Hideyuki Horike

Angiotensin II type 1 receptor blocker ameliorates uncoupled endothelial nitric oxide synthase in rats with experimental diabetic nephropathy

Background Recent studies showed that angiotensin II type 1 receptor blocker (ARB) slows progression of chronic renal disease in patients with type 2 diabetes, regardless of changes in blood pressure. We showed that the imbalance of nitric oxide (NO) and reactive oxygen species (ROS) due to endothelial NO synthase (eNOS) uncoupling contributed to renal dysfunction in the diabetic nephropathy. The aim of this study was to determine the effects of ARB on uncoupled eNOS in rat diabetic nephropathy. Methods. Diabetes was induced in Sprague-Dawley rats with streptozotocin (65 mg/ kg body weight). After 6 weeks, rats were divided into saline (DM; n = 11) and ARB, losartan groups (DM+Los; n = 11). After 2-week treatment, glomerular ROS production was assessed by 2′,7′-dichlorofluorescin diacetate (DCFH-DA)-derived chemiluminescence. Renal NO and ROS production were imaged by confocal laser microscopy after renal perfusion with DCFH-DA and diaminorhodamine-4M acetoxymethyl ester with l-arginine. The dimeric form of eNOS was measured by low-temperature sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Serum tetrahydrobiopterin (BH4) concentrations were determined by high-performance liquid chromatography. Protein and mRNA expression of GTP cyclohydrolase 1 (GTPCH1), key enzyme of BH4 synthesis, were examined. Results Losartan attenuated glomerular ROS production in DM. Accelerated ROS production and diminished bioavailable NO caused by NOS uncoupling were noted in DM glomeruli. Losartan reversed the decreased GTPCH1 and decreased dimeric form of eNOS and glomerular NO production by increased BH4 bioavailability. Conclusions. ARB improved the NOS uncoupling in diabetic nephropathy by increasing BH4 bioavailability.

Olmesartan Ameliorates Progressive Glomerular Injury in Subtotal Nephrectomized Rats through Suppression of Superoxide Production

Angiotensin type 1 receptor blockers are more effective than other antihypertensive agents in slowing the progression of renal disease. Angiotensin II (Ang II) induces production of NAD(P)H oxidase–dependent superoxide in vascular and mesangial cells, but the direct role of Ang II in glomerular superoxide production remains unknown. Here we examined the effect of Ang II on superoxide production both ex vivo and in vivo. Ang II increased superoxide generation in isolated normal glomeruli in a dose-dependent manner, and co-incubation with olmesartan, an angiotensin type 1 receptor blocker, suppressed such increase. Subtotal nephrectomized rats (Nx, n=8) showed impaired renal function, increased glomerular sclerosis, and significantly high superoxide production in glomeruli. These changes were inhibited in olmesartan-treated (n=8), but not hydralazine-treated (n=8) Nx rats. Oxidative stress and nitrosative stress were observed in Nx glomeruli, as evidenced by increased levels of carbonyl protein and nitrotyrosine formation, respectively. These changes were inhibited by 8-week treatment with olmesartan. The apoptosis observed in Nx glomeruli was also suppressed by olmesartan. Superoxide generation in Nx glomeruli was blocked by an NAD(P)H oxidase inhibitor, diphenylene iodinium. The mRNA expression levels of two NAD(P)H oxidase subunits were increased in Nx, and olmesartan significantly reduced the mRNA expression levels. These results indicate that Ang II directly induced superoxide production through activation of NAD(P)H oxidase, and olmesartan would inhibit superoxide production and oxidative stress independent of its blood pressure–lowering effect. These findings support the notion that superoxide plays a primary role in glomerular injury in chronic kidney disease.

Implication of peritubular capillary loss and altered expression of vascular endothelial growth factor in IgA nephropathy.

