Effects Of IGF-1 On Human Renal Tubular Epithelial Cells Transdifferentiation Mechanism

Objective: To investigate the possible role and mechanisms of PI3K/Akt pathway during the process of Epithelial-mesenchymal transdifferentiation of human renal tubular epithelial cells(HKCs) promoted by insulin-like growth factor I(IGF-1).Methods: HKCs were incubated with Dulbecco’s Modified Eagle Media: Nutrient Mixture F-12 medium supplemented with 10% fetal bovine serum in vitro, and seeded on plastic plates to approximately 60% to 70% confluence, and then shifted to free serum medium for 24 hours. These cells were divided into three groups: ①Control group: The HKCs were cultured with 10% FBS DMEM/F12. ②IGF-1(50ug/L) group: The HKCs were treated with 50ug/L IGF-1.③IGF-1+PI3K/Akt inhibitor LY294002 group: The HKCs were co-incubated IGF-1(50ug/L) and LY294002(15 umol/L). According to the above were cultured 24 h, 48 h for 72 h respectively. The expression of a-SMA 、 Akt and p-Akt proteins were assessed by westernblotting(WB). After pre-treated with different doses of LY294002(5、15、20 umol/L)respectively for one hour, and then adding the same concentration of IGF-1(50ug/L) for 48 hours. The morphological changes of cells were observed under inverted phase contrast microscope. The expression of a-SMA and p-Akt m RNA was measured by Reactive-transcription polymerase chain reaction(RTPCR). DAPI stain was used to detect the effects on cells apoptosis.Results:(1)The results of WB showed that when the 50ug/L IGF-1 at different times, the expression of a-SMA protein was up-regulated(P<0.05), Akt protein was up-regulated(P <0.05), and p-Akt protein was up-regulated(P<0.05). All of them were in a time-dependent manner.(2) Compared with the IGF-1(50ug/L) alone, IGF-1(50ug/L) and LY294002(15umol/L) co-interaction, the expression of a-SMA protein was down-regulated(P<0.05), Akt protein was down-regulated(P<0.05), p-Akt protein was down-regulated(P<0.05). Both of them were in a time-dependent manner.(3) HKCs were incubated with IGF-1(50ug/L), the expression of a-SMA m RNA was up-regulated(P <0.05), and p-Akt protein was up-regulated(P<0.05).(4) The results of RT-PCR showed that different concentrations of LY294002 could make the expression of a-SMA m RNA down-regulated(P<0.05), and p-Akt m RNA down-regulated(P<0.05).Both of them were in a dosedependent manner.(5) IGF-1 stimulated the morphlolgical oval-to-fusiform transdifferentiation of the cells, and increased the cell proliferactive activity in HKCs cells. LY294002 could inhibit these changes.(6)The results of DAPI showed that IGF-1 can inhibit apoptosis and increase the cell proliferactive activity in HKCs cells. But LY294002 induce apoptosis, all of them were in a dose- and time-dependent manner.Conclusions:(1)IGF-1 can induce PI3K/Akt activated in HKCs, further promote the transdifferentiation of the human renal tubular epithelial cells, and involved in the occurrence and development of diabetic nephropathy.(2) The pathway of PI3K/Akt was involved in EMT of HKCs promoted by IGF-1, PI3K/Akt is a IGF-1 downstream mediator in EMT.(3) LY294002, is one kind of specific inhibitors of PI3K/Akt, cansignificantly inhibit EMT induced by IGF-1. It suggests that LY294002 may play an anti-fibrosis effect of kidney protection.