Regression of Existing Glomerulosclerosis by Inhibition of Aldosterone
Jean Claude Aldigier,
Talerngsak Kanjanbuch,
Li-Jun Ma,
Nancy J. Brown and
Agnes B. Fogo
Departments of Pathology and Medicine, Vanderbilt University Medical Center, Nashville, Tennessee
Address correspondence to: Dr. Agnes B. Fogo, MCN C3310, Department of Pathology, Vanderbilt University Medical Center, 21st and Garland Avenue, Nashville, TN 37232-2561. Phone: 615-322-3114; Fax: 615-343-7023; agnes.fogo{at}vanderbilt.edu
Received for publication September 28, 2004.
Accepted for publication August 5, 2005.
In this study, the effects of inhibition of aldosterone on regressionof existing hypertension-related glomerulosclerosis were investigated.Adult male Sprague Dawley rats (220 to 250 g) underwent 5/6nephrectomy (Nx). Severity of glomerulosclerosis was assessedby renal biopsy 8 wk later, and rats were divided into fourgroups with equal biopsy sclerosis and then randomized by groupto 4-wk treatments as follows: Control with no further treatment(CONT; n = 6); spironolactone (SP) alone (200 mg/kg per d, bygavage, n = 6); or SP combined with nonspecific triple antihypertensivedrugs (TRX; reserpine, hydralazine, and hydrochlorothiazidein drinking water; SP+TRX, n = 7) or with angiotensin type 1receptor antagonist (AT1RA; losartan in drinking water; SP+AT1RA,n = 8). When the rats were killed 12 wk after Nx, autopsy glomerulosclerosisindex (SI; 0 to 4+ scale) was compared with biopsy SI in thesame rats. Systolic BP was increased at 8 wk after Nx and continuedto increase at 12 wk after Nx in the CONT and SP groups butnot in SP+TRX- or SP+AT1RA-treated rats. Serum creatinine at12 wk was significantly decreased in all SP-treated groups versusCONT. CONT rats had on average a 157% increase in SI from biopsyto killing at 12 wk, compared with only 84% increase in SP rats,with regression of SI in some rats. The effects on glomerulosclerosisby SP were further enhanced (when systolic BP was controlledby TRX or by AT1RA). It is concluded that inhibition of aldosteroneby SP not only slows development of glomerulosclerosis but alsoinduces regression in some rats of existing glomerulosclerosis.
Interruption of the renin-angiotensin-aldosterone system (RAAS)by angiotensin-converting enzyme inhibition (ACEI) or angiotensinII (Ang II) type 1 receptor antagonism dramatically alters thecourse of renal disease in the remnant kidney model (1,2). Furthermore,ACEI and Ang II receptor antagonists have proved clinicallyeffective in slowing the decline in renal function of severalnephropathies, including diabetic nephropathy (3–6). Theserenoprotective effects are associated with reductions in systemicarterial and glomerular pressures (1,2). Attenuation of growth-promotingand other fibroproliferative effects of Ang II may also contributeto the protection against progressive renal injury (7). Therenoprotective effects of RAAS blockade may also derive fromthe prevention of aldosterone-induced glomerular injury. Aldosteronecontributes to renal injury in the remnant kidney model (8,9);conversely, aldosterone receptor antagonism decreases the developmentof glomerular damage and arteriopathy in the stroke-prone spontaneouslyhypertensive rat (10) and in a radiation model of renal damage,independent of effects on BP (11).
Experimental studies on the renoprotective effects of RAAS blockadehave typically been designed to assess effects on preventionof proteinuria and kidney injury, rather than to examine possibleregression of already established structural change. Althoughprogressive deterioration of renal function has been consideredan inexorable process, recent observations in experimental animalsand humans have indicated that regression of renal structuralinjury may occur. We have found that inhibition of the RAASwith an Ang II type 1 receptor antagonist given at high dosesfor 6 mo caused remodeling of sclerosis in aging rats (12).Ang II antagonism also normalized proteinuria, eliminated inflammatorycell infiltration, and ameliorated glomerular and tubular structuralchanges as shown by Remuzzi’s group in the spontaneousovert nephropathy that develops in male Munich Wistar Fromterrats (13). In humans, regression of chronic renal disease, inferredby effects on proteinuria, was seen in the Ramipril Efficacyin Nephropathy follow-up study (6). Direct evidence of regressionwas seen by repeated renal biopsy in patients with diabeticnephropathy over 10 yr after pancreas transplantation curedtheir diabetes. These biopsies showed a regression of renallesions (14).
