The significance of NAD + metabolites and nicotinamide N-methyltransferase in chronic kidney disease.

Dysregulation of nicotinamide adenine dinucleotide (NAD +) metabolism contributes to the initiation and progression of age-associated diseases, including chronic kidney disease (CKD). Nicotinamide N-methyltransferase (NNMT), a nicotinamide (NAM) metabolizing enzyme, regulates both NAD + and methionine metabolism. Although NNMT is expressed abundantly in the kidney, its role in CKD and renal fibrosis remains unclear. We generated NNMT-deficient mice and a unilateral ureter obstruction (UUO) model and conducted two clinical studies on human CKD to investigate the role of NNMT in CKD and fibrosis. In UUO, renal NNMT expression and the degraded metabolites of NAM increased, while NAD + and NAD + precursors decreased. NNMT deficiency ameliorated renal fibrosis; mechanistically, it (1) increased the DNA methylation of connective tissue growth factor (CTGF), and (2) improved renal inflammation by increasing renal NAD + and Sirt1 and decreasing NF-κB acetylation. In humans, along with CKD progression, a trend toward a decrease in serum NAD + precursors was observed, while the final NAD + metabolites were accumulated, and the level of eGFR was an independent variable for serum NAM. In addition, NNMT was highly expressed in fibrotic areas of human kidney tissues. In conclusion, increased renal NNMT expression induces NAD + and methionine metabolism perturbation and contributes to renal fibrosis.

Home-based aerobic exercise and resistance training for severe chronic kidney disease: a randomized controlled trial.

BACKGROUND: The potential effects of aerobic and resistance training in patients with severe chronic kidney disease (CKD) are not fully elucidated. This study investigated the effects of a home-based exercise programme on physical functioning and health-related quality of life (HRQOL) in patients with Stage 4 CKD, equivalent to estimated glomerular filtration rate of 15-30 mL/min/1.73 m2 . METHODS: Forty-six patients with Stage 4 CKD (median age, 73 years; 33 men) were randomly assigned to exercise (n = 23) and control (n = 23) groups. Exercise group patients performed aerobic exercise at 40-60% peak heart rate thrice weekly and resistance training at 70% of one-repetition maximum twice weekly at home for 6 months. Control patients received no specific intervention. Primary outcomes were distance in incremental shuttle walking test and HRQOL assessed using the Kidney Disease Quality of Life-Short Form questionnaire. Secondary outcomes included kidney function assessed with combined urea and creatinine clearance, urinary biomarkers, and anthropometric and biochemical parameters associated with CKD. RESULTS: Improvement in incremental shuttle walking test was significantly greater in the exercise group compared with controls (39.4 ± 54.6 vs. -21.3 ± 46.1; P < 0.001). Among Kidney Disease Quality of Life domains, significant mean differences were observed between the exercise group and the control group in work status, quality of social interaction, and kidney disease component summary outcomes (12.76 ± 5.76, P = 0.03; 5.97 ± 2.59, P = 0.03; and 4.81 ± 1.71, P = 0.007, respectively). There were greater reductions in natural log (ln)-transformed urinary excretion of liver-type fatty acid-binding protein, ln serum C-reactive protein, and acylcarnitine to free carnitine ratio in the exercise group compared with controls, with significant between-group differences of -0.579 ± 0.217 (P = 0.008), -1.13 ± 0.35 (P = 0.003), and -0. 058 ± 0.024 (P = 0.01), respectively. CONCLUSIONS: Our 6 month home-based exercise programme improved aerobic capacity and HRQOL in patients with Stage 4 CKD, with possible beneficial effects on kidney function and CKD-related parameters.

The effect of aldosterone and aldosterone blockade on the progression of chronic kidney disease: a randomized placebo-controlled clinical trial.

