Application of PPP3CA in treatment of renal fibrosis

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of PPP3CA in renal fibrosis treatment. Researches find that the expression of PPP3CA in fibrotic human renal tubular epithelial cells is up-regulated, and the expression level of PPP3CA in fibrotic kidney tissues of mice is up-regulated; and the renal fibrosis can be obviously relieved by constructing a cell knock-down PPP3CA model and a renal tubule specific PPP3CA knockout mouse to interfere the expression of the renal tubule PPP3CA. Therefore, targeted inhibition of PPP3CA can be applied to prevention, alleviation and treatment of renal fibrosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the application of PPP3CA in the treatment of renal fibrosis. Background Art

[0002] Renal fibrosis is caused by excessive deposition of extracellular matrix (ECM), which leads to the destruction of tissue structure and function, and eventually develops into chronic kidney disease or even end-stage renal disease. Its mechanism of action is complex, but there is currently no targeted drug 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 this target have important research significance and clinical value.

[0003] Calcineurin is a serine / threonine-specific protein phosphatase that is widely present in eukaryotic organisms and regulates a variety of cell-related processes and signaling pathways in the body in a calcium- and calmodulin-dependent manner. PPP3CA, as a type of calcineurin, is mainly enriched in the heart, brain, and kidneys. In the kidney, PPP3CA is mainly expressed in the proximal tubules, which account for 80% of all tubules. The proximal tubule epithelial cells are highly energy-consuming and highly dependent on the energy provided by mitochondrial oxidative phosphorylation. They are the main site of renal energy metabolism and are easily stimulated by ischemia, hypoxia, toxins, and elevated blood sugar. They are the main site of acute kidney injury and chronic kidney disease, and are also the main site of fibrosis. PPP3CA is time-dependently upregulated in the renal tubules of rats with diabetic nephropathy induced by streptozotocin, but whether PPP3CA is involved in the process of renal fibrosis and its role and specific mechanism in renal fibrosis are still unclear. Summary of the invention

[0004] In view of the problems and shortcomings in the prior art, the purpose of the present invention is to provide an application of PPP3CA 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, wherein the biomarker is the PPP3CA gene or the PPP3CA protein.

[0007] Western Blot was used to detect the expression of PPP3CA in human renal tubular epithelial cells (HK-2) that underwent fibrotic changes induced by TGFβ1. It was found that the expression level of PPP3CA 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 immunohistochemical staining were used to detect that the expression level of PPP3CA in the fibrotic renal tissue of UUO (unilateral ureteral obstruction) mice was significantly higher than that in sham-operated mice, and the difference was statistically significant.

[0008] Western Blot and immunohistochemical staining detected that the expression level of PPP3CA in the fibrotic renal tissue of UUO (unilateral ureteral obstruction) mice was significantly higher than that in sham-operated mice, and the difference was statistically significant.

[0009] The second aspect of the present invention provides the use of a detection reagent for PPP3CA gene or PPP3CA protein in the preparation of a renal fibrosis auxiliary diagnosis product.

[0010] According to the above application, preferably, the product detects the expression level of PPP3CA gene or PPP3CA protein in a sample by RT-PCR, real-time quantitative PCR, in situ hybridization, Northern Blot, Western Blot, chip, high-throughput sequencing platform, immunohistochemistry or enzyme-linked immunosorbent assay.

[0011] According to the above application, preferably, the product contains an antibody that specifically binds to the PPP3CA protein, or contains primers that specifically amplify the PPP3CA gene, or contains a probe that specifically detects the PPP3CA gene.

[0012] According to the above application, preferably, the antibody is a monoclonal antibody, a polyclonal antibody or a single domain antibody.

[0013] According to the above application, preferably, the detection sample of the product is cells, tissues or serum.

[0014] According to the above application, preferably, the product is a chip, a preparation or a kit.

[0015] A third aspect of the present invention provides a kit for auxiliary diagnosis of renal fibrosis, wherein the kit contains a detection reagent for the PPP3CA gene or the PPP3CA protein.

[0016] According to the above-mentioned kit, preferably, the detection reagent is a reagent for detecting the expression level of PPP3CA gene or PPP3CA protein in a sample by RT-PCR, real-time quantitative PCR, in situ hybridization, Northern Blot, Western Blot, chip, high-throughput sequencing platform, immunohistochemistry or enzyme-linked immunosorbent assay.

[0017] According to the above-mentioned kit, preferably, the detection reagent is an antibody that specifically binds to the PPP3CA protein, or contains primers that specifically amplify the PPP3CA gene, or contains a probe that specifically detects the PPP3CA gene.

[0018] A fourth aspect of the present invention provides use of a PPP3CA inhibitor in the preparation of a medicament for preventing, alleviating or / and treating renal fibrosis.

[0019] According to the above application, preferably, the PPP3CA inhibitor is one or more substances that inhibit PPP3CA gene expression, silence PPP3CA gene, knock out PPP3CA gene, reduce PPP3CA protein content, and reduce PPP3CA protein activity.

[0020] The inhibition of PPP3CA gene expression, silencing of PPP3CA gene, and knocking out of PPP3CA gene can be achieved by gene mutation, gene silencing, gene knockout, gene editing, or gene knockdown techniques known to those skilled in the art. For example, RNA interference (RNAi) technology can be used to specifically eliminate or shut down the expression of a specific gene; the tool using gene editing technology can be CRISPR / Cas9 technology, zinc finger nuclease (ZFNs) or transcription activator-like effector nuclease (TALENs) technology, etc., but is not limited thereto.

[0021] The technology of inactivating PPP3CA gene expression or silencing the gene at the post-transcriptional level or translational level by using gene knock-down technology is well known to those skilled in the art. The gene knock-down technology includes RNA interference, Morpholino interference, antisense nucleic acid, ribozyme or dominant negative inhibitory mutation but is not limited thereto.

[0022] It is well known to those skilled in the art that PPP3CA gene silencing is performed by inhibiting the expression of PPP3CA gene using shRNA or siRNA expressed by viruses (such as lentivirus, adeno-associated virus).

