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.

Unbiased Human Kidney Tissue Proteomics Identifies Matrix Metalloproteinase 7 as a Kidney Disease Biomarker.

SIGNIFICANCE STATEMENT: Although gene expression changes have been characterized in human diabetic kidney disease (DKD), unbiased tissue proteomics information for this condition is lacking. The authors conducted an unbiased aptamer-based proteomic analysis of samples from patients with DKD and healthy controls, identifying proteins with levels that associate with kidney function (eGFR) or fibrosis, after adjusting for key covariates. Overall, tissue gene expression only modestly correlated with tissue protein levels. Kidney protein and RNA levels of matrix metalloproteinase 7 (MMP7) strongly correlated with fibrosis and with eGFR. Single-cell RNA sequencing indicated that kidney tubule cells are an important source of MMP7. Furthermore, plasma MMP7 levels predicted future kidney function decline. These findings identify kidney tissue MMP7 as a biomarker of fibrosis and blood MMP7 as a biomarker for future kidney function decline. BACKGROUND: Diabetic kidney disease (DKD) is responsible for close to half of all ESKD cases. Although unbiased gene expression changes have been extensively characterized in human kidney tissue samples, unbiased protein-level information is not available. METHODS: We collected human kidney samples from 23 individuals with DKD and ten healthy controls, gathered associated clinical and demographics information, and implemented histologic analysis. We performed unbiased proteomics using the SomaScan platform and quantified the level of 1305 proteins and analyzed gene expression levels by bulk RNA and single-cell RNA sequencing (scRNA-seq). We validated protein levels in a separate cohort of kidney tissue samples as well as in 11,030 blood samples. RESULTS: Globally, human kidney transcript and protein levels showed only modest correlation. Our analysis identified 14 proteins with kidney tissue levels that correlated with eGFR and found that the levels of 152 proteins correlated with interstitial fibrosis. Of the identified proteins, matrix metalloprotease 7 (MMP7) showed the strongest association with both fibrosis and eGFR. The correlation between tissue MMP7 protein expression and kidney function was validated in external datasets. The levels of MMP7 RNA correlated with fibrosis in the primary and validation datasets. Findings from scRNA-seq pointed to proximal tubules, connecting tubules, and principal cells as likely cellular sources of increased tissue MMP7 expression. Furthermore, plasma MMP7 levels correlated not only with kidney function but also associated with prospective kidney function decline. CONCLUSIONS: Our findings, which underscore the value of human kidney tissue proteomics analysis, identify kidney tissue MMP7 as a diagnostic marker of kidney fibrosis and blood MMP7 as a biomarker for future kidney function decline.

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.

Role of the Ubiquitin Proteasome System in the Regulation of Blood Pressure: A Review.

The kidney and the vasculature play crucial roles in regulating blood pressure. The ubiquitin proteasome system (UPS), a multienzyme process mediating covalent conjugation of the 76-amino acid polypeptide ubiquitin to a substrate protein followed by proteasomal degradation, is involved in multiple cellular processes by regulating protein turnover in various tissues. Increasing evidence demonstrates the roles of UPS in blood pressure regulation. In the kidney, filtered sodium is reabsorbed through diverse sodium transporters and channels along renal tubules, and studies conducted till date have provided insights into the complex molecular network through which ubiquitin ligases modulate sodium transport in different segments. Components of these pathways include ubiquitin ligase neuronal precursor cell-expressed developmentally downregulated 4-2, Cullin-3, and Kelch-like 3. Moreover, accumulating data indicate the roles of UPS in blood vessels, where it modulates nitric oxide bioavailability and vasoconstriction. Cullin-3 not only regulates renal salt reabsorption but also controls vascular tone using different adaptor proteins that target distinct substrates in vascular smooth muscle cells. In endothelial cells, UPS can also contribute to blood pressure regulation by modulating endothelial nitric oxide synthase. In this review, we summarize current knowledge regarding the role of UPS in blood pressure regulation, focusing on renal sodium reabsorption and vascular function.

