Application of TRAIL in preparation of DKD diagnostic kit and medicine

Abstract

The invention discloses an application of TRAIL in preparation of a DKD diagnostic kit and a medicine, relates to the technical field of diagnosis and treatment of diabetic kidney diseases, and finds that the expression of TRAIL in the kidney tissue of a diabetic nephropathy patient is increased, is in negative correlation with eGFR, and is in positive correlation with UACR and glomerular sclerosis degree. The invention also finds that TRAIL rise is related to podocyte damage, and knock-down of TRAIL can reduce podocyte pan-apoptosis, so that DKD progress can be reflected to a certain extent. The specific mechanism that the TRAIL participates in the foot cell pan apoptosis of the diabetic nephropathy can be further verified through in-vivo and in-vitro experiments subsequently, and a theoretical foundation is laid for the treatment of the diabetic nephropathy and the research and development of targeted drugs.

Description

Technical Field

[0001] The invention relates to the technical field of diagnosis and treatment of diabetic kidney disease, and more specifically to the application of TRAIL in the preparation of DKD diagnostic kits and drugs. Background Art

[0002] Diabetic kidney disease (DKD) is a clinical syndrome characterized by persistent proteinuria and progressive decline in renal function, resulting from metabolic and hemodynamic disorders caused by chronic hyperglycemia. It is one of the most important complications of diabetes and the main cause of end-stage kidney disease (ESKD). The early clinical manifestations of DKD are not typical and difficult to diagnose. Once DKD progresses to the late stage, renal replacement therapy is inevitably required, which increases the economic burden on patients and society. Therefore, it is crucial to identify new diagnostic targets for DKD and develop effective DKD treatment methods.

[0003] Podocytes are terminally differentiated epithelial cells with primary, secondary, and tertiary foot processes. They are attached to the outside of the glomerular basement membrane (GBM) and together with endothelial cells and GBM form the glomerular filtration barrier. A decrease in the number or density of podocytes leads to basement membrane destruction and promotes proteinuria, which in turn leads to glomerular sclerosis and tubulointerstitial fibrosis, exacerbating the progression of DKD. Identifying key proteins that regulate podocyte damage can provide strategies and targets for the treatment of diabetic nephropathy, which has important clinical significance.

[0004] Therefore, finding proteins associated with podocyte injury in diabetic nephropathy and using them as targets for the diagnosis and treatment of DKD is an urgent problem that needs to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention uses TRAIL as a biomarker for the diagnosis and treatment of DKD, laying a theoretical foundation for the development of DKD diagnostic kits and DKD therapeutic drugs.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] Application of substances for detecting TRAIL in the preparation of DKD diagnostic kits.

[0008] In the above scheme, the substance for detecting TRAIL is a primer sequence, specifically:

[0009] TRAIL-F 5'-CGTGATCTTCACAGTGCTCCT-3', SEQ ID NO.1;

[0010] TRAIL-R 5'-CAGCTCGTTGGTAAAGTACACG-3', SEQ ID NO.2;

[0011] β-Actin-F 5'-CATGTACGTTGCTATCCAGGC-3', SEQ ID NO.7;

[0012] β-Actin-R 5'-CTCCTTAATGTCACGCACGAT-3', SEQ ID NO. 8.

[0013] As an inventive concept identical to the above technical solution, the present invention also seeks to protect a DKD diagnostic kit, characterized in that it comprises primers shown in SEQ ID NO.1 to SEQ ID NO.2 and SEQ ID NO.7 to SEQ ID NO.8.

[0014] As an inventive concept identical to the above technical solution, the present invention also requests protection for the use of a substance that knocks down TRAIL expression in the preparation of a drug for treating DKD; the substance that knocks down TRAIL expression is an interfering RNA, the sequence of which is: 5'-CCAUUUCUACAGUUCAAGAAA-3', SEQ ID NO.9.

[0015] In the above technical scheme, compared with the healthy control group, the expression of TRAIL in the renal tissue of patients with diabetic nephropathy was increased, positively correlated with UACR and the degree of glomerular sclerosis, and negatively correlated with eGFR.

[0016] Preferably, compared with the control group, TRAIL expression in podocytes stimulated by TNF-α is increased. TNF-α stimulation can lead to upregulation of pyroptosis-related gene cleaved caspase1, upregulation of apoptosis-related genes cleaved caspase 3, Bax and downregulation of Bcl-2, as well as upregulation of necroptosis-related genes p-MLKL and p-RIPK, promoting pan-apoptosis. Knockdown of TRAIL can alleviate TNF-α-induced pan-apoptosis of podocytes, as manifested by reversing the upregulation of pyroptosis-related gene cleaved caspase1, upregulation of apoptosis-related genes cleaved caspase 3, Bax and downregulation of Bcl-2, and upregulation of necroptosis-related genes p-MLKL and p-RIPK; overexpression of TRAIL is the opposite of the above results, manifested by promoting pan-apoptosis of podocytes and abnormal expression of related genes.