[0023] According to the above application, preferably, the substance is one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, polypeptides, proteins, gene editing vectors, lentiviruses or adeno-associated viruses.

[0024] According to the above application, preferably, the nucleic acid molecule includes shRNA, microRNA, siRNA and / or antisense oligonucleotide.

[0025] According to the above application, preferably, the target sequence of the siRNA is 5′-GCAGTAATAGCAGCAATAT-3′ (as shown in sequence 1)′ or 5′-GCCGTTCCATTTCCACCAA-3′ (as shown in sequence 3).

[0026] According to the above application, preferably, the antibody is an antibody against PPP3CA protein, and the lentivirus or adeno-associated virus is a recombinant lentivirus or recombinant adeno-associated virus expressing the siRNA.

[0027] A fifth aspect of the present invention provides a medicine comprising a PPP3CA inhibitor, and the medicine is used for preventing, alleviating or / and treating renal fibrosis.

[0028] According to the above-mentioned drug, preferably, the PPP3CA inhibitor is one or more substances that inhibit PPP3CA gene expression, silence PPP3CA gene, knock out PPP3CA gene, reduce PPP3CA protein content, and reduce PPP3CA protein activity.

[0029] The inhibition of PPP3CA gene expression, silencing of PPP3CA gene, and knocking out of PPP3CA gene can be achieved by gene mutation, gene silencing, gene knockout, gene editing, or gene knockdown techniques known to those skilled in the art. For example, RNA interference (RNAi) technology can be used to specifically eliminate or shut down the expression of a specific gene; the tool using gene editing technology can be CRISPR / Cas9 technology, zinc finger nuclease (ZFNs) or transcription activator-like effector nuclease (TALENs) technology, etc., but is not limited thereto.

[0030] The technology of inactivating PPP3CA gene expression or silencing the gene at the post-transcriptional level or translational level by using gene knock-down technology is well known to those skilled in the art. The gene knock-down technology includes RNA interference, Morpholino interference, antisense nucleic acid, ribozyme or dominant negative inhibitory mutation but is not limited thereto.

[0031] It is well known to those skilled in the art that PPP3CA gene silencing is performed by inhibiting the expression of PPP3CA gene using shRNA or siRNA expressed by viruses (such as lentivirus, adeno-associated virus).

[0032] According to the above-mentioned medicine, preferably, the substance is one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, peptides, proteins, gene editing vectors, lentiviruses or adeno-associated viruses.

[0033] According to the above-mentioned drug, preferably, the nucleic acid molecule includes shRNA, microRNA, siRNA and / or antisense oligonucleotide.

[0034] According to the above-mentioned drug, preferably, the target sequence of the siRNA is 5′-GCAGTAATAGCAGCAATAT-3′ (as shown in sequence 1)′ or 5′-GCCGTTCCATTTCCACCAA-3′ (as shown in sequence 3).

[0035] According to the above-mentioned drug, preferably, the antibody is an antibody against PPP3CA protein, and the lentivirus or adeno-associated virus is a recombinant lentivirus or recombinant adeno-associated virus expressing the siRNA.

[0036] According to the above-mentioned medicine, preferably, the medicine further comprises one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers may be diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, adsorption carriers, surfactants or lubricants.

[0037] In a sixth aspect, the present invention provides an application of a PPP3CA gene or a PPP3CA protein in screening candidate drugs for preventing, alleviating or / and treating renal fibrosis. The screening method comprises the following steps: screening drugs or agents to be screened using PPP3CA as a target, and selecting drugs or agents that can reduce the expression level of the PPP3CA gene, reduce the content of the PPP3CA protein or reduce the activity of the PPP3CA protein as candidate drugs for preventing, alleviating or / and treating renal fibrosis.

[0038] The seventh aspect of the present invention provides a method for constructing a renal tubule-specific PPP3CA knockout mouse, the construction method comprising: introducing a heterozygous mouse expressing Pax8-rtTA and tet-on LC-1 into a PPP3CA flox / flox Homozygous mice were crossed to generate doxycycline-induced tubule-specific PPP3CA gene knockout mice; the genotype of the tubule-specific PPP3CA gene knockout mice was pax8-cre / PPP3CA flox / flox .

[0039] The renal fibrosis described in the present invention is renal fibrosis caused by various kidney diseases.

[0040] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0041] (1) The present invention first discovered that PPP3CA is upregulated in fibrotic human renal tubular epithelial cells, and that PPP3CA is upregulated in mouse fibrotic renal tissue. Therefore, PPP3CA can be used as an auxiliary diagnostic indicator for renal fibrosis to assist in the diagnosis of renal fibrosis. Moreover, the ROC curve AUC of the present invention using PPP3CA to diagnose and distinguish between renal fibrosis mice and normal control mice can reach 0.778, indicating that PPP3CA has a high diagnostic value for auxiliary diagnosis of renal fibrosis.

[0042] (2) The present invention uses PPP3CA-specific small interfering RNA (si-PPP3CA) to inhibit PPP3CA expression, which can significantly reverse the fibrosis process of human renal tubular epithelial cells induced by TGFβ1 in vitro; moreover, the present invention inhibits PPP3CA expression or activity by constructing renal tubule-specific PPP3CA knockout mice, which can significantly alleviate renal fibrosis. Therefore, PPP3CA can be used as a drug, drug target or target gene in gene therapy, and applied to the prevention, alleviation and treatment of renal fibrosis, which can provide a new strategy for the prevention and treatment of renal fibrosis, and at the same time, it will also provide a new direction for further research on the pathological mechanism and prevention and treatment strategies of renal fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The Western Blot detection results of the expression level of PPP3CA in the TGFβ1-induced human renal tubular epithelial cell fibrosis model; A is a representative band image of Western Blot detection of PPP3CA; B is a figure showing the quantitative analysis results of Western Blot of PPP3CA;