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.

Electrolyte transport in the renal collecting duct and its regulation by the renin-angiotensin-aldosterone system.

Distal nephron of the kidney plays key roles in fluid volume and electrolyte homeostasis by tightly regulating reabsorption and excretion of Na+, K+, and Cl- Studies to date demonstrate the detailed electrolyte transport mechanisms in principal cells of the cortical collecting duct, and their regulation by renin-angiotensin-aldosterone system (RAAS). In recent years, however, accumulating data indicate that intercalated cells, another cell type that is present in the cortical collecting duct, also play active roles in the regulation of blood pressure. Notably, pendrin in ß-intercalated cells not only controls acid/base homeostasis, but is also one of the key components controlling salt and K+ transport in distal nephron. We have recently shown that pendrin is regulated by the co-ordinated action of angiotensin II (AngII) and aldosterone, and at the downstream of AngII, mammalian target of rapamycin (mTOR) signaling regulates pendrin through inhibiting the kinase unc51-like-kinase 1 and promoting dephosphorylation of mineralocorticoid receptor (MR). In this review, we summarize recent advances in the current knowledge on the salt transport mechanisms in the cortical collecting duct, and their regulation by the RAAS.

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.

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.

High-salt in addition to high-fat diet may enhance inflammation and fibrosis in liver steatosis induced by oxidative stress and dyslipidemia in mice.

BACKGROUND: It is widely known that salt is an accelerating factor for the progression of metabolic syndrome and causes cardiovascular diseases, most likely due to its pro-oxidant properties. We hypothesized that excessive salt intake also facilitates the development of nonalcoholic steatohepatitis (NASH), which is frequently associated with metabolic syndrome. METHODS: We examined the exacerbating effect of high-salt diet on high-fat diet-induced liver injury in a susceptible model to oxidative stress, apoE knockout and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) transgenic mice. RESULTS: High-salt diet led to NASH in high-fat diet-fed LOX-1 transgenic/apoE knockout mice without affecting high-fat diet-induced dyslipidemia or hepatic triglyceride accumulation. Additionally, a high-salt and high-fat diet stimulated oxidative stress production and inflammatory reaction to a greater extent than did a high-fat diet in the liver of LOX-1 transgenic/apoE knockout mice. CONCLUSIONS: We demonstrated that high-salt diet exacerbated NASH in high-fat diet-fed LOX-1 transgenic /apoE knockout mice and that this effect was associated with the stimulation of oxidative and inflammatory processes; this is the first study to suggest the important role of excessive salt intake in the development of NASH.

Fibroblast growth factor 23 accelerates phosphate-induced vascular calcification in the absence of Klotho deficiency.

Fibroblast growth factor 23 (FGF23) is a phosphate-regulating hormone that acts primarily on the kidney and parathyroid. With declining kidney function there is an increase in circulating FGF23 levels, which is associated with vascular calcification and mortality in chronic kidney disease. Whether FGF23 exerts direct effects on vasculature is unclear. We evaluated the expression of Klotho and FGF receptors in rat aortic rings and rat aorta vascular smooth muscle cells maintained in culture by reverse transcription-PCR, western blotting, and immunostaining. Signaling pathways underlying FGF23 effects were assessed by western blotting, and effects of FGF23 on osteogenic markers and phosphate transporters were assessed by real-time reverse transcription-PCR. We detected Klotho and FGFR1 in total aorta but not in vascular smooth muscle cells. FGF23 augmented phosphate-induced vascular calcification in the aortic rings from uremic rats and dose dependently increased ERK1/2 phosphorylation in Klotho-overexpressing but not naive vascular smooth muscle cells. FGF23-induced ERK1/2 phosphorylation was inhibited by SU5402 (FGFR1 inhibitor) and U0126 (MEK inhibitor). FGF23 enhanced phosphate-induced calcification in Klotho-overexpressing vascular smooth muscle cells and increased osteoblastic marker expression, which was inhibited by U0126. In contrast, phosphate transporter expression was not affected by phosphate or FGF23. Thus, FGF23 enhances phosphate-induced vascular calcification by promoting osteoblastic differentiation involving the ERK1/2 pathway.

