Application of CNPY2 as new target in diagnosis and treatment of diabetic nephropathy

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of CNPY2 as a new target in diagnosis and treatment of diabetic nephropathy. The invention provides a biomarker CNPY2 gene or CNPY2 protein for auxiliary diagnosis of diabetic nephropathy for the first time. The expression of the CNPY2 in the renal tubule of a DN patient is up-regulated, the expression of the CNPY2 in the renal tubule of a db / db mouse is up-regulated, and the expression of the CNPY2 in an HK-2 cell subjected to in-vitro high-glucose culture is up-regulated; therefore, the CNPY2 can be used as an auxiliary diagnosis index of the diabetic nephropathy for auxiliary diagnosis of the diabetic nephropathy. The invention also provides a kit for auxiliary diagnosis of diabetic nephropathy. The kit contains a detection reagent of the CNPY2 gene or the coded protein. The invention also finds that by knocking down the adeno-associated virus through the CNPY2 and inhibiting the expression or activity of the CNPY2 through the specific small interfering RNA of the adeno-associated virus, the pathological injury of the renal tubules of db / db mice can be obviously improved, the integrity of MAM in the renal tubule epithelial cells is improved, and the ferroptosis, ERS and mitochondrial injury of the renal tubule epithelial cells are alleviated; therefore, the CNPY2 can be used as a drug, a drug target or a target gene in gene therapy, and is applied to prevention, alleviation or / and treatment of diabetic nephropathy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of CNPY2 as a new target in the diagnosis and treatment of diabetic nephropathy. Background Art

[0002] Diabetic nephropathy (DN) is one of the most common and serious microvascular complications of diabetes. Its incidence rate is increasing year by year. It has become the main cause of chronic kidney disease (CKD) in my country and one of the important risk factors for progression to end-stage renal disease (ESRD), with a high mortality rate.

[0003] Mitochondria-associated endoplasmic reticulum membrane (MAM) is a dynamic connection region between the endoplasmic reticulum and mitochondria in morphology and function, which can regulate lipid metabolism, Ca 2+ MAM plays an important role in the process of renal injury in DN.

[0004] As a renal structure rich in mitochondria, the renal tubule is more susceptible to mitochondrial damage. Mitochondrial dysfunction is considered to be one of the important causes of diabetic tubular damage. Under high glucose conditions, mitochondrial reactive oxygen species accumulation and GSH depletion in renal tubular epithelial cells lead to an imbalance in the redox state, ultimately leading to mitochondrial oxidative damage and ferroptosis. Ferroptosis is a new type of programmed cell death, characterized by intracellular ferrous iron retention, reduced glutathione (GSH) depletion, and iron-dependent lipid reactive oxygen species (ROS) accumulation. It is involved in the pathophysiological processes of many diseases, such as cancer, neurodegenerative diseases, metabolic diseases, ischemia-reperfusion injury and other diseases. More and more studies have shown that ferroptosis of renal tubular epithelial cells promotes the occurrence and development of DN, but its specific regulatory mechanism remains unclear. Therefore, elucidating the molecular mechanism of ferroptosis in renal tubular epithelial cells is of great significance for the targeted treatment of DN.

[0005] Canopy FGF signaling regulator 2 (CNPY2) is a new type of angiogenic growth factor located in the endoplasmic reticulum. It plays an important regulatory role in ERS and is involved in the occurrence and development of various diseases including tumors, cardiovascular damage, and liver damage. Previous studies have shown that CNPY2 is a key promoter of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway of unfolded proteins in ERS. It can regulate various cellular processes such as cell growth, migration, and apoptosis by regulating PERK and its mediated downstream signaling pathways, but its role in DN has not been reported. The pathogenesis of DN is complex, lacks specific intervention targets, and there is no effective treatment. Therefore, screening new therapeutic targets for DN is a potential therapeutic method to intervene in its progression. Summary of the invention

[0006] In view of the problems and shortcomings in the prior art, the object of the present invention is to provide application of CNPY2 as a new target in the diagnosis and treatment of diabetic nephropathy.

[0007] Based on the above purpose, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a biomarker that can be used for auxiliary diagnosis of diabetic nephropathy, wherein the biomarker is a CNPY2 gene or a CNPY2 protein.

[0009] The expression of CNPY2 in the renal tissue of DN patients, renal tissue of db / db mice and HK-2 cells cultured in vitro with high glucose was detected by immunohistochemistry, Western blot and other experiments. It was found that CNPY2 was upregulated in the renal tissue of DN patients, renal tissue of db / db mice and HK-2 cells cultured in vitro with high glucose.

[0010] The second aspect of the present invention provides the use of CNPY2 gene or CNPY2 protein as a drug target for screening drugs for preventing, alleviating or treating diabetic nephropathy.

[0011] The third aspect of the present invention provides the use of a substance that inhibits the expression and / or function of CNPY2 in the preparation of a medicament for preventing, alleviating or treating diabetic nephropathy.

[0012] According to the above application, preferably, the substance that inhibits CNPY2 expression and / or function is a CNPY2 knockdown adeno-associated virus, a siRNA specifically targeting the CNPY2 gene, a shRNA specifically targeting the CNPY2 gene, or an inhibitor of the CNPY2 protein.

[0013] A fourth aspect of the present invention provides a medicine for preventing, alleviating or treating diabetic nephropathy, wherein the medicine contains a substance that inhibits the expression and / or function of CNPY2.

[0014] According to the above-mentioned drug, preferably, the substance inhibiting CNPY2 expression and / or function is a CNPY2 knockdown adeno-associated virus, a siRNA specifically targeting the CNPY2 gene, a shRNA specifically targeting the CNPY2 gene, or an inhibitor of the CNPY2 protein.

[0015] According to the above medicine, preferably, the medicine further contains a pharmaceutically acceptable carrier or excipient.

[0016] According to the above-mentioned drugs, preferably, the carrier / excipients include (but are not limited to): diluents, excipients such as lactose, sodium chloride, glucose, urea, starch, water, etc., fillers such as starch, sucrose, etc.; binders such as monosyrup, glucose solution, starch solution, cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; wetting agents such as glycerol; disintegrants such as dry starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate and sodium bicarbonate; absorption promoters quaternary ammonium compounds, sodium lauryl sulfate, etc.; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc.; wetting agents such as glycerol, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin and soap clay, etc.; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, etc.

[0017] A fifth aspect of the present invention provides use of a detection reagent for CNPY2 gene or CNPY2 protein in the preparation of a product for auxiliary diagnosis of diabetic nephropathy.

