Application of substance for detecting DR5 in preparation of DKD diagnostic kit

Abstract

The invention discloses an application of a substance for detecting DR5 in preparation of a DKD diagnostic kit, relates to the technical field of diagnosis and treatment of diabetic kidney diseases, and finds that the expression of DR5 in the kidney tissue of a diabetic nephropathy patient is increased, and co-localization exists between DR5 and podocytes. The invention also finds that the increase of DR5 is related to podocyte damage, and knock-down of DR5 alleviates the occurrence of podocyte pan-apoptosis and slows down the progress of DKD. The specific mechanism that DR5 participates in the occurrence of foot cell pan apoptosis of diabetic nephropathy can be further verified through in-vivo and in-vitro experiments subsequently, and a theoretical foundation is laid for diagnosis of diabetic nephropathy and research and development of targeted therapeutic 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 a substance for detecting DR5 in the preparation of a DKD diagnostic kit. Background Art

[0002] Diabetic kidney disease (DKD) is a chronic kidney disease caused by diabetes. At present, China has become the country with the largest number of diabetes patients in the world. With the sharp increase in the number of diabetes patients, the kidney, as an important target organ, has also suffered more damage. About 40% of diabetic patients may develop DKD. The incidence of DKD has also been gradually increasing in recent years. DKD can cause kidney damage and may eventually progress to end-stage renal disease; it is also prone to a variety of complications, increasing the risk of cardiovascular disease and death in patients. Timely diagnosis and effective management of DKD and delaying the progression of the disease have become major clinical challenges.

[0003] Podocytes, or epithelial cells of the visceral layer of the renal capsule, are attached to the outside of the glomerular basement membrane (GBM). Together with vascular endothelial cells and the glomerular basement membrane, they constitute the glomerular blood filtration barrier, and are essential for maintaining the structural integrity and function of the glomerular filtration barrier. Podocyte damage can lead to glomerular sclerosis and damaged filtration barriers, promote the production of proteinuria, and play a key role in the occurrence and development of chronic kidney diseases such as diabetic nephropathy. Targeting key proteins in podocytes and protecting podocytes to delay the progression of DKD is a new perspective for the treatment of DKD.

[0004] Therefore, screening receptor proteins related to diabetic nephropathy in plantar cells, using them as detection targets as detection markers for diabetic nephropathy, and developing new drugs for the treatment of DKD are technical problems that technicians in this field urgently need to solve. Summary of the invention

[0005] In view of this, the present invention uses DR5 as a marker for diagnosis and targeted treatment of DKD, laying a theoretical foundation for the development of DKD diagnostic kits and DKD targeted therapeutic drugs.

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

[0007] Application of substances for detecting DR5 in the preparation of DKD diagnostic kits.

[0008] In the above scheme, the substance for detecting DR5 is a primer sequence, specifically:

[0009] DR5-F 5'-AGACCCTTGTGCTCGTTGTC-3', SEQ ID NO.1;

[0010] DR5-R 5'-GGGGCTGGACCTCTTTTGTT-3', SEQ ID NO.2;

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

[0012] β-Actin-R 5'-CTCCTTAATGTCACGCACGAT-3', SEQ ID NO. 4.

[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.4.

[0014] As an inventive concept identical to the above technical solution, the present invention further requests protection for the use of a substance for knocking down DR5 expression in preparing a drug for treating DKD; the substance for knocking down DR5 expression is an interfering RNA, the sequence of which is: 5'-CCACAAAGAAUCAGGUACATT-3', SEQ ID NO.5.

[0015] In the above technical scheme, compared with the control group, DR5 expression in the renal tissue of patients with diabetic nephropathy is increased, co-localized with podocytes, and DR5 expression is increased when podocytes are damaged.

[0016] Preferably, compared with the control group, DR5 expression in TNF-α stimulated human immortalized podocytes is increased. TNF-α stimulation can upregulate the expression of podocyte p-MLKL, p-RIPK, cleaved caspase1, cleaved caspase 3, and Bax, and promote pan-apoptosis; knocking down DR5 can alleviate TNF-α-induced pan-apoptosis of podocytes and reverse the abnormal expression of its related genes p-MLKL, p-RIPK, cleaved caspase1, cleaved caspase 3, and Bax.

