAPG can inhibit TGF-β-induced activation of renal fibroblasts. The present invention is of great significance for the pathogenesis and treatment of kidney injury or renal fibrosis.

Description

Use of a substance inhibiting CAPG activity in the preparation of a product for treating renal fibrosis caused by kidney injury TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to use of a substance inhibiting CAPG activity in the preparation of a product for treating renal fibrosis caused by kidney injury. BACKGROUND

[0002] Chronic kidney disease (CKD) is a clinical syndrome characterized by long-term abnormalities in kidney structure and / or function, which can be caused by a variety of causes, such as maladaptive repair after acute kidney injury, glomerulonephritis, diabetes and hypertension, etc. The global prevalence of CKD is about 10%, and the number of patients with CKD in China is about 132 million. CKD can progress to end stage renal disease (ESRD) and require kidney replacement therapy. Renal fibrosis (RF) is a common pathological pathway for all types of CKD to progress to ESRD, and its degree is closely related to the clinical outcome. At present, there is still a lack of effective means for treating CKD and renal fibrosis in clinical practice.

[0003] CAPG (capping actin protein, gelsolin like) is a member of the gelsolin superfamily, which is distributed in the cytoplasm and nucleus. The nuclear localization of CAPG is reported to be involved in the transcriptional regulation of genes. In recent years, studies have found that CAPG is highly expressed in pancreatic cancer, lung cancer, breast cancer, kidney cancer and intestinal cancer, and promotes the invasion and metastasis of cancer cells. The expression and role of CAPG in renal fibrosis caused by kidney injury are still in the blank field.Although CAPG is reported to be up-regulated in renal cancer, since renal cancer and renal fibrosis caused by kidney injury are different pathophysiological processes, the expression and role of CAPG in renal cancer cannot be inferred to renal fibrosis caused by kidney injury. For example, programmed cell death 1 ligand 1 (PD-L1) was found to be expressed at a higher level in renal cancer cells, and PD-1 / L1-based immunotherapy has been recommended for first-line treatment of advanced renal cancer, but the incidence of kidney injury caused by PD-1 / L1 treatment in renal cancer and other tumor patients is as high as 2.2%, and even some patients progress to ESRD (Frank B. Cortazar, Kristen A. Marrone, Megan L. Troxell, et al. Clinicopathological features of acute kidney injury associated with immune checkpoint inhibitors. Kidney International. 2016; 90 (3): 638-647.); sperm-associated antigen 9 (SPAG9) is reported to be expressed at a higher level in renal cancer and is a potential therapeutic target, but the expression of SPAG9 is found to be down-regulated in fibrotic kidney tissue and can inhibit renal fibrosis after kidney injury (Maoqing Tian, Lu Zhang, Meng Zhang, et al. JLP / Foxk1 / N-cadherin axis fosters a partial epithelial-mesenchymal transition state in epithelial tubular cells. iScience. 2023; 26 (4): 106396-106396.), renal fibrosis is a common feature of kidney injury into end-stage renal disease caused by various reasons, and the progression rate of renal fibrosis determines the rate of loss of kidney function, and delaying the progression of renal fibrosis is the key to protecting kidney function, and the expression and role of CAPG in the research of renal fibrosis caused by kidney injury have not been reported, and the research of the present application provides a new way for the treatment of chronic kidney disease. SUMMARY

[0004] An aspect of the main object of the present application is to provide a new treatment scheme for treating renal fibrosis caused by kidney injury, and specifically, the present application proposes an embodiment: a substance that inhibits the activity and / or expression of CAPG gene or protein is used in the preparation of a product for treating renal fibrosis caused by kidney injury.

[0005] In another aspect, the application also provides another embodiment, the use of a reagent for detecting CAPG gene or protein activity and / or expression in the preparation of a product for detecting renal fibrosis caused by kidney injury, in which the expression of CAPG can be detected to assist in evaluating the efficacy of a CAPG-targeted therapeutic product for kidney injury-related diseases.

