doi:10.1152/ajprenal.00147.2006 292:131-139, 2007. First published Jul 11, 2006;Am J Physiol Renal Physiol

Bobadilla Vaidya, Norma Uribe, Joseph V. Bonventre, Gerardo Gamba and Norma A. Jazmin Pérez-Rojas, Jorge A. Blanco, Cristino Cruz, Joyce Trujillo, Vishal S.nephrotoxicity renoprotection in preexisting chronic cyclosporine Mineralocorticoid receptor blockade confers

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Mineralocorticoid receptor blockade confers renoprotection in preexistingchronic cyclosporine nephrotoxicity

Jazmin Perez-Rojas,1,2 Jorge A. Blanco,1,2 Cristino Cruz,1,2 Joyce Trujillo,1,2 Vishal S. Vaidya,3

Norma Uribe,2 Joseph V. Bonventre,3 Gerardo Gamba,1,2 and Norma A. Bobadilla1,2

1Molecular Physiology Unit, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico and2Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Mexico City, Mexico; and 3Renal Division,Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts

Submitted 26 April 2006; accepted in final form 3 July 2006

Perez-Rojas J, Blanco JA, Cruz C, Trujillo J, Vaidya VS, Uribe N,Bonventre JV, Gamba G, Bobadilla NA. Mineralocorticoid receptorblockade confers renoprotection in preexisting chronic cyclosporinenephrotoxicity. Am J Physiol Renal Physiol 292: F131–F139, 2007. Firstpublished July 11, 2006; doi:10.1152/ajprenal.00147.2006.—Recentstudies from our laboratory have shown that the mineralocorticoid recep-tor (MR) blockade with spironolactone (Sp) prevented renal dysfunctionand reduced renal injury in both acute and chronic cyclosporine (CsA)nephrotoxicity. This study was designed to evaluate whether Sp admin-istration reduces functional and structural renal damage associated in thesetting of preexisting chronic CsA nephrotoxicity. Twenty eight maleWistar rats were fed a low-sodium diet. Fourteen received vehicle (V)and the others were treated with CsA (15 mg/kg sc). After 18 days onehalf of each group received Sp (20 mg/kg po) for the subsequent 18 days.Creatinine clearance, arteriolopathy, tubulointerstitial fibrosis, arteriolarthickening, glomerular diameter, apoptosis index and TGF-�, pro-caspase-3, and kidney injury molecule 1 (Kim-1) mRNA levels as well asKim-1 shedding in urine were evaluated. Sp reduced the progression ofrenal dysfunction and tubulointerstitial fibrosis in preexisting chronicCsA nephrotoxicity. There was a significant reduction of arteriolarthickening in the CsA�Sp group that was associated with greater glo-merular diameter and reduction of apoptosis index. These renoprotectiveeffects were associated with reduction of TGF-�, procaspase-3, andKim-1 mRNA levels as well as Kim-1 shedding into the urine. Inconclusion, MR blockade with Sp prevented the progression of renalinjury in preexisting chronic CsA nephropathy. These results suggest thatSp may reduce CsA-induced established nephrotoxicity in patients.

apoptosis; kidney injury molecule 1; transforming growth factor-�;glomerular diameter; fibrosis; procaspase-3

IN RECENT YEARS THERE HAS been growing interest in the role ofaldosterone and mineralocorticoid receptors (MR) in the patho-physiology of cardiovascular and renal diseases. The role ofaldosterone in promoting cardiovascular injury is underlinedby the randomized Aldactone evaluation study (RALES) (32)and the Eplerenone Post-Acute Myocardial Infarction HeartFailure Efficacy and Survival Study (EPHESUS) (31) trials. Inthese studies it was demonstrated that addition of the MRblockers spironolactone or eplerenone to standard therapy inheart failure patients and patients with myocardial infarctionresulted in reduced cardiac mortality that could not be ex-plained solely by blood pressure reduction. In addition, it hasbeen reported that aldosterone infusion in hypertensive patientscan induce endothelial dysfunction (10).

