The Effect Of Chai Ling Tang On RAS System In Chronic Cyclosporine A Nephrotoxicity Rats

Objective: To observe renin activity(PRA), angiotensin II(AngⅡ), angiotensin-convertion enzyme(ACE), Renal angiotensin I(AngⅠ) mRNA, angiotensin II(AngⅡ)mRNA and mineralocor- ticoid receptor(MR) mRNA in rats with cyclosporine A. Expore the mechanism of Chai Ling Tang on chronic CsA toxicity .CsA which used in organ transplantation is currently the main immunosuppressant, because of its toxicity such as renal toxicity which is limited using clinical application. There are two types of renal toxicity by CsA.One is acute nephrotoxicity, and the other is chronic nephrotoxicity. Acute nephrotoxicity is relative to drugs which induced renal afferent artery contraction and it often hasn't lasting pathologic changes. When CsA is reduced or disabled disorder of renal hemodynamics is improved, renal function return to normal. Mechanism of chronic CsA nephrotoxicity is still unclear now. The character of CsA in clinic is showed with progressive renal dysfunction, irreversible interstitial fibrosis, tubular atrophy and afferent arteries hyaline. It affects long-term survival of patients, and it is currently lack of satisfactory treatment.Chronic cyclosporine A nephropathy is similar to some diseases in traditonal chinese medcine, such as Edema, Lumbago, Dysuria and others. It is resulted from drug toxin, spleen and kidney deficiency. Inducing diuresis for removing edema and reducing toxicity are appropriate treatments, and appropriate prescription is Chai Ling Tang. Chai Ling Tang is originated from Shanghan Zabing Lun and composed of Xiao Chai Hu Tang and Wu Ling San. The prescription first appeared in the Han Dynasty of Shi Yi De Xiao Fang, and then heritaged to the world by means of the Yuan Dynasty of Dan Xi Xin Fa. Chai Ling Tang is composed of Bupleurum, Pinellia, Scutellaria, jujube, ginseng,licorice, ginger, Alisma, Atractylodes, Polyporus, Poria and Cinnamon Twig. It plays an important role in harmonzing liver and kidney. And its mechanism is similar to cyclosporine A nephropathy. As a result, we observed the effect of renin-angiotensin-aldosterone system after the chronic renal injury and it could provide experimental evidence for clinical application.The SD female rats were used in this study with normal diet, CsA30mg.kg^-1.d^-1 was administrated orally for 4 weeks. Chai Ling Tang and valsartan were given orally in treated group. Blood urea nitrogen (BUN), serum creatinine (Scr), creatinine clearance rate (Ccr), renin activity (PRA), angiotensinⅡ(AngⅡ)and other indicators as well as renal AngiotensinⅠ(AngⅠ)mRNA,angiotensinⅡ(AngⅡ)mRNA and miner-alocorticoid receptor (MR) mRNA were detected. Disorders of renin-angiotensin-aldosterone system (RAS) in CsA chronic nephrotoxicity were explored and the role of Chai ling Tang to antagonise chronic CsA nephrotoxicity has been studied.Method: Healthy 40 female SD rats weight 200±10g were used in this study. After one week to adapt, rats randomly divided into 4 groups: Control group, CsA group, Valsartan group and TCM group. Animals with CsA treated were given CsA 30mg.kg^-1.d^-1 by orally. Same volume of olive oil was given in Control group. In addition, valsartan 10mg.kg^-1.d^-1 and Chai Lin Tang 3g. kg^-1.d^-1 that dissolved in tap water were given in treated groups for 28 days. The urine was collected in metabolic cages every weekend and body weight to weigh. After the last administration, the animals were fasted for 12 hours and executed for blood that go through rapid centrifugation. Conservation of samples of serum, and plasma samples to be used for serum creatinine (Scr), blood urea nitrogen (BUN) test, and calculated creatinine Clearance rate (Ccr). Kidneys were fixed by 4% paraformaldehyde for pathological changes. Renin activity (PRA), angiotensinⅡ(AngⅡ) and other indicators as well as renal AngiotensinⅠ(AngⅠ) mRNA, angiotensinⅡ(AngⅡ) mRNA and the mineralocorticoid receptor (MR) mRNA were detected to evaluate the effect of Chai Ling Tang in CsA nephropathy.Result:①Renal function: Compared with the control group, serum creatinine(Scr) and urea nitrogen (BUN) were increased significantly while its creatinine clearance rate (Ccr) was decreased in CsA group (P<0.01). Compared with the CsA group, serum creatinine(Scr) and urea nitrogen(BUN) were significantly lower while creatinine clearance rate (Ccr) was significantly higher (P <0.01, P <0.05) in Val group and TCM group.