ICURT PROCEEDINGS
Recent Topics on Podocytes and Aldosterone Miki Nagase, MD, PhD Podocyte injury is a major cause of proteinuria, a core component of chronic kidney disease. We reported that podocyte impairment underlied the early glomerulopathy in animal models of lifestyle-related diseases, such as hypertension and metabolic syndrome. Accumulating evidence suggests that overactivation of the aldosterone–mineralocorticoid receptor (MR) system has harmful effects on podocytes. We found that MR signaling was enhanced in such lifestyle-related diseases with podocyte injury and proteinuria, which were ameliorated by MR antagonist. Subsequent studies revealed that plasma aldosterone concentrations are not always increased in proteinuric conditions with renal MR activation, and the mechanisms of MR overactivation remained elusive. We recently identified a novel mechanism of Rac1-mediated podocyte impairment using RhoGDIa knockout mice; Rac1 potentiates the activity of MR in a ligand-independent manner, thereby accelerating podocyte injury. We demonstrated that the Rac1–MR pathway contributes to the ligand-independent aberrant MR activation in salt-sensitive hypertension and renal injury models. The importance of the RhoGDIaRac1-MR pathway in human glomerular disease is underscored by the findings that mutations in RhoGDIagene cause nephrotic syndrome. Our results provide evidence that the Rac1-MR signal cascade as a novel therapeutic target for chronic kidney disease. Ó 2015 by the National Kidney Foundation, Inc. All rights reserved.
The Aldosterone/Mineralocorticoid Receptor System and Kidney Damage
ISTORICALLY, IN 1943, Hans Selye,1 the advocate of stress theory, reported that deoxycorticosterone acetate, the first synthetic steroid, together with uninephrectomy and salt loading, induced inflammatory and fibrotic changes in the rat kidney. He considered that these phenotypes would be caused by mineralocorticoid actions of deoxycorticosterone acetate. His keen insight suggested that glucocorticoids have anti-inflammatory actions, whereas mineralocorticoids may have proinflammatory effects in the era even before the identification of aldosterone in 1953. Regarding the aldosterone/mineralocorticoid receptor (MR) system, the ligand aldosterone is the final component of the renin-angiotensin-aldosterone system (RAAS). Its receptor, MR, is a ligand-activated transcription factor that belongs to the steroid receptor superfamily of nuclear receptors, close to glucocorticoid receptor (GR). The physiological main ligand of MR is aldosterone, and in some nonepithelial cells, glucocorticoids also act as endogenous MR ligands. In the kidney, the aldosterone–MR system in the distal nephron has classically been considered to have a pivotal
H
Department of Anatomy and Life Structure, School of Medicine, Juntendo University, Bunkyo-ku, Tokyo, Japan. Financial Disclosure: The author declares that their are no relevant financial interests. Address correspondence to Miki Nagase, MD, PhD, Department of Anatomy and Life Structure, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail: [email protected] Ó 2015 by the National Kidney Foundation, Inc. All rights reserved. 1051-2276/$36.00 http://dx.doi.org/10.1053/j.jrn.2014.10.016
Journal of Renal Nutrition, Vol 25, No 2 (March), 2015: pp 201-204
role in the homeostatic regulation of electrolytes, fluid volume, and blood pressure. On the other hand, growing attention has been focused on the roles of MR in nonclassical cells as a mediator of glomerular and tubulointerstitial damage.2 Previous clinical trials have proven the efficacy of angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II (Ang II) type 1 receptor blockers (ARBs) in reducing proteinuria/albuminuria in diabetic and nondiabetic patients, and Ang II has been regarded as the primary factor responsible for the injurious actions of RAAS in the kidney. Recent studies implicate aldosterone as another pathogenic factor in RAAS-mediated kidney injury, independently of its electrolyte regulatory actions. Indeed, ACEIs and/or ARBs reduce plasma aldosterone levels, and aldosterone infusion together with ACEIs and/or ARBs recapitulated the injury.3 Clinical trials have shown that adding MR antagonists could significantly diminish proteinuria in patients with diabetic and nondiabetic chronic kidney disease (CKD) currently treated with ACEI and/or ARB.4 During these 10 years, our group has been engaged in the research exploring the mechanisms of target organ damage in lifestyle–related diseases, such as hypertension,5,6 metabolic syndrome,7 and type 2 diabetes mellitus,8 and found pivotal roles of MR overactivation.