[0017] Preferably, compared with WT mice, DR5 was expressed more in the glomeruli of STZ-induced DKD mice and co-localized with podocytes. Compared with WT mice, the UACR of STZ group mice increased, accompanied by glomerular hypertrophy and increased mesangial matrix, decreased podocyte number, increased apoptosis, and increased expression of pan-apoptosis-related proteins cleaved caspase1, cleaved caspase3, and p-MLKL, indicating that STZ stimulation can induce kidney damage and pan-apoptosis. Compared with the STZ group, DR5 KOThe above-mentioned injuries were alleviated in the mice in the +STZ group, indicating that podocyte-specific knockout of DR5 can improve STZ-induced kidney injury 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 DR5 in the diagnosis of DKD and targeted drug therapy. The present invention finds that DR5 expression is elevated in the renal tissue of patients with diabetic nephropathy and co-localizes with podocytes. The present invention also finds that elevated DR5 is associated with podocyte damage, and knocking down DR5 reduces the occurrence of podocyte pan-apoptosis and slows down the progression of DKD. The specific mechanism of DR5 involvement in the occurrence of pan-apoptosis of podocytes in diabetic nephropathy can be further verified through in vivo and in vitro experiments, which will help lay a theoretical foundation for the diagnosis of diabetic nephropathy and the development of targeted therapeutic 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 results of immunofluorescence staining of DR5 and podocyte-specific marker synaptopodin in renal tissues of healthy controls and patients with diabetic nephropathy.

[0021] Figure 2 The attached figure shows the expression of DR5 in human immortalized podocytes; a shows the changes in DR5 mRNA expression in human podocytes stimulated by TNF-α; b shows the changes in DR5 protein expression in human podocytes stimulated by TNF-α; c shows the knockdown efficiency detection of DR5 after transfection of human podocytes with DR5 small interfering gene. n=3, ***P<0.001.

[0022] Figure 3 The attached figure shows the protein expression of pan-apoptosis related proteins p-MLKL, MLKL, p-RIPK1, RIPK1, Bax, caspase3, cleaved caspase3, caspase1, and cleaved caspase1 after human podocytes were transfected with DR5 small interfering gene. n=3, *P<0.05, **P<0.01.

[0023] Figure 4The attached figure shows the expression and distribution of DR5 in the renal tissue of STZ-induced diabetic nephropathy mice; a is a schematic diagram of the animal model construction; b is the verification of the podocyte-specific DR5 knockout efficiency after extracting and isolating mouse primary podocytes; c is the immunofluorescence staining results of DR5 and podocyte-specific marker synaptopodin in the glomeruli of wild-type and STZ-induced DKD mice.

[0024] Figure 5 The attached figure shows the effect of podocyte-specific knockout of DR5 on STZ-induced renal injury in DKD mice; a is the urine albumin-creatinine ratio (UACR) of mice in each group, n=6. ***P<0.001vs WT, ## P<0.01vs WT+STZ, ### P<0.001 vs WT+STZ; b is the result of PAS staining and the quantitative analysis of glomerular volume and mesangial area percentage; c is the result of glomerular TUNEL staining and the quantification of TUNEL-positive cells; d is the result of glomerular WT-1 staining and the quantification of WT-1-positive cells. e is the immunofluorescence staining result of cleaved caspase1, cleaved caspase3 and p-MLKL in glomeruli. *P<0.05, ***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 and localization analysis of DR5 (death receptor) in renal tissue of patients with diabetic nephropathy

[0027] Collection of human kidney tissue samples

[0028] A total of 20 renal tissues were collected from the control group, which were derived from normal renal tissues adjacent to the tumor after nephrectomy for renal tumors. A total of 20 renal tissues were collected from the DKD group, which were derived from renal tissues obtained by puncture biopsy in the Department of Nephrology, Provincial Hospital Affiliated to Shandong First Medical University. All samples were obtained with the consent of the patients themselves or their guardians before collection, and the informed consent form was signed. All human specimen collection studies were approved by the Ethics Committee of the Provincial Hospital Affiliated to Shandong First Medical University.

[0029] The expression of DR5 in the renal tissue of patients with diabetic nephropathy and its co-localization with podocytes were detected by immunofluorescence co-staining of DR5 and podocyte marker protein synaptopodin.

[0030] The specific steps are as follows:

[0031] Immunofluorescence staining

[0032] (1) Turn on the baking machine and bake the slices at 65℃ for 1 hour.

