Role of Rac1–mineralocorticoid-receptor signalling in renal and cardiac disease

86 | FEBRUARY 2013 | VOLUME 9 www.nature.com/nrneph

Division of Chronic Kidney Disease, Department of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine, 7‑3‑1 Hongo, Bunkyo‑ku, Tokyo 113‑8655, Japan (M. Nagase). Division of Clinical Epigenetics, Research Center for Advanced Science and Technology, The University of Tokyo, 4‑6‑1 Komaba, Meguro‑ku, Tokyo 153‑0041, Japan (T. Fujita).

Correspondence to: T. Fujita toshiro.fujita@ rcast.u‑tokyo.ac.jp

Role of Rac1–mineralocorticoid-receptor signalling in renal and cardiac diseaseMiki Nagase and Toshiro Fujita

Abstract | The Rho‑family small GTPase, Ras‑related C3 botulinum toxin substrate 1 (Rac1), has been implicated in renal and cardiac disease. Rac1 activation in podocytes has been shown in several models of proteinuric kidney disease and a concept involving motile podocytes has been proposed. Evidence also exists for a critical role of Rac1‑mediated oxidative stress in cardiac hypertrophy, cardiomyopathy and arrhythmia, and of the aldosterone–mineralocorticoid‑receptor system in proteinuria and cardiac disorders. However, plasma aldosterone concentrations are not always increased in these conditions and the mechanisms of mineralocorticoid‑receptor overactivation are difficult to determine. Using knockout mice, we identified a novel mechanism of Rac1‑mediated podocyte impairment; Rac1 potentiates the activity of the mineralocorticoid receptor, thereby accelerating podocyte injury. We subsequently demonstrated that the Rac1–mineralocorticoid‑receptor pathway contributes to ligand‑independent mineralocorticoid‑receptor activation in several animal models of kidney and cardiac injury. Hyperkalaemia is a major concern associated with the use of mineralocorticoid‑receptor antagonists; however, agents that modulate the activity of the Rac1–mineralocorticoid‑receptor pathway in target cells, such as cell‑type‑specific Rac inhibitors and selective mineralocorticoid‑receptor modulators, could potentially be novel therapeutic candidates with high efficacy and a low risk of adverse effects in patients with renal and cardiac diseases.

Nagase, M. & Fujita, T. Nat. Rev. Nephrol. 9, 86–98 (2013); published online 8 January 2013; doi:10.1038/nrneph.2012.282

IntroductionRas-related C3 botulinum toxin substrate (Rac) GTPases, such as Rac1, regulate diverse cellular functions, includ-ing organization of the actin cytoskeleton (formation of lamellipodia and membrane ruffles), generation of reactive oxygen species (ROS), gene transcription, cell migration and adhesion, apoptosis, cell cycle progression and vesicle trafficking (Figure 1).1,2 A role of Rac1 activa-tion in renal and cardiac disease has been suggested.3–6 In the kidney, Rac1 activation is associated with podo-cyte foot process effacement, which leads to an increase in glomerular permeability and proteinuria,3,4 whereas in the heart, activation of Rac1 contributes to myo-cardial hyper trophy, cardiomyopathy and arrhythmia.5,6 Activation of reduced nicotinamide-adenine dinucleo-tide phosphate (NADPH) oxidase and overproduction of ROS might be involved in these processes.5,6

Experimental studies and several large-scale, random-ized controlled trials have shown that mineralocorticoid-receptor activation also has an important role in renal and cardiac diseases, such as those involving podocyte injury, glomerulosclerosis, left ventricular hypertrophy, arrhythmia and heart failure.7,8 Interestingly, plasma aldosterone concentrations are not always increased in patients with these diseases or in animal models of these conditions,9–11 suggesting that the mineralo corticoid receptor might be activated by factors other than its

cognate ligands. However, potential mechanisms of ligand-independent mineralocorticoid-receptor acti-vation have only recently been elucidated. In a study using Rho GDP-dissociation inhibitor 1 (RhoGDI1, also known as RhoGDIα)-deficient mice (Arhgdia–/–), we identified a novel mechanism of Rac1-mediated podo-cyte injury; Rac1 enhances the activity of the mineralo-corticoid receptor in a ligand-independent manner, thereby accelerating podocyte impairment.12 We sub-sequently found evidence of Rac1–mineralocorticoid-receptor crosstalk in more established animal models of kidney and heart disease.13–15

In this Review, we summarize the available evidence for a role of Rac1 and mineralocorticoid-receptor activation in renal and cardiac dysfunction. We describe the studies in which Rac1 was identified as an alternative, ligand-independent activator of the mineralocorticoid recep-tor and the evidence for a role of this activation in renal and cardiac disease; finally, we discuss potential mecha-nisms of Rac1-mediated-mineralocorticoid-receptor activation.

Rac1Three isoforms of Rac GTPases exist, which are encoded by three distinct genes; Rac1 is ubiquitously expressed, Rac2 expression is restricted to haematopoietic cells and Rac3 is preferentially expressed in the brain.2 The consti-tutively active alternative splice variant of Rac1, Rac1b, contains an additional exon 3b and is upregulated in

Competing interestsThe authors declare no competing interests.

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NATURE REVIEWS | NEPHROLOGY VOLUME 9 | FEBRUARY 2013 | 87

colon and breast cancers.16 In mice, Rac1 gene ablation leads to gastrulation defects and embryonic lethality.17

ActivationRac1 functions as a molecular switch by cycling between an inactive GDP-bound and an active GTP-bound state (Figure 2). This transition is tightly controlled by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and GDP-dissociation inhibi-tors (GDIs).18,19 GEFs activate Rho family proteins by promoting the exchange of G-protein-bound GDP for GTP, whereas GAPs turn off signalling by stimulating the intrinsic GTP hydrolysis activity of small G proteins. GDIs bind to inactive GTPases and sequester them in the cytosol, preventing their translocation and subse-quent activation. In the mammalian genome, more than 90 GEFs (including Vav, T-lymphoma invasion and metastasis- inducing protein 1 [Tiam-1], son of seven less and Rho guanine nucleotide exchange factor 7) and ~70 GAPs regulate the activation of Rho family GTPases.18

Rac1 is activated by a variety of stimuli, including shear stress,20 mechanical stretch,21 ROS,15 inflam-matory cytokines,22 growth factors,23 integrins,24 high glucose concentrations,25,26 homocysteine,27 NaCl or osmotic stress,28–31 angiotensin II32–34 and aldosterone35,36 (Figure 1). Activation occurs via stimulation of GEFs or inactivation of GAPs in a cell-type-specific and stimulus- specific manner. However, little is known about which GEFs or GAPs are involved in the responses to indi-vidual stimuli in specific cell types. Regulation of GEF activation is highly complex and can involve interactions with heterotrimeric G proteins, activation of phospho-inositide 3-kinase (PI3K) or direct phosphorylation via Src, Ca2+/calmodulin-dependent protein kinase II (CaMKII) or protein kinase C.37