[0018] According to the above application, preferably, the product detects the expression level of CNPY2 gene or its encoded protein in the sample by RT-PCR, real-time quantitative PCR, in situ hybridization, Northern blot, Western blot, chip, high-throughput sequencing platform, immunohistochemistry, tissue immunofluorescence or enzyme-linked immunosorbent assay.

[0019] According to the above application, preferably, the detection sample of the product is a cell or a tissue; and the product is a chip, a preparation or a kit.

[0020] A sixth aspect of the present invention provides a kit for auxiliary diagnosis of diabetic nephropathy, wherein the kit contains a detection reagent for the CNPY2 gene or the encoded protein.

[0021] According to the above kit, preferably, the detection reagent detects the expression level of CNPY2 gene or its encoded protein in the sample by RT-PCR, real-time quantitative PCR, in situ hybridization, Northern blot, Western blot, chip, high-throughput sequencing platform, immunohistochemistry, tissue immunofluorescence or enzyme-linked immunosorbent assay.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention first discovered that CNPY2 is upregulated in the renal tubules of DN patients, CNPY2 is upregulated in the renal tubules of db / db mice, and CNPY2 is upregulated in HK-2 cells cultured in high glucose in vitro; therefore, CNPY2 can be used as an auxiliary diagnostic indicator for diabetic nephropathy to assist in the diagnosis of diabetic nephropathy.

[0024] (2) The present invention uses CNPY2 knockdown adeno-associated virus and its specific small interfering RNA to inhibit CNPY2 expression or activity, which can significantly improve the pathological damage of the renal tubules in db / db mice, improve the integrity of MAM in renal tubular epithelial cells, and reduce ferroptosis, ERS and mitochondrial damage in renal tubular epithelial cells; therefore, CNPY2 can be used as a drug, drug target or target gene in gene therapy, and applied to the prevention, alleviation and / or treatment of diabetic nephropathy, which can provide a new strategy for the prevention and treatment of diabetic nephropathy. At the same time, it will also provide a new direction for further research on the etiology and pathological mechanism of diabetic nephropathy and the corresponding prevention and treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The results of the detection of CNPY2 expression levels in the renal tubules of renal tissues of DN patients and normal controls; A is the immunohistochemical staining of renal tissues in the two groups, the upper scale is 100μm, and the lower scale is 50μm; B is the semi-quantitative analysis result of Figure A, Control group n=11, DN group n=22, compared with the Control group, ****P<00001; C is the correlation analysis of the semi-quantitative results of CNPY2 immunohistochemistry and its eGFR, r=-06324, ****P<00001, n=33;

[0026] Figure 2The results of the detection of CNPY2 expression levels in the renal tubules of mice in the db / db group and the db / m group; A is the HE and PAS pathological staining of mouse renal tissue and the immunohistochemical staining of KIM-1 and CNPY2 in renal tissue, the scale is 50μm; B is the line graph of the changes in mouse body weight, n=6; C is the line graph of the changes in blood glucose in mice, n=6; D is the bar graph of the changes in blood creatinine levels in mice, n=6; E is the line graph of the changes in UACR levels in the two groups of mice, n=5; F is the Western blot results of the renal tissues of the two groups of mice, β-actin is the internal reference; G is the grayscale analysis bar graph of the Western blot bands in Figure F, n=5, compared with db / m mice, *P<005, **P<001;

[0027] Figure 3 The results of the detection of CNPY2 expression level in HK-2 cells cultured in vitro with high glucose; A is the results of Western blot test of HK-2 cell proteins in different groups, with β-actin as the internal reference; B is the grayscale analysis result of the Western blot bands in Figure A, the experiment was repeated 3 times, compared with the NG group and HM group, *P<005;

[0028] Figure 4 The results of the detection of renal tubular injury in db / db mice overexpressing CNPY2; A is the results of HE and PAS pathological staining of renal tissues of the three groups of mice, with a scale of 50 μm; B is a line graph of weight changes of mice in each group, n=5; C is a line graph of blood glucose changes of mice in each group, n=5; D is a Western blot result of renal tissues of mice in each group, with β-actin as an internal reference; E is a grayscale analysis result of the Western blot bands in Figure D, n=5, compared with the db / db+Vehicle group of mice, *P<005, ***P<0001;

[0029] Figure 5 The results of the integrity test of MAM in renal tubular epithelial cells of db / db mice overexpressing CNPY2; A is the transmission electron microscopy TEM (upper figure) and PLA test (lower figure) of renal tissue of mice in each group, the scale bar is 1μm (magnification 8000 times), and the lower scale bar is 10μm; B is the PLA quantitative analysis results of renal tissue of each group, n=5, compared with the db / db+Vehicle group and db / db group mice, **P<001;

[0030] Figure 6 Figure 2 is the result of the detection of ferroptosis in renal tubular epithelial cells of db / db mice overexpressing CNPY2; A is the electron microscopy image (scale bar is 1 μm, magnification is 8000 times) and immunohistochemical staining (scale bar is 50 μm) of the renal tissue of each group of mice; B is the Fe 2+Content bar graph, n=5, compared with db / db+Vehicle group and db / db mice, *P<005; C is the bar graph of GSH content in kidney tissue of each group, n=5, compared with db / db+Vehicle group and db / db mice, *P<005; D is the bar graph of MDA content in kidney tissue of each group, n=5, compared with db / db+ehicle group and db / db mice, *P<005; E is the Western blot bands of ATF4, CHAC1, p-PERK, TFAM proteins and ferroptosis-related proteins GPX4 and TFR-1 in kidney tissues of each group of mice, β-actin is the internal reference; F is the grayscale analysis result of Western blot bands in Figure E, n=5, compared with db / db+Vehicle group and db / db mice, *P<005, **P<001, ***P<0001;

[0031] Figure 7 The results of the detection of renal tubular injury in db / db mice with knockdown of CNPY2; A is the HE and PAS pathological staining of renal tissues of the three groups of mice, with a scale of 50 μm; B is the line graph of weight changes of mice in each group, n=5; C is the blood glucose changes of mice in each group, n=5; D is the Western blot results of renal tissues of mice in each group, with β-actin as the internal reference; E is the grayscale analysis result of the Western blot band in Figure D, n=5, compared with the db / db+Vehicle group and db / db mice, *P<005;

[0032] Figure 8 The results of MAM integrity test of renal tubular epithelial cells in db / db mice with knockdown of CNPY2; A is the transmission electron microscopy (upper figure) and PLA test (lower figure) of renal tissues of mice in each group, the upper scale is 1 μm (magnification 8000 times), and the lower scale is 10 μm; B is the results of PLA quantitative analysis of renal tubules in mice in each group, n=5, compared with db / db+Vehicle group and db / db mice, *P<005;