The progression of chronic kidney disease (CKD) cannot be completely inhibited. We first explored factors contributing to CKD progression in patients with CKD in a prospective observational study. In the next phase, we focused on the effects of aldosterone, conducting a single-blinded placebo-controlled study using the selective mineralocorticoid receptor antagonist (MRA), eplerenone (25 mg/day). We recruited patients with CKD stage 2 and 3 whose plasma aldosterone concentration was above 15 ng/dL based on the prior data of a prospective observational study. In the CKD cohort study (n = 141), baseline plasma aldosterone concentration was identified as an independent contributory factor for the future rate of change in estimated glomerular filtration rate (eGFR). When the cut-off value for aldosterone was set at 14.5 ng/dL, the decline rate was significantly higher in patients with higher plasma aldosterone concentration (- 1.22 ± 0.39 ml/min/1.73 m2/year vs. 0.39 ± 0.40 ml/min/1.73 m2/year, p = 0.0047). In the final intervention study, in the eplerenone group, eGFR dropped at 6 months after the initiation of the study, and thereafter eGFR was maintained until the end of the study. At 24 months and 36 months, eGFR was significantly higher in the eplerenone group than in the placebo group. In conclusion, MRA can be an effective strategy in preventing CKD progression, especially in patients with high plasma aldosterone.

Proton pump inhibitors may hinder hypophosphatemic effect of lanthanum carbonate, but not of ferric citrate hydrate or sucroferric oxyhydroxide, in hemodialysis patients.

Because end-stage renal disease patients undergoing hemodialysis frequently take acid suppressants for the treatment or prevention of gastrointestinal diseases, it is important to clarify the drug-interactions between acid suppressants and phosphate binders on the control of serum phosphate levels. In the present study, we examined whether the phosphate-lowering effects of three phosphate binders, lanthanum carbonate (LC), ferric citrate hydrate (FCH), and sucroferric oxyhydroxide (SFOH), were affected by proton pump inhibitors (PPIs) in maintenance hemodialysis patients. Laboratory data for 71 patients who had been newly prescribed one of the three phosphate binders were examined. LC at a dosage of 500 ± 217 mg/day significantly decreased serum phosphate levels by -18% in the absence of a PPI (n = 9), while a dosage of 700 ± 230 mg/day only decreased it by -3% in the presence of a PPI (n = 10). Thus, the efficacy of LC in reducing serum phosphate levels was significantly hindered by the presence of PPIs. FCH significantly decreased serum phosphate levels by -18% in the absence of a PPI (n = 7, FCH: 571 ± 189 mg/day) and by -17% in the presence of a PPI (n = 20, FCH: 638 ± 151 mg/day). The decrease in serum phosphate levels by SFOH (393 ± 197 mg/day) was -7% in the absence of a PPI (n = 7), and SFOH at a dosage of 556 ± 316 mg/day significantly decreased serum phosphate levels by -13% in the presence of a PPI (n = 18). These results suggest that the phosphate-lowering effect of LC, but not of FCH or SFOH, is diminished in the presence of PPIs in hemodialysis patients.

Safety and efficacy of plasma exchange via direct femoral vein puncture in autoimmune blistering diseases.

Plasma exchange (PE) is performed for patients with autoimmune blistering diseases by using multiple vascular access routes. We retrospectively examined the safety and the efficacy of PE using direct femoral vein puncture (FVP) technique, by comparing with that using double-lumen catheter (DLC). The troubles related to vascular route, such as catheter occlusion, insufficient blood flow and hematoma, were not different between the FVP group (4.6%) and the DLC group (6.7%), whereas access-related infections occurred more frequently in the DLC group (6.7%) than the FVP group (0.4%). Regarding the efficacy, the removal rate of autoantibodies in PE using the FVP technique was similar or lower, as compared with that using the DLC. These results suggest that PE with the FVP technique is able to be performed safely in patients with autoimmune blistering diseases, although the removal of autoantibodies is not superior to that using the DLC.

Circulating FGF23 is not associated with cardiac dysfunction, atherosclerosis, infection or inflammation in hemodialysis patients.

Fibroblast growth factor (FGF) 23 is a bone-derived hormone regulating serum inorganic phosphate (Pi) concentration. FGF23 is also involved in the development of chronic kidney disease (CKD)-mineral and bone disorder. Serum FGF23 concentration begins to increase early in the progression of CKD and can be remarkably high in hemodialysis patients with end-stage renal disease. It has been reported that high FGF23 concentration is a risk factor for cardiac dysfunction, atherosclerosis, infection or systemic inflammation in CKD patients. FGF23 was also shown to induce cardiac hypertrophy directly acting on cardiomyocytes. However, it is still controversial whether high FGF23 is causing cardiac dysfunction, atherosclerosis, infection or systemic inflammation in CKD patients. In the current study, we investigated whether FGF23 concentration is associated with cardiac dysfunction, atherosclerosis, infection or systemic inflammation in Japanese hemodialysis patients. We recruited 119 hemodialysis patients and examined the association between serum FGF23 concentration and several parameters concerning mineral metabolism, cardiac dysfunction, atherosclerosis, infection, and systemic inflammation. Serum FGF23 concentration was independently associated with serum calcium and Pi concentration (ß = 0.276, p < 0.001; ß = 0.689, p < 0.001). However, serum FGF23 concentration was not associated with parameters of cardiac dysfunction, atherosclerosis, infection, and systemic inflammation, either. Our results do not support the hypothesis that high FGF23 in dialysis patients is the cause of cardiac dysfunction, atherosclerosis, infection or systemic inflammation.

