Atsunori Kuwabara

Renal denervation reduces glomerular injury by suppressing NAD(P)H oxidase activity in Dahl salt-sensitive rats

BACKGROUND Renal sympathetic nerve activity has important effects on renal function in chronic kidney disease. Recent studies indicated that beta agonists directly stimulate NAD(P)H oxidase in endothelial cells. Therefore, we investigated whether renal denervation protects renal function through an anti-oxidative effect. METHODS The right kidney was removed from Dahl salt-sensitive hypertensive rats. Two weeks later, the rats underwent either left renal denervation (Nx-RDNx; n = 10) or a sham operation (Nx-Sham; n = 10). After a further 6 weeks, kidney function and renal tissue were assessed. In this ex vivo study, using isolated glomeruli from Sprague-Dawley rats, the direct effects of catecholamine on NAD(P)H oxidase activity were assessed. RESULTS After the Nx-RDNx or Nx-Sham surgery, urinary albumin excretion and the histologic glomerular sclerosis index were lower in the Nx-RDNx group than in the Nx-Sham group. Fluorescence staining for reactive oxygen species in isolated glomeruli was significantly weaker in the Nx-RDNx group. A lucigenin assay of NAD(P)H oxidase activity in isolated glomeruli indicated that renal denervation may have caused the reduction in reactive oxygen species through suppression of the activity of NAD(P)H oxidase. The levels of mRNA for NAD(P)H oxidase components and the levels of rac1 were higher in glomeruli from the Nx-Sham group than from the Nx-RDNx group. In this ex vivo study, although the NAD(P)H oxidase activity did not change with administration of either the alpha- or beta2-agonist, it increased with the beta1-agonist. CONCLUSIONS Renal sympathetic denervation helps to protect against glomerular sclerosis, possibly by suppressing NAD(P)H oxidase activity, thereby decreasing glomerular reactive oxygen species.

Oral adsorbent AST-120 ameliorates endothelial dysfunction independent of renal function in rats with subtotal nephrectomy.

It is important to consider a strategy to halt the development of cardiovascular disease (CVD) in patients with chronic kidney disease (CKD). Oral adsorbent AST-120 retards deterioration in renal function, reducing indoxyl sulfate (IS) accumulation. The aim of this study was to determine whether AST-120 improves endothelial dysfunction by reducing oxidative/nitrative stress in a rat-CKD model. Subtotally nephrectomized (Nx) rats aged 17 weeks were divided into two groups: control rats and rats orally treated with AST-120. Two weeks after initiation of AST-120, serum and urinary IS levels, renal histological scores and endothelium-dependent vascular responses (EDVRs) in the aorta were investigated. EDVR in 5-h incubation with 250 μg ml−1 IS was also examined in normal rat aortas. Nitrotyrosine content, mRNA expression of p47phox, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase component, and expression and phosphorylation (serine-1177) of endothelial nitric oxide synthase (eNOS) in the aorta were examined in untreated and treated Nx rats. At the end of treatment, renal function and histological scores were not different in the two groups. AST-120 prevented the elevation of serum IS level in Nx rats, reducing urinary IS excretion, and ameliorated decreased EDVR in Nx rats. Incubation with IS tended to reduce EDVR in normal aortas, albeit insignificantly. AST-120 also suppressed nitrotyrosine accumulation and inhibited p47phox expression in Nx rats. The eNOS expression and phosphorylation were similar in the two groups. In conclusion, AST-120 ameliorated endothelial dysfunction and alleviated oxidative/nitrative stress in the aorta through reduced accumulation of IS, independent of renal function, in a rat CKD model.

Tacrolimus induces glomerular injury via endothelial dysfunction caused by reactive oxygen species and inflammatory change.

