Jianpi Qushi Heluo Formula Up Regulates Mitochondrial Autophagy And Reduces Podocyte Apoptosis In Rats With Passive Heymann Nephritis

Idiopathic membranous nephropathy(IMN),recognized as the leading cause of adult nephrotic syndrome,has an ever-increasing incidence rate.Worse still,1/3 IMN patients may progress into end-stage renal disease within 5-10 years of disease onset even with active treatment.Therefore,novel and safe strategies for IMN treatment are urgently needed.Persistent massive proteinuria is a risk factor for renal function progression in IMN.Glomerular filtration barrier and proteinuria are mainly caused by podocyte injury or depletion.On the other hand,podocyte apoptosis is validated as the main form of podocyte injury.Hence,it is of great significance to reduce podocyte apoptosis for the treatment of IMN proteinuria.Jianpi qushi heluo formula(JQHF)helps to reduce proteinuria and delay the progress of renal function,showing a strong clinical effect.However,whether JQHF reduces proteinuria by inhibiting podocyte apoptosis and improving glomerular filtration membrane injury remains to be elucidated.Additionally,IMN is mainly pathologically characterized by the deposition of immune complexes in the glomerulus.Immune complex can activate complement system to produce the sub soluble C5b-9,which then inserts into podocyte membrane,resulting in local ROS production.When ROS accumulation exceeds the antioxidant capacity of mitochondria in cells,the oxidative stress induces mitochondrial dysfunction,followed by mitochondria-mediated apoptosis.Mitochondrial autophagy is important to degrade and recover the injured and redundant mitochondria,thereby helping to maintain cell survival.There are three main regulatory pathways of mitochondrial autophagy in mammals:PINK1/Parkin pathway,BNIP3L pathway and FUNDC1 pathway.Among them,the activation of PINK1 is considered to be the initial event that induces mitochondrial autophagy,and it plays an important role in the process of mitochondrial autophagy.In this experiment,we started to study the regulation mechanism of PINK 1/Parkin pathway on cell apoptosis.This study aimed to investigate whether JQHF exerted protective effects on renal function by clearing the injured mitochondria and reducing podocyte apoptosis through PINK1/Parkin-mediated mitochondrial autophagy.The main contents are as follows.ObjectiveIn this study,a rat model of passive Heymann nephritis(PHN)was established to simulate the mechanism of renal pathological injury in human IMN,with the purpose to explore;1.The renal protective effect of JQHF on PHN rats;2.The effect of JQHF on mitochondrial dysfunction and podocyte apoptosis in PHN rats;3.The mechanism of JQHF in protecting podocytes from apoptosis by regulating the PINK1/Parkin mitochondrial autophagy pathway.MethodsThe PHN model was established using sheep anti-rat FX1A serum.Rats were allocated into the model group,benazepril hydrochloride group,JQHF groups(low-dose/high-dose),JQHF(low dose)+3-MA group,and blank group.Rats in each group were given the relevant drugs by intragastric administration.After drug intervention at 0,2,4 and 6 weeks,the 24h urine samples were collected,and serum and after drug intervention at 6 weeks,the serum and renal tissue samples of rats were collected.The detection indicators and methods are as follows.1.Serum and urine sample detection:24h urinary total protein(24h UTP)quantitation,ALB,TC,TG,AST,ALT,BUN and Scr were detected using the automatic biochemical analyzer.2.Renal pathology detection:IgG and C5b-9 depositions in glomerulus were observed by immunofluorescence staining;the pathological changes of rat’s kidney were observed by electron and light microscope,and HE,PAS and Masson staining.3.Mitochondrial function detection:the ultrastructural changes of mitochondria were observed by electron microscope;mitochondrial membrane potential and intracellular ROS level were detected using flow cytometry.4.Apoptosis detection:glomerular and renal tubular cell apoptosis were detected by TUNEL;the expression of podocyte marker proteins(WT-1 and Nephrin)was detected by immunohistochemistry.5.Detection of the PINK 1/Parkin pathway-related proteins:the co-localization of PINK1-Parkin and COX Ⅳ-LC3 in glomerulus was detected by immunofluorescence staining;the protein expressions of PINK1,LC3-Ⅱ/Ⅰ,Cleaved caspase-3,Cyt c,Cyto-Parkin and Mito-Parkin in renal tissues were detected using Western bolt.Results:1.JQHF had renal protective effects on PHN rats.