Metabolic Syndrome and Chronic Kidney Disease: Role of Aldosterone/MR System Clinical studies demonstrated that metabolic syndrome is an important modifiable risk factor for proteinuria and CKD. However, the mechanism linking metabolic syndrome to CKD has not been clearly elucidated. Several studies reported high plasma aldosterone levels in metabolic syndrome, and that aldosterone is a potent inducer of proteinuria. Focusing on the close relationships among 201
202
NAGASE
metabolic syndrome, proteinuria, and aldosterone, we demonstrated that the aldosterone/MR signaling plays a significant role in the process linking metabolic syndrome to CKD.7 We used SHR/cp, a derivative of spontaneously hypertensive rats (SHR) with leptin-receptor gene mutation as a rat model of metabolic syndrome. SHR/cp exhibited extreme9 obesity, hypertension, hyperinsulinemia, and dyslipidemia. This metabolic syndrome rat strain were prone to podocyte injury and proteinuria compared with SHR without obesity. Serum aldosterone was elevated in this model, and enhanced aldosterone/MR signaling was found in the kidney and glomerular fraction of SHR/cp. Proteinuria and podocyte injuries were ameliorated by MR antagonist. L-type calcium channel antagonist did not improve podocyte injury suggesting that MR activation rather than pressor effect contributes to podocyte injury. Salt is a worsening factor in metabolic syndrome. Highsalt diet markedly promoted proteinuria and podocyte injuries in SHR/cp.10 Interestingly, MR antagonist perfectly inhibited the salt-induced exacerbation, suggesting the involvement of aldosterone/MR cascade in salt-mediated mechanism. We also found MR-dependent proteinuria and podocyte injuries in uninephrectomized high-salt–fed rats infused with aldosterone (0.75 mg/h via an osmotic minipump).11 This is an established chronic mineralocorticoid excess model in which plasma aldosterone levels rise to those seen in human congestive heart failure, whereas plasma renin activity and circulating Ang II are quickly suppressed. Podocytes were already impaired at 2 weeks of aldosterone infusion, when proteinuria was modestly increased. Proteinuria and podocyte damages were completely reversed by MR antagonist, but not by hydralazine. MR was actually expressed in glomerular podocytes. These findings suggest that podocyte injury underlies the pathogenesis of proteinuria caused by MR activation.
MR Activation and Kidney Injury in Low Serum Aldosterone Model Subsequently, we found that MR activation could play a crucial role in the target organ damage even in normal or low aldosterone states. For example, Dahl salt–hypertensive rats develop podocyte injury, proteinuria, and glomerulosclerosis under high-salt diet.5 Although the circulating aldosterone level was low, MR was paradoxically activated in the kidney, and eplerenone dramatically retarded the progression of renal diseases. Indeed, clinical studies have shown that plasma reninaldosterone profiles are not predictive of the antihypertensive efficacy of MR blockade. Accordingly, the antialbuminuric effect of MR antagonists may also not be predicted by plasma aldosterone concentration. These findings raise the possibility that molecules other than aldosterone may activate MR.
Mechanisms of MR Activation MR belongs to nuclear receptor superfamily acting as a transcription factor; on ligand binding, the ligandreceptor complex translocates into the nucleus, where it interacts with the mineralocorticoid response element to activate gene transcription. From this viewpoint, MR activity should be modulated by multiple factors in addition to ligand level, such as the amount of MR, nuclear translocation, epigenetic modification, and recruitment of coregulators.12
Ligand-independent MR Activation by Rac1 We identified Rac1 as an alternative activator of MR signaling in podocyte injury, through the analysis of RhoGDIa KO mice, in which Rac1 is constitutively activated (CA).13 These mice showed Rac1-dependent massive albuminuria, podocyte injury, and glomerulosclerosis. Notably, these abnormalities were completely reversed by MR antagonist, indicating that MR signaling is critically involved in the pathogenesis of this model. The KO mice exhibited enhanced MR signaling despite normal aldosterone level, suggesting aldosterone-independent MR activation. Rac1 inhibitor suppressed MR activation and reduced albuminuria, indicating that Rac1 is an upstream regulator of MR signaling. These results indicate that Rac1mediated MR activation plays a central role in the renal phenotype of RhoGDIa KO mice. We also performed in vitro transfection experiments. In luciferase reporter assays, MR-dependent transcriptional activity in response to aldosterone was potentiated by overexpression of CARac1. Furthermore, CA-Rac1 promoted the nuclear translocation of green fluorescent protein-tagged MR, both with and without aldosterone. These results indicate that active Rac1 causes MR activation. The biological functions of Rac1 include reorganization of actin cytoskeleton and cell motility, reactive oxygen species generation, and nuclear translocation of transcription factors.12 Among these, we consider that enhanced MR nuclear translocation may be one mechanisms of Rac1-mediated MR activation.