[0017] Preferably, TRAIL expression is increased in STZ-induced DKD mice. Compared with wild-type WT mice, STZ group mice had increased UACR, glomerular hypertrophy, increased mesangial matrix, thickened basement membrane, and widened podocyte foot pads; the number of glomerular podocytes decreased, apoptosis increased, and the expression of pan-apoptosis-related proteins cleaved caspase1, cleaved caspase3, and p-MLKL increased, indicating that podocytes and glomeruli were damaged in the STZ group. Compared with the STZ group, Trail KO The above-mentioned podocyte and glomerular injuries were improved in the +STZ group mice, indicating that podocyte-specific knockout of TRAIL can alleviate STZ-induced podocyte and glomerular injuries in DKD mice.

[0018] It can be seen from the above technical solution that compared with the prior art, the present invention discloses the role of TRAIL in DKD diagnosis and targeted drug therapy. The present invention finds that TRAIL expression is increased in renal tissue of patients with diabetic nephropathy, negatively correlated with eGFR, and positively correlated with UACR and degree of glomerular sclerosis. The present invention also finds that TRAIL increase is related to podocyte injury, and knocking down TRAIL reduces podocyte pan-apoptosis, which can reflect the progress of DKD to a certain extent. The specific mechanism of TRAIL involvement in pan-apoptosis of podocytes in diabetic nephropathy can be further verified by in vivo and in vitro experiments, which will help to lay a theoretical foundation for the treatment of diabetic nephropathy and the development of targeted drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 The attached figure shows the expression and distribution of TRAIL in patients with diabetic nephropathy; a is the immunohistochemical staining of TRAIL in renal tissues of DKD patients and healthy controls, n=20; b is the correlation analysis of glomerular TRAIL expression with eGFR, UACR and percentage of glomerular sclerosis; c is the immunofluorescence co-staining result of TRAIL and podocyte-specific marker synaptopodin in the glomeruli of DKD patients.

[0021] Figure 2The attached figure shows the expression of TRAIL in human immortalized podocytes; a is the protein and mRNA expression of TRAIL in human immortalized podocytes stimulated by TNF-α; b is the knockdown efficiency detection of TRAIL after transfection of human immortalized podocytes with TRAIL small interfering gene; c is the overexpression efficiency detection of TRAIL after infection of human immortalized podocytes with TRAIL overexpression lentivirus. n=3, **P<0.01, ***P<0.001.

[0022] Figure 3 The attached figure shows the expression of pan-apoptosis-related proteins detected by Western blot after human immortalized podocytes were infected with TRAIL small interfering virus and TRAIL overexpressing lentivirus, respectively. n=3, *P<0.05, **P<0.01, ***P<0.001.

[0023] Figure 4 The attached figure shows the expression and distribution of TRAIL in the renal tissue of STZ mice; a is a diagram of the animal model construction; b is the verification of the podocyte-specific TRAIL knockout efficiency; c is the immunofluorescence co-staining result of TRAIL and podocyte-specific marker synaptopodin in the glomeruli of STZ-induced DKD mice.

[0024] Figure 5 The attached figure shows the effect of podocyte-specific knockout of TRAIL on STZ-induced diabetic nephropathy in mice; a is the analysis of urine albumin to creatinine ratio (UACR), n = 6. ***P < 0.001 vs WT, ### P<0.001 vs WT+STZ; b is the result of PAS staining of kidney, and the quantitative analysis of glomerular volume and mesangial area percentage; c is the transmission electron microscopy image of mouse kidney and the quantitative analysis of foot process width and GBM thickness; d is the representative image of TUNEL staining and the quantification of TUNEL positive cells in each glomerular cross section of each mouse; e is the WT-1 staining of mouse glomerulus and the quantification of WT-1 positive cells in each glomerular cross section. f is the immunofluorescence staining image of cleaved caspase1, cleaved caspase3 and p-MLKL in glomerulus. *P<0.05, **P<0.01, ***P<0.001, n=6. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1 Expression of TRAIL protein in renal tissue of patients with diabetic nephropathy and its correlation with clinical indicators

[0027] Collecting kidney tissue samples

[0028] A total of 20 DKD patient renal tissue specimens were obtained from renal tissues that underwent puncture biopsy in the Department of Nephrology, Provincial Hospital Affiliated to Shandong First Medical University from May 2021 to May 2024; a total of 20 normal control samples were obtained from normal renal tissue adjacent to the cancer that underwent nephrectomy due to tumors. Before all samples were collected, the patient or his / her guardian agreed and signed an informed consent form. All human specimen collection studies were approved by the Ethics Committee of the Provincial Hospital Affiliated to Shandong First Medical University.

