Aberrant proximal tubule DNA methylation underlies phenotypic changes related to kidney dysfunction in patients with diabetes.

Epigenetic mechanisms are considered to contribute to diabetic nephropathy by maintaining memory of poor glycemic control during the early stages of diabetes. However, DNA methylation changes in the human kidney are poorly characterized, because of the lack of cell type-specific analysis. We examined DNA methylation in proximal tubules (PTs) purified from patients with diabetic nephropathy and identified differentially methylated CpG sites, given the critical role of proximal tubules in the kidney injury. Hypermethylation was observed at CpG sites annotated to genes responsible for proximal tubule functions, including gluconeogenesis, nicotinamide adenine dinucleotide synthesis, transporters of glucose, water, phosphate, and drugs, in diabetic kidneys, whereas genes involved in oxidative stress and the cytoskeleton exhibited demethylation. Methylation levels of CpG sites annotated to ACTN1, BCAR1, MYH9, UBE4B, AFMID, TRAF2, TXNIP, FOXO3, and HNF4A were correlated with the estimated glomerular filtration rate, whereas methylation of the CpG site in RUNX1 was associated with interstitial fibrosis and tubular atrophy. Hypermethylation of G6PC and HNF4A was accompanied by decreased expression in diabetic kidneys. Proximal tubule-specific hypomethylation of metabolic genes related to HNF4A observed in control kidneys was compromised in diabetic kidneys, suggesting a role for aberrant DNA methylation in the dedifferentiation process. Multiple genes with aberrant DNA methylation in diabetes overlapped genes with altered expressions in maladaptive proximal tubule cells, including transcription factors PPARA and RREB1. In conclusion, DNA methylation derangement in the proximal tubules of patients with diabetes may drive phenotypic changes, characterized by inflammatory and fibrotic features, along with impaired function in metabolism and transport.NEW & NOTEWORTHY Cell type-specific DNA methylation patterns in the human kidney are not known. We examined DNA methylation in proximal tubules of patients with diabetic nephropathy and revealed that oxidative stress, cytoskeleton, and metabolism genes were aberrantly methylated. The results indicate that aberrant DNA methylation in proximal tubules underlies kidney dysfunction in diabetic nephropathy. Aberrant methylation could be a target for reversing memory of poor glycemic control.

Klotho Supplementation Reverses Renal Dysfunction and Interstitial Fibrosis in Remnant Kidney.

INTRODUCTION: While recent investigations show that klotho exerts renoprotective actions, it has not been fully addressed whether klotho protein supplementation reverses renal damage. METHODS: The impacts of subcutaneous klotho supplementation on rats with subtotal nephrectomy were examined. Animals were divided into 3 groups: group 1 (short remnant [SR]): remnant kidney for 4 weeks, group 2 (long remnant [LR]): remnant kidney for 12 weeks, and group 3 (klotho supplementation [KL]): klotho protein (20 µg/kg/day) supplementation on the remnant kidney. Blood pressure, blood and urine compositions with conventional methods such as enzyme-linked immunosorbent assay and radioimmunoassay, kidney histology, and renal expressions of various genes were analyzed. In vitro studies were also performed to support in vivo findings. RESULTS: Klotho protein supplementation decreased albuminuria (-43%), systolic blood pressure (-16%), fibroblast growth factor (FGF) 23 (-51%) and serum phosphate levels (-19%), renal angiotensin II concentration (-43%), fibrosis index (-70%), renal expressions of collagen I (-55%), and transforming growth factor ß (-59%) (p < 0.05 for all). Klotho supplementation enhanced fractional excretion of phosphate (+45%), glomerular filtration rate (+76%), renal expressions of klotho (+148%), superoxide dismutase (+124%), and bone morphogenetic protein (BMP) 7 (+174%) (p < 0.05 for all). CONCLUSION: Our data indicated that klotho protein supplementation inactivated renal renin-angiotensin system, reducing blood pressure and albuminuria in remnant kidney. Furthermore, exogenous klotho protein supplementation elevated endogenous klotho expression to increase phosphate excretion with resultant reductions in FGF23 and serum phosphate. Finally, klotho supplementation reversed renal dysfunction and fibrosis in association with improved BMP7 in remnant kidney.

Rac1 deficiency impairs postnatal development of the renal papilla.

Development of the renal medulla continues after birth to form mature renal papilla and obtain urine-concentrating ability. Here, we found that a small GTPase, Rac1, plays a critical role in the postnatal development of renal papilla. Mice with distal tubule-specific deletion of Rac1 reached adulthood but showed polydipsia and polyuria with an impaired ability to concentrate urine. The elongation of renal papilla that occurs in the first weeks after birth was impaired in the Rac1-deficient infants, resulting in shortening and damage of the renal papilla. Moreover, the osmoprotective signaling mediated by nuclear factor of activated T cells 5, which is a key molecule of osmotic response to osmotic stress in renal medulla, was significantly impaired in the kidneys of the Rac1-deficient infants. These results demonstrate that Rac1 plays an important role in the development of renal papilla in the postnatal period, and suggested a potential link between Rac1 and osmotic response.

