* Department of Nephrology and Endocrinology and Department of Clinical and Molecular Epidemiology, 22nd Century Medical and Research Center, University of Tokyo Graduate School of Medicine, and Clinical Research Center, National Hospital Organization, Murayama Medical Center, Tokyo, Japan
Address correspondence to: Dr. Miki Nagase, Department of Nephrology and Endocrinology, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Phone: +81-3-5800-9168; Fax: +81-3-5800-9169; E-mail: mnagase-tky{at}umin.ac.jp
Received for publication August 30, 2006.
Accepted for publication September 26, 2006.
Metabolic syndrome is an important risk factor for proteinuriaand chronic kidney disease independent of diabetes and hypertension;however, the underlying mechanisms have not been elucidated.Aldosterone is implicated in target organ injury of obesity-relateddisorders. This study investigated the role of aldosterone inthe early nephropathy of 17-wk-old SHR/NDmcr-cp, a rat modelof metabolic syndrome. Proteinuria was prominent in SHR/NDmcr-cpcompared with nonobese SHR, which was accompanied by podocyteinjury as evidenced by foot process effacement, induction ofdesmin and attenuation of nephrin. Serum aldosterone level,renal and glomerular expressions of aldosterone effector kinaseSgk1, and oxidative stress markers all were elevated in SHR/NDmcr-cp.Mineralocorticoid receptors were expressed in glomerular podocytes.Eplerenone, a selective aldosterone blocker, effectively improvedpodocyte damage, proteinuria, Sgk1, and oxidant stress. An antioxidanttempol also alleviated podocyte impairment and proteinuria,along with inhibition of Sgk1. As for the mechanisms of aldosteroneexcess, visceral adipocytes that were isolated from SHR/NDmcr-cpsecreted substances that stimulate aldosterone production inadrenocortical cells. The aldosterone-releasing activity ofadipocytes was not inhibited by candesartan. Adipocytes fromnonobese SHR did not show such activity. In conclusion, SHR/NDmcr-cpexhibit enhanced aldosterone signaling, podocyte injury, andproteinuria, which are ameliorated by eplerenone or tempol.The data also suggest that adipocyte-derived factors other thanangiotensin II might contribute to the aldosterone excess ofthis model.
Metabolic syndrome, a constellation of comorbidities that includevisceral obesity, hypertension, glucose intolerance, and dyslipidemia,is a highly predisposing condition for cardiovascular disease(1). Recent clinical studies revealed that metabolic syndromealso increases the risk for proteinuria and chronic kidney disease(CKD) (2,3). Management of proteinuria is critically importantin this syndrome, because proteinuria accelerates the progressionof CKD and increases the prevalence of cardiovascular events(4,5). Glomerular podocytes and their slit diaphragm are majorcomponents of the glomerular filtration barrier to prevent urinaryprotein loss (6). Podocyte injury plays a pivotal role in theproteinuria and glomerulosclerosis of diabetic and hypertensivenephropathy (7–9). Notably, rats with metabolic syndromewere shown to be more prone to podocyte damage than streptozotocin-induceddiabetic rats (10). However, the underlying mechanisms and efficienttherapy of nephropathy that are associated with metabolic syndromehave been poorly elucidated.
Accumulating evidence suggests that aldosterone is a potentinducer of proteinuria and podocyte injury. Greene et al. (11)demonstrated that antiproteinuric effects of angiotensin II(AngII) blockage in the remnant kidney rats were reversed byaldosterone infusion. Indeed, proteinuria is enhanced in patientswith primary aldosteronism and rats that receive infusion ofaldosterone (12–14). We previously showed that the selectivealdosterone blocker eplerenone dramatically ameliorated proteinuriaand podocyte injury in Dahl salt hypertensive rats, in whichaldosterone signaling is augmented (9,15). Enhanced aldosteronesignaling also is found in the kidneys of patients with heavyproteinuria (16). Several reports indicated that the deleteriouseffects of aldosterone may be mediated by oxidative stress (14).
SHR/NDmcr-cp (SHR/cp) is a rat model of metabolic syndrome thatmanifests hypertension (derived from SHR), obesity (as a resultof nonsense mutation in the leptin receptor gene), glucose intolerance,and hyperlipidemia (17,18). Aldosterone excess is describedin patients with obesity hypertension or metabolic syndrome(19–22), which may be renin independent (22). It is intriguingthat recent works suggested that adipocyte-derived substancesmay stimulate adrenal aldosterone synthesis (23,24), putativelymediating aldosterone excess in obesity-related disorders. Onthe basis of these findings, we postulated a hypothesis thatcirculating aldosterone level and its signaling in the kidneyare enhanced in metabolic syndrome, which causes podocyte injuryvia oxidative stress. In this study, we analyzed proteinuria,podocyte injury, and aldosterone and its effector Sgk1 in SHR/cpand nonobese SHR and examined the effects of eplerenone andthe antioxidant tempol. We also explored the possible role offat-derived aldosterone-releasing factors in SHR/cp.
Animals
Male SHR/cp (n = 44) and SHR (n = 32) were purchased from JapanSLC (Shizuoka, Japan). All animal procedures were in accordancewith the guidelines for the care and use of laboratory animalsapproved by University of Tokyo Graduate School of Medicine.SHR/cp and SHR at 13 wk of age were fed a normal rat diet for4 wk. Some SHR/cp were treated with eplerenone (1.25 g/kg food)or tempol (6 mM in tap water; Sigma, St. Louis, MO). For time-courseanalysis, SHR/cp and SHR at the indicated ages were used. Thefinal number of rats for each group was four to eight.
Systolic BP was measured by the tail-cuff method (9). Rats wereplaced in metabolic cages for 24-h urine collection. After fastingfor 16 h, rats were anesthetized with ether, and kidneys, adrenals,and epididymal fats were harvested. Glomerular fraction wasisolated by the graded sieving method (25). Biochemical andhormonal data in plasma, serum, and urine were measured at SRL(Tokyo, Japan).
