Pharmacological Targeting The Podocyte-related Proteins And Cytoskeletal Structure With Wenyang-Huoxue-Lishui Formula Confers Protection Against Podocytopathy Induced By PNA

Objective:This study aimed to investigate the effect of warming yang serum on correlative proteins of podocytes in podocytes of mouse with puromycin-induced injury,including the Cathepsin L,transcription factor Dendrin,membrane-associated guanylate kinase inverted 2,synaptopodin and RhoA as well as the distribution of cytoskeletal protein actin and intermediate filament in the podocyte.We attemptted to explore the possible molecular mechanism underlying the therapeutic effect of warming yang serum on proteinuria in patients with nephrotic syndrome.Methods:The podocytes of mouse were incubated with 45mg/L puromycin for 12 hours.The normal control group and the serum blank control group were established and divided into:A.normal control group;B.model group;C.the Chinese medicine serum group;D.dexamethasone group(1umol/l);E.10% Chinese medicine serum group;F.20% Chinese medicine serum group.MAGI-2,synaptopodin,CatL and RhoA protein mRNA were detected by fluorescence quantitative PCR.The protein expression of MAGI-2,synaptopodin,CatL and RhoA was quantitatively observed by western blotting.F-actin was dyed with fluorescein isothiocyanate-phalloidin toobserved the expression and structural changes of the filament.Intermediate filament in cytoskeletal structure was dyed with vimentin to observe the expression and structural changes of the intermediate filament.Results:1.After puromycin aminonucleoside(PAN)-induced podocyte injury for 12 hours,the expression of CatL mRNA was significantly increased(compared with normal control group,P<0.01),the expression of synaptopodin mRNA did not change significantly(compared with normal control group,P>0.05).The expression of CatL mRNA was decreased after treatment with dexamethasone,10% and 20% of Chinese medicine(compared with model group,P<0.01).The expression of mRNA in synaptopodin didn't change(compared with model group,P>0.05).2.After puromycin aminonucleoside(PAN)-induced podocyte injury for 12 hours,the expression of MAGI-2,RhoA and synaptopodin protein decreased significantly(compared with normal group,all P<0.01),and the expression of CatL protein was significantly increased compared with that of normal group(P < 0.01).The expression of MAGI-2,RhoA and synaptopodin protein was increased after treatment with dexamethasone,10% and 20% of Chinese medicine(P < 0.01),and the expression of CatL protein was decreased(Compared with the model group,P<0.01),but did not recover to normal level(MAGI-2,RhoA,synaptopodin,CatL protein in each treatment group compared with that in the normal group,all P<0.01).3.In the model groups,by immunofluorescent staining and observed under the scanning confocal microscope,the percentage of nuclear translocation of dendrin was increased(compared with thatof normal group,P<0.01);after the podocytes were co-incubated with dexamethasone,10% or 20% Chinese medicine,the percentage of nuclear translocation of dendrin was decreased(compared with model group,P<0.01),but didn't recover to normal level(compared with model group,P<0.01).There was no significant difference in the dendrin nucleus relocation between the treatment groups(compared with each other,P>0.05).4.By immunofluorescent staining and observed under the scanning confocal microscope,the microfilament and the intermediate filament were distinct in structure and well-aligned in arrangement.Outstretched pseudopod can be seen.After puromycin aminonucleoside(PAN)-induced podocyte injury for 12 hours,the structure of podocytes became abnormality.The pseudopod retracted and shortened.The cells became around or oval.The structure and arrangement of microfilament and intermediate filament were in disorder.The microfilament and the intermediate filament recovered in dexamethasone group,10% and 20% Chinese medicine serum groups,and the cytoskeleton structure became better as compared with model group.Pseudopod appeared.Conclusion:After damage with puromycin,dendrin nucleus relocation happened;CatL level in podocytes increased;meanwhile,synaptopodin of CatL decreased.But mRNA of synaptopodin didn't change.That implied the decreasing of synaptopodin didn't result from decreasing of transcribing,but result from strengthening of synaptopodin enzymolysis.Meanwhile,dowmstream RhoA of synaptopodin decreased.Wenyang-Huoxue-Lishui formula is able to upregulate the expression of MAGI-2,and then to decrease nuclear import of dendrin,in turn to decrease CatL level,consequently toincrease synaptopodin and RhoA and stabilize the cytoskeletal structure,ultimately to decrease foot process fusion and proteinuria.