Modification of mineralocorticoid receptor function byRac1 GTPase: implication in proteinuric kidney diseaseShigeru Shibata1,6, Miki Nagase1,6, Shigetaka Yoshida1, Wakako Kawarazaki1, Hidetake Kurihara2,Hirotoshi Tanaka3, Jun Miyoshi4, Yoshimi Takai5 & Toshiro Fujita1

Blockade of mineralocorticoid receptor has been shown to

improve the clinical outcomes of proteinuric kidney diseases1,2.

However, little is known about the regulation of

mineralocorticoid receptor–dependent transcriptional activity in

renal disease. Here we identify a new role for Rac1, a member

of the Rho family GTPases, as a potent activator of

mineralocorticoid receptor signal transduction both in vitro and

in vivo. Transient transfection assays in HEK 293 cells revealed

that constitutively active Rac1 (CA-Rac1) enhanced

mineralocorticoid receptor–dependent reporter activity, which

was accompanied by increased nuclear translocation of

mineralocorticoid receptor. CA-Rac1 facilitated

mineralocorticoid receptor nuclear accumulation also in

podocytes via p21-activated kinase phosphorylation. In mice

lacking Rho GDP-dissociation inhibitor-a (Arhgdia–/– mice)3,

renal abnormalities, including heavy albuminuria and podocyte

damage, were associated with increased Rac1 (but not RhoA)

and mineralocorticoid receptor signaling in the kidney, without

alteration in systemic aldosterone status. Pharmacological

intervention with a Rac-specific small-molecule inhibitor4,5

diminished mineralocorticoid receptor overactivity and renal

damage in this model. Furthermore, albuminuria and

histological changes in Arhgdia–/– mice were suppressed by

mineralocorticoid receptor blockade, confirming the pathological

role of Rac1-mineralocorticoid receptor interaction. Our results

provide evidence that signaling cross-talk between Rac1

and mineralocorticoid receptor modulates mineralocorticoid

receptor activity and identify Rac1 as a therapeutic target for

chronic kidney disease.

Steroid receptors are ligand-activated nuclear transcription factors. Inaddition to hormone-mediated regulation of the receptor activity,recent studies have revealed cross-talk between steroid receptors andintracellular signaling pathways6,7. One such example is their interac-tion with the Rho family of small GTPases, which regulate diversebiological processes8,9. Rho family members and their regulatory

proteins are involved in the transactivation of several steroid recep-tors10–12. Mineralocorticoid receptor, a member of the steroid receptorfamily, has been shown to have a major pathophysiological role in theprogression of kidney diseases13, and the inhibition of mineralocorti-coid receptor signaling considerably reduces proteinuria in subjectswith chronic kidney disease1,2. Although several lines of evidenceindicate that the biological activity of mineralocorticoid receptor isinfluenced by molecules other than its ligand14–16, the precise mechan-isms regulating mineralocorticoid receptor transactivation potentialremain largely unknown.

To address the possibility that Rho GTPases could influence thefunction of mineralocorticoid receptor, we first performed in vitrotransfection assays in HEK 293 cells. Pull-down assays revealed thatconstitutively active mutants had more active GTPases than controls(Fig. 1a). In luciferase reporter assays, mineralocorticoid receptor–mediated transcriptional activity was upregulated in response toaldosterone (Fig. 1b), and overexpression of CA-Rac1 potentiatedthis response further (Fig. 1b). In contrast, CA-RhoA suppressedaldosterone-stimulated reporter activity, whereas other mutant con-structs, including CA-Cdc42, had no effect (Fig. 1b). In addition,CA-Rac1 facilitated mineralocorticoid receptor transcriptional activityeven without aldosterone (Fig. 1c). The reason for the transrepressionis unclear, but several studies have shown reciprocal functions of Rac1and RhoA17.

We further assessed nuclear trafficking of GFP-tagged mineralocor-ticoid receptor. Without aldosterone treatment, GFP fluorescence wasdistributed mainly in the cytoplasm; upon activation by aldosterone,mineralocorticoid receptor–GFP was promptly targeted to the nucleus(Fig. 1d). Immunoblotting showed that CA-Rac1 substantiallyincreased the amount of nuclear mineralocorticoid receptor–GFP,both in the absence and in the presence of aldosterone (Fig. 1e).CA-Rac1 also promoted nuclear translocation of mineralocorticoidreceptor–GFP in cultured podocytes, the glomerular visceral epithelialcells that serve as the filtration barrier of the kidney (Fig. 1f). CA-Rac1overexpression in podocytes facilitated phosphorylation of p21-activated kinase (PAK) and LIM-kinase (LIMK) (Fig. 1g), possible

Received 5 August; accepted 22 September; published online 23 November 2008; doi:10.1038/nm.1879

1Department of Nephrology and Endocrinology, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. 2Department ofAnatomy, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. 3Division of Clinical Immunology, Advanced Clinical ResearchCenter, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. 4Department of Molecular Biology, Osaka MedicalCenter for Cancer and Cardiovascular Diseases, 1-3-2 Nakamichi, Higashinari-ku, Osaka 537-8511, Japan. 5Department of Biochemistry and Molecular Biology, KobeUniversity Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. 6These authors contributed equally to this work. Correspondenceshould be addressed to T.F. (fujita-dis@h.u-tokyo.ac.jp).