(1)Comparison of 24h UTP:after administration at 6 weeks,rats in the model group showed a notably increased 24h UTP as compared to that before administration;the 24h UTP of rats in the JQHF low-dose group was obviously decreased compared with those in the model group(P<0.01),benazepril hydrochloride group(P<0.05)and JQHF+3-MA group(P<0.05).Rats in the JQHF high-dose group also exhibited decreased 24h UTP relative to those in the model group(P<0.01).(2)Comparison of renal function,blood lipid and liver function:decreased ALB and increased TC were observed in rats in the model group compared with those in the blank group;ALB and TC of rats in the JQHF low-dose group were significantly improved,relative to those in the model group(P<0.01,P<0.05),while combination with 3-MA dramatically inhibited the improving effects of JQHF on ALB(P<0.01).There was no significant difference in TG,Scr,ALT and AST among the groups.BUN in JQHF low-dose and high-dose groups was decreased(P<0.05,P<0.01),as compared to that in the model group,with no difference between the other groups.(3)Comparison of renal pathological injury:compared with those in the blank group,rats in the model group exhibited an aggravated renal pathological injury;relative to those in the model group,the renal tubulointerstitial injury score,increased glomeruli numbers and IgG deposition were significantly improved in the JQHF low-dose and high-dose groups,while C5b-9 deposition was only notably reduced in the JQHF low-dose group(P<0.01).The JQHF low-dose group had a remarkably reduced renal tubulointerstitial injury score and glomeruli numbers compared with the benazepril hydrochloride group(P<0.05,P<0.05).The 3-MA+JQHF group exhibited aggravated renal pathological injury relative to the JQHF low-dose group.The above indexes were significantly different.2.The effect of JQHF on mitochondrial dysfunction and podocyte apoptosis in PHN rats.(1)Mitochondrial ultrastructure and membrane potential:compared with that in the blank group,the mitochondria were swelled and became round,and thickened outer membrane,vacuolization,cristae vague,disordered arrangement,and decreased mitochondrial membrane potential were found in podocyte in the model group(P<0.01).The ultrastructure of mitochondria in each drug treatment group was improved in varying degrees as compared to the model group.In the JQHF low-dose and high-dose group,the cristae structure was basically normal and the mitochondrial membrane potential was remarkably increased(P<0.01,P<0.05).3-MA obviously inhibited the improving effects of JQHF on mitochondrial ultrastructure and membrane potential.(2)Intracellular ROS level:the model group showed a notably higher intracellular ROS level than the blank group.However,the ROS level was obviously decreased in the benazepril hydrochloride group,JQHF low-dose and high-dose groups(P<0.05,P<0.001,P<0.01)compared with the model group.JQHF low-dose showed a better effect on ROS scavenging than benazepril hydrochloride and 3-MA+JQHF(P<0.05,P<0.001).(3)Apoptosis of glomerular and tubular cells:the apoptosis rates of renal tubular and glomerular cells in the model group,benazepril hydrochloride group and 3-MA+JQHF group were significantly increased(P<0.001,P<0.05,P<0.01)compared with that in the blank group.No significant difference was found between JQHF low-dose and high-dose groups.The JQHF low-dose group exhibited a notably lower apoptosis rate of renal glomerular cell than the model group(P<0.05).