The aim of this study was to investigate whether an aldosteronereceptor antagonist could contribute to regression of existingglomerulosclerosis in the remnant kidney model of sclerosis.Our results show that the aldosterone receptor antagonist notonly slows progression of glomerulosclerosis but also can induceregression of existing glomerulosclerosis in some rats.
Experimental Design and Animals
Adult male Sprague Dawley rats (250 to 300 g; Charles River,Nashville, TN) were studied. Rats were housed under normal conditionswith a 12-h light/dark cycle at 70°F with 40% humidity and12 air exchanges per hour and received standard rat powdereddiet (Purina Rodent "5001" meal, 23.4% protein, 4.5% fat, 6%fiber, 0.40% sodium; Tusculum Feed Center, Nashville, TN) andwater. Rats underwent 5/6 nephrectomy (Nx) by right Nx and ligationof two or three main branches of the left renal artery by silkligature to remove approximately 5/6 renal mass. The surgerywas performed under anesthesia with sodium pentobarbital (50mg/kg body wt, intraperitoneally). Eight weeks after reductionin renal mass, rats underwent a renal biopsy by shave biopsyunder anesthesia by laparotomy approach to determine the severityof existing sclerosis (15). The number of glomeruli availablefor biopsy analysis was, on average 30 (range 11 to 59). Ratsthen were stratified on the basis of this analysis and assignedto one of four treatment groups with initial sclerosis indexequivalent in all groups (Figure 1). The four treatment groupswere (1) control, no further treatment (n = 6); (2) spironolactone200 mg/kg per d solubilized in peanut oil, by gavage (SP; n= 6); (3) SP + antihypertensive triple therapy (reserpine 5mg/L drinking water [DW], hydralazine 80 mg/L DW, and hydrochlorothiazide[HCTZ] 25 mg/L DW; SP+TRX; n = 7); or (4) SP as above combinedwith angiotensin type 1 receptor antagonist (losartan 80 mg/LDW; SP+AT1RA; n = 8). The dose of losartan is four-fold higherthan the usual antihypertensive dose and regressed sclerosisin this model in two thirds of rats in a previous study (15).This dose of TRX was chosen as it normalizes systemic BP, aswe and others have previously shown in previous studies in thismodel, but does not protect against glomerulosclerosis (1,16).The dose of SP is based on our previous study (11). These treatmentswere continued for the next 4 wk. Body weight, BP, and proteinuriawere evaluated at weeks 0, 4, 8, and 12. At 12 wk, animals werekilled and kidneys were harvested for analysis of morphologicand molecular parameters. The average number of glomeruli availablefor analysis in the remnant kidney at killing was 84 (range53 to 123).
On the basis of initial observations in the above SP+TRX group,we also studied the effects of the diuretic HCTZ component ofthe TRX on plasminogen activator inhibitor-1 (PAI-1) expressionin normal rats, as the HCTZ could influence the RAAS axis. Wethus hypothesized on the basis of our initial results showinghigher PAI-1 in the group with added TRX than in other groupsthat the well-known diuretic-induced RAAS activation could haveactivated PAI-1. Therefore, we treated an additional group ofnormal male Sprague Dawley rats (n = 10) without 5/6 Nx withhydrochlorothiazide 50 mg/L DW and killed them at day 3 andat weeks 4, 8, and 12. Effects of treatment on plasma concentrationof PAI-1 and renal PAI-1 mRNA were assessed.
