Experimental Investigation Of The Protective Effect Of Spironolactone On Rat Kidney After Unilateral Obstruction

Objective: Renal interstitial fibrosis is the common way to final renal failure. Renal interstitial fibrosis play more dangerous role than glomerulosclerosis in the kidney. We pay more and more attention to the onset and the progression as well as prevention and therapy. Studies show that all kind of growth factors and cytokines play an important role in the onset and progression of renal interstitial fibrosis. Transforming growth factor-beta1(TGF-β₁) and tissue inhibitor of metalloproteinase –1(TIMP-1) are important factors inducing fibrosis. Both increasing synthesis and decreasing degradation of extracellular matrix(ECM) are thought to be involved in ECM accumulation. ECM accumulation is one of the most important mechanisms of renal interstitial fibrosis. TGF-β₁ promotes ECM accumulation through increasing components of ECM such as Collagen â… ,II,III,IV and fibronectin. TGF-β₁ also can decrease catabolism of ECM through increasing synthesis of TIMP-1 which is one of the most important enzymes of degrading ECM.So, downregulating expression of TGF-β₁ ,TIMP-1 and suppressing their action in the development of renal interstitial fibrosis is a effective method to inhibit fibrosis. In the renin-angiotensin-aldosterone system(RAAS), angiotensin II receptor blocker has protective action to kidney. Recently, several lines of experimental evidence demonstrate that aldosterone,independent of renin-angiotensin,reduces fibrosis of heart ,liver and lungs. Recent evidence also implicates aldosterone as an important pathogenetic factor in progressive renal fibrosis. It is reported that aldosterone induced renal injury and fibrosis has inflammatory components involving macrophage infiltration and cytokine up-regulation in the hypertensive rat model and induced proteinuria and glomerulosclerosis in the subtotally nephrectomized rat model. Studies show that aldosterone induced renal dysfunction and up-regulation of TGF-β₁ and ECM proteins mRNA in the pathogenesis of chronic CsA nephropathy. These findings also indicate that aldosterone receptor antagonist can attenuate renal damage and inflammation, it prevented the fall in renal function and TGF-β₁,collagenâ… ,and fibronectin up-regulation,together with a reduction of tubulointerstitial fibrosis. It is reported that aldosterone has obvious effect on liver fibrosis, it might improve the degradation of liver ECM through inhibiting the activity of TIMP-1. However, there is no report that aldosterone receptor antagonist could alleviate the progression of renal interstitial fibrosis by decreasing the level of TIMP-1. Our studies aim to find one of the mechismes how aldosterone receptor antagonist protects kidney. Rat UUO models was used in our studies. We administed aldosterone antagonistspironolactone to UUO rats. Through HE stain, routine immunohistochemical, RT-PCR method, we researched the changes of expression of TGF-β₁ and TIMP-1 proteins, mRNA.. We hope our studies can make the actions of TGF-β₁, TIMP-1 and aldosterone receptor antagonist clearer. Methods: 54 male Wistar rats weighing 180-200g were randomly divided into 3 groups: sham-operated group (A),UUO group (B),UUO with spironolactone treated group (C), (n=18). Each group was also randomly divided into 7,14,21 groups in term of the days after surgery (n=6). Under pentobarbitale sodium anesthesia, the rat,s left ureter was ligated with 0 silk at two points and cut between the ligatures. Then the unilateral ureteral obstructive rat model was established. Sham-operated rats had their ureters manipulated but not ligated. Spironolactone began to administed 2 days before surgery. UUO and sham rats were received equal volume water by daily gastric gavage. All rats were allowed free access to food and water. Rats were killed after 7,14,21 days. All of the items such as serum creatinine(Scr) ,Blood urea nitrogen(BUN) ,serum potassium(K⁺),natrium(Na⁺),chlorine(Cl^-) were measured. In obstructed kidney, use HE stain to examine the area of pathological changes. The changes of expression of TGF-β₁ and TIMP-1 proteins were analyzed by routine immunohistochemical method. At the same time, TGF-β₁ and TIMP-1 mRNA were detected by RT-PCR. The results were analyzed semi-quantitatively with the pathological imageanalysis system. The experimental data was demonstrated in mean±standard deviation. The analysis of variance test and the relation about datas were used by SPSS 10.0, a statistic software. Results: Compared with group A, the level of Scr,BUN,K⁺,Na⁺,Cl-in group B and group C showed no difference. There were different degrees of interstitial fibrosis, interstitial leukocyte infiltration, tubular dilation, and the relative area of renal tubulointerstitium expansion in the obstructed kidneys of groups B7,B14,B21days after UUO.The relative area of renal tubulointerstitium was significantly increased in the obstructed kidneys compared with that of group A.( P<0.01). However, the relative area of renal tubulointerstitium of group C significantly decreased compared with that of group B (P<0.01). by routine immunohistochemical method, the protein level of TGF-β₁and TIMP-1 in groups B₇,B₁₄,B₂₁ was various and significantly higher than that in groups A₇,A₁₄,A₂₁(P<0.05, P<0.01). In groupsC₇,C₁₄,C₂₁, the protein level of TGF-β1and TIMP-1 was significantly lower than that in groups B₇,B₁₄,B₂₁ (P<0.05, P<0.01). The results of RT-PCR showed, the expression of TGF-β₁and TIMP-1 mRNA in groups B₇,B₁₄,B₂₁ was various and significantly higher than that in groups A₇,A₁₄,A₂₁ (P<0.05, P<0.01). In groupsC₇,C₁₄,C₂₁, the expression of TGF-β₁and TIMP-1 mRNA was significantly lower than that in groups B₇,B₁₄,B₂₁ (P<0.05, P<0.01). Conclusion: TGF-β1and TIMP-1 play important role in...