(4)Number of podocytes and expression of marker proteins:the number of podocytes in the model group was obviously decreased relative to that in the blank group.The benazepril hydrochloride group and JQHF low-dose and high-dose groups showed remarkably increased number of podocytes as compared to the model group(P<0.001).The number of podocytes in the JQHF group was also notably increased(P<0.001)compared with the benazepril hydrochloride group and the 3-MA+JQHF group.In terms of Nephrin protein expression,compared with the model group,the expression of Nephrin was noticeably increased in the JQHF low-dose group,while 3-MA inhibited Nephrin upregulation by JQHF.3.JQHF protected podocytes from apoptosis by regulating the PINK1/Parkin mitochondrial autophagy pathway(1)Expression of PINK1 and Parkin protein:PINK1 and Mito-Parkin protein expressions were decreased(P<0.001,P<0.05),Cyto-Parkin expression was increased(P<0.01),and the PINK1-Parkin fluorescence co-localization was decreased(P<0.001)in the glomerulus of rats in the model group relative to those in the blank group.PINK1,Mito-Parkin and Cyto-Parkin expressions and PINK1-Parkin co-localization were dramatically improved in the benazepril hydrochloride group,and JQHF low-dose and high-dose groups,compared with the model group,with JQHF low-dose showing better improving effects than benazepril hydrochloride.3-MA could inhibit autophagy and block JQHF’s regulation in PINK1 and Parkin.(2)Expressions of LC3 and COX Ⅳ proteins:relative to that in the blank group,the co-localization of LC3-COX Ⅳ in glomeruli of the model group and JQHF+3MA group was significantly decreased.The JQHF low-dose group showed increased LC3-COX IV co-localization compared with the model group(P<0.05).The LC3-II/I ratio in the model group was significantly lower than that in the blank group;the LC3-II/I ratio in the JQHF low-dose and high-dose groups was obviously higher than that in the model group and benazepril hydrochloride group(P<0.001,P<0.001).3-MA suppressed the regulation of LC3-Ⅱ/Ⅰ ratio by JQHF.(3)Expression of Cleaved caspase-3 and Cyt c:the expression of Cleaved caspase-3 and Cyt c in the model group was noticeably increased compared with the blank group(P<0.001,P<0.001).The expression of Cleaved caspase-3 and Cyt c was remarkably decreased in the benazepril hydrochloride group,and JQHF low-dose and high-dose groups relative to that in the model group.JQHF low-dose exhibited a better regulatory effect than benazepril hydrochloride and 3-MA+JQHF.Conclusion(1)JQHF can reduce 24h UTP and increase serum ALB in PHN rats,showing a better pharmacodynamic effect than benazepril hydrochloride.JQHF can reduce renal pathological injury,as manifested by reducing IgG and C5b-9 immunofluorescence deposition in glomerulus,alleviating the interstitial injury of renal tubule and improving the increase of cells in glomerulus.(2)ROS accumulation and mitochondrial dysfunction exist in the podocytes of PHN rats.JQHF can effectively reduce ROS accumulation,improve the mitochondrial ultrastructure in the podocytes,inhibit the decline of mitochondrial membrane potential,and decrease the apoptosis of podocytes and renal tubular cells.(3)JQHF can significantly upregulate PINK1 protein expression in glomerulus,promote cytoplasmic Parkin transfer to mitochondria,increase LC3-Ⅰ transformation to LC3-Ⅱ,and LC3-Ⅱ combination with mitochondria,and improve the intracellular mitochondrial autophagy flow,thereby downregulating the expression of apoptosis controlling factors(Cyt c and cleaved Caspase-3)and reducing podocyte apoptosis.(4)3-MA can remarkably block the activation of the PINK1/Parkin pathway by JQHF,and inhibit the improving effects of JQHF on podocyte apoptosis,24h UTP and renal pathological injury in PHN rats.It is confirmed that JQHF improves mitochondrial dysfunction and promotes the clearance of injured mitochondria via activating the PINK1/Parkin pathway-mediated mitochondrial autophagy,so as to inhibit the release of apoptosis factors(Cyt c and cleaved Caspase-3),thereby suppressing podocyte injury,reducing proteinuria and exerting renal protective effects.