1370 VOLUME 14 [ NUMBER 12 [ DECEMBER 2008 NATURE MEDICINE

L E T T ERS©

2008

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

atur

emed

icin

e

downstream targets of Rac1 (ref. 18). PAK is known to regulatetransactivation of several steroid receptors19. Notably, CA-Rac1–mediated nuclear translocation of mineralocorticoid receptor–GFPwas significantly prevented in the presence of PAK18, an inhibitor ofPAK20 (Fig. 1h). These results indicate that Rac1 promotes nuclearshuttling of mineralocorticoid receptor in podocytes via PAK.

We next carried out in vivo experiments. Rho GDP dissociationinhibitor (RhoGDI) interacts with the GDP-bound inactive RhoGTPases and prevents them from being converted to the activeGTP-bound forms8. Three distinct isoforms exist in mammals:GDI-a, GDI-b and GDI-g. Among these, RhoGDI-a could have amajor role in the kidney, because Arhgdia–/– mice develop pro-gressive renal disease3. We characterized the renal pathology andinvestigated whether Rac1–mineralocorticoid receptor signaling isinvolved in this model. Arhgdia–/– mice showed mild albuminuriaat 1 week of age, progressing to unselective proteinuria at around4 weeks of age (Supplementary Fig. 1a online). Although nephro-genesis and renal histology at 1 week of age were mostly normal bylight microscopy, transmission electron microscopy (TEM)revealed focal effacement of the podocyte foot processes at thisstage (Supplementary Fig. 1b,c), which could explain the protein-uria in Arhgdia–/– mice. Concomitantly, proximal tubular cellscontained an increased number of cytoplasmic vacuoles (Supple-mentary Fig. 1c), which may be a primary effect of RhoGDI-adeletion or secondary to glomerular damage and increased pro-tein reabsorption.

At 12 weeks of age, Arhgdia–/– mice showed massive albuminuria(Fig. 2a), resembling human nephrotic syndrome3. Renal histology

showed glomerular lesions with focal and segmental sclerosis, alongwith prominent intratubular casts and luminal dilatation (Fig. 2b).TEM revealed extensive foot process effacement (Fig. 2c), indicatingsevere podocyte damage. The frequency of filtration slits per milli-meter was reduced by 68% in Arhgdia–/– mice compared with wild-type mice (744.3 ± 79.4 versus 2,317.8 ± 89.1 filtration slits permillimeter of the glomerular basement membrane; n ¼ 4 each group;P o 0.01). Tubular epithelial cells showed degenerative changes withbasement membrane thickening (Supplementary Fig. 1d).

We next investigated the activity of GTPases and the aldosterone–mineralocorticoid receptor cascade in the kidney. RhoGDI-a defi-ciency led to increased levels of active Rac1, whereas, in contrast, it didnot affect RhoA activity (Fig. 2d,e). Total expression of both Rac1 andRhoA was reduced (Fig. 2e). Serum aldosterone concentration andblood pressure were not altered in Arhgdia–/– mice (Fig. 2f,g).However, in the kidneys both the cellular messenger RNA and theprotein in the nuclear fraction representing the mineralocorticoidreceptor were increased in Arhgdia–/– mice (Fig. 2h,i). The expressionof Sgk1, a mineralocorticoid receptor–dependent gene, was alsoupregulated (Fig. 2j).

We next evaluated the role of Rac1 in the renal phenotype ofArhgdia–/– mice. NSC23766 is a unique Rac-specific inhibitor thatdoes not block Rho or Cdc42 (ref. 4). NSC23766 was administered toArhgdia–/– mice from 6 weeks of age for 6 weeks. Untreated Arhgdia–/–

mice showed heavy albuminuria throughout the experiment, whereasadministration of NSC23766 significantly reduced the albuminuria(Fig. 3a,b) concomitantly with repression of renal Rac1 activity(Fig. 3c). Histological damage in Arhgdia–/– mice was substantially

Vehicle

hMR

-GF

P

Aldo

Empt

y

GTP-boundRac1

Total Rac1

CA-Rac

1

WT-R

ac1

DN-Rac

1

hMR-GFP(nuclear)

CREB

Empt

y

CA-Rac

1

Vehicle

Empt

y

CA-Rac

1

Aldo

**

**

0

5

10

Nuc

lear

hM

R-G

FP

/CR

EB

hMR-GFP(nuclear)

CREB

CA-Rac

1

CA-Rac

1

+ PA

K18

Aldo

0

0.5

1

*

Nuc

lear

hMR

-GF

P/C

RE

B

hMR-GFP(nuclear)

CREB

Empt

y

CA-Rac

1

Aldo

**

0

1

3

2

Nuc

lear

hM

R-G

FP

/CR

EB

Empty

Phospho-PAK

Total PAK

Phospho-LIMK

Total LIMK

CA-Rac1

a

e f g h

d

Luci

fera

se a

ctiv

ity(w

ithou

t ald

oste

rone

)

0

1

2

Empt

yCA

WT

Rac1

DNEm

pty

CAW

TDN

Empt

yCA

WT

DN

RhoA Cdc42

**c

Luci

fera

se a

ctiv

ity(w

ith a

ldos

tero

ne)

