Kidney Tissues Of Aldosterone Role In Chronic Cyclosporin A Nephrotoxicity

BackgroundCyclosporine A(CsA) is an immunosuppressive drug used to prevent tissue allograft rejection.However,its long-term utilization is limited due to chronic nephrotoxicity for which no prevention is available.In the development of chronic CsA nephrotoxicity the renin-angiotensin-aldosterone system(RAAS) seems to play a crucial role.Not only does activation of RAAS impair renal hemodynamics,but enhance tubulointerstitial fibrosis.In recent years there has been a growing interest in the role of aldosterone in the pathophysiology of cardiovascular and renal diseases.It seems aldosterone plays an important role in chronic CsA nephrotoxicity because researchers reported spironolactone,which was aldosterone antagonist,not only reduced structural injury in chronic CsA nephrotoxicity,but also prevented renal dysfunction.These facts suggest that aldosterone acts as a key role in chronic CsA nephrotoxicity.The aldosterone synthesized outside adrenal gland has been called local or tissue aldosterone in order to make it different from circulatory aldosterone.Besides the target organ of aldostone,kidney is a producer of local aldosterone.However,it is not clear the role of local renal aldosterone in chronic CsA nephrotoxicity, which was this research's focus.ObjectivesFirst,we made a new chronic CsA nephrotoxity rat model by adrenalectomy without low sodium diet in order to observe the role of renal local aldosterone after circulatory aldosterone was eliminated.Then we evaluated the effect of local renal aldosterone on the progession of chronic CsA nephrotoxicity and the protective effect of eplerenone,a selective aldosterone antagonist,on renal function and structural alterations induced by CsA.Also we assessed whether the protective effect was associated with a reduction of transforming growth factor-β1(TGF-β1),connective tissue growth factor(CTGF) or plasminogen activator inhibitor-1(PAI-1).Finally the contribution of renal local aldosterone to cirulatory aldosterone and electrolyte balance.Methods32 male Sprague-Dawley rats weighing 300～320g were divided randomly into four groups:sham-ADX,ADX,CsA or CsA+Epl.Bilateral adrenalectomy was performed in all rats except rats in sham-ADX group. The second day after surgery,serum and urine aldosterone level were measured to confirm a completely bilateral adrenalectomy.After adrenalectomy,rats received dexamethasone 12μg/kg/d for replacement therapy to maintain normal glomerular filtration rate and fasting blood glucose.Normal sodium diet without 0.9%saline was provided.2 weeks after ADX,placebo,CsA(25mg/kg/d,subcutaneously) or CsA+Epl (100mg/kg/d,by gavage) were administrated to rats in different groups respectively.6 weeks later,all rats were sacrified and the following indicators were evaluated:(1) urine protein excretion(Bradford method) and creatinine clearance (C_cr,picric acid method,by autoanalyzer)(2) aldosterone level in serum and urine(by enzyme immunoassay), serum natrium and potassium,urine natrium and potassium excretion(by autoanalyzer)(3) tubulointerstitial fibrosis by masson trichrome stain(4)TGF-β1,CTGF,PAI-land Col-ⅠmRNA level and protein expression in kidney by real-time quantitative PCR and immunohistochemistry.Also renal tissue aldosterone synthase CYP 11B2 mRNA expression and renal tissue aldosterone were measured.Results and discussion(1) The second day after ADX,serum and urine aldosterone were undetectable,with natriuresis(p＜0.05),hyponatremia(p＜0.01),decreased urine potassium excretion(p＜0.05) and hyperpotassaemia(p＜0.05). These data showed adrenals were removed intactly and no regeneration and the adrenalectomy was successful.(2) Rats in CsA group showed increased urine protein,decreased C_cr, tubulointerstitial fibrosis and there was statistic difference(p＜0.01). These data demonstrated a successful model for chronic CsA nephrotoxity,which was similar with classic low sodium diet model.In renal tissues,these extracellular matrix and cytokines were seen upregulated,including Col-Ⅰ,TGF-β1,CTGF and PAI-1,which indicated that these cytokines maybe participate in tubulointerstitialfibrosis.(3) After treatment by eplerenone,renal aldosterone synthase CYP11B2 mRNA and renal tissue aldosterone expression were upregulated(p＜0.05),which proved eplerenone inhibited aldosterone receptor.Urine protein excretion and tubulointerstitial fibrosis relieved by eplerenone(p＜0.01,p＜0.05 respectively) and clearance of creatinine was restored slightly although there was no significant difference(p＞0.05). Gene and protein expression of Col-Ⅰ,TGF-β1,CTGF and PAI-1 were downregulated although there was no no significant difference of CTGF expression(p＞0.05).(4) At the endpoint,serum potassium,serum aldosterone,urine potassium and urine aldosterone excretion retrieved partially.Natrium in serum and urine was no significant difference compared with sham-ADX group(p＞0.05).Renal local aldosterone and its gene expression significantly upregulated(p＜0.01,p＜0.05 respectively).Based on these data,we hypothesized elevated renal local aldosterone in kidney after adrenalectomy maybe compensate for circulatory aldosterone to maintain electrolyte balance.Conclusions(1) We established a new model for chronic CsA nephtoxity by bilateral adrenalectomy without low sodium diet,which was used for renal local aldosterone research.(2) Eplerenone reduced proteinuria,tubulointerstitial fibrosis by inhibition renal local aldosterone in this animal model.It indicated that renal local aldosterone played an important role in this chronic CsA nephrotoxity model.(3) Renal local aldosterone maybe compensate for circulatory aldosterone to maintain electrolyte balance after adrenalectomy.