Kyoko Yoshioka

Sirt1 protects against oxidative stress-induced renal tubular cell apoptosis by the bidirectional regulation of catalase expression

NAD(+)-dependent protein deacetylase Sirt1 regulates cellular apoptosis. We examined the role of Sirt1 in renal tubular cell apoptosis by using HK-2 cells, proximal tubular cell lines with or without reactive oxygen species (ROS), H(2)O(2). Without any ROS, Sirt1 inhibitors enhanced apoptosis and the expression of ROS scavenger, catalase, and Sirt1 overexpression downregulated catalase. When apoptosis was induced with H(2)O(2), Sirt1 was upregulated with the concomitant increase in catalase expression. Sirt1 overexpression rescued H(2)O(2)-induced apoptosis through the upregulation of catalase. H(2)O(2) induced the nuclear accumulation of forkhead transcription factor, FoxO3a and the gene silencing of FoxO3a enhanced H(2)O(2)-induced apoptosis. In conclusion, endogenous Sirt1 maintains cell survival by regulating catalase expression and by preventing the depletion of ROS required for cell survival. In contrast, excess ROS upregulates Sirt1, which activates FoxO3a and catalase leading to rescuing apoptosis. Thus, Sirt1 constitutes a determinant of renal tubular cell apoptosis by regulating cellular ROS levels.

Kidney-specific overexpression of Sirt1 protects against acute kidney injury by retaining peroxisome function

Sirt1, a NAD-dependent protein deacetylase, is reported to regulate intracellular metabolism and attenuate reactive oxidative species (ROS)-induced apoptosis leading to longevity and acute stress resistance. We created transgenic (TG) mice with kidney-specific overexpression of Sirt1 using the promoter sodium-phosphate cotransporter IIa (Npt2) driven specifically in proximal tubules and investigated the kidney-specific role of Sirt1 in the protection against acute kidney injury (AKI). We also elucidated the role of number or function of peroxisome and mitochondria in mediating the mechanisms for renal protective effects of Sirt1 in AKI. Cisplatin-induced AKI decreased the number and function of peroxisomes as well as mitochondria and led to increased local levels of ROS production and renal tubular apoptotic cells. TG mice treated with cisplatin mitigated AKI, local ROS, and renal tubular apoptotic tubular cells. Consistent with these results, TG mice treated with cisplatin also exhibited recovery of peroxisome number and function, as well as rescued mitochondrial function; however, mitochondrial number was not recovered. Immunoelectron microscopic findings consistently demonstrated that the decrease in peroxisome number by cisplatin in wild type mice was restored in transgenic mice. In HK-2 cells, a cultured proximal tubule cell line, overexpression of Sirt1 rescued the cisplatin-induced cell apoptosis through the restoration of peroxisome number, although the mitochondria number was not restored. These results indicate that Sirt1 overexpression in proximal tubules rescues cisplatin-induced AKI by maintaining peroxisomes number and function, concomitant up-regulation of catalase, and elimination of renal ROS levels. Renal Sirt1 can be a potential therapeutic target for the treatment of AKI.

Role of Asymmetric Dimethylarginine in Vascular Injury in Transgenic Mice Overexpressing Dimethylarginie Dimethylaminohydrolase 2

Dimethylarginie dimethylaminohydrolase (DDAH) degrades asymmetric dimethylarginine (ADMA), an endogenous nitric oxide (NO) synthase inhibitor, and comprises 2 isoforms, DDAH1 and DDAH2. To investigate the in vivo role of DDAH2, we generated trangenic mice overexpressing DDAH2. The transgenic mice manifested reductions in plasma ADMA and elevations in cardiac NO levels but no changes in systemic blood pressure (SBP), compared with the wild-type mice. When infused into wild-type mice for 4 weeks, ADMA elevated SBP and caused marked medial thickening and perivascular fibrosis in coronary microvessels, which were accompanied by ACE protein upregulation and cardiac oxidative stress. The treatment with amlodipine reduced SBP but failed to ameliorate the ADMA-induced histological changes. In contrast, these changes were abolished in transgenic mice, with a reduction in plasma ADMA. In coronary artery endothelial cells, ADMA activated p38 MAP kinase and the ADMA-induced ACE upregulation was suppressed by p38 MAP kinase inhibition by SB203580. In wild-type mice, long-term treatment with angiotensin II increased plasma ADMA and cardiac oxidative stress and caused similar vascular injury. In transgenic mice, these changes were attenuated. The present study suggests that DDAH2/ADMA regulates cardiac NO levels but has modest effect on SBP in normal conditions. Under the circumstances where plasma ADMA are elevated, including angiotensin II–activated conditions, ADMA serves to contribute to the development of vascular injury and increased cardiac oxidative stress, and the overexpression of DDAH2 attenuates these abnormalities. Collectively, the DDAH2/ADMA pathway can be a novel therapeutic target for vasculopathy in the ADMA or angiotensin II–induced pathophysiological conditions.