[0029] Immunohistochemical staining was used to detect the expression level of TRAIL in the renal tissue of DKD patients, and its correlation with eGFR, UACR, and the degree of glomerular sclerosis was further analyzed. The co-localization of TRAIL and podocytes was detected by immunofluorescence co-staining of TRAIL and podocyte marker protein synaptopodin.

[0030] The specific steps are as follows:

[0031] Immunohistochemical staining

[0032] (1) Bake the slices at 65°C for 60-90 minutes.

[0033] (2) Dewaxing and hydration: The paraffin sections were transferred to a copper rack and sequentially treated with dewaxing agent I for 15 min, dewaxing agent II for 15 min, anhydrous ethanol I for 8 min, anhydrous ethanol II for 8 min, 95% ethanol for 5 min, 85% ethanol for 5 min, and 70% ethanol for 5 min.

[0034] (3) Washing: Soak in distilled water three times, 5 min each time; soak in PBS once, 5 min each time.

[0035] (4) Antigen retrieval: Prepare antigen retrieval solution and perform high pressure heat retrieval for 3 minutes.

[0036] (5) Washing: After the repair solution returns to room temperature, soak the tissue sections in PBS three times, each time for 5 min.

[0037] (6) Inactivation: Add endogenous peroxidase blocker to the tissue sections and incubate in a 37°C incubator for 30 min.

[0038] (7) Washing: Soak in PBS three times, 5 min each time.

[0039] (8) Blocking: Add 5% BSA blocking solution to the tissue sections and incubate in a 37°C incubator for 30 min.

[0040] (9) Incubation with primary antibody: discard the blocking solution, add TRAIL (Proteintech, 27064-1-AP) primary antibody working solution, and incubate at 4°C overnight.

[0041] (10) Rewarming: The next day, transfer the slices to a 37°C incubator and rewarm for 30 min.

[0042] (11) Washing: Soak in PBS three times, 5 min each time.

[0043] (12) Add reaction enhancement solution to the tissue sections and incubate in a 37°C incubator for 30 min.

[0044] (13) Washing: Soak in PBS three times, 5 min each time.

[0045] (14) Incubation with secondary antibody: Add enhanced enzyme-labeled goat anti-mouse / rabbit IgG working solution and incubate in a 37°C incubator for 30 min.

[0046] (15) Washing: Soak in PBS three times, 5 min each time.

[0047] (16) DAB color development: Prepare DAB reaction solution and add it dropwise to the tissue sections for reaction. Rinse with running water for 10 min to terminate the color development reaction.

[0048] (17) Restaining: Add hematoxylin solution to the tissue sections, incubate for 3-5 minutes, and then rinse with running water.

[0049] (18) Differentiation: Add hydrochloric acid alcohol differentiation solution to the tissue sections, incubate for a few seconds, and then rinse with running water.

[0050] (19) Dehydration and transparency: The sections were transferred to a copper rack and sequentially placed in 95% ethanol for 30 s, anhydrous ethanol I for 30 s, anhydrous ethanol II for 1 min, xylene I for 1 min, and xylene II for 2 min.

[0051] (20) Sealing: After sealing with neutral gum, observe under a microscope.

[0052] Immunofluorescence staining

[0053] (1) Baking, dewaxing, and hydration: same as above.

[0054] (2) Washing: Soak in distilled water three times, 5 min each time; soak in PBS once, 5 min each time.

[0055] (3) Antigen retrieval: Prepare antigen retrieval solution and perform high pressure heat retrieval for 3 minutes.

[0056] (4) Washing: After the repair solution returns to room temperature, soak the tissue sections in PBS three times, each time for 5 min.

[0057] (5) Permeabilization: 0.5% Triton permeabilization for 5 min, and PBS soaking for 3 times, 5 min each time.

[0058] (6) Blocking: Add 5% BSA blocking solution to the tissue sections and incubate in a 37°C incubator for 30 min.

[0059] (7) Incubation with primary antibody: discard the blocking solution, add TRAIL (Proteintech, 27064-1-AP) and synaptopodin (Proteintech, 67339-1-Ig) primary antibody working solution, and incubate at 4°C overnight.

[0060] (8) Rewarming: Same as above.