Although the role of ANG II in mediating progressive renaldisease has been documented extensively, recent clinical andexperimental evidence has supported the role of aldoste-rone/MR in the progression of renal injury that is independentof ANG II. Patients with primary hyperaldosteronism exhibit ahigher prevalence of proteinuria (8, 28) and severe arteriolarsclerosis and interstitial fibrosis was observed in 50% of 32renal biopsies from patients with aldosterone-producing ade-nomas (13). In animals on high sodium intake, renal lesions ofmalignant nephroesclerosis are observed following unilateralnephrectomy and prolonged treatment with DOCA (22). Inaddition, the effectiveness of MR antagonism in amelioratingrenal injury has also been documented. Pilot studies in humansshowed that addition of spironolactone to ACE inhibitors hadno hemodynamic effects but markedly reduced proteinuria inpatients with renal failure (4) and in patients with type 2diabetes (39). The protective effect of spironolactone in pa-tients with mild renal insufficiency has recently been corrob-orated by a large double blind, placebo-controlled randomizedtrial (7). In rats, MR antagonists had no effect on systemicblood pressure but markedly ameliorated glomerular and/ortubulointerstitial injury in several models of nephropathy in-cluding spontaneously hypertensive stroke-prone rats (33, 34),ANG II and nitric oxide synthase inhibitor-treated rats (35),aldosterone-treated rats (16), and in a model of unilateralureteral obstruction model (46). Moreover, Aldigier et al. (1)reported that MR blockade not only reduced the developmentof glomerulosclerosis but also induced regression of existingglomerulosclerosis in rats after 5/6 nephrectomy. All thesestudies together emphasize the beneficial effect of MR antag-onism in progressive renal diseases.

We previously observed that aldosterone also plays an im-portant role in the toxicity induced by the immunosuppressantcyclosporine A (CsA), an agent that is extensively used for theprevention of allograft rejection (11, 29). The therapeuticbenefits of CsA for transplantation and autoimmune diseaseshave been limited by the occurrence of acute and chronicnephrotoxicity. Acute CsA nephrotoxicity is characterized byrenal vasoconstriction induced by an imbalance of vasoactivesubstance release which, in turn, produced a fall in renalfunction. This form of toxicity is reversible. In contrast,chronic CsA nephrotoxicity is characterized by both renalvasoconstriction and the development of arteriolopathy andscarring tubulointerstitial fibrosis that are irreversible (2, 21,

Address for reprint requests and other correspondence: N. A. Bobadilla,Unidad de Fisiologıa Molecular, Vasco de Quiroga No. 15, Tlalpan, 14000Mexico City, Mexico (e-mail: nab@biomedicas.unam.mx).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Renal Physiol 292: F131–F139, 2007.First published July 11, 2006; doi:10.1152/ajprenal.00147.2006.

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27). Both forms of nephrotoxicity can be reproduced in the ratby administration of repeated doses of CsA. However, toinduce the chronic model, in addition to CsA administration,the animals should be fed a low-sodium diet (2, 9, 11, 19,29, 41).

In recent studies from our laboratory, we observed that MRblockade with spironolactone completely prevented renal dys-function induced by CsA in both acute and chronic CsAnephrotoxicity and significantly reduced renal structural dam-age (11, 29). Because acute kidney injury induced by CsA isassociated with afferent arteriole vasoconstriction, these datasuggested that aldosterone is not only implicated in chronicstructural renal damage, but also in the regulation of vasculartone (29). In these studies, spironolactone was administeredsimultaneously with CsA from the first day of the experimentalperiod. Thus, these observations indicate that spironolactone isan effective prophylactic agent to prevent the development ofCsA nephrotoxicity. It is not known, however, if MR blockadecan contribute to prevent the progression of the already exist-ing tubulointerstitial injury and renal dysfunction in the modelof chronic CsA nephropathy.

In the present study, we show that spironolactone reducedthe progression of existing tubulointerstitial injury and arterio-lar thickening in a model of nephropathy in which chronic CsAnephrotoxicity was already established. The renoprotectionwas associated with a significant reduction of CsA-inducedapoptosis, TGF-�, procaspase-3, and kidney injury molecule 1(Kim-1) expression.

METHODS

Four groups of seven male Wistar rats each weighing 300–330 gwere used. All groups were fed a low-salt diet (0.02%). As shown inFig. 1, one group received 0.1 ml sc per day of olive oil as vehicle (V)and another group received CsA 15 mg �kg�1 �day�1 sc for 36 days(CsA); this dose of CsA and the percentage of sodium in the diet usedin this study have been previously demonstrated to induce chronicCsA nephrotoxicity in the rat (2, 9, 11, 19, 29, 41). To test the effectof spironolactone in preexisting chronic CsA nephrotoxicity, twoadditional groups were included. One group received vehicle for 18days and then vehicle plus spironolactone at 20 mg �kg�1 �day�1 bygastric gavage for another 18 days (Sp) and the other group wastreated with CsA for 18 days followed by CsA plus spironolactone foranother 18 days (CsA�Sp). The dose of spironolactone has beenproved to be enough to blockade MR in the rat (5, 12, 25, 49).

