The Effect Of TXNIP In Renal Tubulointerstitial Fibrosis With UUO In Mice

Objectives: Renal interstitial fibrosis is a crucial pathology of end-stage renal disease. Epithelial to mesenchymal transition(EMT) and the accumulation and deposition of extracellular matrix(ECM) are the important process of pathological changes. Many molecular mechanisms and signaling pathways included the activation of TGF-β1, EMT and accumulation and deposition of extracellular matrix protein fibronectin, collagen(type I, III, IV, V and VII) contribute to interstitial fibrosis. Thioredoxin interacting protein(TXNIP) was the endogenous inhibitor of cellular thioredoxin(TRX), inactivating its anti-oxidative function by binding to the redox-active cysteine residues. In HK-2 cells, knockdown of the expression of TXNIP can inhibit ROS generation, the activation of TGF-β1 and reduce the synthesis of fibronectin. In mesangial cells, knockdown of TXNIP can reduce the expression of fibronectin under high glucose condition. A new report shows that TXNIP has a critical role in high glucose-induced EMT in HK-2 cells. The recent reports demonstrate that TXNIP played important role in renal fibrosis in diabetic nephropathy, and it may be a potential therapeutic target for treatment of progressive fibrosis in diabetic nephropathy. Many studies have showed that oxdative stress was contributed to the renal fibrosis. Up to now, the effects of TXNIP in nondiabetic renal interstitial fibrosis are still unknown.In this study, we use the unilateral ureteral obstruction(UUO) model to investigate the expression of TXNIP in process of renal interstitial fibrosis and observe the role of TXNIP in renal interstitial fibrosis using TXNIP knockout mice, and to find a new target for the treatment of interstitial fibrosis.Methods:1 Animal model:54 male C57BL/6(20g-25g)mice were randomly divided into 2 groups:UUO model group and sham-operated control group. The mice were sacrificed at 3 d, 5 d, 7 d and 14 d after operation. We observed the expression of TXNIP at different time after UUO. TXNIP-/- mice(n=6) were sacrificed at day 14 after UUO operation. The UUO models of renal interstitial fibrosis were established in mice by unilateral ureteral obstruction and sham control group mice didn’t ligate ureters.2 Collection of renal tissues and detection of contents:Mice were sacrificed at 0 d, 3 d, 5 d, 7 d and 14 d after operation, and TXNIP-/- mice just collected renal tissues at day 14 after UUO operation. Partial renal tissues were fixed by 4% formaldehyde for routine light microscopic observation(HE, Masson) and immunohistochemical staining for the detection of α-smooth muscle actin(α-SMA), fibronectin(FN), type I collagen(Col I), type IV collagen(Col Ⅳ)expression. Partial renal tissues were used to abstract total protein and RNA. The expression of TXNIP, α-SMA, Smad2, p-Smad2, FN, Col I and Col IV was detected by Western blot. The m RNA expression of TXNIP and α-SMA was examined by Real-time PCR.Results:1 The expession of TXNIP in the UUO kidneys at different time.Western blot and Real-time PCR results showed that the expression levels of TXNIP protein and m RNA markedly increased in UUO group at day 14 after UUO compared with control group mice. However, no significant change was observed in TXNIP protein and m RNA expression at day 3, 5 and 7 after UUO compared with control group mice.2 Morphological changes in renal tissues.There were no abnormal changes in sham control group including kidney size, shape, color and capsula. The kidnes were enlarged, pale and tight capsula in UUO group. The HE and Masson staining results showed that the kidney structure, glomeruli and tubules were normal, and there no inflammation and renal fibrosis in sham group mice. In UUO group, the kidney structure was destroyed, the paritial tubules were enlarged, paritial tubules were atrophy and fibrosis and with inflammatory cells infiltration. The above renal pathological changes were improved in TXNIP(-/-) group mice.3 The expression of extracellular matrix protein FN, Col I and Col IV in different groups.Compared with sham control group, the expression of FN, Col I and Col IV increased significantly in UUO group; whereas the expression levels of FN, Col I and Col IV were significantly suppressed in TXNIP(-/-) group.4 The protein and m RNA expression of α-SMA in different groups.Compared with sham control group, the expression of α-SMA increased significantly in the interstitial tissues and tubular cells in UUO group mice; whereas the expression of α-SMA was significantly suppressed in TXNIP(-/-) group. Real-time PCR results showed that the expression of α-SMA m RNA was upregulated significantly in UUO group, and the increase expression of α-SMA m RNA was inhibited in TXNIP(-/-) group.5 The expression of TGF-β1 and activation of Smad2 signal pathway.Compared with sham control group, the expression of TGF-β1 and p-Smad2 was significantly increased in UUO group; whereas those of these were significantly suppressed in TXNIP(-/-) group.Conclusion:1 The expression of TXNIP was significantly increased at day 14 in kidneys after UUO operation.2 Knockout of TXNIP can improve tubular interstitial fibrosis, reduce α-SMA expression and inhibit extracellular matrix protein fibronectin, Col I and Col IV synthesis in kidneys of UUO mice.3 The protective role of TXNIP knockout to UUO-induced tubular interstitial fibrosis may be via inhibition of TGF-β1/Smad2 signal pathway.