Activation of Rac1-Mineralocorticoid Receptor Pathway Contributes to Renal Injury in Salt-Loaded db/db Mice.

[Figure: see text].

Klotho supplementation attenuates blood pressure and albuminuria in murine model of IgA nephropathy.

BACKGROUND: Klotho interacts with various membrane proteins, such as transforming growth factor-ß (TGFß) and insulin-like growth factor (IGF) receptors. The renal expression of klotho is diminished in chronic kidney disease. METHOD: In this study, we assessed the effects of klotho supplementation on a murine model of IgA nephropathy. Twenty-four-week-old hyper serum IgA (HIGA) mice were subcutaneously injected daily with recombinant human klotho protein (20 µg/kg per day) or the vehicle. After 2 months, the mice were killed using an anesthesia overdose and their kidneys were harvested for analysis. RESULTS: Supplementation of exogenous klotho protein reduced SBP, albuminuria, 8-epi-prostaglandin F2α excretion, glomerular filtration rate, renal angiotensin II concentration, and angiotensinogen expression in HIGA mice. Additionally, it enhanced renal expression of superoxide dismutase (SOD) and renal klotho itself. The findings using laser-manipulated microdissection demonstrated that klotho supplementation reduced the glomerular expression of TGFß, fibronectin, and IGF, and increased the glomerular expression of connexin (Cx) 40. CONCLUSION: These results indicate that klotho supplementation reduces blood pressure by suppressing the renin--angiotensin system in HIGA mice. Klotho inhibits IGF signaling to preserve glomerular Cx40 levels, ameliorating albuminuria in HIGA mice. Klotho protein supplementation attenuates mesangial expansion by inhibiting TGFß signaling in HIGA mice.

Methylation pattern of urinary DNA as a marker of kidney function decline in diabetes.

INTRODUCTION: Renal tubular injury contributes to the decline in kidney function in patients with diabetes. Cell type-specific DNA methylation patterns have been used to calculate proportions of particular cell types. In this study, we developed a method to detect renal tubular injury in patients with diabetes by detecting exfoliated tubular cells shed into the urine based on tubular cell-specific DNA methylation patterns. RESEARCH DESIGN AND METHODS: We identified DNA methylation patterns specific for human renal proximal tubular cells through compartment-specific methylome analysis. We next determined the methylation levels of proximal tubule-specific loci in urine sediment of patients with diabetes and analyzed correlation with clinical variables. RESULTS: We identified genomic loci in SMTNL2 and G6PC to be selectively unmethylated in human proximal tubular cells. The methylation levels of SMTNL2 and G6PC in urine sediment, deemed to reflect the proportion of exfoliated proximal tubular cells due to injury, correlated well with each other. Methylation levels of SMTNL2 in urine sediment significantly correlated with the annual decline in estimated glomerular filtration rate. Moreover, addition of urinary SMTNL2 methylation to a model containing known risk factors significantly improved discrimination of patients with diabetes with faster estimated glomerular filtration rate decline. CONCLUSIONS: This study demonstrates that patients with diabetes with continual loss in kidney function may be stratified by a specific DNA methylation signature through epigenetic urinalysis and provides further evidence at the level of exfoliated cells in the urine that injury of proximal tubular cells may contribute to pathogenesis of diabetic kidney disease.

PGI2 Analog Attenuates Salt-Induced Renal Injury through the Inhibition of Inflammation and Rac1-MR Activation.

Renal inflammation is known to be involved in salt-induced renal damage, leading to end-stage renal disease. This study aims to evaluate the role of inflammation in anti-inflammatory and renoprotective effects of beraprost sodium (BPS), a prostaglandin I2 (PGI2) analog, in Dahl salt-sensitive (DS) rats. Five-week-old male DS rats were fed a normal-salt diet (0.5% NaCl), a high-salt diet (8% NaCl), or a high-salt diet plus BPS treatment for 3 weeks. BPS treatment could inhibit marked proteinuria and renal injury in salt-loaded DS rats with elevated blood pressure, accompanied by renal inflammation suppression. Notably, high salt increased renal expression of active Rac1, followed by increased Sgk1 expressions, a downstream molecule of mineralocorticoid receptor (MR) signal, indicating salt-induced activation of Rac1-MR pathway. However, BPS administration inhibited salt-induced Rac1-MR activation as well as renal inflammation and damage, suggesting that Rac1-MR pathway is involved in anti-inflammatory and renoprotective effects of PGI2. Based upon Rac1 activated by inflammation, moreover, BPS inhibited salt-induced activation of Rac1-MR pathway by renal inflammation suppression, resulting in the attenuation of renal damage in salt-loaded DS rats. Thus, BPS is efficacious for the treatment of salt-induced renal injury.