[0033] Fig. 9 The results of the detection of ferroptosis in renal tubular epithelial cells of db / db mice with knockdown of CNPY2; A is the electron microscopy images of renal tissues of mice in each group (scale bar is 1 μm, magnified 8000 times) and immunohistochemical staining (scale bar is 50 μm); B is the Fe 2+A is a bar graph of GSH content in renal tissues of each group, n=5, compared with db / db+Vehicle group and db / db mice, *P<005; C is a bar graph of GSH content in renal tissues of each group, n=5, compared with db / db+Vehicle group and db / db mice, *P<005; D is a bar graph of MDA content in renal tissues of each group, n=5, compared with db / db+Vehicle group and db / db mice, *P<005; E is the Western blot bands of ATF4, CHAC1, p-PERK, TFAM and ferroptosis-related proteins GPX4 and TFR-1 in renal tissues of each group, with β-actin as the internal reference; F is the grayscale analysis result of the Western blot bands in Figure E, n=5, compared with db / db+Vehicle group and db / db mice, *P<005. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below through embodiments in combination with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] The test methods described in the following examples are all conventional techniques in the art, or are based on the conditions recommended by the manufacturers unless otherwise specified; the reagents, materials and instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0036] Example 1: Study on the expression of CNPY2 in renal tubules of DN patients

[0037] Immunohistochemical staining was used to detect the expression of CNPY2 in renal tubules of DN patients.

[0038] 1. Source of test materials

[0039] Diabetic nephropathy (DN group) renal tissue (clinical sample-related experimental research was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Ethics Approval No.: 2023-KY-0501-004). Diabetic nephropathy (DN group) renal tissue was derived from paraffin sections of the Renal Pathology Department of our hospital. The inclusion criteria were adult diabetic nephropathy patients aged ≥18 years with a clear clinical diagnosis and confirmed by renal puncture biopsy); normal control renal tissue (derived from the biological sample bank of our hospital, the National Human Genetic Resources Sharing Service Platform (Platform No.: 2005DKA21300), which was normal renal tissue adjacent to the cancer removed during renal tumor surgery); clinical data of the enrolled patients (from the hospital archived medical records).

[0040] 2. Test methods

[0041] 2.1 Immunohistochemical staining

[0042] (1) Tissue dehydration: The renal tissue fixed with 10% tissue fixative was dehydrated using a tissue dehydrator.

[0043] (2) Paraffin embedding: The dehydrated renal tissue is embedded using a paraffin embedding machine.

[0044] (3) Sectioning: Paraffin wax was sliced ​​into 2 μm thick sections and baked in an oven at 60°C for 2 h. The sections were stored at room temperature and baked in an oven at 60°C for 30 min before use.

[0045] (4) Dewaxing: The sections were treated with xylene solution for 10 min × 3 times, anhydrous ethanol for 5 min × 3 times, 95% alcohol for 5 min × 1 time, 85% alcohol for 5 min × 1 time, 75% alcohol for 5 min × 1 time, and PBS for 5 min × 3 times.

[0046] (5) Antigen retrieval: Place the slices in 1× citric acid buffer and microwave on high for 1 minute, medium for 3 minutes, and low for 10 minutes. Allow to cool naturally at room temperature, then repeat the above steps and allow to cool naturally to room temperature.

[0047] (6) Peroxidase quenching: Wash with PBS buffer for 5 minutes × 3 times, circle the tissue area on the slide with a hydrophobic pen, add 1 drop of 3% hydrogen peroxide solution, and incubate at room temperature for 20 minutes.

[0048] (7) Tissue permeabilization: flick off the hydrogen peroxide solution, wash with PBS buffer for 5 minutes × 3 times, and permeabilize with 0.1% Triton 100 solution at room temperature for 15 minutes.

[0049] (8) Blocking: flick off the above solution, wash with PBS buffer for 5 minutes × 3 times, add 1 drop of goat serum blocking solution, and incubate at room temperature for 20 minutes.

[0050] (9) Primary antibody incubation: Prepare the primary antibody to an appropriate concentration using antibody diluent, add it dropwise to each sample, and incubate it in a humidified chamber at 4°C overnight.

[0051] (10) Take out the wet box from the 4°C refrigerator, place it at room temperature for at least 30 min, and wash it with PBS buffer for 5 min × 3 times.

[0052] (11) Add one drop of reaction enhancement solution to the specimen, incubate at 37°C for 20 min, and wash with PBS buffer for 5 min × 3 times.

[0053] (12) Add 1 drop of enhanced enzyme-labeled anti-rabbit / mouse IgG polymer, incubate at 37°C for 20 min, and wash with PBS buffer for 5 min × 3 times.

[0054] (13) DAB color development: Add 50 μL of the concentrate to 1 mL of the DAB substrate solution to prepare the DAB color development solution. Observe the color development under a microscope and rinse with tap water to terminate the color development in time.

[0055] (14) Nuclear staining: Add 1 drop of hematoxylin solution to stain the cell nucleus for about 2-3 seconds, and rinse with running water for 10 minutes.

[0056] (15) Dehydration: Immerse the slides in 75% ethanol once for 2 minutes, 85% ethanol once for 2 minutes, 95% ethanol once for 2 minutes, anhydrous ethanol twice for 2 minutes, and xylene twice for 2 minutes.

[0057] (16) Sealing: Seal the slides with neutral resin, taking care to avoid creating bubbles, and allow to dry naturally in a fume hood.

[0058] (17) X-ray: Take 20× images of the renal tubulointerstitium under a microscope.

[0059] 2.2 Statistical analysis

[0060] ImageJ software was used to semi-quantitatively analyze the relative protein expression of immunohistochemistry or immunofluorescence and Western blot bands. GraphPad Prism 8.0 software was used for statistical analysis of the experimental data. The Student's t test or nonparametric test was used for comparison between the two groups. All normally distributed data were expressed as mean ± standard deviation (SD), and non-normally distributed data were expressed as median (interquartile range). Pearson correlation coefficient was used to test the linear correlation between parameters. P < 0.05 was considered statistically significant.

[0061] 3. Test results

[0062] This example study included 22 DN patients and 11 normal controls. The general clinical data of all cases are shown in Table 1.

[0063] Table 1 Comparison of general data between the control group and DN patients

[0064]

[0065] Note: HbA1c: glycosylated hemoglobin; eGFR: estimated glomerular filtration rate; UACR: urine albumin / creatinine; *P<0.05 compared with the Control group.