[0061] (9) Washing: Soak in PBS three times, 5 min each time.

[0062] (10) Incubate with secondary antibody: Protect from light and add appropriate concentration of Alexa Fluor 594-conjugated donkeyanti-rabbit IgG (ab150076, abcam) and Alexa 488-conjugated goat anti-mouse IgG (ab150113, abcam) secondary antibody working solution was added and incubated at 37°C for 60 min.

[0063] (11) Washing: Protect from light and soak in PBS three times, 5 min each time.

[0064] (12) Protect from light, add DAPI to the tissue sections and incubate for 10 min.

[0065] (13) Washing: Protect from light and soak in PBS three times, 5 min each time.

[0066] (14) Sealing: Protect from light. Use anti-fluorescence fading sealing medium to seal the slices and observe under a fluorescence microscope.

[0067] The results showed that: Immunohistochemistry results showed that compared with the healthy control group, TRAIL expression in the renal tissue of patients with diabetic nephropathy was increased, which was positively correlated with UACR (r = 0.5327, P = 0.0156) and glomerular sclerosis (r = 0.5066, P = 0.0226), and negatively correlated with eGFR (r = -0.4648, P = 0.038); Immunofluorescence results showed that TRAIL and podocytes were co-localized in the renal tissue of DKD patients. The above results suggest that TRAIL may be related to podocyte damage in DKD ( Figure 1 ).

[0068] Example 2 The role of TRAIL in the occurrence of pan-apoptosis of podocytes

[0069] Primer sequences of target gene and internal reference gene:

[0070] TRAIL

[0071] F 5'-CGTGATCTTCACAGTGCTCCT-3', SEQ ID NO.1;

[0072] R 5'-CAGCTCGTTGGTAAAGTACACG-3', SEQ ID NO.2;

[0073] Bax

[0074] F 5'-GAGGTCTTTTTCGAGTGGCA-3', SEQ ID NO.3;

[0075] R 5'-GGCAAAGTAGAAAAGGGCGAC-3', SEQ ID NO.4;

[0076] Bcl-2

[0077] F 5'-GTGAACTGGGGGAGGATTGT-3', SEQ ID NO.5;

[0078] R 5'-GCCCAGACTCACATCACCAAG-3', SEQ ID NO.6;

[0079] β-actin

[0080] F 5'-CATGTACGTTGCTATCCAGGC-3', SEQ ID NO.7;

[0081] R 5'-CTCCTTAATGTCACGCACGAT-3', SEQ ID NO.8;

[0082] Primary antibodies and product numbers used in Western blot:

[0083] TRAIL (Proteintech, 27064-1-AP), cleaved caspase-3Rabbit mAb (Cell Signaling Technology, 9664), caspase-3antibody (Cell Signaling Technology, 9662), caspase-1Rabbit mAb (Cell Signaling Technology, 3866), anti-MLKL (phosphor S358)(abcam,ab187091),MLKL Monoclonal antibody(Proteintech,66675-1-Ig),Phospho-RIPK1(Ser166)RabbitmAb(Cell Signaling Technology,44950),RIPK1 RabbitmAb(CellSignaling Technology,3493),Bax polyclonal antibody(Proteintech,50599-2-Ig),and anti-bcl-2antibody (abcam, ab182858), β-actin (Proteintech, 66009-1-Ig).

[0084] In order to clarify the role of TRAIL in the occurrence of pan-apoptosis of podocytes, we first stimulated podocytes with TNF-α to simulate the in vitro diabetic nephropathy environment, divided the podocytes into a control group (con) and a TNF-α stimulation group (TNF-α), and detected the protein and mRNA expression of TRAIL in podocytes by real-time PCR and Western blot. We further transfected podocytes with the constructed TRAIL small interfering and overexpressing lentiviruses, and the Western blot results showed that good knockdown and overexpression efficiency was obtained ( Figure 2 ).

[0085] TRAIL siRNA:

[0086] TRAIL siRNA 5'-CCAUUUCUACAGUUCAAGAAA-3', SEQ ID NO.9. NC siRNA 5'-UUCUCCGAACGUGUCACGU-3', SEQ ID NO.10;

[0087] After podocytes were transfected with TRAIL small interference or overexpression lentivirus, the expressions of pyroptosis-related gene cleaved caspase1, apoptosis-related genes cleaved caspase3, Bax and Bcl-2, and necroptosis-related genes p-MLKL and p-RIPK were detected by real-time PCR and Western blot. Figure 3 ).