0

10

20

30

Empt

yCA

WT

Rac1

DNEm

pty

CAW

TDN

Empt

yCA

WT

DN

RhoA Cdc42

**

**b

Figure 1 Active Rac1 enhances mineralocorticoid receptor (MR) transcriptional activity and nuclear translocation. (a) GTP-bound active and total Rac1

expression in human kidney-derived HEK 293 cells transfected with CA-, wild-type (WT)- or dominant-negative (DN)-Rac1. Similar results were obtained

for RhoA and Cdc42 (data not shown). (b,c) Ability of Rho GTPase variants to enhance MR-mediated transcriptional activity in the presence (b) or absence

(c) of 1 nM aldosterone, as determined by luciferase reporter assay in HEK 293 cells. Values represent the luciferase activity relative to that of cells

transfected with empty vector without aldosterone (n ¼ 8). (d) Representative GFP fluorescence images of human MR-GFP (hMR-GFP)-transfected HEK293 cells treated with vehicle or 1 nM aldosterone for 1 h. Scale bar, 10 mm. (e) Nuclear expression of hMR-GFP in HEK 293 cells transfected with empty

vector or CA-Rac1, in the absence (Vehicle) and presence of 1 nM aldosterone (Aldo), as quantified by western blotting (n ¼ 7). Cyclic AMP response

element–binding protein (CREB) served as a loading control of nuclear lysates. (f) Effect of CA-Rac1 on nuclear translocation of MR in cultured podocytes in

the presence of 1 nM aldosterone (n ¼ 6). (g) Western analyses of phosphorylated or total PAK and LIMK expression in podocytes transfected with CA-Rac1.

(h) Effect of PAK inhibitor (PAK18) on the nuclear expression of MR in CA-Rac1–transfected podocytes (n ¼ 8). Data are expressed as means ± s.e.m.

* P o 0.05; ** P o 0.01.

L E T T ERS

NATURE MEDICINE VOLUME 14 [ NUMBER 12 [ DECEMBER 2008 1371

©20

08 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

urem

edic

ine

ameliorated by NSC23766 treatment (Fig. 3d), which was semiquan-titatively evaluated by previously described scoring methods21,22

(Fig. 3e). Elevated blood urea nitrogen in Arhgdia–/– mice wasconsiderably reduced in the treated mice (Fig. 3f), and the favorableeffect of NSC23766 on podocyte morphology was confirmed by TEM(Fig. 3g). Fasudil, a specific inhibitor of the Rho kinase pathway, hadno protective effects in Arhgdia–/– mice (Supplementary Fig. 2online). We also found that neither apocynin nor tempol couldeffectively ameliorate the kidney damage in Arhgdia–/– mice (datanot shown). Therefore, oxidative stress does not seem to mediate renaldamage in Arhgdia–/– mice.

To further characterize the renoprotective action of NSC23766,we additionally evaluated the role of Rac1 in Dahl rats, a modelfor hypertensive glomerulosclerosis. Salt loading triggers glomerulardamage in this model, which is associated with increased mineralo-corticoid receptor signaling23. Rac1 was activated in the kidneys ofsalt-loaded Dahl salt-sensitive rats (Supplementary Fig. 3a online).Moreover, administration of NSC23766 for 4 weeks clearly suppressedsevere proteinuria and glomerulosclerosis in this model (Supplemen-tary Fig. 3b–d), supporting a pathogenic role for Rac1 activation inglomerular damage.

NSC23766 also diminished the enhanced mineralocorticoid recep-tor signaling in the kidneys of Arhgdia–/– mice, as assessed by Sgk1induction (Fig. 3h). Gene expression of Serpine1 (encoding plasmino-gen activator inhibitor-1) and Ccl2 (encoding monocyte chemoattrac-tant protein-1), putative downstream mediators of mineralocorticoidreceptor signaling causing renal inflammation and sclerosis24, wasmarkedly elevated in the kidneys of Arhgdia–/– mice and wasalso suppressed by NSC23766 (Fig. 3i). These results corroborate

our in vitro data linking Rac1 activation with mineralocorticoidreceptor signaling.

Finally, we treated Arhgdia–/– mice with eplerenone, a selectiveantagonist of mineralocorticoid receptor, to clarify the contribution ofthe enhanced mineralocorticoid receptor signaling. Eplerenone givenfrom 4 weeks of age markedly decreased albuminuria in Arhgdia–/–

mice (Fig. 4a), and urinary albumin almost completely disappearedafter 8 weeks of treatment (11.76 ± 2.28 mg per mg creatinine in thenontreated group versus 0.17 ± 0.07 mg per mg creatinine in thetreatment group, Po 0.005). Under light microscopy, it was clear thatglomerulosclerosis and tubulointerstitial damage were ameliorated(Fig. 4b,c). Morphological analysis under TEM as well as immuno-histochemical studies indicated that mineralocorticoid receptor block-ade conferred podocyte protection (Fig. 4d–f). Eplerenone alsosuppressed the upregulation of Serpine1 and Ccl2 (Fig. 4g).

