Motoaki Komatsu

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.—Hasegawa, K, Wakino, S., Kimoto, M, Minakuchi, H., Fujimura, K., Hosoya, K., Komatsu, M., Kaneko, Y., Kanda, T., Tokuyama, H., Hayashi, K., Itoh, H. The hydrolase DDAH2 enhances pancreatic insulin secretion by transcriptional regulation of secretagogin through a Sirt1‐dependent mechanism in mice. FASEB J. 27, 2301–2315 (2013). www.fasebj.org

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-β (TGF-β) 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-β 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.

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.

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.

LB02.03: EVALUATION OF DAY-BY-DAY BLOOD PRESSURE VARIABILITY IN CLINIC (DO WE STILL NEED STANDARD DEVIATION?).

Objective: Blood pressure (BP) variability correlates with cardio-vascular disease as BP level itself. There is not known easy way to evaluate the BP variability in clinic. To evaluate the usefulness of maximum-minimum difference (MMD) of BP in a month compared to standard deviation (SD), as an index of BP variability. Design and method: Study-1: Twelve patients (age 65.9 ± 12.1 y/o) were enrolled. Measurements of home systolic (S) BP were required in the morning. The 12 months consecutive data and at least 3 times measurements a month were required for including. (Mean 29.0 ± 4.5 times/month in the morning). We checked the correlation between MMD and SD. Study-2: Six hemodialized patients monitored with i-TECHO system (J of Hypertens 2007: 25: 2353–2358) for longer than one year were analyzed. As in study-1, we analyzed the correlation between SD and MMD of SBP. Measurements: 17.4 ± 11.9 times per month. Study-3: The data from our previous study (FUJIYAM study Clin. Exp Hypertens 2014: 36:508–16) were extracted. 1524 patient-month morning BP data were calculated as in study-1. Picking up data measuring more than 24 times a month, 517 patient-month BP data were analyzed. We compared the ratio to 25 times measured data of SD and MMD, in the setting 5, 10, 15, 20 times measured data. Results: Study-1: SBP, MMD was correlated very well to SD (p < 0.0001, R=0.923). Equation of SBPSD = 1.275+ 0.208xMMD. Study-2: R = 0.884 (P < 0.0001) SBPSD = 2.17+ 0.22xMMD. Study-3: R = 0.842(P < 0.0001), if we used all data measurements > 2 times. If data were extracted (measurements>24 times), correlation was 0.927 (P < 0.0001). The equation of SBPSD = 1.520+ 0.201xMMD. The ratios of SD to 25 times were as follows; 0.956 in 5 times, 0.956 in 10, 0.979 in 15, 0.991 in 20 times. The ratios of MMD to 25 times were as follows; 0.558 in 5, 0.761 in 10, 0.874 in 15, 0.944 in 20. Conclusions: We can assume SD easily by measuring MMD as an index of day-by-day BP variability of a month. The equation formulas were very similar though the patients’ groups were different. But we have to be careful how many times patients measure in a month.

NNMT activation can contribute to the development of fatty liver disease by modulating the NAD + metabolism

Nicotinamide N-methyltransferase (NNMT) catalyses the reaction between nicotinamide (NAM) and S-adenosylmethionine to produce 1-methylnicotinamide and S-adenosylhomocysteine. Recently, this enzyme has also been reported to modulate hepatic nutrient metabolism, but its role in the liver has not been fully elucidated. We developed transgenic mice overexpressing NNMT to elucidate its role in hepatic nutrient metabolism. When fed a high fat diet containing NAM, a precursor for nicotinamide adenine dinucleotide (NAD)+, these NNMT-overexpressing mice exhibit fatty liver deterioration following increased expression of the genes mediating fatty acid uptake and decreased very low-density lipoprotein secretion. NNMT overactivation decreased the NAD+ content in the liver and also decreased gene activity related to fatty acid oxidation by inhibiting NAD+–dependent deacetylase Sirt3 function. Moreover, the transgenic mice showed liver fibrosis, with the induction of inflammatory and fibrosis genes. Induced NNMT expression decreased the tissue methylation capacity, thereby reducing methylation of the connective tissue growth factor (CTGF) gene promoter, resulting in increased CTGF expression. These data indicate that NNMT links the NAD+ and methionine metabolic pathways and promotes liver steatosis and fibrosis. Therefore, targeting NNMT may serve as a therapeutic strategy for treating fatty liver and fibrosis.

