Flounce Of Different Concentrations Of Insulin On Phenotypic Transformation Of HK-2

Objective:To study the effects of different concentrations of insulin on the phenotypic transformation of human kidney proximal tubular epithelial cell (HK-2).Methods: The HK-2 cells were cultured in vitro with PRMI-1640 medium supplemented with 10% fetal bovine serum, and seeded on 24 plastic plates to approximately 60% to 70% confluence, and then shifted to serum-free medium for 12 hours. These cells were divided into four groups: Control group(CG): The HK-2 cells were treated respectively with free serum medium. Normal concentration group (NG): The HK-2 cells were cultured in the concentration (5mU/L) of insulin. High concentration group (HG): The HK-2 cells were cultured in the concentration (125mU/L) of insulin. Super - concentration group (SG): The HK-2 cells were cultured in the concentration (400mU/L) of insulin. Cells were observed the morphological change by inverted microscope and electronic microscopy. Immunocytochemistry was used to examine the expression of E-cadherin andα-SMA. The expression of E-cadherin mRNA andα-SMA mRNA in 72h were detected by RT-PCR. The concentrations of fibronection and type Ⅰcollagen in culture supernatant were detected with ELISA.Results:1. Inverted microscope observation: The shape of HK-2 cells showed a classic cobblestone morphology at 24h, 48h and 72h in control and normal group. Hyperinsulinemia induced morphological changes, with cells becoming elongated in shape, disassociating from neighboring cells, and losing their cobblestone monolayer pattern in high concentration and super - concentration group.2. Electronic microscopy observation: Cells were full of microvilli, with little rough endoplasmic reticulum in control and normal group. Microvilli decreased, rough endoplasmic reticulum increased in high concentration and super - concentration group.3. Immunocytochemistry: E-cadherin was positive expression in control and normal group, the expression of E-cadherin in high concentration and super - concentration group were also positive, but gradually attenuated. Insulin (125mU/L and 400mU/L) decreased the expression of E-cadherin in HK-2 cells at 24h 48h and 72h.α-SMA was negative expression in control and normal group, while insulin (125mU/L and 400mU/L) inducedα-SMA expression at 48h and 72h.4. RT-PCR: There were certain extent expressions of E-cadherin mRNA in control and normal group so did high concentration and super - concentration group, but the brightness of expressions of E-cadherin mRNA in high concentration and super - concentration group were gradually descended. The expression ofα-SMA mRNA was negative in control and normal group, while insulin (125mU/L and 400mU/L) inducedα-SMA mRNA expression.5. ELISA: The concentrations of Collagen TypeⅠand Fibronectin in culture supernatant treated with insulin (125mU/L and 400mU/L) were more notable than those treated with insulin (5mU/L) or no insulin. Meanwhile insulin may time-dependently improve the accumulation of Collagen TypeⅠin high concentration and super - concentration group, accumulation of Fibronectin in each group.Conclusion: Hyperinsulinemia (≥125mU/L ) caused phenotypic transformation of HK-2 , which concerned with the time and concentration of insulin,conclude follow aspects:1.Hyperinsulinemia (≥125mU/L ) caused the cell shape changed, meanwhile the alteration of cell ultrastructure happened.2.Hyperinsulinemia (≥125mU/L ) decreased the expression of E-cadherin in HK-2 cells ,while inducedα-SMA expression.3.Hyperinsulinemia (≥125mU/L ) dose-dependently and time-dependently improve the accumulation of Collagen TypeⅠand Fibronectin in culture supernatant.