Background/Aims: To determine the roles of peritubular capillary (PTC) loss and expression of vascular endothelial growth factor (VEGF) and its transcription factor, hypoxia-inducible factor-1 (HIF-1), in the progression of IgA nephropathy (IgAN), we analyzed the expression of VEGF and HIF-1, and the number of PTCs in patients with variable severity of IgAN. Methods: Renal biopsy specimens from patients with IgAN (n = 23) were classified according to interstitial injury score: grade 0 (0%), grade 1 (1–25%), grade 2 (25–50%) and grade 3 (50–100%). We examined the immunohistochemical expression of CD34, VEGF and HIF-1α. Results: VEGF was expressed in the cytoplasm of tubular epithelia, and VEGF-positive area significantly expanded in grades 1 (35.5 ± 5.9%, mean ± SD) and 2 (32.5 ± 5.9%) compared with grade 0 (23.4 ± 4.5%). The numbers of PTCs were significantly lower in grades 2 (559 ± 49/mm2) and 3 (510 ± 56/mm2) than grade 0 (708 ± 49/mm2). HIF-1α was weakly expressed in tubular epithelia in grade 0, increased with progression to grade 2, and markedly decreased in grade 3. It was also increased in pericapsular interstitial area in grade 1. The expression pattern of HIF-1α did not parallel that of VEGF. In renal biopsies of 5 control patients with minor glomerular abnormality, glomerular expression levels of VEGF and HIF-1α were similar to those of IgAN grade 0 kidneys. Conclusion: VEGF production was accelerated in the early stage of IgAN but it did not protect against PTC injury/loss. The lack of correlation between VEGF and HIF-1α expression suggests HIF-independent VEGF production in IgAN.

Zinc deficiency anemia and effects of zinc therapy in maintenance hemodialysis patients.

Quantitative adjuvant zinc therapy using polaprezinc was performed to examine the correlation between zinc concentration and anemia in maintenance hemodialysis patients to propose appropriate treatment. Anemia and serum zinc concentration were measured in 117 patients with chronic renal failure receiving outpatient maintenance hemodialysis at Tsuyama Chuo Kinen Hospital. Two bags of polaprezinc (containing zinc 34 mg/day) were administered to 58 patients with lower than normal zinc levels (Zn < 80 mg/dl) as adjuvant zinc therapy to assess anemia improvement. Zinc concentration and all anemia parameters showed significant positive correlation, indicating that anemia improves in patients with high serum zinc levels. Regarding the effects of adjuvant zinc therapy for improving anemia, hemoglobin levels were found to increase significantly to the highest value at 3 weeks. During treatment, the dosage of erythropoietin was reduced significantly from baseline at all assessment points. No zinc poisoning from therapy was seen, but two patients had diarrhea (1.9%). Zinc‐treated patients required iron therapy due to the development of iron deficiency. Most maintenance hemodialysis patients suffer from zinc deficiency anemia, and zinc‐based polaprezinc has been confirmed to be an effective and safe adjuvant zinc treatment. Most patients diagnosed as refractory anemia with no response to erythropoietin also suffer from zinc deficiency anemia, many of whom are expected to benefit from zinc therapy to improve their anemia. Possible zinc deficiency anemia should be considered in the treatment of refractory anemia with no response to erythropoietin.

Mitochondrial damage-induced impairment of angiogenesis in the aging rat kidney

Decreased expression of vascular endothelial growth factor (VEGF) in the renal tubules is thought to cause progressive loss of the renal microvasculature with age. Mitochondrial dysfunction may be a principal phenomenon underlying the process of aging. The relation between VEGF expression and mitochondrial dysfunction in aging is not fully understood. We hypothesized that mitochondrial dysfunction blocks VEGF expression and contributes to impaired angiogenesis in the aging kidney. The aim of this study was to assess the role of mitochondria in VEGF expression in the aging rat kidney. We evaluated the accumulation of 8-hydroxy-2′-deoxyguanosine in mitochondrial DNA, as well as mitochondrial dysfunction, as assessed by electron microscopy of mitochondrial structure and histochemical staining for respiratory chain complex IV, in aging rat kidney. An increase in hypoxic area and a decrease in peritubular capillaries were detected in the cortex of aging rat kidneys; however, upregulation of VEGF expression was not observed. The expression of VEGF in proximal tubular epithelial cells in response to hypoxia was suppressed by the mitochondrial electron transfer inhibitor myxothiazol. Mitochondrial DNA-deficient cells also failed to upregulate VEGF expression under hypoxic conditions. These results indicate that impairment of VEGF upregulation, possibly as a result of mitochondrial dysfunction, contributes to impaired angiogenesis, which in turn leads to renal injury in the aging rat kidney.