PS 11-09 EVALUATION OF DAY-BY-DAY BLOOD PRESSURE VARIABILITY IN CLINIC

Objective: To evaluate the usefulness of maximum-minimum difference (MMD) of BP in a month compared to standard deviation (SD), as an index of BP variability. Design and Method: Study-1: Twelve patients (age 65.9 ± 12.1 y/o) were enrolled. Measurements of home systolic (S) BP were required in the morning. The 12 months consecutive data and at least 3 times measurements a month were required for including. (Mean 29.0 ± 4.5 times/month in the morning). We checked the correlation between MMD and SD. Study-2: Six hemodialized patients monitored with i-TECHO system (J of Hypertens 2007: 25: 2353–2358) for longer than one year were analyzed. As in study-1, we analyzed the correlation between SD and MMD of SBP. Measurements: 17.4 ± 11.9 times per month. Study-3: The data from our previous study (FUJIYAM study Clin. Exp Hypertens 2014: 36:508–16) were extracted. 1524 patient-month morning BP data were calculated as in study-1. Picking up data measuring more than 24 times a month, 517 patient-month BP data were analyzed. We compared the ratio to 25 times measured data of SD and MMD, in the setting 5, 10, 15, 20 times measured data. Results: Study-1: In SBP, DBP, pulse, MMD were correlated very well to SD (p < 0.0001, R = 0.923 in SBP). Equation of SBPSD = 1.275 + 0.208xMMD. Study-2: R = 0.884 (P < 0.0001) SBPSD = 2.17 + 0.22xMMD. Study-3: If data were extracted (measurements >24 times), correlation was 0.927 (P < 0.0001). The equation of SBPSD = 1.520 + 0.201xMMD. As in study 1, the correlations between MMD and SD were very strong either in SBP, BP, pulse. The ratios of SD to 25 times were as follows; 0.956 in 5 times, 0.956 in 10, 0.979 in 15, 0.991 in 20 times. The ratios of MMD to 25 times were as follows; 0.558 in 5, 0.761 in 10, 0.874 in 15, 0.944 in 20. Conclusions: SD is easily assumed by MMD as an index of day-by-day BP variability of a month.

ISH NIA PS 01-01 Ghrelin receptor in the endothelial cells regulates blood pressure and metabolic responses in mice

Objective: Ghrelin, a gastric derived peptide, reduces blood pressure and increases adiposity. We and others have reported that growth hormone secretagogue-receptor (GHSR) deficiency leads to high blood pressure and less adiposity compared to wild-type mice. Endothelial cells play roles in the adiposity by lipid uptake in white adipose tissue as well as in blood pressure. In this study, we examined the role of endogenous Ghrelin/GHSR in the endothelium in blood pressure and metabolic responses on high fat feeding. Design and Method: We generated transgenic mice expressing GHSR selectively in the endothelial cells (GHSR-EC mice) in the background of GHSR null mice by using Cre-LoxP system, and compared these mice with GHSR null mice and wild-type mice. Blood pressure was measured by tail cuff method. Serum free fatty acid (FFA) and triglyceride (TG) concentrations after olive oil gavage and the effect of high fat diet on weight gain were examined. Results: GHSR-null mice showed increase in blood pressure compared to wild-type mice (142.1 ± 3.6 mmHg vs 126.7 ± 2.6 mmHg, p < 0.05). In GHSR-EC mice, blood pressure levels were 132.9 ± 2.6 mmHg, and there was not significant difference between GHSR-EC mice and wild-type mice. In GHSR-null mice, serum FFA and TG levels after olive oil gavage were significantly increased compared with wild-type mice. The weight of white adipose tissue was significantly decreased in GHSR-null mice treated with high fat diet, suggesting that lipid uptake is reduced in GHSR-null mice. Re-expressing of GHSR in endothelial cells in GHSR null mice restored lipid uptake in WAT and decreased serum lipid levels. Conclusions: These data suggest that Ghrelin/GHSR pathway in the endothelium play an important role of endothelial function and regulate blood pressure and metabolic responses.