[0006] Further, the kidney injury includes acute kidney injury (AKI) or chronic kidney disease (CKD).

[0007] Further, the pathogenic factors of chronic kidney disease include Minimal Change Disease (MCD), IgA Nephropathy (IgAN), Lupus Nephritis (LN), diabetic kidney disease (DKD), Hypertensive Renal Disease (HRD), and other diseases that cause renal fibrosis.

[0008] Further, the kidney injury includes folate intake or TGF-β signaling pathway activation-induced kidney injury.

[0009] Further, the substance for inhibiting CAPG gene expression is a substance for silencing, interfering, knocking out or knocking down CAPG gene expression.

[0010] Further, under the scheme proposed in the application, the use of existing technologies such as some embodiments of miRNA, siRNA, dsRNA or shRNA designed according to the CAPG gene as a substance for interfering or silencing CAPG gene expression can achieve the purpose of inhibiting CAPG gene expression and treating renal fibrosis caused by kidney injury.

[0011] Further, as another specific embodiment, the substance for knocking out CAPG gene expression is a gene editing tool such as ZFN gene editing system, TALENs gene editing system or CRISPR / Cas9 gene editing system.

[0012] Further, as some embodiments, the present application provides a product for detecting renal fibrosis caused by kidney injury, which comprises a kit, a test paper or a gene chip. The kit has a physical form, for example, the kit can be a container with one or more spaces for holding controls, standards, reagents, materials or devices. The reagents, tools and / or instructions for performing the methods described herein can be provided in the kit. The kit can also include one or more reagents for performing gene expression analysis, such as reagents for performing RT-PCR, qPCR, northern blotting, proteomic analysis or immunohistochemistry to determine the expression level of a gene or protein of interest in a patient sample.

[0013] Further, as some embodiments, the reagent comprises a reagent for detecting the expression level of CAPG in the sample of the subject by immunohistochemistry, in situ molecular hybridization, Western blotting, Northern blotting, PCR, RT-PCR or biochip method;

[0014] Preferably, the immunohistochemistry comprises an immunofluorescence analysis, a reverse enzyme-linked immunosorbent assay or an immunogold colloid method.

[0015] Further, the subject comprises a murine, a rabbit, a pig, a non-human primate or a human; and the sample comprises blood, tissue, a cell sample, urine or feces from the subject. The term "subject" includes a patient and a non-patient. "Subject" and "individual" are used interchangeably. The term "patient" refers to an individual afflicted with or likely to be afflicted with a medical condition, such as inflammation or an inflammatory disease, while a "non-patient" refers to an individual who is not afflicted or likely to be afflicted with the medical condition. A "non-patient" includes a healthy individual, an individual who is not ill, and / or an individual who does not have the medical condition. The term "subject" includes mammals, in particular humans and animals. Animals include murines, rabbits, pigs, non-human primates, etc. "Murine" refers to any mammal from the family Muridae, such as a mouse, a rat, etc.

[0016] The terms "sample" and "biological sample" and "test sample" are used interchangeably herein to refer to any material, biological fluid, tissue or cells obtained or otherwise derived from an individual, such as blood, tissue, a cell sample, urine or feces, etc.

[0017] Technical effects achieved by the present application:

