Nobuhiro Ayuzawa

Rac1 GTPase in rodent kidneys is essential for salt-sensitive hypertension via a mineralocorticoid receptor–dependent pathway

Hypertension is a leading contributor to cardiovascular mortality worldwide. Despite this, its underlying mechanism(s) and the role of excess salt in cardiorenal dysfunction are unclear. Previously, we have identified cross-talk between mineralocorticoid receptor (MR), a nuclear transcription factor regulated by the steroid aldosterone, and the small GTPase Rac1, which is implicated in proteinuric kidney disease. We here show that high-salt loading activates Rac1 in the kidneys in rodent models of salt-sensitive hypertension, leading to blood pressure elevation and renal injury via an MR-dependent pathway. We found that a high-salt diet caused renal Rac1 upregulation in salt-sensitive Dahl (Dahl-S) rats and downregulation in salt-insensitive Dahl (Dahl-R) rats. Despite a reduction of serum aldosterone levels, salt-loaded Dahl-S rats showed increased MR signaling in the kidneys, and Rac1 inhibition prevented hypertension and renal damage with MR repression. We further demonstrated in aldosterone-infused rats as well as adrenalectomized Dahl-S rats with aldosterone supplementation that salt-induced Rac1 and aldosterone acted interdependently to cause MR overactivity and hypertension. Finally, we confirmed the key role of Rac1 in modulating salt susceptibility in mice lacking Rho GDP-dissociation inhibitor α. Therefore, our data identify Rac1 as a determinant of salt sensitivity and provide insights into the mechanism of salt-induced hypertension and kidney injury.

Angiotensin II- and Salt-Induced Kidney Injury through Rac1-Mediated Mineralocorticoid Receptor Activation

Experiments with hyperaldosteronemic animals suggest that, despite lowering plasma aldosterone, salt worsens renal injury by paradoxical activation of the mineralocorticoid receptor (MR). Salt and aldosterone synergistically contribute to renal impairment through Rac1-mediated activation of the MR, but whether angiotensin II also promotes renal injury through this mechanism is unknown. Here, we placed angiotensin II-overproducing double transgenic Tsukuba hypertensive mice on a low- or high-salt intake for 6 weeks and treated some animals with adrenalectomy, the MR antagonist eplerenone, the Rac inhibitor EHT1864, or hydralazine. High-salt intake, but not low-salt intake, led to hypertension and prominent kidney injury. Adrenalectomy prevented angiotensin II/salt-induced nephropathy in mice receiving high-salt intake, which was recapitulated by aldosterone supplementation, suggesting the involvement of aldosterone/MR signaling. Plasma aldosterone levels, however, were lower in high- than low-salt conditions. Instead, angiotensin II/salt-evoked MR activation associated with Rac1 activation and was not dependent on plasma aldosterone level. Both EHT1864 and eplerenone repressed the augmented MR signaling and mitigated kidney injury with partial but significant reduction in BP with high-salt intake. Hydralazine similarly reduced BP, but it neither suppressed the Rac1-MR pathway nor ameliorated the nephropathy. Taken together, these results show that angiotensin II and salt accelerate kidney injury through Rac1-mediated MR activation. Rac inhibition may be a promising strategy for the treatment of CKD.

Oxidative Stress Causes Mineralocorticoid Receptor Activation in Rat Cardiomyocytes: Role of Small GTPase Rac1