Salt causes aging-associated hypertension via vascular Wnt5a under Klotho deficiency.

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.

Two Mineralocorticoid Receptor-Mediated Mechanisms of Pendrin Activation in Distal Nephrons.

BACKGROUND: Regulation of sodium chloride transport in the aldosterone-sensitive distal nephron is essential for fluid homeostasis and BP control. The chloride-bicarbonate exchanger pendrin in ß-intercalated cells, along with sodium chloride cotransporter (NCC) in distal convoluted tubules, complementarily regulate sodium chloride handling, which is controlled by the renin-angiotensin-aldosterone system. METHODS: Using mice with mineralocorticoid receptor deletion in intercalated cells, we examined the mechanism and roles of pendrin upregulation via mineralocorticoid receptor in two different models of renin-angiotensin-aldosterone system activation. We also used aldosterone-treated NCC knockout mice to examine the role of pendrin regulation in salt-sensitive hypertension. RESULTS: Deletion of mineralocorticoid receptor in intercalated cells suppressed the increase in renal pendrin expression induced by either exogenous angiotensin II infusion or endogenous angiotensin II upregulation via salt restriction. When fed a low-salt diet, intercalated cell-specific mineralocorticoid receptor knockout mice with suppression of pendrin upregulation showed BP reduction that was attenuated by compensatory activation of NCC. In contrast, upregulation of pendrin induced by aldosterone excess combined with a high-salt diet was scarcely affected by deletion of mineralocorticoid receptor in intercalated cells, but depended instead on hypokalemic alkalosis through the activated mineralocorticoid receptor-epithelial sodium channel cascade in principal cells. In aldosterone-treated NCC knockout mice showing upregulation of pendrin, potassium supplementation corrected alkalosis and inhibited the pendrin upregulation, thereby lowering BP. CONCLUSIONS: In conjunction with NCC, the two pathways of pendrin upregulation, induced by angiotensin II through mineralocorticoid receptor activation in intercalated cells and by alkalosis through mineralocorticoid receptor activation in principal cells, play important roles in fluid homeostasis during salt depletion and salt-sensitive hypertension mediated by aldosterone excess.

Mineralocorticoid receptor blockade suppresses dietary salt-induced ACEI/ARB-resistant albuminuria in non-diabetic hypertension: a sub-analysis of evaluate study.

Excessive dietary salt intake can counteract the renoprotective effects of renin-angiotensin system (RAS) blockade in hypertensive patients with chronic kidney disease (CKD). In rodents, salt loading induces hypertension and renal damage by activating the mineralocorticoid receptor (MR) independently of plasma aldosterone levels. Thus, high salt-induced resistance to RAS blockade may be mediated by MR activation. To test this, a post hoc analysis of the Eplerenone Combination Versus Conventional Agents to Lower Blood Pressure on Urinary Antialbuminuric Treatment Effect (EVALUATE) trial was conducted. Thus, 304 non-diabetic hypertensive patients on RAS-blocking therapy were divided into tertiles according to salt intake (estimated 24-h urinary sodium excretion at baseline) and compared in terms of percent reduction in urinary albumin-to-creatinine ratio (UACR) at 52 weeks relative to baseline. The eplerenone-treated patients in the highest sodium excretion tertile exhibited significantly greater reduction in UACR than the placebo subjects in the same tertile (-22.5% vs. +21.8%, p = 0.02). This disparity was not observed in the lowest (-10.2% vs. -0.84%, p = 0.65) or middle (-19.5% vs. +9.5%, p = 0.22) tertiles. Similar systolic blood pressure changes were observed. In the whole cohort, reduction in UACR correlated positively with reduction in systolic blood pressure (r2 = 0.04, p = 0.02). These results support the hypothesis that excessive salt intake can enhance resistance to RAS blockade by activating MR. They also suggest that eplerenone plus RAS blockade may be effective for CKD in hypertensive patients, especially those with excessive salt intake.

Aberrant DNA methylation of Tgfb1 in diabetic kidney mesangial cells.