[0018] The present application firstly discovers that CAPG is expressed at a low level in normal kidney tissue, and the expression level is increased in kidney tissue of patients with acute kidney injury, minimal change nephropathy, IgA nephropathy, lupus nephritis and diabetic nephropathy, indicating that the expression of CAPG may be involved in the occurrence and development of kidney fibrosis caused by kidney injury. Then further through the construction of a CAPG knockout mouse model, it is found that the knockout of CAPG can effectively reduce the progression of kidney fibrosis, and through the in vitro culture of kidney fibroblasts, it is found that silencing or knocking down CAPG can inhibit the activation of kidney fibroblasts induced by TGF-β. The present application has important significance for the pathogenesis and treatment of kidney injury or kidney fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a schematic diagram of the expression level of CAPG in the kidney tissue of CKD patients and the correlation with the degree of kidney fibrosis; A, CAPG immunohistochemical staining of kidney tissue sections of healthy controls (CTL), acute kidney injury (AKI) and various etiological CKD patients (minimal change nephropathy (MCD), IgA nephropathy (IgAN), lupus nephritis (LN) and diabetic nephropathy (DKD)); B, comparison of the expression amount of CAPG in kidney tissue of different degrees of fibrosis.

[0020] Figure 2 is a graph of the expression level of CAPG in the kidney of folate-induced CKD mice. A, immunohistochemical staining of CAPG at different times after folate induction (Figure 2A); B, the cumulative optical density in Figure 2A is used to reflect the protein expression level of CAPG: comparison of the expression amount of CAPG in the kidney at different times after folate induction.

[0021] Figure 3 is a graph of the effect of CAPG knockdown / knockout on kidney fibrosis in folate-induced CKD mice. Wild type control mice and CAPG knockdown / knockout mice were given intraperitoneal injection of folate to induce CKD, and the mice were sacrificed 28 days after induction to take the kidney for analysis; A, immunofluorescence detection of Capg + / + protein expression level of CAPG in the kidney of wild type mice, Capg + / - knockdown and Capg - / - knockout mice; B, Masson and Sirius red staining of the kidney to detect the collagen deposition in the kidney of Capg + / + wild type mice, Capg + / - knockdown and Capg - / - knockout mice; C, western blot detection of Capg + / + protein expression level of FN, Col I and CAPG in the kidney tissue of wild type mice and Capg + / - knockdown mice; D, western blot detection of Capg + / + protein expression level of FN, Col I and CAPG in the kidney tissue of wild type mice and Capg - / -Knockout the expression level of FN and α-SMA in mouse kidney tissue.

[0022] Figure 4 is a diagram of detection of kidney fibroblast CAPG knockdown. (A) After transfection of three CAPG-siRNAs (si#1, si#2, si#3), transfection reagent control Lipo, negative control Ctrl, positive control Pos and blank control NC into kidney fibroblast cell line NRK-49F, the protein expression level of CAPG was detected after 36h or 48h culture.

[0023] Figure 5 is a diagram of the effect of kidney fibroblast CAPG knockdown on fibroblast fibrosis. (A) After CAPG-siRNA was given to kidney fibroblast cell line NRK-49F to down-regulate CAPG expression, the protein level changes of FN, Col I and α-SMA in NRK-49F cells with CAPG knockdown; (B) Under TGF-β stimulation, the protein level changes of FN, Col I and α-SMA in NRK-49F cells with CAPG knockdown.

[0024] Figure 6 is a diagram of the design of using CRISPR / Cas9 technology to edit CAPG gene. DETAILED DESCRIPTION

[0025] The concept and the technical effects produced by the present application will be described below in conjunction with the embodiments for a clear and complete description, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0026] In the experimental method, the purchased commodities are carried out according to the conventional conditions or the conditions recommended by the manufacturer, and the reagents or instruments used without specifying the manufacturer can be obtained by purchasing the conventional products on the market.

[0027] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure.

[0028] Example 1 CAPG is highly expressed in the kidney tissues of patients with acute kidney injury and chronic kidney disease

[0029] I. CAPG is highly expressed in the kidney tissues of patients with acute kidney injury and chronic kidney disease caused by multiple causes

[0030] Experimental materials and methods

[0031] Renal biopsy tissues from patients with pathologic diagnosis of "acute kidney injury" or primary or secondary kidney disease were collected from the First Affiliated Hospital of Sun Yat-Sen University. Normal kidney tissues were used as controls. CAPG immunohistochemical analysis was performed on kidney tissue sections.