Recent studies have elucidated the importance of apoptosis in thedevelopment of glomerular diseases25,26. We assessed apoptosis inArhgdia–/– mice because mineralocorticoid receptor signaling is con-sidered to cause organ dysfunction partly through apoptosis induc-tion27. Although apoptosis was almost undetectable in the glomeruliof wild-type mice, we occasionally identified TUNEL-positive cells inthe glomeruli of Arhgdia–/– mice, including podocytes (Fig. 4h). Thenumber of apoptotic cells was also clearly increased in the tubuloin-terstitium of Arhgdia–/– mice (Fig. 4i). Moreover, eplerenone treat-ment substantially reduced apoptosis in Arhgdia–/– mice (Fig. 4j),supporting the importance of mineralocorticoid receptor–mediatedapoptosis in this model. These data confirmed that enhanced mineral-ocorticoid receptor signaling in the kidney has a central role in thedevelopment of proteinuria and renal damage in Arhgdia–/– mice.

kDa

WT

12 weeks

Arhgdia–/–

WT Arhgdia–/– WT

Arhgdia–/–

100

75

50

37

WT

RhoGDlα

GAPDH

Arhgdia–/–

WT0

Arhgdia–/–

50100150

Ald

oste

rone

(pg

ml–1

)

200250300350 NS

WT0

20406080

100120140

Arhgdia–/–B

lood

pre

ssur

e(m

m H

g)

NS

WT

Arhgdia

–/–

0

0.5

1

1.5

2

Nr3c2

/Gap

dh

*

WT Arhgdia–/–0

0.5

1

1.5

2

MR(nucleus)

Nuc

lear

MR

(rel

ativ

e de

nsity

) *

WT

Arhgdia

–/–

0

0.5

1

1.5

2

Sgk

1/Gap

dh

**

a b

d

c

GTP-bound

Total

WT

Rac1

Arhgdia–/–

GTP-bound

Total

RhoA

WT Arhgdia–/–

e

f hg ji

Figure 2 Renal damage, activity of Rho family GTPases and MR signaling in Arhgdia–/– mice. (a) SDS-PAGE analysis of mouse urine at 12 weeks of age.

(b) PAS-stained kidney sections of WT and Arhgdia–/– mice at 12 weeks of age. Arhgdia–/– mice showed focal and segmental glomerulosclerosis with

prominent proteinaceous casts and tubular dilatation. Scale bars, 100 mm. (c) Ultrastructure of glomerular filtration barrier showing extensive effacement

of podocyte foot processes in Arhgdia–/– mice. Scale bar, 1 mm. (d) RhoGDI-a protein expression in the kidneys of WT and Arhgdia–/– mice. GAPDH,

glyceraldehyde 3-phosphate dehydrogenase. (e) Rac1 and RhoA activity in the kidney. Experiments were replicated at least three times. (f,g) Serum

aldosterone concentrations (n ¼ 7; f) and systolic blood pressure (n ¼ 5; g) in WT and Arhgdia–/– mice. (h) Quantitative analysis of Nr3c2 (encoding MR)

gene expression in the kidney (n ¼ 9). (i) MR abundance in the nuclear fraction of the kidney in WT and Arhgdia–/– mice (n ¼ 7). (j) Quantitative analysis

of the MR downstream effector Sgk1 gene expression in the kidney (n ¼ 7). NS, not significant. Data are expressed as means ± s.e.m. *P o 0.05, and

**P o 0.01 compared with WT mice.

L E T TERS

1372 VOLUME 14 [ NUMBER 12 [ DECEMBER 2008 NATURE MEDICINE

©20

08 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

urem

edic

ine

Here we have identified a new role for the small GTPase Rac1 as amodulator of mineralocorticoid receptor transactivation potential.In vitro, CA-Rac1 enhances nuclear translocation and transcriptionalactivity of mineralocorticoid receptor. It should be noted that a recentstudy likewise reported an essential role for Rac1 in the nuclearlocalization of b-catenin28, a transcription factor involved incanonical Wnt signaling. We have also shown that Rac1-dependentmineralocorticoid receptor activation plays a major part in thepathogenesis of renal damage in vivo. We consider these findingsappealing because clinical studies have shown that serum aldosteronelevels are not necessarily predictive of the efficacy of mineral-ocorticoid receptor blockade2, suggesting the existence of alternativepathways responsible for mineralocorticoid receptor overactivity. Onthe basis of our results, we propose a model in which Rac1 servesas a key regulator of nonaldosterone-mediated mineralocorticoidreceptor activation.

What would be the pathological stimuli for Rac1-mediated miner-alocorticoid receptor activation? In this study, we demonstrated thatNSC23766 effectively ameliorates proteinuria and glomerular damagein Dahl salt-sensitive rats. Recently, mechanical stretch and fluid shearstress were reported to induce Rac1 activation9,29. Considering theimpaired autoregulation of glomerular capillary pressure and glomer-ular hyperfiltration in Dahl salt-sensitive rats30, increased mechanicalstress in the glomeruli may lead to Rac1 activation in this model. In

addition, we reported previously that enhanced mineralocorticoidreceptor signaling has a crucial role in the development of podocytedamage and glomerulosclerosis in this low-aldosterone model23. Thesefindings imply that Rac1 activation might contribute to the nonaldos-terone-mediated mineralocorticoid receptor activation in Dahl model.

The current study also provides, to our knowledge, the first in vivoevidence that inhibition of Rac1 can be beneficial in preserving kidneyfunction. In our experiments, eplerenone almost completely reversedthe kidney damage, whereas NSC23766 had partial effects. We proposethree reasons to account for this observation. First, NSC23766 did notfully inhibit Rac1 overactivation in Arhgdia–/– mice. Second, thedelayed initiation of NSC23766 treatment might have affected itsefficacy. Third, other unknown pathways may also be involved in theenhanced mineralocorticoid receptor signaling in Arhgdia–/– mice, inaddition to the Rac1-mediated pathway. Clinically, there is limitedevidence for the role of Rac1 GTPase in kidney diseases. Consideringthat NSC23766 successfully ameliorates glomerular damage inArhgdia–/– mice and Dahl rats, Rac1 inhibition might be useful intreating human glomerular disease.