A case of de novo focal segmental glomerulosclerosis occurred one and half years after kidney transplantation supposed to be caused by calcineurin inhibitor.

A 64-yr-old man with end-stage kidney disease caused by hypertensive nephrosclerosis underwent living-unrelated ABO-identical kidney transplantation (KTx) at the age of 60 yr from his 60-yr-old wife. Maintenance trough concentration of cyclosporine A (CsA) was 100 ± 30 ng/mL. Five months after KTx, proteinuria gradually increased to around 1 g/d. TRBx at eight months after KTx revealed the new-onset alteration of mild arteriolosclerosis with intimal hyalinosis, which might reflect calcineurin inhibitor (CNI)-associated arteriopathy (CAA). Nearly one and half years after KTx, urinary protein excretion became nearly 2 g/d. TRBx revealed the advanced CAA and findings of focal segmental glomerulosclerosis (FSGS). Then, CNI was switched from CsA to tacrolimus (TAC). TRBx at two and half years after KTx revealed progressed arteriolar transmural thickening and striped fibrosis, which were supposed to be induced by an increase in serum TAC concentration because of acute enterocolitis. Then, TAC dose was reduced to serum trough concentration 5-8 ng/mL, but urinary protein excretion was increased up to 10 g/d. Reduction of TAC to trough concentration 2.0 ± 0.5 ng/mL reduced urinary protein excretion. Attempts to elevate TAC trough concentration within normal range (4-8 ng/mL) reproducibly induced the recurrence of an increase in sCr or urinary protein excretion. All these findings supported the etiology of graft dysfunction, and proteinuria of this case was FSGS.

Selenium Associates With Response to Erythropoiesis-Stimulating Agents in Hemodialysis Patients.

Introduction: Impaired response to erythropoiesis-stimulating agents (ESAs) is associated with increased mortality in patients with end-stage kidney disease. However, the underlying mechanisms are not fully elucidated. Accumulating data reveal that selenium (Se), a trace element, plays a key role in stress erythropoiesis and erythrocyte homeostasis. We evaluated the relationship between serum Se levels and the response to ESAs in hemodialysis patients. Methods: In this cross-sectional study, we determined serum Se levels in 173 hemodialysis patients. We analyzed the association of serum Se with ESA responsiveness, as defined by ESA resistance index. Results: Of the study participants, 50% had lower Se levels than the population-based reference values. We found that serum Se levels were significantly and inversely correlated with erythropoiesis resistance index (ERI) but not transferrin saturation (TSAT) or ferritin levels. Multiple regression analyses confirmed the association between Se levels and ESA hyporesponsiveness, independently of other known factors, such as iron status, being female, and dialysis vintage (ß = -0.11, P < 0.001). When patients were divided according to Se levels and iron status, both low serum Se (<10.5 µg/dl) and iron deficiency significantly affected the response to ESA. Conversely, serum Se levels were significantly different among groups when patients were divided according to ERI quartiles. The association of low serum Se with ESA hyporesponsiveness persisted after adjustment of confounding variables. Conclusion: Serum Se levels are associated with the response to ESAs and can predict ESA resistance independently of iron status in Japanese hemodialysis patients. These data open the possibility to test whether Se supplementation reduces ESA demand.

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

[Figure: see text].

A Ruptured Jejunal Arterial Aneurysm in a Young Woman Undergoing Chronic Hemodialysis Due to Myeloperoxidase-antineutrophil Cytoplasmic Antibody-associated Vasculitis.