[0032] Test results

[0033] As shown in Figure 1, the results of immunohistochemical analysis showed that CAPG was expressed at a low level in normal kidney tissues, but the expression level was increased in kidney tissues from patients with acute kidney injury and minimal change disease, IgA nephropathy, lupus nephritis and diabetic nephropathy. The above results suggest that the expression of CAPG can be involved in the occurrence and development of kidney fibrosis caused by kidney injury.

[0034] Example 2 High expression of CAPG in kidney tissues of a mouse model of chronic kidney disease

[0035] I. Construction of a folate-induced mouse model of chronic kidney disease

[0036] Test materials and methods

[0037] 6-8 week old wild type C57BL / 6 male mice were used as test materials, and the method in the literature "Suppressed Mitochondrial Biogenesis in Folic Acid-Induced Acute Kidney Injury and Early Fibrosis (DOI: 10.1016 / j.toxlet.2013.11.014)" was used to inject folate (folic acid, FA) solution intraperitoneally to induce kidney fibrosis after kidney injury, and a FA-induced kidney fibrosis mouse model was obtained. Mice injected intraperitoneally with 0.3M sodium bicarbonate solution were used as a control group.

[0038] The mice were sacrificed 2 days, 7 days and 28 days after injection of folate, and the kidney tissues were taken to detect the expression level of CAPG in the kidney of the FA mouse model.

[0039] Test results

[0040] As shown in Figure 2, compared with the normal control, in the folate-induced chronic kidney disease mouse model, the expression of CAPG in the kidney tissue gradually increased with the progression of kidney fibrosis, and was mainly highly expressed in the nucleus; using Image J for cumulative optical density analysis, the expression of CAPG in the kidney tissue gradually increased over time after FA induction.

[0041] The above research results show that the expression of CAPG in the kidney tissue of the FA-induced kidney fibrosis mouse model is increased.

[0042] Example 3 Inhibition of CAPG improves FA-induced chronic kidney disease

[0043] I. Origin and construction of CAPG knockdown and knockout mice

[0044] 1. Capg + / - Origin of knockdown mice

[0045] Capg + / - Knockdown mice were purchased from Jiangsu Jizui Yekang Biotechnology Co., Ltd. (Nanjing) with the strain name C57BL / 6JGpt-Capgem3Cd10097 / Gpt and the product code T029492, and were bred by Jiangsu Jizui Yekang Biotechnology Co., Ltd.

[0046] The mice were edited using CRISPR / Cas9 technology to edit the Capg gene. The Capg gene has 11 transcripts, as shown in Figure 6. According to the structure of the Capg gene, the 2nd to 9th exons of the CAPG-202 (ENSMUST00000114071.7) transcript were used as the knockout region, which contains the start codon ATG. Knocking out this region will result in the loss of CAPG protein function.

[0047] 2. Capg - / - Construction of knockout mice

[0048] The Capg + / - Mice were interbred to obtain wild-type mice and Capg - / - knockout mice (homozygous) born in the same litter.

[0049] 3. Identification of Capg knockdown / knockout mice

[0050] (1) Cut off about 0.5 cm of the mouse tail, place the mouse tail in a numbered 200 μL centrifuge tube and cover the tube with a lid.

[0051] (2) Add 100 μL lysis buffer and 2 μL protease, digest in a 55°C water bath / metal bath for 15 min, then incubate the sample in a 95°C water bath / metal bath for 5 min to inactivate the protease activity in the digestion solution. Centrifuge at 12000 rpm for 5 minutes, and take the supernatant as the PCR template.

[0052] (3) Prepare the PCR system

[0053] Table 1 Primer information

[0054]

[0055] Table 2 PCR system

[0056]

[0057] Table 3 PCR procedure

[0058]

[0059] (4) PCR product identification

[0060] PCR products were spotted for agarose gel electrophoresis, only PCR product ① was homozygous, only PCR product ② was wild type, and PCR products ① and ② were both heterozygous.

