Application of GLP-1RA in preparation of medicine for improving podocyte damage and / or reducing urine protein

Abstract

The invention relates to the field of biological medicine, and discloses application of GLP-1RA in preparation of a medicine for improving podocyte damage and / or reducing urine protein. According to the application, an FSGS mouse model induced by adriamycin is used, the model has no hyperglycemia phenotype, the blood sugar reducing effect of liraglutide can be excluded, the protection effect of liraglutide on podocytes independent of blood sugar reducing can be better verified, and meanwhile, in-vivo and in-vitro GLP1R overexpression models are constructed to observe the effect. Results show that liraglutide can significantly reduce the level of doxorubicin-induced urine protein and improve podocyte injury, and liraglutide may play a positive role in non-diabetic nephropathy, and provides a new idea for comprehensive treatment of kidney diseases.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and more specifically, to the use of GLP-1RA in the preparation of a drug for improving podocyte injury and / or reducing urinary protein. Background Art

[0002] Podocytes are highly specific cells on the glomerulus and are an important component of the glomerular filtration barrier. The causes of podocyte injury include various immune complex glomerulonephritis with different histopathological changes, such as membranous nephropathy, and subepithelial immune complex deposition, which can directly damage podocytes and cause massive proteinuria. The renal pathological changes of podocyte injury without immune complex deposition can be divided into four types: diffuse mesangial sclerosis (DMS), characterized by mesangial matrix expansion and podocyte hypertrophy; minimal change disease (also known as minimal change nephropathy), mainly seen in children, and histopathological ultrastructural abnormalities can only be seen under electron microscopy; focal segmental glomerulosclerosis (FSGS) lesions, sclerotic lesions involving glomerular segments; collapsing glomerulopathy, characterized by collapse of glomerular capillaries, proliferation of parietal epithelial cells and migration to capillary loops, manifested as "pseudocrescents". Molecular diagnostic studies of monogenic causes of proteinuria or nephrotic syndrome have shown that the histomorphological changes in DMS, minimal change disease, FSGS, or collapsing glomerulopathy are nonspecific lesions, which are merely different forms of podocyte damage and cannot identify the specific cause or diagnosis, nor can they provide a basis for treatment.

[0003] Many systemic diseases are accompanied by podocyte injury, which is closely related to the progression of diabetic nephropathy (DKD). It is independent of glomerular lesions, appears early, promotes the progression of DKD, and affects the renal prognosis of DKD, inducing glomerular injury through complex mechanisms.

[0004] Many patients with DKD use GLP-1 receptor agonists (GLP-1RA) for glucose-lowering therapy. GLP-1RA is a class of incretin GLP-1 analogs that are resistant to exogenous DPP4 enzyme degradation and can lower blood sugar while exerting unique organ protection functions. The renal protective effect of GLP-1RA in DKD has been widely recognized. Although most current studies focus on the use of GLP-1RA in DKD, there is increasing evidence that GLP-1RA may also play a positive role in non-diabetic nephropathy.

[0005] Therefore, studying the protective mechanism of GLP-1RA on podocytes independent of hypoglycemic effect can provide new ideas for the comprehensive treatment of kidney disease. Summary of the invention

[0006] The purpose of the present invention is to study the effects of GLP-1RA on improving podocyte damage and / or reducing urine protein independent of lowering blood sugar.

[0007] Albuminuria and podocyte injury are known to be key links in the progression of diabetic nephropathy, but studies on animal or cell models of diabetic nephropathy cannot well explain the protective effect of liraglutide on podocytes. It may be the effect of GLP1R after lowering blood sugar. Therefore, we used the FSGS mouse model induced by doxorubicin. This model has no hyperglycemia phenotype, which can exclude the hypoglycemic effect of liraglutide and better verify the protective effect of liraglutide on podocytes independent of hypoglycemia. Therefore, we also constructed in vivo and in vitro GLP1R overexpression models to observe this effect. The results showed that liraglutide can significantly reduce the level of urine protein induced by doxorubicin and improve podocyte injury. Liraglutide may also play a positive role in non-diabetic nephropathy, providing new ideas for the comprehensive treatment of kidney disease.

[0008] In order to achieve the above invention objectives, this application adopts the following technical solutions:

[0009] In a first aspect, the present application provides the use of GLP-1RA in the preparation of a drug for improving podocyte injury.

[0010] Furthermore, the GLP-1RA protects podocytes independently of lowering blood sugar and improves podocyte damage.

[0011] Furthermore, the GLP-1RA includes liraglutide.