Rac1-Mediated MR Activation in CKD Models RhoGDIa KO mice are rather artificial CKD model. We subsequently demonstrated that the Rac1–MR pathway contributes to ligand-independent MR activation in more established CKD models, such as Dahl-S rats,14 and Tsukuba hypertensive mice with high Ang II and aldosterone model because of human renin and angiotensinogen double transgenes.15 In Tsukuba hypertensive mice, salt loading induced albuminuria and podocyte injury, along with MR activation, whereas under strict salt restriction, the mice did not exhibit podocyte injury or proteinuria.
203
PODOCYTES AND ALDOSTERONE
It should be noted that serum aldosterone level was lower and that Rac1 was more activated in salt-loaded group, suggesting Rac1-mediated MR activation. Both MR antagonist and Rac inhibitor ameliorated the nephropathy. Another example is obese diabetic KKAy mice, a high aldosterone model.8 Guanosine triphosphate-bound active Rac1 was increased in the kidney of this model and treatment with Rac inhibitor significantly alleviated albuminuria, associated with correction of MR activation. In cultured cells, high glucose activated Rac1 and MR, both of which were suppressed by Rac inhibitor. Thus, both aldosterone-dependent and aldosterone-independent mechanisms contributed to the MR activation in this model.
MR Downstream Pathways in Renal Distal Tubular Cells and Glomerular Podocytes Next, we examined what kind of genes are induced on nuclear MR translocation in the podocytes and distal tubular cells. We performed chromatin immunoprecipitation with deep sequencing (ChIP)-seq analysis and compared the genome-wide MR binding sites on ligand stimulation in cultured podocytes and distal tubular cells. The two cell types displayed quite distinct transcriptional profiles, supporting the idea that Rac1-MR activation would trigger cell-specific molecular events and phenotypes.16 Data from conditional MR knockout mice in the macrophage, cardiomyocyte, and vascular smooth muscle cells have indicated cell-specific unique downstream pathways on MR activation and their roles in the development of target organ damage and hypertension. Interestingly, both MR and GR are thought to bind to mineralocorticoid response element/glucocorticoid response element in the promoter region of target genes. GR agonists, glucocorticoids, are widely used for the treatment of glomerular podocyte injury in steroid-sensitive nephrotic syndrome, whereas MR antagonists are also effective in reducing proteinuria/albuminuria. The physiological and pathophysiological roles of GR and MR and their signaling cascades in the podocytes, however, have still been elusive.
Rac1 Activation and Motile Podocyte Phenotype Cause Podocyte Impairment As for Rac1 in podocyte injury, recent articles have highlighted pivotal roles of Rac1 in podocyte injury. Mundel et al.17 proposed ‘‘motile podocyte’’ hypothesis. According to this hypothesis, normal static phenotype is associated with RhoA activation, whereas injured podocytes have motile phenotype, which is associated with Rac1 activation. Indeed, Rac1 was shown to be activated by several proteinuric stimuli such as Ang II, diabetic condition, and lipopolysaccharide, causing motile phenotype and foot process effacement. According to our results, MR signaling
DM PAN, LPS
Ang II
uPAR integrin
HIV Nef
genetic mutation Arhgap24 RhoGDIα etc.
TRPC5
Rac1 activation
motile podocyte
MR activation
foot process effacement
Figure 1. Rac1 activation and podocyte injury. Rac1 was shown to be activated by several proteinuric stimuli such as angiotensin II (Ang II), diabetic condition (DM), lipopolysaccharide (LPS), HIV infection, and genetic mutation, causing motile phenotype and foot process effacement. Our results suggest additional mechanism, Rac1-mediated MR activation. HIV, human immunodeficiency virus; MR, mineralocorticoid receptor.
may be activated in such situations and contributed to the Rac1-induced podocyte injury (Fig. 1).13 In summary, I showed that (1) podocyte injury was shown to underlie the nephropathy of SHR/cp, a rat model of MetS, and the aldosterone/MR signal was involved in the process. (2) MR is activated both ligand dependently and independently. We identified Rac1 as a novel activator of MR. Rac1-mediated MR activation was shown to cause podocyte injury in several CKD models. (3) Deletion of RhoGDIa resulted in Rac1- and MR-dependent podocyte injury. (4) Various proteinuric stimuli were shown to cause Rac1 activation in the podocyte, causing motile phenotype and foot process effacement. MR signaling may also be involved in the Rac1-induced podocyte injury. I expect the development of cell-specific MR antagonist that specifically block MR signaling responsible for target organ damage in the near future. The importance of the RhoGDIa-Rac1-MR pathway in human glomerular disease is underscored by the findings that mutations in RhoGDIa and Arhgap24 genes cause nephrotic syndrome and familial focal and segmental glomerulosclerosis, respectively.18-20 Our results provide evidence that the Rac1-MR signal cascade as a novel therapeutic target for CKD.
