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

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.