[0012] In a second aspect, the present application provides a drug for improving podocyte injury, wherein the drug comprises a therapeutically effective amount of GLP-1RA as the sole active ingredient.

[0013] In a second aspect, the present application provides a drug for improving podocyte injury, wherein the drug is a pharmaceutical composition, which includes, in addition to a therapeutically effective amount of GLP-1RA as an active ingredient, other pharmaceutical ingredients for improving podocyte injury.

[0014] In a fourth aspect, the present application provides the use of GLP-1RA in the preparation of a drug for treating FSGS.

[0015] Furthermore, the drug for treating FSGS refers to a drug that reduces urine protein and / or improves podocyte damage.

[0016] Furthermore, the GLP-1RA includes liraglutide.

[0017] In a fifth aspect, the present application provides a drug for treating FSGS, wherein the drug comprises a therapeutically effective amount of GLP-1RA as the sole active ingredient.

[0018] In a sixth aspect, the present application provides a drug for treating FSGS, wherein the drug is a pharmaceutical composition, which includes, in addition to a therapeutically effective amount of GLP-1RA as an active ingredient, other pharmaceutical ingredients for treating FSGS. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 :Schematic diagram of the modeling process of FSGS mouse model;

[0020] Figure 2 :Changes in mouse urine protein at different time points after tail vein injection of doxorubicin (the ones marked before are the baseline urine of mice, the ones marked after are the urine at the end point, the blank control is the model without modeling, and ADR is the model with tail vein injection of doxorubicin);

[0021] Figure 3 : Electron microscopic results of different animal groups (a and b are WT+saline group, c and d are WT+LIRA group, e and f are ADR+saline group, g and h are ADR+LIRA group);

[0022] Figure 4 :Electron microscopic results after HE staining and PAS staining;

[0023] Figure 5 , Figure 6 :Effect of liraglutide on urinary protein in ADR mice (Balb / c mice, 8-10 weeks old, samples were collected 28 days after tail vein injection of doxorubicin, WT: blank control group, no modeling, only injected with normal saline every day; WT+LIRA: negative control group, no modeling, injected with liraglutide (300ug / kg) every day; ADR: ADR modeling group (12mg / kg), only injected with normal saline every day; ADR+LIRA: intervention group, injected with liraglutide (300ug / kg) every day after successful ADR modeling);

[0024] Figure 7 : Phalloidin staining of the cytoskeleton after different concentrations of liraglutide intervened in PAN-induced podocyte injury (red is phalloidin, blue is DAPI. 1640 group: negative control group; DMSO group: this group was added because PAN was dissolved in DMSO; PAN group was the modeling group, and the concentration of aminonucleoside puromycin was 100 μg / mL; 10LIRA group: 10 nmol liraglutide intervention group; 50LIRA group: 50 nmol liraglutide intervention group; 100LIRA group: 100 nmol liraglutide intervention group. Fixed at 24 hours and 48 hours, respectively). DETAILED DESCRIPTION

[0025] The technical scheme and effects of the present application are further described in detail below in conjunction with the embodiments. It is to be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention. It should also be noted that the experimental methods used in the following embodiments are conventional methods unless otherwise specified; the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial sources.

[0026] Main reagents:

[0027]

[0028]

[0029] Main experimental instruments:

[0030] Instrument Name source <![CDATA[OxyCyler Dynamic O2 Controller]]> Biospherix, Inc. Low temperature high speed desktop centrifuge Thermo Fisher Scientific, USA Dehydrator Wuhan Junjie Electronics Co., Ltd. Embedding machine Wuhan Junjie Electronics Co., Ltd. Microplate Reader Biotek, Inc. Vertical electrophoresis apparatus Bio-Rad Corporation Vertical electrophoresis tank Bio-Rad Corporation Cyclotron Oscillator Thermo Fisher Scientific, USA Fully automatic chemiluminescence / fluorescence image analysis system Shanghai Tianneng Technology Co., Ltd.

[0031] Statistical analysis

[0032] GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, US) and SPSS22.0 software (IBM, Armonk, NY, US) were used for statistical analysis and drawing. Quantitative data were expressed as mean ± standard error (Mean ± SEM). All data were tested for normality and homogeneity of variance using the Shapiro-Wilk test and Levene test. Two-way ANOVA and Tukey's multiple comparison method and two-sided unpaired t test were used for difference analysis for data with normal distribution and homogeneity of means; Kruskal-Wallis test and Dunn's multiple comparison method and Mann-Whitney U test were used for nonparametric statistics between groups for data that did not meet the normal distribution. P < 0.05 was considered statistically significant.

