Application of substances that inhibit CAPG activity in preparing products for treating renal fibrosis caused by renal injury

Abstract

The present invention belongs to the field of biomedical technology and specifically discloses the use of a substance that inhibits the activity and / or expression of a CAPG gene or protein in preparing a product for treating renal fibrosis caused by renal injury. The present invention first discovered through research that CAPG is expressed at a low level in normal renal tissue, but its expression level is elevated in renal tissue of patients with acute renal injury, minimal change disease, IgA nephropathy, lupus nephritis, and diabetic nephropathy, indicating that CAPG expression may be involved in the occurrence and development of renal fibrosis caused by renal injury. Then, by constructing a CAPG knockout mouse model, it was found that knocking out CAPG can effectively reduce the progression of renal fibrosis. By culturing renal fibroblasts in vitro, it was found that silencing or knocking down CAPG can inhibit TGF-β-induced renal fibroblast activation. The research of the present invention is of great significance for the pathogenesis and treatment of renal injury or renal fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to the use of a substance that inhibits CAPG activity in the preparation of a product for treating renal fibrosis caused by renal injury. Background Art

[0002] Chronic kidney disease (CKD) is a clinical syndrome characterized by long-term abnormalities in renal structure and / or function. It can be caused by a variety of etiologies, including maladaptive renal repair after acute kidney injury, glomerulonephritis, diabetes, and hypertension. The global prevalence of CKD is approximately 10%. CKD can progress to end-stage renal disease (ESRD), necessitating renal replacement therapy. Renal fibrosis (RF) is a common pathological pathway in the progression of all types of CKD to ESRD, and its extent is closely correlated with clinical outcomes. Currently, there is a lack of effective treatments for CKD and renal fibrosis.

[0003] Capping actin protein (CAPG), a member of the gelsolin superfamily, is distributed in both the cytoplasm and the nucleus. Nuclear-localized CAPG has been reported to participate in gene transcriptional regulation. Recent studies have found that CAPG is highly expressed in pancreatic, lung, breast, kidney, and intestinal cancers, promoting cancer cell invasion and metastasis. However, the expression and role of CAPG in renal fibrosis caused by renal injury remain understudied.Although CAPG has been reported to be upregulated in renal cancer, the expression and role of CAPG in renal cancer cannot be inferred to renal fibrosis caused by renal injury because renal cancer and renal fibrosis caused by renal injury are different pathophysiological processes. For example, programmed cell death 1 ligand 1 (PD-L1) was found to be expressed at an elevated level in renal cancer cells, and PD-1 / L1-based immunotherapy has been recommended for the first-line treatment of advanced renal cancer. However, the incidence of renal injury caused by PD-1 / L1 treatment in patients with renal cancer and other tumors is as high as 2.2%, and some patients even 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. KidneyInternational. 2016;90 (3):638-647.); Sperm-associated antigen 9 (Sperm-associated antigen 9) CAPG antigen 9 (SPAG9) has been reported to be overexpressed in renal cancer and is a potential therapeutic target. However, in fibrotic renal tissue, SPAG9 expression was found to be downregulated and could inhibit renal fibrosis after renal injury (Maoqing Tian, ​​Lu Zhang, MengZhang, 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 end-stage renal disease caused by various causes of renal damage, and the rate of progression of renal fibrosis determines the rate of renal function loss. Delaying the progression of renal fibrosis is the key to protecting renal function. There have been no reports on the expression and role of CAPG in the study of renal fibrosis caused by renal injury. The research of this invention provides a new approach for the treatment of chronic kidney disease. Summary of the Invention

[0004] The main purpose of the present invention is to provide a new treatment plan for treating renal fibrosis caused by renal injury. Specifically, the present invention proposes an embodiment: the use of a substance that inhibits the activity and / or expression of the CAPG gene or protein in the preparation of a product for treating renal fibrosis caused by renal injury.

