Empirical Study Of The Transdifferentiation In Renal Tubular Epithelial Cells In Diabetic Nephropathy And Effect Of Rosiglitazone

Empirical Study of the Transdifferentiation in Renal Tubular Epithelial Cells in Diabetic Nephropathy and Effect of RosiglitazoneObjective: In this study, we found the model in vitro of human proximal renal tubular epithelial cells (HK-2) stimulated by high glucose and in vivo of diabetic nephropathy rats to observe the changes in the morphology and proliferation of renal tubular epithelial cells, the correlated marker proteins in epithelial- mesenchymat transition (EMT), and the effect of rosiglitazone(RGZ) on HK-2 stimulated by high glucose and renal tubules in diabetic nephropathy rats, and to explore the pathway by which rosiglitazone took effect on EMT. By investigating how high glucose as a pathological factor affected in the process of EMT and the molecular mechanism of PPARΥexcitomotor treating diabetic nephropathy, it possibly provides a new strategy and theoretical basis for treating diabetic nephropathy(DN).Methods: in vitro experiment. The third generation cell line of HK-2 were randomly divided into five groups, normal control group (N Group) cultured in 5.5mmol/L glucose, high glucose group (H Group) cultured in 25mmol/L glucose, high osmotic pressure group (M Group) cultured in 5.5mmol/L glucose and 19.5mmol/L mannitol, high glucose DMEM and low concentration RGZ group (HR5 Group) cultured in 25mmol/L glucose and 5μmol/L rosiglitazone, high glucose DMEM and high concentration RGZ group(HR10) cultured in 25mmol/L glucose and 10μmol/L rosiglitazone. Cell proliferation was examed by MTT, and the morphology was observed by light microscopy and electron microscopy. The expression of mRNA and protein level of FSP 1,α-SMA,E-cad were tested by RT-PCR and Western blot respectively. The concentration of TGF-β1 in supematant liquid of the cells was examed by ELISA.in vivo experiment: Eighteen clean Wistar male rats were randomly divided into three groups, normal control group (N Group), diabetic nephropathy model group (D Group) and rosiglitazone(Avandia) treating group (R Group) with six rats each. Urine protein was quantitated by biuret method, urinary NAG enzyme activity was examed by nitrophenol colorimetric method, serum creatinine (Scr) and urine creatinine of twenty four hours were checked by automatic biochemistry analyzer. The nephridial tissue were collected for HE and MASSON staining, and immunohistochemically stained for FSP1,α-SMA, Col I and CK-18. RT-PCR was applied to check the mRNA expression level of FSP1,α-SMA and CK- 18.Results: in vitro experiment. (1) Compared with N Group,the cells in H Group showed: fusiform shape; under electron microscope, rough endoplasmic reticulum(RER) significantly increased, mitochondria and microvilli decreased, and muscle microfilament emerged; the mRNA and protein expressions of FSP1 and a-SMA increased (P＜0.01), but those of E-cad decreased (P＜0.01); the concentration of TGF-β1 in supernatant liquid of these cells increased (P＜0.01). (2) Compared with H Group, the cells treated with RGZ displayed: polygon and round; under electron microscope, mitochondria and microvilli increased and RER decreased; the mRNA and protein levels of FSP1 and a-SMA decreased (P＜0.05 or 0.01), but the mRNA and protein levels of E-cad increased (P＜0.01); the concentration of TGF-β1 in supematant liquid of these cells decreased (P＜0.05 or 0.01), and high concentration RGZ had more significant effect on the changes of mRNA and protein of FSP1,α-SMA,E-cad (P＜0.01) and the decrease of the concentration of TGF-β1 (P＜0.01). (3) Compared with N Group, the cells proliferation in H Group was faster, and the quantity of cell proliferation was 1.19 times at 24h, 1.31 times at 48h and 1.51 times at 72h of N Group (P＜0.05 or 0.01). Compared with H Group, cells proliferation was inhibited when treated with RGZ (P＜0.05 or 0.01). High concentration RGZ had more significant inhibitory effect on cell proliferation than low concentration at 48h and 72h (P＜0.05).in vivo experiment: (1) Compared with N Group, the rats in D Group showed: urine protein, urine NAG enzyme activity and Scr significantly increased, and Ccr decreased (P＜0.01); destructure of renal tubules and interstitial fibrosis were obvious; the expressions of mRNA and protein of FSP1, a-SMA increased (P＜0.01), and those of CK- 18 decreased (P＜0.01), the expression of protein of Col I increased (P＜0.01). (2) Compared with D Group, the rats in R Group displayed: urine protein, urine NAG enzyme activity and Scr decreased, and Ccr increased (P＜0.01); pathologic changes of renal tubules and interstitial fibrosis were alleviated; the expressions of mRNA and protein of FSP1, a-SMA decreased (P＜0.01), and those of CK-18 increased (P＜0.05 or 0.01), the expression of protein of Col I also significantly decreased (P＜0.05).Conclusions: (1) HK-2 stimulated by high glucose and diabetic nephropathy rats both displayed transdifferentiation of renal tubular epithelial cell, which indicated EMT in DN. (2) The changes of biochemistic indexes in blood and urine, and Col I of nephridial tissue indicated EMT played an important role in occurrence and development of renal fibrosis. (3) Rosiglitazone could reverse EMT in high glucose stimulated HK-2 and diabetic nephropathy rats to alleviate renal fibrosis and protect renal function. (4) EMT of HK-2 under high glucose were possibly correlated with the increase of TGFβ1, and rosigtitazone may inhibit TGFβ1 to prohibit the development of EMT.