Mechanism of Kemeng Fang's Inhibition of Podocyte Apoptosis in Rats with Membranous Nephropathy through the PI3K/AKT Signaling Pathway

Summary

The present protocol describes the establishment of a membranous nephropathy (MN) animal model, and how Kemeng Fang's inhibition reduces MN rat podocyte apoptosis by activating the PI3K/AKT signaling pathway.

Abstract

Membranous nephropathy (MN) is a common pathological type of adult nephrotic syndrome. Up to 20% of patients with MN develop end-stage renal disease (ESRD). Podocytes have an important function in maintaining the glomerular filtration barrier and play a crucial role in the occurrence and development of proteinuria and MN. PI3K/AKT signaling pathway is involved in the entire process of podocyte growth, differentiation, and apoptosis. Kemeng Fang (KMF) is a traditional Chinese medicine formula that has been used to delay kidney injury. However, the therapeutic mechanism of KMF in MN is unclear. Here, the MN rat model was established by axillary, inguinal, and tail vein injections of cationized bovine serum albumin (C-BSA), and then KMF and PI3K inhibitor (LY294002) were administered. The data of liver function, kidney function, blood lipid, renal pathology, podocyte function, expression level of PI3K/AKT signaling pathway, and transcriptomics of rats demonstrated that KMF has a protective effect on the podocytes of MN rats by activating the PI3K/AKT signaling pathway, and it can effectively prevent the progression of MN.

Introduction

Membranous nephropathy (MN) is a common pathological type of adult nephrotic syndrome, with an annual incidence rate of approximately 5-10 per 100,000 individuals. It mostly occurs between the ages of 30 and 50 and is rare in children (about 5%). It is significantly more prevalent in men than women (2:1). Moreover, up to 20% of patients with MN develop the end-stage renal disease (ESRD). Furthermore, there is an increasing trend where patients with MN develop ESRD year by year^1,2,3. The pathological feature of MN is that granular immunoglobin G (IgG) and complement system membrane attack complex (MAC) are heavily deposited in the glomerular basement membrane (GBM) adjacent to the podocytes. This deposition leads to the thickening of the GBM and disruption of the glomerular filtration barrier integrity, ultimately leading to proteinuria⁴.

Supportive therapy, immunosuppressants, and targeted monoclonal antibodies are the main methods for treating MN. Although these interventions can significantly reduce proteinuria and delay the progression of renal deterioration, they also have many shortcomings. First, supportive therapy is only suitable for low-risk patients⁵. Second, immunosuppressants can cause adverse reactions, such as femoral head necrosis, secondary infection, and inhibition of bone marrow hematopoietic function⁶. Third, extensive randomized-controlled trial research is needed to provide evidence-based medicine for the use of monoclonal antibodies such as ofamizumab, otuzumab, daretozumab, and isatuximab^7,8,9. Therefore, actively seeking effective treatment methods for MN has great significance in delaying the onset of ESRD and improving the quality of life of patients with MN.

Podocytes, also known as glomerular visceral epithelial cells, are attached to the outer side of the GBM, and the GBM capillary endothelium together form the glomerular blood filtration barrier. They have important functions, such as maintaining the glomerular protein filtration barrier, synthesizing normal basement membrane, and providing structural support for the glomerular capillary plexus^10,11. Research has shown that apoptosis of podocytes plays a crucial role in the occurrence and development of proteinuria and MN, and the PI3K/AKT signaling pathway is involved in the entire process of podocyte growth, differentiation, and apoptosis^12,13,14.

An increasing number of studies have shown that Chinese medicine has significant advantages in the treatment of MN, which can significantly reduce blood creatinine, proteinuria, and delayed kidney injury^15,16. KMF is a traditional Chinese medicine compound with ingredients derived from 13 plants: Codonopsis pilosula (Franch.) Nannf. (Dangshen, DS); Astragalus membranaceus (Fisch.) Bunge. (Huangqi, HQ); Coptis chinensis Franch. (Huanglian, HL); Perilla frutescens (L.) Britt. (Suye, SY); Rehmannia glutinosa (Gaertn.) DC. (Shudihuang, SDH); Ligusticum chuanxiong Hort. (Chuanxiong, CX); Euryale ferox Salisb. (Qianshi, QS); Sabia japonica Maxim. (Qinfengteng, QFT); Rhus chinensis Mill. (Wubeizi, WBZ); Lobelia chinensis Lour. (Banbianlian, BBL); Oldenlandia diffusa (Willd.) Roxb. (Baihuasheshecao, BHSSC; Table 1). KFM has many functions, such as tonifying the kidney (enhancement of renal function), enhancing qi (strengthening immunity), promoting diuresis, and dredging collaterals (promoting blood circulation). However, the therapeutic mechanism of KMF in MN is unclear^17,18.