Pioglitazone Lowers Systemic Asymmetric Dimethylarginine by Inducing Dimethylarginine Dimethylaminohydrolase in Rats

Peroxisome proliferator activated receptor-γ (PPARγ) ligands increase nitric oxide (NO) production and reduce systemic blood pressure. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor degraded by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which has two isoforms, DDAH-I and -II. In order to elucidate the mechanism whereby PPARγ ligands affect NO metabolism, their effects on the DDAH-ADMA pathway were investigated. Six-week-old male Wister-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were maintained with or without pioglitazone (PIO), a PPARγ ligand. After 4 weeks, serum ADMA levels and urinary daily NO excretion were analyzed. Tissue DDAH expression was examined by real-time polymerase chain reaction (PCR), immunoblotting, and immunohistochemistry. The results showed that PIO decreased serum ADMA and increased urinary NO excretion in both WKY and SHR. Also in both strains, the expression level of DDAH-II in the kidney was increased at transcriptional levels, although the DDAH-I level was unaffected. PIO lowered blood pressure in SHR, but not in WKY. We also demonstrated that PIO induced DDAH-II protein expression in Marbin-Dubin Canine Kidney (MDCK) cells, a renal tubular cell line. In conclusion, a PPARγ ligand was here found to increase NO production partly by upregulating tissue DDAH-II expression and decreasing systemic ADMA levels. This mechanism constitutes a direct action on renal tubular cells, but is less likely to be responsible for the blood pressure-lowering effects of PPARγ ligands. Since ADMA is one of the risk factors for cardiovascular events, this study provides compelling evidence that PPARγ ligands have the potential for reducing cardiovascular risks.

Peroxisome Proliferator-Activated Receptor γ Ligands Inhibit Rho/Rho Kinase Pathway by Inducing Protein Tyrosine Phosphatase SHP-2

Although peroxisome proliferator-activated receptor γ (PPARγ) ligands have an antihypertensive effect in vivo, the precise mechanism has not been fully elucidated. We examined their effects on Rho/Rho kinase pathway, a key regulator of vascular tone. In cultured rat aortic smooth muscle cells (RASMC), Rho kinase stimulated by angiotensin II was suppressed by the pretreatment with pioglitazone and troglitazone, and these effects were explained by the inhibition of the Rho translocation to the cell membrane. We evaluated the role of Vav, a GTP/GDP exchange factor upregulating Rho kinase activity, and Src homology region 2–containing protein tyrosine phosphatase-2 (SHP-2), a protein tyrosine phosphatase that dephosphorylated Vav and subsequently inactivated Rho kinase. Both pioglitazone and troglitazone upregulated SHP-2, particularly in the cytosolic fraction, and the SHP-2-bound Vav, and reduced the phosphorylation of Vav. Furthermore, 4-week treatment with pioglitazone lowered systolic blood pressure in spontaneously hypertensive rats (SHR) and suppressed the Rho/Rho kinase activity in aortic tissues isolated from SHR. Consistently, the expression of SHP-2 was upregulated in vascular tissues from pioglitazone-treated SHR. The phosphorylated Vav was increased in SHR, compared with that in normotensive Wistar–Kyoto rats (WKY), which was mitigated by pioglitazone. Finally, both basal and angiotensin II–stimulated levels of Rho kinase activity were greater in RASMC from SHR than those from WKY, and the enhanced Rho kinase activity was blocked by pioglitazone or troglitazone in both strains. Collectively, PPARγ ligands inhibit the Rho/Rho kinase pathway through upregulation of cytosolic SHP-2 expression and inactivation of Vav, and may contribute to the hemodynamic, in addition to metabolic, action in hypertensive metabolic syndrome. The full text of this article is available online at http://circres.ahajournals.org.