In summary, the present study reveals a previously undescribedsignaling interaction between mineralocorticoid receptor and Rac1GTPase. Our data extend the current understanding of the mechanismfor the efficacy of mineralocorticoid receptor antagonists and under-score the clinical value of mineralocorticoid receptor blockade in

*

#

WT

Arhgdia

–/–

Arhgdia

–/–

+ NSC

TI i

njur

y sc

ore

0

0.5

1

1.5

2

*

#

0

GS

sco

re

WT

Arhgdia

–/–

Arhgdia

–/–

+ NSC

0.2

0.4

0.6

0.8

1 **

##

0

BU

N (

mg

dl–1

)

WT

Arhgdia

–/–

Arhgdia

–/–

+ NSC

10

20

30

40e fArhgdia–/– Arhgdia–/– + NSC

g

**

**#

##

**

##

WT Arhgdia–/–0

0.5

0Sgk

1/Gap

dh

Serpine

1/Gap

dh

WT

Arhgdia

–/–

Arhgdia

–/–

+ NSC

1

1.5

2

2.5

12345678

0

Ccl2/Gap

dh

12345678

Arhgdia–/–

+ NSC

h i

WT

Rac1

GTP-bound

Total

Arhgdia

–/–

Arhgdia

–/–

+ NSC

#

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

GT

P-R

ac1

(rel

ativ

e to

WT

)

Arhgdia

–/–

Arhgdia

–/–

+ NSC

WT Arhgdia–/– Arhgdia–/– + NSC

WTArhgdia–/–

Arhgdia–/– + NSC

00

2Duration of treatment (weeks)

4 6

1

2

3

4

5

Urin

ary

albu

min

(m

g d–1

)

**

#

a

**

#

0

3

6

Urin

ary

albu

min

(mg

per

mg

Cr) 9

12

WT

Arhgdia

–/–

Arhgdia

–/–

+ NSC

b d

c

Figure 3 Effects of NSC23766 on renal damage of Arhgdia–/– mice. (a) Urinary albumin excretion in

WT, Arhgdia–/– and Arhgdia–/– mice treated with NSC23766 (NSC) from 6 weeks until 12 weeks

of age. (b) Urinary albumin normalized to creatinine (Cr) concentration at the end of the

experiment (12 weeks of age). (c) Expression of GTP-bound Rac1 in the kidney. The bar graph

shows the result of densitometric analysis for GTP-Rac1. (d) Representative photomicrographs of

PAS-stained kidney sections. Scale bars, 100 mm. (e) Histological analyses of glomerulosclerosis

and tubulointerstitial injury by semiquantitative morphometric evaluation. GS, glomerulosclerosis;

TI, tubulointerstitial. (f) Serum blood urea nitrogen (BUN) from WT, Arhgdia–/– and Arhgdia–/–

mice treated with NSC23766. (g) Electron micrographs showing preserved foot processes in

NSC23766-treated Arhgdia–/– mice. Scale bar, 1 mm. (h) Quantitative analysis of Sgk1 geneexpression in the kidney. (i) Serpine1 (PAI-1) and Ccl2 (MCP-1) gene expression in the kidney. Data are expressed as means ± s.e.m.; n ¼ 4 each group for

a–c, e, f, h, i. *P o 0.05 and **P o 0.01 compared with WT mice; #P o 0.05 and ##P o 0.01 compared with Arhgdia–/– mice.

L E T T ERS

NATURE MEDICINE VOLUME 14 [ NUMBER 12 [ DECEMBER 2008 1373

©20

08 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

urem

edic

ine

treating proteinuric kidney diseases. Our data also highlight the Rac-mediated signaling pathway as an alternative target for therapeuticintervention in individuals with chronic kidney disease.

METHODSCell culture. We purchased HEK 293 cells from RIKEN Cell Bank and

incubated them in minimum essential medium supplemented with nonessen-

tial amino acids, 10% FBS, and antibiotics at 37 1C in a humidified atmo-

sphere of 5% CO2. The rat podocyte cell line (2DNA1D7) was established as

previously described31. Before steroid treatment, we replaced FBS with

charcoal-stripped FBS.

Transient transfection and cell treatment. The pEF-BOS-Myc expression

plasmids containing Rac1, RhoA, Cdc42 and the constitutively active mutants

(G12V Rac1, G14V RhoA, G12V Cdc42), and dominant-negative mutants

(T17N Rac1, T19N RhoA, T17N Cdc42) were described previously32. CA-Rac1

with the G12V mutation (Supplementary Fig. 4a online) has decreased

intrinsic GTPase activity and is unresponsive to GTPase-activating proteins33.