At present, there are many ways to construct MN models, Including Heymann nephritis model, C-BSA nephritis model, α3NC1 mouse model, in which Heymann nephritis model, the main pathogenic antigen megalin protein is not found in human MN, so it is different from the pathogenesis of human MN, α3NC1 mouse model, only DBA/1 genetic background of the mouse model success rate is higher, the rest of the mice were less successful in modeling, or even unable to be modeled^19,20,21. The C-BSA nephritis model is cost-effective and simple to operate, and its pathogenesis is highly similar to that of the human MN animal model¹⁹. The basic principle is that because the GBM is negatively charged, and C-BSA is positively charged, it can easily cross the GBM to become a planted antigen, which induces circulating antibodies to accumulate there to form an in situ immune complex, thereby constructing an MN model^22,23. The aim of this study was to observe the therapeutic effect of KMF on MN and its molecular mechanism by a combination of transcriptomics and molecular biology and to provide a reliable scientific basis for the treatment of MN with KMF.

Protocol

This study was reviewed and approved by the Experimental Animal Management and Use Committee of the Hubei Provincial Center for Disease Control and Prevention (ID number: 202220144). The rats underwent a 12 h light/dark cycle under non-pathogenic conditions of 23 ± 1 °C and 50%-60% atmospheric humidity. We procured 100 male 8-week-old Sprague-Dawley rats from the Hubei Provincial Center for Disease Control and Prevention (license number: SYXK [E] 2022-0065), and they were subjected to adaptive feeding in a specific pathogen-free grade environment for 1 week with a normal maintenance feed and drinking sterile water.

1. Drug preparation

1. Preparation of KMF
   1. The Chinese medicines used were provided by the hospital affiliated to Changchun University of Traditional Chinese Medicine, the specific composition and dosage are shown in Table 1. To prepare, use a total of 147 g of raw medicines-Codonopsis pilosula (20 g), Astragalus membranaceus (30 g), Coptis chinensis (3 g), Perilla frutescens (6 g), Rehmannia glutinosa (15 g), Ligusticum chuanxiong (15 g), Euryale ferox (15 g), Sabia japonica (10 g), Rhus chinensis (3 g), Lobelia chinensis (15 g), Oldenlandia diffusa (15 g). Mix and soak in 1470 mL of distilled water for 30 min.
   2. Place the herbal mixture in a ceramic pot and heat at 100 °C for 90 min for decoction. Filter through two layers of medical gauze and store at room temperature.
   3. Add distilled water (1470 mL) again to the leftover herbal mixture and repeat the above decoction operation once more, as described in step 1.1.2. Store the filtrate at room temperature.
   4. Mix the above two filtrates and oven-dry at 80 °C for 10 h until the aqueous solution has completely evaporated and only the solute is retained in the form of powder.
   5. Weigh the powder and dissolve it in saline to make a solution containing 1.323 g, 2.646 g, and 5.292 g of the drug per 4 mL (the concentrations were 0.331 g/mL, 0.662 g/mL, and 1.323 g/mL, respectively), which is the daily dosage of the drug administered to rats.
2. Preparation of Benadryl hydrochloride
   1. Add Benadryl hydrochloride tablet (90 g) into 100 mL of saline and shake well to completely dissolve.
3. Preparation of PI3K inhibitor LY294002
   1. Add 82.3 mg of LY294002 powder into 1.1 mL of DMSO and shake well to completely dissolve it. Next, add 198.9 mL of saline to dilute it to obtain a concentration of 0.41 mg/mL.
4. Preparation of C-BSA emulsifier
   1. Add 67 mL of anhydrous ethylenediamine to 500 mL of double-distilled water and mix. Slowly add 350 mL of 6 M hydrochloric acid and adjust the pH to 4.75. Maintain the temperature of the final solution at 25 °C.
   2. Dissolve 5 g of natural bovine serum albumin in 25 mL of double-distilled water and maintain the solution at a constant temperature of 25 °C with constant stirring. Add 1.8 g of carbodiimide hydrochloride and 30 mL of 4 M acetic acid buffer with pH 4.75 after 2 h to obtain the C-BSA solution.
   3. Dialyze the obtained solution in double-distilled water at 4 °C for 72 h (with the water changed every 3-5 h) using a selenite paper and freeze-dry to obtain C-BSA lyophilized powder, Store at -80 °C²⁴.
   4. Add 100 mg of C-BSA dried powder in 50 mL of saline to form a C-BSA solution. Mix this with an equal volume of Incomplete Freund's adjuvant for complete emulsification; its concentration is 1 mg/mL.
5. Preparation of C-BSA solution
   1. Add 640 mg of C-BSA dry powder in 100 mL of PBS and shake well to fully dissolve it; its concentration is 6.4 mg/mL.

2. Establishment of MN animal model

NOTE: The experiment was divided into eight groups: normal control group (CON), model group (MOD), benazepril hydrochloride group (BEN), KMF low-dose group (KM-L), KMF medium-dose group (KM-M), KMF high-dose group (KM-H), PI3K inhibitor group (PI3K), and PI3K inhibitor + KMF medium-dose group (PI3K + KM-M). Except for the normal control group, all groups were administered C-BSA to produce the MN model.