[0088] The specific steps are as follows:

[0089] Real-time PCR

[0090] (1) RNA extraction

[0091] Total RNA was extracted from cultured podocytes using the SteadyPure RNA extraction kit (Acrobio, China) as follows:

[0092] ① Aspirate the culture medium in the six-well plate when the cell confluence is above 80%, and add PBS buffer to rinse the cells once.

[0093] ② Aspirate the PBS washing solution, add 500 μl of RNA lysis solution to each well, and gently shake the six-well plate to make the RNA lysis solution evenly cover the cell surface.

[0094] ③ Use a pipette to repeatedly blow and mix until the cells fall off the attached surface, then transfer the lysate containing the cells to an RNase-free EP tube, and continue to blow and mix with a pipette until the lysate is clear and there is no obvious precipitation.

[0095] ④ After standing at room temperature for 5 minutes, add 500 μl of 70% ethanol to the lysate and mix well with a pipette.

[0096] ⑤ Transfer the above mixed solution to the RNA Mini Column and centrifuge at 12000 rpm at room temperature for 1 minute.

[0097] ⑥Discard the filtrate, add 600μl of Buffer RW1 to the RNA Mini Column, and centrifuge at 12000rpm at room temperature for 1min.

[0098] ⑦Discard the filtrate, add 650 μl of Buffer RW2 to the RNA Mini Column, and centrifuge at 12000 rpm at room temperature for 1 min.

[0099] ⑧Discard the filtrate, add 650 μl of Buffer RW2 to the RNA Mini Column, and centrifuge again at 12000 rpm at room temperature for 1 min.

[0100] ⑨Discard the filtrate, place the RNA Mini Column in a new RNase-free EP tube, and centrifuge at 12000 rpm at room temperature for 2 minutes.

[0101] ⑩ Place the RNA Mini Column on a new RNase-free EP tube and add an appropriate amount of DEPC water to the center of the RNA Mini Column membrane.

[0102] Let stand at room temperature for 5 minutes, centrifuge at 12000 rpm for 2 minutes at room temperature, and the liquid in the EP tube is the RNA solution. This RNA can be directly used in the next experiment or stored at -80℃.

[0103] (2) Reverse transcription

[0104] The extracted RNA was tested for purity using a NanoDrop-2000 spectrophotometer. 1 μg of total RNA was reverse transcribed into cDNA using the Evo M-MLV reverse transcription kit (Aikuri Biotech, China) according to the manufacturer's instructions. The cDNA samples can be directly used for real-time PCR detection or stored at -20°C.

[0105] The reverse transcription reaction system is shown in Table 1;

[0106] Table 1

[0107]

[0108] (3) Fluorescence quantitative PCR analysis

[0109] Boshan Bio is responsible for synthesizing the primers used in Real-time PCR. Before use, the primer freeze-dried powder needs to be centrifuged at 4°C and 12000rpm for 2 minutes, and an appropriate amount of DEPC water is added to completely dissolve the freeze-dried powder to a final concentration of 10μM.

[0110] The SYBR Green Pro Taq HS premixed qPCR kit (Acori Biotech, China) was used to prepare the PCR reaction system on ice according to the manufacturer's instructions. The reaction system was added to a 96-well plate and sealed according to the experimental design. The plate was centrifuged at 3000 rpm for 2 min at room temperature. The quantitative expression level of the target gene was detected by qPCR using a Roche 480 PCR instrument. The gene expression level was normalized to β-actin and 2 -ΔΔCt Method for analysis.

[0111] The qPCR reaction system is shown in Table 2;

[0112] Table 2

[0113]

[0114] Western blot

[0115] (1) Total cell protein extraction

[0116] ① Aspirate the culture medium in the six-well plate when the cell confluence is above 80%, and add PBS buffer to rinse the cells twice.

[0117] ② Aspirate the PBS washing solution, add 100 μl of protein lysis solution (prepared according to protein lysis solution: protease inhibitor: phosphatase inhibitor = 100:1:1) to each well, gently shake the six-well plate to make the protein lysis solution evenly cover the cell surface, and place the six-well plate on ice for 30 minutes.

[0118] ③ Scrape the lysed cells and transfer them to an RNase-free EP tube, and centrifuge at 4°C and 12,000 rpm for 30 min.

[0119] ④ Transfer the supernatant to a new RNase-free EP tube.

[0120] (2) Protein concentration determination

[0121] ① Prepare BCA working solution according to BCA reagent A:B=50:1, and ensure that the two are fully mixed.

[0122] ② Add 0-20 μl of protein standard solution and sample solution to be tested to the standard wells respectively, and then add PBS buffer to fill the volume of each well to 20 μl to ensure that the total amount of liquid in each well is consistent.

