Tubulointerstitial expression of thrombosondin-1 in 5/6 renal ablation rats

Objective:Progressive tubulointerstitial fibrosis is proved to be assoicated with tubulointerstitial expression of TGF-β_1 and thrombosondin-1(TSP1). In this study, we established rat model of 5/6 renal ablation, and examined the expression of TGF-β_1 TSP1 in the tubulointerstitial area. We futher explored the effects of angiotensin II receptor blocker on the mRNA and protein level of TGF-β_1 and TSP1 expression in vitro HK-2 cell line. Methodology:Progressive tubulointerstitial fibrosis was induced in rats by 5/6 renal ablation and sham-operated rats set as the control. The expression of TSP1, TGF-β_1, fibronectin ( FN, an extracellular matrix component) was determined by immunohistochemistry or immunofluorescence at multiple time points(1 week, 4 weeks, 8 weeks and 12 weeks after disease induction). Co-localization of TSP1/TGF-β_1 was observed by double-label staining analysis. The mRNA level of TSP1, TGF-β_1 and FN in each group was followed by RT-PCR. Using cultured human tubular epithelial cell line (HK-3), we examined the effects of angiotensin II receptor blocker(losartan) on TSP-1/ TGF-β_1 mRNA and protein level undergoing angiotensin II stimulation. FN expression was detected by RT-PCR, immunofluorescence and Western blot. Active and total levels of TGF-β_1 in supernatants were measured by ELISA. Results:De novo expression of TSP1 was observed in tubular cells, myofibroblasts and some macrophages in areas of tubulointerstitial injury, while not nor even rarely in sham control rats. Increased TSP1 expression was sustained during the development of tubulointerstitial fibrosis, and frequently localized at sites of increased expression of TGF-β_1. The mRNA level of TSP1、TGF-β_1 in 5/6 renal ablation rats were up-regulated 1.94 folds and 1.97 folds comparing with control. Increased TSP1 expression was correlated closely with the up-regulated TGF-β_1, renal function loss and the development of tubulointerstitial fibrosis. In vitro study showed that angotensin II enhanced mRNA and protein levels of TSP1, TGF-β_1 and FN in HK-2 cell, while pretreatment with losartan prevented the up-regulated production of TGF-1,TSP1 and FN in HK-2 cells as well as the active and total level of TGF-β_1 in culture supernatants. Conclusion:In the remnant kidney model, the presence and location of TSP1 expression induced by angiotensin II are consistent with the expression of TGF-β_1. TSP1 correlated well with progressive renal function loss. Angiotensin II receptor blocker prevented the up-regulated TSP1 expression in the area of renal tubulointerstitial fibrosis, and may furthermore down-regulated ECM over-deposition.