1. Pre-immunization: Multiple subcutaneous injections of C-BSA emulsifier in the axilla and groin
   1. Grasp the back skin of the rats with the left hand and turn their abdomen upward with the abdominal skin tightened. Inject the C-BSA emulsifier subcutaneously into the axillae and groin of the rats using a 10 mL syringe at the dose of 1 mL/400 g once every other day for 1 week.
2. Formal immunization: Tail vein injection of C-BSA solution
   1. Mount the rats in a fixator with the lid tightly closed and the exposed tail facing outward.
   2. Wipe the rat's tail with an alcohol cotton ball and pinch both sides of the rat's tail with the thumb and forefinger of the left hand to fill the vein and keep the vein facing up.
   3. Hold a 1 mL syringe in the right hand so that the needle is at 30° to the tail vein. Keep the tip of the needle beveled upward and the needle parallel to the vessel immediately after gently pricking into the skin. If there is any return of blood from the needle, inject it back along with a C-BSA dose of 2.5 mL/kg 3x a week for 4 weeks.
   4. After completion of the injection, use a dry cotton ball to press the injection point for approximately 1 min to stop the bleeding. Remove the mice from the immobilizer and return to the cage.

3. Analysis of KMF

1. Weigh 1 g sample into a 2 mL centrifuge tube, add 600 µL of MeOH (stored at -20 °C containing 2-Amino-3-(2-chloro-phenyl)-propionic acid (4 ppm)), and vortex the mixture for 30 s.
2. Add approximately 100 mg of glass bead and place the mixture in a tissue grinder for 90 s at 60 Hz.
3. Perform ultrasound at 40 kHz for 15 min at room temperature.
4. Centrifuge for 10 min at 15,984 x g, 4 °C, and filter the supernatant through a 0.22 µm membrane. Transfer a detection bottle for LC-MS detection.

4. Drug treatments

NOTE: Adult humans need 147 g KMF per day. According to the conversion formula of experimental rat and human drug dose, the equivalent experimental dose for rat (g/kg) = human dose (g)/body weight (70 kg) x 6.3, the daily dose of the rat was approximately 13.23 g/kg.

1. Using the left hand, grasp the skin on the back of the rats, turn their abdomen upward, and tighten the abdominal skin. Hold the needle of a 10 mL syringe in the right hand and insert it into the mouth from one side, sliding along the palate and the posterior wall of the pharynx and further along to the stomach using the swallowing action.
2. Administer the drug slowly using the index finger of the right hand and pull the gastric needle out after the administration is complete. The dosage of the administered drug is 10 mL/kg for a total duration of 4 weeks.
   1. For the CON and MOD groups, administer 10 mL/kg/day saline by gavage.
   2. For the BEN group, administer 9 mg/kg/day Benadryl hydrochloride aqueous solution by gavage.
   3. For the KM-L group, administer 3.3075 g/kg/day aqueous extract of KMF by gavage. For the KM-M group, administer 6.615 g/kg/day aqueous extract of KMF by gavage. For the KM-H group, administer 13.23 g/kg/day aqueous extract of KMF by gavage.
   4. For the PI3K group, administer 2.1 mg/kg/day LY294002 by gavage.
   5. For the PI3K+KM-M group, administer 2.1 mg/kg/day LY294002 (5 mL/kg) + 6.615 g/kg/day KMF aqueous extract (5 mL/kg) by gavage.