Because CsA-treated animals reduced their food consumption and lostbody weight, control animals were pair fed with their correspondingCsA group. To confirm the presence of chronic CsA nephrotoxicity atthe middle of the study, an additional group of six animals was treatedwith CsA for 18 days and killed to evaluate the renal structuraldamage. All animal procedures followed were in accordance with ourinstitutional guidelines.

Functional and histological studies. At the beginning, middle, andend of the study (0, 18, and 36 days), rats were placed in metaboliccages and urine that was spontaneously voided during 24 h wascollected. Serum and urine creatinine concentration were measuredwith an autoanalyzer (Technicon RA-1000, Bayer Tarrytown, NY).Renal creatinine clearances were calculated by the standard formulaC � U*V/P, where U is the concentration in urine, V is urine flowrate, and P is plasma concentration. Serum potassium levels weremeasured at the end of the study.

At the end of the study, rats were anesthetized by intraperitonealinjection of pentobarbital sodium (30 mg/kg ip) and placed on ahomeothermic table to maintain core body temperature at 37°C, bymeans of a rectal probe attached to a temperature regulator which is,in turn, attached to a homeothermic blanket. Trachea, jugular veins,and femoral arteries were cannulated with polyethylene tubing PE-240 and PE-50. Mean arterial pressure (MAP) was monitored with apressure transducer (model p23 db, Gould, Puerto Rico) and recordedon a polygraph (Grass Instruments, Quincy, MA). A midline laparot-omy was made, right renal artery was ligated, and the right kidney wasexcised, macroscopically divided into cortex and medulla, frozen inliquid nitrogen, and kept at �80°C until used. The left kidney wasperfused through the femoral catheter with phosphate buffer preserv-ing the MAP of each animal. Following blanching of the kidney, theperfusate was replaced by 10% freshly prepared formalin buffer andthe perfusion was continued until fixation was completed. Afterappropriate dehydration, kidney slices were embedded in paraffin,sectioned at 4 �m, and stained with routine methods: hematoxylin/eosin, periodic acid-Schiff, and Masson trichromic. The slides wereanalyzed blindly. In at least 50 arterioles per rat, arteriolopathy wasquantified as a characteristic lesion of this model. Arteriolopathy wascounted as present or nonpresent (dichotomic variable), thus resultsare expressed as percentage of affected arterioles over total number ofarterioles. Arteriolar thickening was evaluated in at least 25 arteriolesper animal from recorded digital microphotographs, using a digitalcamera incorporated in a Nikon microscope. Three different measure-ments in each arteriole were made by using eclipse net software(magnification �400). In addition, the glomerulus size was evaluatedby measuring glomerular diameter. For this purpose, 10 to 15 imagesof different renal cortex fields were recorded and the diameter of atleast 500 glomeruli was measured in the digitalized microphotographs(magnification �100). Moreover, in Masson-stained sections 10 sub-cortical periglomerular fields per section were randomly selected inkidneys from the different groups to evaluate the degree of tubuloin-terstitial fibrosis by morphometry. Tubulointerstitial fibrosis consistedof extracellular matrix expansion with collagen deposition togetherwith distortion and collapse of the tubules; fibrosis was evidenced byblue coloration in Masson stain. Fifteen images were recorded, theaffected area was delimited, and the percentage of tubulointerstitialfibrosis was calculated by dividing the fibrotic area by the total fieldarea, excluding the glomerular area.

TUNEL assay. Apoptosis in kidney sections was determined byTUNEL assay using the ApopTag in situ apoptosis detection kit(S7101, Chemicon International, Temecula, CA). The slides wereprepared following the procedures described previously (50). A min-imum of 10 fields per kidney were evaluated and all kidney tissueswere examined (magnification �400). Only tubular cells that con-tained TUNEL-positive nuclei with the characteristic morphology ofapoptosis, including nuclear fragmentation and nuclear condensation,were quantified. The number of TUNEL-positive tubules was counted

Fig. 1. Experimental protocol. Male Wistar rats fed a low-sodium diet (0.02%)were included and 4 groups were formed: vehicle (V), cyclosporine CsA,vehicle plus spironolactone (Sp), and CsA plus Sp (CsA�Sp). Vehicle andCsA were given during 36 days. In the Sp and CsA�Sp groups, spironolactonewas started at the middle of the protocol (day 18) and continued to the end ofthe study.