[0044] Figure 2 The results of the expression of PPP3CA in the kidney of a mouse fibrosis model induced by unilateral ureteral obstruction are shown in Figure 2A. Figure 2A is a HE staining image of kidney tissue. Figure 2B is a Masson staining image of kidney tissue. Figure 2C is an immunohistochemical image of PPP3CA in kidney tissue. Figure 2D is a representative band image of Western Blot detection of PPP3CA and fibrosis marker α-SMA. Figure 2 EF is the quantitative analysis diagram of Western Blot detection of α-SMA and PPP3CA;

[0045] Figure 3 The ROC curve diagram for the value evaluation of PPP3CA in the diagnosis of renal fibrosis;

[0046] Figure 4 This is the result of Western Blot detection of PPP3CA knockdown;

[0047] Figure 5The experimental results show the effect of knocking down PPP3CA on the TGFβ1-induced human renal tubular epithelial cell fibrosis model; Figure A shows the representative band images of Western Blot detection of PPP3CA, PPP3CB, FN1, vimentin, and α-SMA expression in the TGFβ1-induced human renal tubular epithelial cell fibrosis model after knocking down PPP3CA; Figures BF show the quantitative analysis of Western Blot detection of PPP3CA, PPP3CB, FN1, vimentin, and α-SMA; Figure G shows the immunofluorescence detection result of FN1;

[0048] Figure 6 The construction and verification results of tubular-specific PPP3CA knockout mice: A is a schematic diagram of the hybridization process for the construction of tubular-specific PPP3CA knockout mice; B is a diagram showing the genotype identification results of tubular-specific PPP3CA knockout mice by agarose gel electrophoresis; C is a representative band image of Western Blot detection of PPP3CA in the kidney of tubular-specific PPP3CA knockout mice; Figure D is a representative band image of Western Blot detection of PPP3CA in the heart, liver, spleen, and lung of tubular-specific PPP3CA knockout mice; Figure E is a diagram showing the immunofluorescence results of PPP3CA in the kidney of tubular-specific PPP3CA knockout mice;

[0049] Figure 7 The results of renal pathological changes in tubular-specific PPP3CA knockout mice after unilateral ureteral obstruction; A is a schematic diagram of the design of the UUO modeling experiment in tubular-specific PPP3CA knockout mice; B is the results of HE, Masson, and Sirius Red pathological staining in tubular-specific PPP3CA knockout mice after unilateral ureteral obstruction;

[0050] Figure 8 These are immunofluorescence images and immunohistochemistry images of PPP3CA expression and changes in fibrosis phenotype indicators in the kidneys of tubular-specific PPP3CA knockout mice after unilateral ureteral obstruction, among which A is the immunofluorescence result of PPP3CA, FN1, and α-SMA in tubular-specific PPP3CA knockout mice after UUO; B is the immunohistochemistry result of Collegen I and Collegen III in tubular-specific PPP3CA knockout mice after UUO;

[0051] Fig. 9The Western Blot results of the changes in PPP3CA expression and fibrosis phenotype indicators in the kidneys of mice with tubular-specific PPP3CA knockout after unilateral ureteral obstruction are shown in Figure 1. Figure A is a representative band image of PPP3CA, FN1, α-SMA, and vimentin detected by Western Blot in mice with tubular-specific PPP3CA knockout after UUO. Figure BE is a quantitative analysis of FN1, α-SMA, vimentin, and PPP3CA detected by Western Blot.

[0052] Fig.10 The figure shows the effect of tubular-specific PPP3CA knockout on the kidneys of the mouse model of renal fibrosis induced by ischemia-reperfusion; A is the design diagram of the IRI modeling experiment in tubular-specific PPP3CA knockout mice; B is a representative picture of Western Blot detection of FN1 in tubular-specific PPP3CA knockout mice after IRI; Figure C is a quantitative analysis of Western Blot detection of FN1; D is the immunofluorescence detection results of FN1 and α-SMA in tubular-specific PPP3CA knockout mice after IRI. DETAILED DESCRIPTION

[0053] 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 in conjunction with specific embodiments.

[0054] 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 meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0055] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also 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, it indicates the presence of features, steps, operations, components and / or combinations thereof.

[0056] The experimental methods in the following examples without specifying specific conditions all adopt conventional techniques in the art or follow the conditions recommended by the manufacturers; the reagents or instruments used without specifying the manufacturers are all conventional products that can be obtained commercially.

[0057] Example 1: Study on the expression level of PPP3CA in TGFβ1-induced human renal tubular epithelial cells

[0058] Western Blot was used to detect the expression level of PPP3CA in TGFβ1-induced human renal tubular epithelial cells and normal human renal tubular epithelial cells.

[0059] 1. Human renal tubular epithelial cell culture:

[0060] Human renal tubular epithelial cells HK-2 were purchased from the Shanghai ATCC database. When the cell density reached 80%-90%, the original culture medium in the culture dish was discarded, and the cells were rinsed twice with 1ml PBS. 1ml 0.25% trypsin was added for digestion for 30s-1min. Most of the cells were blown slightly with a gun until they fell off, and then transferred to a 15ml centrifuge tube. Then 1-2ml complete culture medium was added to the culture dish, and the cells were added to the 15ml centrifuge tube after continuous blowing to terminate digestion. The centrifuge was centrifuged at 1000rpm for 5min. The supernatant was discarded, and 2ml complete culture medium was added to resuspend the cells. The cells were inoculated in a 60mm culture dish according to the proportion, and the cells were gently shaken horizontally in a clean bench to mix well. The cells were transferred to a 37°C incubator with 5% CO2 for further culture.