References 1. Selye H, Hall C. Pathologic changes induced in various species by overdosage with desoxycorticosterone. Arch Pathol. 1943;36:19-31. 2. Nagase M. Activation of the aldosterone/mineralocorticoid receptor system in chronic kidney disease and metabolic syndrome. Clin Exp Nephrol. 2010;14:303-314. 3. Greene EL, Kren S, Hostetter TH. Role of aldosterone in the remnant kidney model in the rat. J Clin Invest. 1996;98:1063-1068. 4. Navaneethan SD, Nigwekar SU, Sehgal AR, Strippoli GF. Aldosterone antagonists for preventing the progression of chronic kidney disease: a systematic review and meta-analysis. Clin J Am Soc Nephrol. 2009;4:542-551.
204
NAGASE
5. Nagase M, Shibata S, Yoshida S, Nagase T, Gotoda T, Fujita T. Podocyte injury underlies the glomerulopathy of Dahl salt-hypertensive rats and is reversed by aldosterone blocker. Hypertension. 2006;47:1084-1093. 6. Nagase M. Role of Rac1 GTPase in salt-sensitive hypertension. Curr Opin Nephrol Hypertens. 2013;22:148-155. 7. Nagase M, Yoshida S, Shibata S, et al. Enhanced aldosterone signaling in the early nephropathy of rats with metabolic syndrome: possible contribution of fat-derived factors. J Am Soc Nephrol. 2006;17:3438-3446. 8. Yoshida S, Ishizawa K, Ayuzawa N, et al. Local mineralocorticoid receptor activation and the role of Rac1 in obesity-related diabetic kidney disease. Nephron Exp Nephrol. 2014;126:16-24. 9. Nagase M, Fujita T. Aldosterone and glomerular podocyte injury. Clin Exp Nephrol. 2008;12:233-242. 10. Nagase M, Matsui H, Shibata S, Gotoda T, Fujita T. Salt-induced nephropathy in obese spontaneously hypertensive rats via paradoxical activation of the mineralocorticoid receptor: role of oxidative stress. Hypertension. 2007;50:877-883. 11. Shibata S, Nagase M, Yoshida S, Kawachi H, Fujita T. Podocyte as the target for aldosterone: roles of oxidative stress and Sgk1. Hypertension. 2007;49:355-364. 12. Nagase M, Fujita T. Role of Rac1-mineralocorticoid-receptor signalling in renal and cardiac disease. Nature reviews. Nephrology. 2013;9:86-98.
13. Shibata S, Nagase M, Yoshida S, et al. Modification of mineralocorticoid receptor function by Rac1 GTPase: implication in proteinuric kidney disease. Nat Med. 2008;14:1370-1376. 14. Shibata S, Mu S, Kawarazaki H, et al. Rac1 GTPase in rodent kidneys is essential for salt-sensitive hypertension via a mineralocorticoid receptordependent pathway. J Clin Invest. 2011;121:3233-3243. 15. Kawarazaki W, Nagase M, Yoshida S, et al. Angiotensin II- and saltinduced kidney injury through Rac1-mediated mineralocorticoid receptor activation. J Am Soc Nephrol. 2012;23:997-1007. 16. Ueda K, Fujiki K, Shirahige K, et al. Genome-wide analysis of murine renal distal convoluted tubular cells for the target genes of mineralocorticoid receptor. Biochem Biophys Res Commun. 2014;445:132-137. 17. Mundel P, Reiser J. Proteinuria: an enzymatic disease of the podocyte? Kidney Int. 2010;77:571-580. 18. Gee HY, Saisawat P, Ashraf S, et al. ARHGDIA mutations cause nephrotic syndrome via defective RHO GTPase signaling. J Clin Invest. 2013;123:3243-3253. 19. Gupta IR, Baldwin C, Auguste D, et al. ARHGDIA: a novel gene implicated in nephrotic syndrome. J Med Genet. 2013;50:330-338. 20. Akilesh S, Suleiman H, Yu H, et al. Arhgap24 inactivates Rac1 in mouse podocytes, and a mutant form is associated with familial focal segmental glomerulosclerosis. J Clin Invest. 2011;121:4127-4137.