[0033] (2) Place the paraffin sections on a copper rack and place them in the following cylinders: 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) Soak in distilled water three times, 5 min each time; soak in PBS once, 5 min each time.

[0035] (4) Prepare an appropriate antigen retrieval solution according to the instructions and perform high-pressure heat retrieval for 3 minutes.

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

[0037] (6) The tissue sections were permeabilized with 0.5% Triton for 5 min and soaked in PBS three times for 5 min each time.

[0038] (7) Add 5% BSA blocking solution to the tissue sections and incubate at room temperature for 30 min.

[0039] (8) Discard the blocking solution, add DR5 (diagbio, db6178) and synaptopodin (Proteintech, 21064-1-AP), and incubate at 4°C overnight.

[0040] (9) The next day, transfer the slices to a 37°C incubator and incubate for 30 min.

[0041] (10) Soak in PBS three times, 5 min each time.

[0042] (11) Protect from light and add appropriate concentration of Alexa 594-conjugated donkey anti-rabbitIgG (ab150076, abcam) and Alexa 488-conjugated goat anti-mouse IgG (ab150113, abcam) secondary antibody working solution was used and incubated at 37°C for 60 min.

[0043] (12) Protect from light and soak in PBS three times, 5 min each time.

[0044] (13) Protect from light, add DAPI, and incubate for 5 min.

[0045] (14) Protect from light and soak in PBS three times, 5 min each time.

[0046] (15) Protect from light, seal the slides with an anti-fluorescence quencher, and observe and photograph them under a fluorescence microscope.

[0047] The results showed that compared with the healthy control group, DR5 expression in the renal tissue of patients with diabetic nephropathy was significantly increased and co-localized with podocytes. When podocytes in the DKD group were damaged, DR5 expression was upregulated. This suggests that DR5 may be related to podocyte damage in DKD ( Figure 1 ).

[0048] Example 2 Role of DR5 in podocyte pan-apoptosis

[0049] Target gene and internal reference gene primer sequences:

[0050] DR5

[0051] F 5'-AGACCCTGTGCCTCGTTGTC-3', SEQ ID NO.1;

[0052] R 5'-GGGGCTGGACCTCTTTTGTT-3', SEQ ID NO.2;

[0053] β-actin

[0054] F 5'-CATGTACGTTGCTATCCAGGC-3', SEQ ID NO.3;

[0055] R 5'-CTCCTTAATGTCACGCACGAT-3', SEQ ID NO.4;

[0056] Primary antibodies and product numbers used in Western blot:

[0057] DR5(diagbio,db6178),cleaved caspase-3Rabbit mAb(Cell Signaling Technology,9664),caspase-3antibody(Cell Signaling Technology,9662),caspase-1RabbitmAb(Cell Signaling Technology,3866),anti-MLKL(phosphor S358)(abcam,ab187091),MLKL Monoclonal antibody(Proteintech,66675-1-Ig),Phospho-RIPK1(Ser166)Rabbit mAb(Cell Signaling Technology,44950),RIPK1 RabbitmAb(CellSignaling Technology,3493),Bax polyclonal antibody (Proteintech, 50599-2-Ig), and β-actin (Proteintech, 66009-1-Ig).

[0058] We stimulated human immortalized podocytes with TNF-α to simulate the in vitro diabetic nephropathy environment. In order to clarify the role of DR5 in the occurrence of pan-apoptosis of podocytes induced by TNF-α stimulation, we first detected the mRNA and protein expression of DR5 in podocytes by real-time PCR and Western blot. We further constructed DR5 siRNA to transfect podocytes, and the Western blot results showed that the knockdown sequence had a good knockdown efficiency ( Figure 2 ).

[0059] DR5 small interfering sequence:

[0060] DR5 siRNA 5'-CCACAAAGAAUCAGGGUACATT-3', SEQ ID NO. 5.

[0061] NC siRNA 5'-UUCUCCGAACGUGUCACGU-3', SEQ ID NO.6;

[0062] After DR5 siRNA was transfected into human podocytes, the expression of apoptosis-related genes cleaved caspase1, apoptosis-related genes cleaved caspase 3 and Bax, and necroptosis-related genes p-MLKL and p-RIPK were detected by Western blot ( Figure 3 ).