Role of Nampt-Sirt6 Axis in Renal Proximal Tubules in Extracellular Matrix Deposition in Diabetic Nephropathy.

Nicotinamide adenine dinucleotide (NAD+) metabolism plays a critical role in kidneys. We previously reported that decreased secretion of a NAD+ precursor, nicotinamide mononucleotide (NMN), from proximal tubules (PTs) can trigger diabetic albuminuria. In the present study, we investigated the role of NMN-producing enzyme nicotinamide phosphoribosyltransferase (Nampt) in diabetic nephropathy. The expression of Nampt in PTs was downregulated in streptozotocin (STZ)-treated diabetic mice when they exhibited albuminuria. This albuminuria was ameliorated in PT-specific Nampt-overexpressing transgenic (TG) mice. PT-specific Nampt-conditional knockout (Nampt CKO) mice exhibited TBM thickening and collagen deposition, which were associated with the upregulation of the profibrogenic gene TIMP-1. Nampt CKO mice also exhibited the downregulation of sirtuins, particularly in Sirt6. PT-specific Sirt6-knockout mice exhibited enhanced fibrotic phenotype resembling that of Nampt CKO mice with increased Timp1 expression. In conclusion, the Nampt-Sirt6 axis in PTs serves as a key player in fibrogenic extracellular matrix remodeling in diabetic nephropathy.

High Basolateral Glucose Increases Sodium-Glucose Cotransporter 2 and Reduces Sirtuin-1 in Renal Tubules through Glucose Transporter-2 Detection.

Under diabetic conditions, sodium-glucose cotransporter 2 (SGLT2) for glucose uptake in proximal tubules (PTs) increases, whereas NAD+-dependent protein deacetylase silent mating type information regulation 2 homolog 1 (Sirtuin-1; SIRT1) for PT survival decreases. Therefore, we hypothesized that increased glucose influx by SGLT2 reduces SIRT1 expression. To test this hypothesis, db/db mice with diabetes and high-glucose (HG)-cultured porcine PT LLC-PK1 cells in a two-chamber system were treated with the SGLT2 inhibitor canagliflozin. We also examined SIRT1 and SGLT2 expression in human kidney biopsies. In db/db mice, SGLT2 expression increased with concomitant decreases in SIRT1, but was inhibited by canagliflozin. For determination of the polarity of SGLT2 and SIRT1 expression, LLC-PK1 cells were seeded into Transwell chambers (pore size, 0.4 µm; Becton Dickinson, Oxford, UK). HG medium was added to either or to both of the upper and lower chambers, which corresponded to the apical and basolateral sides of the cells, respectively. In this system, the lower chamber with HG showed increased SGLT2 and decreased SIRT1 expression. Canagliflozin reversed HG-induced SIRT1 downregulation. Gene silencing and inhibitors for glucose transporter 2 (GLUT2) blocked HG-induced SGLT2 expression upregulation. Gene silencing for the hepatic nuclear factor-1α (HNF-1α), whose nuclear translocation was enhanced by HG, blocked HG-induced SGLT2 expression upregulation. Similarly, gene silencing for importin-α1, a chaperone protein bound to GLUT2, blocked HG-induced HNF-1α nuclear translocation and SGLT2 expression upregulation. In human kidney, SIRT1 immunostaining was negatively correlated with SGLT2 immunostaining. Thus, under diabetic conditions, SIRT1 expression in PTs was downregulated by an increase in SGLT2 expression, which was stimulated by basolateral HG through activation of the GLUT2/importin-α1/HNF-1α pathway.

Aldosterone and Insulin Resistance: Vicious Combination in Patients on Maintenance Hemodialysis.