Epigenetic modulation may underlie the progression of diabetic nephropathy (DN). Involvement of TGFB1 in mesangial fibrosis of DN led us to hypothesize that Tgfb1 DNA demethylation contributes to progression of DN. In primary mesangial cells from diabetic (db/db) mouse kidneys, demethylation of Tgfb1 DNA and upregulation of Tgfb1 mRNA progressed simultaneously. USF1 binding site in Tgfb1 promoter region were demethylated, and binding of USF1 increased, with decreased binding of DNMT1 in db/db compared with control. Given downregulation of Tgfb1 expression by folic acid, antioxidant Tempol reversed DNA demethylation, with increased and decreased recruitment of DNMT1 and USF1 to the promoter, resulting in decreased Tgfb1 expression in db/db mice. Addition of H2O2 to mesangial cells induced DNA demethylation and upregulated Tgfb1 expression. Finally, Tempol attenuated mesangial fibrosis in db/db mice. We conclude that aberrant DNA methylation of Tgfb1 due to ROS overproduction play a key to mesangial fibrosis during DN progression.

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 ß-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 ß-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.

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-α 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-α and phosphorylated Na+-Cl- cotransporter expression in the knockout kidney. Our data suggest that kidney-specific deficiency of 11ß-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.

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.

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.

Immunomodulation with eicosapentaenoic acid supports the treatment of autoimmune small-vessel vasculitis.

Small-vessel vasculitis is a life-threatening autoimmune disease that is frequently associated with anti-neutrophil cytoplasmic antibodies (ANCAs). Conventional immunotherapy including steroids and cyclophosphamide can cause serious adverse events, limiting the efficacy and safety of treatment. Eicosapentaenoic acid (EPA), a key component of fish oil, is an omega-3 polyunsaturated fatty acid widely known to be cardioprotective and beneficial for vascular function. We report two elderly patients with systemic ANCA-associated vasculitis (AAV) in whom the administration of EPA in concert with steroids safely induced and maintained remission, without the use of additioal immunosuppressants. To explore the mechanisms by which EPA enhances the treatment of AAV, we employed SCG/Kj mice as a spontaneous murine model of AAV. Dietary enrichment with EPA significantly delayed the onset of crescentic glomerulonephritis and prolonged the overall survival. EPA-derived anti-inflammatory lipid mediators and their precursors were present in the kidney, plasma, spleen, and lungs in the EPA-treated mice. Furthermore, a decrease in ANCA production and CD4/CD8-double negative T cells, and an increase in Foxp3(+) regulatory T cells in the lymph nodes of the kidney were observed in the EPA-treated mice. These clinical and experimental observations suggest that EPA can safely support and augment conventional therapy for treating autoimmune small-vessel vasculitis.

Notch signaling regulates nucleocytoplasmic Olig2 translocation in reactive astrocytes differentiation after ischemic stroke.

Treatment with DAPT, an inhibitor of the Notch-activating enzyme, γ-secretase is known to reduce damage to ischemic brain. However, the molecular mechanisms supporting this therapeutic effect are not fully understood. Here we demonstrated that Notch/RBP-J signaling is activated in NG2(+) glial progenitors and reactive astrocytes such as GFAP(+) cells, Nestin(+) cells and RC2(+) cells, using Notch/RBP-J signaling reporter mice. 3-day DAPT treatment reduced the number of reactive astrocytes but not NG2(+) glial progenitors. BrdU labeling experiments have shown that this reduction was due to decreased proliferation of reactive astrocytes. DAPT inhibited nuclear-translocation of Olig2, which is indispensable for proliferation and differentiation of reactive astrocytes. These findings suggest that Notch signaling might promote proliferation and differentiation of reactive astrocytes through the regulation of nucleo-cytoplasmic translocation of Olig2.

Transplantation of telencephalic neural progenitors induced from embryonic stem cells into subacute phase of focal cerebral ischemia.

Cerebral ischemia causes neuronal death and disruption of neural circuits in the central nervous system. Various neurological disorders caused by cerebral infarction can severely impair quality of life and are potentially fatal. Functional recovery in the chronic stage mainly depends on physical treatment and rehabilitation. We aim to establish cell therapy for cerebral ischemia using embryonic stem (ES) cells, which have self-renewing and pluripotent capacities. We previously reported that the transplanted monkey and mouse ES cell-derived neural progenitors, by stromal cell-derived inducing activity method, could survive and differentiate into various types of neurons and glial cells, and form the neuronal network in basal ganglia. In this report, we induced the differentiation of the neural progenitors from mouse ES cells using the serum-free suspension culture method and confirmed the expression of various basal ganglial neuronal markers and neurotransmitter-related markers both in vitro and in vivo, which was thought to be suitable for replacing damaged striatum after middle cerebral artery occlusion. This is the first report that used selectively induced telencephalic neural progenitors into ischemia model. Furthermore, we purified the progenitors expressing the neural progenitor marker Sox1 by fluorescence-activated cell sorting and Sox1-positive neural progenitors prevented tumor formation in ischemic brain for 2 months. We also analyzed survival and differentiation of transplanted cells and functional recovery from ischemic damage.