[0033] Example 1: Establishment of doxorubicin-induced FSGS mouse model

[0034] SPF grade 8-week-old male Balb / c mice were purchased from Saiye Biotechnology Co., Ltd. and raised in the Animal Experiment Center of the Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. The mice were randomly divided into a control group and an adriamycin intervention group (n=8). The control group was injected with normal saline through the tail vein, and the model group was injected with adriamycin (ADR, 12 mg / kg) through the tail vein to construct the FSGS model. Urine was collected from each group every 7 days, and the mice were killed after 28 days. Urine, blood, and kidney specimens were collected from the mice. The Coomassie Brilliant Blue method was used to detect mouse urine protein, and the changes in urine protein before and after modeling and the differences between the groups were compared. The modeling schematic diagram is shown in the figure. Figure 1 shown.

[0035] Verification of the mouse FSGS model and selection of intervention time: During the entire experiment, 2 of the 8 mice in the model group died, and 6 mice were finally selected for the model group and 8 for the control group. The model group mice began to show a gradual increase in urine protein 7 days after modeling. The difference between the experimental group and the control group was obvious on the 7th day after modeling, and reached a peak on the 28th day, indicating that the model group mice had renal tissue damage. Therefore, the subsequent experiments used 28 days as the model intervention time. The changes in mouse urine protein at different time points are shown in the figure below. Figure 2 shown.

[0036] Example 2: GLP-1RA (using liraglutide (lira) as an example) significantly improves doxorubicin-induced renal pathology

[0037] (1) Animal grouping and intervention:

[0038] SPF grade 8-week-old male Balb / c mice were purchased from Saiye Biotechnology Co., Ltd. and raised in the Animal Experiment Center of the Sixth People's Hospital Affiliated to Shanghai Jiaotong University School of Medicine. The mice were randomly divided into a control group and a model group. At 8 weeks of age, the model group was injected with adriamycin (ADR, 12 mg / kg) through the tail vein to establish the FSGS model; the control group and the model group were given saline or liraglutide intervention, namely: WT+LIRA group (300ug / kg, QD), ADR+LIRA group (300ug / kg, QD), WT+saline group, and ADR+saline group. After 4 weeks of intervention, urine, blood, and kidney specimens of the mice were collected.

[0039] (2) Collection of tissue and body fluid specimens:

[0040] Before the mice were killed, 24-hour urine was collected from all mice using a mouse-specific metabolic cage and a sterile 15 mL centrifuge tube. The urine was centrifuged at 4°C (3500 rpm, 10 minutes), the supernatant was collected and the urine sediment was discarded, and stored at -80°C for subsequent detection of relevant biochemical indicators.

[0041] After modeling and drug administration for 28 days and collecting urine for 8 hours, the mice were anesthetized with 1% sodium pentobarbital, and then the chest and abdominal cavities of the mice were opened, and blood was collected from the abdominal aorta of the mice with a 1mL syringe. The blood samples were left to stand at room temperature for 30 minutes and then centrifuged at 4°C (3500rpm, 15 minutes), and the upper serum was collected and stored at -80°C for subsequent related experimental tests.

[0042] After collecting blood samples from mice, the mice were perfused by heart with 0.9% saline. After observing that the kidneys turned from red to white, the perfused kidneys were removed and the renal capsules were carefully separated, and the weight of the kidneys was measured. The kidneys were then dissected, and two samples were kept for 4% paraformaldehyde fixation and OCT embedding. The remaining samples were separated from the cortex and frozen at -80°C for later use.

[0043] (3) Paraffin-embedded mouse kidney tissue:

[0044] ① Fixation: Place the freshly excised mouse kidney into a 4% paraformaldehyde solution 10 times the volume of the kidney and shake it at room temperature for at least 24 hours.

[0045] ② Dehydration, transparency, and wax impregnation: After trimming the kidney tissue section, use a dehydrator to dehydrate the kidney tissue with gradient alcohol, transparentize it with xylene, and wax impregnate it with melted paraffin (65°C). The processing steps are set as follows:

[0046] Reagents Processing time 75% alcohol 4 hours 85% alcohol 2 hours 90% alcohol 2 hours 95% alcohol 1 hour Anhydrous ethanol 1 30 minutes Anhydrous ethanol 2 30 minutes Alcohol benzene 10 minutes Xylene 1 10 minutes Xylene 2 10 minutes Melt paraffin1 1 hour Melted paraffin 2 1 hour Melt paraffin 3 1 hour

[0047] ③Embedding: Embed the wax-impregnated tissue using an embedding machine. Place the fully expanded tissue into an embedding box, cool the solidified wax block in a freezer (-20°C) and trim it.