Rho-kinase as a molecular target for insulin resistance and hypertension

Rho‐kinase plays an important role in hypertension and is reported to interfere with insulin signaling through serine phosphorylation of insulin receptor substrate‐1 (IRS‐1) in cultured vascular smooth muscle cells. We therefore examined the role of Rho‐kinase in the development of insulin resistance in Zucker obese rats. In skeletal muscles and aortic tissues of Zucker obese rats, activation of RhoA/Rho‐kinase was observed. Long‐term Rho‐kinase inhibition by 4 wk treatment with fasudil (a Rho‐kinase inhibitor) not only reduced blood pressure but corrected glucose and lipid metabolism, with improvement in serine phosphorylation of IRS‐1 and insulin signaling in skeletal muscles. Direct visualization of skeletal muscle arterioles with an intravital CCD videomicroscope demonstrated that both acetylcholine‐ and sodium nitroprusside‐induced vasodilations were blunted, which were restored by the fasudil treatment. Furthermore, both fasudil and Y‐27632 prevented the serine phosphorylation of IRS‐1 induced by insulin and/or tumor necrosis factor‐α in skeletal muscle cells. Collectively, Rho‐kinase is responsible for the impairment of insulin signaling and may constitute a critical mediator linking between metabolic and hemodynamic abnormalities in insulin resistance.

Dimethylarginine Dimethylaminohydrolase 2 Increases Vascular Endothelial Growth Factor Expression Through Sp1 Transcription Factor in Endothelial Cells

Objectives—Dimethylarginie dimethylaminohydrolase (DDAH) is a degrading enzyme for asymmetrical dimethylarginine, an endogenous NO synthase inhibitor. The molecular mechanism for DDAH-induced vascular endothelial growth factor (VEGF) expression was examined. Methods and Results—Although the transfection of expression vectors for 2 isoforms of DDAH, DDAH1, or DDAH2 increased DDAH activity in bovine aortic endothelial cells and human umbilical vein endothelial cells, expression and secretion of VEGF were increased only in DDAH2-transfected cells. Knocking down the DDAH2 gene reduced VEGF production, and DDAH2 overexpression enhanced both proliferation and migration of endothelial cells. The VEGF promoter activity was increased by DDAH2 transfection, which was not blocked by an NO synthase (NOS) inhibitor but required the Sp1 sites. DDAH2 overexpression increased nuclear protein levels bound to Sp1 oligonucleotides in endothelial cells. Sp1 small interfering RNA blocked DDAH2-induced upregulation of VEGF. DDAH2 transfection increased nuclear and threonine-phosphorylation levels of Sp1 in a protein kinase A (PKA)–dependent manner. Protein–protein interaction between DDAH2 and PKA was enhanced in DDAH2-transfected cells. Conclusions—DDAH2 upregulated the expression of VEGF through Sp1-dependent and NO/NOS system-independent promoter activation. DDAH2-increased Sp1 DNA binding activity was PKA dependent. These mechanisms may provide a novel therapeutic strategy for VEGF-related vasculopathies such as atherosclerosis.

Rho and Rho-Kinase Activity in Adipocytes Contributes to a Vicious Cycle in Obesity That May Involve Mechanical Stretch

Mechanical stretch activates Rho-kinase in adipocytes, promoting obesity and obesity-related complications. Stopping the Obesity Cycle Obesity is associated with an increase in lipid storage in adipocytes and a consequent increase in adipocyte size. Changes in cell size affect the cytoskeleton, which is regulated by molecules such as the guanosine triphosphatase Rho and its effector Rho-kinase. Noting that mechanical stretch can lead to activation of the Rho to Rho-kinase (Rho–Rho-kinase) signaling pathway, Hara et al. investigated the role of Rho–Rho-kinase signaling in obesity and its complications. They found that adipocyte Rho-kinase signaling was increased in obese mice fed a high-fat diet. Rho-kinase activity in adipocytes increased with increasing cell size and was also activated by mechanical stretch. Inhibition of Rho-kinase signaling—either systemically or specifically in adipocytes—inhibited the development of an inflammatory obesity-related phenotype in adipose tissue. Moreover, it decreased weight gain in mice fed a high-fat diet and attenuated such pathophysiological complications of obesity as insulin resistance and glucose intolerance. The authors thus propose that adipocyte stretch may contribute to obesity and its complications through activation of Rho–Rho-kinase signaling and that inhibition of this signaling pathway may provide a mechanism for disrupting this cycle. 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.