[0061] 2. Experimental methods:

[0062] Human renal tubular epithelial cells HK-2 were collected and evenly plated in 6-well plates, with 1×10 5 cells. The cells were divided into 5 groups: Control group, TGFβ1 treatment 6h group, TGFβ1 treatment 12h group, TGFβ1 treatment 24h group, TGFβ1 treatment 48h group. On the second day, after the cell morphology was completely restored, TGFβ1 solution was added (the solvent was citric acid, the concentration of TGFβ1 solution was 10ug / mL, it needed to be diluted 1000 times, and the final concentration was 10ng / mL). The same dose of control solvent (citric acid) was added to the Control group. After the corresponding time, the cells were collected and Western Blot was used to detect the changes in the protein expression of PPP3CA in the cells of the Control group, TGFβ1 treatment 6h group, TGFβ1 treatment 12h group, TGFβ1 treatment 24h group, and TGFβ1 treatment 48h group. Among them, the TGFβ1 solution was prepared as follows: TGFβ1 powder was centrifuged to the bottom of the tube instantly, 100μl of citric acid was added first, and after it was dissolved, 900μl of trehalose was added and mixed evenly. The steps of Western Blot detection are as follows: prepare 10% concentration electrophoresis gel, load the sample, perform electrophoresis at a constant voltage of 120V for 1 hour, transfer the membrane at a constant current of 300mA for 1 hour, block with 5% milk for 1 hour, incubate with primary and secondary antibodies, and expose with an odyssey protein developer.

[0063] 3. Experimental results:

[0064] Western Blot test results Figure 1As shown in the figure, PPP3CA was upregulated in a time-dependent manner in the TGFβ1-induced human renal tubular epithelial cell fibrosis model, and the difference was statistically significant, indicating that PPP3CA was involved in the TGFβ1-induced fibrosis process of human renal tubular epithelial cells.

[0065] Example 2: Study on the expression level of PPP3CA in the kidney tissue of mice with renal fibrosis induced by unilateral ureteral obstruction

[0066] 1. Selection of experimental animals and feeding conditions:

[0067] SPF grade 6-7 week old C57BL / 6 male mice weighing about 18 g were selected and purchased from Southern Model Organisms Science Co., Ltd. The animals had free access to food and water, 12 hours of light and 12 hours of darkness per day, temperature controlled at 18-23°C, humidity maintained at 40%-60%, and were fed with normal mouse chow.

[0068] 2. Experimental groups and experimental methods:

[0069] 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.

[0070] Method for constructing a mouse model of renal fibrosis due to unilateral ureteral obstruction: anesthetize the mouse with an isoflurane gas anesthesia machine, make it lie on its back, disinfect the abdominal waist area with alcohol, pick up the skin on the left waist with curved forceps, and cut the skin and subcutaneous tissue vertically in sequence (about 2-3 cm). Find the ureter at the lower end of the left kidney, separate the surrounding tissue with toothless curved forceps, pass the pre-cut 6-0 suture through the ureter, ligate the upper and lower parts of the middle section of the ureter, and cut the ureter between the two ligature points. Use a suture needle to suture the subcutaneous tissue and skin in sequence, and keep the suture as short as possible to prevent the mouse from biting. Disinfect the abdominal waist again with alcohol to remove the bloody smell. Place the mouse on a 37°C heating pad, and put it in a cage after it recovers from anesthesia. Observe the mouse's condition on the night of surgery and the next day.

[0071] One week after the model was established, mice in each group were killed and kidney tissues were collected. Part of the kidney tissue was fixed with 4% paraformaldehyde for 48 h and then embedded in paraffin. The tissues were cut into 4 μM thick sections and HE and Masson staining were performed to detect the fibrosis of the kidney tissues of UUO mice to evaluate whether the construction of the UUO-induced mouse renal fibrosis model was successful. The remaining kidney tissues were quickly frozen in liquid nitrogen and stored at -80 °C. Western Blot was used to detect the expression of PPP3CA and fibrosis marker α-SMA in the fibrotic kidney tissues of UUO mice. Immunohistochemical staining was used to detect the expression changes of PPP3CA in the fibrotic kidney tissues of UUO mice.

[0072] 3. Experimental results:

[0073] (1) HE staining, Masson staining:

[0074] The results of HE staining and Masson staining are as follows Figure 2 As shown in A and B. Figure 2 As shown in Figures A and B, compared with the mice in the Sham group, the renal tubules of the mice in the UUO group were significantly dilated, a large number of inflammatory cells infiltrated the tubular interstitium, and obvious collagen fiber deposition, indicating that the renal fibrosis model of UUO mice was successfully established.

[0075] (2) Immunohistochemical staining:

[0076] The results of immunohistochemical staining to detect the expression changes of PPP3CA in the renal tissue of UUO mice with fibrosis are as follows: Figure 2 As shown in C.

[0077] Depend on Figure 2 As shown in middle C, after UUO treatment, PPP3CA expression increased mainly in the renal tubules of mice.

[0078] (3) Western Blot detection of PPP3CA and fibrosis marker α-SMA expression:

[0079] The results of Western Blot detection of PPP3CA and fibrosis marker α-SMA in kidney tissue of UUO mice are shown in the figure. Figure 2 As shown in D and E.

[0080] Depend on Figure 2 As shown in Figures D and E, compared with the Sham group mice, the expression of fibrosis marker α-SMA 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; at the same time, compared with the Sham group mice, the expression of PPP3CA in the kidney tissue of UUO mice was significantly increased, and the difference was statistically significant, indicating that PPP3CA may be involved in the process of renal fibrosis in vivo.

[0081] Example 3: Evaluation of the value of PPP3CA for the diagnosis of renal fibrosis

[0082] 1. Selection of experimental animals and feeding conditions:

[0083] SPF grade 6-7 week old C57BL / 6 male mice weighing about 18 g were selected and purchased from Southern Model Organisms Science Co., Ltd. The animals had free access to food and water, 12 hours of light and 12 hours of darkness per day, temperature controlled at 18-23°C, humidity maintained at 40%-60%, and were fed with normal mouse chow.

[0084] 2. Experimental groups and experimental methods:

[0085] 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 6 mice in each group. The UUO modeling method was the same as that in Example 2.