Overactivation of the mineralocorticoid receptor signaling is implicated in cardiovascular disease, including hypertensive heart disease. Oxidative stress is suggested to augment mineralocorticoid receptor signal transduction, but the precise mechanisms remain unclear. Mineralocorticoid receptor activity is regulated by multiple factors, in addition to plasma ligand levels. We previously identified Rac1 GTPase as a modulator of mineralocorticoid receptor activity. Here we show that oxidative stress induces mineralocorticoid receptor activation in a ligand-independent, Rac1-depenent manner in cardiomyocytes. Oxidant stress was induced in rat cultured cardiomyocytes (H9c2) by L-buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. BSO depleted intracellular glutathione and concomitantly increased reactive oxygen species (199%; P<0.01). BSO significantly enhanced the corticosterone-induced, mineralocorticoid receptor–dependent luciferase reporter activity (186%; P<0.01) and basal luciferase activity without ligand stimulation. These effects were inhibited by the antioxidant N-acetylcysteine. The ligand independency of BSO action was indicated using a mutant mineralocorticoid receptor that does not bind ligands. With this mutant mineralocorticoid receptor, BSO-evoked mineralocorticoid receptor activation remained intact, whereas ligand-induced mineralocorticoid receptor activation was abolished. We next examined the involvement of Rac1. BSO increased active Rac1 in a redox-dependent fashion, and Rac inhibition suppressed the enhancing effect of BSO. Constitutively active Rac1, indeed, potentiated mineralocorticoid receptor transactivation. Furthermore, mineralocorticoid receptor transactivation by BSO was accompanied by enhanced nuclear accumulation of mineralocorticoid receptor. We conclude that alteration of redox state modulates mineralocorticoid receptor–dependent transcriptional activity via Rac1 in the heart. This redox-sensitive, ligand-independent mineralocorticoid receptor activation may contribute to the processes by which oxidant stress promotes cardiac injury.

Peritoneal Morphology After Long-Term Peritoneal Dialysis with Biocompatible Fluid: Recent Clinical Practice in Japan

♦ Background: Morphology changes of the peritoneal membrane after long-term peritoneal dialysis (PD) consist of denudation of peritoneal mesothelial cells, interstitial sclerosis, and hyalinizing vasculopathy. Those changes are considered to be the result of uremia and bioincompatible effects of conventional acidic lactate-buffered dialysate with glucose degradation products (GDPs). In the last decade, biocompatible dialysate with neutral pH and low GDPs has become widely used. Clinical practice has been modified in Japan, especially for anuric patients, and now includes the use of hybrid therapy. The impact on peritoneal morphology has not been well reported. ♦ Objective: The aim of the present study was to investigate the long-term effect on peritoneal morphology and function of biocompatible fluid use and current clinical practice in Japan, including hybrid dialysis therapy. ♦ Methods: We evaluated peritoneal biopsy specimens from patients who had undergone PD for more than 3 years. We used the average peritoneal thickness (APT) of the submesothelial compact zone as a marker of interstitial sclerosis and the lumen/vessel diameter ratio (L/V ratio) at postcapillary venules as a marker of hyalinizing vasculopathy. Demography and other data for the patients, including dialysate-to-plasma (D/P) ratio of creatinine, were obtained at baseline and every 6 months by peritoneal equilibration test. ♦ Results: Between 2002 and 2009, 110 patients started PD therapy with biocompatible dialysate at Tokyo University Hospital. Among them, 11 patients (8 men, 3 women; age: 54.2 ± 11.8 years; 1 with diabetes mellitus) were enrolled into this morphology study. The mean duration of PD in this group was 61 ± 11.3 months, and the mean time to peritoneal biopsy was 58 ± 15.1 months. The median APT was 180 μm (96 – 1424 μm), and the median L/V ratio was 0.66 (0.46 – 0.74). No obvious correlations between APT, L/V ratio, and PD duration were detected. The D/P creatinine of the 11 patients was maintained at a favorably low value, comparable with that of the other 99 patients. ♦ Conclusions: Peritoneal dialysis therapy using biocompatible dialysate in conjunction with modification of clinical practice may minimize the progression of peritoneal interstitial sclerosis and hyalinizing vasculopathy, preserving favorable peritoneal function for more than 3 years.