5. Evaluation of KMF efficacy

1. Detection of blood and urine biochemical indices
   1. Collect urine every 2 weeks after the start of the experiment. Place a single rat in a metabolic cage for 24 h. Restrict diet and provide free access to drinking water. Collect urine using a 50 mL centrifuge tube placed below the metabolic cage.
   2. Centrifuge the urine at 15,984 x g for 10 min and use the supernatant to estimate the level of urinary protein according to the instructions of the Urinary Protein Test Kit.
   3. After 4 weeks of continuous administration, anesthetize rats by inhaling 2% isoflurane. Assess anesthesia depth by an absence of reflex response. Using vet ointment on eyes to prevent dryness while under anesthesia. Grasp the skin on the back of the rats with the left hand and trim the whiskers of the rats with scissors.
   4. Disinfect the skin around the eyeballs with ethanol and quickly remove the eyeballs using hemostatic forceps. Collect the blood dropped into the centrifuge tube and centrifuge for 10 min at 15,984 x g after standing at room temperature for 30 min. Collect the supernatant.
   5. Detect Alanine aminotransferase (ALT), Aspartate transaminase (AST), Albumin (ALB), Triglyceride (TG), Total cholesterol (TC), Blood urea nitrogen (BUN), Serum creatinine (Scr), TotalProtein (TP) and other blood indices using an automatic biochemical analyzer.
2. Separation of renal tissues
   1. After the blood is taken from the rats, fix them on the surgical plate, and slowly cut the fur and muscular layer at the midline of the abdomen, pushing the intestinal tubes open to expose the abdominal aorta and kidneys²⁵.
   2. Block the abdominal aorta above the right kidney using surgical artery clamps. Attach a scalp needle to a 5 mL syringe to puncture below the block and perfuse PBS buffer to irrigate the kidneys until both kidneys become white or pale in color.
   3. Remove the kidneys and bluntly separate the periosteum. Divide the right kidney into freeze-storage tubes and store in the refrigerator at -80 °C. Fix the left kidney in 4% paraformaldehyde solution.
   4. After 24 h of fixation, remove the kidney. Perform dehydration, transparency, and paraffin embedding. After the wax block solidifies, slice with a tissue slicer at a thickness of 3 µm, fish out with an anti-dehiscence slide, bake the slices at 98 °C for 20 min, and store for spare²⁶.
3. Renal histopathological analysis
   1. Subject the sections to gradient xylene dewaxing and anhydrous ethanol dehydration and rinse using distilled water. Subject to xylene for 15 min, followed by fresh xylene solution for 15 min. Switch to anhydrous ethanol for 5 min, change to fresh anhydrous ethanol for 5 min, followed by 90% ethanol for 5 min, then 80% ethanol for 5 min, then 70% ethanol for 5 min, and finally flush with distilled water.
   2. Follow the instructions for hematoxylin-eosin staining (H&E), periodic acid-Schiff (PAS), and Masson staining kits for sample staining.
   3. Perform gradient anhydrous ethanol dehydration and xylene clear again.
   4. Place the slices in a constant temperature incubator, set the temperature to 90 °C, and bake for 20 min. After baking, remove the slices and add neutral gum dropwise. Cover them with a cover glass.
   5. Observe under a light microscope at a magnification of 200x and collect images.
4. Immunofluorescence (IF) analysis of renal tissues
   1. Dewax sections and dehydrate as described in step 5.3.1. Place samples in 0.01 M EDTA buffer solution for high-pressure, high-heat (125 °C at 103 KPa) fixation for 15 min. Let the sample cool naturally, and wash 3x for 3 min each in PBS.
   2. Place the sections in a 3% hydrogen peroxide solution and incubate for 10 min. Wash the samples 3x for 5 min each in PBS. Then, place the sections in 10% goat serum, incubate for 30 min, and wash 3x for 3 min each in PBS.
   3. Add primary antibody IgG (1:100) and C3 (1:100) dropwise and incubate in a wet box at 4 °C overnight. The primary antibody dilution ratio is shown in Table 2. Remove the sections from the refrigerator and wash with TBST 3x for 3 min each.
   4. Add fluorescent secondary antibody (1:100) dropwise, incubate at 37 °C for 1 h, and wash with TBST 3x for 3 min each.
      NOTE: This step and all subsequent steps should be carried out in the dark^27,28.
   5. Incubate with 4',6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature and avoid light, wash with tris-buffered saline with tween 20 (TBST) 3x for 5 min each to remove excess DAPI.
   6. Add autofluorescence quencher in a circle, incubate for 5 min, and rinse with running water for 10 min. Shake dry the sections and seal with anti-fluorescence quenching sealer. Observe under a fluorescence microscope at 200x and collect images.
5. Immunohistochemical (IHC) analysis of kidney tissue
   1. Dewax sections and dehydrate as described in step 5.3.1. Repair under high pressure (125 °C · 103KPa) in 0.01M EDTA buffer solution for 15 minutes. After natural cooling, wash with PBS 3x for 3 min each.
   2. Block endogenous peroxidase and serum sealing operation method as described in step 5.4.2.
   3. Add primary antibody WT-1 (1:200) and Nephrin (1:100) dropwise and incubate in a wet box in the refrigerator at 4 °C overnight²⁹.
   4. Remove the sections from the 4 °C refrigerator, wash with PBS 3x for 5 min each. Add secondary antibody (1:200) dropwise, incubate at 37 °C for 30 min, and wash with PBS 3x for 3 min each.
   5. Add drops of freshly prepared diaminobenzidine (DAB) chromogenic solution and observe under the light microscope at 200x. The positive signal is brownish yellow or brownish brown. Wash the staining solution immediately with tap water when a color change in the section is observed.
   6. Use hematoxylin to restain for 3 min, then add 1% hydrochloric acid alcohol for differentiation and rinse with tap water for 10 min.
   7. Dehydrate the sections, transparent, and seal, as described in step 5.2.4. Observe under the light microscope at 200x and collect images.
6. Analysis of terminal deoxynucleotidyltransferase-mediated dUTP nick-end Labeling (TUNEL) staining of renal tissue