[0061] II. Effect of CAPG knockdown / knockout on renal fibrosis in a mouse model of folate-induced chronic kidney disease

[0062] 1. Test materials and methods

[0063] 6-8 week old wild type control male mice (Capg + / + ), Capg + / - knockdown male mice, and Capg - / - knockout male mice were used as test materials to construct a mouse model of FA-induced renal fibrosis. The mice were sacrificed 28 days after injection of folate, and the kidney tissues were taken to detect and analyze the effect of CAPG knockdown or knockout on FA-induced renal fibrosis.

[0064] 2. Test results

[0065] As shown in Figure 3, Masson and Sirius red staining showed that compared with Capg + / + control mice, collagen deposition in Capg + / - and Capg - / - mice was significantly reduced. Immunoblotting showed that the expression levels of fibrosis markers in Capg + / - and Capg - / - mice were down-regulated compared with Capg + / + control mice, including FN, Col I and a-SMA.

[0066] The above results suggest that knocking down / knocking out CAPG can effectively reduce the progression of FA-induced renal fibrosis.

[0067] Example 4. Renal fibroblast-specific knockdown of CAPG improves renal fibrosis

[0068] I. Knockdown of CAPG in renal fibroblasts

[0069] 1. Test materials and methods

[0070] According to the reference "Decoding myofibroblast origins in human kidney fibrosis (DOI: 10.1038 / s41586-020-2941-1)," myofibroblasts are the main source of extracellular matrix (ECM) in the process of kidney fibrosis, and their cellular origin is mainly fibroblasts and pericytes.

[0071] To investigate the specific mechanism of action of CAPG in renal fibrosis, this invention, through design and preliminary optimization, selected three optimal siRNAs targeting the rat Capg gene. These siRNAs were then transfected into the fibroblast cell line NRK-49F to obtain siRNAs with good interference effects to knock down CAPG in renal fibroblasts. The specific process is as follows:

[0072] Cell seeding: Seed well-grown NRK-49F cells into 6-well plates and culture overnight.

[0073] Cell transfection: Before cell transfection, the culture medium in the wells was replaced with serum-free medium. Then, the three synthesized siRNAs targeting the CAPG gene (si#1, si#2, si#3), the negative control siRNA (Ctrl), and the positive control siRNA (Pos) were incubated with Lipofectamine 3000 to prepare complexes. After incubation at room temperature for 15 min, the complexes were added to the corresponding wells. Simultaneously, the untreated wells were set as blank control groups (NC), and Lipofectamine 3000 (Lipo) alone was used as the transfection reagent control group.

[0074] Six hours after continuous transfection, the culture medium in the wells was aspirated, and 1 ml of fresh culture medium containing 10% fetal bovine serum was added. The cells were then cultured for another 36 or 48 hours before scraping them for immunoblotting experiments.

[0075] The siRNA used in this invention is from Tuoran Biotechnology Co., Ltd. The siRNA sequence is shown in Table 4 below:

[0076] Table 4 siRNA sequence information

[0077]

[0078] 2. Test Results

[0079] As shown in Figure 4, Western blotting showed that, under normal culture conditions, the protein expression levels of CAPG in the three groups transfected with CAPG siRNA (si#1, si#2, si#3) were significantly decreased compared with the control groups (blank control NC, transfection reagent control Lipo, negative control Ctrl, positive control Pos), and the CAPG knockdown efficiency at 48h was better than at 36h, and the CAPG knockdown efficiency of si#2 was better than that of si#1 and si#3.

[0080] The above results suggest that the three siRNAs targeting the CAPG gene can all down-regulate the protein expression level of CAPG in NRK-49F cells, and the silencing efficiency of si#2 is the highest after transfection of NRK-49F cells for 48h, and this siRNA is selected as the effective siRNA for subsequent experiments.