Functions and effector proteinsRac1 activates downstream effector and signalling pro-teins (Figure 1), including p21-activated kinase (PAK) and LIM kinase, which are involved in gene regulation and actin reorganization; Wiskott-Aldrich syndrome protein family verprolin-homologous protein complex, actin-related protein 2/3 and mammalian diaphanous-2, which are involved in actin polymerization; PI3K, which is involved in cell migration; and IQ motif containing GTPase activating protein (IQGAP1, also known as Ras GTPase-activating-like protein), which is involved in cell–cell adhesion and actin reorganization.2 Rac1 also activates mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1/2, p38 MAPK, and c-jun N-terminal kinase (JNK).1

Unlike other Rho-family members, Rac GTPases stim-ulate ROS production by activating NADPH oxidase.38–41 This effect is mediated by Rac2 in neutrophils38 and by Rac1 in macrophages39 and nonphagocytic cells.40,41 Structural analyses have shown that effector proteins interact with distinct residues within the Rac1 mol-ecule; the amino acid Phe37 interacts with proteins that have a role in cytoskeletal changes, amino acid Tyr40 is responsible for activation of PAK and JNK, and the α3’

Key points

■ Rac1 activation has been implicated in podocyte injury, cardiac dysfunction and other diseases

■ Rac1 activation in glomerular podocytes might be associated with increased podocyte motility and foot process effacement

■ Rac1 potentiates the activity of the mineralocorticoid receptor in a ligand‑independent manner, thereby accelerating podocyte impairment

■ Ligand‑independent mineralocorticoid‑receptor activation is mediated by Rac1 in several animal models of salt‑sensitive hypertension and kidney disease

■ Rac1–mineralocorticoid‑receptor crosstalk contributes to the cardiac injury induced by reactive oxygen species

■ Rac inhibitors and selective mineralocorticoid‑receptor modulators that regulate the activity of the Rac1–mineralocorticoid‑receptor pathway might be novel candidates for the treatment of renal and cardiac disease

helix has a role in ROS production. The generation of mutant forms of Rac1 with limited effector functions is, therefore, possible.42

Role in kidney diseaseThe motile podocyte hypothesisPodocytes are highly specialized epithelial cells that line the outer surface of the glomerular capillaries in the kidney and serve as the final barrier to urinary protein loss.43,44 The podocyte cell body extends long pro trusions called major processes from which branch numerous foot processes. The foot processes of neighbouring podocytes interdigitate and are interposed by modified adherens junctions termed slit diaphragms. Podocytes typically undergo foot process effacement (retraction of foot processes) in response to damage. Persistence of insults culminates in podocyte loss as a result of apop tosis, necrosis or detachment, and focal segmental glomerulo sclerosis (FSGS).43

Podocyte foot process effacement is a dynamic and reversible process. The Rho family small GTPases, RhoA, Rac1 and cell division control protein 42 (Cdc42) are master regulators of the actin cytoskeleton. They have a vital role in maintaining the morphological and func-tional integrity of foot processes under normal condi-tions, and their imbalance results in abnormalities.4 A concept of motile podocytes has been suggested, whereby healthy, differentiated podocytes have a RhoA-dependent stationary phenotype and injured podocytes undergo a phenotypic shift to a Rac1-dependent and Cdc42-dependent motile state with suppressed RhoA signalling.3 The latter phenotype is associated with increased podocyte motility in vitro and foot process effacement, slit diaphragm remodelling and protein-uria in vivo.3 Motile podocytes with high turnover of the actin cytoskeleton might eventually become worn out, resulting in podocyte depletion and FSGS. Supporting this concept, Rac1 activation and increased podocyte motility have been shown in several animal models of proteinuric kidney disease, which are discussed below.

Angiotensin-II-induced podocyte injuryA Rac1-dependent switch, whereby podocytes switch from a stationary phenotype to a motile pheno-type has been clearly shown in rodent models of

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angiotensin-II-induced podocyte injury.45 In cultured mouse podocytes with stable type 1 angiotensin II (AT1)-receptor expression, angiotensin II activated the Rac1–NADPH oxidase–ROS cascade, resulting in rearrange ment of the actin cytoskeleton from the filo-podia/stress fibre type to the lamellipodia/cortical actin ring type, which has increased migratory capacity.45 In addition, increased expression of Rac1 was shown in the glomeruli of rats with podocyte-specific over expression of the AT1 receptor—a model of effaced foot processes, podocyte depletion and FSGS.45,46 Angiotensin II acti-vated Rac1 but did not affect Cdc42 activity and the effects of Rac1 and ROS were mediated via phospho-rylation of ezrin/radixin/moesin proteins.45 Thus, the researchers proposed that Rac1 inhibitors might protect podocytes from excess ROS generation and cytoskeletal rearrangement.45

A subsequent study further clarified the mechanism by which angiotensin II regulates the Rac1–RhoA balance.47 In mouse podocytes, angiotensin II stimulated Ca2+ influx via transient receptor potential canonical (TRPC) 5 and TRPC6 channels.47 Interestingly, TRPC5 formed a molecular complex with Rac1, whereas TRPC6 associated with RhoA. Ca2+ influx via TRPC5 channels, therefore, activates Rac1 and stimulates podocytes to undergo conversion to a motile phenotype, whereas Ca2+ influx via TRPC6 channels activates RhoA and elicits a stationary podocyte phenotype.

uPAR–integrin–FAK-mediated podocyte injuryA role of Rac1 in podocyte injury has also been shown in animal models of urokinase plasminogen activa-tor receptor (uPAR)-mediated podocyte injury. uPAR is upregulated in many human cancers and promotes extracellular matrix proteolysis by binding and activating urokinase. In addition to its role as a proteinase receptor, uPAR binds the signalling coreceptors αvβ3 integrins and activates the focal adhesion kinase (FAK)–Rac1 cascade, resulting in cytoskeleton reorganization, increased motil-ity and cancer cell invasion.48 Angiotensin-II-induced migration of vascular smooth muscle cells also involves a uPAR-mediated migratory phenotype.49

As cancer cell motility during invasion and podocyte motility during proteinuric conditions are analogous processes, Wei et al. investigated whether uPAR– integrin signalling had a role in podocyte injury.50 They showed that uPAR was induced and activated in podocytes in response to proteinuric stimuli, such as lipopoly-saccharide and puromycin aminonucleoside. In addition, uPAR stimulated β3 integrin, which in turn increased podocyte motility through activation of Rac1 and Cdc42. Both podocyte-specific deletion of uPAR and αvβ3 inte-grin blockade efficiently reduced podocyte motility and proteinuria, suggesting a role for uPAR–integrin–Rac1 signalling and the Cdc42 cascade in these processes.