Analysis of Kidney Function
Systolic BP (SBP) was measured using tail-cuff plethysmographyin unanesthetized prewarmed trained rats at ambient temperatureof 29°C. Animals were placed in metabolic cages for 24 hfor urine collection, and urine protein was measured by Bio-RadProtein Assay Kit (Bio-Rad Laboratories, Hercules, CA). Serumcreatinine was measured by Vitros CREA slides (Johnson &Johnson Clinical Diagnostics Inc., Rochester, NY). The plasmaconcentrations of PAI-1 were determined using a double antibodyenzyme immunoassay (Innotest; Byk-Sangtec, Dietzenbach, Germany).
Structural Analysis
Kidney tissue from rats was immersion-fixed in 4% paraformaldehyde/PBSsolution and processed routinely, and 4-µm sections werestained with periodic acid-Schiff and Masson’s trichrome.A semiquantitative sclerosis index (SI) score was used to evaluatethe degree of glomerular sclerosis. The severity of sclerosisfor each glomerulus was graded from 0 to 4+ as follows: 0, nolesion; 1+, sclerosis of up to 25% of the glomerulus; while2+, 3+, and 4+, sclerosis of >25 to 50%, >50 to 75%, and>75% of the glomerulus, respectively (16). A whole kidneyaverage SI was obtained by averaging scores from all glomerulion one section. Tubulointerstitial fibrosis and vascular lesionswere assessed qualitatively. All sections were examined withoutknowledge of the treatment protocol.
Portions of kidney tissues from paraffin blocks from kidneysat killing from representative rats from the above studies wereprocessed for electron microscopy (EM). We chose three to fourrats from each group, representative of the average proteinuriafor that group. Nonsclerotic glomeruli from the middle of eachscout section then were chosen for ultrastructural examination.EM sections were examined directly using a Fei Morgagni electronmicroscope, without knowledge of the treatment protocol or degreeof proteinuria, and foot process effacement was assessed byestimating degree of effacement for each grid square and averagingfor all portions.
Northern Blot Hybridization
Total RNA from renal cortex was extracted by the RNAzol B method(Tel-Test, Inc., Friendswood, TX). Twenty micrograms of totalRNA was loaded and fractionated by electrophoresis in 1% agarosegel and transferred to a nylon membrane. Mouse PAI-1 and TGF-cDNA were labeled with 32PdCTP, and hybridization was performedin buffer (4x SSCP, 1x Denhardt’s, 1% SDS, 100 µg/mldenatured salmon sperm DNA, and 10% dextran sulfate) overnightat 65°C. The membrane was washed, air-dried, and exposedto XAR film (Eastman Kodak, Rochester, NY) in intensifying screenat –70°C for 3 to 5 d. Autoradiographs were scannedby image scanner JX-330 (Sharp, Tokyo, Japan), and the intensityof the signals was measured by National Institutes of Healthimage (Bethesda, MD). The ratio of specific message to the housekeepinggene glyceraldehyde-3-phosphate dehydrogenase was used to quantifythe expression for each tissue sample.
In Situ Hybridization
[35S]-labeled sense and antisense riboprobes for PAI-1 and TGF-were prepared as described previously (16). Briefly, sectionswere treated with proteinase K and triethanolamine/acetic anhydride,and hybridization was done at 50°C. Sections were washedin buffer with 5x SSC and 20 mmol/L -mercapto-ethanol at 50°Cfor 15 min, then washed in 2x SSC, 50% formamide at 68°Cfor 20 min, in TEN twice at 37°C for 10 min and incubatedtwice in 2x SSC and 0.1x SSC at 68°C for 15 min. Sectionsthen were dehydrated in ethanol and air-dried, dipped in photographicemulsion, and exposed at 4°C for 10 d. The sections weredeveloped with D-19 developer (Eastman Kodak) and counterstainedwith toluidine blue. Negative control in situ hybridizationswere done with sense probes and showed no specific signal.
Statistical Analyses
Results are expressed as mean ± SEM. Statistical differencewas assessed by a single factor variance followed by unpairedt test or Mann-Whitney U test for nonparametric data. Podocytefoot process effacement and proteinuria were compared by Spearmancorrelation coefficient. P < 0.05 was considered to be significant.