[0066] The results of immunohistochemical staining for the expression of CNPY2 in the renal tubules of DN patients and normal controls are shown in Figure 1As shown. Figure 1 As can be seen in Figures A and B, compared with the Control group, the expression level of CNPY2 in the renal tubules of DN patients was significantly increased. Figure 1 As shown in Figure C, CNPY2 and eGFR have a significant negative correlation, suggesting that increased expression levels of CNPY2 may be involved in the renal function impairment in DN patients.

[0067] Example 2: Study on mouse modeling and expression of CNPY2 in mouse renal tubules

[0068] Western blot was used to detect the expression of CNPY2 and tubular injury molecule KIM-1 in the renal tubules of diabetic mouse models.

[0069] 1. Animal husbandry and grouping

[0070] The experiment set up two groups, db / db and db / m. The db / db group consisted of 8-week-old db / db diabetic male mice (at least 5 mice), and the db / m group consisted of 8-week-old db / m control male mice (at least 5 mice); db / db diabetic male mice and db / m control males were purchased from the Model Animal Research Center of Nanjing University and raised in the Experimental Animal Center of Zhengzhou University. All animals were raised under standard feeding conditions. After one week of adaptive feeding, blood glucose and body weight were tested every other week, and random urine of mice was collected. All mice were euthanized at 22 weeks of age, and kidney and blood specimens were quickly collected.

[0071] 2. Test methods

[0072] 2.1 Mouse blood glucose detection

[0073] The blood glucose level of each group of mice was tested every 2 weeks. Specific steps: Wipe the tail with alcohol cotton pad, quickly insert the blood collection needle into the tail vein, draw blood with blood glucose test strips, measure the blood glucose level, and keep records.

[0074] 2.2 Random urine collection from mice and determination of urine albumin to creatinine ratio (UACR)

[0075] Random urine was collected from each group of mice every 2 weeks, centrifuged at low speed, and stored at -80°C for later use. Finally, the UACR value was determined in the Nephrology Laboratory of the First Affiliated Hospital of Zhengzhou University.

[0076] 2.3 Collection of mouse serum

[0077] The mice were placed on an inhalation anesthesia machine for continuous inhalation anesthesia. The mice were dissected and the hearts were exposed. Blood was drawn from the right ventricle. After standing at room temperature for 1 hour, the blood was centrifuged at 3000 rpm and 4°C for 10 minutes. The supernatant was drawn and stored at -80°C for later use.

[0078] 2.4 Determination of serum creatinine in mice

[0079] After the collected serum was dissolved on ice, the blood creatinine was determined using a creatinine determination kit. The specific steps were as follows: 6 μL of sample serum, standard substance, and double distilled water were added to the sample well, standard well, and blank well, respectively. 180 μL of enzyme solution A was added to all wells, incubated at 37°C for 5 minutes, and the absorbance at 546 nm was measured using an enzyme reader as A1. Then 60 μL of enzyme solution B was quickly added to each well, incubated at 37°C for 5 minutes, and the absorbance at 546 nm was measured using an enzyme reader as A2. The blood creatinine value (μmol / L) of each group of samples was calculated according to the formula in the instruction manual.

[0080] 2.5 Collection of Mouse Kidney Tissue

[0081] The mice were placed on an inhalation anesthesia machine for continuous inhalation anesthesia, the mice were dissected and the hearts were exposed, blood was drawn from the right ventricle, the right atrial appendage was cut open, and PBS buffer was uniformly injected into the mouse from the apex of the heart using a syringe pump for renal perfusion. The perfusion was stopped after the kidneys turned white, and the kidneys on both sides were quickly removed and rinsed in pre-cooled PBS buffer. The renal outer membrane was quickly peeled off on ice. According to subsequent experiments, the kidneys were divided into electron microscopy part (electron microscopy solution fixation), paraffin embedded part (10% tissue fixative fixation), OCT embedded, and renal tissue protein extraction part (placed in cryopreservation tubes, quick-frozen in liquid nitrogen, and stored at -80°C for later use).

[0082] 2.6 HE staining

[0083] (1) Dewaxing and hydration: Place the paraffin sections in a 60°C oven for 30 minutes; then dewax in three cylinders of xylene at room temperature, with each cylinder dewaxing for 15 minutes. Then, wash the xylene into distilled water in 100%, 100%, 95%, 85%, and 75% alcohol, with each cylinder soaking for 5 minutes; finally, wash in distilled water three times.

[0084] (2) Hematoxylin staining: Add hematoxylin to the sliced ​​tissue for 5 minutes, add hydrochloric acid ethanol differentiation solution for 3 seconds, then rinse with tap water for 15 minutes to return to blue.

[0085] (3) Eosin staining: Dehydrate the sections in 70% and 90% ethanol for 5 minutes. After removing the sections, add drops of eosin staining solution for 3 minutes.

[0086] (4) Dehydration and sealing: Dehydrate the slices from graded alcohol to xylene, and immerse them in 95% and 100% alcohol twice, each for 1 minute. Finally, immerse them in 3 cylinders of xylene in sequence, each for 3 minutes; remove the slices from xylene, slowly cover them with coverslips, and seal them with neutral resin.

[0087] 2.7 PAS staining

[0088] (1) Dewaxing and hydration: Place the paraffin sections in a 60°C oven for 30 minutes; then dewax in three cylinders of xylene at room temperature, with each cylinder dewaxing for 15 minutes. Then, wash the xylene into distilled water in 100%, 100%, 95%, 85%, and 75% alcohol, with each cylinder soaking for 5 minutes; finally, wash in distilled water three times.

[0089] (2) Periodic acid reaction: Soak in Periodic Acid solution for 5 minutes at room temperature, and rinse with distilled water twice, 5 minutes each time.

[0090] (3) Schiff reaction: Soak in Schiff reagent for 15 minutes and rinse with tap water for 10 minutes.

[0091] (4) Hematoxylin nuclear staining: drop hematoxylin on the sliced ​​tissue for 5 minutes, add hydrochloric acid ethanol differentiation solution for 3 seconds, and then place the slice in tap water to rinse and blue for 15 minutes.

[0092] (5) Dehydration and sealing: Dehydrate the slices from graded alcohol to xylene, and immerse them in 95% and 100% alcohol twice, each for 1 minute. Finally, immerse them in 3 cylinders of xylene in sequence, each for 3 minutes; remove the slices from xylene, slowly cover them with coverslips, and seal them with neutral resin.

[0093] 2.8 Immunohistochemical staining

[0094] The test method is as in 2.1 of Example 1.

[0095] 2.9 Western blot

[0096] 2.9.1 Extraction of tissue proteins

[0097] (1) Prepare RIPA lysis buffer: add RIPA lysis buffer, protease inhibitor, phosphatase inhibitor, and PMSF in a ratio of 100:1:1:1, mix well, and place on ice until ready for use.