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and the results were expressed as the number of TUNEL-positivenuclei per square millimeter.

RNA isolation. Total RNA was isolated from each renal cortexfollowing the guanidine isothyocianate-cesium chloride method (38).Integrity of isolated total RNA was examined by 1% agarose gelelectrophoresis and RNA concentration was determined by UV-lightabsorbance at 260 nm (Beckman DU640, Brea, CA). Reverse tran-scription (RT) was carried out using 2.5 �g of total RNA from renalcortex of each rat. RT was performed at 37°C for 60 min in a totalvolume of 20 �l using 200 U of the Moloney murine leukemia virusreverse transcriptase (Invitrogen), 100 pmol of random hexamers(Invitrogen), 0.5 mM of each dNTP (Sigma, St. Louis, MO), and 1�RT buffer (75 mM KCl, 50 mM Tris �HCl, 3 mM MgCl2, 10 mMDTT, pH 8.3).

Real-time PCR. The mRNA levels of TGF-�, Kim-1, and pro-caspase-3 were quantified by real-time PCR on the ABI Prism 7300Sequence Detection System (TaqMan, Applied Biosystems ABI,Foster City, CA). FAM or VIC dye-labeled probes were selected fromthe Applied Biosystems Assays-on-Demand ABI product line andwere specifically used to detect and quantify cDNA sequences withoutdetecting genomic DNA. For Kim-1, procaspase-3 and TGF-� ex-pression FAM probes and for eukaryotic 18S rRNA expression VICprobe were used. The FAM (6-carboxyfluorescein) and VIC wereused as fluorescent reporter dies to detect amplification products.Primers and probes for TGF-�, Kim-1, and procaspase-3 were orderedas kits: Rn00572010_m1, RN00597703_m1, and Rn00563902_m1(Assays-on-Demand, ABI). Validation of amplification efficiency wasmade for every primer/probe set and was calculated for each run. Asendogenous control, we used eukaryotic 18S rRNA (predesignedassay reagent Appplied by ABI, external run) to correct for potentialvariation in RNA loading or efficiency of the amplification reaction.Standard curves for each primer/probe were computed from a series ofserial template dilutions from 0.187 through 187 ng. PCR was carriedout in 96-well plates on cDNA equivalent to 3.5 ng of total RNAisolated individually from each renal cortex. Thermal cycling condi-tions were 10 min at 95°C followed with 40 cycles at 95°C for 1 minand 60°C for 1 min. Data were collected using the ABI PRISM 7300SDS analytical thermal cycler (Applied Biosystems). Each individualsample was tested in triplicate.

The relative quantification of TGF-�, Kim-1, and procaspase-3gene expression was performed using the comparative CT method(23). The threshold cycle (CT) is defined as the fractional cyclenumber at which the reporter fluorescence reaches a certain level (i.e.,usually 10 times the standard deviation of the baseline). In allexperiments, 18S eukaryotic rRNA was used as control. Negativecontrols were included in the reaction plate.

Urinary Kim-1. Kim-1 protein in urine was measured by Micro-sphere-based Luminex xMAP technology (47). This technique is anadaptation of the recently developed and validated sandwich ELISAassay (48). For quantitation of urinary Kim-1 ectodomain, 30 �l of24-h urine were analyzed in duplicate.

Statistical analysis. Results are presented as means � SE. Thesignificance of the differences among groups was tested by ANOVAcomparison using Bonferroni’s correction for multiple comparisons.The differences in the ranks of glomerular diameters among the

groups were evaluated by a contingency analysis and the differenceswere tested by �-square test with Yates correction. Statistical signif-icance was defined when P value was 0.05.

RESULTS

Animal body weight during the study as well as MAP andserum potassium levels at the end of the study are shown inTable 1. At the beginning, all rats had similar body weight, butit was significantly reduced at the end of the study, because ofthe little food consumption of CsA rats and the pair feeding ofthe control groups. No differences among the groups wereobserved in MAP, thus spironolactone effect was pressureindependent. Serum potassium levels tended to be higher inCsA group, but the difference was not statistically different andit was not enhanced by spironolactone administration.