Real-Time PCR
Total RNA was extracted using an RNeasy kit or RNeasy lipidtissue kit (Qiagen, Hilden, Germany). Gene expression was determinedby real-time quantitative reverse transcripion PCR using ABIPRISM 7000, TaqMan chemistry, and assay-on-demand primers andprobe sets, as described previously (Applied Biosystems, FosterCity, CA) (9).
Western Blotting
Western blotting was performed as described previously (9).The membrane was immunoblotted with rabbit anti-rat nephrin(1:10000; a gift from Dr. Kawachi, Niigata University, Niigata,Japan), rabbit anti-human Sgk1 (1:1000; Cell Signaling Technology,Danvers, MA), or rabbit anti-actin (1:500; Sigma).
Immunohistochemistry and Periodic Acid-Schiff Staining
Immunohistochemistry and semiquantitative analysis of desminwere performed as described (9). Immunofluorescence double stainingwas carried out as follows: For mineralocorticoid receptor (MR)and synaptopodin double staining, cryosections (4 µm)were boiled for antigen retrieval, incubated with rabbit anti-ratMR (1:1000; antibody raised against amino acids 103 to 507 ofrat MR; a gift from Dr. Kawata, Kyoto Prefectural Universityof Medicine, Kyoto, Japan) (26), peroxidase-conjugated anti-rabbitIgG, and Cy3-tyramide (PerkinElmer Life Sciences, Boston, MA).Samples then were immunolabeled with mouse anti-rat synaptopodin(1:10; Research Diagnostics, Flanders, NJ) and FITC-conjugatedanti-mouse IgG. For MR and WT-1 immunostaining, pretreated sectionswere incubated with mouse anti-human MR (1:200; antibody againstamino acids 2 to 99 of human MR; Perseus Proteomics, Tokyo,Japan), peroxidase-conjugated anti-mouse IgG, biotinyl-tyramide,and streptavidin-FITC. Subsequently, samples were immunolabeledwith rabbit anti-human WT-1 (1:500; Santa Cruz Biotechnology,Santa Cruz, CA) and Cy3-conjugated anti-rabbit IgG. The specificityof anti-rat MR was described in detail previously (26). We confirmedthe specificity of these two MR antibodies as follows: Westernblotting using the antibodies revealed a band with molecularmass of >100 kD (compatible with the size of 106 kD), andimmunohistochemistry without primary antibody resulted in negativestaining. Periodic acid-Schiff staining was performed as described(9).
Electron Microscopy
Ultrastructure of glomerular podocytes was analyzed using Hitachitransmission electron microscope H-7000 (Tokyo, Japan), as describedpreviously (25). Morphology of epididymal fats was examinedusing Hitachi scanning electron microscope S-3500N, as describedpreviously (27).
Urinary 8-OHdG Excretion
Urine was ultrafiltrated using Microcon YM-10 (Millipore, Billerica,MA) to separate interfering substances. Then urinary 8-hydroxy-2'-deoxyguanosine(8-OHdG) concentration was measured using the 8-OHdG ELISA Kit(Japan Institute for the Control of Aging, Shizuoka, Japan).
Aldosterone Releasing Activity of Adipocytes
Isolation of adipocytes and preparation of fat cell–conditionedmedium (FCCM) were performed according to the method by Ehrhart-Bornsteinet al. (23). Briefly, fresh adipose tissues were minced immediatelyinto small pieces in Krebs Ringer Bicarbonate buffer that contained3% BSA and 4 mM glucose, digested with 0.05% collagenase typeII (Sigma) for 60 min at 37°C, filtered through a nylonmesh (425 µm), and washed. Isolated adipocytes were culturedin 4 vol of DMEM/F12 supplemented with antibiotics and 10% FBSat 37°C for 24 h. The FCCM was collected and kept frozenat –20°C until use.
NCI-H295R human adrenocortical cells were purchased from AmericanType Culture Collection (Manassas, VA). Cells were maintainedin DMEM/F12 supplemented with insulin (66 nM), hydrocortisone(10 nM), 17-estradiol (10 nM), transferrin (10 µg/ml),selenite (30 nM), antibiotics, and 2% FBS at 37°C. For aldosteronesecretion assay, cells were plated at a density of 70,000 cells/cm2and precultured for 96 h. Then the medium was replaced withFCCM that contained previously mentioned supplements, and cellswere incubated for 36 h. Aldosterone concentration in the mediumwas determined by RIA using SPAC-S aldosterone kit (TFB, Tokyo,Japan). In some experiments, aldosterone-releasing activitywas evaluated after pretreatment with AngII type 1 receptorantagonist candesartan (1 x 10–5 mol/L).
Statistical Analyses
Data are expressed as mean ± SEM. Statistical analyseswere performed by unpaired t test, ANOVA and subsequent Tukeypost hoc test, or Mann-Whitney test. P < 0.05 was consideredto be statistically significant.
Metabolic Parameters Table 1 summarizes the metabolic parameters of SHR and SHR/cpat 17 wk of age. SHR/cp were obese, and serum insulin, triglycerides,and free fatty acids were markedly higher in SHR/cp than SHR(P < 0.01), whereas there was no difference in fasting bloodglucose. Systolic BP was comparably elevated in both strains.Therefore, SHR/cp rats can be considered as a model of metabolicsyndrome. Table 2 shows the temporal changes in BP and fastingglucose concentration in SHR/cp.
Table 2. Temporal profile of BP and fasting blood glucose concentration in SHR/cpa
Urinary Protein Excretion Is Exaggerated in SHR/cp
We first compared the profile of proteinuria between SHR andSHR/cp (Figure 1). Urinary protein excretion remained low innonobese SHR. By contrast, the metabolic syndrome model SHR/cphad exaggerated proteinuria despite similar BP elevation. Urinaryprotein was already increased at 12 wk of age (P < 0.01),which was increased progressively thereafter.