[0005] Based on the treatment scheme proposed by the present invention, on the other hand, the present invention also proposes another embodiment, which is the use of a reagent for detecting the activity and / or expression of the CAPG gene or protein in the preparation of a product for detecting renal fibrosis caused by renal injury. In this embodiment, the detection of CAPG expression can assist in evaluating the efficacy of targeted CAPG treatment products for diseases related to renal fibrosis caused by renal injury.

[0006] Furthermore, the type of renal injury includes acute kidney injury (AKI) or chronic kidney disease (CKD).

[0007] Furthermore, the causative factors of chronic kidney disease include diseases that lead to renal fibrosis, such as minimal change disease (MCD), IgA nephropathy (IgAN), lupus nephritis (LN), diabetic kidney disease (DKD) or hypertensive renal disease (HRD).

[0008] Furthermore, the renal injury includes renal injury caused by folic acid intake or activation of the TGF-β signaling pathway.

[0009] Furthermore, the substance that inhibits CAPG gene expression is a substance that silences, interferes with, knocks out or knocks down CAPG gene expression.

[0010] Furthermore, under the scheme proposed by the present invention, the use of existing technologies such as some embodiments of miRNA, siRNA, dsRNA or shRNA designed according to the CAPG gene as substances that interfere with or silence CAPG gene expression can achieve the purpose of the present invention of inhibiting CAPG gene expression and thus treating renal fibrosis caused by kidney injury.

[0011] Furthermore, as other specific embodiments, the substance for knocking out the expression of the CAPG gene is a gene editing tool such as a ZFN gene editing system, a TALENs gene editing system or a CRISPR / Cas9 gene editing system.

[0012] Further, as some embodiments, the present invention prepares a product for detecting renal fibrosis caused by renal injury, including a test kit, a test paper or a gene chip. The test kit has a physical form, for example, the test kit can be a container with one or more spaces, and the one or more spaces are used to accommodate materials or devices for controls, standards, and detection reagents. Reagents, tools and / or instructions for performing the methods described herein can be provided in the test kit. The test 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 the target gene or protein in the patient sample.

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

[0014] Preferably, the immunohistochemistry method includes: immunofluorescence analysis, reverse enzyme-linked immunosorbent assay or immunocolloidal gold method and other detection techniques.

[0015] Furthermore, the subject includes rodents, rabbits, pigs, non-human primates or humans; the sample includes blood, tissue, cell samples, urine or feces from the subject. In the present invention, the term "subject" includes patients and non-patients. "Subject" and "individual" are used interchangeably. The term "patient" refers to an individual who suffers from or may suffer from a medical condition such as inflammation or inflammatory disease, while "non-patient" refers to an individual who does not suffer from or may not suffer from the medical condition. "Non-patients" include healthy individuals, non-diseased individuals, and / or individuals without the medical condition. The term "subject" includes mammals, specifically humans and animals. Animals include rodents, rabbits, pigs, non-human primates, etc. "Rodent" refers to any mammal from the family Muridae, such as mice, rats, etc.

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

[0017] The technical effects achieved by the present invention are:

[0018] The present invention first discovered through research that CAPG is expressed at low levels in normal renal tissue, but is expressed at elevated levels in the renal tissue of patients with acute kidney injury, minimal change disease, IgA nephropathy, lupus nephritis, and diabetic nephropathy, suggesting that CAPG expression may be involved in the development and progression of renal fibrosis caused by renal injury. Further, by constructing a CAPG knockout mouse model, it was found that knocking out CAPG effectively mitigated the progression of renal fibrosis. In vitro culture of renal fibroblasts revealed that silencing or knocking down CAPG inhibited TGF-β-induced renal fibroblast activation. This invention has important implications for the pathogenesis and treatment of renal injury or renal fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0020] Figure 2 The expression level of CAPG in the kidney of CKD mice induced by folic acid. A. Immunohistochemical staining of CAPG at different times after folic acid induction ( Figure 2 A); B. Statistics Figure 2 The cumulative optical density in A reflects the protein expression level of CAPG: comparison of the expression levels of CAPG in the kidney at different times after folic acid induction.