The percentage of animals surviving after 36 days of treat-ment was 90% in the CsA�Sp-treated group vs. 67% in theCsA group (Fig. 2A). Thus, spironolactone administrationimproved survival rate despite the delay in administration to

Table 1. Effect of CsA and MR blockade on BW, mean arterial pressure and potassium levels

Group BW, g0 Days

BW, g18 Days

BW, g36 Days

MAP,mmHg

K�, meq/l

V 313�11 306�7 270�8* 110.0�6.2 4.7�0.3Sp 310�13 314�11 289�8* 95.2�6.6 5.4�0.2CsA 321�6 269�11 261�13* 91.2�2.7 5.8�0.5CsA�Sp 319�4 268�12 271�16* 97.6�6.1 5.4�0.4

Values are means � SE. V, vehicle; Sp, spironolactone; CsA, cyclosporine; MR, mineralocorticoid receptor; BW, body weight; MAP, mean arterial pressure;K�, serum potassium levels. *P 0.05 vs. their respective groups at the beginning of the study.

Fig. 2. A: survival percentage of CsA- and CsA�Sp-treated rats along thestudy. B: creatinine clearance in V (■ ), Sp (�), CsA (F), and CsA�Sp (E).*P 0.05 vs. the same group at 0 days. **P 0.05 vs. the same group at 18days.

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CsA-treated animals. The creatinine clearance for the fourgroups studied at 0, 18, and 36 days is shown in Fig. 2B. At thebeginning, all groups had similar normal renal function thatwas not modified in both control groups along the study. Ratsreceiving CsA showed a significant and similar reduction ofcreatinine clearance at 18 days. Creatinine clearance was 0.7 �0.1 and 0.8 � 0.1 ml/min in CsA and CsA�Sp groups,respectively. From this day, the spironolactone administrationreduced the rate of progression of renal dysfunction. At the endof the study, the creatinine clearance in CsA-treated animalsfurther decreased to 0.4 � 0.1 ml/min (P � 0.006 vs. CsAalone at day 18). In contrast, creatinine clearance in theCsA�Sp remained unchanged at 0.9 � 0.1 ml/min at day 36(P � NS vs. CsA�Sp at day 18).

After 18 days of CsA administration, six rats were killed andtheir kidneys were fixed to determine the presence of arteriol-opathy and tubulointerstitial fibrosis thereby evaluating thepresence of chronic CsA nephropathy at this time of the study.We observed that this group presented structural renal injurywith arteriolopathy in 20.4 � 1.6% of arterioles. In addition,20.2% of the analyzed area was affected by tubulointerstitialfibrosis. These results together with the measurement of cre-atinine clearance after 18 days of CsA administration corrob-orated that at this time, the rats had already established chronicCsA nephrotoxicity. When spironolactone was started at day18 in the CsA�Sp group, the chronic CsA nephropathy did notprogress. Represented in Fig. 3 is the percentage of the areaaffected by fibrosis, together with representative photomicro-graphs, at the end of the study. In rats that received CsA, thepercentage area affected by fibrosis was enhanced at 36 days toa value of 45.6 � 3.8% (P 0.05 vs. 18 days). In contrast, theprogression of renal fibrosis was reduced in rats that receivedspironolactone at the middle of the study (30.8 � 2.3% inCsA�Sp vs. 45.6 � 3.8% in CsA, P 0.005). In Fig. 4 arerepresentative images of arteriolopathy in CsA and CsA�Spgroups together with the analysis of the percentage of arteriol-opathy and arteriolar thickening in these groups. In theCsA�Sp group, arteriolopathy percentage tended to be minor,but the difference did not reach statistical significance. Incontrast, MR blockade produced a significant reduction ofarteriolar thickening, since mean values for CsA group were9.3 � 0.3 �m vs. those of CsA�Sp group that were 8.1 � 0.3�m (P 0.05).

The glomerular diameters were evaluated in rats that re-ceived vehicle, CsA, and CsA�Sp. In digitalized images, thediameter size was evaluated in at least 80 glomeruli per rat anddistributed by rank. Figure 5 shows the normal diameter sizedistribution of the control group, in which it is possible toappreciate that most of the glomerulus are in the rank of 101 to125 �m (56.9%) and a minor proportion are in the rank of 76to 100 �m (25.7%) and 126 to 150 �m (13.9%). The histogramin control group presents a typical bell-shaped Gaussian dis-tribution. In contrast, in chronic CsA-treated rats, the distribu-tion of diameter size was shifted to the left, reflecting glomer-ular constriction. Accordingly, the glomerular diameters weresmaller: 13.8% from 50 to 75 �m and 36.8% from 76 to 100�m (25.7%). In addition, a lower proportion of glomeruli werein the diameter ranges from 101 to 125 �m (40.9%) and 126 to150 �m (7.1%). All these differences were statistically signif-icant by using a contingency analysis. The MR blockade wasassociated with less glomerular constriction in progressive

chronic CsA nephrotoxicity (CsA�Sp), evidenced by a glo-merular size distribution that was similar to control group.CsA�Sp group exhibited a greater percentage of glomerulardiameters in the range from 101 to 125 �m (49.3%) and lesserproportion in the range from 50 to 75 �m (4.2%). Bothdifferences were statistically significant when compared withthe group treated with CsA alone.