Recently, we demonstrated that plasma aldosterone contributed to insulin resistance in chronic kidney disease. The aim of this study is the clinical impact of this relationship in hemodialysis patients. In a cross section study using a total of 128 hemodialysis patients, multiple regression analysis revealed that plasma aldosterone levels were independently associated with HOMA-IR, insulin resistance index. This association was found to be more stringent in diabetic patients than in non-diabetic patients. Aldosterone levels were associated with cardiac hypertrophy and carotid artery stenosis. HOMA-IR was associated with cardiac hypertrophy. The patients whose aldosterone and HOMA-IR were above the top tertile of each parameter in this cohort showed more severe cardiac hypertrophy and lower contractile function as compared with the patients whose aldosterone levels and HOMA-IR are below the lowest tertile of each parameter. In conclusion, in hemodialysis patients, aldosterone levels and insulin resistance are closely interrelated and the constellation of the two is related to severe cardiovascular tissue damages.

Obesity-induced kidney injury is attenuated by amelioration of aberrant PHD2 activation in proximal tubules.

The involvement of tissue ischemia in obesity-induced kidney injury remains to be elucidated. Compared with low fat diet (LFD)-mice, high fat diet (HFD)-fed mice became obese with tubular enlargement, glomerulomegaly and peritubular capillary rarefaction, and exhibited both tubular and glomerular damages. In HFD-fed mice, despite the increase in renal pimonidazole-positive areas, the expressions of the hypoxia-responsive genes such as Prolyl-hydroxylase PHD2, a dominant oxygen sensor, and VEGFA were unchanged indicating impaired hypoxic response. Tamoxifen inducible proximal tubules (PT)-specific Phd2 knockout (Phd2-cKO) mice and their littermate control mice (Control) were created and fed HFD or LFD. Control mice on HFD (Control HFD) exhibited renal damages and renal ischemia with impaired hypoxic response compared with those on LFD. After tamoxifen treatment, HFD-fed knockout mice (Phd2-cKO HFD) had increased peritubular capillaries and the increased expressions of hypoxia responsive genes compared to Control HFD mice. Phd2-cKO HFD also exhibited the mitigation of tubular damages, albuminuria and glomerulomegaly. In human PT cells, the increased expressions of hypoxia-inducible genes in hypoxic condition were attenuated by free fatty acids. Thus, aberrant hypoxic responses due to dysfunction of PHD2 caused both glomerular and tubular damages in HFD-induced obese mice. Phd2-inactivation provides a novel strategy against obesity-induced kidney injury.

Communication from Tubular Epithelial Cells to Podocytes through Sirt1 and Nicotinic Acid Metabolism.

We have recently published that tubular epithelial cells affect the podocyte epigenome though nicotinic acid metabolism in diabetic nephropathy (DN), and we have named this relationship "proximal tubule-podocyte communication". In this review, we describe this novel mechanism in the early stage of DN, focusing on the function of renal tubular Sirt1 and Sirt1-related nicotinic acid metabolism. Mainly, we discuss the following three findings. First, we described the details of proximal tubule-podocyte communication. Second, we explained how Sirt1 regulates albuminuria via epigenetic mechanisms. This means that repeated high glucose stress triggers the initial changes in proximal tubules, which lead to the epigenetically irreversible glomerular damages. However, proximal tubular Sirt1 overexpression can rescue these changes. Our previous data indicated that the decrease in Sirt1 expression in proximal tubules caused the reduction in glomerular Sirt1 and the subsequent increase in glomerular Claudin-1. It seemed plausible that some humoral mediator is released from proximal tubules, migrates to podocytes and glomeruli, and affects Sirt1 expression in podocytes. Third, we mentioned a mediator connecting this communication, nicotinamide mononucleotide (NMN). We suggest the potential of Sirt1 or NMN as not only a therapeutic target but also as a prognostic marker of very early stage DN.

The role of adipose tissue asymmetric dimethylarginine/dimethylarginine dimethylaminohydrolase pathway in adipose tissue phenotype and metabolic abnormalities in subtotally nephrectomized rats.