[0086] One week after the model was established, the mice in each group were killed and the kidney tissues were collected. A part of the kidney tissues was fixed with 4% paraformaldehyde for 48 hours and then embedded in paraffin. The sections were cut into 4 μM thick sections and immunohistochemical staining was used to detect the expression changes of PPP3CA in the kidney tissues of the UUO group and the Sham group. The experimental results were statistically analyzed, the ROC curve was drawn, and the AUC value was calculated to evaluate the value of PPP3CA in the diagnosis of renal fibrosis.

[0087] 3. Experimental results:

[0088] The immunohistochemical staining results of PPP3CA were statistically analyzed, and the ROC curve of PPP3CA diagnosis and differentiation between UUO group mice and Sham group mice was drawn. Figure 3 shown.

[0089] Depend on Figure 3 It can be seen that the ROC curve AUC value of PPP3CA for distinguishing the UUO group and the Sham group reached 0.778, which can effectively distinguish renal fibrosis mice from normal control mice. This shows that PPP3CA can be used for auxiliary diagnosis of renal fibrosis and has a high diagnostic value.

[0090] Example 4: Effect of knockdown of PPP3CA on TGFβ1-induced human renal tubular epithelial cell fibrosis model

[0091] 1. Knockdown of PPP3CA in human renal tubular epithelial cells:

[0092] (1) Experimental cells and culture methods

[0093] 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 discard, add 2 mL of 0.25% trypsin preheated at 37°C and digest for 2-3 minutes. Gently shake the culture dish to make the cells fall off, add 2 mL of fresh DMEM / F12 complete culture medium (10% FBS + 1% double antibody) to stop digestion. Collect the cells, centrifuge at 1000 rpm for 5 minutes at room temperature, discard the supernatant, resuspend the cells with fresh complete culture medium, add to the culture dish, and continue to culture in a 37°C, 5% CO2 incubator.

[0094] (2) PPP3CA knockdown experimental method

[0095] PPP3CA-specific small interfering RNA (si-PPP3CA) and negative control (si-Ctrl) were synthesized by Ruibo Biotechnology Co., Ltd.

[0096] The target sequence of si-PPP3CA-1 is: 5′-GCAGTAATAGCAGCAATAT-3′ (as shown in Sequence 1).

[0097] The target sequence of si-PPP3CA-2 is: 5′-GCCGTACTGATGTGGCTAT-3′ (as shown in Sequence 2).

[0098] The target sequence of si-PPP3CA-3 is: 5′-GCCGTTCCATTTCCACCAA-3′ (as shown in Sequence 3).

[0099] The target sequence of the negative control si-Ctrl is: 5′-GCGCTACTGTTGAGGCTAT-3′ (as shown in Sequence 4).

[0100] Taking the knockdown of PPP3CA gene by si-PPP3CA-1 as an example, the specific experimental method is as follows:

[0101] 1) Centrifuge the above si-PPP3CA-1 powder at 3000-4000rpm for 1min, add an appropriate amount of DEPC water to dissolve it according to the instructions to obtain si-PPP3CA-1 dilution. Add 150μl of serum-free and dual antibody-free culture medium to a 1.5ml EP tube, add 6μl of transfection reagent, mix gently, and let stand at room temperature for 5min. Add the culture medium with transfection reagent to the culture medium containing si-PPP3CA-1 dilution, mix gently, and let stand at room temperature for 18min to obtain transfection solution.

[0102] 2) Human renal tubular epithelial cells HK-2 were cultured in a 37°C, 5% carbon dioxide incubator using DMEM / F12 (10% FBS, 100 U / ml penicillin, 100 μg / mL streptomycin) medium. When the cell density reached 80%, the cells were digested with trypsin, resuspended in fresh medium, counted, and plated in 6-well plates. The number of cells per well was 1×10 5 , and culture in a cell culture incubator overnight. Aspirate the original culture medium in the 6-well plate, add 1 ml of culture medium without double antibody but containing serum, add the previously mixed transfection solution to each well, shake gently to mix, and transfer to a 37°C incubator with 5% CO2. On the second day (at least > 8h), replace with normal complete culture medium and observe whether the transfection is successful under a fluorescence microscope.

[0103] The experimental method for knocking down the PPP3CA gene using si-PPP3CA-2 and si-PPP3CA-3 is the same as the above-mentioned method for knocking down the PPP3CA gene using si-PPP3CA-1.

[0104] (3) Experimental results

[0105] The results of Western Blot detection of PPP3CA knockdown are as follows Figure 4 shown.

[0106] Depend on Figure 4 It can be seen that compared with si-Ctrl, the PPP3CA protein expression in the si-PPP3CA-1 and si-PPP3CA-3 treatment groups decreased significantly, indicating that PPP3CA was successfully knocked down in HK-2 cells. Among them, the PPP3CA knockdown efficiency in the si-PPP3CA treatment group was the highest. Therefore, si-PPP3CA was used to knock down the PPP3CA gene in subsequent experiments. 2. Effect of PPP3CA knockdown on the expression of FN1, α-SMA, and vimentin proteins, fibrosis-related phenotypic indicators induced by TGFβ1 in HK-2 cells:

[0107] (1) Experimental methods

[0108] The experimental groups were as follows: the HK-2 cells treated with PPP3CA knockdown were divided into PPP3CA knockdown group (si-PPP3CA) and PPP3CA knockdown + TGFβ1 treatment group (si-PPP3CA + TGFβ1), and the HK-2 cells treated with si-Ctrl were divided into control group (si-Ctrl) and TGFβ1 treatment group (si-Ctrl + TGFβ1). TGFβ1 solution (the solvent was citric acid, the concentration of TGFβ1 solution was 10ug / mL, it needed to be diluted 1000 times, and the final effective concentration was 10ng / mL) was added to the si-PPP3CA + TGFβ1 group and the si-Ctrl + TGFβ1 group, and the same dose of control solvent (citric acid) was added to the si-Ctrl group and the si-PPP3CA group, and the incubation continued for 24 hours. The cells were collected and lysed by RIPA and the total protein concentration was detected by BCA protein quantification kit. Western Blot was used to detect the effect of PPP3CA knockdown on TGFβ1-induced PPP3CA, PPP3CB, FN1, α-SMA, and vimentin protein expressions in HK-2 cells. Cell immunofluorescence experiments were used to detect the effect of PPP3CA knockdown and TGFβ1 addition on the changes in FN1 protein expression.