Recent Topics on Podocytes and Aldosterone Miki Nagase, MD, PhD Podocyte injury is a major cause of proteinuria, a core component of chronic kidney disease. We reported that podocyte impairment underlied the early glomerulopathy in animal models of lifestyle-related diseases, such as hypertension and metabolic syndrome. Accumulating evidence suggests that overactivation of the aldosterone–mineralocorticoid receptor (MR) system has harmful effects on podocytes. We found that MR signaling was enhanced in such lifestyle-related diseases with podocyte injury and proteinuria, which were ameliorated by MR antagonist. Subsequent studies revealed that plasma aldosterone concentrations are not always increased in proteinuric conditions with renal MR activation, and the mechanisms of MR overactivation remained elusive. We recently identified a novel mechanism of Rac1-mediated podocyte impairment using RhoGDIa knockout mice; Rac1 potentiates the activity of MR in a ligand-independent manner, thereby accelerating podocyte injury. We demonstrated that the Rac1–MR pathway contributes to the ligand-independent aberrant MR activation in salt-sensitive hypertension and renal injury models. The importance of the RhoGDIaRac1-MR pathway in human glomerular disease is underscored by the findings that mutations in RhoGDIagene cause nephrotic syndrome. Our results provide evidence that the Rac1-MR signal cascade as a novel therapeutic target for chronic kidney disease. Ó 2015 by the National Kidney Foundation, Inc. All rights reserved.
The Aldosterone/Mineralocorticoid Receptor System and Kidney Damage
ISTORICALLY, IN 1943, Hans Selye,1 the advocate of stress theory, reported that deoxycorticosterone acetate, the first synthetic steroid, together with uninephrectomy and salt loading, induced inflammatory and fibrotic changes in the rat kidney. He considered that these phenotypes would be caused by mineralocorticoid actions of deoxycorticosterone acetate. His keen insight suggested that glucocorticoids have anti-inflammatory actions, whereas mineralocorticoids may have proinflammatory effects in the era even before the identification of aldosterone in 1953. Regarding the aldosterone/mineralocorticoid receptor (MR) system, the ligand aldosterone is the final component of the renin-angiotensin-aldosterone system (RAAS). Its receptor, MR, is a ligand-activated transcription factor that belongs to the steroid receptor superfamily of nuclear receptors, close to glucocorticoid receptor (GR). The physiological main ligand of MR is aldosterone, and in some nonepithelial cells, glucocorticoids also act as endogenous MR ligands. In the kidney, the aldosterone–MR system in the distal nephron has classically been considered to have a pivotal
H
Department of Anatomy and Life Structure, School of Medicine, Juntendo University, Bunkyo-ku, Tokyo, Japan. Financial Disclosure: The author declares that their are no relevant financial interests. Address correspondence to Miki Nagase, MD, PhD, Department of Anatomy and Life Structure, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail: [email protected] Ó 2015 by the National Kidney Foundation, Inc. All rights reserved. 1051-2276/$36.00 http://dx.doi.org/10.1053/j.jrn.2014.10.016
Journal of Renal Nutrition, Vol 25, No 2 (March), 2015: pp 201-204
role in the homeostatic regulation of electrolytes, fluid volume, and blood pressure. On the other hand, growing attention has been focused on the roles of MR in nonclassical cells as a mediator of glomerular and tubulointerstitial damage.2 Previous clinical trials have proven the efficacy of angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II (Ang II) type 1 receptor blockers (ARBs) in reducing proteinuria/albuminuria in diabetic and nondiabetic patients, and Ang II has been regarded as the primary factor responsible for the injurious actions of RAAS in the kidney. Recent studies implicate aldosterone as another pathogenic factor in RAAS-mediated kidney injury, independently of its electrolyte regulatory actions. Indeed, ACEIs and/or ARBs reduce plasma aldosterone levels, and aldosterone infusion together with ACEIs and/or ARBs recapitulated the injury.3 Clinical trials have shown that adding MR antagonists could significantly diminish proteinuria in patients with diabetic and nondiabetic chronic kidney disease (CKD) currently treated with ACEI and/or ARB.4 During these 10 years, our group has been engaged in the research exploring the mechanisms of target organ damage in lifestyle–related diseases, such as hypertension,5,6 metabolic syndrome,7 and type 2 diabetes mellitus,8 and found pivotal roles of MR overactivation.