Figure 1. Temporal profile of proteinuria in nonobese SHR () and SHR/cp () at indicated ages. Data are mean ± SEM (n = 4 per group). **P < 0.01 versus age-matched SHR.
Podocyte Injury Is an Early Event in the Nephropathy of SHR/cp
We assessed podocyte damage and tubulointerstitial injury aspossible causes of proteinuria in young SHR/cp. Renal histologicchanges were not apparent under periodic acid-Schiff–stainedlight micrograph in 17-wk-old SHR/cp (Figure 2A). Gene expressionsof osteopontin and macrophage chemotactic protein-1 were notincreased in the whole kidney samples of SHR/cp, suggestingthe absence of tubulointerstitial proinflammatory responses(Figure 2B).
Figure 2. (A) Representative photomicrograph of periodic acid-Schiff–stained renal section from 17-wk-old SHR/cp. Bar = 100 µm. (B) Quantitative analysis of osteopontin and macrophage chemotactic protein-1 (MCP-1) mRNA expressions in the kidneys of SHR and SHR/cp determined by real-time PCR (n = 8 per group). (C) Western blotting of nephrin in the glomeruli of 17-wk-old SHR and SHR/cp. (Top) Representative bands for nephrin and control actin. (Bottom) Result of densitometric analysis (n = 3 per group). (D) Glomerular mRNA expression of nephrin determined by real-time PCR. Comparison between 17-wk-old SHR and SHR/cp (left: n = 8 per group) and time-course analysis in SHR/cp (right: n = 4 per group). (E) Representative immunostaining for desmin in the kidneys of SHR (left) and SHR/cp (right). Bars = 100 µm. (F) Representative transmission electron micrographs of glomeruli from SHR (left) and SHR/cp (right). Bars = 1 µm. *P < 0.05, **P < 0.01 versus SHR; #P < 0.05, ##P < 0.01 versus 6 wk.
Conversely, the glomerular expression of nephrin, a slit diaphragm–associatedprotein in the podocytes, was significantly reduced in SHR/cpcompared with SHR (Figure 2C). Nephrin expression also was diminishedat the mRNA level, which was decreased in parallel with thetemporal profile of proteinuria (Figure 2D). Expression of desmin,an injured podocyte marker, was induced in some glomeruli ofSHR/cp but not of SHR (Figure 2E). Electron microscopy revealedpodocyte foot process effacement, vacuolization, and accumulationof dense deposits in 17-wk-old SHR/cp. SHR had intact ultrastructureof podocytes (Figure 2F). These findings suggest that podocyteinjury underlies the cause of early proteinuria in SHR/cp.
Circulating Aldosterone Level and Expression of Its Effector Sgk1 Are Elevated in SHR/cp
Recent studies suggested that aldosterone is a potential mediatorof proteinuria and that it often is overproduced in obesityhypertension. Therefore, we evaluated aldosterone and its effectorsin SHR/cp. Serum aldosterone concentration was elevated significantlyin obese SHR/cp compared with nonobese SHR at 17 wk of age (P< 0.05; Figure 3A). Time-course analysis revealed that serumaldosterone increased in an age-dependent manner (P < 0.05;Figure 3B). There was a positive correlation between circulatingaldosterone concentration and proteinuria (r2 = 0.44; Figure 3C).
Figure 3. (A) Serum aldosterone concentration in 17-wk-old SHR and SHR/cp (n = 8 per group). (B) Time-course analysis of serum aldosterone in SHR/cp (n = 3 to 4 per group). (C) Relationship between serum aldosterone level and proteinuria in SHR/cp (n = 23). (D) Gene expression of Sgk1 in the whole kidney (left) and glomerular fraction (right) of 17-wk-old SHR and SHR/cp (n = 8 per group). (E) Western blotting of Sgk1 in the kidneys of SHR and SHR/cp (n = 3 per group). **P < 0.01 versus SHR.
Sgk1 is a widely known effector of aldosterone and an indexof MR activation. The Sgk1 mRNA expression was increased inthe kidneys as well as glomerular fraction of SHR/cp comparedwith those of SHR (Figure 3D). Sgk1 upregulation also was observedat the protein level (Figure 3E). These results suggest thataldosterone and its signaling in the kidney and glomeruli areenhanced in SHR/cp.
MR Is Expressed in Podocytes
Several reports demonstrate the presence of MR in cultured renalcells. However, the precise in vivo localization of MR withinthe kidney has not been shown clearly. Therefore, we performedimmunohistochemical analysis of MR in the kidney of SHR/cp (Figure 4).As previously reported, MR was localized predominantly in thenuclei in the in vivo condition (26,28). Intense signals weredetected in the distal nephron and also perivascular regions(Figure 4A). Distinct staining also was present in the glomeruli(Figure 4A, arrows). Double immunostaining of MR and WT-1, whichis expressed in the podocyte nuclei, yielded a considerablenumber of double-positive cells (Figure 4B). Double immunostainingof MR and synaptopodin, which is present in the podocyte cytoplasms,indicated that many MR-positive cells are located just outsidethe synaptopodin-positive podocyte cytoplasms (Figure 4C). Theseresults suggest that podocytes constitute substantial portionsof the MR-expressing cells within the glomeruli.
Figure 4. (A) Localization of mineralocorticoid receptor (MR) in the kidney of SHR/cp. MR was expressed not only in the distal nephron but also in the glomeruli (arrows). Bar = 100 µm. (B) Double immunofluorescence stainings for MR and WT-1 (a marker for podocyte nuclei). Bars = 50 µm. (C) Double immunofluorescence stainings for MR and synaptopodin (Syn; a marker for podocyte cytoplasms). Bars = 50 µm.