Focal and segmental glomerulosclerosisA mutation (Gln158Arg) in the GAP domain of the Arhgap24 gene, which encodes the RhoA-activated, Rac1 GAP, Rho GTPase-activating protein 24 (RhoGAP24), has been identified in patients with FSGS.51 These patients had a Rac1-induced motile podocyte phenotype. RhoGAP24 is a regulator of the activity of RhoA and Rac1, which often act antago-nistically.52 When RhoA is activated, RhoGAP24 is phosphorylated by Rho kinase. This phosphoryla-tion increases the GAP activity of RhoGAP24, result-ing in inactivation of Rac1 and Cdc42.52 In wild-type mouse podocytes, RhoGAP24 was upregulated during differentiation and conferred a static phenotype.51 Conversely, knockdown of the Arhgap24 gene in mouse podocytes resulted in activation of Rac1 and Cdc42 and a switch to the motile phenotype.51 As the expression of RhoGAP24 is mainly restricted to podocytes, this protein is a promising therapeutic target for proteinuric kidney disease. Therapies that target RhoGAP24 could potentially be used to specifically modulate the activity

IntegrinsMechanical

stress CytokinesGrowthfactors

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WAVE complexmDia2

PAK, LIM kinase

Actin reorganization(lamellipodia, ruf�es)

Increased podocyte motilitySarcomeric structures

In�ammation, �brosis,apoptosis, gene regulation

Podocyte injury, proteinuria, FSGSCardiac hypertrophy, cardiomyopathy, heart failure, arrhythmia

NefGlucose

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Kinase cascade■ ERK■ JNK■ p38 MAPK■ JAK/STAT■ PI3KActivation ofcalcineurin–NFATpathwayNuclear translocationof transcriptionfactorsModulation of nuclearreceptor function

Figure 1 | Mechanisms of Rac1‑induced renal and cardiac disease. Rac1 is activated by various stimuli (such as angiotensin II, adrenaline, endothelin‑1, integrins, Nef, high glucose or salt levels, mechanical stress, oxidative stress, cytokines and growth factors) and stimulates downstream effector and signalling proteins, including WAVE complex, mDia2, PAK and LIM kinase, which have a role in actin reorganization and motility. Rac1 also activates kinases and transcription factors (resulting in the activation of signalling pathways) and stimulates the production of ROS via NADPH oxidase and mitochondrial pathways. ROS activate NFκB, leading to inflammation, fibrosis, apoptosis and gene regulation. These processes culminate in podocyte injury, proteinuria, and FSGS in the kidney, and cardiac hypertrophy, cardiomyopathy, heart failure, and arrhythmia in the heart. Abbreviations: ERK, extracellular signal‑regulated kinase; FSGS, focal segmental glomerulosclerosis; JAK, Janus kinase; JNK, c‑jun N‑terminal kinase; MAPK, mitogen‑activated protein kinase; mDia2, mammalian diaphanous‑2; NADPH, reduced nicotinamide‑adenine dinucleotide phosphate; NFAT, nuclear factor of activated T cells; NFκB, nuclear factor κB; PAK, p21‑activated kinase; PI3K, phosphoinositide 3‑kinase; Rac1, Ras‑related C3 botulinum toxin substrate 1; ROS, reactive oxygen species; STAT, signal transducer and activator of transcription; WAVE, Wiskott–Aldrich syndrome protein family verprolin‑homologous protein.

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of RhoA and Rac1 in podocytes with minimal systemic adverse effects.

HIV nephropathyAberrant Rac1 signalling and a change in podocyte pheno type also occurs in HIV-associated nephro-pathy.53 The HIV adaptor protein Nef upregulates Rac1 signalling via Src and Vav2, resulting in podocyte con-version to the motile phenotype, podocyte injury and collapsing FSGS.53

Role in heart diseaseIn the heart, aberrant Rac1 activation has been reported to cause cardiomyocyte hypertrophy, cardiomyopathy, heart failure and arrhythmia.5,6 Rac1 activation has also been identified in human hypertrophic hearts,54 in failing left ventricular myocardium,55 and in the left atria of patients with atrial fibrillation.56 The majority of these studies showed that Rac1 activation was associated with increased NADPH oxidase activity, and the resultant ROS and nuclear factor κB (NFκB) activation have been proposed as critical mediators of cardiac patho biology.5 However, activation of the calcineurin–NFAT signalling pathway and MAPK phosphorylation cascade have also been shown to have a role in the induction of cardiac hypertrophy.6,57 In addition to their cholesterol-lowering effects, statins suppress the activity of Rac1 by reduc-ing its isoprenylation and translocation to the cell mem-brane.58 Statin therapy was shown to ameliorate cardiac hypertrophy in a rat model33 and reduce the incidence of atrial fibrillation in aged mice with cardiac-specific over-expression of constitutively active Rac1.56 Rac1 inhibi-tor therapy might have similar therapeutic benefits in patients with heart disease.

Myocardial hypertrophyMechanical overload21,59 and hypertrophic agonists such as adrenaline-receptor activators,60–62 endothelin-1,61 and angiotensin II,33,34,63,64 have been shown to activate Rac1 in vitro in rodent cardiomyocytes and/or in vivo in rodent hearts. A critical role of Rac1 in the signal-ling pathway that leads to cardiomyocyte hypertrophy was also shown in an in vitro study in which the effects of adenoviral-mediated gene transfer of constitutively active or dominant-negative Rac1 on rat neonatal ven-tricular myocytes were investigated.60 Gene transfer of constitutively active Rac1 resulted in morphological and biochemical hypertrophic effects, such as reorganization of the actin cytoskeleton into sarcomeric structures, an increase in cell surface area, increased secretion of atrial natriuretic peptide, and protein synthesis. By contrast, expression of dominant-negative Rac1 led to inhibition of ligand-stimulated and pressure-stimulated hypertrophic responses.21,33,60,61

Cardiomyocyte-specific Rac1 transgenic or knockout mice were used to investigate the role of Rac1 in cardiac pathology in vivo. Specific overexpression of consti-tutively active Rac1 in murine myocardium resulted in two distinct phenotypes: rapid-onset lethal dilated cardiomyopathy and slow-onset cardiac hypertrophy

in juvenile mice that resolved with age.65 Conversely, cardio myocyte-specific ablation of Rac1 in mice reduced angiotensin-II-induced myocardial hypertrophy, NADPH oxidase activity and oxidative stress.63 These results support a critical role of cardiomyocyte Rac1 in the pathogenesis of myocardial hypertrophy.

Diabetic cardiomyopathyCardiomyocyte apoptosis is a characteristic feature of diabetic cardiomyopathy. In cardiomyocytes and in the hearts of mice with type 1 or type 2 diabetes, high glucose levels activated Rac1 and induced myocardial apoptosis through NADPH-oxidase-mediated and mitochondria-mediated ROS production.26 These effects were blocked by dominant-negative Rac1 expression, cardiomyocyte-specific Rac1 deletion or administration of the Rac inhibitor NSC23766.26

Diabetes is also associated with cardiac hypertrophy, inflammation and fibrosis. In a mouse model of chronic type 1 diabetes, deletion of Rac1 abrogated cardiac hyper-trophy and fibrotic remodelling, possibly via suppression of inflammation and endoplasmic reticulum stress.66 Similarly, in a rat model of diabetes, statin treatment normalized Rac1 and RhoA activation, ROS produc-tion, and p38 MAPK phosphorylation and ameliorated hyperglycaemia-induced cardiac inflammation and fibro-sis.67 However, the individual effects of Rac1 and RhoA on cardiac inflammation and fibrosis were not elucidated.

