Transdifferentiation Of Cultured Human Renal Tubular Epithelial Cells And The Expression Of Integrin-linked Kinase Induced By High Glucose

Background: Tubulointerstitial lesion is an essential pathological basis in the development of diabetic nephropathy (DN) to end-stage renal disease (ESRD), the basic pathological characteristic of which is excessive extracellular matrix (ECM) accumulation in the renal interstitium. Currently, it is considered that the renal interstitial ECM is mainly secreted by the myofibroblast (MyoF). Researches have shown that tubular epithelial-myofibroblast transdifferentiation (TEMT) can occur to the renal tubular epithelial cells in pathological condition. During TEMT, renal tubular epithelial cells lose their original phenotypic characteristics and gain characteristics of MyoF, which is a principal source of renal interstitial MyoF. Multitude in vivo and in vitro researches indicated that TEMT participated in the progression of renal interstitial fibrosis in many chronic renal diseases. Our studies have showed that in diabetic rats TEMT was observed in tubulointerstitial fibrosis. However, it is unknown whether high glucose can induce TEMT.TGF-β1 is an essential factor in the initiation and promotion of EMT. The biological functions of TGF-β1 are extensive and complex, which is shown through the diversity of positive and negative effects. Inhibiting only TGF-β1 as an upstream factor could bring negative side effects together with positive side effects. Therefore, researchers' attention has been turned to a new hot spot: looking for more specific and effective downstream factors or segments. Integrin-linked kinase(ILK) is a multifunctional cytoplasmic serin/threonine protein kinase identified in 1996. ILK, a key downstream effector molecule of TGF-β1, played an important role in TEMT and was the one of the major regulatory factors both in TEMT and in fibronectin(FN) accumulation. Our studies have showed that expression of ILK increased in renal tubular epithelial cells of diabetic rats. It is also unknown whether high glucose can induce ILK expression in vitro.Objective: To observe TEMT and expression of ILK in HK-2 induced by high glucose in vitro. To approach the role and mechanism of ILK in TEMT.Methods: HK-2 cells were cultured in vitro and induced by high glucose culture fluid (30mmol D-glucose), compared with low glucose culture fluid (5.5mmol D-glucose) and manicol culture fluid(24.5mmol D-manicol + 5.5mmol D-glucose). Shape of all cells was observed through inverted phase contrast microscope. At Oh, 12h, 24h, 48h, 72h disposal cells were collected and total protein was extracted. Experiment of each group was repeated for three times. Protein expressions of ct-SMA, E-cadherin, ILK and FN in HK-2 were detected by Western Blotting.Results: 1. Protein expression of E-cadherin was high in group N and group M and was elevated with no difference (P>0.05) in five time points from Oh to 72h. Protein expression of E-cadherin decreased with time gradually, reached perigee in 72h and was time-dependent in group H. 2. In group N and group M, HK-2 had no expression ofα-SMA nearly and expression of FN was low. They were elevated with no difference (P>0.05) in five time points. In group H expression ofα-SMA and ILK increased with time gradually , reached peak in 72h and were time-dependent. 3. Expression of ILK was very low, protein expression in five time points was elevated with no difference (P>0.05) from Oh to 72h in group N and group M. Protein expression of ILK increased with time gradually, reached peak in 72h and was time-dependent in group H. In group H, there were direct correlation not only between protein expression of ILK andα-SMA(r=0.84, P<0.01), but also between protein expression of ILK and FN(r=0.93, P<0.01). There were inverse correlation between protein expression of ILK and E-cadherin(r = -0.85, P<0.01) in group H.Conclusions: 1. In HK-2 induced by high glucose, expression of E-cadherin decreased and expression ofα-SMA and FN increased in time-dependent manner, indicating that high glucose, as an independent factor, can induce tubular epithelial-myofibroblast transdifferentiation in vitro. 2. Expression of ILK was up-regulated in HK-2 induced by high glucose, which had inverse correlation with E-cadherin, and had direct correlation withα-SMA and FN, implying that ILK may participate and play a positive control role in the process of TEMT.