[0063] The specific steps are as follows:

[0064] Real-time PCR

[0065] (1) RNA extraction

[0066] RNA was extracted from cultured podocytes using the SteadyPure RNA extraction kit (Acrobio, China) as follows:

[0067] ①Discard the culture medium in the 6-well plate and wash the cells once with PBS buffer.

[0068] ② Aspirate PBS, add 500 μl of lysis solution to each well, and shake gently to ensure that the lysis solution in the six-well plate evenly covers the cell surface.

[0069] 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 a 1.5 ml EP tube without RNase and blow and mix well.

[0070] ④ After standing at room temperature for 5 minutes, add 500 μl of 70% ethanol and mix well with a pipette.

[0071] ⑤ Transfer the above mixed solution to the RNA Mini Column and centrifuge at 12000 rpm at room temperature for 1 minute.

[0072] ⑥Discard the filtrate, add 600μl of Buffer RW1 to the RNA Mini Column, and centrifuge at 12000rpm at room temperature for 1min.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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℃.

[0078] (2) Reverse transcription

[0079] The RNA concentration and purity were detected using a NanoDrop-2000 spectrophotometer. Reverse transcription was performed using the Evo M-MLV reverse transcription kit (Acrobio, China). The cDNA samples after reverse transcription can be directly used for fluorescence quantitative PCR detection or stored in a -20°C refrigerator.

[0080] The reverse transcription reaction system is shown in Table 1;

[0081] Table 1

[0082]

[0083] (3) Fluorescence quantitative PCR analysis

[0084] The primers used in the fluorescent quantitative PCR were designed and synthesized by Boshan Biotechnology Co., Ltd. Before use, the primers were centrifuged at 4°C and 12,000 rpm for 2 min, and DEPC water was added according to the instructions to completely dissolve them to a final concentration of 10 μM.

[0085] According to the instructions of SYBR Green Pro Taq HS premixed qPCR kit (Acori Biotech, China), the PCR reaction system was prepared on ice and added to a 96-well plate. After sealing, the plate was centrifuged at 3000 rpm for 2 min at room temperature. The target gene was detected using a Roche 480 PCR instrument. -ΔΔCt The method was used for analysis, and β-actin was used as the internal reference gene.

[0086] The qPCR reaction system is shown in Table 2;

[0087] Table 2

[0088]

[0089]

[0090] The PCR reaction program is shown in Table 3;

[0091] Table 3

[0092]

[0093] Western blot

[0094] (1) Total cell protein extraction

[0095] ①Discard the culture medium in the six-well plate and add PBS buffer to rinse the cells twice.

[0096] ② Prepare protein lysis solution according to the ratio of protein lysis buffer: protease inhibitor: phosphatase inhibitor = 100:1:1. Aspirate the PBS washing solution, add 100 μl of the above lysis system to each well, gently shake the six-well plate to make the protein lysis solution evenly cover the cell surface, place on ice, and lyse for 30 minutes.

[0097] ③ Use a cell scraper to scrape the lysed cells, transfer them to an RNase-free EP tube with a pipette, and centrifuge at 4°C and 12,000 rpm for 30 min.

[0098] ④ Aspirate the supernatant and transfer it to a new RNase-free EP tube.

[0099] (2) Protein concentration determination

[0100] The protein concentration was determined using the BCA protein concentration assay kit.

[0101] ① Prepare BCA working solution according to BCA reagent A:B=50:1 and mix it thoroughly.

[0102] 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.

[0103] ③ Add 200 μl of BCA working solution to all wells and incubate in a 37°C incubator for 30 min.

[0104] ④ Use a microplate reader to measure the absorbance value of each well at wavelength A562.

[0105] ⑤ Draw a standard curve based on the measured absorbance values, and calculate the protein concentration of each sample based on this curve.

[0106] (3) Protein denaturation

[0107] Add loading buffer to the supernatant at a ratio of 3:1 between protein supernatant and 4×SDS-PAGE loading buffer. Use a pipette to mix the two, and heat at 95°C for 10 min to fully denature the protein.