[0109] (3) Experimental results:

[0110] The results of Western Blot detection of the effect of PPP3CA knockdown on TGFβ1-induced protein expression of PPP3CA, PPP3CB, FN1, α-SMA, and vimentin in HK-2 cells are shown in the figure. Figure 5 Shown in AF.

[0111] Depend on Figure 5 It can be seen from AF that compared with the si-ctrl group, the expression of PPP3CA and fibrosis-related phenotype indicators FN1, α-SMA, and vimentin proteins in HK-2 cells in the si-ctrl+TGFβ1 group were significantly increased, and the difference was statistically significant, indicating that PPP3CA was upregulated in HK-2 cells induced by TGFβ1-induced fibrosis; at the same time, compared with the si-ctrl+TGFβ1 group, after knocking down PPP3CA, the expression of fibrosis-related phenotype indicators FN1, α-SMA, and vimentin proteins in HK-2 cells was significantly decreased, and the difference was statistically significant, indicating that PPP3CA knockdown can significantly reduce the expression of FN1 and α-SMA proteins induced by TGFβ1.

[0112] The results of cell immunofluorescence assay are shown in Figure 5 As shown in G. Figure 5 As shown in G, the fluorescence intensity of FN1 increased in the si-ctrl+TGFβ1 group, while that in the si-PPP3CA+TGFβ1 group decreased. The experiment showed that PPP3CA knockdown could significantly reverse the fibrosis process of HK-2 cells induced by TGFβ1 in vitro.

[0113] Example 5: Changes in PPP3CA and fibrosis phenotype indicators in the renal fibrosis model induced by unilateral ureteral obstruction in renal tubule-specific PPP3CA knockout mice

[0114] 1. Construction of renal tubule-specific PPP3CA knockout mice:

[0115] SPF-grade heterozygous mice expressing Pax8-rtTA and tet-on LC-1 on a C57BL / 6 background (the heterozygous mice were donated by the research group of Wenhui Wang at New York Medical College, USA; in addition, the heterozygous mice can be purchased from the Jacksonlaboratory, strain #007176) were crossed with PPP3CA flox / flox Homozygous mice (PPP3CA flox / flox Homozygous mice were constructed according to the method described in patent application number 202210579625.2) and hybridized (such as Figure 6 doxycycline-induced tubule-specific PPP3CA knockout mice (denoted as pax8-cre / PPP3CA flox / floxAt 6-8 weeks of age, pax8-cre / PPP3CA flox / flox Mice (hereafter referred to as KO mice) and PPP3CA flox / flox Doxycycline was added to the drinking water of homozygous mice (hereinafter referred to as flox mice) (the concentration of doxycycline in the drinking water was 2 mg / ml, and the drinking water also contained sucrose at a concentration of 2%), and the mice were fed for 2 weeks to induce tubule-specific PPP3CA gene knockout. The mice were then left alone for another 2 weeks before relevant experiments.

[0116] 2. Genotyping of tubule-specific PPP3CA knockout mice:

[0117] (1) To extract mouse DNA, cut off 2 mm of the tail tissue of the breeding mouse and place it in a labeled sterile, enzyme-free 1.5 ml EP tube. Centrifuge at 5000 rpm for 1 min to remove the mouse tail to the bottom of the tube. Preheat the metal bath to 98°C in advance and set aside. Prepare PH5 neutralizing buffer and PH12 lysis buffer as needed. Add 75 μl of PH12 lysis buffer to each tube, heat it in a 98°C metal bath for 15 min, shake and mix it, centrifuge it at 10000 rpm for 1 min, and then heat it in a metal bath for 15 min. Add 75 μl of PH5 neutralizing buffer to each tube of sample, shake and mix it, centrifuge it, and store it in a 4°C refrigerator for later use.

[0118] (2) PCR primer synthesis and amplification. In this experiment, Ppp3ca flox / flox , Pax8-rtTA and LC1-Cre primer sequences are as follows:

[0119] The Ppp3ca primer sequence is:

[0120] F1:5'-AATTGACTCTTGCTAAAGGGAGGG-3',

[0121] R1:5′-TCCACCATGAACAGATAATGGCTA-3′.

[0122] The Pax8-rtTA primer sequence is:

[0123] F1:5'-CCATGTCTAGACTGGACAAGA-3',

[0124] R1:5′-CAGAAAGTCTTGCCATGACT-3′.

[0125] The LC1-Cre primer sequences are:

[0126] F1:5'-TTTCCCGCAGAACCTGAAGATG-3',

[0127] R1:5′-TCACCGGCATCAACGTTTTTCTT-3′.

[0128] The PCR reaction system and reaction conditions are shown in Tables 1 and 2.

[0129] Table 1 PCR reaction system

[0130] Reaction components Reaction system (25 μl) 2×TaqMastermix 12.5 <![CDATA[ddH2O]]> 9 F1 1 R1 1 DNA 1.5

[0131] Table 2 PCR reaction conditions

[0132] Temperature(℃) Time Cycles 95 3min 1 95 15s 35 60 15s 35 72 60s 35 72 5min 1 12 hold 1

[0133] (3) DNA agarose gel electrophoresis: Place the gel plate in the gel tank, insert the sample comb, and pour an appropriate amount of 1×TAE solution into the electrophoresis tank. Weigh 2g of agarose into a beaker, pour in 100ml of 1×TAE solution, shake slightly to mix, heat in a microwave oven on high heat for 1min, take out and shake evenly, and heat for another 1min until the agarose is completely melted and transparent. Pour the melted agarose into two 50ml centrifuge tubes, add 5μl of nucleic acid dye to each tube, mix upside down (you can rinse it under the faucet to cool it down), pour it into the gel plate, avoid bubbles, and wait for 30min to cool and solidify. Pull out the sample comb, take out the solidified gel plate, and put it into the electrophoresis tank so that the TAE solution completely covers the gel. Add 5μl of DNA marker to the first well, and then add 20μl of amplified DNA sample in sequence. Depending on the type of sample, DNA markers can be added in the middle to separate different samples. Start the electrophoresis instrument and run the electrophoresis at 100V for 30min-40min. After electrophoresis is completed, take out the gel and scan it in a gel imaging system to observe the bands (such as Figure 6 (shown in B).