ArrhythmiaA potential role of Rac1 in atrial fibrillation has also been shown. In one study, mice with cardiac-specific

RhoGDI

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NSC23766EHop-016

P

GDP

GEFsGAPs

GDP

InactiveRac1

GTP

ActiveRac1

GTP

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Figure 2 | Regulation of Rac1 activity. Rac1 cycles between an inactive GDP‑bound and an active GTP‑bound state. GEFs such as TIAM‑1, Trio, Vav1–3, RhoGEF7, SOS‑1 and SOS‑2, activate Rac1 by promoting the exchange of Rac1‑bound GDP for GTP, whereas GAPs, such as RhoGAP24, inactivate Rac1 by stimulating its GTPase activity. Binding of RhoGDI to GDP‑bound inactive Rac1 prevents Rac1 activation. The sites of action of the Rac1 inhibitors, NSC23766, EHop‑016, and EHT1864, are shown. Abbreviations: GAPs, GTPase activating proteins; GEFs, guanine nucleotide exchange factors; P, phosphorus; Rac1, Ras‑related C3 botulinum toxin substrate 1; RhoGAP24, Rho GTPase activating protein 24; RhoGDI, Rho GDP‑dissociation inhibitor; RhoGEF7, Rho guanine nucleotide exchange factor 7; SOS, son of sevenless; TIAM‑1, T‑lymphoma invasion and metastasis‑inducing protein 1; Trio, Triple functional domain protein.

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expression of constitutively active Rac1 developed atrial fibrillation with age.56 Statin treatment suppressed cardiac Rac1 and NADPH oxidase activity and reduced the incidence of atrial fibrillation in these mice but had no effect on atrial size or the extent of fibrosis. A sub-sequent study identified a role of the connective tissue growth factor–connexin-43–N-cadherin pathway in the development of atrial fibrillation induced by Rac1 and ROS.68 However, data from another study suggested an alternative mechanism of Rac1-induced arrhythmia. In atrial myocytes and fibroblasts, but not in ventricles, angiotensin II stimulation activated Rac1, leading to activation of the JAK–STAT signalling pathway, myocyte hypertrophy and fibrosis.64 In atrial myocytes, activation of STAT3 by Rac1 was mediated by their direct associa-tion, whereas in atrial fibroblasts, STAT3 activation was mediated via an indirect paracrine effect. Consistent with these findings, angiotensin II concentrations and phospho-STAT3 levels were increased in tissue samples from patients with atrial fibrillation.64

Role in other diseasesIn addition to renal and cardiac disorders, Rac1 activa-tion has been implicated in other diseases. Hemizygous knockout of Rac1 in vascular endothelium protected mice against brain injury after focal cerebral ischae-mia, possibly by promoting endothelial barrier integ-rity and neurotrophic activity via upregulation of the neurotrophic factor artemin.69 A role for Rac1 in the midline crossing of commissural axons in the brain has also been reported.70 In murine models, inactivation of Rac1 using adenovirus-mediated gene delivery of dominant-negative Rac1 ameliorated diabetic vascular complications71 and ischaemia–reperfusion-induced liver injury.72 Beneficial effects of the specific inhibitors of Rac1, NSC23766, EHT1864 and EHop-016, have been shown in animal models of pancreatitis and associated lung injury,73 Alzheimer disease74 and metastatic cancer cell migration.75

RhoA and Cdc42Role in kidney diseaseAccording to the motile podocyte hypothesis, increased Rac1 and Cdc42 activity and decreased RhoA signalling are associated with podocyte foot process effacement and proteinuric kidney disease.3 However, podocyte injury and FSGS have been shown in transgenic mice with podo-cyte-specific expression of constitutively active RhoA as well as in mice that express dominant-negative RhoA76,77 or have podocyte-specific knockout of Cdc42.78 In addi-tion, the temporospatial pattern of Rac1, RhoA and Cdc42 activation within cells might be important in determining cellular function.79 These data suggest that both too much and too little motility are detrimental to podocytes and that properly balanced Rac1–Cdc42–RhoA signalling is essential for normal podocyte function.80

Role in heart diseaseContrary to the established hypertrophic role of Rac1, Cdc42 was shown to function as an antihypertrophic

molecular switch in the heart by antagonizing NFAT activity via JNK activation.81 PAK1, an effector of Rac1 and Cdc42, also acts as an antihypertrophic regula-tor82 and the PAK1 activator FTY720 prevented pres-sure-overload-induced hypertrophy in wild-type mice without compromising cardiac function.83 In mice, cardiac- specific overexpression of constitutively active RhoA did not cause hypertrophy but did induce arrhyth-mia.84 In addition, expression of constitutively active RhoA conferred cardioprotection against ischaemia–reperfusion injury via a mechanism involving activation of protein kinase D.85

The mineralocorticoid receptorThe mineralocorticoid receptor is a ligand-activated transcription factor that belongs to the steroid recep-tor superfamily of nuclear receptors. The principle physio logical ligand of the receptor is aldosterone; however, in nonepithelial cells that do not express the enzyme 11β-hydroxysteroid dehydrogenase 2, gluco-corticoids might also act as endogenous mineralo-corticoid-receptor ligands.10 In the distal nephron of the kidney, the aldosterone–mineralocorticoid-receptor system has conventionally been considered to have a pivotal role in the homeostatic regulation of electro-lytes, fluid volume and blood pressure. However, attention is increasingly being focused on the role of the mineralo corticoid receptor in nonclassical cells (including macrophages) as a mediator of organ injury mainly through induction of inflammatory and oxida-tive processes. Data from recent studies have indicated a role for mineralocorticoid- receptor activation in the develop ment of angiotensin-II-induced cardiovascular and kidney damage.86–89

Role in renal and cardiac diseasePodocyte injuryAccumulating evidence suggests that mineralocorticoid- receptor activation has harmful effects on podocytes. Uninephrectomized salt-loaded rats treated with aldo-sterone or deoxycorticosterone showed prominent podocyte injury, proteinuria and FSGS, which was at least partially attributable to the nonhaemodynamic effects of mineralocorticoid-receptor activation.35,90 Conversely, mineralocorticoid-receptor antagonists conferred protection against podocyte injury and/or proteinuria and albuminuria in animal models of dia-betic nephro pathy,91–94 hypertension,11,95 metabolic syndrome,96 antiglomerular basement membrane antibody-induced nephritis,97 anti-Thy-1 nephritis,98 lupus nephritis,99 puromycin aminonucleoside nephro-sis100 and adriamycin nephrosis101 as well as in remnant kidney models.102 A systematic review and meta-analysis confirmed the efficacy of mineralocorticoid-receptor antagonists for the reduction of proteinuria in patients with chronic kidney disease (CKD) who were receiving angio tensin II-inhibitor therapy.103

NADPH oxidase-derived ROS,35,94 mitochondria-derived ROS,104 mitochondrial dysfunction, a reduc-tion in peroxisome proliferator-activated receptor-γ

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coactivator-1α levels,105 MAPK phosphorylation106 and apoptosis12,107 have all been reported to have a role in aldosterone-mediated and mineralocorticoid- receptor-mediated podocyte injury. However, the spe-cific mechanisms leading to injury, in particular which target genes are induced through the genomic actions of the mineralo corticoid receptor, have not yet been fully elucidated.