Body Weight and Renal Function
Body weights were similar in all groups at 8 wk after 5/6 Nx(CONT 336.0 ± 8.4, SP 338.9 ± 5.8, SP+TRX 347.0± 10.9, SP+AT1RA 341.1 ± 7.1 g). However, by 12wk, body weight was higher in treated rats versus untreatedcontrol, primarily as a result of weight loss in the controls,likely reflecting severe uremia (CONT 310.7 ± 7.5, SP339.2 + 9.9, SP+TRX 339.1 ± 22.9, SP+AT1RA 362.8 ±6.1 g; CONT versus all treated groups, P < 0.05).
SBP was increased significantly in all groups by 4 wk versusbaseline (115.2 ± 2.9 mmHg; P < 0.01) after 5/6 Nxand was similar at 8 wk (CONT 180.7 ± 7.2, SP 209.5 ±9.7, SP+TRX 203.9 ± 9.6, SP+AT1RA 202.2 ± 8.8mmHg). After randomization to treatment groups, SBP decreasedsignificantly compared with control (213.3 ± 5.0 mmHg)from 8 to 12 wk in the SP+TRX (172.3 ± 13.0 mmHg; P <0.01) and SP+AT1RA groups (186.8 ± 7.9 mmHg; P < 0.01)but not in the SP alone group (225 ± 2.8 mmHg; Figure 2).
Figure 2. Systolic BP (SBP) changes after 5/6 nephrectomy (Nx). SBP decreased significantly in treatment groups, except spironolactone (SP) alone, versus control with no further treatment (CONT) at 12 wk after 5/6 Nx. *P < 0.01 versus CONT.
Serum creatinine increased over 12 wk in untreated control 5/6Nx rats. In all treated groups, renal function was better preserved(serum creatinine SP 0.88 ± 0.13, SP+TRX 0.86 ±0.05, SP + AT1RA 0.97 ± 0.08 versus CONT 2.90 ±0.58 mg/dl; P < 0.05; Figure 3).
Figure 3. Serum creatinine at 12 wk after 5/6 Nx. Renal function, assessed by serum creatinine, was improved in all treatment groups versus CONT.
Urinary protein excretion was dramatically increased by 4 wkafter 5/6 Nx (167.0 ± 17.6 versus baseline 5.6 ±0.8 mg/24 h; P < 0.001) and continued to increase (at 8 wk182.0 ± 13.8 mg/24 h; P < 0.001 versus baseline).Untreated control rats had further increased proteinuria at12 wk (397.6 ± 47 mg/24 h; P < 0.001 compared withbaseline). SP alone or in combination did not significantlyaffect the course of proteinuria (at 12 wk: SP 295.2 ±44.0, SP+TRX 364.7 ± 81.5, SP+AT1RA 331.1 ± 31.3mg/24 h; NS versus CONT; Figure 4).
Figure 4. Proteinuria changes after 5/6 NX. Proteinuria was less in all treated groups versus CONT at 12 wk after 5/6 Nx.