[0081] II. Effect of CAPG Knockdown on TGF-β-induced Fibroblast Fibrosis Phenotype

[0082] 1. Test materials and methods

[0083] 1) Normal culture conditions: The negative control siRNA (Ctrl) and CAPG siRNA were transfected into NRK-49F cells, and 6-8 hours later, the normal culture medium was replaced, and 48 hours later, the cells were scraped for Western blotting experiment;

[0084] 2) TGF-β stimulated NRK-49F cell to construct in vitro fibroblast activation model conditions: The negative control siRNA (Ctrl) and CAPG siRNA were transfected into NRK-49F cells, and 6-8 hours later, the normal culture medium was replaced, and 36 hours later, recombinant TGF-β (10 ng / mL) was used to stimulate NRK-49F cells for 12h to construct in vitro fibroblast activation model, and the cells were collected for detection and analysis of the effect of CAPG knockdown on TGF-β-induced fibroblast fibrosis phenotype.

[0085] 2. Test results

[0086] As shown in Figure 5, Western blotting showed that, under normal culture conditions, the protein expression level of CAPG in NRK-49F cells transfected with CAPG siRNA was significantly decreased compared with the control groups. And the expression of FN and Col I in NRK-49F cells with CAPG knockdown by siRNA was significantly lower than that in the control groups, and the expression of a-SMA showed no significant difference; under TGF-β stimulation, the FN, Col I and a-SMA in the cells with CAPG knockdown were significantly lower than those in the control groups. It is shown that down-regulation of CAPG can inhibit TGF-β-induced fibroblast activation.

[0087] The above results suggest that under normal culture conditions, specific knockdown of CAPG in fibroblasts can down-regulate the expression of FN and Col I in fibroblasts, and has no obvious effect on the expression of α-SMA. Under the stimulation of TGF-β, specific knockdown of CAPG in fibroblasts can inhibit the activation of fibroblasts induced by TGF-β.

Claims

1. The use of substances that inhibit the activity and / or expression of the CAPG gene or protein in the preparation of products for the treatment of renal fibrosis caused by kidney injury.

2. Application of reagents for detecting CAPG gene or protein activity and / or expression in the preparation of products for detecting renal fibrosis caused by kidney injury.

3. The application according to claim 1 or 2, characterized in that, The kidney injury mentioned includes acute kidney injury (AKI) or chronic kidney disease (CKD).

4. The application according to claim 3, characterized in that, The pathogenic factors of the chronic kidney disease include minimal change disease (MCD), IgA nephropathy (IgAN), lupus nephritis (LN), diabetic nephropathy (DKD), or hypertensive nephropathy (HRD).

5. The application according to claim 1 or 2, characterized in that, The kidney injury mentioned includes kidney injury caused by folic acid intake or activation of the TGF-β signaling pathway.

6. The application according to claim 1, characterized in that, The substance that inhibits CAPG gene expression is a substance that silences, interferes with, knocks out, or down-expresses the CAPG gene.

7. The application according to claim 6, characterized in that, Substances that silence or interfere with CAPG gene expression include miRNA, siRNA, dsRNA, or shRNA; substances that knock out CAPG gene expression include ZFN gene editing systems, TALENs gene editing systems, or CRISPR / Cas9 gene editing systems.

8. The application according to claim 2, characterized in that, The products include reagent kits, test strips, or gene chips.

9. The application according to claim 2, characterized in that, The reagents include those for detecting CAPG expression levels in subject samples using immunohistochemistry, in situ molecular hybridization, Western blotting, Northern blotting, PCR, RT-PCR, or microarray methods. Preferably, the immunohistochemical method includes: immunofluorescence analysis, reverse enzyme-linked immunosorbent assay (ELISA), or immunogold immunoassay.

10. The application according to claim 9, characterized in that, The subjects include rodents, rabbits, pigs, non-human primates, or humans; the samples include blood, tissue, cell samples, urine, or feces from the subjects.

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