[0048] ④ Paraffin sectioning: Use a microtome to cut the wax block into 4 μm thin slices along the cross section of the renal tissue. After ironing, quickly place it on a slide treated with anti-shedding and put it in a 37°C oven to dry overnight.

[0049] (4) Kidney hematoxylin-eosin (HE) staining:

[0050] ① Baking: Place the paraffin sections of kidney tissue in a 65°C oven and bake for 45 minutes to melt the paraffin on the surface of the tissue.

[0051] ② Dewaxing and hydration: Dewaxing and hydration with gradient alcohol are carried out in a fume hood. The processing steps are as follows:

[0052] Reagents Processing time Xylene 1 20 minutes Xylene 2 20 minutes Anhydrous ethanol 1 10 minutes Anhydrous ethanol 2 10 minutes 95% Alcohol 1 5 minutes 95% Alcohol 2 5 minutes 90% alcohol 5 minutes 75% alcohol 5 minutes Distilled water 1 minute

[0053] ③Hematoxylin staining: Place the dewaxed and hydrated sections in hematoxylin staining solution for 5 minutes, then wash with distilled water for 1 minute.

[0054] ④ Differentiation: Differentiate the sections with hematoxylin differentiation solution for 2 seconds, and then wash with distilled water for 10 seconds.

[0055] ⑤ Reblueing: Reblue the sections with hematoxylin bluing solution for 2 seconds, then wash with distilled water for 10 seconds.

[0056] ⑥Eosin staining: Dehydrate the blue sections with 95% alcohol for 5 minutes, and then stain them in eosin solution for 5 minutes.

[0057] ⑦ Dehydration and transparency: Dehydration with gradient alcohol and transparency with xylene are carried out in a fume hood. The processing steps are as follows:

[0058] Reagents Processing time 75% alcohol 5 minutes 90% alcohol 5 minutes 95% Alcohol 1 5 minutes 95% Alcohol 2 5 minutes Anhydrous ethanol 1 5 minutes Anhydrous ethanol 2 5 minutes Xylene 1 5 minutes Xylene 2 5 minutes

[0059] ⑧ Sealing: Let the dehydrated transparent slices dry, and seal them with neutral gum, taking care to remove bubbles. Once the slides are completely dry, you can start observing.

[0060] (5) Kidney periodic acid-Schiff (PAS) staining:

[0061] ① Baking, dewaxing and hydration: The experimental method is the same as HE staining.

[0062] ②Periodic acid staining: Place the dewaxed and hydrated sections in periodic acid staining solution for 15 minutes, then wash three times with distilled water.

[0063] ③ Schiff staining: Protect from light. Stain the sections in Schiff stain for 30 minutes, then wash with running water for 10 minutes.

[0064] ④ Nuclear staining: Stain the sections with hematoxylin for 5 minutes, then wash and differentiate for 2 seconds, wash again and turn blue for 2 seconds, then rinse with distilled water. The experimental method is the same as HE staining.

[0065] ⑤ Dehydration, transparency and sealing: The experimental method is the same as HE staining.

[0066] (6) Conclusion:

[0067] The renal pathological changes of mice in each experimental group were compared by electron microscopy. The electron microscopy results of different groups are as follows: Figure 3 The electron microscopic results after HE staining and PAS staining are shown in Figure 4As shown in the figure, in the WT+normal saline group, the capillary endothelial cells in the glomeruli were not significantly swollen, no obvious endothelial cell proliferation was observed, and the capillary loops were open. There was no obvious thickening of the basement membrane. The podocytes were slightly swollen. The foot processes were well preserved and no obvious fusion was observed. There was no obvious proliferation of mesangial cells and matrix in the mesangial region. No definite electron-dense material deposition was observed. In the WT+LIRA group, the capillary endothelial cells in the glomeruli were not significantly swollen, no obvious endothelial cell proliferation was observed, and the capillary loops were open. There was no obvious thickening of the basement membrane. The podocytes were slightly swollen. The foot processes were well preserved and no obvious fusion was observed. There was no obvious proliferation of mesangial cells and matrix in the mesangial region. No definite electron-dense material deposition was observed. In the ADR model group, the capillary endothelial cells in the glomeruli were significantly swollen, no obvious endothelial cell proliferation was observed, and some capillary loops were poorly opened. There was no obvious thickening of the basement membrane and segmental shrinkage. The podocytes were obviously swollen, and podocytes were occasionally detached and bare basement membranes were formed. Some podocytes had mitochondrial swelling, vacuolar degeneration, and increased membrane density. The foot processes were diffusely fused, and microfilamentous plaques (cytoskeleton condensation) were observed in a few podocytes. There was no obvious proliferation of mesangial cells and matrix in the mesangial region. No definite electron-dense material deposition was observed. In the ADR+LIRA group, there was no obvious swelling of glomerular capillary endothelial cells, no obvious endothelial cell proliferation, and open capillary loops. There was no obvious thickening of the basement membrane. The podocytes were slightly swollen. The foot processes were well preserved, and mild fusion was occasionally observed locally. There was no obvious proliferation of mesangial cells and matrix in the mesangial region. No definite electron-dense material deposition was observed.