In contrast to the detrimental effects of mineralo-corticoid-receptor activators, glucocorticoid-receptor agonists exert direct beneficial effects on podocytes. For example, dexamethasone treatment increased actin polymerization, stabilized actin filaments (possibly via RhoA activation),108 prevented podocyte apoptosis,109 inhibited NFκB activity and altered the expression of nephrin and inflammatory cytokines in cultured podo-cytes.110,111 In addition, we found that mineralocorticoid- receptor stimulation induced Rac1 activation in the rat kidney and glomerulus.14,35 Mineralocorticoid receptors might, therefore, antagonize the effects of the gluco corticoid receptor on the actin cyto skeleton. It is generally thought that activation of both the mineralo corticoid receptor and glucocorticoid receptor enhance generation.112

Mineralocorticoid receptors and glucocorticoid receptors derive from a common ancestral corticoid receptor,113 share a high level of homology (especially in their DNA-binding and ligand-binding domains) and bind to the same DNA consensus sequence. However, they have opposite effects on podocyte func-tion. The mineralo corticoid receptor is a promiscu-ous nuclear receptor that has a high affinity for both aldosterone and glucocorticoids.10 Studies suggest that differential patterns of coregulator recruitment might explain the distinct effects of glucocorticoid recep-tors and mineralo corticoid receptors to aldosterone or gluco corticoid stimulation.114 Selective nuclear recep-tor modulators that activate only a subset of receptor functions or act in a cell-type-specific manner are now being developed.115,116

Cardiac injuryAnimals with an augmented mineralocorticoid- receptor signalling cascade, such as transgenic mice with cardiomyocyte- specific expression of 11β-hydroxysteroid dehydrogenase 2,117 aldo sterone synthase118 or the mineralo corticoid receptor,119 and uninephrectomized rats receiving aldosterone and a high-salt diet,120 exhibit cardiac hypertrophy, heart failure, coronary dysfunction, arrhythmia, peri vascular inflammation and fibrosis. Conversely, mice with cardiomyocyte- specific knockout of the mineralo-corticoid receptor are protected from myocardial infarc-tion121 and chronic pressure overload.122 High circulating levels of the mineralocorticoid-receptor ligands aldoster-one and cortisol were reportedly associated with poor prognosis in patients with heart failure and myocardial infarction,123,124 and large-scale random ized controlled trials have shown beneficial effects of mineralo corticoid-receptor blockade on morbidity and mortality in patients

with systolic heart failure.125–127 The indications for which mineralocorticoid-receptor- antagonist therapy is recommended were, therefore, greatly expanded in the 2012 European Society of Cardiology Guidelines for the Diagnosis and Treatment of Acute and Chronic Heart Failure.128 Furthermore, a preliminary report of the Aldosterone Receptor Blockade in Diastolic Heart Failure Trial showed that compared with placebo, treat-ment with the aldosterone antagonist spironolactone improved diastolic function and decreased N-terminal probrain natriuretic peptide levels in patients with diastolic heart failure.129 The effect of treatment with spironolactone on morbidity, mortality and quality of life in patients with heart failure and preserved systolic function is currently being evaluated in the Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist Trial.130

The mechanisms by which mineralocorticoid- receptor activation leads to cardiac injury have been investigated in several studies. Aldosterone has been shown to induce cardiomyocyte injury via the NADPH-oxidase-dependent oxidative activation of CaMKII, which promotes matrix metalloproteinase 9 expression and cardiac rupture in mice after myo-cardial infarction.131 Moreover, studies using cell-type-specific mineralocorticoid- receptor-knockout mice showed that the receptor activates distinct signalling events in cardio myocytes, macrophages, and cardiac fibroblasts and has unique roles in the development of cardiac injury in different cell types. In macrophages the mineralo corticoid receptor has a role in ROS generation, inflammation and fibrotic changes,88,132 whereas activation of the mineralocorticoid receptor in cardio myocytes has a role in arrhythmogenic changes, left ventricular remodelling, and the transition from cardiac hypertrophy to heart failure.121,122 Interestingly, activation of the mineralocorticoid receptor in cardiac fibroblasts had no overt effects on fibrosis.122

The Rac1–mineralocorticoid-receptor pathwayThe RALES trial showed that addition of mineralo-corticoid- receptor antagonists to standard therapy dra-matically improved the survival of patients with heart failure.125 The trial was designed based on the assump-tion that plasma aldosterone levels might be high in patients receiving long-term angiotensin II blockade as a result of aldosterone breakthrough.125 However, the plasma aldosterone levels of the RALES partici-pants were normal.10 Increasing evidence suggests that mineralo corticoid receptors might be activated in target organs even when circulating aldosterone con-centrations are normal or low.9–11 These findings raise the possibility that the receptors might be activated by factors other than their cognate ligands. As described below, we identified an alternative pathway of Rac1-mediated mineralocorticoid- receptor activation in Arhgdia–/– mice12 and showed that this pathway contrib-utes to ligand-independent mineralocorticoid- receptor activation in several animal models of kidney and cardiac injury.

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Identification of the pathwayIn 1999, Togawa et al. reported that Arhgdia–/– mice developed proteinuric kidney disease.133 However, the detailed mechanisms leading to disease, including the activation profiles of Rho family members, were not elucidated. As RhoGDI1 interacts with GDP-bound inactive GTPases and maintains them in an inactive state, we hypothesized that the RhoGDI1 deficiency in Arhgdia–/– mice might result in overactivation of Rho family members. We found that the mice had podocyte impairment and albuminuria at 1 week of age, which pro-gressed to nephritic-range albuminuria, severe podocyte injury and FSGS at 12 weeks of age.12 GTP-bound active Rac1, but not active RhoA, was markedly increased in the kidney of Arhgdia–/– mice and treatment with the Rac-specific inhibitor NSC23766 significantly reduced albuminuria, podocyte dysfunction, and FSGS, con-comitantly with repression of Rac1 activity. By contrast, the Rho-kinase inhibitor fasudil had no renoprotective effects in Arhgdia–/– mice.12 These results clearly indi-cated that RhoGDI1 deletion caused Rac1-dependent podocyte injury in Arhgdia–/– mice.