   1. Use step 5.3.1 to deparaffinize and dehydrate the sections. Draw circles around the tissues using an immunohistochemistry pen and add 100 µL of Proteinase K working solution dropwise to each sample. Incubate at 37 °C for 20 min, wash with PBS 3x for 5 min each, and store the treated samples in a wet box.
   2. Place the sections in 3% hydrogen peroxide solution, incubate for 10 min, and wash with PBS 3x for 5 min each.
   3. Add equilibrium buffer (100 µL) dropwise to the circle to cover the tissue and incubate for 20 min at room temperature.
   4. Add 50 µL of labeling working solution (equilibrium solution: fluorescent labeling solution: TDT enzyme = 35 µL:10 µL:5 µL) dropwise to each sample, incubate in a wet box at 37 °C for 60 min, protected from light, and wash with PBS 3x for 5 min each.
   5. Add DAPI and incubate with DAPI for 5 min at room temperature under light, wash with PBS 3x for 5 min each.
   6. After the sections were shaken dry and sealed with anti-fluorescence quenching sealer, observe under a fluorescence microscope at 200x and collect images.
7. Sample preparation for electron microscopic analysis of renal tissue
   1. Fix the kidney tissues with 2.5% glutaraldehyde at 4 °C for 2-4 h and wash 3x for 15 min each in PBS.
   2. Fix in osmium acid (1%) at room temperature away from light for 2 h and wash with PBS 3x for 15 min each.
   3. Perform gradient alcohol and acetone dehydration, using acetone and embedding agent Immerse in 1:1, 1:2, and 1:31 gradients for 2 h, 4 h, and 8 h, respectively. After this, use pure embedding agent and embed the samples at 37 °C overnight.
   4. After 8 h, place the embedded plate in a 60 °C oven for polymerization for 48 h. Slice the resin block processed above in an ultra-thin slicer, with a slice thickness set to 60-80 nm; use copper mesh to dislodge slices.
   5. Use a 2% hydrogen peroxide acetate and alcohol solution for staining for 8 min, followed by a 70% ethanol and ultrapure water wash 3x. Complete by washing with 2.6% ethanol and ultrapure water.
   6. Observe and acquire images under a transmission electron microscope.
8. Quantitative real-time polymerase chain reaction (qRT-PCR) assay analysis
   1. Add 20 mg of frozen kidney tissue to the microcentrifuge tube and grind thoroughly with a high-speed tissue grinder until there is no visible tissue mass.
   2. Collect the supernatant after centrifugation at 15,984 x g for 10 min. Add 250 µL of chloroform and invert the tube for 15 s; the solution is mixed thoroughly. Leave to stand at room temperature for 3 min, then centrifuge at 15,984 x g for 10 min at 4 °C.
   3. Collect the upper aqueous phase and transfer to a new RNase Free centrifuge tube. Add an equal volume of 70% ethanol (RNase-free water preparation), invert, and mix.
   4. Add the solution to the adsorbent column loaded into the collection tube for RNA washing. After Buffer RW washing, leave the column at room temperature for 5 min to dry.
   5. Place the column in a new RNase-free centrifuge tube, add 30-50 µL of RNase-free water to the middle of the column, allow to stand at room temperature for 1 min, and then centrifuge at 15,984 x g for 1 min. Collect the RNA solution and store it at -80 °C.
   6. Remove the RNA solution from the freezer and pre-denature at 90 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 35 s, and 72 °C for 25 s, and finally lysed in the order of 95 °C for 15 s, 60 °C for 60 s, 95 °C for 15 s (Supplementary Table 1).
   7. Using β-actin as an internal reference, calculate the relative expression of WT-1 and Nephrin using the 2¬-ΔΔCt method based on the Ct value of each sample (ΔΔCt = Ct value of target gene - Ct value of internal reference gene)³⁰. Detailed information on primers is shown in Table 3.
9. Western blot (WB) analysis
   1. Prepare the lysate according to the ratio of protease inhibitor:phosphatase inhibitor:RIPA= 1:1:100, and add 500 µL of lysate into a microcentrifuge tube.
   2. Add 100 mg of frozen kidney tissue to the microcentrifuge tube and grind thoroughly with a high-speed tissue grinder until there is no visible tissue mass.
   3. Remove the microcentrifuge tube, lyse on ice for 30 min, and then centrifuge at 15,984 x g for 10 min. Aspirate the supernatant and store it at -80 °C.
   4. Detect the protein concentration according to the instructions of the bicinchoninic acid (BCA) Protein Concentration Assay Kit and add PBS solution dropwise to ensure consistent protein concentration in each group.
   5. Add 5x protein up-sampling buffer to the microcentrifuge tube, incubate at 100 °C for 15 min to fully denature it, and remove after cooling.
   6. Separate the proteins by 12.5% SDS-PAGE gel electrophoresis, first using 80 V, causing all the samples to be pressed into a flat blue line, and then adjusting the voltage to 130 V, until the bromophenol blue runs out from the bottom of the gel plate. Stop the electrophoresis.
   7. Remove the gel. Wet the filter paper with membrane transfer buffer. Activate the polyvinylidene fluoride (PVDF) membrane in methanol for 30 seconds. Then, arrange everything in the following order: sponge mesh/filter paper/gel/PVDF membrane/filter paper/filter paper/sponge mesh in the clip used for transferring the membrane.
   8. Place the gel in the negative pole, the PVDF membrane in the positive pole, and transfer the membrane with a current of 200 MA for 90 min.
   9. Remove the PVDF membrane and wash with TBST once for 5 min. Add the closure solution at room temperature while shaking for 90 min. Wash with TBST 3x for 5 min each.
   10. Add primary antibodies PI3K (1:1000), PIK3CA (1:1000), AKT (1:1000), P-AKT (1:1000), BAD (1:1000), P-BAD (1:1000), BCL-2 (1:1000), bax (1:4000), and c-caspase3 (1:1000) and incubate in a wet box overnight at 4 °C in the refrigerator^31,32.
   11. Remove the PVDF membrane from the 4 °C refrigerator, wash with TBST 3x for 5 min each. Add secondary antibody (1:10,000) dropwise, incubate at 37 °C for 90 min, and wash with TBST 3x for 3 min each.
   12. Prepare the developing solution according to the ratio (A: B=1:1) and develop them on the machine as per the instructions.