[0123] ③ Add 200 μl of BCA working solution to all wells, and then place the entire plate in a 37°C incubator and incubate for 20-30 minutes.

[0124] ④ Use a microplate reader to measure the absorbance value of each well at wavelength A562.

[0125] ⑤ Draw a standard curve based on the measured absorbance values, and calculate the protein concentration of each sample based on this curve.

[0126] ⑥ According to the ratio of protein supernatant and 4×SDS-PAGE loading buffer 3:1, mix the two, blow thoroughly to make them evenly distributed, and heat them in a metal bath at 95℃ for 10 minutes to fully denature the protein.

[0127] (3) Immunoblotting

[0128] Protein samples were separated by 10% or 12.5% ​​SDS-PAGE, transferred to PVDF membrane (Millipore, USA), blocked with fast blocking solution for 10-15 min, and incubated with primary antibody at 4°C overnight. The membrane was washed with TBS-T the next day and incubated with the corresponding secondary antibody at room temperature for 1-2 h. Protein bands were visualized using ECL luminescent solution and imaged using Amersham Imager680 (GE, USA). Quantification was performed using ImageJ.

[0129] The results showed that compared with the control group, TRAIL expression was increased in podocytes stimulated by TNF-α. TNF-α stimulation can lead to upregulation of pyroptosis-related gene cleaved caspase1, upregulation of apoptosis-related genes cleaved caspase 3 and Bax, downregulation of Bcl-2, and upregulation of necroptosis-related genes p-MLKL and p-RIPK, promoting pan-apoptosis. Knockdown of TRAIL can alleviate TNF-α-induced pan-apoptosis of podocytes, which is manifested by reversing the upregulation of pyroptosis-related gene cleaved caspase1, upregulation of apoptosis-related genes cleaved caspase 3 and Bax, downregulation of Bcl-2, and upregulation of necroptosis-related genes p-MLKL and p-RIPK; overexpression of TRAIL is the opposite of the above results, which is manifested by promoting pan-apoptosis of podocytes and abnormal expression of related genes. It suggests that TRAIL is involved in the occurrence of TNF-α-induced pan-apoptosis of podocytes.

[0130] Example 3 Effect of TRAIL on podocyte and glomerular damage in STZ-induced diabetic nephropathy mice

[0131] C57BL / 6 mice were provided by the Experimental Animal Center of Shandong First Medical University. All animal studies were reviewed and approved by the Experimental Animal Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University.

[0132] Construction of podocyte-specific TRAIL knockout mice

[0133] First, (flox / +) heterozygous mice were mated to obtain (flox / flox) homozygous mice. Subsequently, NPHS2-Cre tool mice were mated with (flox / +) heterozygous mice or (flox / flox) homozygous mice to obtain [flox / +,Cre(+ / -)] mice. Finally, [flox / +,Cre(+ / -)] mice were mated with (flox / +) or (flox / flox) to obtain [flox / flox,Cre(+ / -)] mice, i.e., podocyte-specific TRAIL knockout mice. Eight-week-old male mice were further randomly divided into (WT, n=6), podocyte-specific TRAIL knockout group (TRAIL KO, n = 6), STZ injection group (STZ, n = 6), podocyte-specific TRAIL knockout + STZ injection group (STZ + TRAIL KO ,n=6).

[0134] Building the DKD model

[0135] STZ group: 8-week-old male C57BL / 6J mice were fed a high-fat diet for 4 weeks and then intraperitoneally injected with STZ 50 mg / kg for 5 consecutive days; the control group (WT group) mice were intraperitoneally injected with the same dose of citrate buffer.

[0136] After 20 weeks of STZ or citrate buffer injection, mice were killed and kidney tissues of mice in each group were collected: ① Primary podocytes were isolated and cultured, and the expression of TRAIL in primary podocytes was detected by Western blot to verify the efficiency of podocyte knockout; ② TRAIL and podocyte marker protein synaptopodin were co-stained with immunofluorescence to detect the expression and distribution of TRAIL in the kidney tissue of STZ mice ( Figure 4 ).

[0137] The specific steps are as follows:

[0138] Isolation and culture of primary podocytes

[0139] The glomeruli were separated by mesh sieve method. The mouse kidneys were stripped of their capsules and cut into small pieces. They were placed in a digestion solution containing collagenase and pronase E and digested at 37°C for 15 minutes. Complete medium was added to terminate the digestion. The digested tissues were sequentially passed through a 50-mesh sieve, a 100μm sieve, and a 400-mesh sieve to collect the glomeruli. The collected glomeruli were cultured in an incubator for 5 days, and then the podocytes that crawled out were digested and passaged. After trypsin digestion, the completely digested cells were passed through a 40μm sieve to remove the glomerular core, and inoculated into a 6-well plate. After maturation, the podocytes were identified using the podocyte-specific marker molecule WT-1.