Diabetes Induces Aberrant DNA Methylation in the Proximal Tubules of the Kidney

Epigenetic mechanisms may underlie the progression of diabetic kidney disease. Because the kidney is a heterogeneous organ with different cell types, we investigated DNA methylation status of the kidney in a cell type-specific manner. We first identified genes specifically demethylated in the normal proximal tubules obtained from control db/m mice, and next delineated the candidate disease-modifying genes bearing aberrant DNA methylation induced by diabetes using db/db mice. Genes involved in glucose metabolism, including Sglt2, Pck1, and G6pc, were selectively hypomethylated in the proximal tubules in control mice. Hnf4a, a transcription factor regulating transporters for reabsorption, was also selectively demethylated. In diabetic mice, aberrant hypomethylation of Agt, Abcc4, Cyp4a10, Glut5, and Met and hypermethylation of Kif20b, Cldn18, and Slco1a1 were observed. Time-dependent demethylation of Agt, a marker of diabetic kidney disease, was accompanied by histone modification changes. Furthermore, inhibition of DNA methyltransferase or histone deacetylase increased Agt mRNA in cultured human proximal tubular cells. Aberrant DNA methylation and concomitant changes in histone modifications and mRNA expression in the diabetic kidney were resistant to antidiabetic treatment with pioglitazone. These results suggest that an epigenetic switch involving aberrant DNA methylation causes persistent mRNA expression of select genes that may lead to phenotype changes of the proximal tubules in diabetic kidney disease.

Rac1-Mediated Activation of Mineralocorticoid Receptor in Pressure Overload–Induced Cardiac Injury

There is increasing evidence for a crucial role of aberrant mineralocorticoid receptor (MR) activation in heart failure, with clinical studies showing beneficial effects of MR blockade. However, the mechanisms of MR activation in heart failure remain unclear. In this study, we observed that the small GTPase Rac1 contributes to myocardial MR activation, whereas Rac1-MR pathway activation leads to cardiac dysfunction. Mouse hearts subjected to chronic pressure overload induced by transverse aortic constriction showed Rac1 activation and increased nuclear accumulation of MR and expression of MR target genes, suggesting MR activation. Pharmacological inhibition of Rac1 and heterozygous deletion of Rac1 in cardiomyocytes suppressed Rac1-induced MR signaling and reduced NADPH oxidase 4 gene induction and reactive oxygen species overproduction, which attenuated transverse aortic constriction–induced cardiac hypertrophy and dysfunction. Consistently, treatment with the selective MR antagonist eplerenone blocked transverse aortic constriction–induced MR signaling and NADPH oxidase 4 gene upregulation, which improved cardiac hypertrophy and dysfunction. These findings suggest that Rac1-MR pathway activation in the myocardium is involved in development of heart failure induced by pressure load via recruitment of the responsible isoform of NADPH oxidase. Thus, the cardiac Rac1-MR-NADPH oxidase 4 pathway may be a therapeutic target for treatment of the pressure-overloaded heart.

Local Mineralocorticoid Receptor Activation and the Role of Rac1 in Obesity-Related Diabetic Kidney Disease

Background/Aims: Obesity and diabetes are intimately interrelated, and are independent risk factors for kidney disease. Overactivation of mineralocorticoid receptor (MR) is implicated in end organ damage of both pathologies. But the underlying mechanism of MR activation in kidney remains uncertain. We explored the involvement of Rac1, which we previously identified as a ligand-independent MR activator, in renal MR activation in vitro and in vivo. Methods: We evaluated the MR activity and Rac1 activity under high-glucose stimulation using luciferase reporter system and glutathione S-transferase pull-down assay in cultured mesangial cells. To elucidate the role of Rac1 in vivo, we employed KKAy, a mouse model of obesity-related type 2 diabetes, which spontaneously developed massive albuminuria and distinct glomerular lesions accompanied by increased plasma aldosterone concentration. Results: High-glucose stimulation increased Rac1 activity and MR transcriptional activity in cultured mesangial cells. Overexpression of constitutively active Rac1 activated MR, and glucose-induced MR activation was suppressed by overexpression of dominant negative Rac1 or Rac inhibitor EHT1864. In KKAy, renal Rac1 was activated, and nuclear MR was increased. EHT1864 treatment suppressed renal Rac1 and MR activity and mitigated renal pathology of KKAy without changing plasma aldosterone concentration. Conclusion: Our results suggest that MR activation plays an important role in the nephropathy of KKAy mice, and that glucose-induced Rac1 activation, in addition to hyperaldosteronemia, contributes to their renal MR activation. Along with MR blockade, Rac inhibition may potentially be a preferred option in the treatment of nephropathy in obesity-related diabetic patients.