Discussion

This study aimed to observe the pharmacological effects of KMF and explore its specific mechanism of inhibiting apoptosis of MN rat podocytes. First, it demonstrated in vivo that KMF can alleviate podocyte apoptosis and delay MN progression by activating the PI3K/AKT signaling pathway. Second, transcriptomic results showed that KMF may exert its effects through the PPAR, PI3K/AKT, and p53 signaling pathways, ECM receptor interaction, etc. Among them, the PI3K/AKT signaling pathway has been validated in experiments. These findings may provide a scientific basis for the clinical use of KMF as a potential treatment option for patients with MN.

Research has shown that podocyte apoptosis is one of the key factors leading to the gradual progression of MN. Furthermore, MN limits the division and proliferation ability of podocytes; consequently, once damaged or lost, this sequela seriously affects renal function. When the number of podocytes decreases to the point where they cannot fully cover the GBM, the GBM is completely exposed and adheres to Bowman's capsule, causing compression or even collapse of the glomerular capillary loop, ultimately promoting MN to develop into ESRD^33,34,35,36. Therefore, further research on the specific mechanism of podocyte apoptosis and methods to block or inhibit podocyte apoptosis is key to delaying the progression of MN. Research has shown that the PI3K/AKT signaling pathway has multiple functions in regulating cell apoptosis, oxidative stress, and inflammatory response and plays an important regulatory role in the occurrence and development of MN. WT-1 and Nephrin are pore membrane proteins expressed on podocytes, which not only play an important role in maintaining the normal structure and function of the pore membrane but also initiate PI3K/AKT-dependent signaling pathways and participate in podocyte signaling. The decrease in expression levels of these proteins often indicates damage to podocytes^37,38,39.

PI3K is a dimer composed of a regulatory subunit, p85, and a catalytic subunit, p110, which can be activated by various growth factors and complexes. It is a key and initiating factor in this pathway⁴⁰. The PIK3CA gene is located on chromosome 3 and has a total of 20 exons. Its main function is to encode one of the catalytic subunits of the PI3K enzyme, p110 α Protein; hence, changes in PIK3CA can cause the PI3K enzyme to remain in a sustained activated state⁴¹. Once PI3K is activated, the second messenger, PIP3, will generate and continuously stimulate the downstream AKT signaling pathway, while p-AKT promotes the phosphorylation of the pro-apoptotic molecule, Bad, dissociating the pro-apoptotic complex of Bad with Bcl-2 and Bcl-xL, and forming a complex with the 14-3-3 protein in the cytoplasm, thereby losing its pro-apoptotic function. The anti-apoptotic molecules, BCL-2 and Bcl-xL, can be fully dissociated and exert an inhibitory effect on podocyte apoptosis^42,43. Caspase-3 is a protease that plays a core role in the execution phase of cell apoptosis, ultimately leading to cell apoptosis by cleaving the DNA repair enzyme PARP into small fragments⁴⁴. The induction of cytochrome c release from mitochondria to the cytoplasm by the pro-apoptotic protein Bad is a key step in activating caspase, and the complex of P-Bad binding to the 14-3-3 protein inhibits this process, thereby preventing the occurrence of the apoptotic cascade^45,46,47.

Transcriptomic results indicate that KMF treatment for MN is closely related to the PI3K/AKT, PPAR, and p53 signaling pathways. Validated the PI3K/AKT signaling pathway through WB analysis. The experimental results showed that the PI3K/AKT signaling pathway in MN rats was significantly inhibited, while KMF could significantly activate the PI3K/AKT signaling pathway, reducing the generation of the pro-apoptotic molecules Bad and Bax and promoting the generation of the anti-apoptotic molecule BCL-2, thereby increasing the expression levels of podocyte hiatal membrane proteins, WT-1 and Nephrin, and reducing the incidence of podocyte apoptosis. Therefore, KMF reduces podocyte apoptosis by activating the PI3K/AKT signaling pathway and was found to have a protective effect on MN model rats.

However, this study has some limitations. First, this study only explored the mechanism of KMF inhibition of podocyte apoptosis at the level of in vivo animal experiments, which needs to be verified by in vitro cellular experiments as well as in-depth explorations of the mechanism; second, podocyte injury is also closely related to autophagy, immune-inflammation, and pyroptosis, and it needs to be further explored whether KMF can affect MN by regulating autophagy, immune-inflammation, and pyroptosis^48,49.