Because it has been suggested that apoptosis plays a role inaldosterone-induced target organ damage (30), we evaluated

Fig. 3. A: percentage area affected by tubulointerstitial fibrosis in the 4 groupsincluded at the end of the study (36 days): V (open bars), Sp (hatched bars),CsA (filled bars), and CsA�Sp (gray bars). *P 0.05 vs. V and Sp groups.†P 0.05 vs. CsA group. B and C: representative images showing tubuloin-terstitial fibrosis in CsA and CsA�Sp groups, respectively. *P 0.05 vs. CsAgroup.

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whether aldosterone receptor blockade modifies the apoptosisthat is known to be induced by CsA during chronic nephrop-athy (43, 45). The presence of apoptotic cells in renal cortex inthe CsA and CsA�Sp groups was evaluated with the TUNELtechnique. Representative microphotographs of TUNEL stain-

ing in a rat treated with CsA for 36 days and a rat that receivedspironolactone after day 18 of CsA treatment up to the end ofthe study are shown in Fig. 6, A and B, respectively. About 60digitalized images from renal cortex sections from CsA andCsA�Sp groups were obtained using Eclipse net software and

Fig. 4. A: percentage of arteriolopathy. B: arteriolar thickening in CsA (filled bars) and CsA�Sp groups (gray bars). C and D: representative images ofarteriolopathy showing arteriolar thickening in CsA and CsA�Sp groups, respectively (magnification �400). *P 0.05 vs. CsA group.

Fig. 5. Glomerular diameter distribution in vehicle group represented by open bars, CsA-treated rats in filled bars, and CsA�Sp in gray bars. The significancewas tested by contingency analysis and �2. *P 0.05 vs. the same rank in V group. ** P 0.05 vs. the same rank in CsA-treated rats.

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the positive nuclei per square millimeter per kidney werequantified. The results obtained are graphically expressed inFig. 6C. Consistent with previous observations (43, 45), after36 days of CsA treatment there was evident tubular cellapoptosis degree quantified as 698 � 196 positive nuclei/mm2.A significant reduction in the number of apoptotic cells (174 �31 positive nuclei/mm2) was observed in rats treated with CsAin which the MR was blocked starting on day 18 (CsA�Spgroup). In addition to these findings, we observed that CsA-treated rats exhibited a 2.5-fold increase in renal cortex pro-caspase 3 mRNA expression when compared with controlgroup (Fig. 6D). Spironolactone administration also signifi-cantly reduced the expression of procaspase 3. These resultsrevealed that CsA induced significant degree of apoptosis inthe kidney that was reduced by MR blockade.

The renoprotective effect of spironolactone was also cor-roborated with the evaluation of the expression of Kim-1that we demonstrated to be a sensitive marker of renal injury

(48). Figure 7, A and B, shows cortical mRNA levels andurinary protein Kim-1, respectively. Chronic CsA nephro-toxicity was associated with a marked increase in Kim-1mRNA and Kim-1 protein levels by 40- and 8-fold, respec-tively. The Kim-1 upregulation was partially prevented bythe use of a MR inhibitor. An inversely proportional andsignificant correlation between Kim-1 urine levels and cre-atinine clearance was observed (r � 0.8441), thus, withgreater Kim-1 expression is associated with a greater reduc-tion in creatinine clearance.

We previously showed that prevention of chronic CsAtoxicity when spironolactone was administered since the firstday of treatment with CsA was associated with significantreduction in the CsA-induced upregulation of TGF-�-mRNA(11). As shown in Fig. 8, the chronic CsA-treated groupexhibited a threefold increase in TGF-� mRNA levels normal-ized to 18S (3.1 � 0.2), compared with control group. Admin-istration of spironolactone to rats with existing chronic CsA

Fig. 6. A and B: representative microphoto-graphs showing apoptotic cells by TUNELassay of CsA and CsA�Sp groups, respec-tively. C: apoptotic-positive nuclei per mm2

in CsA and CsA�Sp groups. D: pro-caspase-3 mRNA levels in the 4 groups in-cluded at the end of the study (36 days). V(open bars), Sp (hatched bars), CsA (filledbars), and CsA�Sp (gray bars). *P 0.05vs. all the groups. **P 0.05 vs. the CsAgroup.