Metabolic Syndrome and Chronic Kidney Disease: Role of Aldosterone/MR System Clinical studies demonstrated that metabolic syndrome is an important modifiable risk factor for proteinuria and CKD. However, the mechanism linking metabolic syndrome to CKD has not been clearly elucidated. Several studies reported high plasma aldosterone levels in metabolic syndrome, and that aldosterone is a potent inducer of proteinuria. Focusing on the close relationships among 201
202
NAGASE
metabolic syndrome, proteinuria, and aldosterone, we demonstrated that the aldosterone/MR signaling plays a significant role in the process linking metabolic syndrome to CKD.7 We used SHR/cp, a derivative of spontaneously hypertensive rats (SHR) with leptin-receptor gene mutation as a rat model of metabolic syndrome. SHR/cp exhibited extreme9 obesity, hypertension, hyperinsulinemia, and dyslipidemia. This metabolic syndrome rat strain were prone to podocyte injury and proteinuria compared with SHR without obesity. Serum aldosterone was elevated in this model, and enhanced aldosterone/MR signaling was found in the kidney and glomerular fraction of SHR/cp. Proteinuria and podocyte injuries were ameliorated by MR antagonist. L-type calcium channel antagonist did not improve podocyte injury suggesting that MR activation rather than pressor effect contributes to podocyte injury. Salt is a worsening factor in metabolic syndrome. Highsalt diet markedly promoted proteinuria and podocyte injuries in SHR/cp.10 Interestingly, MR antagonist perfectly inhibited the salt-induced exacerbation, suggesting the involvement of aldosterone/MR cascade in salt-mediated mechanism. We also found MR-dependent proteinuria and podocyte injuries in uninephrectomized high-salt–fed rats infused with aldosterone (0.75 mg/h via an osmotic minipump).11 This is an established chronic mineralocorticoid excess model in which plasma aldosterone levels rise to those seen in human congestive heart failure, whereas plasma renin activity and circulating Ang II are quickly suppressed. Podocytes were already impaired at 2 weeks of aldosterone infusion, when proteinuria was modestly increased. Proteinuria and podocyte damages were completely reversed by MR antagonist, but not by hydralazine. MR was actually expressed in glomerular podocytes. These findings suggest that podocyte injury underlies the pathogenesis of proteinuria caused by MR activation.
MR Activation and Kidney Injury in Low Serum Aldosterone Model Subsequently, we found that MR activation could play a crucial role in the target organ damage even in normal or low aldosterone states. For example, Dahl salt–hypertensive rats develop podocyte injury, proteinuria, and glomerulosclerosis under high-salt diet.5 Although the circulating aldosterone level was low, MR was paradoxically activated in the kidney, and eplerenone dramatically retarded the progression of renal diseases. Indeed, clinical studies have shown that plasma reninaldosterone profiles are not predictive of the antihypertensive efficacy of MR blockade. Accordingly, the antialbuminuric effect of MR antagonists may also not be predicted by plasma aldosterone concentration. These findings raise the possibility that molecules other than aldosterone may activate MR.
Mechanisms of MR Activation MR belongs to nuclear receptor superfamily acting as a transcription factor; on ligand binding, the ligandreceptor complex translocates into the nucleus, where it interacts with the mineralocorticoid response element to activate gene transcription. From this viewpoint, MR activity should be modulated by multiple factors in addition to ligand level, such as the amount of MR, nuclear translocation, epigenetic modification, and recruitment of coregulators.12
Ligand-independent MR Activation by Rac1 We identified Rac1 as an alternative activator of MR signaling in podocyte injury, through the analysis of RhoGDIa KO mice, in which Rac1 is constitutively activated (CA).13 These mice showed Rac1-dependent massive albuminuria, podocyte injury, and glomerulosclerosis. Notably, these abnormalities were completely reversed by MR antagonist, indicating that MR signaling is critically involved in the pathogenesis of this model. The KO mice exhibited enhanced MR signaling despite normal aldosterone level, suggesting aldosterone-independent MR activation. Rac1 inhibitor suppressed MR activation and reduced albuminuria, indicating that Rac1 is an upstream regulator of MR signaling. These results indicate that Rac1mediated MR activation plays a central role in the renal phenotype of RhoGDIa KO mice. We also performed in vitro transfection experiments. In luciferase reporter assays, MR-dependent transcriptional activity in response to aldosterone was potentiated by overexpression of CARac1. Furthermore, CA-Rac1 promoted the nuclear translocation of green fluorescent protein-tagged MR, both with and without aldosterone. These results indicate that active Rac1 causes MR activation. The biological functions of Rac1 include reorganization of actin cytoskeleton and cell motility, reactive oxygen species generation, and nuclear translocation of transcription factors.12 Among these, we consider that enhanced MR nuclear translocation may be one mechanisms of Rac1-mediated MR activation.
