The Role And Significant Of PI3K/AKT Pathway On Epithelial-mesenchymal Transdifferentiation Of HKCs Promoted By TGF-β₁

Objective: To investigate the possible role and mechanisms of PI3K/Akt pathway during the process of Epithelial-mesenchymal transdifferentiation of HKCs(human kidney proximal tubular epithelial cell line,HKCs)promoted by transforming growth factorβ1 (TGF-β1) .Methods: The HKC cells were cultured in vitro with Dulbecco's modified Eagle's medium/F12 medium supplemented with 10% fetal bovine serum, and seeded on plastic plates to approximately 60% to 70% confluence, and then shifted to serum-free medium for 12 hours. These cells were divided into three groups:①Control (C) group:The HKC cells were treated respectively with free serum medium.②TGF-β1 (T) group:The HKC cells were cultured in the different concentration (5,10,15,20ng/mL) of TGF-β1.③TGF-β1 + PI3K inhibitor wortmannin group(T+W):The HKC cells were co-incubated TGF-β1 (10ng/mL) and wortmannin (10nmol/L) with free serum medium. Cells were observed the morphological change by microscope, the expression of total– Akt(t-akt) and phosphor-Akt (p-Akt) was assessed by western blot. At the time ,the expression of E-cadherin, alpha-smooth muscle actin(α-SMA) were detected by reverse transcription polymerase chain reaction(RT-PCR) and western blot assays., Indirect immunofluorescence andr immunocytochemistry. were also used at dectecting the expression of E-cadherin,α-SMA and p-AktResults: 1.Cell shape observation:①C group: The shape of HKC cells showed a classic cobblestone morphology.②T group: 10,15,20ng/ml TGF-β1 induced profound morphologic changes, with cells becoming elongated in shape, disassociating from neighboring cells, and losing their cobblestone monolayer pattern ; But incubated with 5ng/ml TGF-β1, only a few cells become elongated, the others maintained cobblestone morphology.③T+W group: with PI3K inhibitors wortmannin, TGF-β1 could not induce HKC cells morphologic changes, cells showed a classic cobblestone morphology.