A 21-year-old woman was admitted to our hospital because of massive intestinal bleeding. She started hemodialysis due to myeloperoxidase antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) at 18 years of age. Her ANCA titers remained stable; however, her C-reactive protein increased on 5 mg/day prednisolone before admission. Computed tomography angiography revealed a ruptured jejunal arterial aneurysm. Transcatheter arterial embolization, blood transfusion and the reinforcement of steroid therapy resolved her symptoms of AAV. Our case of a young patient with AAV and medium-sized arterial vasculitis is rare and emphasizes that the ANCA titer does not always rise, especially in patients with nonrenal vasculitis flare-ups.

Characterization of pendrin in urinary extracellular vesicles in a rat model of aldosterone excess and in human primary aldosteronism.

Pendrin is a Cl-/HCO3- exchanger selectively present in the intercalated cells of the kidney. Although experimental studies have demonstrated that pendrin regulates blood pressure downstream of the renin-angiotensin-aldosterone system, its role in human hypertension remains unclear. Here, we analyzed the quantitative changes in pendrin in urinary extracellular vesicles (uEVs) isolated from a total of 30 patients with primary aldosteronism (PA) and from a rat model of aldosterone excess. Western blot analysis revealed that pendrin is present in dimeric and monomeric forms in uEVs in humans and rats. In a rodent model that received continuous infusion of aldosterone with or without concomitant administration of the selective mineralocorticoid receptor (MR) antagonist esaxerenone, pendrin levels in uEVs, as well as those of epithelial Na+ channel (ENaC) and Na-Cl-cotransporter (NCC), were highly correlated with renal abundance. In patients with PA, pendrin levels in uEVs were reduced by 49% from baseline by adrenalectomy or pharmacological MR blockade. Correlation analysis revealed that the magnitude of pendrin reduction after treatment significantly correlated with the baseline aldosterone-renin ratio (ARR). Finally, a cross-sectional analysis of patients with PA confirmed a significant correlation between the ARR and pendrin levels in uEVs. These data are consistent with experimental studies showing the role of pendrin in aldosterone excess and suggest that pendrin abundance is attenuated by therapeutic interventions in human PA. Our study also indicates that pendrin analysis in uEVs, along with other proteins, can be useful to understand the pathophysiology of hypertensive disorders.

Development of myeloperoxidase-antineutrophil cytoplasmic antibody-associated renal vasculitis in a patient receiving treatment with anti-tumor necrosis factor-α.

We report on a 33-year-old woman who presented with positive myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA) during rheumatoid arthritis treatment with infliximab. She had a history of worsening arthralgia, and urinalysis showed the new occurrence of hematuria and proteinuria. Renal biopsy showed necrotizing crescentic glomerulonephritis. Immunosuppressive therapy and discontinuation of the infliximab therapy alleviated her arthralgia and improved the urinalysis results. We report this rare case in which an anti-tumor necrosis factor-α (TNF-α) agent for ANCA-associated systemic vasculitis was studied.

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.

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.

Evaluation of the pathophysiological mechanisms of salt-sensitive hypertension.

The currently available data have indicated that dietary salt is directly correlated with blood pressure (BP) and the occurrence of hypertension. However, the salt sensitivity of BP is different in each individual. Genetic factors and environmental factors influence the salt sensitivity of BP. Obesity, stress, and aging are strongly associated with increased BP salt sensitivity. Indeed, a complex and interactive genetic and environmental system can determine an individual's BP salt sensitivity. However, the genetic/epigenetic determinants leading to salt sensitivity of BP are still challenging to identify primarily because lifestyle-related diseases, including hypertension, usually become a medical problem during adulthood, although their causes may be attributed to the earlier stages of ontogeny. The association between distinct developmental periods involves changes in gene expression, which include epigenetic phenomena. The role of epigenetic modification in the development of salt-sensitive hypertension is presently under investigation. Recently, we identified aberrant DNA methylation in the context of prenatally programmed hypertension. In this review, we summarize the existing knowledge regarding the pathophysiological mechanisms of salt-sensitive hypertension. Additionally, we discuss the contribution of epigenetic mechanisms in the development of salt-sensitive hypertension.

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.