②Pathologic change: The renal pathologic changes were measured with HE and Masson staining: It was no change in control group, regularly arranged tubular epithelial cells in tubule and little inflammative cells in interstitium, interstitial edema had not showed in control rats. In CsA group, a large number of inflammative cells and edema had seen in interstitial space, renal tubular epithelial cells were denatured and necrosis in CsA treated rats. There were some inflammation and renal tubular epithelial cells were turgid in Val group, but it was lighter than CsA group. There were a little of inflammation and turgid in renal interstitial infiltration of TCM group, but it was significantly reduced compared with CsA group. Masson staining showed that tubular membrane was not ruptured, its structure was clear and collagen was not increased in control group. The structure was disordered and renal tubular interstitial collagen was significantly increased in CsA group. There were some degree of deposition of tubular interstitial collagen in TCM group and Val group, but it was significantly reduced compared with the CsA group.③To detect the expression of serum renin activity (PRA) and angiotensinⅡ(AngⅡ) with radioimmunoassay: compared with control group, the level of serum PRA and AngⅡwas significantly higher in CsA group (P<0.01). Compared with CsA group, the level of serum PRA and AngⅡwas decreased in Val group and TCM group (P <0.01, P<0.05).④T o detect the expression of tissue's activity (PRA) and angiotensinⅡ(AngⅡ) with radioimmunoassay: compared with control group, the level of tissue's PRA and AngⅡwas significantly higher in CsA group (P <0.01). Compared with CsA group, the level of tissue's PRA and AngⅡwas decreased in Val group and TCM group(P<0.01, P<0.05).⑤The expression of ACE: the expression of ACE was detected by immunohistochemistry, the result showed that weakly in control group and significantly up-regulated by CsA, mainly expressed in tubular epithelial cells. Both of Valsartan and Chai Ling Tang had the role to down-regulated the expression of ACE.⑥RT-PCR detection of renal angiotensinⅠ(AngⅠ) mRNA, angiotensinⅡ(AngⅡ) mRNA and mineralocorticoid receptor (MR) mRNA expression: Compared with control group, the expression of AngⅠmRNA and AngⅡmRNA was significantly up-regulated in CsA group (P<0.01). Compared with CsA group, the expression of AngⅠmRNA was decreased significantly in Val group and TCM group(P<0.01).Compared with Val group, the expression of AngⅠmRNA was reduced significantly in TCM group(P<0.05).Compared with CsA group, the expression of AngⅡmRNA was decreased in Val group, but it had no significant difference(P>0.05), while the expression of AngⅡmRNA was down-regulated in TCM group(P<0.01). Compared with Val group, the expression of AngⅡmRNA was significantly lower in TCM group (P<0.05). It had no statistically significance about MR mRNA expression between different groups (P> 0.05).Conclusion: 1 CsA induced renal interstitial injury and contributed to the increase of blood urea nitrogen(Scr), serum creatinine(BUN) and decrease of creatinine clearance(Ccr). Renal tubulointerstitial pathology showed a significance of renal toxicity damage. It contained the increase of position of collagen and infiltration of inflammatory cell .2 Chai Ling Tang had the role to reduce the blood urea nitrogen(BUN) and serum creatinine(Scr), increase creatinine clearance rate (Ccr) and improve renal function.3 Chai Ling Tang had the role to reduce the level of the serum and tissue of PRA, AngⅡand the expression of ACE. It played an important role in improving the glomerular filtration rate and renal protective effect through the block of RAS system.4 Chai Ling Tang could reduce the expression of AngⅠmRNA and AngⅡmRNA in the kidney.