Naoki Washida

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.

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.

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)-β, 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-β 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.

Role of mineralocorticoid receptor/Rho/Rho-kinase pathway in obesity-related renal injury

OBJECTIVE:We examined whether aldosterone/Rho/Rho-kinase pathway contributed to obesity-associated nephropathy.SUBJECTS:C57BL/6J mice were fed a high fat or low fat diet, and mice on a high fat diet were treated with a mineralocorticoid receptor antagonist, eplerenone.RESULTS:The mice on a high fat diet not only developed obesity, but also manifested renal histological changes, including glomerular hypercellularity and increased mesangial matrix, which paralleled the increase in albuminuria. Furthermore, enhanced Rho-kinase activity was noted in kidneys from high fat diet-fed mice, as well as increased expressions of inflammatory chemokines. All of these changes were attenuated by eplerenone. In high fat diet-fed mice, mineralocorticoid receptor protein levels in the nuclear fraction and SGK1, an effector of aldosterone, were upregulated in kidneys, although serum aldosterone levels were unaltered. Furthermore, aldosterone and 3β-hydroxysteroid dehydrogenase in renal tissues were upregulated in high fat diet-fed mice. Finally, in cultured mesangial cells, stimulation with aldosterone enhanced Rho-kinase activity, and pre-incubation with eplerenone prevented the aldosterone-induced activation of Rho kinase.CONCLUSION:Excess fat intake causes obesity and renal injury in C57BL/6J mice, and these changes are mediated by an enhanced mineralocorticoid receptor/Rho/Rho-kinase pathway and inflammatory process. Mineralocorticoid receptor activation in the kidney tissue and the subsequent Rho-kinase stimulation are likely to participate in the development of obesity-associated nephropathy without elevation in serum aldosterone levels.

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.

Impact of Switching From Darbepoetin Alfa to Epoetin Beta Pegol on Iron Utilization and Blood Pressure in Peritoneal Dialysis Patients.

New erythropoiesis‐stimulating agents with a longer half‐life have been developed for the treatment of anemia in patients with end‐stage renal disease. This study evaluated the efficacy of darbepoetin alfa (DA) and long‐acting epoetin beta pegol (continuous erythropoietin receptor activator, CERA) in patients on peritoneal dialysis (PD). Twenty‐nine patients who had undergone PD for at least 6 months and were iron replacement‐naïve and negative for inflammatory parameters were enrolled. Hemoglobin (Hgb) levels and blood pressure were evaluated before and after switching from DA to CERA. Percent transferrin saturation (TSAT), serum ferritin levels and blood pressure were also assessed. Twenty‐eight patients were subject to the analysis, excluding one patient with a decrease in Hgb by ≥10%. Switching from DA to CERA did not alter Hgb levels. The doses of DA and CERA after 12 month treatment of each agent were 118.48 ± 79.63 and 89.88 ± 47.50 μg/4 weeks, respectively (conversion ratio, 1:0.76). The CERA dose administered during the final 6 months was abated, compared with that given during the initial 6 months (P = 0.035). The frequency of CERA injection over a 12‐month period was less than that of DA (10.0 ± 3.0 vs. 16.4 ± 5.0, P < 0.01). The conversion from DA to CERA did not alter TSAT, but decreased serum ferritin levels (from 202.69 ± 132.57 to 150.15 ± 110.07 ng/mL, P = 0.012) and systolic blood pressure (from 133.8 ± 17.3 to 129.5 ± 11.3 mm Hg, P = 0.024). In PD patients, lower doses and less frequent injection of CERA are sufficient to maintain Hgb at levels similar to those achieved by DA therapy, with improved iron utilization and reduced blood pressure.

Policy for developing clinical practice guidelines of Japanese Society for Dialysis Therapy

BackgroundThe Scientific Academy Committee of Japanese Society for Dialysis Therapy (JSDT) has developed 14 clinical practice guidelines (CPGs) and their revised editions independently or in cooperation with other organizations and translated many of them into English to make them available to the world. These guidelines were presented in a user-friendly textbook-like format and were useful for many healthcare professionals engaged in dialysis therapy. However, because the definition and the process for developing CPGs have recently become more rigorous worldwide, the conventional process of development and the format of JSDT no longer meet the requirements for CPGs.MethodSince 2012, JSDT, with its Guideline Developing Working Group (formerly Guideline Subcommittee), had evaluated several CPGs developing systems. The working group evaluated the advantages and disadvantages of several CPGs developing systems. The most important point of them which JSDT would adopt for their own system was to be based on evidence and be able to receive international recognition.ResultsIn Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) system, the quality of evidence was evaluated by systematic review on each clinical outcome by systematic review panel. After the report from the systematic review panel, the CPG panel grades the strength of recommendation on each CPG statement. GRADE system was the most frequently used for developing CPGs by many human healthcare societies in the world. If JSDT adopt GRADE system, it is necessary for us to learn how to assess the quality of evidence through a systematic review, to develop a system for determining the strength of recommendation on the basis of a systematic review, and to decide how to manage any conflicts of interest.ConclusionsJSDT should provide the useful information from Japanese experiences on chronic dialysis to the world. In order to achieve this aim, we concluded that the most reasonable approach is to utilize the data from JSDT Renal Data Registry in a more creative manner, publish such data for the world as evidence, and develop CPGs in accordance with a globally recognized methodology, GRADE.NotificationThe current manuscript is the position statement of JSDT for developing future CPGs and the second publication of “Policy for Developing Clinical Practice Guidelines (CPGs) of the Japanese Society for Dialysis Therapy” on J Jpn Soc Dial Ther 2016; 49: 453-62 (in Japanese), with permission.

Non-tuberculous mycobacterial infections related to peritoneal dialysis

Most infections related to peritoneal dialysis (PD) are caused by common bacteria, and non-tuberculous mycobacteria are rare. The clinical characteristics and prognosis of PD patients with non-tuberculous mycobacterial infections were investigated at our hospital. Non-tuberculous mycobacteria were detected in 11 patients (exit-site infection, tunnel infection, and peritonitis in 3, 5, and 3 patients, respectively). Mycobacterium fortuitum, Mycobacterium chelonae, and Mycobacterium abscessus were identified in 4, 2, and 2 patients, respectively. Most patients with peritonitis or tunnel infection required catheter removal. During the study period (2007 – 2017), peritonitis occurred in 44 patients, including 3 patients (6.8%) with non-tuberculous mycobacterial peritonitis. When non-tuberculous mycobacterial infection occurs, multi-agent antibiotic therapy, unroofing surgery, and/or catheter replacement should be performed to prevent peritonitis.