Eplerenone Ameliorates Proteinuria and Podocyte Damage in SHR/cp: Possible Role of Oxidative Stress
The above findings suggest that excessive aldosterone signalingmay contribute to the increased susceptibility to proteinuriaand podocyte injury in SHR/cp. Therefore, we examined the effectof selective aldosterone blocker eplerenone. After 4 wk of treatment,eplerenone significantly reduced proteinuria in SHR/cp (P <0.05; Figure 5A). In association with the decrease in proteinuria,eplerenone prevented the reduction of nephrin protein and mRNAand alleviated the induction of desmin (Figure 5, B throughD). Eplerenone also inhibited the upregulated expression ofSgk1 in the kidneys and glomeruli of SHR/cp (Figure 5E). Eplerenonetended to reduce BP (167 ± 4 mmHg; 0.05 < P < 0.1)but did not change fasting blood glucose (203 ± 25 mg/dl)or cause hyperkalemia (4.6 ± 0.1 mEq/L).
Figure 5. Effects of eplerenone (Epl) on proteinuria, podocyte injury, Sgk1, and oxidative stress in SHR/cp. SHR/cp at 13 wk of age were treated with Epl for 4 wk (SHR/cp+Epl). (A) Proteinuria in SHR/cp and SHR/cp+Epl (n = 4 per group). (B) Western blotting of nephrin in the glomeruli (n = 3 per group). (C) Glomerular nephrin mRNA expression (n = 4 per group). (D) Representative immunostaining for desmin in the kidneys of SHR/cp (left) and SHR/cp+Epl (center). Bars = 100 µm. Immunostaining score of desmin in the glomeruli (right: n = 4 per group). (E) Western blotting of Sgk1 in the kidneys (left) and glomeruli (right) of SHR/cp and SHR/cp+Epl (n = 3 per group). (F) Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) excretion of SHR, SHR/cp, and SHR/cp+Epl (n = 4 per group). (G) Gene expression of p22phox (left) and gp91phox (right) in the glomeruli (n = 4 per group). #P < 0.05, ##P < 0.01 versus untreated SHR/cp; *P < 0.05, **P < 0.01 versus SHR.
We tested the role of oxidative stress in this process, becauseinduction of reactive oxygen species (ROS) is proposed to bea mediator of injurious effects of aldosterone (14). Oxidativestress markers such as urinary 8-OHdG and glomerular expressionsof NADPH oxidase components p22phox and gp91phox were increasedin SHR/cp, which was completely inhibited by eplerenone (Figure 5,F and G). These results suggest that endogenous aldosteroneexcess, via induction of oxidative stress, causes podocyte injuryand proteinuria in SHR/cp.
Effect of Tempol on Proteinuria, Podocyte Injury, and Sgk1 Expression in SHR/cp
To confirm the contribution of oxidative stress to the nephropathyof SHR/cp, we treated these rats with the antioxidant tempol.Administration of tempol for 4 wk significantly reduced proteinuria(P < 0.05; Figure 6A). Reduced expression of nephrin andupregulation of desmin also were alleviated by tempol (Figure 6,B through D). It is interesting that the upregulated expressionof Sgk1 in SHR/cp also was abrogated by tempol (Figure 6, Eand F). Tempol did not affect BP (184 ± 13 mmHg) andfasting glucose (186 ± 22 mg/dl).
Figure 6. Effects of tempol (Temp) on proteinuria, podocyte injury, and Sgk1 expression in SHR/cp. SHR/cp at 13 wk of age were treated with Temp for 4 wk (SHR/cp+Temp). (A) Proteinuria in SHR/cp and SHR/cp+Temp (n = 4 per group). (B) Western blotting of nephrin in the glomeruli (n = 3 per group). (C) Glomerular nephrin mRNA expression (n = 4 per group). (D) Immunostaining score of desmin in the glomeruli (n = 4 per group). (E) Western blotting of Sgk1 (n = 3 per group). (F) Real-time PCR of Sgk1 (n = 4 per group). *P < 0.05, **P < 0.01 versus SHR/cp.
Fat-Derived Factors May Contribute to Hyperaldosteronism in SHR/cp
Finally, we explored the mechanisms of high aldosterone statein SHR/cp. Expression of aldosterone synthase was enhanced inthe adrenal glands from SHR/cp but was below the detection levelin the kidney (Figure 7A), suggesting that aldosterone productionin the adrenals is responsible for high circulating aldosteronein SHR/cp. SHR/cp displayed lower plasma renin activity (Figure 7B),suggesting that this is not the responsible factor. Ehrhart-Bornsteinet al. (23) reported adipocyte-derived substances that stimulateadrenal aldosterone production. Scanning electron micrographsshowed markedly enlarged visceral adipocytes in SHR/cp (Figure 7C),implicating the pathogenetic role for adipocytes.
Figure 7. Mechanisms of aldosterone excess state in SHR/cp. (A) Gene expression of aldosterone synthase in the adrenals and kidneys of SHR and SHR/cp. (B) Plasma renin activity. (C) Representative scanning electron micrographs of epididymal adipose tissues of SHR (left) and SHR/cp (right). Bars = 100 µm. Note markedly enlarged adipocytes in SHR/cp. (D) Aldosterone secretion from H295R adrenocortical cells that were subjected to control medium and fat cell–conditioned medium (FCCM) from SHR and that from SHR/cp. (E) Effects of angiotensin II receptor antagonist (ARB) on aldosterone secretion from H295R cells that were subjected to control medium and FCCM from SHR/cp. (F) Angiotensinogen gene expression in the epididymal adipose tissues. (G and H) Gene expression of aldosterone synthase (G) and steroidogenic acute regulatory protein (StAR; H) in H295R cells that were exposed to control medium and FCCM from SHR and that from SHR/cp. (I) StAR mRNA expression in the adrenal glands from SHR and SHR/cp. n = 4 per group for A and D through I; n = 8 per group for B. n.d., not detected. *P < 0.05, **P < 0.01 versus SHR; ##P < 0.01 versus control medium.