Fig. 7. mRNA levels of kidney injury mole-cule (Kim-1; A) and urinary Kim-1 shedding(B) in the 4 groups included at the end of thestudy (36 days). *P 0.05 vs. all the groups.**P 0.05 vs. CsA group.

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nephrotoxicity partially prevented the increase in TGF-�mRNA levels (1.9 � 0.1, P 0.05).

DISCUSSION

In the present study, we found that MR blockade withspironolactone increases rat survival and prevents the progres-sion of renal dysfunction, tubulointerstitial fibrosis, and arte-riolar thickening. Furthermore, MR blockade reduces theamounts of apoptotic cells seen with preexisting chronic CsAnephrotoxicity. MR blockade mitigates the reduction of glo-merular diameter and reduction of TGF-� and procaspase-3mRNA levels, as well as Kim-1 expression seen with contin-ued CsA treatment.

Distal convoluted tubule cells and principal cells of collect-ing duct were considered for many years to be the main cellulartarget of aldosterone actions. In the last decade, however, thelocalization of MR in several epithelial and nonepithelialtissues has provided evidence that aldosterone plays a role in avariety of physiological and pathophysiological processes (4,14, 20, 31–37, 40). These studies support the hypothesis that inaddition to its physiological role in maintaining extracellularsalt, potassium and water homeostasis, aldosterone seems toplay an additional role as a profibrotic hormone, thus contrib-uting to organ damage when produced in excess. For example,it has been reported that treatment of patients with chronicheart disease with spironolactone or eplerenone improves thesurvival rates due, at least in part, to the reduction in myocar-dial fibrosis (5, 32). In this regard, recent studies from ourlaboratory have also demonstrated that aldosterone plays apivotal role in both renal functional and structural toxic effectsof CsA (11, 29). Our previous studies, however, were designedto assess the prevention of renal injury with MR blockade,rather than to examine possible prevention of progressionand/or regression of already established renal injury.

Once chronic CsA was established after 18 days of treatmentand confirmed through renal function and structural analysis,one group of rats received simultaneously CsA and spirono-lactone administration during 18 days more and was comparedwith the group that received only CsA for 36 days. Weobserved that renal dysfunction already established in CsA-treated rats was not reverted by MR blockade. However,spironolactone administration beginning on day 18 of CsAtreatment avoided further renal function deterioration. At thestructural level, spironolactone produced significant renal pro-tection associated with a reduction of arteriolar thickening,

apoptosis, and the area affected by tubulointerstitial fibrosis,despite the continuous administration of CsA. Because it isknown that tubulointerstitial fibrosis is associated with higherrates of progression of renal disease (for a review, see Ref. 26),the reduction observed in fibrosis in the CsA�Sp group couldbe responsible for the reduction in renal injury.

In the present study, we observed that the degree of tubulo-interstitial fibrosis but not arteriolopathy was lower after MRblockade, suggesting that spironolactone beneficial effect wasmore evident for tubulointerstitial fibrosis. However, in ratstreated only with CsA, the degree of arteriolopathy observed at18 days was similar to that at 36 days. That is, the percentageof arteriolopathy did not increase after 18 days. In contrast, thedegree of fibrosis at 18 days was lower than at 36 days. Thus,the area affected by fibrosis progressed but the percentage ofarteriolopathy did not. In this regard, however, it is worth thatalthough reduction of arteriolopathy induced by spironolactonewas not significant, there was a significant decrease in arterio-lar thickening in the CsA�Sp group that was associated withgreater glomerular size. We observed that distribution of glo-merular diameters by rank in the CsA�Sp group was similar tocontrol group, while CsA alone-treated animals exhibited areduced glomerular size distribution, suggesting that MRblockade maintains a better renal perfusion state, counteractingCsA-inducing vasoconstriction. In this regard, we previouslyobserved that spironolactone completely prevented the renaldysfunction and reduction of renal blood flow induced by CsAduring acute nephropathy in which afferent arteriolar vasocon-striction is an important component (29), suggesting that aldo-sterone plays a role in the regulation of vascular tone. Themechanism of aldosterone-inducing vasoconstriction in CsAnephrotoxicity is not well understood. We have observed thatCsA upregulates the expression of prorenin and modulates theexpression of certain receptors that mediate vasoconstrictor orvasodilator actions that together could be responsible for renalvasoconstriction. These effects of CsA were partially pre-vented by spironolactone (29).