[0108] (4) SDS-PAGE gel electrophoresis

[0109] ① Glue preparation: prepare glass plates, and make sure that the lower edges of the two glass plates are level and without any gaps. Wash the glass plates and place them on the glue rack. Note that the inside is longer and the outside is shorter during installation. Press the glass plates and the vertical groove glue rack with your hands. Prepare the corresponding concentration of glue according to the molecular weight of the target protein. Prepare the separation gel at 5 ml per piece. Mix gently and add it to the gap between the large and small glass plates to about 2 / 3 of the height. Then add distilled water to flatten the separation gel surface. After the dividing line appears, discard the upper distilled water. Prepare the concentrated gel at 1.5 ml per piece, add it to the gap between the glass plates, and insert a comb of appropriate size. Note that the operation should be quick and horizontal, and no bubbles should be generated. After the upper layer of gel solidifies, it can be used immediately.

[0110] ② Loading: According to the BCA assay results, 20 μg of protein was loaded into each well, and 3 μL of protein marker was added as a reference.

[0111] ③Electrophoresis: Use low voltage constant voltage electrophoresis for the upper gel, 80V, 30min; when bromophenol blue enters the lower gel, use high voltage constant voltage electrophoresis, 120V, and stop electrophoresis when bromophenol blue reaches the bottom of the gel.

[0112] ④ Transfer: Prepare the transfer buffer in advance and precool it to 4°C; cut the matching PVDF membrane as needed and soak it in methanol for later use; gently pry open the glass plate and cut off the concentrated gel and the surrounding unnecessary areas. Put the gel into the transfer "sandwich", clamp it into the transfer tank, and add enough transfer buffer. Set the transfer current to constant current, 220mA, and set the transfer time according to the size of the protein. Transfer the target protein for 1min per 1KD.

[0113] (4) Immunoblotting

[0114] ① Block with 5% skim milk at room temperature for 1-2 hours, and wash with TBST buffer three times, 10 minutes each time.

[0115] ②Primary antibody incubation: Incubate in primary antibody working solution overnight on a shaker at 4°C.

[0116] ③ Secondary antibody incubation: The next day, wash with TBST buffer 3 times, 10 min each time. Prepare secondary antibody working solution at a ratio of 1:5000, incubate on a shaker at room temperature for 1 h. Wash with TBST buffer 3 times, 10 min each time.

[0117] ④ Development: Mix Western ECL luminescent solution A and B in equal proportions, drop an appropriate amount on the PVDF membrane, place it in a developer, and expose it to form an image.

[0118] ⑤Image processing: Image J was used to analyze and process images.

[0119] The results showed that the mRNA and protein levels of DR5 were increased in human immortalized podocytes stimulated by TNF-α compared with the control group. TNF-α stimulation can upregulate the expression of p-MLKL, p-RIPK, cleaved caspase1, cleaved caspase 3, and Bax in podocytes and promote pan-apoptosis; while knocking down DR5 can alleviate TNF-α-induced pan-apoptosis of podocytes and reverse the abnormal expression of related genes p-MLKL, p-RIPK, cleaved caspase1, cleaved caspase 3, and Bax. The above results suggest that DR5 is involved in the occurrence of TNF-α-induced pan-apoptosis of podocytes.

[0120] Example 3 Role of DR5 in renal damage in STZ-induced diabetic nephropathy mice

[0121] Animal husbandry and animal experiments were performed with approval from the Medical Ethics Committee of Shandong Provincial Hospital.

[0122] Construction of podocyte-specific DR5 knockout mice

[0123] Podocyte-specific DR5 knockout mice were constructed by Saiye Biotechnology Co., Ltd. The specific construction method is as follows: First, (flox / +) heterozygous mice were mated to obtain (flox / flox) homozygous mice, then NPHS2-Cre tool mice were mated with (flox / +) heterozygous mice or (flox / flox) homozygous mice to obtain [flox / +, Cre(+ / -)] mice, and finally, [flox / +, Cre(+ / -)] mice were mated with (flox / +) or (flox / flox) to obtain [flox / flox, Cre(+ / -)] mice, i.e., podocyte-specific DR5 knockout mice. 8-week-old male mice were further randomly divided into (WT, n=6), podocyte-specific DR5 knockout group (DR5 KO , n = 6), STZ injection group (STZ, n = 6), podocyte-specific DR5 knockout + STZ injection group (STZ + DR5 KO ,n=6).