The PPAR family (PPAR α, PPAR β/δ, PPAR γ) is a nuclear hormone receptor that relies on ligand activation and has important functions such as participating in energy metabolism, regulating cell apoptosis, and inflammatory response^50,51. They affect gene transcription by forming heterodimerization with retinoic acid X receptor (RXR), where PPAR γ both regulate the inflammatory factor NF- κB. The key to B activation lies in its function of protecting podocytes from damage^52,53,54. Multiple studies have shown that PPAR γ agonists (TZD, such as pioglitazone) have renal protective effects independent of hypoglycemic effects, directly protecting podocytes from damage and reducing proteinuria and glomerular damage in various kidney disease animal models^{55,56,57,58,59}. There are also reports indicating that PPAR can reduce podocyte apoptosis by inhibiting the activation of Caspase-3⁶⁰. The latest research indicates a new type of PPAR γ, the selective regulator GQ-16, is more effective than TZD in reducing proteinuria and nephrotic syndrome-related complications, which also brings dawn to the treatment of kidney disease⁶¹. KMF has a protective effect on the apoptosis of MN rat podocytes by activating the PI3K/AKT signaling pathway. Based on the important role of the PPAR signaling pathway in the kidneys, further in-depth exploration of the relationship between KMF, PPAR, and MN is required in the future.