We confirmed that CA-Rac1 plasmid induces Rac1 activation (Fig. 1a and

5

4

3

Urin

ary

albu

min

(m

g d–1

)

2

1

0

0

0 4Duration of treatment (weeks)

WT

8

##

**0.2

0.4

0.6

GS

sco

re

0

0.5##

##

TI i

njur

y sc

ore

0.8

1

1.2

Arhgdia

–/–

Arhgdia–/–

Arhgdia–/– Arhgdia–/– + Epl

Arhgdia

–/–

+ Epl

0

42

68

1012

*

#14

WT

1-po

sitiv

e ce

lls

0

4

2

6

8

# ##

10

**

**

Serpine

1/Gap

dh

0

4

2

6

8

10

Ccl2/Gap

dh

Arhgdia

–/–

WT

Arhgdia

–/–

+ Epl

Arhgdia

–/–

Arhgdia

–/–

+ Epl

Arhgdia–/– Arhgdia–/– + Epl

Arhgdia–/– + Epl

1

1.5

2

2.5

WT Arhgdia–/– Arhgdia–/– + Epl

WTArhgdia–/–

Arhgdia–/– + Epl

a

e

Arhgdia–/– Arhgdia–/– + Eplh Arhgdia–/– Arhgdia–/– + Epli

0

4

2

6

8

10

## ##

****

TU

NE

L-po

sitiv

e ce

llspe

r 10

0 gl

omer

uli

0

4

2

6

8

10

TU

NE

L-po

sitiv

e ce

llspe

r vi

sual

fiel

d

Arhgdia

–/–

WT

Arhgdia

–/–

+ Epl

Arhgdia

–/–

WT

Arhgdia

–/–

+ Epl

j

f g

b c

d

Figure 4 MR signaling mediates albuminuria and renal injury in Arhgdia–/– mice. (a) Albuminuria in WT (n ¼ 9), Arhgdia–/– (n ¼ 13) and Arhgdia–/– micetreated with eplerenone (n ¼ 9) from 4 weeks until 12 weeks of age. (b) PAS-stained kidney sections from Arhgdia–/– and Arhgdia–/– mice treated with

eplerenone (Epl). Scale bars, 100 mm. (c) Morphometric analyses of glomerulosclerosis and tubulointerstitial injury in non-treated and treated Arhgdia–/–

mice (n ¼ 9). (d) Electron micrographs of glomeruli showing protected foot process structure in eplerenone-treated Arhgdia–/– mice. Scale bar, 1 mm.

(e) Immunohistochemical study for the podocyte marker WT1 (arrows) in the kidney. Scale bar, 50 mm. (f) Number of podocytes, determined as WT1-

expressing nuclei per glomerulus (n ¼ 4). (g) Quantitative analysis of Serpine1 and Ccl2 expression in the kidney (n ¼ 9). (h,i) Representative images of

TUNEL staining in the glomeruli (h) and tubules (i). Arrows indicate TUNEL-positive cells. Scale bars, 50 mm. (j) Quantitative analysis of TUNEL-positive

cells in WT, Arhgdia–/–,and Arhgdia–/– mice treated with eplerenone (n ¼ 5). Data are expressed as means ± s.e.m. *P o 0.05 and **P o 0.01 compared

with WT mice; #P o 0.05 and ##P o 0.01 compared with Arhgdia–/– mice.

Supplementary Fig. 4b). We added the PAK inhibitor PAK18 (Calbiochem) to

the medium at 4 h after transfection. PAK18 at a concentration of 10 mM

reduced PAK phosphorylation in podocytes (Supplementary Fig. 4c).

The expression plasmids encoding human mineralocorticoid receptor

(pCMX-FLAG-hMR), the chimeric construct of human mineralocorticoid

receptor and GFP (pCMX-hMR-GFP) and the mineralocorticoid response

element-driven luciferase reporter (pMRE-LUC) were described previously34.

We performed transient transfection experiments with LipofectAMINE 2000

reagent (Invitrogen). We treated cells with 1 nM aldosterone or ethanol vehicle

12 h after transfection for 24 h.

Luciferase assay. We assayed luciferase activity with a PicaGene kit (Toyo Ink)

and a luminometer (MiniLumat LB9506, Berthold). We normalized

the reporter assay to protein concentration and expressed values as the

relative activity.

Visualization of green fluorescence protein. We transfected HEK 293 cells

grown on cover slips with pCMX-hMR-GFP and cultured them for 48 h. We

then fixed the cells in 4% paraformaldehyde. We visualized GFP fluorescence

with a fluorescence microscope equipped with a FITC filter (Olympus BX51).

L E T TERS

1374 VOLUME 14 [ NUMBER 12 [ DECEMBER 2008 NATURE MEDICINE

©20

08 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

urem

edic

ine

Animals. Arhgdia–/– mice were generated as previously described3. All animal

procedures conducted were approved by the University of Tokyo Ethics

Committee for Animal Experiments. We subcutaneously infused NSC23766

(Calbiochem) via an osmotic minipump (Alza) from 6 weeks of age. Our

preliminary study revealed that NSC23766 at a dose of 10 mg kg–1 d–1 reduced

Rac1 activity in the kidney without apparent evidence of organ toxicity in mice.

Administration at a lower dose did not efficiently reduce renal Rac1 activity

(data not shown). We administered eplerenone (1.67 mg g–1 chow) from 4 weeks

of age. We measured systolic blood pressure by the tail-cuff method22. We fed

4-week-old male Dahl salt-sensitive rats (Japan SLC) an 8% or a 0.3% NaCl diet

for 4 weeks. We subcutaneously administered NSC23766 at 8 mg kg–1 d–1.

