Mitsuhiro Nishimoto

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.

Renal mechanisms of salt-sensitive hypertension: contribution of two steroid receptor-associated pathways

Although salt is a major environmental factor in the development of hypertension, the degree of salt sensitivity varies widely among individuals. The mechanisms responsible for this variation remain to be elucidated. Recent studies have revealed the involvement of two important signaling pathways in renal tubules that play key roles in electrolyte balance and the maintenance of normal blood pressure: the β2-adrenergic stimulant-glucocorticoid receptor (GR)-with-no-lysine kinase (WNK)4-Na(+)-Cl(-) cotransporter pathway, which is active in distal convoluted tubule (DCT)1, and the Ras-related C3 botulinum toxin substrate (Rac)1-mineralocorticoid receptor (MR) pathway, which is active in DCT2, connecting tubules, and collecting ducts. β2-Adrenergic stimulation due to increased renal sympathetic activity in obesity- and salt-induced hypertension suppresses histone deacetylase 8 activity via cAMP/PKA signaling, increasing the accessibility of GRs to the negative GR response element in the WNK4 promoter. This results in the suppression of WNK4 transcription followed by the activation of Na(+)-Cl(-) cotransporters in the DCT and elevated Na(+) retention and blood pressure upon salt loading. Rac1 activates MRs, even in the absence of ligand binding, with this activity increased in the presence of ligand. In salt-sensitive animals, Rac1 activation due to salt loading activates MRs in DCT2, connecting tubules, and collecting ducts. Thus, GRs and MRs are independently involved in two pathways responsible for renal Na(+) handling and salt-sensitive hypertension. These findings suggest novel therapeutic targets and may lead to the development of diagnostic tools to determine salt sensitivity in hypertensive patients.

FRET-based sensor analysis reveals caveolae are spatially distinct Ca2+ stores in endothelial cells

Ca2+-regulating and Ca2+-dependent molecules enriched in caveolae are typically shaped as plasmalemmal invaginations or vesicles. Caveolae structure and subcellular distribution are critical for Ca2+ release from endoplasmic reticulum Ca2+ stores and for Ca2+ influx from the extracellular space into the cell. However, Ca2+ dynamics inside caveolae have never been directly measured and remain uncharacterized. To target the fluorescence resonance energy transfer (FRET)-based Ca2+ sensing protein D1, a mutant of cameleon, to the intra-caveolar space, we made a cDNA construct encoding a chimeric protein of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) and D1 (LOXD1). Immunofluorescence and immunoelectron microscopy confirmed that a significant portion of LOXD1 was localized with caveolin-1 at morphologically apparent caveolar vesicles in endothelial cells. LOXD1 detected ATP-induced transient Ca2+ decreases by confocal FRET imaging in the presence or absence of extracellular Ca2+. This ATP-induced Ca2+ decrease was abolished following knockdown of caveoin-1, suggesting an association with caveolae. The X-ray spectra obtained by the spot analysis of electron-opaque pyroantimonate precipitates further confirmed that ATP-induced calcium decreases in intra-caveolar vesicles. In conclusion, subplasmalemmal caveolae function as Ca2+-releasable Ca2+ stores in response to ATP. This intracellular local Ca2+ delivery system may contribute to the complex spatiotemporal organization of Ca2+ signaling.

Stromal interaction molecule 1 modulates blood pressure via NO production in vascular endothelial cells

In vascular endothelial cells, store-operated calcium entry (SOCE) activates endothelial NO synthase (eNOS) and regulates nitric oxide (NO) production as well as flow-dependent mechanical stimuli. Stromal interaction molecule 1, or STIM1, was recently identified to be essential for SOCE, acting as a calcium sensor for intracellular calcium stores. However, how STIM1 affects endothelial function and blood pressure (BP) remains unclear. We generated STIM1 fl/fl mice and vascular endothelial cell-specific STIM1 knockout mice using the Cre–loxP system, and conducted experiments using these mice to clarify the physiological role of STIM1 in vascular endothelial function and BP as follows: (1) SOCE was analyzed in isolated aortic endothelial cells by calcium add-back with fluorescent Ca2+ indicators. Phosphorylation of eNOS and NO production were evaluated by immunoblotting and the NO indicator, respectively. (2) Tension of aortic rings was measured in 10-week-old mice in response to acetylcholine. (3) BP was measured in 10-week-old mice by the telemetry system. The results were: (1) SOCE, eNOS activation, and NO production were suppressed by ~50–60% in endothelial cells from STIM1 knockout. (2) Endothelium-dependent vasodilation was decreased in aortic rings from STIM1 knockout mice, whereas endothelium-independent relaxation was not altered. (3) STIM1 knockout mice exhibited significant BP elevation, especially in nighttime. (124.3 ± 2.5/99.2 ± 3.9 vs. 114.1 ± 3.2/83.6 ± 1.7 (nighttime, mmHg), 109.7 ± 1.7/83.0 ± 3.0 vs. 104.8 ± 3.3/73.7 ± 1.6 (daytime, mmHg), knockout vs. control, respectively). In conclusion, STIM1 in vascular endothelial cell modulates vascular function through NO production and has a major role in regulating BP, especially in the active time.