[0098] (2) Weigh about 10-20 mg of kidney tissue, add 1 mL of the above-prepared RIPA lysis buffer to 50 mg of kidney tissue, add grinding beads, and grind the tissue using a high-throughput tissue grinder at 40 Hz for 90 s.

[0099] (3) The above grinding solution was centrifuged at 4°C, 12000 rpm for 10 minutes, the supernatant was aspirated and placed on ice for later use.

[0100] 2.9.2 Protein concentration determination using BCA kit

[0101] (1) Preparation of standard: Dilute the BCA standard stock solution according to the instructions to a final concentration of 0.5 mg / mL. Set the standard concentration gradient to 0 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, and place on ice for later use.

[0102] (2) Preparation of BCA working solution: Mix solution A and solution B in the kit at a ratio of 50:1, prepare before use, and stabilize at room temperature for 24 hours.

[0103] (3) The extracted tissue protein supernatant was diluted 20 times for protein concentration determination.

[0104] (4) Add 20 μL of the standard and diluted sample into a 96-well plate, then add 200 μL of BCA working solution to all wells and vortex to mix well to avoid bubbles.

[0105] (5) Incubate in a 37°C incubator for 30 min.

[0106] (6) Use an ELISA reader to measure the absorbance at 562 nm. Draw a standard curve and calculate the concentration of the sample based on the standard curve.

[0107] (7) Select an appropriate concentration to balance the protein concentration of the above samples, add 5× loading buffer and mix well, place in a metal dry bath pot at 100°C for 10 minutes, cool naturally and store in a -80°C refrigerator for later use.

[0108] 2.9.3 Preparation of 10% and 15% gels

[0109] (1) Clean the glass plate with double distilled water, dry it, and align and fix it on the gel-making rack.

[0110] (2) Prepare the lower layer of separation gel: prepare a small beaker, add appropriate volumes of double distilled water, 30% gel preparation buffer, separation gel solution, 10% ammonium persulfate, and TEMED in sequence, mix quickly, add to two layers of glass plates, and finally add about 1 ml of anhydrous ethanol to make the separation gel flat and free of bubbles.

[0111] (3) Prepare the upper concentrated gel: Similarly, add appropriate volumes of double distilled water, 30% gel preparation buffer, separation gel solution, 10% ammonium persulfate, and TEMED in sequence, mix quickly, and add to the glass plate, taking care to avoid bubbles. Then quickly insert a comb and wait for the concentrated gel to solidify. Wrap the prepared gel with dry toilet paper, moisten it with double distilled water, and store it in a sealable bag at 4°C.

[0112] 2.9.4 SDS-PAGE gel electrophoresis

[0113] (1) Sample loading and electrophoresis: After the concentrated gel has solidified, take out the glass plate and put it into the electrophoresis tank (the small glass plate is on the inside and the large glass plate is on the outside. At the same time, in order to ensure the airtightness of the electrophoresis tank, glass plates or plastic plates should be placed on both sides of the electrophoresis tank). Then fill it with the prepared 1× Running Buffer electrophoresis solution and slowly pull out the comb. Take out the protein sample to be tested, add about 20μg / μL and 3μL of pre-stained protein marker to each well, cover the lid after loading the sample, and pay attention to the positive and negative electrodes. Set the concentrated gel part to a constant voltage of 80V. After seeing the protein marker separation, adjust the voltage to 120V and terminate the electrophoresis when it reaches the bottom layer of the separation gel.

[0114] (2) Transfer: Depending on the molecular weight of the protein, soak a 0.45μm or 0.22μm PVDF membrane in a methanol solution. Remove the glass plate, carefully cut off the corresponding separation gel colloid, and place the colloid on filter paper soaked in 1× Transfer Buffer solution in the order of black gel and white membrane, and then place a PVDF membrane of the same size, taking care not to have bubbles. After fastening the transfer clamp, place it in the transfer tank (black to black, white to white), and fill it with pre-cooled 1× Transfer Buffer solution, then place the transfer tank in an ice box filled with ice cubes, determine the positive and negative directions, and connect the power supply. Set the current to 250mA, and determine the transfer time according to the size of the target protein for transfer.

[0115] (3) Blocking: After transfer, quickly place the PVDF membrane in freshly prepared 5% skim milk and place it on a shaker at room temperature for 1-1.5 hours.

[0116] (4) Primary antibody incubation: Remove the blocked PVDF membrane and wash the membrane twice for 5 minutes with 1×TBST solution. Dilute the primary antibody in a certain ratio with antibody diluent and incubate at 4°C on a shaker overnight.

[0117] (5) Secondary antibody incubation: Recover the primary antibody and wash the membrane 4 times for 5 minutes with 1×TSBT solution. Then, place the membrane in the dilution solution of the same species of secondary antibody prepared with 1×TSBT and incubate at room temperature for 1 hour.

[0118] (6) Exposure: Aspirate and discard the secondary antibody, wash the membrane with 1× TSBT solution 4 times for 5 minutes, and then expose the protein bands using a chemiluminescence imager.

[0119] 2.10 Statistical analysis

[0120] Refer to 2.2 in Example 1 for the test method.

[0121] 3. Test results

[0122] The results of HE and PAS pathological staining are as follows Figure 2As shown in A, compared with db / m control mice, the kidneys of db / db spontaneous type 2 diabetic mice showed pathological changes such as enlarged glomeruli, increased mesangial matrix, dilated tubules, and reduced or disappeared brush borders.

[0123] Depend on Figure 2 BE showed that the body weight of db / db mice was significantly increased compared with that of db / m control mice, and blood glucose, urinary albumin / creatinine ratio (UACR) and serum creatinine were significantly increased. These results indicated that the model was successfully established.

[0124] The results of immunohistochemical staining were Figure 2 As shown in A, it was observed that the expressions of kidney injury molecule 1 (KIM-1) and CNPY2 in the renal tubules of db / db mice were significantly increased compared with those of db / m mice.

[0125] Western blot test results are as follows Figure 2 As shown in F and G, it can be seen that the expression of KIM-1 and CNPY2 in the kidney tissue of db / db mice was significantly higher than that in the kidney tissue of db / m mice.

[0126] The results of this example indicate that CNPY2 may be involved in renal tubular injury in DN.

[0127] Example 3: Study on the expression of CNPY2 in HK-2 cells under different sugar concentrations

[0128] Western blot was used to detect the expression of CNPY2 in HK-2 cells under different sugar concentrations.