[0140] Immunofluorescence staining as in Example 1

[0141] In order to clarify the effect of podocyte-specific knockout of TRAIL on STZ-induced diabetic nephropathy in mice, the levels of UACR in mice were collected and detected at 0W, 4W, 8W, 12W, 16W, and 20W after STZ or citrate buffer injection; the mice were killed at 20W, and the renal tissues were collected for PAS staining, transmission electron microscopy, TUNEL staining, and immunofluorescence staining (WT-1 to detect the number of podocytes; cleaved caspase1, cleaved caspase3, and p-MLKL to detect glomerular apoptosis) ( Figure 5 ).

[0142] PAS staining

[0143] (1) Baking, dewaxing, and hydration: same as above.

[0144] (2) Washing: Soak in distilled water three times, 5 min each time; soak in PBS once, 5 min each time.

[0145] (3) Add oxidant to the tissue sections, incubate for 8 minutes, and then rinse with running water.

[0146] (4) Washing: Soak in distilled water twice, 5 min each time.

[0147] (5) Add Schiff staining solution to the tissue sections, incubate for 15 minutes, and then rinse with running water for 10 minutes.

[0148] (6) Add hematoxylin solution to the tissue sections, incubate for 3 minutes, and then rinse with running water.

[0149] (7) Differentiation: same as above.

[0150] (8) Dehydration and transparency: same as above.

[0151] (9) Sealing: After sealing with neutral gum, observe under a microscope.

[0152] Transmission electron microscopy

[0153] (1) Cut the kidney tissue into 1 mm 3 Place the tissue blocks of about 100 μg / cm2 in pre-cooled glutaraldehyde solution and fix them in a 4°C refrigerator for 2-4 h.

[0154] (2) The tissue was thoroughly washed with PBS buffer and fixed in 1% osmium hydroxide for 1-2 h.

[0155] (3) After the tissue was thoroughly washed with PBS buffer, it was immersed in 30%, 50%, 70%, 90% and 100% acetone for gradient dehydration.

[0156] (4) After embedding and polymerization, the tissue block was cut into slices with a thickness of 50 nm using an ultrathin slicer.

[0157] (5) Tissue sections were stained with uranyl acetate and lead citrate, observed under a transmission electron microscope, and photographed.

[0158] TUNEL staining

[0159] The procedure was performed according to the instructions of the Rapid One-Step TUNEL Cell Apoptosis Detection Kit (KeyGen Biotech Co., Ltd., Jiangsu).

[0160] (1) Bake mouse kidney tissue slices on a 65°C oven for 30-60 min.

[0161] (2) Dewaxing and hydration: Soak in two xylene tanks for 15 min each; two anhydrous ethanol tanks for 8 min each; a 95% ethanol tank for 5 min; an 80% ethanol tank for 5 min; and a 75% ethanol tank for 5 min.

[0162] (3) Soak in distilled water for 3 times, 5 min each time; soak in PBS for 5 min.

[0163] (4) Add 100 μl of Proteinase K working solution to each slice and incubate at 37°C for 30 min. Then soak in PBS three times, 5 min each time.

[0164] (5) Select a slice and add 100 μl of DNase I reaction solution and incubate at 37°C for 30 min (to make positive slices). Soak in PBS three times, 5 min each time.

[0165] (6) Incubate the slices with the prepared TUNEL detection solution at 37°C in the dark for 1 h, and wash with PBS three times, 5 min each time.

[0166] (7) DAPI nucleus staining for 5 min.

[0167] (8) Wash with PBS three times, 5 min each time.

[0168] (9) Seal the slides with a fluorescence quencher and observe the fluorescence expression using a fluorescence microscope.

[0169] Immunofluorescence staining of WT-1, cleaved caspase1, cleaved caspase3, and p-MLKL

[0170] (1) Baking, dewaxing, and hydration: same as above.

[0171] (2) Washing: Soak in distilled water three times, 5 min each time; soak in PBS once, 5 min each time.

[0172] (3) Antigen retrieval: Prepare antigen retrieval solution and perform high pressure heat retrieval for 3 minutes.

[0173] (4) Washing: After the repair solution returns to room temperature, soak the tissue sections in PBS three times, each time for 5 min.

[0174] (5) Permeabilization: 0.5% Triton permeabilization for 5 min, and PBS soaking for 3 times, 5 min each time.

[0175] (6) Blocking: Add 5% BSA blocking solution to the tissue sections and incubate in a 37°C incubator for 30 min.