[0021] Figure 3 The figure shows the effect of CAPG knockdown / knockout on renal fibrosis in folic acid-induced CKD mice. Wild-type control mice and CAPG knockdown / knockout mice were intraperitoneally injected with folic acid to induce CKD. The mice were killed 28 days after induction and the kidneys were harvested for analysis; A, Immunofluorescence detection of CAPG + / + Wild-type mice, Capg + / - Knockdown and Capg - / - The protein expression level of CAPG in the kidney of knockout mice. B. Masson and Sirius red staining of kidney to detect CAPG + / + Wild-type mice, Capg + / - Knockdown and Capg - / - Collagen deposition in the kidneys of knockout mice; C, western blot detection of Capg + / + Wild-type mice and Capg + / - Knockdown of FN and Col in mouse kidney tissue , CAPG expression level; D, western blot detection of Capg + / + Wild-type mice and Capg - / - The expression levels of FN and α-SMA in the kidney tissue of knockout mice.

[0022] Figure 4 Figure 1 shows the detection of CAPG knockdown in renal fibroblasts. (A) The renal fibroblast line NRK-49F was transfected with three CAPG-siRNAs (si#1, si#2, and si#3), the transfection reagent control Lipo, the negative control Ctrl, the positive control Pos, and the blank control NC, and then cultured for 36 or 48 hours to determine the protein expression level of CAPG.

[0023] Figure 5 Figure 1 shows the effect of CAPG knockdown on fibroblast fibrosis in renal fibroblasts. (A) CAPG expression was downregulated in the renal fibroblast line NRK-49F by CAPG-siRNA administration, and the changes in FN, Col I, and α-SMA protein levels in CAPG-knockdown NRK-49F cells; (B) Changes in FN, Col I, and α-SMA protein levels in CAPG-knockdown NRK-49F cells under TGF-β stimulation.

[0024] Figure 6 Schematic diagram of the design for editing the CAPG gene using CRISPR / Cas9 technology. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the concept and technical effects of this application in conjunction with the embodiments to fully understand the purpose, features and effects of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of this application.

[0026] For purchased commodities in the test method, if the specific conditions are not specified, the test shall be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they can be conventional products obtained from the market.

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

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

[0029] 1. CAPG is highly expressed in renal tissue of patients with acute kidney injury and chronic kidney disease caused by various etiologies

[0030] 1. Experimental materials and methods

[0031] Renal biopsies from patients with a pathological diagnosis of acute kidney injury or primary or secondary renal disease were collected from the First Affiliated Hospital of Sun Yat-sen University. Normal renal tissue served as a control. Immunohistochemical analysis of CAPG was performed on renal tissue sections.

[0032] 2. Test results

[0033] like Figure 1 As shown in Figure 2, immunohistochemical analysis revealed that CAPG was expressed at low levels in normal renal tissue, but its expression was elevated in renal tissue from patients with acute kidney injury, minimal change disease, IgA nephropathy, lupus nephritis, and diabetic nephropathy. These results suggest that CAPG expression may be involved in the development and progression of renal fibrosis caused by renal injury.

[0034] Example 2: CAPG is highly expressed in the kidney tissue of a chronic kidney disease mouse model

[0035] 1. Construction of a folic acid-induced chronic kidney disease mouse model

[0036] 1. Experimental materials and methods

[0037] 6-8 week-old wild-type C57BL / 6 male mice were used as experimental materials. Following the method described in the literature, "Suppressed Mitochondrial Biogenesis in Folic Acid-Induced Acute Kidney Injury and Early Fibrosis" (DOI: 10.1016 / j.toxlet.2013.11.014), folic acid (FA) solution was injected intraperitoneally to induce renal fibrosis following renal injury. This FA-induced renal fibrosis mouse model was established. Mice that received an intraperitoneal injection of 0.3 M sodium bicarbonate solution served as a control group.

[0038] The mice were killed 2 days, 7 days and 28 days after folic acid injection and kidney tissues were obtained to detect the expression level of CAPG in the kidneys of FA mouse model.

[0039] 2. Test results

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

[0041] The above research results showed that CAPG expression increased in the renal tissue of mice with FA-induced renal fibrosis.

