The Protection Effects Of Adiponectin On MTOR-mediated Early Diabetic Nephropathy

PART1Adiponectin inhibits PDGF-induced Mesangial cell proliferation:Regulation of mammalian target of rapamycin-MediatedSurvival Pathway by Adenosine5-Monophosphate-Activated Protein KinaseObjective: To investigate the effects of human globular adiponectinrecombinant protein on platelet-derived growth factor-BB(PDGF-BB)induced human mesangial cells(HMCs) proliferation and intracellularsignaling pathway.Methods: HMCs were treated with or without PDGF, Rapamycin,adiponectin, and compound C(a AMPK inhibitor) at the indicatedconcentrations for the indicated periods of time. Cell proliferation wasassessed by using cell count and [3H]thymidine incorporation assay. Thenthe cells were randomly assigned into six groups:the control group, thePDGF-BB group, the adiponectin group, PDGF-BB+adiponectin group,PDGF-BB+Rapamycin group, PDGF-BB+adiponectin+compound Cgroup. The phosphorylation of PI3K, AMPKα, TSC2, mTOR, S6K1weredetected by western blot. The interaction between adiponectin and PDGFβ receptor was detected by immunoprecipitation.Results: PDGF-induced HMCs proliferation was significantlyinhibited by the co-treatment of adiponectin. Adiponectin alone had noeffect on HMC proliferation. The mTOR and S6K1were activated byPDGF stimulation in HMCs. PDGF-induced mTOR and S6K1phosphorylations were significantly attenuated by the co-treatment ofadiponectin in HMCs. Adiponectin alone had no effects onPDGF-receptor autophosphorylation by PDGF. We also confirmed thatthe inhibitory effect of adiponectin on PDGF-induced HMCs proliferationwas significantly suppressed by compound C, AMPK inhibitor.Conclusion: adiponectin inhibited PDGF-induced HMCsproliferation and could attenuate renal dysfunction associated with MCdisorders. Mechanistic insights into this phenotype suggest thatadiponectin attenuated PDGF-induced phosphorylation of mTOR andS6K1via AMPK activation. PART2Effects of recombinant lentivirus encoding human apM1gene onplatelet-derived growth factor-BB induced mesangial cellproliferation and extracellular matrix protein synthesisObjective: To investigate the effects of recombinant lentivirusencoding human apM1gene on platelet-derived growth factor inducedhuman mesangial cells(HMCs) proliferation and extracellularmatrix protein synthesis.Methods: The mRNA expression of apM1in HMCs wasdetected by RT-PCR. Protein expression of apM1in cell culturesupernatant of HMCs transfected with Lenti-apM1-EGFP was detectedby ELISA．The effects of human adiponectin on cell proliferation and cellcycle was assessed by [3H]thymidine incorporation assay and Flowcytometry respectively. The expression of type IV collagen, laminin andfibronectin were detected by western blot.Results: Lenti-apM1-EGFP had no significant toxicity on HMCs.MOI of50of the Lenti-apM1-EGFP efficiently infected HMCs, andmade it stable expression of adiponectin protein (149.6±12.8) μg/l.PDGF-induced HMCs proliferation was significantly inhibited byadiponectin. PDGF-induced type IV collagen, laminin and fibronectinsynthesis in HMCs were also inhibited by adiponectin.Conclusion: Human apM1gene can be effectively expressed inHMCs and do not influence HMCs growth. Adiponectin antagonizesstimulatory effects of PDGF-BB on HMCs proliferation and extracellularmatrix protein synthesis. From these findings, it is implied thatLenti-apM1-EGFP could attenuate renal dysfunction associated with MCs disorders. PART3Effect of lentivirus-mediated adiponectin gene transfection onstreptozocin-induced diabetic nephropathymice and its mechanismObjective: To investigate the effect of recombinant lentivirusencoding adiponectin gene on streptozocin-induced diabetic nephropathymice and and its potential mechanism.Methods: Forty C57BL/6mice were randomly divided into normalcontrol group (NC group,n=10), diabetic nephropathy group (DN group,n=10), Lenti-IRES-EGFP treatment group (DL group, n=10) andLenti-Acdc-IRES-EGFP treatment group (DA group, n=10). Mousediabetic nephropathy models were induced by high-lipids andhigh-sucrose feeding plus STZ intraperitoneal injection. After8weeks ofrecombinant lentivirus injection, kidney-to-body weight ratio(KW/BW),mean glomerular volume(MGV), fractional mesangial area(FMA),Twenty-four hours urinary protein excretion(UTP), serum creatinine(Cr),serum urea nitrogen(BUN), serum albumin and serum adiponectin weremeasured. The renal pathological changes were evaluated by Light microscopy and electron microscopy respectively. Proliferation ofglomerular and tubulointerstitial cells was assessed by using an antibodyagainst PCNA by immunohistochemistry. The expression of p-AMPKαand p-mTOR in mouse kidneys were detected by immunohistochemistry.Results: we successfully used a lentivirus to overexpressadiponectin in STZ-induced diabetic nephropathy mice. KW/BW ratio,MGV, FMA and UTP were significantly decreased in the mice of DAgroup compared to those of DN group and DL group(P<0.05). DA groupanimals had significantly fewer PCNA-positive cells per view field thanDN group and DL group(P<0.01). DA group animals had a higherp-AMPK levels and a lower p-mTOR levels compared to that of DNgroup and DL group(P<0.01).Conclusion: Over-expression of adiponectin has beneficial effectson early stage diabetic nephropathy and mechanistic insights into thisphenotype suggest that adiponectin attenuated aberrant proliferation ofrenal cells.