Rac1-Mediated MR Activation in CKD Models RhoGDIa KO mice are rather artificial CKD model. We subsequently demonstrated that the Rac1–MR pathway contributes to ligand-independent MR activation in more established CKD models, such as Dahl-S rats,14 and Tsukuba hypertensive mice with high Ang II and aldosterone model because of human renin and angiotensinogen double transgenes.15 In Tsukuba hypertensive mice, salt loading induced albuminuria and podocyte injury, along with MR activation, whereas under strict salt restriction, the mice did not exhibit podocyte injury or proteinuria.
203
PODOCYTES AND ALDOSTERONE
It should be noted that serum aldosterone level was lower and that Rac1 was more activated in salt-loaded group, suggesting Rac1-mediated MR activation. Both MR antagonist and Rac inhibitor ameliorated the nephropathy. Another example is obese diabetic KKAy mice, a high aldosterone model.8 Guanosine triphosphate-bound active Rac1 was increased in the kidney of this model and treatment with Rac inhibitor significantly alleviated albuminuria, associated with correction of MR activation. In cultured cells, high glucose activated Rac1 and MR, both of which were suppressed by Rac inhibitor. Thus, both aldosterone-dependent and aldosterone-independent mechanisms contributed to the MR activation in this model.
MR Downstream Pathways in Renal Distal Tubular Cells and Glomerular Podocytes Next, we examined what kind of genes are induced on nuclear MR translocation in the podocytes and distal tubular cells. We performed chromatin immunoprecipitation with deep sequencing (ChIP)-seq analysis and compared the genome-wide MR binding sites on ligand stimulation in cultured podocytes and distal tubular cells. The two cell types displayed quite distinct transcriptional profiles, supporting the idea that Rac1-MR activation would trigger cell-specific molecular events and phenotypes.16 Data from conditional MR knockout mice in the macrophage, cardiomyocyte, and vascular smooth muscle cells have indicated cell-specific unique downstream pathways on MR activation and their roles in the development of target organ damage and hypertension. Interestingly, both MR and GR are thought to bind to mineralocorticoid response element/glucocorticoid response element in the promoter region of target genes. GR agonists, glucocorticoids, are widely used for the treatment of glomerular podocyte injury in steroid-sensitive nephrotic syndrome, whereas MR antagonists are also effective in reducing proteinuria/albuminuria. The physiological and pathophysiological roles of GR and MR and their signaling cascades in the podocytes, however, have still been elusive.
Rac1 Activation and Motile Podocyte Phenotype Cause Podocyte Impairment As for Rac1 in podocyte injury, recent articles have highlighted pivotal roles of Rac1 in podocyte injury. Mundel et al.17 proposed ‘‘motile podocyte’’ hypothesis. According to this hypothesis, normal static phenotype is associated with RhoA activation, whereas injured podocytes have motile phenotype, which is associated with Rac1 activation. Indeed, Rac1 was shown to be activated by several proteinuric stimuli such as Ang II, diabetic condition, and lipopolysaccharide, causing motile phenotype and foot process effacement. According to our results, MR signaling
DM PAN, LPS
Ang II
uPAR integrin
HIV Nef
genetic mutation Arhgap24 RhoGDIα etc.
TRPC5
Rac1 activation
motile podocyte
MR activation
foot process effacement
Figure 1. Rac1 activation and podocyte injury. Rac1 was shown to be activated by several proteinuric stimuli such as angiotensin II (Ang II), diabetic condition (DM), lipopolysaccharide (LPS), HIV infection, and genetic mutation, causing motile phenotype and foot process effacement. Our results suggest additional mechanism, Rac1-mediated MR activation. HIV, human immunodeficiency virus; MR, mineralocorticoid receptor.
may be activated in such situations and contributed to the Rac1-induced podocyte injury (Fig. 1).13 In summary, I showed that (1) podocyte injury was shown to underlie the nephropathy of SHR/cp, a rat model of MetS, and the aldosterone/MR signal was involved in the process. (2) MR is activated both ligand dependently and independently. We identified Rac1 as a novel activator of MR. Rac1-mediated MR activation was shown to cause podocyte injury in several CKD models. (3) Deletion of RhoGDIa resulted in Rac1- and MR-dependent podocyte injury. (4) Various proteinuric stimuli were shown to cause Rac1 activation in the podocyte, causing motile phenotype and foot process effacement. MR signaling may also be involved in the Rac1-induced podocyte injury. I expect the development of cell-specific MR antagonist that specifically block MR signaling responsible for target organ damage in the near future. The importance of the RhoGDIa-Rac1-MR pathway in human glomerular disease is underscored by the findings that mutations in RhoGDIa and Arhgap24 genes cause nephrotic syndrome and familial focal and segmental glomerulosclerosis, respectively.18-20 Our results provide evidence that the Rac1-MR signal cascade as a novel therapeutic target for CKD.