Azelnidipine exerts renoprotective effects by improvement of renal microcirculation in angiotensin II infusion rats

BACKGROUND Hypoxia-induced tubulointerstitial injury caused by loss of peritubular capillary (PTC) blood flow may be associated with progressive renal disease. Therefore, the maintenance of blood flow in PTCs may protect against loss of renal function. A long-acting calcium channel blocker, azelnidipine, has been shown to be useful in the treatment of progressive renal disease. However, its mechanism of action remains unclear. The aim of the present study was to elucidate whether azelnidipine maintains PTC blood flow and to compare it to nifedipine in its ability to improve tubulointerstitial injury caused by angiotensin II (AII) infusion in rats. METHODS PTC blood flow was initially monitored using a pencil-lens interval microscope before and after intravenous AII (30 ng/kg/min) infusion with or without azelnidipine (10 microg/kg/min). Next, Wistar rats were treated with chronic infusion of AII (500 ng/kg/min) via an osmotic minipump with or without azelnidipine (3 mg/kg/day, orally) or nifedipine (60 mg/kg/day, orally) for 14 days, and tubulointerstitial damage (PTC loss, interstitial fibrosis, tubular atrophy) was examined. RESULTS PTC blood flow was reduced after AII infusion but improved after a bolus injection of azelnidipine. Tubulointerstitial damage observed in chronically AII-treated kidneys was associated with hypoxic conditions, as indicated by the measurement of hypoxia biomarkers (intracellular hypoxyprobe-1 adducts). These tubulointerstitial injuries in AII-infused rats were more effectively reduced by azelnidipine than by nifedipine. The area showing hypoxic conditions in the kidney was also more reduced with azelnidipine than nifedipine treatment. CONCLUSIONS Azelnidipine may increase PTC blood flow and improve renal hypoxia and tubulointerstitial injury induced by AII infusion.

Co-occurrence of poststreptococcal reactive arthritis and acute glomerulonephritis

We report a 16-year-old patient who developed concurrent poststreptococcal reactive arthritis and acute glomerulonephritis. A high titer of antistreptolysin O antibody confirmed the preceding streptococcal infection. The patient presented with symmetric persistent tenosynovitis of hands and feet. Renal biopsy showed typical findings of acute glomerulonephritis with crescent formation. Physicians who treat patients with arthritis of acute onset, especially after throat infection, should be aware of possible urinary abnormalities or renal dysfunction.

Clear Correlation of Tetrahydrobiopterin with Nitric Oxide Bioavailability in Continuous Ambulatory Peritoneal Dialysis

have been no investigations on the roles of NO and BH 4 in CAPD. Therefore, we measured both NO and BH 4 concentrations in spent dialysates of CAPD patients to investigate the relation between the two. The NO concentration was measured by an NO sensor [6, 12] and BH 4 and BH 2 concentrations were measured fluorometrically by the HPLC-based method [13] . This study protocol was approved by the Ethical Committee of Kawasaki Medical School. Written informed consent was obtained from each patient. Nitric oxide (NO) plays diverse physiological roles including vasodilatory and anti-atherogenic actions [1] . In continuous ambulatory peritoneal dialysis (CAPD), NO has been thought to regulate permeability of the peritoneal membrane and suppress angiogenesis in the peritoneum [2–4] . Thus, abnormal production and/or metabolism of NO would lead to peritoneal dysfunctions. Previous studies suggested that changes in NO bioavailability induce the dysfunction of peritoneal membranes [3, 5] . Recently, we demonstrated that NO exists in spent dialysates at measurable levels over a wide range [6] . Tetrahydrobiopterin (BH 4 ) is a cofactor for NO synthase (NOS) and plays an important role in maintaining the NOS activity. Depletion of BH 4 would result in NOS uncoupling and NOS-derived superoxide release [1, 7, 8] . Yokoyama et al. [9] reported that a significant positive correlation between creatinine clearance and the BH 4 /BH 2 ratio (BH 2 = biopterin, an oxidized form of BH 4 ) in the patients with chronic renal failure. Yamamizu et al. [10] demonstrated the involvement of BH 4 deficiency in endothelial dysfunction in rats with chronic renal failure. Recently, we also reported the possible involvement of BH 4 in the NO production rate of hemodialysis (HD) patients [11] . However, there Published online: July 13, 2010