Subsequently, we investigated the mechanism by which Rac1 activation caused podocyte damage. ROS did not seem to be responsible for the damage because Rac1 acti-vation was not accompanied by increased NADPH oxidase activity, and neither the NADPH oxidase inhibitor apo-cynin nor the superoxide scavenger tempol ameliorated podocyte injury in Arhgdia–/– mice.12 Moreover, accumu-lating evidence indicated that Rho GTPases and their asso-ciated proteins modulate nuclear receptor functions.134–138 We previously showed that mineralocorticoid- receptor overactivation leads to podocyte dysfunction,11,35,96,139 and that the mineralocorticoid receptor might some-times be activated by factors other than aldosterone.11,139 These findings led us to hypothesize that Rac1 might act as a ligand-independent modifier of mineralocorticoid- receptor function. To investigate this hypothesis, we transiently transfected HEK 293 cells with constitutively active Rac1 and evaluated the transcriptional activity of the mineralo corticoid receptor as well as the nuclear translocation of green fluorescent protein (GFP)-tagged mineralo corticoid receptor. We found that mineralo-corticoid-receptor-dependent transcriptional activity was increased in response to aldosterone and that the transfection of constitutively active Rac1 potentiated this response.12 In addition, constitutively active Rac1 facili-tated the mineralocorticoid- receptor-dependent transcrip-tion of target genes and enhanced the nuclear translocation of the mineralocorticoid receptor both in the absence and in the presence of aldosterone.12 These data indicate that Rac1 activation increases mineralocorticoid- receptor activity. Similarly, constitutively active Rac1 promoted the nuclear translocation of GFP-tagged mineralo corticoid receptor in cultured podocytes.12 Rac1 activation was accompanied by increased phosphorylation of the effector proteins PAK and LIM kinase. Notably, the PAK inhibitor PAK18 substantially reduced the Rac1-mediated increase in mineralocorticoid- receptor translocation, indicating that Rac1 acts via activation of PAK.

Next we evaluated the contribution of the Rac1–mineralocorticoid-receptor pathway to nephropathy in Arhgdia–/– mice. Although serum aldosterone concen-trations were not increased, the amount of mineralo-corticoid receptor in the nuclear fraction was upregulated in the kidneys of these mice, suggesting ligand- independent activation and nuclear accumulation of the mineralocorticoid receptor.12 Treatment with the mineralocorticoid-receptor antagonist eplerenone pre-vented albuminuria, podocyte injury and FSGS, and the Rac-specific small-molecule inhibitor NSC23766 inhib-ited mineralocorticoid-receptor activation in Arhgdia–/– mice. Levels of the potential downstream effectors of the Rac1–mineralocorticoid-receptor pathway, Sgk1, plasminogen activator inhibitor-1 and monocyte chemo attractant protein-1, as well as levels of apop tosis, were increased in Arhgdia–/– mice and suppressed by NSC23766 and eplerenone.12 These in vivo results cor-roborate our in vitro data indicating cross-talk between Rac1 and mineralocorticoid-receptor signalling,12 and suggest that Rac1-mediated mineralocorticoid-receptor activation has a central role in podocyte damage and FSGS in Arhgdia–/– mice.

Role in cardiac and renal diseaseAs the Arhgdia–/– mouse is an artificial model of kidney injury created using gene deletion, we next examined whether the Rac1–mineralocorticoid-receptor activa-tion pathway has a pathogenic role in more commonly encountered renal and cardiac diseases.

Angiotensin II/salt-induced renal injuryTsukuba hypertensive mice (THM) are a transgenic strain that express both human renin and human angiotensino-gen genes and produce high levels of angio tensin II.13 We found that despite the elevated renin–angiotensin– aldosterone levels caused by transgene expression, THM did not show renal injury under salt-restricted conditions. However, salt-loaded THM developed prominent kidney injury (Figure 3). As both angio-tensin II and aldosterone are excessively produced in THM we investigated which factor predominates as the cause of renal injury in the salt-loaded mice.

Aldosterone removal by adrenalectomy prevented nephropathy in salt-loaded THM despite persistent angiotensin II and salt excess, whereas aldosterone sup-plementation recapitulated renal impairment in the adrenalectomized mice.13 These findings suggest the involvement of the aldosterone–mineralocorticoid-receptor pathway in the development of salt-induced renal injury. In THM, salt-loading reduced plasma aldosterone levels and resulted in activation of Rac1 and the mineralocorticoid-receptor cascade in the kidney (Figure 3b). These data suggest that Rac1 was responsible for the ligand-independent activation of the mineralo corticoid receptor. Treatment with either the Rac inhibitor EHT1864 or eplerenone normalized mineralo-corticoid-receptor signalling and reduced kidney injury in salt-loaded THM mice. Both treatments also resulted in a slight decrease in blood pressure although the mice

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remained hypertensive.13 The direct vasodilator hydrala-zine similarly lowered blood pressure but did not suppress the Rac1–mineralocorticoid-receptor pathway, nor ame-liorate nephropathy in salt-loaded THM mice.13 Plasma or tissue angiotensin II levels are increased in several clin-ical conditions, such as obesity, metabolic syndrome and CKD.140 Kidney injury in patients with these conditions might be worsened by high-salt intake via Rac1-mediated mineralocortocoid-receptor activation. Rac inhibi-tion and reduction of salt intake might, therefore, be a promising therapeutic strategy in these patients.

Salt-sensitive hypertensionWe used Dahl rats to investigate the role of the Rac1–mineralocorticoid-receptor pathway in salt-sensitive hypertension.14 In Dahl salt-sensitive (Dahl-S) rats, a high-salt diet increased blood pressure and caused renal damage, whereas salt did not affect blood pressure or cause renal damage in Dahl salt-resistant (Dahl-R) rats.14 Interestingly, salt loading reduced serum aldosterone concentrations to a similar extent in both rat strains but had opposite effects on mineralocorticoid-receptor signalling; salt loading activated the mineralocorticoid receptor in Dahl-S rats and inactivated the mineralo-corticoid receptor in Dahl-R rats. We found that Rac1 activation determined the differential response of the mineralocorticoid-receptor cascade to salt loading in the two rat strains. In Dahl-S rats, salt triggered Rac1 activation and the consequent mineralocorticoid- receptor stimulation, whereas in Dahl-R rats, salt loading resulted in downregulation of Rac1 and inactivation of the mineralo corticoid receptor.