References 1. Selye H, Hall C. Pathologic changes induced in various species by overdosage with desoxycorticosterone. Arch Pathol. 1943;36:19-31. 2. Nagase M. Activation of the aldosterone/mineralocorticoid receptor system in chronic kidney disease and metabolic syndrome. Clin Exp Nephrol. 2010;14:303-314. 3. Greene EL, Kren S, Hostetter TH. Role of aldosterone in the remnant kidney model in the rat. J Clin Invest. 1996;98:1063-1068. 4. Navaneethan SD, Nigwekar SU, Sehgal AR, Strippoli GF. Aldosterone antagonists for preventing the progression of chronic kidney disease: a systematic review and meta-analysis. Clin J Am Soc Nephrol. 2009;4:542-551.
204
NAGASE
5. Nagase M, Shibata S, Yoshida S, Nagase T, Gotoda T, Fujita T. Podocyte injury underlies the glomerulopathy of Dahl salt-hypertensive rats and is reversed by aldosterone blocker. Hypertension. 2006;47:1084-1093. 6. Nagase M. Role of Rac1 GTPase in salt-sensitive hypertension. Curr Opin Nephrol Hypertens. 2013;22:148-155. 7. Nagase M, Yoshida S, Shibata S, et al. Enhanced aldosterone signaling in the early nephropathy of rats with metabolic syndrome: possible contribution of fat-derived factors. J Am Soc Nephrol. 2006;17:3438-3446. 8. Yoshida S, Ishizawa K, Ayuzawa N, et al. Local mineralocorticoid receptor activation and the role of Rac1 in obesity-related diabetic kidney disease. Nephron Exp Nephrol. 2014;126:16-24. 9. Nagase M, Fujita T. Aldosterone and glomerular podocyte injury. Clin Exp Nephrol. 2008;12:233-242. 10. Nagase M, Matsui H, Shibata S, Gotoda T, Fujita T. Salt-induced nephropathy in obese spontaneously hypertensive rats via paradoxical activation of the mineralocorticoid receptor: role of oxidative stress. Hypertension. 2007;50:877-883. 11. Shibata S, Nagase M, Yoshida S, Kawachi H, Fujita T. Podocyte as the target for aldosterone: roles of oxidative stress and Sgk1. Hypertension. 2007;49:355-364. 12. Nagase M, Fujita T. Role of Rac1-mineralocorticoid-receptor signalling in renal and cardiac disease. Nature reviews. Nephrology. 2013;9:86-98.
13. Shibata S, Nagase M, Yoshida S, et al. Modification of mineralocorticoid receptor function by Rac1 GTPase: implication in proteinuric kidney disease. Nat Med. 2008;14:1370-1376. 14. Shibata S, Mu S, Kawarazaki H, et al. Rac1 GTPase in rodent kidneys is essential for salt-sensitive hypertension via a mineralocorticoid receptordependent pathway. J Clin Invest. 2011;121:3233-3243. 15. Kawarazaki W, Nagase M, Yoshida S, et al. Angiotensin II- and saltinduced kidney injury through Rac1-mediated mineralocorticoid receptor activation. J Am Soc Nephrol. 2012;23:997-1007. 16. Ueda K, Fujiki K, Shirahige K, et al. Genome-wide analysis of murine renal distal convoluted tubular cells for the target genes of mineralocorticoid receptor. Biochem Biophys Res Commun. 2014;445:132-137. 17. Mundel P, Reiser J. Proteinuria: an enzymatic disease of the podocyte? Kidney Int. 2010;77:571-580. 18. Gee HY, Saisawat P, Ashraf S, et al. ARHGDIA mutations cause nephrotic syndrome via defective RHO GTPase signaling. J Clin Invest. 2013;123:3243-3253. 19. Gupta IR, Baldwin C, Auguste D, et al. ARHGDIA: a novel gene implicated in nephrotic syndrome. J Med Genet. 2013;50:330-338. 20. Akilesh S, Suleiman H, Yu H, et al. Arhgap24 inactivates Rac1 in mouse podocytes, and a mutant form is associated with familial focal segmental glomerulosclerosis. J Clin Invest. 2011;121:4127-4137.
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