The Protective Effect And Mechanisms Of Kushenin On Renal Interstitial Fibrosis After Unilateral Ureteral Obstruction In Rats

The progression of chronic kidney disease (CKD) is considered to be an irreversible process that eventually leads to end-stage renal failure. Renal interstitial fibrosis (RIF) is the common pathologic pathway to end-stage renal failure in almost all renal disease. Many animal and clinical experiments have demonstrated that the deterioration of renal function is largely determined by the extent and severity of interstitial lesions in many forms of renal disease. The degree of the tubular and interstitial damage is the most important index which can reflect the declined renal function and judge the prognosis of disease. Nowadays, as population of the patients with end-stage renal disease is increasing at a rate of approximately 7% per year, the economic burden of CKD to the families and society alike is enormous. It has significant meaning that studying further the molecular mechanisms of RIF and searching effective therapeutic strategy to slow the progression of CKD and prolong the life of the patients.Renal interstitial fibrosis is characterized by atrophy of tubule, proliferation of myofibroblast (MoyF) and relentless accumulation and deposition of extracellular matrix (ECM). During the last several years, the role of tubular epithelial-mesenchymal transformation (EMT) and activation of main effective cells has been emphasized. Of the many factors that regulate EMT in different ways, transforming growth factor- β1 is the most potent inducer that is capable of initiating and completing the entire EMT course. The complete TGF- β1/smad intracellular signal pathway mediates EMT. TGF-β1 can promote the tubular epithelial cells and activate smad2 and smad3. Upon TGF-β1 stimulation, smad2 and smad3 are phosphorylated and then induce tubular epithelial cell to undergo phenotypic conversion to α-SMA postive myofibroblast. The tubular epithelial cells loss the epithelial maker E-cadherin and produce I ,III, IV type collagen, and finally induce renal interstitial fibrosis.Kushenin is a kind of compound which mainly contained oxymatrine and derived from Sophraflavescens Ait or S.alopecuroides L. Recently, pharmacologic and clinical studies have found that besides the role of anti-inflammation, anti-virus, anti-tumor and elevating leukocyte, the Kushenin can inhibit fibroblast proliferation, collagen biosythesis, inflammatory cells accumulation and can also prevent liver fibrosis in experimental rats. But, till now, little is known that whether kushenin can interfere with renal interstitial inflammation and fibrosis.To explore the effect and probable mechanism of kushenin on renal interstitial fibrosis and search a new method to prevent and treat it, the renal interstitial fibrosis model was established by unilateral ureteral obstruction (UUO) in rats. The changes of renal pathologic features and serum biochemical indexes were observed. Reverse transcription polymerase chain reaction (RT-PCR) was used to examine the expression of TGF-β1 and α— SMA mRNA. Immunohistochemistry method was performed to investigate the expression of TGF-β1,α—SMA ,smad3 and Col I in kidney. Western-blot was performed to examine the expression of TGF-β1 and α—SMA protein.Section one The effects of kushenin on renal lesions in UUO ratsObjective To study the effect of kushenin on the renal lesion following unilateral ureteral obstruction in rats.Methods Sprague-Dawley male rats were randomly assigned to 3 groups: shame operation group, UUO group and treatment group. The rats of shame operation group were just separated left ureter while the rats of UUO group and treatment group were performed operation by left ureter double ligature. The rats of treatment group were treated with daily injection intraperitoneal of 100mg/Kg of kushenin after operation. Rats of each group were killed at 7,14,21,28 days after operation respectively. And the rats of sham operation were killed at 28 days after surgery. On the day before the rats were killed, the urine samples of 24h were collected. On the operation day, the samples of vein blood and renal tissue were collected. The weight of left kidney and body of rats were measured. Automatic biochemical analysis machine was used to examine the level of BUN, Scr ,ALB, urinary protein excretion of 24h. Morphological changes of renal tissue were observed by PAS and Masson stain and were investigated through light microscope. The method of percentage evaluation was used to evaluate the degree of tubular damage and renal fibrosis. The number of the inflammatory cells which accumulate in renal interstitial was calculated. Immunohistochemistry method was performed to investigate the protein expression of Col I in kidney. Results1 Compared with UUO group, the degree of swelling and hydrocele of the obstructive renal in the treatment group was milder. The color of latter was redder.2 The levels of BUN and Scr in UUO group and treatment group began to increase at 14 day after operation and were much higher than that in shame operation group (p<0.05) and then decreased to normal at 21 day after surgery. There was no significant difference between UUO group and treatment group.3 The degree of tubular damage and renal fibrosis in treatment group was much lower than that in UUO group at every time point (p<0.05).4 In both UUO and treatment groups, the number of the inflammatory cells which accumulated in renal interstitial increased markedly in time-dependent manner from 7 to 21 days after operation and then decreased. Compared to UUO group, the number of the inflammatory cells was lower in treatment group from 7-21 day after operation (p<0.05). 5 The protein expression of Col I increased in time-dependent manner in both UUO group and treatment group, while, it was significantly lower in treatment group at every time point (p<0.05).6 There was no significant difference in the level of ALB, urinary protein excretion of 24h among three groups.Conclusion kushenin could suppress markedly the accumulation of inflammatory cells, downregulate the protein expression of Col I , ameliorate the degree of tubular damage and renal interstitial fibrosis. It played a protective role in the renal interstitial fibrosis. Section two The effects of Kushenin on the expression of TGF-β1, smad3 and α—SMA in UUO ratsObjective To explore the mechanism of kushenin to ameliorate the degree of renalinterstitial fibrosis.Methods Sprague-Dawley male rats were randomly assigned to 3 groups: shameoperation group, UUO group and treatment group. The rats of shame operation group werejust separated left ureter while the rats of UUO group and treatment group were performedoperation by left ureter double ligature. The rats of treatment group were treated with dailyinjection intraperitoneal of 100mg/Kg of kushenin after operation. Rats of each groupwere killed at 7,14,21,28 days after operation, respectively. And the rats of sham operationwere killed at 28 days after surgery. Immuno- histochemistry method was performed toinvestigate the expression of TGF-β1, smad3 and α — SMA in kidney. Reversetranscription polymerase chain reaction (RT-PCR) was used to examine the expression ofTGF-β1 and α—SMA mRNA.Western-blot was performed to examine the expression ofTGF-β1 and α-SMA protein.Results1 The results of immunohistochemistry showed that there was little expression of TGF-β1, smad3 and α—SMA in shame operation group .2 The expression of TGF-β1 and α—SMA increased in time-dependent manner in both UUO and treatment group while they were significantly less in the latter (p<0.05).3 The expression of smad3 in UUO group increased and reached to the climax at 21 day then decreased at 28 day. The expression of smad3 in treatment group increased in time-dependent manner and was much lower than UUO group at 7, 14, 21 day after operation, respectively (p<0.05). On the contrary, at 28 day after operation, the expression of smad3 in treatment group was higher than UUO group (p<0.05).4 The results of RT-PCR and Western-blot showed that the expression of TGF-β1 and α— SMA mRNA or protein increased since operation. The expression of TGF-β1 and α— SMA mRNA or protein in treatment group markedly lower than UUO group (p<0.05 ).Conclusion kushenin could inhibit tubular epithelial-mesenchymal transformation (EMT), suppress the activation and proliferation of myofibroblast , reduce the accumulation and deposition of ECM by downregulate the expression of TGF-β1 . The probable mechanism was to intervene with the TGF-β1/smad3 signal pathway by downregulate, the expression of smad3 and reduce the production of target protein, ameliorate renal interstitial fibrosis.