In the heart, it has been recognized that MR activationcontributes to the pathophysiology of heart failure by inducingcardiomyocyte apoptosis that can be prevented by MR block-ade, a finding that may explain the beneficial effects of MRblockade on ventricular remodeling following myocardial in-farction (6, 24). Moreover, in recent studies, Burniston et al.(6) and Williams (51) demonstrated that aldosterone increasedapoptosis also in skeletal muscle and human vascular endothe-lial cells through a mechanism that implicated activation ofcaspase-3. In the present study, we extended these findings tothe kidney. Chronic CsA administration in rats fed with alow-sodium diet produced a significant increase in pro-caspase-3 mRNA levels and cellular apoptosis. These effectswere reduced by MR blockade, suggesting that aldosteronepromotes cell death by apoptosis in chronic CsA toxicity.

Previous studies from us and others have shown that chronictoxicity induced by CsA is associated with TGF-� overexpres-sion and pharmacological agents that reduced the expression ofthis profibrotic cytokine-reduced renal injury (11, 42, 44, 52).Therefore, these studies emphasize the role of TGF-� inpromoting renal structural injury. Indeed, renal protection con-ferred by spironolactone in preexisting chronic CsA nephro-toxicity was associated with reduction of TGF-� mRNA levels,confirming our previous finding that TGF-� upregulation by

Fig. 8. mRNA levels of transforming growth factor (TGF-�) in the 4 groupsincluded at the end of the study (36 days). V (open bars), Sp (hatched bars),CsA (filled bars), and CsA�Sp groups (gray bars). *P 0.05 vs. all thegroups. **P 0.05 vs. CsA group.

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cyclosporine is at least partially responsible for renal injuryobserved in this model (11). In this regard, Juknevicius et al.(20) observed that aldosterone infusion to normal rats resultedin twofold increase of TGF-� urinary excretion withoutchanges at transcriptional level. Thus, in the model of CsAadministration plus low-salt diet, two independent stimuli toincrease TGF-� expression concur and the fact that spirono-lactone prevented the increase in TGF-� mRNA suggests thatthe mechanism by which CsA induced this upregulation in-volves the activity of the MR receptor.

Kim-1, a recently discovered type 1 transmembrane glyco-protein, is undetectable in normal kidneys but is upregulated10- to 100-fold following renal proximal tubular damage inhumans, rats, and mice (3, 15, 17, 18, 48). The function ofKim-1 is unclear, but it is implicated in damage processes andit has been proposed as a kidney injury marker. The Kim-1ectodomain is cleaved by metalloproteinases and is detectablein urine. In chronic CsA nephropathy characterized by tubularinjury, we observed a significant Kim-1 overexpression, evi-denced by a 40-fold increase of Kim-1 mRNA levels and an8-fold increase in Kim-1 ectodomain amount detected in theurine. Interestingly, the renal structural protection conferred byspironolactone was associated with reduction of urinaryKim-1, supporting the notion that Kim-1 could be also amarker of renal injury in chronic nephropathies.

In summary, our data show that the aldosterone/MR path-way plays an important role in the pathophysiology of chronicCsA nephropathy and reveal that spironolactone administrationdecreases the slope of renal function deterioration and struc-tural damage in preexisting chronic CsA nephrotoxicity, de-spite continuous CsA administration. Our data suggest thepotential use of spironolactone to reduce chronic nephropathyin patients that are receiving this immunosuppressive agent andsuggest that MR blockade could also be useful in other chronicnephropathies in which the insult cannot be avoided.

ACKNOWLEDGMENTS

We thank N. Vazquez for technical assistance.

GRANTS

Part of this work was presented at the Experimental Biology Meeting 2006,San Francisco, CA. This work was supported by research Grant 208602–3from the Mexican Council of Science and Technology (CONACYT) and bythe grant DGAPA IN228206–3 from National University of Mexico (to N. A.Bobadilla); National Institutes of Health Grants DK-39773, DK-72381, andDK-74099 (to J. V. Bonventre); and American Heart Association ScientistDevelopment Grant 0535492T (to V. S. Vaidya). J. P. Rojas is supported byscholarship grants from CONACYT and DGAPA.

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