[0068] Example 3: GLP-1RA (using liraglutide (lira) as an example) significantly reduces the urine protein level induced by doxorubicin

[0069] The animal grouping and intervention in this example are the same as those in Example 2.

[0070] (1) Extraction of mouse kidney tissue protein:

[0071] ① Prepare protein lysis working solution: Mix 100× protease inhibitor complex, 100× phosphatase inhibitor complex and RIPA lysis buffer in a ratio of 1:1:98 to prepare lysis working solution, and pre-cool on ice. Each sample requires approximately 500 μl.

[0072] ② Tissue grinding: Weigh 10±1 mg of mouse renal cortical tissue, add 500 ul of pre-cooled protein lysis working solution, and then grind the tissue thoroughly using a grinder until the liquid is clear and there are no obvious tissue blocks.

[0073] ③ Lysis: Rotate the ground tissue sample at 4°C for 1 hour to fully lyse it.

[0074] ④ Centrifugation and sample collection: Centrifuge the lysed tissue at 4°C, 14000 rpm for 20 minutes. Collect the supernatant, which is the obtained protein lysis sample.

[0075] (2) Detection of protein expression by immunoblotting (Western Blot, WB):

[0076] ① Prepare electrophoresis gel: According to the molecular weight of the target protein to be detected, use the PAGE gel rapid preparation kit (Yazyme) to prepare separation gel and concentrated gel of different concentrations. Wash and assemble the gel preparation plate and glass plate, add the separation gel liquid to the gel preparation plate, seal it with liquid and let it stand at room temperature for 30 minutes to solidify, then prepare the upper concentrated gel and insert the 15-hole comb teeth, let it stand at room temperature for 30 minutes to solidify.

[0077] ② Protein loading and electrophoresis: Assemble the electrophoresis gel and the vertical electrophoresis tank and electrophoresis gel, and pour in 800ml of electrophoresis buffer. Pull out the comb teeth, wash the wells, add a total of about 30μg of protein sample to each lane, and add 5μl of protein marker at both ends for positioning. Then turn on the electrophoresis instrument, set the voltage to 80V for 40 minutes, and after confirming that all proteins have entered the separation gel, set the voltage to 120V and continue electrophoresis for about 1 hour. Turn off the electrophoresis instrument when the sample containing bromophenol blue is electrophoresed to the bottom of the instrument.

[0078] ③ Activate PVDF membrane: Cut the PVDF membrane of appropriate size according to the electrophoresis separation gel, and soak the membrane in pre-cooled methanol for 1 minute to fully activate it. Then take out the membrane and place it in PVDF membrane equilibration solution for 1 minute.

[0079] ④Transfer (semi-dry transfer): using eBlot TM L1 fast wet transfer instrument (GenScript L00686C) was used for transfer. Add sufficient 1×PVDF transfer working solution and coolant (ultrapure water) into the instrument. Lay the dry sponge pad, balanced PVDF membrane, and electrophoresis separation gel on the transfer clip in sequence. Use the transfer roller to remove bubbles and lay the second dry sponge pad. Close the transfer clip and insert it into the transfer instrument channel. Select the appropriate transfer time according to the molecular weight of the target protein to be detected and start the transfer (about 10 to 20 minutes).

[0080] ⑤ Blocking: Weigh 2g BSA and dissolve it in 40ml TBST buffer to prepare 5% BSA blocking solution. Separate the transferred PVDF membrane and electrophoresis gel naturally in water, place the membrane in the blocking solution and block it at room temperature for 90 minutes using a gyroscopic oscillator (shaking table).

[0081] ⑥ Incubate with primary antibody: Dilute the primary antibody in 5% BSA TBST buffer according to the antibody instructions and pre-experiments. Place the blocked PVDF membrane in the primary antibody solution and incubate overnight at 4°C with shaking.

[0082] ⑦ Wash the membrane: Recover the primary antibody solution for reuse. Place the membrane in sufficient TBST buffer and shake at room temperature for 3 times, 5 minutes each time.