Background/Aims: The immunosuppressive drug tacrolimus (FK506) is used clinically to reduce the rejection rate in patients with kidney transplantation; however, the resultant nephrotoxicity remains a serious problem. In the present study we attempted to elucidate the mechanisms of glomerular injury induced by FK506 and the renoprotective effects of the angiotensin II receptor blocker telmisartan. Methods: Seven-week-old male Wistar rats were divided into three groups: vehicle group, FK506 group, and FK506 + telmisartan group. After 8 weeks, we assessed kidney function and renal morphological changes including oxidative stress. We also assessed the effect of FK506 in human glomerular endothelial cells (hGECs) with regard to reactive oxygen species (ROS). Results: FK506 induced ROS production via activation of NAD(P)H oxidase in the glomeruli. Expression of ICAM mRNA was increased in glomeruli from the FK506 group. These effects resulted in macrophage infiltration into the glomeruli. FK506 directly promoted NAD(P)H oxidase activity and accelerated production of ROS in hGECs. Conversely, cotreatment with telmisartan inhibited both NAD(P)H oxidase activity and production of ROS. Conclusion: These findings suggest that glomerular injury resulting from FK506 is caused by oxidative stress mediated by activation of NAD(P)H oxidase and that telmisartan exerts a renoprotective effect via antioxidative activity.

In Vivo Visualization of Glomerular Microcirculation and Hyperfiltration in Streptozotocin‐Induced Diabetic Rats

Microcirculation (2010) 17, 1–10. doi: 10.1111/j.1549‐8719.2009.00010.x

Overexpression of klotho protein modulates uninephrectomy-induced compensatory renal hypertrophy by suppressing IGF-I signals

The klotho gene is highly expressed in the distal convoluted tubule of the kidney, while its encoded protein has many physiological and pathophysiological renal roles. We investigated the effect of klotho protein on physiological compensatory renal hypertrophy after nephrectomy in klotho transgenic (KLTG) mice. Renal hypertrophy was suppressed in KLTG mice compared with wild-type mice, and this was associated with suppression of insulin growth factor-1 (IGF-1) signaling by klotho protein. In vitro, IGF-1 signaling was suppressed in human proximal tubular cells transfected with the klotho plasmid. Our data suggest that klotho modulates compensatory renal hypertrophy after nephrectomy via suppression of the IGF-1 signaling pathway, indicating a novel physiological role for klotho protein in the kidney.

Angiotensin II regulates islet microcirculation and insulin secretion in mice.

Angiotensin II causes potent increases in systemic and local pressure through its vasoconstrictive effect. Despite the importance of angiotensin II for local blood flow regulation, whether angiotensin II regulates the pancreatic islet microcirculation remains incompletely understood. We hypothesized that angiotensin II directly regulates the pancreatic islet microcirculation and thereby regulates insulin secretion. The aims of this study were to develop a new technique to visualize pancreatic islet hemodynamic changes in vivo and to analyze changes in islet circulation induced by angiotensin II or an angiotensin type 1 receptor blocker.

Establishment of a Blood Purification System for Renal Failure Rats Using Small‐Size Dialyzer Membranes

Hemodialysis techniques for small animals have not been established because no small dialysis apparatus has been available. We recently developed a small‐size dialyzer and established an appropriate blood purification system for small animals. To confirm the appropriate dialysate flow rate, bovine blood was dialyzed for 60 min at a fixed blood flow rate of 1.0 mL/min and variable dialysate flow rates. Blood urea nitrogen and creatinine levels decreased significantly at a dialysate flow rate of 5 mL/min (from 13.7 ± 0.2 to 10.3 ± 1.2 mg/dL and 1.07 ± 0.15 to 0.61 ± 0.12 mg/dL, respectively, P < 0.05). To determine the appropriate in vivo conditions, extracorporeal circulation was performed in anesthetized male Sprague‐Dawley rats at a dialysate flow rate of 0.0 mL/min, for 240 min, and at variable blood flow rates. Extracorporeal circulation was successful at a blood flow rate of 1.0 mL/min, but not 1.5 mL/min. To establish in vivo hemodialysis conditions, we used the animal model of end stage renal failure. Sprague‐Dawley rats were fed a 0.75% adenine‐containing diet for 3 weeks, after which they received hemodialysis for 120 min at a dialysate and blood flow rate of 5.0 and 1.0 mL/min, respectively. There were no significant changes in systolic blood pressure or heart rate during dialysis. Thus, this blood purification system can be safely used for small animals at a dialysate flow rate of 5.0 mL/min and a blood flow rate of 1.0 mL/min. This system provides a basis for further research on hemodialysis therapy.