Role of Nitric Oxide–Producing and –Degrading Pathways in Coronary Endothelial Dysfunction in Chronic Kidney Disease

Cardiovascular events are accelerated in chronic kidney disease (CKD). Although deranged nitric oxide (NO) pathways and asymmetric dimethylarginine (ADMA) cause endothelial dysfunction, no direct evidence for coronary artery endothelial dysfunction in CKD has been documented. CKD was induced in male dogs by heminephrectomy (1/2Nx) or five-sixths nephrectomy (5/6Nx). After 4 wk, renal ablation reduced GFR (control 76 [54 to 85]; 1/2Nx 38 [29 to 47]; 5/6Nx 15 [12 to 46] ml/min) and elevated plasma ADMA (control 1.88 [1.68 to 2.54]; 1/2Nx 2.51 [2.11 to 3.55]; 5/6Nx 3.84 [2.16 to 3.95] micromol/L). Coronary circulatory responses to acetylcholine revealed marked increases in coronary blood flow in control group (83 +/- 17% increment) but blunted responses in 1/2Nx (34 +/- 8% increment) and 5/6Nx (20 +/- 4% increment). The acetylcholine-induced changes in epicardial arteriolar diameter, using needle-lens probe charge-coupled device videomicroscopy, showed similar results. The responsiveness to sodium nitroprusside did not differ among three groups. Plasma nitrite/nitrate levels decreased in 1/2Nx and 5/6Nx, and the mRNA expressions of dimethylarginine dimethylaminohydrolase-II (DDAH-II), ADMA-degrading enzyme, and endothelial NO synthase (eNOS) in coronary arteries were downregulated in 1/2Nx and 5/6Nx. Finally, 4-wk treatment with all-trans retinoic acid restored the impaired endothelium-dependent vasodilation and reversed the expression of eNOS but not DDAH-II. Coronary endothelial function is impaired in the early stage of CKD. The dysfunction is attributed to the downregulation of eNOS and/or DDAH-II in coronary arteries. Furthermore, the manipulation of NO pathways may constitute a therapeutic strategy for the prevention of coronary dysfunction in CKD.

Role of Rho-Kinase and p27 in Angiotensin II-Induced Vascular Injury

Angiotensin II enhances the development of atherosclerotic lesion in which cellular proliferation and/or migration are critical steps. Although cyclin-dependent kinase inhibitor, p27, and Rho/Rho-kinase pathway have recently been implicated as factors regulating these events cooperatively, their role in vivo has not been fully elucidated. We evaluated the contribution of p27 and Rho-kinase to angiotensin II-induced vascular injury using p27-deficient mice. Two-week angiotensin II (1500 ng/kg per minute SC) infusion elicited similar degrees of elevation in systolic blood pressure in wild-type mice (159±5 mm Hg) and p27-deficient mice (157±5 mm Hg; P>0.05). Angiotensin II infusion to wild-type mice resulted in increases in the medial thickness of aorta, proliferating cell number, and monocyte/macrophage infiltration within the vasculature. In p27-deficient mice, however, these changes were more prominent than those in wild-type mice. Treatment of wild-type mice with fasudil, a selective Rho-kinase inhibitor, did not alter blood pressure but significantly upregulated p27 expression, decreased medial thickness of aorta, reduced proliferating cell number, and prevented monocyte/macrophage infiltration. These protective effects of fasudil were attenuated in p27-deficient mice. In conclusion, p27 constitutes an important modulator of angiotensin II–induced monocyte/macrophage infiltration and vascular remodeling, which is mediated in part by Rho-kinase stimulation. Inhibition of Rho-kinase activity improves angiotensin II–induced vascular injury through p27-dependent and p27-independent mechanisms.