[0083] ⑧ Incubate with secondary antibody: dilute the secondary antibody in TBST buffer at 1:5000. Place the washed PVDF membrane in the secondary antibody solution and incubate at room temperature for 1 hour with shaking. Wash the membrane 3 times after incubation, 5 minutes each time.

[0084] ⑨ Development: Protect from light. Mix ECL developer A and B in a 1:1 ratio to prepare the working solution. Use it within 30 minutes. Place the dried PVDF membrane in the imager and drip the working solution on the membrane. Incubate for 1 minute. Choose the appropriate exposure time to develop the bands and take pictures.

[0085] ⑩ Image analysis: ImageJ software (National Institutes of Health, Bethesda, MD) was used to perform quantitative grayscale analysis on all protein bands obtained, and the grayscale value of the sample's internal reference protein β-actin or other appropriate proteins was used for standardization. The results were expressed as relative band intensity.

[0086] (3) Mouse urine creatinine detection:

[0087] Materials: Cayman 500701 creatinine assay kit (containing 96-well plate, 20 mg / dl creatinine standard (10005314), 1.2% picric acid color developer (10005315), 1 M sodium hydroxide (10005316), sulfuric acid + acetic acid mixed acid solution (10005317), sodium borate (10008477), surfactant (10008478)), horizontal shaker, urine sample to be tested, 1.5 mL ep tube, sample tank, discharge gun, dd water, and microplate reader (absorbance 490-500 nm).

[0088] Experimental steps:

[0089] 1. Use dd water to dilute the sample at a ratio of 1:10-1:20. Add 15 μL of diluted sample to each reaction well;

[0090] 2. Prepare the alkaline picrate solution (APS): 150 μL of colorimetric solution is required in each reaction well. For example, prepare a 96-well plate reaction system (calculated as 1.5X): 2 mL of sodium borate + 6 mL of surfactant + 10 mL of picric acid colorimetric agent + 3.6 mL of sodium hydroxide. The system for each reaction well is as shown in the table below. The total volume can be calculated based on 1.2-1.5 times the volume. When preparing APS, it needs to be kept away from light and does not need to be placed on ice. After the APS is prepared, it can be stably stored at room temperature and away from light for 1 week. Except for the sample, all reagents must be equilibrated at room temperature before starting the assay. Before aspirating each reagent with a pipette, it is recommended to equilibrate the tip in the reagent (i.e., slowly fill the tip and gently discharge the contents, repeat several times). Do not expose the tip to the reagent already in the reaction well.

[0091] Reagents Volume per well (μL) Sodium Borate 13.9 Surfactant 41.7 Color Reagent 69.4 Sodium Hydroxide 25.0 Total volume: 150

[0092] 3. Configure the standard gradient according to the table below:

[0093]

[0094]

[0095] 4. Add 15 μL of standard / sample to each reaction well. It is recommended to make 2-3 replicates of standard and sample. Then add 150 μL of alkaline picrate solution to each reaction well to start the reaction.

[0096] 5. Cover the plate, place on a shaker, and react at room temperature for 10 minutes.

[0097] 6. Remove the plate cover and read the absorbance at 490-500nm using an ELISA reader. This absorbance is the initial absorbance reading (I abs ).

[0098] 7. Add 5 μL of mixed acid solution to all reaction wells, cover the plate, place on a shaker, and react at room temperature for 20 minutes.

[0099] 8. Remove the plate cover and read the absorbance at 490-500nm using an ELISA reader. This absorbance is the final absorbance reading (Final absorbance reading abs ).

[0100] 9. Calculate the average initial absorbance reading (I abs ), calculate the average final absorbance reading (F) for each standard and sample abs), and the average initial absorbance minus the average final absorbance is used to obtain the corrected absorbance. That is:

[0101] ΔCorrected absorbance=Ave(I abs )-Ave(F abs ).

[0102] 10. Use the corrected absorbance of each standard / sample to subtract the average corrected absorbance of standard A. Get the adjusted absorbance. That is: ΔΔAdjusted absorbance=ΔSample / Standard cor abs -Standard A cor abs .

[0103] 11. Use ΔΔAdjusted absorbance and the corresponding standard gradient concentration to calculate the regression equation to draw a standard curve, and use the linear regression equation of the standard curve to calculate the urine creatinine concentration of the sample. Creatinine (mg / dl) = [sample absorbance - (y intercept) / slope] × sample dilution. Note: To convert the unit from mg / dl to μmol / l, multiply the creatinine value by 88.4.