[0134] The DNA agarose gel electrophoresis results are as follows Figure 6 As shown in B. Figure 6 From B, we can see that the genotype is pax8-cre / ppp3ca flox / flox The mice are a doxycycline-induced conditional tubule-specific knockout mouse model of PPP3CA.

[0135] 3. PPP3CA protein verification in renal tubule-specific PPP3CA knockout mice:

[0136] Western Blot was used to detect the expression level of PPP3CA in the kidney, heart, liver, spleen and lung of KO mice and flox mice, and immunofluorescence was used to detect the expression changes of PPP3CA protein in the renal tubules of KO mice and flox mice.

[0137] The results of Western Blot detection of PPP3CA expression levels in kidney, heart, liver, spleen, and lung of KO mice and flox mice are shown in Figure 6 As shown in C and D. Figure 6 As shown in Figures C and D, compared with flox mice, the expression of PPP3CA protein in the kidney tissue of KO mice was significantly downregulated, and the expression levels of PPP3CA protein in the heart, liver, spleen, and lung tissues of KO mice were not significantly different from those of flox mice.

[0138] The results of immunofluorescence detection of PPP3CA protein expression changes in renal tubules of KO mice and flox mice are shown in Figure 6 As shown in E. Figure 6 As shown in Figure E, compared with flox mice, the fluorescence expression of PPP3CA in the renal tubules of KO mice was significantly weakened, indicating that renal tubule-specific PPP3CA knockout mice were successfully constructed.

[0139] 4. Effect of tubule-specific PPP3CA knockout on renal fibrosis in mice after unilateral ureteral obstruction:

[0140] (1) Experimental groups and methods:

[0141] The KO mice constructed above were divided into KO-sham group and KO-UUO group, and the flox mice constructed above were divided into flox-sham group and flox-UUO group. The mice in KO-UUO group and flox-UUO group were treated according to the method for constructing a mouse model of unilateral ureteral obstruction and renal fibrosis described in Example 2. The KO-sham group and flox-sham group were subjected to sham surgery. After 11 days, the mice in each group were killed and the kidney tissues of the mice were collected (such as Figure 7 A part of the kidney tissue was fixed with 4% paraformaldehyde for 48 hours and then embedded in paraffin. The tissue was cut into 4 μM thick sections and HE, Masson and Sirius red staining were used to detect the fibrosis of mouse kidney tissue. Immunofluorescence was used to detect the fluorescence expression of PPP3CA, FN1 and α-SMA proteins in mouse kidney tissue. Immunohistochemistry was used to detect the expression of CollegenⅠ and CollegenⅢ in mouse kidney tissue. The remaining kidney tissue was quickly frozen in liquid nitrogen and stored at -80℃. Western Blot was used to detect the expression of PPP3CA protein and fibrosis phenotype indicators FN1, α-SMA and vimentin protein.

[0142] (2) Experimental results:

[0143] The experimental results of HE staining, Masson staining, and Sirius red staining are as follows Figure 7 As shown in B. Figure 7 As shown in Figure B, the renal tubules of flox mice were significantly dilated, with inflammatory cell infiltration, brush border shedding, and obvious collagen fiber deposition after UUO (unilateral ureteral obstruction), while the renal tubular dilation and inflammatory cell infiltration and collagen fiber deposition were reduced in KO mice, indicating that tubule-specific PPP3CA knockout can alleviate renal fibrosis.

[0144] The results of immunofluorescence experiments to detect the fluorescence expression of PPP3CA, FN1, and α-SMA proteins in mouse kidney tissue are as follows Figure 8 As shown in A. Figure 8 As shown in Figure A, compared with the flox-sham group mice, the fluorescence intensities of PPP3CA, FN1, and α-SMA in the flox-UUO group mice were significantly enhanced, while the fluorescence intensities of FN1 and α-SMA in the KO-UUO group mice were significantly weakened.

[0145] The results of immunohistochemical experiments to detect the expression of Collegen I and Collegen III in mouse kidney tissue are as follows Figure 8 As shown in B. Figure 8 As shown in Figure B, compared with the flox-sham group mice, the expression of Collegen I and Collegen III in the flox-UUO group mice was significantly increased, while the expression of Collegen I and Collegen III in the KO-UUO group mice was significantly decreased.

[0146] The results of Western Blot detection of PPP3CA protein and fibrosis phenotype indicators FN1, α-SMA, and vimentin protein expression are shown in the figure. Fig. 9As shown in the figure, after UUO surgery, the expression of PPP3CA in the flox-UUO group mice was upregulated compared with that in the flox-sham group mice, which further proved that PPP3CA was involved in renal fibrosis; in addition, the expression levels of fibrosis-related phenotypic indicators FN1, α-SMA, and vimentin proteins in the renal tissues of the flox-sham group mice and the KO-sham group mice were low, and there was no significant difference between the two groups, while the expression of fibrosis-related phenotypic indicators FN1, α-SMA, and vimentin proteins in the renal tissues of the flox-UUO group mice was significantly increased compared with those in the flox-sham group mice, indicating that the UUO model was successfully established; moreover, the expression of fibrosis-related phenotypic indicators FN1, α-SMA, and vimentin proteins in the renal tissues of the KO-UUO group mice was downregulated compared with those in the flox-UUO group mice, and the difference was statistically significant.

[0147] The above results indicate that knocking out PPP3CA in renal tubules at the animal level can delay renal fibrosis.

