The Role Of Rho/Rock Pathway In Pathogenesis Of Rat Renal Interstitial Fibrosis And Ameliorative Effect Of Sodium Ferulate On The Fibrosis

The renal interstitial fibrosis is the common pathway of various kinds of chronic renal diseases developing to renal failure, and its severity has a close correlation with impairment of the renal function. It is characterized by renal interstitial fibroblasts proliferation and excess extracellular matrix (ECM) accumulation in the renal interstitium. The renal interstitial fibrosis is the results of ECM accumulation caused by interaction of several kinds of cells, cytokines, and growth factors that induce increased ECM synthesis and decreased degradation. The regulation of components and amounts of ECM is related with the dynamic balance between ECM synthesis and degradation. However, the mechanism and influencing factors of ECM disequilibrium are not clarified yet. Therefore, it is significant to investigate the mechanisms of ECM accumulation further and approach the prevention and cure measures of decreased ECM accumulation.The pathogenesis of renal interstitial fibrosis is very complex, which is the result of interaction of several kinds of factors. Ttransforming growth factor-β(TGF-β)and connective tissue growth factor(CTGF)are two kinds of the most important cytokines that cause fibrosis. In the recent years, in order to alleviate tissue fibrosis, a series of intervention study have be approached to blockade TGF-βexpression and activity, including antibody neutralization, antisense oligonucleotide probe, RNA interference, and so on. However, after complete blockade of TGF-βusing the measures, the uncontrolled cell growth, immune disturbance and serious inflammation may be caused, because TGF-βwhich has many kinds of target cells and many complex functions, it can not only induce cell multiplication and tissue fibrosis, but also resist inflammation and cell multiplication. Therefore, it is urgent to find a more specific anti-fibrosis therapeutic target. CTGF, the downstream of TGF-β, is a new discovered fibrogenesis factor with single biological effect. It only mediates fibrogenesis effects of TGF-βand does not influence anti-inflammatory and anti-proliferation of TGF-β. Accordingly, it is more specific and effective to blocking CTGF expression or inhibiting its activity. In addition, the recent studies have shown that Rho play a key role in the gene regulation of TGF-βand CTGF. Rho and its effector molecule, Rho kinase (Rock), constitute Rho/Rock pathway. In vitro, an important cell mechanism in the renal interstitial fibrosis, epithelial cell-mesenchymal transdifferentiation, is mediated by the pathway. In vivo, the renal tubule interstitial fibrosis can be inhibited after blockaded Rho/Rock pathway. NF-κB also plays an important role in the renal interstitial fibrosis. The latest researches indicated that refraining Rho/Rock pathway may decrease CTGF expression through down regulating NF-κB in smooth muscle cell in radiation enterocolitis. This result suggests that the relationship between Rho/Rock pathway with CTGF is mediated by NF-κB. Therefore, the profound researches about Rho/Rock pathway and CTGF are helpful to provide effective treatment pathway for renal interstitial fibrosis. Approaching medicine that inhibits Rho/Rock pathway and CTGF may supply ideal new target for the renal interstitial fibrosis.Sodium ferulate (SF) which is hydrosoluble sodium salt with multi- pharmacological effects such as distending blood vessel, inhibiting platelet aggregation, improve minicirculation, suppressing inflammatory reaction, resisting oxidative stress, and so on. Actually, many biological effects of SF have not been discovered. At the present, the most researches for SF focus on its antioxidation and antagonism for endothelin receptor through which SF protects kidney. However, as to renal interstitial fibrosis caused by unilateral ureteral occlusion (UUO), the effects of SF on renal function and structure, TGF-βas well as CTGF expression, ECM accumulation, and the role of Rho/Rock in CTGF expression have not been reported.In the present study, the renal interstitial fibrosis model induced by UUO in rats was established and the rat renal interstitium fibroblasts were cultured in vitro. The light microscope, immunohistochemistry, cytobiology, RT-PCR, Western blot and ELISA techniques were employed to investigate the following issues:①the protection of SF on the renal function and structure in rats with renal interstitial fibrosis;②the expression of TGF-β, CTGF, RhoA, RockⅠm RNA and