[0129] 1. Test methods

[0130] 1.1 Cultivation of immortalized human proximal tubular epithelial cell line (HK-2)

[0131] (1) Preparation of HK-2 cell culture medium: DMEM basal medium and F12 medium were mixed in a ratio of 1:1, and then 5% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution were added in sequence to prepare the basal culture medium for HK-2 cells (containing 5.6 mM glucose). The culture medium was aliquoted and stored at 4°C.

[0132] (2) HK-2 cell recovery: Take out the HK-2 cells from the liquid nitrogen tank and quickly put them into a 37°C water bath for rapid rewarming. After they are completely dissolved, transfer them to a 15 mL centrifuge tube and add 10 mL of the prepared culture medium. Centrifuge at 1000 rpm for 5 min at room temperature and discard the supernatant. The cell pellet can be seen at the bottom. Resuspend the cell pellet with 4 mL of fresh culture medium and transfer it to a culture flask. Culture it in a 37°C, 5% CO2 incubator and replace it with fresh culture medium every other day.

[0133] (3) HK-2 cell passaging: When the cells are almost fully grown, they are digested with trypsin and centrifuged at 1000 rpm for 5 min at room temperature. The cell pellet is resuspended in fresh culture medium and inoculated into a culture flask at an appropriate ratio for further culture.

[0134] (4) Cell cryopreservation: Select cells with faster growth rate and better cell status. When the cells grow to about 90%, digest them with trypsin, collect the cell pellets, resuspend them in serum-free cell cryopreservation solution, divide them into 2 mL cryopreservation tubes, store them at -80°C, and transfer them to a liquid nitrogen tank for long-term storage after at least 24 hours.

[0135] 1.2 HK-2 cell culture and grouping

[0136] HK-2 cells were divided into NG group, hypertonic (HM) group and HG group. In NG group, HK-2 cells were cultured in a medium containing 5.6 mM glucose, with the medium changed every other day; in HM group, HK-2 cells were cultured in a medium containing 5.6 mM glucose and 24.4 mM mannitol, with the medium changed every other day; in HG group, HK-2 cells were cultured in a medium containing 30 mM glucose, with the medium changed every other day.

[0137] 1.3 Western blot

[0138] Refer to 2.9 in Example 2 for the test method.

[0139] 1.4 Statistical analysis

[0140] Refer to 2.10 in Example 2 for the test method.

[0141] 2. Test results

[0142] Western blot results Figure 3 As shown, compared with the NG group and the HM group, the expression of CNPY2 in HK-2 cells cultured in a high glucose environment was significantly increased, and the expression of the renal tubular injury molecule KIM-1 was also increased.

[0143] The results of this example indicate that CNPY2 is overexpressed in renal tubular epithelial cells cultured in a high glucose environment.

[0144] Example 4: Study on the damage of renal tubules in db / db mice by overexpression of CNPY2

[0145] Through the research contents of Examples 1 to 3, we preliminarily verified that the expression level of CNPY2 in DN renal tubules is upregulated, which may be involved in DN renal tubular injury. Therefore, in order to further explore the mechanism of CNPY2 regulating DN renal tubular injury, this example injected CNPY2 overexpressing adeno-associated virus (db / db+CNPY2 OE) and control virus (db / db+Vehicle) into db / db mice through the tail vein to observe the renal tubular injury of db / db mice.

[0146] 1. Virus Information

[0147] The mouse-derived renal tubular epithelial cell-specific promoter-mediated CNPY2 overexpression adeno-associated virus and the corresponding control virus were constructed and synthesized by Shanghai GeneCare Gene Co., Ltd.

[0148] 2. Animal husbandry and grouping

[0149] All mouse-related studies in this application were approved by the Animal Ethics Committee of Zhengzhou University (ZZULAC20230526). Thirty 8-week-old db / db diabetic male mice were purchased from the Model Animal Research Center of Nanjing University and housed in the Experimental Animal Center of Zhengzhou University, with at least 5 mice per group. All animals were housed under standard feeding conditions.

[0150] Animal grouping: After one week of adaptive feeding, the mice were randomly divided into the following three groups: ①db / db control group, ②db / db+Vehicle group (CNPY2 OE empty vector control group), and at 10 weeks of age, the overexpression adeno-associated control virus targeting renal tubular epithelial cells was injected through the tail vein, with no less than 1×10 11 vg, ③db / db+CNPY2 OE group, at 10 weeks of age, renal tubule-targeted adeno-associated virus overexpression was injected into the tail vein, with no less than 1×10 11 vg; Blood glucose and body weight of mice in each group were tested every other week, and urine of mice was collected randomly. Mice in each group were euthanized at 22 weeks of age, and kidney and blood samples were collected quickly.

[0151] 3. Test methods

[0152] 3.1 Blood glucose detection in mice

[0153] The test method is as in 2.1 of Example 2.

[0154] 3.2 Collection of mouse kidney tissue

[0155] Refer to 2.5 in Example 2 for the test method.

[0156] 3.3HE staining

[0157] Refer to 2.6 in Example 2 for the test method.

[0158] 3.4 PAS staining

[0159] Refer to 2.7 in Example 2 for the test method.

[0160] 3.5 Western blot

[0161] Refer to 2.9 in Example 2 for the test method.

[0162] 3.6 Tissue TEM sample preparation and imaging

[0163] (1) Take the renal cortex and cut it into 1 mm 3 Tissue blocks of different sizes were fixed in electron microscopy solution at room temperature for at least 2 hours and stored in a 4°C refrigerator overnight.

[0164] (2) After washing with PBS buffer, the cells were fixed with osmium tetroxide for 90 minutes.

[0165] (3) Dehydration: The tissue is dehydrated by soaking in graded alcohol and acetone in sequence.

[0166] (4) Embedding: The samples after the above treatment were embedded in epoxy resin overnight.

[0167] (5) Sectioning: Use an ultra-thin slicer to slice the tissue and place it on a copper mesh guncotton film.

[0168] (6) Staining: Use lead citrate and uranyl acetate solution for staining.

[0169] (7) Filming: Observe the ultrastructure and take pictures under a transmission electron microscope.

[0170] 3.7 Proximity in situ detection test

[0171] use In situ detection reagent bright field kit (Cat. No.: DU092012, Sigma-Aldrich, USA), in situ PLA probe anti-rabbit PLUS (Cat. No.: DU092002, Sigma-Aldrich, USA), in situ PLA probe anti-mouse MINUS (Cat. No.: DU092004, Sigma-Aldrich, USA) were used to perform adjacent in situ detection (PLA) test on mouse kidney tissue. The specific steps were carried out according to the instructions of the kit.

[0172] 3.8 Immunohistochemical staining

[0173] Refer to 2.8 in Example 2 for the test method.