Renal Morphologic Changes
In all 5/6 Nx rats, glomerulosclerosis, tubular atrophy, dilation,and interstitial fibrosis were present at 8 wk. By study design,all groups had similar levels of sclerosis at 8 wk. Sclerosisprogressed further by 12 wk in untreated 5/6 Nx controls. SPalone or in combination with either TRX or AT1RA significantlyameliorated the development of glomerulosclerosis compared withcontrol 5/6 Nx (SI at 12 wk: CONT 2.11 ± 0.24 versusSP 0.89 ± 0.15, SP+TRX 0.40 ± 0.09, SP+AT1RA 0.82± 0.32; P < 0.01; Figure 5). In all untreated controlrats, structural abnormalities worsened; over time, sclerosisinvolved more glomeruli and was more severe in affected glomeruli.In these untreated rats, glomeruli without lesions composedon average 50% at biopsy, decreasing to 29% at autopsy. Treatmentwith SP was associated with regression of glomerulosclerosisin 33% of rats (two of six) when used alone, 60% (four of seven)when used with TRX, and 50% (four of eight) when used with losartan(Figure 6). Glomeruli without lesions on average for all ratsdecreased slightly from biopsy to autopsy for SP alone but increasedin SP+TRX and SP+AT1RA (Figure 7). Thus, regression was increasedwhen SP was associated with effective systemic antihypertensivetherapy (SP+TRX versus SP and SP+AT1RA versus SP; P < 0.01).We next compared beginning severity of sclerosis in animalswith regression response with those with continued progression.This analysis showed that in rats with regression in responseto treatment, average SI of glomeruli at biopsy was <1.2.In rats with progression despite treatment, SI was >1.2 atbiopsy. Tubulointerstitial fibrosis and vascular lesions improvedin parallel with sclerosis. Electron microscopy showed variablebut more severe foot process effacement in control versus alltreated rats (n = 3; average 62%; 15, 85, and 85% in individualrats). In rats that were treated only with SP, foot processeffacement was on average 13% (n = 4; 25, 7.5, 15, and 5% inindividual rats). In rats that were treated with SP+TRX, tissuefor EM was inadequate in one. The remaining two rats showed45 and 7.5% foot process effacement. Best foot process restorationwas observed in rats that were treated with SP+AT1RA, in whichaverage foot process effacement was only 5% (n = 3; 5, 10, and0% in individual rats; Figure 8). However, for all rats examined,degree of foot process effacement did not correlate significantlywith degree of proteinuria (r = 0.204, NS). Further comparisonof degree of proteinuria in all rats with regression pooledversus all rats with continued progression, regardless of treatmentgroup, showed no difference in proteinuria (361.1 versus 332.6mg/24 h, respectively; NS).
Figure 5. Kidney histopathologic changes after 5/6 Nx. (A) SP alone or in combination with triple antihypertensive drugs (TRX; reserpine, hydralazine, and hydrochlorothiazide in drinking water) or angiotensin type 1 receptor antagonist (AT1RA) significantly decreased glomerulosclerosis (assessed by sclerosis index [SI]) versus untreated 5/6 Nx CONT. (B) Biopsy (left) at week 8 and autopsy (right) at week 12 from the same animal in untreated CONT with progression, SP-induced regression, or SP+AT1RA–induced regression. Magnification, x400, periodic acid-Schiff.
Figure 6. Changes in SI from biopsy to autopsy. Each circle represents the change in SI from biopsy at week 8 to autopsy at week 12 after 5/6 Nx in an individual rat. SI was increased in untreated CONT rats from biopsy to autopsy. Regression, with less sclerosis at autopsy than at biopsy, occurred in some rats in all treated groups. *P < 0.01 versus CONT; #P < 0.01 versus SP.
Figure 8. Foot process effacement was extensive in untreated control 5/6 Nx rats at week 12 (left) compared with partial restoration of foot processes in treated rats, most prominent in SP+losartan-treated rats (right). Magnification, x3000, transmission electron microscopy.
TGF-1 mRNA Expression
TGF- mRNA levels that were assessed by Northern blot analysisin the renal cortex were increased in untreated control 5/6Nx and were not significantly influenced by treatment (CONT0.76 ± 0.27 versus SP 0.55 ± 0.09, SP+TRX 0.69± 0.08, SP+AT1RA 0.67 ± 0.09; NS CONT versus alltreatment groups; Figure 6, A and C). By in situ hybridization,signal was marked in areas with increased tubulointerstitialfibrosis, surrounding large arteries, with focal increase inmesangial areas, and rare localization suggestive of podocytesignals. Bowman’s capsule also showed increased signal,especially in sclerotic glomeruli. With SP treatment, TGF- signalwas equally strong in areas with tubulointerstitial fibrosis,with remaining moderate to strong signals in glomerular mesangialareas and in Bowman’s capsule. Similar pattern and intensitywere present in SP+TRX rats in the tubulointerstitium, but therewas in addition mild to moderate signals in the tubules of thedeep cortex and at the corticomedullary junction and only minimalsignal in glomeruli, with remaining signal in Bowman’scapsule. With SP+AT1RA, there was mild to moderate signal infibrotic areas, focal mild signal in mesangial areas and Bowman’scapsule, and moderate signal in artery adventitia (Figure 9).