Indeed, aldosterone production in H295R adrenocortical cellswas increased markedly by FCCM from SHR/cp but not that fromnonobese SHR (Figure 7D). Aldosterone-releasing activity ofFCCM from SHR/cp was not mediated by AngII, because it was notinhibited by candesartan (Figure 7E), and angiotensinogen mRNAexpression in the adipose tissues was lower in SHR/cp (Figure 7F).The activity was not recapitulated by the known adipocytokines(data not shown). FCCM from SHR/cp but not from SHR stimulatedthe expression of aldosterone synthase in H295R cells (Figure 7G).FCCM from SHR/cp upregulated mRNA expression of steroidogenicacute regulatory protein (StAR), another key factor in aldosteronesynthesis that mediates transfer of cholesterol to mitochondria(Figure 7H). Adrenal steroidogenic acute regulatory protein(StAR) expression also was enhanced in SHR/cp compared withSHR (Figure 7I). These findings suggest the involvement of adipocyte-derivedsubstances other than AngII in hyperaldosteronism in SHR/cp.
Our study demonstrates that SHR/cp, a rat model of metabolicsyndrome, exhibit enhanced proteinuria as well as glomerularpodocyte injury. Circulating aldosterone level, glomerular expressionof Sgk1, and oxidative stress markers were increased in SHR/cpcompared with SHR. Eplerenone effectively ameliorated podocytedamage and inhibited elevated oxidative stress and Sgk1 expression.Tempol also alleviated podocyte impairment along with inhibitionof Sgk1 expression. Furthermore, adipocytes from obese SHR/cpsecreted substances that stimulate aldosterone production inthe adrenocortical cells. Our findings suggest that enhancedaldosterone signaling plays a key role in podocyte injury inSHR/cp via induction of oxidative stress and that adipocyte-derivedfactors might contribute to the aldosterone excess in this model.
Although metabolic syndrome is associated with proteinuria independentof diabetes and hypertension (2,3), the underlying mechanismshave not been elucidated. In this study, we examined proteinuriain SHR/cp, a derivative of SHR with spontaneous nonsense mutationin the leptin receptor gene (17,18). This strain manifesteda clustering of abdominal obesity, hypertension, hyperinsulinemia,hypertriglyceridemia, and elevated free fatty acids at 17 wkof age, fulfilling the criteria of metabolic syndrome. Podocyteinjury was reported previously in this animal model, but theanalysis was made at a later stage, when other abnormalitiessuch as tubulointerstitial changes are evident (10,29). Ourfindings indicate that podocyte injury should be an early keymanifestation in the nephropathy of this model, because at thisphase, we did not detect apparent renal morphologic changesor tubulointerstitial inflammatory alterations.
Multiple factors are proposed to be involved in the initiationof renal injury in metabolic syndrome: Enhanced renin-angiotensin-aldosteronesystem, insulin resistance, sympathetic nerve overactivation,and hyper-hemodynamics (30). We consider that enhanced aldosteronesignaling plays a critical role in the proteinuria and podocyteinjury of this model for the following reasons. First, circulatingaldosterone level was elevated in SHR/cp along with aldosteroneeffectors such as oxidant stress and Sgk1, and aldosterone levelswere correlated with the degree of proteinuria. Second, eplerenonecould reverse the proteinuria and podocyte damage of this model,together with suppression of oxidative stress and Sgk1. Finally,we found that aldosterone/salt rats develop marked podocyteinjury and massive proteinuria (Shibata et al. unpublished observation,2006). Aldosterone has been implicated as an important mediatorof proteinuria and glomerular damage in CKD or diabetic or hypertensivenephropathy (9,31–33). Our study would be the first reportto show the involvement of aldosterone in the early nephropathyof the metabolic syndrome model.
To date, few studies have demonstrated clearly the precise localizationof MR within the kidney. Our data showed immunolocalizationof MR on glomerular cells, including podocytes. Glomerular expressionof MR also was described by Gomez-Sanchez et al. (28). We confirmedthe presence of MR as well as activation of MR signaling byexposure to aldosterone using a cultured podocyte cell line(Shibata et al., unpublished observation, 2006). These findingssuggest that the proteinuric effects of aldosterone should bemediated at least in part through direct action on podocytes,although hemodynamic alteration by aldosterone also might beinvolved (34).
We observed that Sgk1 expression is enhanced in the kidneysof SHR/cp. Sgk1 is a transcriptionally regulated serine threoninekinase and considered as one of the main effectors of aldosterone(35–37). Sgk1 is induced by aldosterone not only in distaltubular cells but also in vascular cells (37). Aldosterone infusionincreased Sgk1 expression in the whole kidney and glomeruli(38). Studies have demonstrated the presence of Sgk1 in bothmesangial cells and podocytes (39,40). Quinkler et al. (16)demonstrated increased expression of aldosterone effectors,including Sgk1, in kidney biopsies of patients with heavy proteinuria,implicating the close relationship between Sgk1 and proteinuria.The pivotal role of Sgk1 in the pathogenesis of proteinuriaalso is suggested by the report that gene targeting of Sgk1protects against DOCA/salt-induced albuminuria (41).