BACKGROUND: The lipodystrophy-like phenotype has been suggested in early chronic kidney disease (CKD). It includes adipose tissue atrophy, systemic insulin resistance (IR), dyslipidemia and ectopic lipid accumulation. To elucidate its pathogenesis, we investigated the role of two uremic toxins that affect insulin sensitivity: an endogenous nitric oxide synthase inhibitor, and asymmetric dimethylarginine (ADMA) and indoxyl sulfate (IS). METHODS: Six-week-old Sprague-Dawley rats were rendered CKD by subtotal nephrectomy (Nx) and compared with sham-operated rats. Cultured 3T3-L1 fibroblasts were differentiated into mature adipocytes with or without ADMA. Transgenic (Tg) mice overexpressing each isoform of ADMA degrading enzyme, dimethylarginine dimethylaminohydrolase 1 (DDAH1) and DDAH2 were subject to Nx and their phenotypes were investigated. RESULTS: In Nx rats, IR was evident and insulin stimulation failed to activate insulin signaling in adipose tissues. Adipose tissue weight, adipocyte size and adipocyte differentiation marker expressions decreased as a consequence of IR in Nx. Tissue lipid content in the liver and muscle increased in Nx rats. Tissue levels of ADMA, IS and oxidative stress increased in the adipose tissue of Nx rats. Both DDAH1 and DDAH2 expressions decreased, and a putative IS receptor, aryl hydrocarbon receptor, expression increased in the adipose tissue of Nx rats. ADMA inhibited adipocyte differentiation, triglyceride accumulation and insulin signaling, which were reversed by pretreatment with cGMP. In each type of Tg mice overexpressing DDAH1 or DDAH2, all lipodystrophy-like phenotypes induced by Nx were reversed. CONCLUSIONS: In mild CKD, dysregulation of the ADMA/DDAH pathway in adipose tissue triggers lipodystrophy-like phenotype changes, including ectopic fat depositions.

Insulin resistance in chronic kidney disease is ameliorated by spironolactone in rats and humans.

In this study, we examined the association between chronic kidney disease (CKD) and insulin resistance. In a patient cohort with nondiabetic stages 2-5 CKD, estimated glomerular filtration rate (eGFR) was negatively correlated and the plasma aldosterone concentration was independently associated with the homeostasis model assessment of insulin resistance. Treatment with the mineralocorticoid receptor blocker spironolactone ameliorated insulin resistance in patients, and impaired glucose tolerance was partially reversed in fifth/sixth nephrectomized rats. In these rats, insulin-induced signal transduction was attenuated, especially in the adipose tissue. In the adipose tissue of nephrectomized rats, nuclear mineralocorticoid receptor expression, expression of the mineralocorticoid receptor target molecule SGK-1, tissue aldosterone content, and expression of the aldosterone-producing enzyme CYP11B2 increased. Mineralocorticoid receptor activation in the adipose tissue was reversed by spironolactone. In the adipose tissue of nephrectomized rats, asymmetric dimethylarginine (ADMA; an uremic substance linking uremia and insulin resistance) increased, the expression of the ADMA-degrading enzymes DDAH1 and DDAH2 decreased, and the oxidative stress increased. All of these changes were reversed by spironolactone. In mature adipocytes, aldosterone downregulated both DDAH1 and DDAH2 expression, and ADMA inhibited the insulin-induced cellular signaling. Thus, activation of mineralocorticoid receptor and resultant ADMA accumulation in adipose tissue has, in part, a relevant role in the development of insulin resistance in CKD.

Ghrelin protects against renal damages induced by angiotensin-II via an antioxidative stress mechanism in mice.

We explored the renal protective effects by a gut peptide, Ghrelin. Daily peritoneal injection with Ghrelin ameliorated renal damages in continuously angiotensin II (AngII)-infused C57BL/6 mice as assessed by urinary excretion of protein and renal tubular markers. AngII-induced increase in reactive oxygen species (ROS) levels and senescent changes were attenuated by Ghrelin. Ghrelin also inhibited AngII-induced upregulations of transforming growth factor-ß (TGF-ß) and plasminogen activator inhibitor-1 (PAI-1), ameliorating renal fibrotic changes. These effects were accompanied by concomitant increase in mitochondria uncoupling protein, UCP2 as well as in a key regulator of mitochondria biosynthesis, PGC1α. In renal proximal cell line, HK-2 cells, Ghrelin reduced mitochondria membrane potential and mitochondria-derived ROS. The transfection of UCP2 siRNA abolished the decrease in mitochondria-derived ROS by Ghrelin. Ghrelin ameliorated AngII-induced renal tubular cell senescent changes and AngII-induced TGF-ß and PAI-1 expressions. Finally, Ghrelin receptor, growth hormone secretagogue receptor (GHSR)-null mice exhibited an increase in tubular damages, renal ROS levels, renal senescent changes and fibrosis complicated with renal dysfunction. GHSR-null mice harbored elongated mitochondria in the proximal tubules. In conclusion, Ghrelin suppressed AngII-induced renal damages through its UCP2 dependent anti-oxidative stress effect and mitochondria maintenance. Ghrelin/GHSR pathway played an important role in the maintenance of ROS levels in the kidney.