[0174] 3.9 Determination of malondialdehyde (MDA) content

[0175] The malondialdehyde (MDA) detection kit (Cat. No. S0131S, manufactured by Beyotime Biotech, China) was used to detect the malondialdehyde (MDA) content in mouse kidney tissue. The specific steps were performed according to the instructions of the kit.

[0176] 3.10 Detection of reduced glutathione (GSH) content

[0177] The reduced glutathione (GSH) detection kit (Cat. No. BC1175, Solebao Co., Ltd., China) was used to detect the reduced glutathione (GSH) content in mouse kidney tissue. The specific steps were performed according to the instructions of the kit.

[0178] 3.11 Iron ion content detection (Iron Assay kit)

[0179] The iron ion detection kit (Iron Assay Kit) (Cat. No.: MAK025, Sigma-Aldrich, USA) was used to detect the iron ion content in mouse kidney tissue, and the specific steps were performed according to the instructions of the kit.

[0180] 3.12 Statistical analysis

[0181] The test method is as in 1.4 of Example 3.

[0182] 4. Test results

[0183] 4.1 Overexpression of CNPY2 aggravates renal tubular pathological damage in db / db mice

[0184] The results of HE and PAS staining are as follows Figure 4 As shown in A, it can be seen that the db / db+CNPY2 OE group showed more severe tubular dilatation, disappearance of brush border and other pathological changes of renal tubular injury compared with the db / db+Vehicle group. Figure 4 As shown in B and C, overexpression of CNPY2 did not affect the body weight and blood glucose levels of db / db mice.

[0185] Western blot test results Figure 4 As shown in DE, compared with the db / db+Vehicle group and db / db group mice, the expression of CNPY2 in the CNPY2 overexpression group was significantly increased, and the expression level of the renal tubular injury marker KIM-1 was also significantly increased.

[0186] The above results indicate that overexpression of CNPY2 aggravates renal tubular damage in db / db mice.

[0187] 4.2 Overexpression of CNPY2 disrupts the integrity of MAM in renal tubular epithelial cells of db / db mice

[0188] Transmission electron microscopy (TEM) Figure 5 As shown in Figure A, the red dotted line represents the MAM area. It can be seen that after overexpression of CNPY2, the MAM area is significantly reduced.

[0189] The results of the PLA test are as follows Figure 5 As shown in the figure below, the dark red spots represent the MAM area. After overexpression of CNPY2, the MAM area is also significantly reduced. Figure 5 The PLA semi-quantitative results shown in B show that the number of MAMs in the renal tubular epithelial cells of the db / db+CNPY2 OE group was significantly reduced compared with the db / db+Vehicle and db / db groups.

[0190] The above results indicate that CNPY2 can regulate the expression level of MAM in DN renal tubular epithelial cells.

[0191] 4.3 Overexpression of CNPY2 aggravates ferroptosis, ERS, and mitochondrial damage in renal tubular epithelial cells of db / db mice

[0192] Transmission electron microscopy results Figure 6 As shown in A, compared with the db / db+Vehicle group and db / db group mice, the renal tubular epithelial cells of the db / db+CNPY2 OE group mice showed a more obvious reduction or disappearance of mitochondrial cristae. Similarly, the immunohistochemistry results also showed that in the db / db+CNPY2 OE group, the expression level of ferroptosis-related protein TFR-1 was significantly increased compared with the previous two groups, while the expression level of GPX4 was significantly decreased.

[0193] Depend on Figure 6 BD showed that compared with the db / db+Vehicle group and db / db mice, the Fe 2+ The content of glutathione increased significantly, the content of GSH decreased significantly, and the content of MDA increased significantly.

[0194] Western blot test results Figure 6 As shown in EF, the expression of ERS protein p-PERK and downstream ATF4 and CHAC1 proteins in the kidney tissue of db / db+CNPY2 OE mice was increased compared with the db / db+Vehicle group and db / db group, the expression of mitochondria-related protein TFAM was decreased, the expression of ferroptosis-related protein TFR1 was upregulated, and the expression of GPX4 protein was downregulated.

[0195] The above experimental results show that overexpression of CNPY2 can aggravate the level of ferroptosis in renal tubular epithelial cells of DN mice, accompanied by pathological changes such as ERS and aggravated mitochondrial damage.

[0196] Example 5: Study on the effect of knockdown of CNPY2 on renal tubular damage in db / db mice

[0197] Example 4 verified that overexpression of CNPY2 can destroy the integrity of MAM in renal tubular epithelial cells of db / db mice, further reducing their number, and aggravating the ferroptosis of renal tubular epithelial cells, ultimately leading to aggravated renal tubular damage in db / db mice. In order to further verify the regulatory mechanism of CNPY2 in DN renal tubular damage, this example injected CNPY2 knockdown adeno-associated virus (db / db+CNPY2 KD) and control virus (db / db+Vehicle) into db / db mice through the tail vein to observe whether the renal tubular damage in db / db mice can be improved.

[0198] 1. Virus Information

[0199] The mouse-derived renal tubular epithelial cell-specific promoter CNPY2 knockdown adeno-associated virus and the corresponding control virus were constructed and synthesized by Shanghai GeneCare Gene Co., Ltd.

[0200] 2. Animal husbandry and grouping

[0201] All mouse-related studies in this application were approved by the Animal Ethics Committee of Zhengzhou University (ZZULAC20230526). Thirty 8-week-old db / db diabetic male mice were purchased from the Model Animal Research Center of Nanjing University and kept in the Experimental Animal Center of Zhengzhou University, with at least 5 mice in each group. All animals were raised under standard feeding conditions. Grouping of experimental animals: After one week of adaptive feeding, the mice were randomly divided into the following 5 groups: ①db / db control group, ②db / db+Vehicle group (CNPY2 KD empty vector control group), and at 10 weeks of age, the renal tubule-targeted knockdown adeno-associated control virus was injected through the tail vein, with no less than 1×10 11 vg, ③db / db+CNPY2 KD group, at 10 weeks of age, adeno-associated virus targeting renal tubules was injected via tail vein, with no less than 1×10 11 vg, blood glucose and body weight were tested every other week, and urine samples were collected from mice randomly. All mice were euthanized at 22 weeks of age, and kidney and blood samples were collected quickly.

[0202] 3. Test methods

[0203] 3.1 Blood glucose detection in mice

[0204] The test method is as in 3.1 of Example 4.

[0205] 3.2 Collection of mouse kidney tissue

[0206] Refer to 3.2 in Example 4 for the test method.

[0207] 3.3HE staining

[0208] Refer to 3.3 in Example 4 for the test method.

[0209] 3.4 PAS staining

[0210] Refer to 3.4 in Example 4 for the test method.