[0124] Building the DKD model

[0125] 8-week-old adult male SPF C57BL / 6J mice were selected. The mice were fasted but not watered for 8-10 hours before intraperitoneal injection of STZ. After 4 weeks of high-fat diet feeding, the mice were intraperitoneally injected with STZ at a dose of 50 mg / kg for 5 consecutive days, which was the STZ group; the control group (WT group) was injected with an equal amount of citric acid buffer. Blood was collected from the tail vein of the mice, and the model was successfully established if the blood sugar level remained ≥16.7 mmol / l.

[0126] After 20 weeks of STZ or citrate buffer injection, the mice were killed and the renal tissues of each group of mice were collected: ① The primary podocytes were isolated and cultured, and the expression of DR5 in the primary podocytes was detected by Western blot to verify the DR5 knockout efficiency of podocytes; ② DR5 and podocyte marker protein synaptopodin were co-stained with immunofluorescence to detect the expression and distribution of DR5 in the renal tissues of STZ mice ( Figure 4 ).

[0127] The specific steps are as follows:

[0128] Isolation and culture of primary podocytes

[0129] After the mice were anesthetized by intraperitoneal injection of 2.5% pentobarbital, the skin was disinfected with 75% ethanol, and the abdominal cavity and thoracic cavity were exposed in turn. After cardiac perfusion, the bilateral kidneys were quickly removed and placed in D-Hank's solution. The capsule was removed, the renal cortex was separated, the cortex was cut into pieces, the renal cortex was gently ground, and it was continuously rinsed with D-Hank's solution. The rinse solution was passed through 80 mesh, 150 mesh, and 300 mesh stainless steel sieves in turn. The separated material was collected to make a suspension, and after standing for 10 minutes, it was centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, the purified glomerular precipitate was collected, and RPMI 1640 culture medium containing 10% fetal bovine serum was added to make a glomerular suspension. 2×10 4 The cells were inoculated at a density of 100 μg / well in a six-well plate and cultured in a 37°C, 5% CO2 incubator. On the 7th to 10th day of inoculation, a large number of cells crawled out around all the adherent glomeruli, which were podocytes. After the cells matured, the podocyte-specific marker WT1 was used to identify podocytes.

[0130] Immunofluorescence staining as in Example 1

[0131] To further clarify the effect of podocyte-specific knockout of DR5 on STZ-induced renal injury in DKD mice, the levels of UACR in each group of mice were collected and detected at 0W, 4W, 8W, 12W, 16W, and 20W after successful DKD mouse modeling; the mice were killed at 20W, and renal tissues were collected for PAS staining, TUNEL staining, WT-1 immunofluorescence staining, and pan-apoptotic protein immunofluorescence staining ( Figure 5 ).

[0132] PAS staining

[0133] (1) Baking, dewaxing, and hydration: same as above.

[0134] (2) Soak in distilled water three times, 5 min each time; soak in PBS once, 5 min each time.

[0135] (3) Add oxidant dropwise, leave at room temperature for 5-8 minutes, and rinse with running water.

[0136] (4) Soak in distilled water twice, 5 min each time.

[0137] (5) Pipette an appropriate amount of Schiff reagent onto the slice, protect from light, incubate for 15 minutes, and then rinse with running water for 10 minutes.

[0138] (6) Add hematoxylin solution, incubate for 3 min, and then rinse with running water.

[0139] (7) Differentiation 1-2s.

[0140] (8) Dehydration and transparency: same as above.

[0141] (9) After sealing with neutral gum, observe and photograph under a microscope.

[0142] TUNEL staining

[0143] The procedure was performed according to the instructions of the Rapid One-Step TUNEL Cell Apoptosis Detection Kit (KeyGen Biotech Co., Ltd., Jiangsu).

[0144] (1) Turn on the baking machine and bake the slices at 65℃ for 1 hour.

[0145] (2) Soak the tissue sections in xylene I for 15 min, xylene 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.

[0146] (3) Soak in distilled water for 3 times, 5 min each time; soak in PBS for 5 min.

[0147] (4) Prepare Proteinase K working solution and add 100 μl of Proteinase K working solution to the slices. Incubate at 37°C for 30 min and soak in PBS three times for 5 min each time.

[0148] (5) Select a slice to make a positive slice: add 100 μl of DNase I reaction solution, incubate at 37°C for 30 min, and soak in PBS three times, each time for 5 min.