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

[0043] 1. Source and Construction of CAPG Knockdown and Knockout Mice

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

[0045] Capg + / - The knockdown mice were purchased from Jiangsu Jicui Yaokang Biotechnology (Nanjing) and the strain was named C57BL / 6JGpt-Capg em3Cd10097 / Gpt, item number T029492, and bred by Jiangsu Jicui Yaokang Biological.

[0046] The mouse uses CRISPR / Cas9 technology to edit the Capg gene, which has 11 transcripts, such as Figure 6 As shown, according to Capg Regarding the gene structure, exon 2 to exon 9 of the CAPG-202 (ENSMUST00000114071.7) transcript was used as the knockout region. This region contains the start codon ATG, and knocking out this region will lead to the loss of CAPG protein function.

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

[0048] Capg + / - Mice were self-crossed to obtain wild-type mice and Capg mice born from the same litter. - / - Knockout mice (homozygous).

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

[0050] (1) Cut off about 0.5 cm from the end of the mouse's tail, place the tail into a numbered 200 μL centrifuge tube and cover it.

[0051] (2) Add 100 μL of lysis buffer and 2 μL of 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 buffer. Centrifuge at 12,000 rpm for 5 minutes, and use the supernatant as a PCR template.

[0052] (3) Preparation of PCR system

[0053] Table 1 Primer information

[0054]

[0055] Table 2 PCR system

[0056] PCR reaction components 25μL reaction system (μL) ddH2O 9.5 Forward primer (10 μM) 1 Reverse primer (10 μM) 1 Template (digestion product) 1 2 x M-PCR OPTI Mix 12.5

[0057] Table 3 PCR program

[0058]

[0059] (4) PCR product identification

[0060] The PCR products were spotted and subjected to agarose gel electrophoresis. The cells with only PCR product ① were homozygous, those with only PCR product ② were wild type, and those with both PCR products ① and ② were heterozygous.

[0061] Effects of CAPG knockdown / knockout on renal fibrosis in a folic acid-induced chronic kidney disease mouse model

[0062] 1. Experimental materials and methods

[0063] Wild-type male control mice (Capg + / + )、Capg + / - Knockdown of male mice, Capg - / - A mouse model of FA-induced renal fibrosis was established using knockout male mice. Twenty-eight days after folic acid injection, mice were sacrificed and kidney tissue was obtained to examine and analyze the effects of CAPG knockdown or knockout on FA-induced renal fibrosis.

[0064] 2. Test results

[0065] like Figure 3 As shown, Masson and Sirius red staining can be seen, and Capg + / + Compared with control mice, Capg + / - and Capg - / - The collagen deposition of mice was significantly reduced. + / - and Capg - / - The expression levels of fibrosis markers in mice were higher than those in Capg + / +Downregulation of proteins in control mice included FN, Col I, and α-SMA.

[0066] The above results suggest that knockdown / knockout of CAPG can effectively alleviate the progression of FA-induced renal fibrosis.

[0067] Example 4: Knockdown of CAPG in renal fibroblasts improves renal fibrosis

[0068] 1. Knockdown of CAPG in Renal Fibroblasts

[0069] 1. Experimental materials and methods

[0070] 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 renal fibrosis, and their main cell sources are fibroblasts and pericytes.

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

[0072] 1) Cell plating: Seed well-grown NRK-49F cells into 6-well plates and culture overnight.

[0073] 2) Cell Transfection: Prior to cell transfection, the culture medium in the wells was replaced with serum-free medium. Three synthetic siRNAs targeting the CAPG gene (si#1, si#2, and si#3), a negative control siRNA (Ctrl), and a positive control siRNA (Pos) were then incubated with Lipofectamine 3000 to prepare complexes. The complexes were incubated at room temperature for 15 minutes before addition to the corresponding wells. Untreated wells were designated as blank controls (NC), and Lipofectamine 3000 (Lipo) alone served as the transfection reagent control.