[0211] 3.5 Western blot

[0212] Refer to 3.5 in Example 4 for the test method.

[0213] 3.6 Tissue TEM sample preparation and imaging

[0214] Refer to 3.6 in Example 4 for the test method.

[0215] 3.7 Proximity in situ detection test

[0216] Refer to 3.7 in Example 4 for the test method.

[0217] 3.8 Immunohistochemical staining

[0218] Refer to 3.8 in Example 4 for the test method.

[0219] 3.9 Determination of malondialdehyde (MDA) content

[0220] Refer to 3.9 in Example 4 for the test method.

[0221] 3.10 Detection of reduced glutathione (GSH) content

[0222] Refer to 3.10 in Example 4 for the test method.

[0223] 3.11 Iron ion content detection (Iron Assay kit)

[0224] Refer to 3.11 in Example 4 for the test method.

[0225] 3.12 Statistical analysis

[0226] Refer to 3.12 in Example 3 for the test method.

[0227] 4. Test results

[0228] 4.1 Knockdown of CNPY2 improves renal tubular pathological damage in db / db mice

[0229] The results of HE and PAS staining are as follows Figure 7 As shown in A, it can be seen that the db / db+CNPY2 KD group showed less tubular damage, such as tubular dilatation and disappearance of brush border, compared with the db / db+Vehicle group and the db / db group. Figure 7 As shown in B and C, knocking down CNPY2 did not affect the body weight and blood glucose levels of db / db mice.

[0230] Western blot test results Figure 7 As shown in DE, compared with the db / db+Vehicle group and db / db group mice, the expression of CNPY2 in the CNPY2KD group was significantly reduced, and the expression level of the renal tubular injury marker KIM-1 was also downregulated.

[0231] The above results indicate that knocking down CNPY2 can alleviate renal tubular damage in DN mice.

[0232] 4.2 Knockdown of CNPY2 improves MAM integrity in renal tubular epithelial cells of db / db mice

[0233] Transmission electron microscopy Figure 8 As shown in Figure A, the red dotted line represents the MAM area. It can be seen that after knocking down CNPY2, the MAM area was improved.

[0234] The results of the PLA test are as follows Figure 8 As shown in the figure below, the dark red spots represent the MAM area. It can be seen that after knocking down CNPY2, the MAM area was also improved. Figure 8 The semi-quantitative results of PLA shown in B showed that the number of MAMs in renal tubular epithelial cells of mice in the db / db+CNPY2 KD group was increased compared with those in the db / db+Vehicle and db / db groups.

[0235] The above results indicate that knocking down CNPY2 can improve MAM integrity in renal tubular epithelial cells of db / db mice.

[0236] 4.3 Knockdown of CNPY2 alleviates ferroptosis, ERS, and mitochondrial damage in renal tubular epithelial cells of db / db mice

[0237] Transmission electron microscopy results Fig. 9 As shown in A, compared with the db / db+Vehicle group and db / db group mice, the phenomenon of reduced or disappeared mitochondrial cristae in the renal tubular epithelial cells of the db / db+CNPY2 KD group mice was alleviated; the immunohistochemistry results also showed that in the db / db+CNPY2 KD group, the expression level of ferroptosis-related protein TFR-1 was significantly reduced compared with the previous two groups, while the expression of GPX4 was significantly increased.

[0238] Depend on Fig. 9 The BD results showed that compared with the db / db+Vehicle group and the db / db group, the Fe 2+ The content of CNPY2 was significantly decreased, the content of GSH was significantly increased, and the content of MDA was significantly decreased, indicating that after knocking down CNPY2, the level of lipid peroxidation in the kidney tissue of DN mice was alleviated, the accumulation of divalent iron ions was reduced, and the antioxidant capacity was improved.

[0239] Western blot test results Fig. 9 As shown in EF, the expression of ERS-related protein p-PERK and downstream ATF4 and CHAC1 in the kidney tissue of db / db+CNPY2 KD group mice was reduced compared with those in db / db+Vehicle group and db / db group, the expression of mitochondria-related protein TFAM was increased, the expression of ferroptosis-related protein TFR-1 was downregulated, and the expression of GPX4 protein was upregulated.

[0240] The above results indicate that knocking down CNPY2 can reduce the level of ferroptosis as well as ERS and mitochondrial damage in renal tubular epithelial cells of DN mice.

[0241] In summary, CNPY2 may participate in DN renal tubular injury by regulating MAM, ERS and ferroptosis levels, suggesting that targeting CNPY2 is expected to become a potential DN diagnosis and treatment strategy, so the present invention has a high utilization value. The above embodiments serve to illustrate the substantive content of the present invention, but do not limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or equivalently replaced without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. Application of CNPY2 gene or CNPY2 protein as a drug target for screening drugs for preventing, alleviating or treating diabetic nephropathy.

2. Use of a substance that inhibits the expression and / or function of CNPY2 in the preparation of a drug for preventing, alleviating or treating diabetic nephropathy.

3. The use according to claim 2, characterized in that: The substance that inhibits the expression and / or function of CNPY2 is a CNPY2 knockdown adeno-associated virus, a siRNA specifically targeting the CNPY2 gene, a shRNA specifically targeting the CNPY2 gene, or an inhibitor of the CNPY2 protein.

4. A drug, characterized in that The medicine is used for preventing, alleviating or treating diabetic nephropathy, and contains a substance that inhibits the expression and / or function of CNPY2.

5. The drug according to claim 4, characterized in that The substance that inhibits the expression and / or function of CNPY2 is a CNPY2 knockdown adeno-associated virus, a siRNA specifically targeting the CNPY2 gene, a shRNA specifically targeting the CNPY2 gene, or an inhibitor of the CNPY2 protein.

6. The drug according to claim 5, characterized in that The medicine also contains pharmaceutically acceptable carriers or excipients.

7. Use of a detection reagent for CNPY2 gene or CNPY2 protein in the preparation of a product for auxiliary diagnosis of diabetic nephropathy.

8. The use according to claim 7, characterized in that: The product detects the expression level of CNPY2 gene or CNPY2 protein in a sample by RT-PCR, real-time quantitative PCR, in situ hybridization, Northern blot, Western blot, chip, high-throughput sequencing platform, immunohistochemistry, tissue immunofluorescence or enzyme-linked immunosorbent assay.

9. The use according to any one of claims 7 or 8, characterized in that: The detection sample of the product is a cell or tissue; the product is a chip, a preparation or a kit.

10. A kit for auxiliary diagnosis of diabetic nephropathy, comprising a detection reagent for CNPY2 gene or CNPY2 protein.

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