Oxidative stress is postulated to be an important mediator ofaldosterone actions (14). Our data indicated that oxidativestress markers are elevated in SHR/cp and that tempol amelioratedproteinuria and podocyte injury, along with inhibition of theenhanced Sgk1 expression. Importantly, eplerenone suppressedthe elevated oxidative stress markers in SHR/cp. Sgk1 regulationby oxidative stress was demonstrated previously in other cells(42). These results suggest that aldosterone increases ROS generation,which causes Sgk1 upregulation and podocyte injury. Nishiyamaet al. (14,43) demonstrated that aldosterone increases ROS,which in turn activate extracellular signal–regulatedkinase 1/2, c-Jun N-terminal kinase, and big mitogen-activatedprotein kinase (BMK1) but not p38 mitogen-activated proteinkinase in rat renal cortex and cultured mesangial cells. Thus,multiple kinases seem to be involved in the actions of aldosterone.Further studies are necessary to elucidate the mechanisms bywhich Sgk1 causes podocyte injury, including the cross-talkbetween Sgk1 and mitogen-activated protein kinases.
Adipose tissue now is recognized as a dynamic endocrine organthat secretes a number of adipocytokines, not just an inertstorage depot (44). Although AngII is a major regulator of adrenalaldosterone production, aldosterone excess in SHR/cp was notaccompanied by increased renin activity in this study. On thebasis of the comparison between obese SHR/cp and nonobese SHR,we assumed that factors that are responsible for aldosteroneexcess in SHR/cp are likely to reside in adipocytes. Ehrhart-Bornsteinet al. (23) showed that some adipocytokines, although as yetunidentified, stimulate aldosterone secretion from adrenocorticalcells. In this study, we first demonstrated a possible pathogenicrole for these fat-derived products. We found that this aldosterone-releasingactivity of adipocytes was upregulated in the adipocytes fromSHR/cp compared with those from nonobese SHR. It should be notedthat hyperaldosteronism that is caused by these adipocyte-derivedfactors is not inhibitable by angiotensin-converting enzymeinhibitors or AngII receptor antagonists. Thus, eplerenone shouldhave benefit over AngII blockade in situations in which suchfactors are overproduced. Goodfriend et al. (24) reported thatepoxy-keto derivative of linoleic acid, one of the oxidizedproducts of fatty acids, not native linoleic acid, stimulatesaldosterone secretion in rat adrenal cells. Although they originallyhypothesized that the site of oxidative modification might bethe liver and showed that incubation of linoleic acid with hepatocytesgave rise to compounds that enhanced aldosterone productionin adrenal cells, adipocytes also might contribute to the epoxy-ketomodification of linoleic acid in our model. We expect that theseadipocyte-derived aldosterone-releasing factors, if identified,can be a novel target of therapy in metabolic syndrome.
We demonstrated that enhanced aldosterone signaling, such asincreased oxidative stress and Sgk1 upregulation, plays a crucialrole in podocyte injury and proteinuria in metabolic syndromemodel SHR/cp and that adipocyte-derived aldosterone secretagoguesmight be involved in the aldosterone excess. We also showedthat eplerenone effectively ameliorates podocyte injury andproteinuria in this model without causing hyperkalemia. Recentanimal studies revealed that aldosterone blockers are effectivein treating diabetic and hypertensive nephropathy, atherosclerosis,and balloon-induced vascular injury (9,45–48). Althoughfuture studies should be awaited, we believe that aldosteroneblockade would be a clinically promising strategy toward nephropathyin metabolic syndrome as well.
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Researchfrom Japan Society for the Promotion of Science (17590820).
We thank Pfizer (Tokyo, Japan) for providing eplerenone, TakedaPharmaceutical Co. (Osaka, Japan) for providing candesartan,Hiroshi Kawachi and Mitsuhiro Kawata for providing antibodies,and Satoru Fukuda for help in electron microscopic analysis.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Lakka HM, Laaksonen DE, Lakka TA, Niskanen LK, Kumpusalo E, Tuomilehto J, Salonen JT: The metabolic syndrome and total and cardiovascular disease mortality in middle-aged men.
JAMA 288
: 2709
–2716, 2002[Abstract/Free Full Text]
Chen J, Muntner P, Hamm LL, Jones DW, Batuman V, Fonseca V, Whelton PK, He J: The metabolic syndrome and chronic kidney disease in US adults.
Ann Intern Med 140
: 167
–174, 2004[Abstract/Free Full Text]
Kurella M, Lo JC, Chertow GM: Metabolic syndrome and the risk for chronic kidney disease among nondiabetic adults.
J Am Soc Nephrol 16
: 2134
–2140, 2005[Abstract/Free Full Text]
Remuzzi G, Bertani T: Pathophysiology of progressive nephropathies.
N Engl J Med 339
: 1448
–1456, 1998[Free Full Text]
Go AS, Chertow GM, Fan D, McCulloch CE, Hsu CY: Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization.
N Engl J Med 351
: 1296
–1305, 2004[Abstract/Free Full Text]
Pavenstadt H, Kriz W, Kretzler M: Cell biology of the glomerular podocyte.
Physiol Rev 83
: 253
–307, 2003[Abstract/Free Full Text]
Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG, Coplon NS, Sun L, Meyer TW: Podocyte loss and progressive glomerular injury in type II diabetes.
J Clin Invest 99
: 342
–348, 1997[Medline]
Kretzler M, Koeppen-Hagemann I, Kriz W: Podocyte damage is a critical step in the development of glomerulosclerosis in the uninephrectomised-desoxycorticosterone hypertensive rat.
Virchows Arch 425
: 181
–193, 1994[Medline]
Nagase M, Shibata S, Yoshida S, Nagase T, Gotoda T, Fujita T: Podocyte injury underlies the glomerulopathy of Dahl salt-hypertensive rats and is reversed by aldosterone blocker.
Hypertension 47
: 1084
–1093, 2006[Abstract/Free Full Text]
Gross ML, Ritz E, Schoof A, Adamczak M, Koch A, Tulp O, Parkman A, El-Shakmak A, Szabo A, Amann K: Comparison of renal morphology in the Streptozotocin and the SHR/N-cp models of diabetes.