Relowering of Serum Na for Osmotic Demyelinating Syndrome

We report a case in whom slow correction of hyponatremia (5 mmol/day for 3 days) induced central pontine myelinolysis (CPM). After the diagnosis was confirmed by imaging, we started to relower serum Na that completely recovered the sign and symptoms of CPM. Rapid correction of serum sodium is known to be associated with CPM. However, it may occur even after slow correction of hyponatremia. Currently, there is no standard therapy for CPM other than supportive therapy. Other therapy includes sterioid, plasmaphresis and IVIG, but these therapies have not been shown to be particularly effective. The pathophysiology of CPM is related to a relative dehydration of the brain during the correction of hyponatremia, resulting in cell death and demyelination, therefore gentle rehydration with lowering serum sodium may not be an unreasonable therapy. The present case provides supportive evidence that reinduction of hyponatremia is effective in treating CPM if started immediately after the diagnosis is suggested. The present case tells us that severe chronic hyponatremia must be managed with extreme care especially in patients with chronic debilitating illness and that relowering serum Na is a treatment of choice when CPM is suggested.

Activation of Mineralocorticoid Receptor in Salt-Sensitive Hypertension

The impaired capacity of the kidney to excrete sodium plays an essential role in the development of hypertension. Adrenal corticosteroids control renal handling of sodium by regulating tubular sodium reabsorption in the distal nephron where both mineralocorticoid receptors (MR) and glucocorticoid receptors are expressed. In addition, cell type- and segment-specific expression of 11β-HSD2 and sodium transporters such as Na–Cl cotransporter (NCC), epithelial sodium channel (ENaC), and pendrin/Na+-driven Cl−/HCO3− exchanger (NDCBE) builds a distinctive model of sodium transport in the aldosterone-sensitive distal nephron. Aberrant MR activation in the distal nephron triggers salt-sensitive hypertension and hypokalemia through inappropriate sodium reabsorption and potassium secretion. However, MR activity is not necessarily modulated by the ligand alone. Recently, several lines of evidence revealed alternative mechanisms that regulate the activity of MR in a ligand-independent manner or through ligand binding modulation. This review summarizes the disorders related to MR activation in individual tubular cells and highlights the renal mechanism of salt-sensitive hypertension and new approaches for the prevention and treatment of this disease.

Renal Dysfunction Induced by Kidney-Specific Gene Deletion of Hsd11b2 as a Primary Cause of Salt-Dependent Hypertension

Genome-wide analysis of renal sodium-transporting system has identified specific variations of Mendelian hypertensive disorders, including HSD11B2 gene variants in apparent mineralocorticoid excess. However, these genetic variations in extrarenal tissue can be involved in developing hypertension, as demonstrated in former studies using global and brain-specific Hsd11b2 knockout rodents. To re-examine the importance of renal dysfunction on developing hypertension, we generated kidney-specific Hsd11b2 knockout mice. The knockout mice exhibited systemic hypertension, which was abolished by reducing salt intake, suggesting its salt-dependency. In addition, we detected an increase in renal membrane expressions of cleaved epithelial sodium channel-&agr; and T53-phosphorylated Na+-Cl− cotransporter in the knockout mice. Acute intraperitoneal administration of amiloride-induced natriuresis and increased urinary sodium/potassium ratio more in the knockout mice compared with those in the wild-type control mice. Chronic administration of amiloride and high-KCl diet significantly decreased mean blood pressure in the knockout mice, which was accompanied with the correction of hypokalemia and the resultant decrease in Na+-Cl− cotransporter phosphorylation. Accordingly, a Na+-Cl− cotransporter blocker hydrochlorothiazide significantly decreased mean blood pressure in the knockout mice. Chronic administration of mineralocorticoid receptor antagonist spironolactone significantly decreased mean blood pressure of the knockout mice along with downregulation of cleaved epithelial sodium channel-&agr; and phosphorylated Na+-Cl− cotransporter expression in the knockout kidney. Our data suggest that kidney-specific deficiency of 11&bgr;-HSD2 leads to salt-dependent hypertension, which is attributed to mineralocorticoid receptor–epithelial sodium channel–Na+-Cl− cotransporter activation in the kidney, and provides evidence that renal dysfunction is essential for developing the phenotype of apparent mineralocorticoid excess.