Urinalysis. We collected urine for 24 h with an individual metabolic cage

(Natsume). We determined urine albumin levels with a mouse albumin ELISA

(Shibayagi). For SDS-PAGE analysis, we electrophoresed 2 ml of the urine and

stained the gel with Coomassie brilliant blue.

Histomorphometric analysis and transmission electron microscopy. We

fixed kidneys in 4% paraformaldehyde solution and embedded them in paraffin.

In some mice, we performed in vivo perfusion fixation. For morphologic

evaluations, we stained transverse sections (4 mm) with periodic acid-Schiff

(PAS) reagents. We semiquantitatively assessed the degrees of tubulointerstitial

injury and glomerulosclerosis according to an established scoring system21,22.

We performed TEM analysis according to standard protocols21. We determined

the filtration slit frequency by counting the number of filtration slits divided by

the area of the underlying glomerular basement membrane in millimeters. We

counted more than 300 (for wild-type mice) or 200 (for Arhgdia–/– mice)

filtration slits for each mouse.

Western blot analysis. We performed western blotting as previously described20.

We prepared the nuclear extracts with commercially available kits (BioVision).

The primary antibodies used included antibodies to RhoGDI-a, RhoA, Cdc42,

Myc (all Santa Cruz Biotechnology), Rac1 (Upstate Biotechnology), GFP

(MBL), mineralocorticoid receptor (Perseus Proteomics), cyclic AMP response

element–binding protein (Millipore), total PAK, phosphorylated PAK, phos-

phorylated LIMK (all Cell Signaling), total LIMK and GAPDH (both Abcam).

Rho GTPase activation assay. We assessed the activities of RhoA, Rac1 and

Cdc42 with commercially available kits (Upstate Biotechnology). We homo-

genized samples in the magnesium lysis buffer and incubated at 4 1C for 60 min

with glutathione beads coupled with GST fusion protein corresponding to the

p21-binding domain of human PAK1 (for Rac1 and Cdc42) or the Rho-binding

domain of mouse rhotekin (for RhoA) (Upstate Biotechnology). We deter-

mined the RhoA, Rac1 and Cdc42 content in these samples by SDS-PAGE

and immunoblotting21.

Quantitative RT-PCR. We performed TaqMan real-time RT-PCR as previously

described21.

Immunohistochemistry. We performed immunostaining with a Wilm’s tumor

homolog-1 (WT1)-specific antibody (Santa Cruz Biotechnology) following a

standard protocol21. We calculated the number of podocytes per glomerulus as

WT1-expressing nuclei per glomerulus by counting 430 glomeruli per

kidney. We detected apoptosis with a commercially available kit (Chemicon).

We counterstained the sections with 0.5% methyl green. We counted

TUNEL-positive cells in more than 60 glomeruli for glomerular cell apoptosis

and in 10 randomly selected visual fields for tubular cell apoptosis in

each mouse.

Statistics. The data are shown as means ± s.e.m. We used an unpaired t test for

comparisons between two groups. For multiple comparisons, we used analysis

of variance with the Tukey-Kramer post hoc test. We analyzed histological data

by nonparametric analysis with the Kruskal-Wallis test followed by the Mann-

Whitney U test. P values o 0.05 were considered to be significant.

Note: Supplementary information is available on the Nature Medicine website.

ACKNOWLEDGMENTSWe are grateful to S. Fukuda for help in electron microscopic analysis. We thankPfizer for providing eplerenone and Asahi Kasei Pharma for providing fasudil.

AUTHOR CONTRIBUTIONSS.S. and M.N. planned and performed experiments and wrote the manuscript.S.Y. and W.K. helped with experimental procedures and contributed to datadiscussion. H.K. provided the podocyte cell line and advised on the technicalproposal. H.T. provided mineralocorticoid receptor plasmids and advised on theexperimental approach and writing. J.M. and Y.T. generated Arhgdia–/– mice,provided expression plasmids containing wild-type and mutant Rho GTPases,advised on the experimental approach and contributed to data discussion. T.F.planned and directed the project and reviewed the manuscript.

Published online at http://www.nature.com/naturemedicine/

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

1. Chrysostomou, A. & Becker, G. Spironolactone in addition to ACE inhibition to reduceproteinuria in patients with chronic renal disease. N. Engl. J. Med. 345, 925–926(2001).

2. Williams, G.H. et al. Efficacy of eplerenone versus enalapril as monotherapy insystemic hypertension. Am. J. Cardiol. 93, 990–996 (2004).

3. Togawa, A. et al. Progressive impairment of kidneys and reproductive organs in micelacking Rho GDIalpha. Oncogene 18, 5373–5380 (1999).

4. Gao, Y., Dickerson, J.B., Guo, F., Zheng, J. & Zheng, Y. Rational design andcharacterization of a Rac GTPase–specific small molecule inhibitor. Proc. Natl Acad.Sci. USA 101, 7618–7623 (2004).

5. Cancelas, J.A. et al. Rac GTPases differentially integrate signals regulating hemato-poietic stem cell localization. Nat. Med. 11, 886–891 (2005).

6. Kato, S. et al. Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270, 1491–1494 (1995).

7. Tronche, F. et al. Glucocorticoid receptor function in hepatocytes is essential topromote postnatal body growth. Genes Dev. 18, 492–497 (2004).

8. Takai, Y., Sasaki, T. & Matozaki, T. Small GTP-binding proteins. Physiol. Rev. 81,153–208 (2001).

9. Tzima, E. et al. Activation of Rac1 by shear stress in endothelial cells mediates bothcytoskeletal reorganization and effects on gene expression. EMBO J. 21, 6791–6800(2002).