Lab Invest 84
: 452
–464, 2004[CrossRef][Medline]
Greene EL, Kren S, Hostetter TH: Role of aldosterone in the remnant kidney model in the rat.
J Clin Invest 98
: 1063
–1068, 1996[Medline]
Ribstein J, Du Cailar G, Fesler P, Mimran A: Relative glomerular hyperfiltration in primary aldosteronism.
J Am Soc Nephrol 16
: 1320
–1325, 2005[Abstract/Free Full Text]
Rossi GP, Bernini G, Desideri G, Fabris B, Ferri C, Giacchetti G, Letizia C, Maccario M, Mannelli M, Matterello MJ, Montemurro D, Palumbo G, Rizzoni D, Rossi E, Pessina AC, Mantero F: Renal damage in primary aldosteronism: Results of the PAPY Study.
Hypertension 48
: 232
–238, 2006[Abstract/Free Full Text]
Nishiyama A, Yao L, Nagai Y, Miyata K, Yoshizumi M, Kagami S, Kondo S, Kiyomoto H, Shokoji T, Kimura S, Kohno M, Abe Y: Possible contributions of reactive oxygen species and mitogen-activated protein kinase to renal injury in aldosterone/salt-induced hypertensive rats.
Hypertension 43
: 841
–848, 2004[Abstract/Free Full Text]
Farjah M, Roxas BP, Geenen DL, Danziger RS: Dietary salt regulates renal SGK1 abundance: Relevance to salt sensitivity in the Dahl rat.
Hypertension 41
: 874
–878, 2003[Abstract/Free Full Text]
Quinkler M, Zehnder D, Eardley KS, Lepenies J, Howie AJ, Hughes SV, Cockwell P, Hewison M, Stewart PM: Increased expression of mineralocorticoid effector mechanisms in kidney biopsies of patients with heavy proteinuria.
Circulation 112
: 1435
–1443, 2005[Abstract/Free Full Text]
Ernsberger P, Koletsky RJ, Friedman JE: Molecular pathology in the obese spontaneous hypertensive Koletsky rat: A model of syndrome X.
Ann N Y Acad Sci 892
: 272
–288, 1999[CrossRef][Medline]
Takaya K, Ogawa Y, Hiraoka J, Hosoda K, Yamori Y, Nakao K, Koletsky RJ: Nonsense mutation of leptin receptor in the obese spontaneously hypertensive Koletsky rat.
Nat Genet 14
: 130
–131, 1996[CrossRef][Medline]
Tuck ML, Sowers J, Dornfeld L, Kledzik G, Maxwell M: The effect of weight reduction on blood pressure, plasma renin activity, and plasma aldosterone levels in obese patients.
N Engl J Med 304
: 930
–933, 1981[Abstract]
Engeli S, Bohnke J, Gorzelniak K, Janke J, Schling P, Bader M, Luft FC, Sharma AM: Weight loss and the renin-angiotensin-aldosterone system.
Hypertension 45
: 356
–362, 2005[Abstract/Free Full Text]
Egan BM, Stepniakowski K, Goodfriend TL: Renin and aldosterone are higher and the hyperinsulinemic effect of salt restriction greater in subjects with risk factors clustering.
Am J Hypertens 7
: 886
–893, 1994[Medline]
Bochud M, Nussberger J, Bovet P, Maillard MR, Elston RC, Paccaud F, Shamlaye C, Burnier M: Plasma aldosterone is independently associated with the metabolic syndrome.
Hypertension 48
: 239
–245, 2006[Abstract/Free Full Text]
Ehrhart-Bornstein M, Lamounier-Zepter V, Schraven A, Langenbach J, Willenberg HS, Barthel A, Hauner H, McCann SM, Scherbaum WA, Bornstein SR: Human adipocytes secrete mineralocorticoid-releasing factors.
Proc Natl Acad Sci U S A 100
: 14211
–14216, 2003[Abstract/Free Full Text]
Shibata S, Nagase M, Fujita T: Fluvastatin ameliorates podocyte injury in proteinuric rats via modulation of excessive Rho signaling.
J Am Soc Nephrol 17
: 754
–764, 2006[Abstract/Free Full Text]
Ito T, Morita N, Nishi M, Kawata M: In vitro and in vivo immunocytochemistry for the distribution of mineralocorticoid receptor with the use of specific antibody.
Neurosci Res 37
: 173
–182, 2000[CrossRef][Medline]
Nagase T, Nagase M, Osumi N, Fukuda S, Nakamura S, Ohsaki K, Harii K, Asato H, Yoshimura K: Craniofacial anomalies of the cultured mouse embryo induced by inhibition of sonic hedgehog signaling: An animal model of holoprosencephaly.
J Craniofac Surg 16
: 80
–88, 2005[CrossRef][Medline]
Gomez-Sanchez CE, de Rodriguez AF, Romero DG, Estess J, Warden MP, Gomez-Sanchez MT, Gomez-Sanchez EP: Development of a panel of monoclonal antibodies against the mineralocorticoid receptor.
Endocrinology 147
: 1343
–1348, 2006[Abstract/Free Full Text]
Nangaku M, Izuhara Y, Usuda N, Inagi R, Shibata T, Sugiyama S, Kurokawa K, van Ypersele de Strihou C, Miyata T: In a type 2 diabetic nephropathy rat model, the improvement of obesity by a low calorie diet reduces oxidative/carbonyl stress and prevents diabetic nephropathy.
Nephrol Dial Transplant 20
: 2661
–2669, 2005[Abstract/Free Full Text]
Zhang R, Liao J, Morse S, Donelon S, Reisin E: Kidney disease and the metabolic syndrome.
Am J Med Sci 330
: 319
–325, 2005[CrossRef][Medline]
Hostetter TH, Ibrahim HN: Aldosterone in chronic kidney and cardiac disease.