RockⅠ, NF-κB protein in rats with renal interstitial fibrosis as well as the intervention of SF;③the alleviation of SF on ECM accumulation in renal interstitium;④t he correlation between RockⅠ, CTGF and ECM accumulation as well as the role of Rho/Rock in the development of the renal interstitial fibrosis. Through these investigations, the role of Rho/Rock pathway in the development of the renal interstitial fibrosis was observed, the alleviation of SF on the renal interstitial fibrosis was investigated, and the beneficial information for clinical treatmen of the renal interstitial fibrosis was provided. The main results of this study are as follows:1. NAG activity (P<0.01), BUN (P<0.01), serum creatinine (P<0.01) levels, and 24 h-urinary protein excretion (P<0.05) were significantly decreased after administration of SF for 2 w. BUN levels were obviously reduced (P<0.05) after SF administration for 4 w. The extent of the renal interstitial fibrosis was significantly improved in the rat treated with SF for 2 w and 4 w (P<0.01).2. Immunohistochemical detection showed thatα-SMA protein expression in the renal tissue in SF group was lower than that of UUO group after administration of SF for 4 w. FN and ColⅠprotein expression in the renal tissue was markedly reduced after treated with SF for 2w and 4w (P<0.01), however, ColⅣprotein expression was markedly decreased after SF administration for 2w only. Protein expression of TGF-β, CTGF and RockⅠwas decreased significantly after administration of SF for 2w and 4w (P<0.01). The cell numbers of NF-κB positive staining in nuclei were reduced after the rats treated with SF for 2w and 4w also (P<0.01).3. TGF-βand CTGF mRNA expression in the renal tissue of SF group was lower than that in UUO group (Ρ<0.05) after SF administration for 2w and 4w. RhoA mRNA expression was obviously decreased after the rats treated with SF for 2w and 4w(Ρ<0.01), and RockⅠmRNA expression was significantly decreased after rats administered with SF for 2w and 4w (P<0.01).4. RockⅠand NF-κB protein expression in the renal interstitium in SF group was lower than that in UUO group (Ρ<0.01) after administration of SF for 2w and 4w.5. Compared with AngⅡgroup, 25, 50 and 500μg/ml groups of SF had lower ColⅠprotein contents in the medium cultured fibroblasts (P<0.05). After treated with SF for 24 h, all concentrations of SF suppressed TGF-βand CTGF protein contents in the medium cultured fibroblasts (P<0.01). After treated with SF for 48 h, only 25 and 50μg/ml of SF reduced TGF-βprotein contents (P<0.05), however, 25, 50 and 500μg/ml of SF inhibited CTGF protein contents in the medium cultured fibroblasts (P<0.05).6. RT-PCR results showed that all concentrations of SF inhibited the overexpression of TGF-β, CTGF, RhoA and RockⅠmRNA in fibroblasts in the renal interstitium (P<0.01).7. RhoA and RockⅠgene expression was closely related with CTGF gene expression in renal interstitial fibroblasts (r=0.987, P=0.001; r=0.934, P=0.006).8. After treated with SF for 48h, all concentrations of SF significantly suppressed RockⅠa nd NF-κB protein expression (P<0.05 and P<0.01 ).The main conclusions are as follows:1. SF improves the renal function and structure in rats with the renal interstitial fibrosis induced by UUO and the extent of the renal interstitial fibrosis is alleviated.2. SF down regulates TGF-β, CTGF, RhoA, and RockⅠmRNA expression, inhibits TGF-βand CTGF protein expression, and ECM accumulation in the renal interstitium is reduced by SF.3. SF shows a decrease in RockⅠand NF-κB protein expression in the interstitium of renal interstitial fibrosis and down regulates ColⅠ, TGF-βand CTGF protein expression in the medium cultured renal interstitial fibroblasts.4. SF reduce AngⅡ-induced TGF-β, CTGF, RhoA and RockⅠmRNA overexpression in fibroblasts of the renal interstitium.5. RhoA and RockⅠgene expression had obviously positive correlations with CTGF gene expression in the renal interstitial fibroblasts.6. SF inhibits AngⅡ-induced RockⅠa nd NF-κB protein overexpression of renal interstitial fibroblasts.7. SF improves the development of the renal interstitial fibrosis through down regulation of CTGF.The main creativities are:Rho/Rock pathway can upregulate CTGF through NF-κB to promote occurrence and development of renal interstitial fibrosis; SF downregulates TGF-β, CTGF mRNA as well as protein expression; SF decreases RhoA, RockⅠgene expression and RockⅠ, NF-κB protein expression; SF possesses multi-target therapeutic effects, including inhibition of Rho/Rock pathway, down-regulation of CTGF expression and protection of kidney.