10. Rubino, D. et al. Characterization of Brx, a novel Dbl family member that modulatesestrogen receptor action. Oncogene 16, 2513–2526 (1998).

11. Su, L.F., Knoblauch, R. & Garabedian, M.J. Rho GTPases as modulators of theestrogen receptor transcriptional response. J. Biol. Chem. 276, 3231–3237(2001).

12. Kino, T. et al. Rho family Guanine nucleotide exchange factor Brx couples extracellularsignals to the glucocorticoid signaling system. J. Biol. Chem. 281, 9118–9126(2006).

13. Le Menuet, D. et al. Alteration of cardiac and renal functions in transgenic miceoverexpressing human mineralocorticoid receptor. J. Biol. Chem. 276, 38911–38920(2001).

14. Massaad, C., Houard, N., Lombes, M. & Barouki, R. Modulation of human miner-alocorticoid receptor function by protein kinase A. Mol. Endocrinol. 13, 57–65(1999).

15. Funder, J.W. Is aldosterone bad for the heart? Trends Endocrinol. Metab. 15, 139–142(2004).

16. Nagase, M., Matsui, H., Shibata, S., Gotoda, T. & Fujita, T. Salt-induced nephro-pathy in obese spontaneously hypertensive rats via paradoxical activation of themineralocorticoid receptor: role of oxidative stress. Hypertension 50, 877–883(2007).

17. Burridge, K. & Wennerberg, K. Rho and Rac take center stage. Cell 116, 167–179(2004).

18. Edwards, D.C., Sanders, L.C., Bokoch, G.M. & Gill, G.N. Activation of LIM-kinase byPak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. Nat. CellBiol. 1, 253–259 (1999).

19. Wang, R.A., Mazumdar, A., Vadlamudi, R.K. & Kumar, R. P21-activated kinase-1phosphorylates and transactivates estrogen receptor-alpha and promotes hyperplasia inmammary epithelium. EMBO J. 21, 5437–5447 (2002).

20. Zhao, L. et al. Role of p21-activated kinase pathway defects in the cognitive deficits ofAlzheimer disease. Nat. Neurosci. 9, 234–242 (2006).

21. Shibata, S., Nagase, M. & Fujita, T. Fluvastatin ameliorates podocyte injury inproteinuric rats via modulation of excessive Rho signaling. J. Am. Soc. Nephrol. 17,754–764 (2006).

22. Shibata, S., Nagase, M., Yoshida, S., Kawachi, H. & Fujita, T. Podocyte as the target foraldosterone: roles of oxidative stress and Sgk1. Hypertension 49, 355–364(2007).

23. Nagase, M. et al. Podocyte injury underlies the glomerulopathy of Dahl salt-hypertensive rats and is reversed by aldosterone blocker. Hypertension 47,1084–1093 (2006).

24. Brown, N.J. et al. Aldosterone modulates plasminogen activator inhibitor-1 andglomerulosclerosis in vivo. Kidney Int. 58, 1219–1227 (2000).

25. Niranjan, T. et al. The Notch pathway in podocytes plays a role in the development ofglomerular disease. Nat. Med. 14, 290–298 (2008).

26. Isermann, B. et al. Activated protein C protects against diabetic nephropathyby inhibiting endothelial and podocyte apoptosis. Nat. Med. 13, 1349–1358(2007).

L E T T ERS

NATURE MEDICINE VOLUME 14 [ NUMBER 12 [ DECEMBER 2008 1375

©20

08 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

urem

edic

ine

27. Pitt, B. & Leopold, J.A. A role for mineralocorticoid receptor blockade in the preventionof apoptosis. J. Mol. Cell. Cardiol. 39, 415–417 (2005).

28. Wu, X. et al. Rac1 activation controls nuclear localization ofb-catenin during canonicalWnt signaling. Cell 133, 340–353 (2008).

29. Liu, W.F., Nelson, C.M., Tan, J.L. & Chen, C.S. Cadherins, RhoA and Rac1 aredifferentially required for stretch-mediated proliferation in endothelial versus smoothmuscle cells. Circ. Res. 101, e44–e52 (2007).

30. Takenaka, T., Forster, H., De Micheli, A. & Epstein, M. Impaired myogenic responsive-ness of renal microvessels in Dahl salt-sensitive rats. Circ. Res. 71, 471–480 (1992).

31. Eto, N. et al. Podocyte protection by darbepoetin: preservation of the cytoskeleton andnephrin expression. Kidney Int. 72, 455–463 (2007).

32. Komuro, R., Sasaki, T., Takaishi, K., Orita, S. & Takai, Y. Involvement of Rho and Racsmall G proteins and Rho GDI in Ca2+-dependent exocytosis from PC12 cells. GenesCells 1, 943–951 (1996).

33. Diekmann, D. et al. Bcr encodes a GTPase-activating protein for p21rac. Nature 351,400–402 (1991).

34. Iida, T. et al. Functional modulation of the mineralocorticoid receptor by cis-diammi-nedichloroplatinum (II). Kidney Int. 58, 1450–1460 (2000).

L E T TERS

1376 VOLUME 14 [ NUMBER 12 [ DECEMBER 2008 NATURE MEDICINE

©20

08 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

urem

edic

ine