WISP1 silencing confers protection against epithelial-mesenchymal transition of renal tubular epithelial cells in rats via inactivation of the wnt/ß-catenin signaling pathway in uremia.

Uremia can affect hepatic metabolism of drugs by regulating the clearance of drugs, but it has not been clarified whether gene silencing could modulate the epithelial-mesenchymal transition (EMT) process in uremia. Hence, we investigated the effect of WISP1 gene silencing on the renal tubular EMT in uremia through the wnt/ß-catenin signaling pathway. Initially, microarray-based gene expression profiling of uremia was used to identify differentially expressed genes. Following the establishment of uremia rat model, serum creatinine, and urea nitrogen of rats were detected. Renal tubular epithelial cells (TECs) were transfected with shRNA-WISP1 lentivirus interference vectors and LiCI (the wnt/ß-catenin signaling pathway activator) to explore the regulatory mechanism of WISP1 in uremia in relation to the wnt/ß-catenin signaling pathway. Then, expression of WISP1, wnt2b, E-cadherin, α-SMA, c-myc, Cyclin D1, MMP-2, and MMP-9 was determined. Furthermore, TEC migration and invasion were evaluated. Results suggested that WISP1 and the wnt/ß-catenin signaling pathway were associated with uremia. Uremic rats exhibited increased serum creatinine and urea nitrogen levels, upregulated WISPl, and activated wnt/ß-catenin signaling pathway. Subsequently, WISP1 silencing decreased wnt2b, c-myc, Cyclin D1, α-SMA, MMP-2, and MMP-9 expression but increased E-cadherin expression, whereas LiCI treatment exhibited the opposite trends. In addition, WISP1 silencing suppressed TEC migration and invasion, whereas LiCI treatment promoted TEC migration and invasion. The findings indicate that WISP1 gene silencing suppresses the activation of the wnt/ß-catenin signaling pathway, thus reducing EMT of renal TECs in uremic rats.

[Significance of imbalance between matrix metalloproteinases and tissue type inhibitor of metalloproteinases in renal tubulointerstitial lesions of aging rats].

OBJECTIVE: To explore the imbalance between the expression of metalloproteinases (MMPs) and that of tissue type inhibitors of metalloproteinase (TIMPs) in the renal tubulointerstitial lesions of aging rats and the potential role of this imbalance. METHODS: Forty-eight male 26-month-old Wistar rats were randomly divided into 2 groups of 24 rats: unilateral ureteral obstruction (UUO) group with the left ureter ligated and excised and false operation group used as control group. Forty-eight male 3-month-old Wistar rats were randomly divided into 2 groups of 24 rats: UUO group and false operation group just as in the 26-month-old rats. Every group was randomly divided into 3 subgroups of 8 rats: 3-day group, 7-day group, and 14-day group to be killed 2, 7, and 14 days after the operation respectively. The 2 kidneys of each rat were taken out. Routine pathological examination and immunofluorescence (IF) examination were made to calculate the area of renal tubulointerstitial fibrosis and the expression of IV type collagen. RT-PCR and Western blotting were used to detect the expressions of mRNA and protein of TIMP-1, TIMP-2, MMP-2, and MMP-9 in the kidney at different time points. Gelatin zymography was used to detect the proteolytic activity of MMP-2 and MMP-9. RESULTS: The renal interstitial lesion was more significantly in the 26-month-old UUO rats than in the 3-month-old UUO rats since the 3rd day after operation. Both the expression of MMP-2 mRNA and the expression of MMP-9 mRNA were not different between the 2 control groups. In the control groups, TIMP-1 and TIMP-2 Both MMP-2 mRNA and MMP-9 mRNA were expressed in both control groups, without significant differences between these 2 control groups. TIMP-1 mRNA and TIMP-2 mRNA were only weakly expressed in the renal tissues of the 3-month-old control group, however, the expressions of both TIMP-1 and TIMP-2 were significantly stronger in the 16-month-old control group than in the 3-month-old control group. The expressions of TIMP-1 mRNA and TIMP-2 mRNA at different time points were significantly stronger in the obstructed side than in the healthy side in the UUO groups, and significantly stronger in the 26-month-old rats than in the 3-month-old rats. MMP-2 protein and MMP-9 protein were expressed in the 3-month-old and 26-month-old controls without difference between them. The expressions of MMP-2 protein and MMP-9 protein at different time points were significantly stronger in the obstructed side than in the healthy side in the UUO groups, without significant difference between the 26-month-old UUO rats and the 3-month-old UUO rats. The expressions of TIMP-1 protein and TIMP-2 protein were very weak at different time points in the renal tissues of the 3-month-old controls and were significantly stronger in the 26-month-old control rats. In the UUO rats the expressions of TIMP-1 protein and TIMP-2 protein in the renal tissues of the obstructed side at different time points were all significantly stronger for both age groups in comparison with the control groups of the same age and were significantly stronger in the 26-month-old UUO rats than in the 3-month-old UUO rats without significant difference between the 26-month-old UUO rats and the 3-month-old UUO rats. The MMP-2 activity and MMP-9 activity of the 26-month-old rats were both significantly lower than those of the 3-month-old control rats. The MMP-2 activity and MMP-9 activity at different time points of the 2 UUO groups were all significantly lower than those of the control groups of the same age, and those of the 26-month-old were significantly lower than those of the 3-month-old rats The MMP-2 activity and MMP-9 activity were negatively correlated with the expressions of TIMP-2 and TIMP-1. CONCLUSION: The abnormal expression of MMPs/TIMPs including higher expression of TIMPs and decreased proteolytic activity of MMPs induced by aging may be one of the factors aggravating the renal tubulointerstitial lesions of aging rats.