Figure 9. TGF- mRNA expression at week 12 was increased in fibrotic tubulointerstitium and focal mesangial areas in untreated 5/6 Nx rats (A), with similar pattern in fibrotic areas in SP-treated rats (B). Remaining fibrotic areas in rats that were treated with SP+TRX (C) or SP+AT1RA (D) also showed TGF- signal, but glomerular signal was decreased (in situ hybridization). Magnification, x200.
PAI-1 mRNA Expression
PAI-1 mRNA levels that were assessed by Northern blot analysisin the renal cortex were increased in untreated control 5/6Nx. The level of PAI-1 mRNA was attenuated after treatment withSP and SP+AT1RA. Rats that were treated with SP+TRX achievedonly a numeric decrease in PAI-1 mRNA expression (CONT 1.50± 0.23 versus SP 0.78 ± 0.23, SP+TRX 1.10 ±0.2, SP+AT1RA 0.66 ± 0.1; P < 0.01 CONT versus SPand SP+AT1RA, NS CONT versus SP+TRX; Figure 10).
Figure 10. Northern blot analysis of kidney mRNA expressions at week 12 for plasminogen activator inhibitor-1 (PAI-1; A and B) and TGF-1 (A and C); n = 3 for normal, n = 4 to 6 for CONT and all treated groups.
In situ hybridization revealed high-level PAI-1 mRNA expressionin untreated 5/6 Nx rats in sclerotic glomeruli and fibrotictubules and interstitium. In contrast, the PAI-1 signal wasmarkedly diminished in SP- and SP+AT1RA-treated rats (Figure 11,A through C). PAI-1 signal was moderately attenuated afterSP+TRX treatment (Figure 11D).
Figure 11. PAI-1 mRNA expression by in situ hybridization. Untreated 5/6 Nx CONT rats at week 12 showed increased PAI-1 mRNA expression in sclerotic glomeruli (A). PAI-1 signals at week 12 were markedly diminished in SP-treated (B), SP+AT1RA-treated (C), and SP+TRX-treated (D) kidneys. Magnification, x400.
Plasma PAI-1 Concentration and Diuretic Treatment
In view of the lack of significant PAI-1 mRNA decrease whenTRX was added to SP, we next examined the effects of the diureticcomponent of the treatment, HCTZ, on PAI-1 levels. Plasma activePAI-1 after chronic HCTZ treatment in normal rats was increased(day 0 11.4 ± 0.55, day 3 18.0 ± 2.3, week 2 13.6± 5.1, week 4 13.6 ± 2.6, week 8 10.2 ±0.94, week 12 42.8 ± 22.8 ng/ml; P < 0.01 week 12versus weeks 2, 4, and 8; Figure 12). However, the source doesnot seem to be predominantly renal, as renal PAI-1 mRNA wasonly mildly increased by Northern blot or in situ hybridizationin these rats (Figure 13).
Figure 12. Plasma PAI-1 activity after diuretic treatment. Plasma active PAI-1 after chronic hydrochlorothiazide treatment in normal rats was increased versus baseline.
Many intervention strategies to slow or even reverse the progressionof glomerulosclerosis have been explored (1,2,13–17).In this study, we demonstrated that the mineralocorticoid antagonistSP alone or even more with added antihypertensive drugs canameliorate progression or even regress glomerulosclerosis insome rats in the hypertensive 5/6 nephrectomy model.
Increased attention has focused on aldosterone as a potentiallyimportant mediator of chronic heart failure and renal disease(18–23). Furthermore, it has been postulated that beneficialeffects of ACEI may be related to a decrease in aldosteronelevel (23). Aldosterone promotes fibrosis and vascular toxicityin a variety of experimental animal models. Prolonged aldosteroneadministration causes myocardial fibrosis and ventricular hypertrophyin rats (24), and aldosterone infusion abrogates the renal protectionin stroke-prone spontaneously hypertensive or 5/6 Nx rats conferredby Ang II inhibition either with ACEI or Ang II type 1 receptorantagonists (8,9,25). However, in past experiments in the remnantkidney model, SP alone did not reduce glomerulosclerosis (8),although selective blockade of aldosterone with eplerenone reducedproteinuria and glomerulosclerosis in L-NAME–treated hypertensiverats (26). SP was effective in preventing arteriolopathy andtubulointerstitial fibrosis in experimental chronic cyclosporineA nephrotoxicity and in attenuating glomerulosclerosis in aradiation-induced nephrosclerosis model by SP, an effect linkedto decreased PAI-1 (11,27).