[0074] 3) After 6 hours of continuous transfection, aspirate the culture medium in the wells and add 1 ml of fresh culture medium containing 10% fetal bovine serum. Continue culturing for 36 hours or 48 hours, then scrape the cells for immunoblotting.

[0075] The siRNA used in the present invention is from Tuoran Biotechnology Co., Ltd. The siRNA sequences are shown in Table 4 below:

[0076] Table 4 siRNA sequence information

[0077]

[0078] 2. Test results

[0079] like Figure 4 As shown, immunoblotting showed that under normal culture conditions, compared with the control groups (blank control NC, transfection reagent control Lipo, negative control Ctrl, positive control Pos), the protein expression levels of CAPG in the three groups transfected with CAPG siRNA (si#1, si#2, si#3) were significantly decreased, and the CAPG knockdown efficiency at 48h was better than that 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 all three siRNAs targeting the CAPG gene can downregulate the protein expression level of CAPG in NRK-49F cells. Among them, sequence si#2 has the highest silencing efficiency after 48 hours of transfection into NRK-49F cells. This siRNA was selected as the effective siRNA in subsequent experiments.

[0081] Effect of CAPG knockdown on TGF-β-induced fibrotic phenotype of fibroblasts

[0082] 1. Experimental materials and methods

[0083] 1) Normal culture conditions: Negative control siRNA (Ctrl) and CAPG siRNA were transfected into NRK-49F cells, respectively. After 6-8 hours, the culture medium was replaced with normal culture medium. After 48 hours, the cells were scraped and used for immunoblotting.

[0084] 2) TGF-β stimulates NRK-49F cells to construct an in vitro fibroblast activation model. Conditions: Negative control siRNA (Ctrl) and CAPG siRNA were transfected into NRK-49F cells, respectively. After 6-8 hours, the culture medium was changed to normal medium. After 36 hours of culture, NRK-49F cells were stimulated with recombinant TGF-β (10 ng / mL) for 12 hours to construct an in vitro fibroblast activation model. The cells were harvested and tested to analyze the effect of CAPG knockdown on the TGF-β-induced fibroblast fibrosis phenotype.

[0085] 2. Test results

[0086] like Figure 5As shown in the figure, immunoblotting revealed that under normal culture conditions, CAPG protein expression levels in NRK-49F cells transfected with CAPG siRNA were significantly decreased compared with the control group. Furthermore, in NRK-49F cells knocked down by CAPG using siRNA, FN and Col I expressions were significantly decreased compared with the control group, while α-SMA expression did not show significant differences. Under TGF-β stimulation, FN, Col I, and α-SMA levels in CAPG knockdown cells were significantly decreased compared with the control group. This suggests that downregulating CAPG can inhibit TGF-β-induced fibroblast activation.

[0087] These results suggest that specific knockdown of CAPG in fibroblasts under normal culture conditions can downregulate the expression of FN and Col I in fibroblasts, but has no significant effect on α-SMA expression. Under TGF-β stimulation, specific knockdown of CAPG in fibroblasts can inhibit TGF-β-induced fibroblast activation.

Claims

1. Use of a substance that inhibits the activity and / or expression of CAPG gene or protein in the preparation of a product for treating renal fibrosis caused by renal injury, characterized in that: The substance is a gene editing system for knocking down / knocking out the CAPG gene using siRNA-370, siRNA-1201, siRNA-853 or CRISPR / Cas9 technology; the siRNA-370 sequence is ACAUAUGGCGUGUGGAGAATT, UUCUCCACACGCCAUAUGUTT; the siRNA-1201 sequence is GGAGAAAAGCCAAUGAGAATT, UUCUCAUUGGCUUUUCUCCTT; and the siRNA-853 sequence is CCAACAUCCUGGAGCGCAATT, UUGCGCUCCAGGAUGUUGGTT.

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

3. The use according to claim 2, characterized in that The causative factors of chronic kidney disease include minimal change disease (MCD), IgA nephropathy (IgAN), lupus nephritis (LN), diabetic nephropathy (DKD) or hypertensive nephropathy (HRD).

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