Serum creatinine and albumin decline predict the contraction of nosocomial aspiration pneumonia in patients undergoing hemodialysis.

Aspiration pneumonia (AP) is prevalent in older adults and the hemodialysis (HD) population has been getting older. Therefore, it is speculated that increasing number of HD patients would suffer from AP. However, the clinical aspects of AP in HD patients have not been elucidated. Consecutive HD patients with nosocomial AP hospitalized in our university hospital from April 2007 to December 2008 were recruited. Their clinical characteristics, risk factors for contraction, and the fatality of AP and treatment options were described. Nineteen out of 356 hospitalized HD patients had AP and 8 out of 19 AP patients died, indicating the incidence rate and fatality rate were 5.34% and 42.1%, respectively. Multiple regression analysis revealed that the risk factors for contracting AP included age, body mass index, serum creatinine levels (Cre) and the monthly decline rate of Cre. It also revealed that serum albumin (Alb) and basal total cholesterol levels, the decline rate of Alb and Cre levels, and the duration of AP were independent risk factors for fatality. Survivors were most often treated with tube feeding. Both contraction rate and fatality of nosocomial AP were high among HD patients. Both the malnutrition as well as the decline rate for nutrition and muscle volume indicated by falls in Alb and Cre, respectively, had clinical relevance in AP. Maintaining nutritional state by tube feeding and muscle volume seems to be the mainstay for the prevention and the treatment of AP in HD patients.

Renal tubular Sirt1 attenuates diabetic albuminuria by epigenetically suppressing Claudin-1 overexpression in podocytes.

Sirtuin 1 (Sirt1), a NAD(+)-regulated deacetylase with numerous known positive effects on cellular and whole-body metabolism, is expressed in the renal cortex and medulla. It is known to have protective effects against age-related disease, including diabetes. Here we investigated the protective role of Sirt1 in diabetic renal damage. We found that Sirt1 in proximal tubules (PTs) was downregulated before albuminuria occurred in streptozotocin-induced or obese (db/db) diabetic mice. PT-specific SIRT1 transgenic and Sirt1 knockout mice showed prevention and aggravation of the glomerular changes that occur in diabetes, respectively, and nondiabetic knockout mice exhibited albuminuria, suggesting that Sirt1 in PTs affects glomerular function. Downregulation of Sirt1 and upregulation of the tight junction protein Claudin-1 by SIRT1-mediated epigenetic regulation in podocytes contributed to albuminuria. We did not observe these phenomena in 5/6 nephrectomized mice. We also demonstrated retrograde interplay from PTs to glomeruli using nicotinamide mononucleotide (NMN) from conditioned medium, measurement of the autofluorescence of photoactivatable NMN and injection of fluorescence-labeled NMN. In human subjects with diabetes, the levels of SIRT1 and Claudin-1 were correlated with proteinuria levels. These results suggest that Sirt1 in PTs protects against albuminuria in diabetes by maintaining NMN concentrations around glomeruli, thus influencing podocyte function.

The hydrolase DDAH2 enhances pancreatic insulin secretion by transcriptional regulation of secretagogin through a Sirt1-dependent mechanism in mice.

The role of dimethylarginine dimethylaminohydrolase 2 (DDAH2) in glucose metabolism is unknown. Here, we generated DDAH2 transgenic (Tg) mice. These mice had lower plasma glucose levels (60 min: 298±32 vs. 418±35 mg/dl; 120 min: 205±15 vs. 284±20 mg/dl) and higher insulin levels (15 min: 2.1±0.2 vs. 1.5±0.1 ng/ml; 30 min: 1.8±0.1 vs. 1.5±0.1 ng/ml) during intraperitoneal glucose tolerance tests when fed a high-fat diet (HFD) compared with HFD-fed wild-type (WT) mice. Glucose-stimulated insulin secretion (GSIS) was increased in Tg islets by 33%. Pancreatic asymmetrical dimethylarginine, nitric oxide, and oxidative stress levels were not correlated with improvements in insulin secretion in Tg mice. Secretagogin, an insulin vesicle docking protein, was up-regulated by 2.7-fold in Tg mice and in pancreatic MIN-6 cells overexpressing DDAH2. GSIS in MIN-6 cells was dependent on DDAH2-induced secretagogin expression. Pancreatic Sirt1, DDAH2, and secretagogin were down-regulated in HFD-fed WT mice by 70, 75, and 85%, respectively. Overexpression of Sirt1 overexpression by 3.9-fold increased DDAH2 and secretagogin expression in MIN-6 cells by 3.2- and 2.5-fold, respectively. DDAH2 overexpression improved GSIS in pancreas-specific Sirt1-deficient mice. In summary, the Sirt1/DDAH2/secretagogin pathway is a novel regulator of GSIS.