[0148] Example 6: Ischemia-reperfusion renal fibrosis model to verify the effect of in vivo inhibition of PPP3CA on renal fibrosis in mice

[0149] 1. Experimental groups and methods:

[0150] The KO mice constructed in Example 5 were divided into a KO-sham group and a KO-IRI group, and the above-mentioned flox mice were divided into a flox-sham group and a flox-IRI group. The mice in the KO-IRI group and the flox-IRI group were treated according to the construction of the ischemia-reperfusion renal fibrosis mouse model. The KO-sham group and the flox-sham group were subjected to sham surgery. After 35 days, the mice in each group were killed and the kidney tissues of the mice were collected (such as Fig.10 A portion of the kidney tissue was quickly frozen in liquid nitrogen and stored at -80°C. Western Blot was used to detect the expression of FN1 protein, a fibrosis phenotype indicator, in the kidney tissue of mice. Another portion of the kidney tissue was fixed with 4% paraformaldehyde for 48 hours and then embedded in paraffin. The sections were cut into 4 μM thick sections, and immunofluorescence was used to detect the fluorescence expression of FN1 and α-SMA proteins in the kidney tissue of mice.

[0151] Among them, the method for constructing a mouse model of ischemia-reperfusion renal fibrosis is as follows: anesthetize the mouse with an isoflurane gas anesthesia machine, make it lie on its back on a heating pad, disinfect the abdominal area with alcohol, pick up the skin in the middle of the abdomen with curved forceps, and cut the skin and subcutaneous tissue vertically in sequence (about 3-4 cm), so that the renal arteries on both sides can be seen clearly, and it should not be too long. Pull out the intestine carefully, and use curved forceps to carefully separate the renal arteries on both sides to avoid damaging the blood vessels. The separation time should not be too long, and use a 1ml syringe to absorb saline to moisten the intestine continuously during the period. Clamp the artery clamp with an artery clamp, and quickly clamp the renal arteries on both sides, and the interval should not be too long. It can be observed that the mouse kidney turns purple-red. Cover the mouse abdomen with gauze moistened with saline in advance, clamp it for 35 minutes, and use a 1ml syringe to absorb saline to moisten the gauze continuously during the period. When the time is up, lift the gauze and quickly remove the arterial clamps on both sides. Observe whether the blood supply to the kidneys on both sides has been restored and the color has turned bright red. Retract the intestines and use a suture needle with thread to suture the subcutaneous tissue and skin in turn. Keep the sutures as short as possible to prevent the mice from biting. Disinfect the abdomen and waist with alcohol again to remove the bloody smell. Place the mice on a 37℃ heating pad and put them in a cage after they recover from anesthesia. Observe the status of the mice on the night of surgery and the next day.

[0152] 2. Experimental results:

[0153] The results of Western Blot detection of the expression of FN1 protein, a fibrosis phenotype indicator, in mouse kidney tissue are as follows: Fig.10 As shown in B and C. Fig.10 As shown in Figures B and C, after IRI modeling, compared with the flox-sham group mice, the FN1 of the flox-IRI group mice was significantly upregulated, while the FN1 of the KO-IRI group mice was downregulated.

[0154] The experimental results of immunofluorescence detection of the fluorescence expression of FN1 and α-SMA proteins in mouse kidney tissue are as follows Fig.10 As shown in D. Fig.10 As shown in Figure D, after IRI modeling in flox mice, the fluorescence of fibrosis-related phenotypic indicators FN1 and α-SMA in the kidneys was enhanced, while the fluorescence intensity in the kidneys of KO mice was weakened.

[0155] The above experimental results show that tubule-specific PPP3CA knockout can significantly improve the renal fibrosis response in mice.

[0156] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned 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 PPP3CA gene or PPP3CA protein detection reagents in the preparation of auxiliary diagnostic products for renal fibrosis.

2. The use according to claim 1, characterized in that: The product detects the expression level of PPP3CA gene or PPP3CA protein in a sample by RT-PCR, real-time quantitative PCR, in situ hybridization, Northern Blot, Western Blot, chip, high-throughput sequencing platform, immunohistochemistry or enzyme-linked immunosorbent assay.

3. The use according to claim 2, characterized in that: The product contains an antibody that specifically binds to the PPP3CA protein, or contains a primer that specifically amplifies the PPP3CA gene, or contains a probe that specifically detects the PPP3CA gene.

4. Use of PPP3CA inhibitors in the preparation of drugs for preventing, alleviating or / and treating renal fibrosis.

5. The use according to claim 4, characterized in that: The PPP3CA inhibitor is one or more substances that inhibit PPP3CA gene expression, silence PPP3CA gene, knock out PPP3CA gene, reduce PPP3CA protein content, and reduce PPP3CA protein activity.

6. The use according to claim 5, characterized in that: The substance is one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, polypeptides, proteins, gene editing vectors, lentiviruses or adeno-associated viruses; the nucleic acid molecules include shRNA, microRNA, siRNA and / or antisense oligonucleotides.

7. The use according to claim 6, characterized in that: The target sequence of the siRNA is 5′-GCAGTAATAGCAGCAATAT-3′ or 5′-GCCGTTCCATTTCCACCAA-3′.

8. A drug, characterized in that The medicine contains the PPP3CA inhibitor according to any one of claims 4 to 8, and is used for preventing, alleviating or / and treating renal fibrosis.

9. Use of the PPP3CA gene or PPP3CA protein in screening candidate drugs for preventing, alleviating or / and treating renal fibrosis, the screening method comprising: screening drugs or agents to be screened using PPP3CA as a target, and selecting drugs or agents that can reduce the expression level of the PPP3CA gene, reduce the content of the PPP3CA protein or reduce the activity of the PPP3CA protein as candidate drugs for preventing, alleviating or / and treating renal fibrosis.

10. A method for constructing a renal tubule-specific PPP3CA knockout mouse, characterized in that: Heterozygous mice expressing Pax8-rtTA and tet-on LC-1 were crossed with PPP3CA flox / flox Homozygous mice were interbred to generate doxycycline-inducible tubule-specific PPP3CA knockout mice.

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