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.

Aberrant DNA methylation of hypothalamic angiotensin receptor in prenatal programmed hypertension

Maternal malnutrition, which causes prenatal exposure to excessive glucocorticoid, induces adverse metabolic programming, leading to hypertension in offspring. In offspring of pregnant rats receiving a low-protein diet or dexamethasone, a synthetic glucocorticoid, mRNA expression of angiotensin receptor type 1a (Agtr1a) in the paraventricular nucleus (PVN) of the hypothalamus was upregulated, concurrent with reduced expression of DNA methyltransferase 3a (Dnmt3a), reduced binding of DNMT3a to the Agtr1a gene, and DNA demethylation. Salt loading increased BP in both types of offspring, suggesting that elevated hypothalamic Agtr1a expression is epigenetically modulated by excessive glucocorticoid and leads to adult-onset salt-sensitive hypertension. Consistent with this, dexamethasone treatment of PVN cells upregulated Agtr1a, while downregulating Dnmt3a, and decreased DNMT3a binding and DNA demethylation at the Agtr1a locus. In addition, Dnmt3a knockdown upregulated Agtr1a independently of dexamethasone. Hypothalamic neuron-specific Dnmt3a-deficient mice exhibited upregulation of Agtr1a in the PVN and salt-induced BP elevation without dexamethasone treatment. By contrast, dexamethasone-treated Agtr1a-deficient mice failed to show salt-induced BP elevation, despite reduced expression of Dnmt3a. Thus, epigenetic modulation of hypothalamic angiotensin signaling contributes to salt-sensitive hypertension induced by prenatal glucocorticoid excess in offspring of mothers that are malnourished during pregnancy.

Aberrant DNA methylation of pregnane X receptor underlies metabolic gene alterations in the diabetic kidney

Epigenetic abnormalities have been suggested to mediate metabolic memory observed in diabetic complications. We have shown that epigenetic alterations may induce persistent phenotypic changes in the proximal tubules of the diabetic kidneys. In this study, we show that pregnane X receptor (PXR), a xenobiotic nuclear receptor, is epigenetically altered and upregulated and may have a possible function in the diabetic kidney. PXR has been shown to play a critical role in metabolic changes in obesity and diabetes; however, its distribution and function in the kidney are unknown. In the normal kidney, Pxr was selectively expressed in the proximal tubular cells with demethylation in the promoter DNA. In db/db mice, significant increases in Pxr mRNA, further demethylation of DNA, and stimulatory histone marks in the promoter were observed. Epigenetic changes are likely to play a causative role in PXR induction, since a DNA methyltransferase inhibitor increased PXR mRNA in cultured human proximal tubular cells. Administration of a PXR agonist increased mRNA levels of solute carrier organic anion transporter family member 2B1 ( Slco2b1), a xenobiotic transporter; response gene to complement 32 ( Rgc32), a molecule known to exert fibrotic effects in the kidney; and phosphoenolpyruvate carboxykinase 1 ( Pck1), a gluconeogenic enzyme in the kidney. The expressions of these genes were inhibited by PXR small interfering RNA in cultured proximal tubular cells. Increased mRNA levels of Slco2b1, Rgc32, and Pck1 were also observed in the kidney of db/db mice. These data indicate that PXR is upregulated in the diabetic kidney with aberrant epigenetic modifications and may modulate the course of diabetic kidney disease through the activation of these genes.

Aldosterone Is Essential for Angiotensin II-Induced Upregulation of Pendrin

The renin-angiotensin-aldosterone system has an important role in the control of fluid homeostasis and BP during volume depletion. Dietary salt restriction elevates circulating angiotensin II (AngII) and aldosterone levels, increasing levels of the Cl-/HCO3- exchanger pendrin in β-intercalated cells and the Na+-Cl- cotransporter (NCC) in distal convoluted tubules. However, the independent roles of AngII and aldosterone in regulating these levels remain unclear. In C57BL/6J mice receiving a low-salt diet or AngII infusion, we evaluated the membrane protein abundance of pendrin and NCC; assessed the phosphorylation of the mineralocorticoid receptor, which selectively inhibits aldosterone binding in intercalated cells; and measured BP by radiotelemetry in pendrin-knockout and wild-type mice. A low-salt diet or AngII infusion upregulated NCC and pendrin levels, decreased the phosphorylation of mineralocorticoid receptor in β-intercalated cells, and increased plasma aldosterone levels. Notably, a low-salt diet did not alter BP in wild-type mice, but significantly decreased BP in pendrin-knockout mice. To dissect the roles of AngII and aldosterone, we performed adrenalectomies in mice to remove aldosterone from the circulation. In adrenalectomized mice, AngII infusion again upregulated NCC expression, but did not affect pendrin expression despite the decreased phosphorylation of mineralocorticoid receptor. By contrast, AngII and aldosterone coadministration markedly elevated pendrin levels in adrenalectomized mice. Our results indicate that aldosterone is necessary for AngII-induced pendrin upregulation, and suggest that pendrin contributes to the maintenance of normal BP in cooperation with NCC during activation of the renin-angiotensin-aldosterone system by dietary salt restriction.