Feasibility of fluorescence energy transfer system for imaging the renoprotective effects of aliskiren in diabetic mice

Introduction: We investigated the feasibility of using a fluorescence resonance energy transfer system to image enzymatic activity in order to evaluate the effects of aliskiren (a direct renin inhibitor) on diabetic nephropathy. Materials and methods: First, we induced diabetes in C57BL/6J mice using streptozotocin, then treated them with either aliskiren (25 mg/kg/day) or the angiotensin type 1 receptor blocker valsartan (15 mg/kg/day) for four weeks. Finally, we utilized renin fluorescence resonance energy transfer substrate to assess renin activity. Results: Renin activity was much higher in the kidneys of diabetic mice compared to those of the non-diabetic control mice. While aliskiren inhibited this activity, valsartan did not. We noted that production of reactive oxygen species intensified and the bioavailability of nitric oxide diminished in the glomeruli of diabetic mice. Aliskiren and valsartan significantly ameliorated these effects. They suppressed glomerular production of reactive oxygen species and urinary albumin excretion. In fact, urinary albumin excretion in diabetic mice treated with aliskiren or valsartan was lower than that in untreated diabetic mice. Furthermore, aliskiren and valsartan significantly reduced glomerular permeability by maintaining the glomerular endothelial surface layer. Conclusion: Fluorescence resonance energy transfer could provide a new tool for evaluating tissue and plasma enzymatic activity.

High dietary protein intake induces endothelial dysfunction in uninephrectomized rats

High dietary protein (HP) intake is a risk factor for chronic kidney disease (CKD). HP intake is associated with the development of albuminuria and glomerulosclerosis in uninephrectomized rats. In such rats, we investigated whether HP intake induces endothelial dysfunction. Male Wistar rats were divided into sham-operated rats fed a standard-protein diet, sham-operated rats fed a high-protein diet, uninephrectomized rats fed a standard-protein diet (NxSP) and uninephrectomized rats fed a high-protein diet (NxHP) (n=8 each). One week after treatment, endothelial function and urinary albumin excretion (UAE) were measured. Protein expression, phosphorylation at serine residue 1177 and uncoupling of endothelial nitric oxide synthase (eNOS), and mRNA expression of NADPH oxidase components were assessed in the aorta. NxHP rats showed hypertriglyceridemia and modest hyperhomocysteinemia. Endothelial function was significantly lower, and UAE was significantly higher in NxHP rats compared with the other groups (P<0.01 each), although there was no difference in creatinine clearance between NxSP and NxHP rats. Expression levels, phosphorylation and the dimer/monomer ratio of eNOS did not differ among the four groups. HP intake did not modify p22phox and p47phox expression levels in uninephrectomized rats. In conclusion, HP intake induced endothelial dysfunction and enhanced albuminuria in uninephrectomized rats, inde-pendent of renal function, suggesting that HP intake may cause the development of cardiovascular disease associated with CKD.

PS 14-36 EFFECT OF SINGLE PILL-BASED COMBINATION THERAPY OF OLMESARTAN AND AZELNIDIPINE ON VARIABILITY OF HOME BLOOD PRESSURE IN HYPERTENSIVE OUTPATIENTS

Objective: Recent studies have shown the association between blood pressure (BP) variability and cardiovascular events. The present study was designed to investigate the effects of the single pill-based combination therapy of olmesartan and azelnidipine on achievement of the target BP and day-by-day home BP variability in outpatients with hypertension. Design and Method: We enrolled hypertensive outpatients if they could not achieve the BP goal (home systolic BP ≥ 135 mmHg and diastolic BP ≥ 85 mmHg) by treatment with olmesartan or azelnidipine monotherapy for over 4 weeks. After the run-in period, eligible patients were given a single pill of olmesartan/azelnidipine tablet (olmesartan medoxomil /azelnidipine; 20/16 mg) in the morning for 16 weeks. Home BP was taken in triplicate in the morning for 5 consecutive days before each visit. Visits occurred at 4-week intervals. Home BP variability was defined as within-individual standard deviation (SD), coefficient of variation (CV), and maxi- mum minus minimum difference (MMD) of the 5-day home BP. Results: The combination therapy with of olmesartan and azelnidipine for 16 weeks significantly decreased both clinic and home BP, and achievement of target BP control was attained in an average of 43% after the combination therapy in spite of the presence of no achievement at baseline. In addition, the combination therapy significantly decreased the SD and MMD, but not CV. Especially, addition of azelnidipine significantly decreased the SD and MMD. Conclusions: Single pill-based combination therapy of olmesartan and azelnidipine is effective to home BP reduction for hypertensive outpatients whose BP is not controlled by each monotherapy. Home BP variability is partially decreased by combination therapy.