[0149] (6) Add the prepared TUNEL detection solution, incubate at 37°C in the dark for 1 h, and wash with PBS three times, 5 min each time.

[0150] (7) DAPI nucleus staining for 5 min.

[0151] (8) Wash with PBS three times, 5 min each time.

[0152] (9) Seal the slides with a fluorescence quencher and observe and photograph them using a fluorescence microscope.

[0153] Immunofluorescence staining of WT-1, cleaved caspase1, cleaved caspase3, and p-MLKL

[0154] (1) Baking, dewaxing, and hydration: same as above.

[0155] (2) Soak in distilled water three times, 5 min each time; soak in PBS once, 5 min each time.

[0156] (3) Antigen repair: Prepare the corresponding antigen repair solution according to the instructions and perform high-pressure heat repair for 3 minutes.

[0157] (4) After the repair solution returns to room temperature, soak the tissue sections in PBS three times, each time for 5 min.

[0158] (5) Place tissue sections in 0.5% Triton, permeabilize for 5 min, and soak in PBS three times, each time for 5 min.

[0159] (6) Add 5% BSA blocking solution to the tissue sections and incubate at room temperature for 30 minutes.

[0160] (7) 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.

[0161] (8) The next day, incubate the tissue sections at 37°C for 30 min.

[0162] (9) Soak in PBS three times, 5 min each time.

[0163] (10) Protect from light and add appropriate concentration of Alexa 594-conjugated donkey anti-rabbitIgG(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.

[0164] (11) Protect from light and soak in PBS three times, 5 min each time.

[0165] (12) Protect from light, add DAPI to the tissue sections and incubate for 10 min.

[0166] (13) Protect from light and soak in PBS three times, 5 min each time.

[0167] (14) Protect from light. After sealing with anti-fluorescence fading sealing medium, observe and photograph under a fluorescence microscope.

[0168] The results showed that: Immunofluorescence results showed that compared with WT mice, DR5 expression was increased in STZ-induced DKD mice, and co-localized with podocytes. In addition, compared with WT mice, the UACR of STZ group mice increased; PAS staining results showed that the glomerular hypertrophy and mesangial matrix of STZ group mice increased; WT-1 immunofluorescence staining showed that the number of WT1-positive podocytes in the glomeruli of the STZ group decreased; TUNEL staining results showed that the number of TUNEL-positive cells and cell apoptosis increased in the STZ group; immunofluorescence results of pan-apoptosis-related proteins showed that the expression of pan-apoptosis-related proteins cleaved caspase1, cleaved caspase3, and p-MLKL in STZ group mice increased. The above results indicate that kidney damage occurred in STZ group mice. Compared with the STZ group, DR5 KO The above-mentioned injuries were all improved in the mice in the +STZ group, indicating that podocyte-specific knockout of DR5 could alleviate STZ-induced kidney injury in DKD mice.

[0169] 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 DR5 in the preparation of DKD diagnostic kits and targeted therapeutic drugs.

2. The use according to claim 1, characterized in that: The substance for detecting DR5 is shown in SEQ ID NO.1 to SEQ ID NO.4: DR5-F 5'-AGACCCTTGTGCTCGTTGTC-3', SEQ ID NO.1; DR5-R 5'-GGGGCTGGACCTCTTTTGTT-3', SEQ ID NO.2; β-Actin-F 5'-CATGTACGTTGCTATCCAGGC-3', SEQ ID NO.3; β-Actin-R 5'-CTCCTTAATGTCACGCACGAT-3', SEQ ID NO.

4.

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

4.

4. Application of substances that knock down DR5 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 DR5 expression is interfering RNA, and the sequence is: 5'-CCACAAAGAAUCAGGUACATT-3', SEQ ID NO.

5.

6. The use according to claim 4, characterized in that: DR5 expression is increased in renal tissue of patients with diabetic nephropathy and co-localizes with podocytes.

7. The use according to claim 4, characterized in that: Knockdown of DR5 alleviated TNF-α-induced podocyte pan-apoptosis and reversed the abnormal expression of its related genes p-MLKL, p-RIPK, cleaved caspase1, cleaved caspase 3, and Bax.

8. The use according to claim 4, characterized in that: DR5 expression was upregulated in STZ-induced DKD mice, and podocyte-specific knockout of DR5 ameliorates renal injury and pan-apoptosis in STZ-induced DKD mice.

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