Our study also showed that the Rac GEF, Tiam-1, contributes to salt-induced Rac1 activation.14 The Rac inhibitor NSC23766, which blocks Rac1–Tiam-1 inter-actions, suppressed both Rac1 and mineralocorticoid-receptor activity and corrected hypertension and renal injury in salt-loaded Dahl-S rats. Although aldosterone production was suppressed by salt in both Dahl-S and Dahl-R rats, aldosterone was indispensable to renal Rac1 activation; adrenalectomy abrogated salt-induced Rac1 activation but this effect could be reversed using aldo sterone infusion.14 Similarly, salt was required for renal Rac1 activation, as aldosterone infusion acti-vated renal Rac1 under high-salt conditions but not in response to salt restriction.14 These results suggest that aldosterone and salt activate Rac1 independently, and that Rac1 and aldosterone synergistically activate the mineralocorticoid receptor.

Cardiac injuryData from several studies suggests that oxidative stress augments mineralocorticoid-receptor signal trans-duction in the heart10,141 and we have found evidence that Rac1–mineralocorticoid-receptor interactions are involved in this process.15 Glutathione is a major antioxidant in the body and L-buthionine sulfoxi-mine (BSO) increases oxidative stress by inhibiting glutathione synthesis. Treatment with BSO increased mineralocorticoid- receptor-dependent transcrip-tional activity both in the absence and presence of mineralocorticoid- receptor ligands. BSO activated Rac1 and BSO-mediated mineralocorticoid- receptor activation was blocked by Rac inhibitors as well as

Rac1

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MR MRactivation

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Figure 3 | The role of Rac1–MR signalling in angiotensin II and salt‑induced renal injury. a | In normal mice, salt loading suppresses angiotensin II production, resulting in a decrease in plasma aldosterone concentration. The low plasma aldosterone concentration leads to Rac1 inactivation and a subsequent reduction in MR activity despite the high salt levels; therefore, renal injury does not occur in response to salt‑loading in normal mice. b | By contrast, in THM fed a high salt diet—a transgenic model of angiotensin II and salt‑induced renal injury—the MR is activated by both aldosterone‑dependent and Rac1‑dependent pathways. Angiotensin II levels are constitutively high in THM as a result of the expression of human renin and angiotensinogen genes; therefore, salt loading does not inhibit angiotensin II production. However, aldosterone concentrations are reduced in these mice in response to high salt levels. Despite this reduction, aldosterone levels in salt‑loaded THM remain inappropriately high in comparison with those of normal mice. The high aldosterone levels augment salt‑induced Rac1 activation, and aldosterone and active Rac1 synergistically stimulate the activity of the MR leading to renal injury. c | In THM fed a low salt diet, salt depletion leads to inactivation of Rac1 and, therefore, results in a reduction in MR activity and prevention of renal injury despite the high aldosterone levels induced by the activated renin–angiotensin system. Abbreviations: MR, mineralocorticoid receptor; Rac1, Ras‑related C3 botulinum toxin substrate 1; THM, Tsukuba hypertensive mice.

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by the antioxidant N-acetylcysteine. BSO-induced mineralocorticoid- receptor transactivation was accom-panied by enhanced nuclear accumulation of the receptor. This redox- sensitive, ligand-independent mineralocorticoid- receptor activation might contrib-ute to the processes by which oxidative stress promotes cardiac injury, such as through alterations in ion channel configuration, reduced contractility, cardiac hypertrophy and apoptosis.142

As discussed above, Rac1 inhibition (via adminis-tration of dominant-negative Rac1, statin therapy or cardiomyocyte- specific Rac1 deletion) protected against angiotensin-II-induced cardiac injury.33,56,63 Similarly, cardiac damage was mitigated by a mineralocorticoid-receptor antagonist in mice treated with angiotensin II143 and in angiotensin II-overproducing transgenic rats,

which expressed human renin and angiotensinogen genes.144 As inhibition of Rac1 and mineralocorticoid-receptor antagonism blocked similar signalling cascades, we speculate that Rac1–mineralocorticoid-receptor sig-nalling might also have a role in angiotensin-II-induced cardiac injury.

Molecular mechanismsUpon ligand binding, the ligand–mineralocorticoid-receptor complex translocates from the cytosol to the nucleus, binds to the hormone response element in the promoter region of target genes and regulates transcrip-tion by recruiting coactivator proteins (Figure 4). The mineralocorticoid receptor is then exported from the nucleus via a process of nucleocytoplasmic shuttling, which terminates transcription. The mineralocorticoid receptor undergoes post-translational modifications, including phosphorylation, ubiquitination, sumoylation and acetylation, some of which might alter the activ-ity of the receptor or target it for proteasomal degrada-tion. In addition to these classic pathways of genomic action, rapid, nongenomic actions of mineralocorticoid- receptor signalling have been described, including increases in intracellular calcium concentrations, ROS production, and activation of protein kinase A, protein kinase C, MAPKs and Rac1.145 These data raise the pos-sibility that Rac1 might stimulate mineralocorticoid-receptor signalling at multiple levels without changing aldosterone concentrations. Rac1-mediated enhance-ment of mineralocorticoid-receptor signalling could result from changes in the amount and phosphorylation of the mineralocorticoid receptor, its nuclear translo-cation, transactivational efficacy (including chromatin remodelling, epigenetic modification and the recruit-ment of coregulators) and cross-talk with intracellular signals (Figure 4).

Nuclear translocationA plausible explanation for Rac1-mediated mineralo-corticoid-receptor activation is that Rac1 facilitates the retrograde transport of the mineralocorticoid receptor to the nucleus. Our data indicate that Rac1-induced transcriptional activation of the mineralo corticoid receptor is accompanied by its enhanced nuclear accu-mulation.12,15 Consistent with these findings, several studies have shown that Rac1 has a pivotal role in the nuclear translocation of transcription factors, including the translocation of NFκB in response to shear stress and cytokines,20,146 the translocation of STAT3 and STAT5A in response to cytokine signalling,147,148 and the translo-cation of β-catenin during canonical Wnt signalling.149 Although the mechanisms have not been fully eluci-dated, in the case of STAT5A, active Rac1 is known to form a complex with Rac GTPase-activating protein 1, which contains a nuclear localization signal and acts as a chaperone.