J Am Soc Nephrol 14
: 2395
–2401, 2003[Abstract/Free Full Text]
Aldigier JC, Kanjanbuch T, Ma LJ, Brown NJ, Fogo AB: Regression of existing glomerulosclerosis by inhibition of aldosterone.
J Am Soc Nephrol 16
: 3306
–3314, 2005[Abstract/Free Full Text]
Sato A, Hayashi K, Naruse M, Saruta T: Effectiveness of aldosterone blockade in patients with diabetic nephropathy.
Hypertension 41
: 64
–68, 2003[Abstract/Free Full Text]
de Paula RB, da Silva AA, Hall JE: Aldosterone antagonism attenuates obesity-induced hypertension and glomerular hyperfiltration.
Hypertension 43
: 41
–47, 2004[Abstract/Free Full Text]
Bhargava A, Fullerton MJ, Myles K, Purdy TM, Funder JW, Pearce D, Cole TJ: The serum- and glucocorticoid-induced kinase is a physiological mediator of aldosterone action.
Endocrinology 142
: 1587
–1594, 2001[Abstract/Free Full Text]
Vallon V, Lang F: New insights into the role of serum- and glucocorticoid-inducible kinase SGK1 in the regulation of renal function and blood pressure.
Curr Opin Nephrol Hypertens 14
: 59
–66, 2005[Medline]
Fuller PJ, Young MJ: Mechanisms of mineralocorticoid action.
Hypertension 46
: 1227
–1235, 2005[Abstract/Free Full Text]
Hou J, Speirs HJ, Seckl JR, Brown RW: Sgk1 gene expression in kidney and its regulation by aldosterone: Spatio-temporal heterogeneity and quantitative analysis.
J Am Soc Nephrol 13
: 1190
–1198, 2002[Abstract/Free Full Text]
Lang F, Klingel K, Wagner CA, Stegen C, Warntges S, Friedrich B, Lanzendorfer M, Melzig J, Moschen I, Steuer S, Waldegger S, Sauter M, Paulmichl M, Gerke V, Risler T, Gamba G, Capasso G, Kandolf R, Hebert SC, Massry SG, Broer S: Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy.
Proc Natl Acad Sci U S A 97
: 8157
–8162, 2000[Abstract/Free Full Text]
Kumar JM, Brooks DP, Olson BA, Laping NJ: Sgk, a putative serine/threonine kinase, is differentially expressed in the kidney of diabetic mice and humans.
J Am Soc Nephrol 10
: 2488
–2494, 1999[Abstract/Free Full Text]
Vallon V, Huang DY, Grahammer F, Wyatt AW, Osswald H, Wulff P, Kuhl D, Lang F: SGK1 as a determinant of kidney function and salt intake in response to mineralocorticoid excess.
Am J Physiol Regul Integr Comp Physiol 289
: R395
–R401, 2005[Abstract/Free Full Text]
Leong ML, Maiyar AC, Kim B, O’Keeffe BA, Firestone GL: Expression of the serum- and glucocorticoid-inducible protein kinase, Sgk, is a cell survival response to multiple types of environmental stress stimuli in mammary epithelial cells.
J Biol Chem 278
: 5871
–5882, 2003[Abstract/Free Full Text]
Nishiyama A, Yao L, Fan Y, Kyaw M, Kataoka N, Hashimoto K, Nagai Y, Nakamura E, Yoshizumi M, Shokoji T, Kimura S, Kiyomoto H, Tsujioka K, Kohno M, Tamaki T, Kajiya F, Abe Y: Involvement of aldosterone and mineralocorticoid receptors in rat mesangial cell proliferation and deformability.
Hypertension 45
: 710
–716, 2005[Abstract/Free Full Text]
Kershaw EE, Flier JS: Adipose tissue as an endocrine organ.
J Clin Endocrinol Metab 89
: 2548
–2556, 2004[Abstract/Free Full Text]
Fujisawa G, Okada K, Muto S, Fujita N, Itabashi N, Kusano E, Ishibashi S: Spironolactone prevents early renal injury in streptozotocin-induced diabetic rats.
Kidney Int 66
: 1493
–1502, 2004[CrossRef][Medline]
Han SY, Kim CH, Kim HS, Jee YH, Song HK, Lee MH, Han KH, Kim HK, Kang YS, Han JY, Kim YS, Cha DR: Spironolactone prevents diabetic nephropathy through an anti-inflammatory mechanism in type 2 diabetic rats.
J Am Soc Nephrol 17
: 1362
–1372, 2006[Abstract/Free Full Text]
Rajagopalan S, Duquaine D, King S, Pitt B, Patel P: Mineralocorticoid receptor antagonism in experimental atherosclerosis.
Circulation 105
: 2212
–2216, 2002[Abstract/Free Full Text]
Ward MR, Kanellakis P, Ramsey D, Funder J, Bobik A: Eplerenone suppresses constrictive remodeling and collagen accumulation after angioplasty in porcine coronary arteries.
Circulation 104
: 467
–472, 2001[Abstract/Free Full Text]
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Top
Abstract
Introduction
-estradiol (10 nM), transferrin (10 µg/ml), selenite (30 nM), antibiotics, and 2% FBS at 37°C. For aldosterone secretion assay, cells were plated at a density of 70,000 cells/cm2 and precultured for 96 h. Then the medium was replaced with FCCM that contained previously mentioned supplements, and cells were incubated for 36 h. Aldosterone concentration in the medium was determined by RIA using SPAC-S aldosterone kit (TFB, Tokyo, Japan). In some experiments, aldosterone-releasing activity was evaluated after pretreatment with AngII type 1 receptor antagonist candesartan (1 x 10–5 mol/L). 
) and SHR/cp (
) at indicated ages. Data are mean ± SEM (n = 4 per group). **P < 0.01 versus age-matched SHR.
