High Glucose-induced Hepertrophy Of Mesangial Cells Is Reversed By Connexin43Overexpression Via PTEN/Akt/mTOR Signaling

Backgroud and objective: Hypertrophy of glomerular mesangial cells(GMC) is one of the earliest pathological abnormalities in diabetic nephropathy,which correlates with eventual glomerulosclerosis. We have previously provedthat this hypertrophy is mediated by downregulation of connexin43(Cx43) anddysfunction of gap junctional intercellular communication (GJIC), but themechanism involved is still unclear. PTEN/Akt/mTOR signaling pathwayinvolved in the regulation of mesangial cell hypertrophy in the intracellularsignaling network. This study aims to investigate whether Akt/mTOR wasinvolved as the downstream molecular signaling of Cx43in regulating highglucose-induced GMC hypertrophy. Thus provide more basis for understandingthe pathogenesis of DN.Methods: GMC were isolated from male Wistar rats at the age of3monthand separated into3groups, cultured in normal glucose, isosmotic mannitol orhigh glucose for48h. Cell volume was analyzed by flow cytometry forwardscatter(FSC). MTT assay was used to detect cell proliferation ability. Flowcytometry was used to analyze cell cycle. Cx43mRNA expression was detectedby RT-PCR. Western blot was used for analysis of Cx43, PTEN/Akt/mTORsignaling protein expressions. Gene transfer technique was used to upregulateand silence Cx43in GMC, which was validated by RT-PCR, western blot andimmunofluorescent staining. Forward scatter of flow cytometry was examinedto testify GMC hypertrophy; western blot was performed to demonstrate thechanges of Cx43and signal protein level; flow cytometry and MTT test werecarried out to check cell cycle and proliferation rate, respectively.Results: GMC exposed to high concentration of glucose48h, cells were bigger in size, FSC average intensity increased, proliferative capacity decreased,the proportion of cells in the G0/G1phase was significantly increased butS-phase cells decreased. Cx43mRNA and protein expression were bothdecreased, PTEN expression was inhibited, triggered Akt and mTORphosphoralation, compared with two control groups these results havetatistically significant. Leading to stagnancy of cell cycle, decline ofproliferation rate and occurrence of hypertrophy. While Cx43overexpressioncould prevent Akt and mTOR phosphoralation, resulting in restoration of cellcycle and proliferation ability, and reversion of GMC hypertrophy. Andsilencing Cx43led to Akt and mTOR activation, cell proliferation abilitydecreased and enlargement of GMC.Conclusions: This study demonstrated that Akt/mTOR signalingstimulated by high concentration of glucose is regulated by Cx43overexpression, which unveils part of the molecular mechanism of Cx43inregulating hyperglycemia-induced hypertrophy.