Rho-kinase inhibition ameliorates peritoneal fibrosis and angiogenesis in a rat model of peritoneal sclerosis.

BACKGROUND: Peritoneal fibrosis (PF) and angiogenesis are typical morphological changes, leading to loss of peritoneal functions in patients undergoing peritoneal dialysis. The small G protein, Rho, and its downstream effector Rho-kinase have been shown to be involved in the tissue fibrosis process. This study was undertaken to investigate the role of Rho-kinase in the pathogenesis of these alterations. METHODS: PF was induced by intraperitoneal administration of chlorhexidine (CHX) in male rats (CHX group). These rats were treated with a Rho-kinase inhibitor, fasudil (Fas group). Human pleural mesothelial cells, MeT-5A cells, were stimulated by glucose with or without another Rho-kinase inhibitor, Y-27632. RESULTS: Peritoneal damage including peritoneal thickening, fibrous changes, macrophage migration and angiogenesis were evident in the CHX group and were ameliorated in the Fas group. The expression of markers of tissue fibrosis, such as transforming growth factor (TGF)-ß, fibronectin and α-smooth muscle cell actin, were increased in the CHX group and were downregulated by fasudil. Similar results were also seen with an inducer of angiogenesis, vascular endothelial growth factor (VEGF). Rho-kinase was activated in the peritoneum of the CHX group, which was inhibited by fasudil. In MeT-5A cells, high glucose increased TGF-ß expression and VEGF secretion, which were blocked by Y-27632. CONCLUSIONS: The activation of Rho-kinase is involved in peritoneal damage at multiple stages including tissue fibrosis and angiogenesis. The inhibition of Rho-kinase constitutes a novel strategy for the treatment of PF.

Rho and Rho-kinase activity in adipocytes contributes to a vicious cycle in obesity that may involve mechanical stretch.

The development of obesity involves multiple mechanisms. Here, we identify adipocyte signaling through the guanosine triphosphatase Rho and its effector Rho-kinase as one such mechanism. Mice fed a high-fat diet (HFD) showed increased Rho-kinase activity in adipose tissue compared to mice fed a low-fat diet. Treatment with the Rho-kinase inhibitor fasudil attenuated weight gain and insulin resistance in mice on a HFD. Transgenic mice overexpressing an adipocyte-specific, dominant-negative form of RhoA (DN-RhoA TG mice) showed decreased Rho-kinase activity in adipocytes, decreased HFD-induced weight gain, and improved glucose metabolism compared to wild-type littermates. Furthermore, compared to HFD-fed wild-type littermates, DN-RhoA TG mice on a HFD showed decreased adipocyte hypertrophy, reduced macrophage recruitment to adipose tissue, and lower expression of mRNAs encoding various adipocytokines. Lipid accumulation in cultured adipocytes was associated with increased Rho-kinase activity and increased abundance of adipocytokine transcripts, which was reversed by a Rho-kinase inhibitor. Direct application of mechanical stretch to mature adipocytes increased Rho-kinase activity and stress fiber formation. Stress fiber formation, which was also observed in adipocytes from HFD-fed mice, was prevented by Rho-kinase inhibition and in DN-RhoA TG mice. Our findings indicate that lipid accumulation in adipocytes activates Rho to Rho-kinase (Rho-Rho-kinase) signaling at least in part through mechanical stretch and implicate Rho-Rho-kinase signaling in inflammatory changes in adipose tissue in obesity. Thus, inhibition of Rho-Rho-kinase signaling may provide a therapeutic strategy for disrupting a vicious cycle of adipocyte stretch, Rho-Rho-kinase signaling, and inflammation of adipose tissue that contributes to and aggravates obesity.