[0104] 12. Measurement range: The range of urine creatinine measured by this kit is 0-15 mg / dl.

[0105] (4) Detection of microalbumin in mouse urine:

[0106] Materials: sodium carbonate, sodium bicarbonate, ultrapure water, Tris, sodium chloride, Tween 20, BSA, enzyme substrate, sulfuric acid, 96-well plate, sample tank, discharge gun, coated antibody (A90-134A), protein standard, HRP-coupled mouse protein antibody (A90-134P), TMB colorimetric reagent, horizontal shaker, urine sample to be tested, 1.5 mL ep tube.

[0107] Prepare various buffer systems:

[0108] 1. Coating Buffer, i.e. 0.05 M bicarbonate buffer: 1.59 g sodium carbonate + 2.93 g sodium bicarbonate dissolved in 1000 mL dd water.

[0109] 2. Wash Solution: 50 mM Tris + 0.14 M sodium chloride + 0.05% Tween 20, dissolved in 1000 mL dd water. PH = 8.0. That is, 6.057 g Tris + 8.1816 mg sodium chloride dissolved in 1000 mL dd water, then add 500 μL Tween 20, PH = 8.0.

[0110] 3. Blocking (post coat) Solution (blocking solution / secondary coating solution), 50mM Tris + 0.14M sodium chloride + 1% BSA, pH = 8.0. That is, 6.057g Tris + 8.1816mg sodium chloride are dissolved in 1000mL dd water, and then 10g BSA is added, pH = 8.0.

[0111] 4. Sample / Conjugate Diluent (enzyme conjugate diluent), 50mM Tris + 0.14M sodium chloride + 1% BSA, pH = 8.0. That is, 6.057g Tris + 8.1816mg sodium chloride are dissolved in 1000mL dd water, and then 10g BSA, 500μL Tween 20 are added, pH = 8.0.

[0112] 5. Enzyme Substrate

[0113] 6. Stopping solution, 2M sulfuric acid.

[0114] Experimental steps:

[0115] 1. Identify the number of test wells to be tested and calculate the required test reagent volume (calculated as 1.5 times).

[0116] 2. Add 100 μL Coating Buffer and 1 μL capture antibody (A90-134A) to each test well, cover the plate, and incubate at room temperature for 60 minutes (if subsequent experiments cannot be performed on the same day, place in a 4°C refrigerator away from light (12 to 18 hours), and continue with subsequent steps the next day);

[0117] 3. Aspirate all the liquid in the wells, then add 300 μl Wash Solution to each well, rinse on a shaker for 3 minutes, repeat 3 times, invert the well plate and tap on absorbent paper to remove residual liquid;

[0118] 4. Add 200 μL Blocking (post coat) Solution to each test well and incubate for 30 minutes;

[0119] 5. Aspirate all the liquid in the wells, then add 300 μl Wash Solution to each well, rinse on a shaker for 3 minutes, repeat 3 times, invert the well plate and tap on absorbent paper to remove residual liquid;

[0120] 6. Prepare standard and sample diluents in Sample / Conjugate Diluent: The storage concentration of the protein standard is 4.5 mg / mL. See the table / figure below for the gradient dilution ratio of the standard. Similarly, the dilution ratio of the urine sample varies depending on the mouse strain. See the table below for the dilution ratio.

[0121] Con (ng / ml) Protein stock solution (4.5 mg / mL) Sampledilute A 10000 2μL 900μL B 500 50μL from A 950μL C 250 500μL from B 500μL D 125 500μL from C 500μL E 62.5 500μL from D 500μL F 31.25 500μL from E 500μL G 15.625 500μL from F 500μL H 7.8 500μL from G 500μL I 0 NONE 500μL

[0122] 7. Add 100 μL of standard or sample to each well. Repeat 2-3 times to reduce the error. Incubate at room temperature for 60 minutes (about 500 rpm).

[0123] 8. Aspirate all the liquid in each well, add 300 μL of washing buffer and rinse on a shaker for 3 minutes, repeat 5 times. After washing, invert the plate and tap on absorbent paper to remove residual liquid.

[0124] 9. Prepare the detection antibody solution by diluting the detection antibody in Sample / Conjugate Diluent. Dilute the HRP-conjugated Mouse Albumin Antibodies (A90-134P) at a ratio of 1:20,000.

[0125] 10. Add 100 μL of detection antibody solution to each well. Incubate at room temperature in the dark for 60 minutes (about 500 rpm);

[0126] 11. Aspirate the liquid from each well and add 300 μL of washing buffer to each well and rinse on a shaker for 3 minutes. Repeat 5 times. After washing, invert the plate and tap gently on absorbent paper to remove residual liquid.