[0176] (7) Incubation with primary antibody: discard the blocking solution, add WT-1 (abcam, ab89901), cleaved caspase-3 (Cell Signaling Technology, 9664), cleaved caspase1 (Invitrogen, PA5-105049), and p-MLKL (Cell Signaling Technology, 37333) primary antibody working solution, and incubate at 4°C overnight.

[0177] (8) Rewarming: Same as above.

[0178] (9) Washing: Soak in PBS three times, 5 min each time.

[0179] (10) Incubate with secondary antibody: Protect from light and add appropriate concentration of Alexa Fluor 594-conjugated donkeyanti-rabbit IgG (ab150076, abcam) or Alexa 488-conjugated goat anti-rabbit IgG H&L (ab150077, abcam) secondary antibody working solution was added and incubated at 37°C for 60 min.

[0180] (11) Washing: Protect from light and soak in PBS three times, 5 min each time.

[0181] (12) Protect from light, add DAPI to the tissue sections and incubate for 10 min.

[0182] (13) Washing: Protect from light and soak in PBS three times, 5 min each time.

[0183] (14) Sealing: Protect from light. Use anti-fluorescence fading sealing medium to seal the slices and observe under a fluorescence microscope.

[0184] The results showed that compared with wild-type WT mice, TRAIL expression was increased in STZ-induced DKD mice, and co-localized with podocytes. In addition, compared with wild-type WT mice, UACR of STZ group mice increased, and PAS staining results showed glomerular hypertrophy and increased mesangial matrix in STZ group mice; transmission electron microscopy results showed thickening of glomerular basement membrane and widening of podocyte foot pads in STZ group mice; WT-1 staining showed a decrease in the number of glomerular podocytes in STZ group; TUNEL results showed increased cell apoptosis in STZ group; immunofluorescence results showed increased expression of pan-apoptosis-related proteins cleaved caspase1, cleaved caspase3, and p-MLKL in STZ group mice. The above results indicate that podocytes and glomeruli of STZ group mice were damaged. Compared with the STZ group, Trail KOThe above-mentioned podocyte and glomerular injuries were all improved in the +STZ group mice, indicating that podocyte-specific knockout of TRAIL could alleviate STZ-induced podocyte and glomerular injuries in DKD mice.

[0185] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same and similar parts between the embodiments can be referred to each other. The above description of the disclosed embodiments enables professionals and technicians in this field to implement or use the present invention. Various modifications to these embodiments will be obvious to professionals and technicians in this field, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will comply with the widest range consistent with the principles and novel features disclosed herein.

Claims

1. Application of substances for detecting TRAIL in the preparation of DKD diagnostic kits.

2. The use according to claim 1, characterized in that: The substances for detecting TRAIL are shown in SEQ ID NO.1 to SEQ ID NO.2 and SEQ ID NO.7 to SEQ ID NO.8: TRAIL-F 5'-CGTGATCTTCACAGTGCTCCT-3', SEQ ID NO.1; TRAIL-R 5'-CAGCTCGTTGGTAAAGTACACG-3', SEQ ID NO.2; β-Actin-F 5'-CATGTACGTTGCTATCCAGGC-3', SEQ ID NO.7; β-Actin-R 5'-CTCCTTAATGTCACGCACGAT-3', SEQ ID NO.

8.

3. A DKD diagnostic kit, characterized in that: It includes primers shown in SEQ ID NO.1 to SEQ ID NO.2 and SEQ ID NO.7 to SEQ ID NO.

8.

4. Application of substances that knock down TRAIL expression in the preparation of drugs for the treatment of DKD.

5. The use according to claim 4, characterized in that: The substance for knocking down TRAIL expression is interfering RNA, and the sequence is: 5'-CCAUUUCUACAGUUCAAGAAA-3', SEQ ID NO.

9.

6. The use according to claim 4, characterized in that: The expression of TRAIL is increased in the renal tissue of patients with diabetic nephropathy and is correlated with renal function indicators.

7. The use according to claim 4, characterized in that: Knockdown of TRAIL alleviated TNF-α-induced podocyte pan-apoptosis and reversed the abnormal expression of its related genes cleaved caspase1, cleaved caspase 3, Bax, Bcl-2, p-MLKL, and p-RIPK; overexpression of TRAIL promoted the occurrence of podocyte pan-apoptosis and the abnormal expression of related genes.

8. The use according to claim 4, characterized in that: TRAIL expression was upregulated in STZ-induced DKD mice, and podocyte-specific knockout of TRAIL alleviated STZ-induced podocyte and glomerular damage in DKD mice.

Images