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.

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.

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.

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.

Aging is associated with a high prevalence of hypertension due to elevated susceptibility of BP to dietary salt, but its mechanism is unknown. Serum levels of Klotho, an anti-aging factor, decline with age. We found that high salt (HS) increased BP in aged mice and young heterozygous Klotho-knockout mice and was associated with increased vascular expression of Wnt5a and p-MYPT1, which indicate RhoA activity. Not only the Wnt inhibitor LGK974 and the Wnt5a antagonist Box5 but Klotho supplementation inhibits HS-induced BP elevation, similarly to the Rho kinase inhibitor fasudil, associated with reduced p-MYPT1 expression in both groups of mice. In cultured vascular smooth muscle cells, Wnt5a and angiotensin II (Ang II) increased p-MYPT1 expression but knockdown of Wnt5a with siRNA abolished Ang II–induced upregulation of p-MYPT1, indicating that Wnt5a is indispensable for Ang II–induced Rho/ROCK activation. Notably, Klotho inhibited Wnt5a- and Ang II–induced upregulation of p-MYPT1. Consistently, Klotho supplementation ameliorated HS-induced augmentation of reduced renal blood flow (RBF) response to intra-arterial infusion of Ang II and the thromboxane A₂ analog U46619, which activated RhoA in both groups of mice and were associated with the inhibition of BP elevation, suggesting that abnormal response of RBF to Ang II contributes to HS-induced BP elevation. Thus, Klotho deficiency underlies aging-associated salt-sensitive hypertension through vascular non-canonical Wnt5a/RhoA activation.