The molecular machinery that regulates nuclear traf-ficking of the mineralocorticoid receptor has been inves-tigated.150 In the unliganded state, the mineralo corticoid receptor is located in the cytosol and is complexed

Nucleus

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Genomicactions

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such as NFκB and AP-1

Dynein

FKBP51

FKBP52

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Figure 4 | Mechanisms of MR activation. The MR is a ligand‑activated transcription factor. In the cytosol, the MR forms a complex with chaperones such as HSPs and FKBP51. Upon ligand binding, FKBP51 is replaced by FKBP52, which enables the complex to interact with the motor protein dynein. The ligand–MR–chaperone–dynein complex then translocates along microtubules from the cytosol to the nucleus and binds to a hormone response element in the promoter region of a target gene. The MR regulates transcription by controlling chromatin remodelling and the recruitment of coactivators (genomic actions). Post‑translational modifications, such as phosphorylation and ubiquitination, might alter the activity of the MR or target the MR for proteasomal degradation. Nongenomic actions of MR signalling have also been described. These actions include increasing intracellular Ca2+ concentrations, production of ROS and activation of PKA, PKC, MAPKs and Rac1. MR signalling can, therefore, be activated independently of aldosterone via modifying the amount of MR and its nuclear translocation and transactivational efficacy, and by cross‑talk with transcription factors or intracellular signals. Abbreviations: A, aldosterone; CASP8AP2, CASP8‑associated protein 2; CBP/p300, cyclic AMP response element binding protein binding protein/p300; ELL, RNA polymerase II elongation factor ELL; FKBP, FK506‑binding protein; GTFs, general transcription factors; HAT, histone acetyltransferase; HDAC, histone deacetylase; HSPs, heat shock proteins; MR, mineralocorticoid receptor; NCoA, nuclear receptor coactivator; PCAF, P300/CBP‑associated factor; ROS, reactive oxygen species; SWI/SNF, switch/sucrose nonfermentable; TFs, transcription factors; TRAP/DRIP, thyroid hormone receptor‑associated proteins/vitamin D receptor‑interacting proteins; UBC9, SUMO‑conjugating enzyme UBC9.

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with chaperones, such as heat shock proteins (HSPs) and immunophilin FK506 binding protein (FKBP) 51. Upon aldosterone binding, FKBP51 is replaced by FKBP52, which enables interaction with the motor protein dynein (Figure 4). The aldosterone–mineralo-corticoid receptor–HSPs–FKBP52–dynein complex is efficiently transported from the cytosol to the nucleus along microtubular tracks, using dynein as a motor.151 Aldosterone binding also exposes the nuclear localiza-tion signal in the mineralocorticoid receptor; thus the whole complex is imported to the nucleus through the nuclear core complex.151

Cytoskeleton-based motor machinery is thought to have an important role in the efficient transport of cargo molecules from the foot process to the nuclei of podocytes. Major foot processes consist predominantly of microtubules and intermediate filaments. The mol-ecular nature of the major processes are still poorly understood, although it has been shown that podocytes utilize a dynein–microtubular system for the retrograde transport of the adherens junction protein, Wilm’s tumor protein 1-interacting protein, to the nucleus in response to lipopolysaccharide.152 Podocytes also express Hsp90, FKBP51 and FKBP52, as well as gluco-corticoid and mineralocorticoid receptors.35,153 Rac1 might activate mineralocorticoid-receptor signalling in podocytes by stimulating its nuclear translocation via cytoskeletal rearrangement. Rac1 binds tubulin in fibroblasts154 and might be necessary for the mol ecular transport system. In myocardium, Rac1 activation increased tubulin expression,155 whereas Rac1 depletion resulted in a reduction of motor protein expression in endo thelial cells.69

Transcriptional activityIn addition to its role in nuclear trafficking, Rac1 might directly enhance the transcriptional activity of the mineralo corticoid receptor, possibly through phos-phorylation.156,157 Denner et al. were among the first to describe hormone-independent activation of the pro-gesterone receptor by protein kinase A.158 Subsequent studies showed that intracellular signalling proteins, such as receptor tyrosine kinase, protein kinase A, protein kinase C and MAPK, modulate the activity of the oestro-gen receptor, androgen receptor and glucocorticoid receptor in the absence of hormones.156 Phosphorylation of these receptors and/or their coregulators might under-lie their ligand-independent activation.157 Receptor or coregulator phosphorylation might also have a role in Rac1-mediated activation of the mineralocorticoid receptor, although there is currently no direct evidence that this is the case. Data on the hormone-independent activation of the mineralocorticoid receptor are limited; however protein kinase A, protein kinase C, UBC9 and nuclear transcription factor Y subunit g have all been reported to modulate its transcriptional activity.159–162

Like other steroid receptors, the mineralocorticoid receptor consists of an N-terminal domain, which con-tains the activation function domains AF-1a and AF-1b, a DNA-binding domain, a hinge region, and a carboxyl

terminal ligand-binding domain, which contains the AF-2 region.163 Coregulators bind to the AF-1 and/or the AF-2 regions; AF-2 has a role in the induction of ligand-dependent transcriptional activity, whereas the AF-1 domain is required for ligand-independent tran-scriptional activity.164 The mineralocorticoid receptor is phosphorylated,160 and most of the phosphorylation sites are located in the AF-1 region,163 suggesting that mineralocorticoid-receptor phosphorylation might alter the function of AF-1. Alternatively, recent studies have shown that actin has a role in transcriptional events as a component of chromatin remodelling complexes.165 Rac1 might modulate these effects of actin.

ConclusionsThis Review summarizes the evidence in support of a role for Rac1 in podocyte injury, in relation to the motile podocyte hypothesis and Rac1-induced cardiac dysfunc-tion and introduces the concept of ligand-independent activation of mineralocorticoid-receptor function by Rac1. Rac1-induced activation of the mineralo corticoid receptor has a critical role in angiotensin II and salt-induced kidney injury, salt-sensitive hyper tension and possibly cardiac injury. The importance of the RhoGDI1–Rac1–mineralocorticoid-receptor pathway in human glomerular disease is underscored by the findings that mutations in Arhgdia and Arhgap24 cause nephrotic syndrome and familial FSGS, respectively. In the future, treatment with Rac1 inhibitors and mineralocorticoid-receptor antagonists might effectively ameliorate these genetic diseases.

Large-scale, long-term, randomized controlled trials are required to assess the efficacy and safety of mineralocorticoid- receptor antagonist therapy in patients with CKD. We are currently carrying out a double- blind, randomized, placebo-controlled trial to evaluate the antialbuminuric effect of low-dose eplerenone in patients with hypertension and albumin-uria.166 Strong evidence supports organ-protective effects of mineralocorticoid-receptor antagonists. Nevertheless, hyperkalaemia and a reduction in glomerular filtration rate remain a concern for the use of mineralocorticoid-receptor antagonists in patients with CKD. We expect that drugs that modulate the activity of the Rac1– mineralocorticoid-receptor pathway, such as Rac inhibi-tors that selectively target GEFs or GAPs in podocytes in response to proteinuric stimuli, or selective mineralo-corticoid-receptor modulators, could be novel thera-peutic candidates for the treatment of CKD and cardiac injury, with a low risk of adverse effects.

Review criteria

We searched PubMed for articles published before October 18, 2012 using the search terms “Rac1”, “mineralocorticoid receptor”, “aldosterone”, “spironolactone”, “eplerenone”, “kidney”, and “heart”. We evaluated all full‑text, English‑language publications, and the reference lists of identified articles for further leads.

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AcknowledgementsThis work was supported in part by Grants‑in‑Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 24390214 [M. Nagase] and No. 21229012 [T. Fujita]).

Author contributionsM. Nagase researched the data for the article and wrote the manuscript. T. Fujita made a substantial contribution to discussions of the content and reviewed and edited the manuscript before submission.

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Role of Rac1–mineralocorticoid-receptor signalling in renal and cardiac disease