Aldosterone also modulates the vascular tone, possibly throughincreased vasoconstrictive effects of catecholamines (28), impairedvasodilation in response to acetylcholine (29), and upregulationof -adrenergic and Ang II receptors (30,31). Additional directaldosterone effects are postulated to be mediated by nongenomicand/or nonhemodynamic mechanisms (24,32–34).
BP is an important factor associated with progression (35).In this study, we observed less renoprotection with SP alonethan when conventional triple antihypertensive therapy, at dosesthat alone do not protect against glomerulosclerosis, or AT1RAwas added. It is interesting that urinary protein excretioncontinued to increase in all groups, demonstrating that effectson proteinuria and glomerulosclerosis may be discordant. Persistenthypertension may have allowed higher levels of persistent proteinuria.We showed previously by micropuncture that proteinuria largelyemanates from glomeruli without sclerosis, as the scleroticglomeruli do not filter and thus cannot give rise to proteinuria(36). These studies further suggest that podocyte injury, reflectedin increased permselectivity, persists even after light microscopicallydetectable sclerosis has regressed. We speculate that the podocytewith its limited regenerative capacity may have less abilityto heal and/or require longer time for regression of injurythan remodeling of the extracellular matrix of the scleroticlesion. Although EM studies showed less foot process effacementin treated rats than in control 5/6 Nx, there was no significantcorrelation with degree of proteinuria, suggesting that additionalfiltration barrier injury remained. Analysis of proteinuriain all rats with progression versus those with regression, regardlessof treatment, also revealed no difference in proteinuria amongall rats with progression versus all with regression. Thus,additional nonhemodynamic factors seem to have contributed toeffects on proteinuria and sclerosis in this study, perhapsreflecting unique characteristics of this rat model (37,38).
The sclerotic segment of the glomerulus is not static. Thereis ongoing cell proliferation and apoptosis, and modulationof matrix. To achieve regression, matrix degradation must exceedmatrix synthesis. This balance is influenced by key factorsthat promote collagen synthesis and degradation. TGF- has beenrecognized as a key mediator of renal fibrogenesis (39). AngII increases TGF- and promotes conversion of TGF- to its activeform (40,41). However, previous data on TGF- and mineralocorticoidsare conflicting. Aldosterone infusion in normal rats increasedTGF- mRNA in the kidney (42). In the uninephrectomized rat,TGF-, ACE, and AT1R expressions were increased in the kidneyafter aldosterone infusion (41). In contrast, in the DOCA-saltmodel, the TGF- level was not changed after treatment with mineralocorticoidantagonist (10). We previously in the radiation-induced injurymodel observed a mildly attenuated TGF- expression by SP (11).In this study, we did not observe decreased TGF- expressionat the whole-kidney level when sclerosis was decreased, suggestingthat modulation of TGF-, at least at the mRNA level, is nota major mechanism for the beneficial effects on sclerosis inthis setting. Expression by in situ hybridization remained prominentafter treatment, especially in SP and SP+TRX, with mild decreasewith SP+AT1RA, thus suggesting that although whole-kidney mRNAlevels of TGF- were not altered, local changes in TGF- couldhave had a modulating influence on sclerosis.