[0127] 12. Mix two substrate reagents in equal volumes (traditional TMB colorimetric reagents are usually provided in the form of AB solution (TMB and H2O2 solutions are divided into two tubes and can be mixed before use), and add 100μL of TMB substrate solution to each well. Incubate the plate at room temperature in the dark for 5-10 minutes (or until the gradient color change can be clearly felt by the naked eye). Add 100μL of stop solution (2M sulfuric acid) to each well.

[0128] 13. The absorbance at 450 nm must be measured within 30 minutes after adding the stop solution.

[0129] 14. Calculate results using log-log or 4-parameter curve fit.

[0130] Example 4: Protective effect of GLP-1RA (using liraglutide (lira) as an example) on podocyte injury induced by puromycin aminonucleoside (PAN)

[0131] Use sterilized cell slides and place them in a 12-well cell culture plate. Pay attention to dripping a small amount of culture medium in each well where the slide is to be placed, so that the cell slide and the culture dish are bonded together by the tension of the culture medium, and then place the cell slide to prevent the cell slide from floating up when the cell suspension is added, resulting in a double-layer cell patch. According to the conventional experimental protocol, after trypsin digestion of adherent cells, complete culture medium is used to prepare kidney podocyte suspension, and the appropriate cell density is selected to inoculate podocytes into 12-well plates. Culture at 33°C, 5% CO2, replace the culture medium every 2 days, and replace the culture medium when the cell confluence is 50% and place at 37°C, 5% CO2 for differentiation. On the 7th day of differentiation, starve the cells with serum-free 1640 culture medium for 12 hours, and then replace it with 1640 culture medium containing 2% FBS, and use 100ug / mL PAN to stimulate podocytes. Different groups were set up as follows: 1640 group, DMSO group, PAN group, 10 nmol liraglutide intervention group, 50 nmol liraglutide intervention group, and 100 nmol liraglutide intervention group, a total of 6 groups. Among them, the intervention group was pretreated with different concentrations of liraglutide for 1 hour before PAN stimulation. After 24 hours and 48 hours after PAN stimulation, the cell culture plate was removed from the incubator, the culture medium was aspirated, and PBS was added to rinse twice. Then 4% paraformaldehyde was added to fix for 10 minutes and rinsed with PBS for 5 minutes. The PBS in the 12-well plate was aspirated with a pipette tip, and 100 μL 0.1% Triton X-100 was added to each well for 5 minutes to increase cell permeability. Then Triton X-100 was aspirated, PBS was washed twice, 5 minutes each time, 100 μL 1X phalloidin was added to each well, incubated at room temperature for 60 minutes, washed 3 times with PBS, phalloidin was removed, and DAPI sealing liquid was used to seal the plate. The cytoskeleton morphology and fluorescence intensity were observed under a fluorescence microscope. The results are as follows Figure 7 As shown, compared with normal cultured cells, after podocytes were stimulated with PAN for 48 hours, the fluorescence intensity of the podocyte cytoskeleton was significantly weakened, while in podocytes intervened with liraglutide, the cytoskeleton fluorescence intensity was improved compared with the PAN group.

[0132] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. Application of GLP-1RA in the preparation of drugs for improving podocyte injury.

2. The use according to claim 1, characterized in that: The GLP-1RA protects podocytes independently of lowering blood sugar and improves podocyte damage.

3. The use according to claim 1, characterized in that: The GLP-1RA includes liraglutide.

4. A drug for improving podocyte injury, characterized in that: The medicament comprises a therapeutically effective amount of GLP-1RA as the only active ingredient.

5. A drug for improving podocyte injury, characterized in that: The drug is a pharmaceutical composition, which includes, in addition to a therapeutically effective amount of GLP-1RA as an active ingredient, other pharmaceutical ingredients for improving podocyte injury.

6. Application of GLP-1RA in the preparation of drugs for the treatment of FSGS.

7. The use according to claim 6, characterized in that: The drug for treating FSGS refers to a drug that reduces urine protein and / or improves podocyte damage.

8. The use according to claim 7, characterized in that: The GLP-1RA includes liraglutide.

9. A drug for treating FSGS, characterized in that: The medicament comprises a therapeutically effective amount of GLP-1RA as the only active ingredient.

10. A drug for treating FSGS, characterized in that: The drug is a pharmaceutical composition, which, in addition to comprising a therapeutically effective amount of GLP-1RA as an active ingredient, also comprises other pharmaceutical ingredients for treating FSGS.

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