Materials

Name	Company	Catalog Number	Comments
2-Amino-3-(2-chloro-phenyl)-propionic acid	Aladdin	103616-89-3
30% H2O2	China National Pharmaceutical Group Chemical Reagent Co., Ltd	10011218
812 embedding agent	SPI	90529-77-4
Acetone	China National Pharmaceutical Group Chemical Reagent Co., Ltd	10000418
Acetonitrile	Thermo	75-05-8
Ammonia	China National Pharmaceutical Group Chemical Reagent Co., Ltd	10002118
Ammonium formate	Sigma	540-69-2
Analytical balance	Changzhou Lucky Electronic Equipment Co., Ltd	FA
Anhydrous ethanol	China National Pharmaceutical Group Chemical Reagent Co., Ltd	10009218
Anti fluorescence quenching and sealing agent	southernbiotech	0100-01
Automatic biochemical analyzer	Rayto Life and Analytical Sciences	Chemray240
BCA Protein Assay Kit	Solarbio	PC0020
Benazepril hydrochloride tablets	Xinya Minhang	H20044840
Blender	Kylin-Bell	BE-2600
Brick and stone cutting blade	Daitome	Ultra45
BSA	ZSGB-BIO	ZLI-9027
Buffer RW	Beijing Baiao Leibo	WK191
Carbodiimide hydrochloride	Hubei Xinghengye Technology	25952-53-8
Cell apoptosis detection kit	Elabscience	E-CK-A322
Chemiluminescence imaging system	Hangzhou Shenhua Technology Co., Ltd	SH-523
Chloroform	China National Pharmaceutical Group Chemical Reagent Co., Ltd	10006818
Constant temperature drying oven	Thermo Fisher	Heto PowerDiy LL3000
Cover glass slide	Jiangsu Shitai Experimental Equipment Co., Ltd	10212450C
CY3 Conjugated AffiniPure Goat Anti-rabbit IgG (H+L)	BOSTER	BA1032
DAB reagent kit	Servicebio	G1212-200T
DAPI	Blue Cloud Sky	C1002
Decolorization shaker	Wuhan Lingsi Biotechnology Co., Ltd	TSY-B
Dehydration machine	Wuhan Junjie Electronics Co., Ltd	JJ-12J
DL2000 DNA Marker	TIANGEN	MD114
DMSO	MCE	HY-Y0320
dNTP	TIANGEN	CD117
EBlot L1 Rapid Wet Rotation Instrument	Kingsray Biotechnology Co., Ltd	L00686C
ECL substrate solution	affinity	KF8003
Electric constant temperature water bath pot	Fisaff Instrument (Hebei) Co., Ltd	DK-20000-IIIL
Electrophoresis instrument power supply	Beijing Longfang Technology Co., Ltd	LF-600S
Embedding machine	Wuhan Junjie Electronics Co., Ltd	JB-P5
Equilibrium buffer	Wuhan Lingsi Biotechnology Co., Ltd	E8090
Ethylenediamine	Ruichengkang Pharmaceutical Technology	107-15-3
FA series multifunctional analytical electronic balance	Changzhou Lucky Electronic Equipment Game Company	FA1204
Filter membrane	Jinteng	Nylon6-0.22μm
Formaldehyde solution	China National Pharmaceutical Group Chemical Reagent Co., Ltd	10010018
Formic acid	TCI	64-18-6
Freezing centrifuge	Xiangyi	H1850-R
Frozen platform	Wuhan Junjie Electronics Co., Ltd	JB-L5
Glacial acetic acid	China National Pharmaceutical Group Chemical Reagent Co., Ltd	10000218
Glass bead	Sigma	G8772-500G
Glass slide	Nantong Meiweide Life Science Co., Ltd	PC2-301
glycine	Biofroxx	30166428
Hematoxylin	Wuhan Lingsi Biotechnology Co., Ltd	G1140
High speed refrigerated centrifuge	Hunan Kecheng Instrument Equipment Co., Ltd	H1-16KR
Horizontal agarose electrophoresis tank	Long Fang	LF-31DS
Horizontal shaker	Jiangsu Haimen Qilin Bell Instrument Manufacturing Co., Ltd	TS-1
HRP Conjugated AffiniPure Goat Anti-rabbit IgG (H+L)	BOSTER	BA1054
hydrochloric acid	China National Pharmaceutical Group Chemical Reagent Co., Ltd	10011028
Imaging system	Nikon	Nikon DS-U3
Incomplete Freund's adjuvant	MCE	ISA-51
Intelligent digital magnetic heating stirrer	Hangzhou Miou Instrument Co., Ltd	TP-350E+
Isoflurane	Sigma	26675-46-7
Isopropanol	China National Pharmaceutical Group Chemical Reagent Co., Ltd	80109218
KCl	China National Pharmaceutical Group Chemical Reagent Co., Ltd	10020318
KH2PO4	China National Pharmaceutical Group Chemical Reagent Co., Ltd	1115GR500
Liquid chromatograph	Thermo	IQLAAAGABHFAPUMBJC
marker(10-250KD)	Mei5bio	MF028-plus-01
marker(20-120KD)	GenScript	M00521
Mass spectrometer	Thermo	IQLAAEGAAPFALGMBDK
Masson staining kit	BASO	BA4079B
Methanol	Thermo	67-56-1
microscope	Nikon	ECLIPSE Ci
microwave oven	Galanz Microwave Oven Electrical Appliance Co., Ltd	P70D20TL-P4
Multi sample tissue grinder	Shanghai Jingxin Industrial Development Co., Ltd	Tissuelyser-24L
Multiskan FC ELISA reader	Thermo scientific	1410101
Na2HPO4.12H2O	China National Pharmaceutical Group Chemical Reagent Co., Ltd	10017618
NaCl	China National Pharmaceutical Group Chemical Reagent Co., Ltd	10000218
Neutral resin	Wuhan Lingsi Biotechnology Co., Ltd	G8590
Normal Goat Serum	Solarbio	SL038
Organizational spreading machine	Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd	KD-P
Osmic acid	Ted Pella Inc	18450
oven	Shanghai Huitai Instrument Manufacturing Co., Ltd	DHG-9140A
Palm centrifuge	Wuhan Lingsi Biotechnology Co., Ltd	D1008E
Paraformaldehyde	Solarbio	P1110
PAS staining kit	BASO	BA4114B
Pathological slicer	Shanghai Leica Instrument Co., Ltd	RM2016
PBS	Solarbio	P1020
PCR instrument	Hangzhou Miou Instrument Co., Ltd	PR-96
PH meter	Sedolis Scientific Instruments (Beijing) Co., Ltd	PB-10
PH meter	Sedolis Scientific Instruments (Beijing) Co., Ltd	2018C132-11
PI3K inhibitor LY294002	MCE	HY-10108
Pipette gun	Dragon	KE0003087/KA0056573
Protein phosphatase inhibitor complex	Meilunbio	MB12707-1
PVDF membrane (0.22 μm)	Solarbio	ISEQ00010
PVDF membrane (0.45 μm)	Solarbio	YA1701
Quick primary/secondary antibody diluent	Solarbio	A1811
Rabbit anti-AKT	Affinity	AF0836
Rabbit anti-BAD	Affinity	AF6471
Rabbit anti-C3	Affinity	DF13224
Rabbit anti-GAPDH	Hangzhou Xianzhi	AB-P-R 001
Rabbit anti-IgG	CST	3900S
Rabbit anti-Nephrin	bioss	bs-10233R
Rabbit anti-P-AKT	Affinity	AF0016
Rabbit anti-p-BAD	invitrogen	PA5-105023
Rabbit anti-PI3K	Affinity	AF6241
Rabbit anti-PIK3CA	Bioss	Bs-2067R
Rabbit anti-WT-1	Affinity	DF6331
Real-Time PCR System	ABI	QuantStudio 6
RIPA Lysis Buffer	Meilunbio	MA0151
SDS	China National Pharmaceutical Group Chemical Reagent Co., Ltd	10019318
Slide and cover glass	Jiangsu Shitai Experimental Equipment Co., Ltd	10212432C
Super pure water instrument	Zhiang Instrument (Shanghai) Co., Ltd	Clever-S15
SYBR Green Master Mix	VAZYME	Q111-02
Taq Plus DNA Polymerase	TIANGEN	ET105-02
Tissue grinder	Beautiful Wall	MB-96
Transmission electron microscope	HITACHI	HT7800/HT7700
Tris-base	Biofroxx	10019318
Trizol	Ambion	15596-026
TUNEL Cell Apoptosis Detection Kit (FITC)	Beijing Baiao Leibo	SY0475
Tween 20	China National Pharmaceutical Group Chemical Reagent Co., Ltd	30189328
Ultra micro UV visible spectrophotometer	Hangzhou Miou Instrument Co., Ltd	ND-100
Ultra thin slicer	Leica	Leica UC7
Ultrasonic cleaner	shumei	KQ- 800DE
Upright optical microscope	Nikon	Nikon Eclipse CI
Urinary Protein Test Kit	Nanjing Jiancheng Bioengineering Research Institute	C035-2
Vertical electrophoresis tank	Beijing 61 Instrument Factory	DYCZ-24DN
Vortex mixer	Wuhan Lingsi Biotechnology Co., Ltd	MX-F
Western Blocking Buffer	Solarbio	SW3010
xylene	China National Pharmaceutical Group Chemical Reagent Co., Ltd	10023418