Amelioration of glomerulosclerosis in our study by SP alonewas linked to downregulated PAI-1 expression. PAI-1 is the majorinhibitor of tissue-type plasminogen activator and urokinase-typeplasminogen activators, which activate plasminogen to yieldplasmin, which in turn degrades both fibrin and matrix and increasesmatrix metalloproteinase production and activates latent matrixmetalloproteinase (43). PAI-1 may also have indirect effectson matrix synthesis, perhaps by increasing macrophage infiltration(44). Ang II induces PAI-1 expression directly through an AT1R-dependentmechanism and independently via TGF- (45–47). Aldosteroneinteracts with Ang II to increase PAI-1 expression through aglucocorticoid responsive element and a serum-inducible elementlocalized in the PAI-1 promoter (48,49). Furthermore, serumaldosterone concentrations correlate with PAI-1 antigen concentrationin humans (50), supporting a role of aldosterone in vivo inregulation of PAI-1 as well. Finally, inhibition of aldosteronedownregulated PAI-1 expression in vivo, and local PAI-1 expressionand sclerosis were tightly linked. These observations supportthe hypothesis that aldosterone induces renal injury at leastin part through its effects on PAI-1 expression (11).
In this model, even SP alone had a modest effect on sclerosisand PAI-1, supporting the hypothesis of a direct effect of aldosteroneon PAI-1 expression, with further benefit when effective BPcontrol was added. One limitation of our study is that the statusof RAAS activity was not defined. Regulated extra-adrenal synthesisof aldosterone has been demonstrated (51–53). However,circulating concentrations of aldosterone do not reflect localtissue level of aldosterone. We thus speculate that SP effectsare very likely secondary to adrenal and extra-adrenal aldosteroneantagonism and that local levels of aldosterone are of primaryimportance for effects on sclerosis.
Our studies further show that chronic diuretic use stimulatesPAI-1 expression. We have observed this effect in humans aswell. Dietary NaCl restriction to 10 mmol/d was associated witha stimulation of the RAAS and increased PAI-1 in healthy volunteers(50). Furthermore, PAI-1 antigen correlated highly with serumaldosterone levels (50). In normal volunteers, diuretic usewas associated with increased PAI-1. Acute administration oflosartan and SP together but not either alone blunted this effect,suggesting that this effect was RAAS mediated (52,54). It isinteresting that salt restriction but not HCTZ protected againstproteinuria, sclerosis, and renal and glomerular hypertrophyin the uninephrectomized SHR rat (55). These results were notdependent on plasma renin or glomerular pressure effects. Classicstudies have shown that "triple therapy" in the same doses usedin our study did not protect against sclerosis, despite normalizingsystemic BP, thought to reflect persistent glomerular hypertension(1). HCTZ itself was not protective against sclerosis (1,55).We speculate that an additional explanation for the above observationscould be that HCTZ, a component of this triple therapy, mayhave prosclerotic effects via PAI-1. Our data also show thatSP had a beneficial effect on sclerosis even with concomitantdiuretic, a treatment that failed to decrease significantlyPAI-1. This observation suggests that mineralocorticoid antagonismmay exert additional beneficial nonhemodynamic effects on sclerosisbeyond PAI-1. Further studies will be necessary to define preciselysuch additional potential mechanisms.
In conclusion, our study demonstrates that inhibition of aldosteronewith SP not only slows the progression of glomerulosclerosisbut also induces regression in some rats of existing glomerulosclerosis.These effects were independent of proteinuria changes and wereenhanced by added BP control.
Acknowledgments
These studies were supported by National Institutes of Healthgrants DK 56942 (A.B.F.) and HL 67308 (N.J.B.).
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Received for publication September 28, 2004.
Accepted for publication August 5, 2005.
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Abstract
Introduction
cDNA were labeled with 32PdCTP, and hybridization was performed in buffer (4x SSCP, 1x Denhardt’s, 1% SDS, 100 µg/ml denatured salmon sperm DNA, and 10% dextran sulfate) overnight at 65°C. The membrane was washed, air-dried, and exposed to XAR film (Eastman Kodak, Rochester, NY) in intensifying screen at –70°C for 3 to 5 d. Autoradiographs were scanned by image scanner JX-330 (Sharp, Tokyo, Japan), and the intensity of the signals was measured by National Institutes of Health image (Bethesda, MD). The ratio of specific message to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was used to quantify the expression for each tissue sample. 
