Megumu Fukunaga

Stimulatory Effect of 8-Epi-PGF2α, An F2-Isoprostane, on Endothelin-1 Release

Summary: 8-Epi-prostaglandin F2α (8-epi-PGF2α) is an F2-isoprostane produced in vivo by a cyclooxygenase-independent, free radical-catalyzed lipid peroxidation mechanism. It exhibits renal vasoconstrictor effects by binding to a receptor related to, but distinct from, that of thromboxane A2 (TxA2). In cultured bovine aortic endothelial cells (BAECs), competitive binding assays using [3H]-8-epi-PGF2α indicated the existence of two distinct binding sites. The Kd values were similar to those of cultured rat aortic smooth-muscle cells, suggesting that the high- and the low-affinity binding sites represent isoprostane and TxA2 receptors, respectively. 8-Epi-PGF2α dose-dependently stimulated endothelin-1 (ET-1) secretion from BAECs. These increases were partially inhibited by a TxA2 receptor antagonist, consistent with the premise that isoprostanes and TxA2 recognize closely related receptors. The presence of specific binding sites for F2-isoprostanes on endothelial cells widens the scope of the possible pathophysiologic significance of these eicosanoids, released during oxidant injury, to include alteration of endothelial cell biology. The release of ET-1 by 8-epi-PGF2α may help to explain the large increases in plasma and urinary concentrations for both ET-1 and 8-epi-PGF2α in patients with hepatorenal syndrome.

Mutation Analysis of 2009 Pandemic Influenza A(H1N1) Viruses Collected in Japan during the Peak Phase of the Pandemic

Background Pandemic influenza A(H1N1) virus infection quickly circulated worldwide in 2009. In Japan, the first case was reported in May 2009, one month after its outbreak in Mexico. Thereafter, A(H1N1) infection spread widely throughout the country. It is of great importance to profile and understand the situation regarding viral mutations and their circulation in Japan to accumulate a knowledge base and to prepare clinical response platforms before a second pandemic (pdm) wave emerges. Methodology A total of 253 swab samples were collected from patients with influenza-like illness in the Osaka, Tokyo, and Chiba areas both in May 2009 and between October 2009 and January 2010. We analyzed partial sequences of the hemagglutinin (HA) and neuraminidase (NA) genes of the 2009 pdm influenza virus in the collected clinical samples. By phylogenetic analysis, we identified major variants of the 2009 pdm influenza virus and critical mutations associated with severe cases, including drug-resistance mutations. Results and Conclusions Our sequence analysis has revealed that both HA-S220T and NA-N248D are major non-synonymous mutations that clearly discriminate the 2009 pdm influenza viruses identified in the very early phase (May 2009) from those found in the peak phase (October 2009 to January 2010) in Japan. By phylogenetic analysis, we found 14 micro-clades within the viruses collected during the peak phase. Among them, 12 were new micro-clades, while two were previously reported. Oseltamivir resistance-related mutations, i.e., NA-H275Y and NA-N295S, were also detected in sporadic cases in Osaka and Tokyo.

Formula and Nomogram for Predicting Creatinine Clearance from Serum Creatinine Concentration

BackgroundQuick estimation of creatinine clearance from serum without calculation or urine measurements may be useful in many bedside clinical circumstances. A novel formula and a nomogram for predicting creatinine clearance are presented here.MethodsWe determined 24-hour creatinine clearance in 155 men and 143 women. A formula for creatinine clearance prediction from serum creatinine (Scr), age, body weight (BW), and body mass index (BMI) was developed [men: creatinine clearance=(−0.065 Age−0.493BMI+33)BW/Scr/14.4; women: creatinine clearance=(−0.052 Age−0.202BMI+21)BW/Scr/14.4]. Variables in this formula were evaluated for suitability by multiple regression analysis. The correlation between creatinine clearance measured and creatinine clearance predicted using serum creatinine from the same subject group was high (r=0.882). A nomogram for creatinine clearance prediction was derived from an equation that included the logarithmic transformation of age, body weight, height, and creatinine excretion instead of the original, nonlogarithmic values.ResultsCorrelation between measured and estimated creatinine clearance values for the nomogram was slightly lower (r=0.879) than ther value (0.882) obtained using the original formula. However, the nomogram still has considerable reliability in creatinine clearance prediction compared several other methods previously reported.ConclusionThe improved formula and nomogram for creatinine clearance presented here show high correlation with measured creatinine clearance. However, almost all the subjects in this study were nondiabetic patients. Therefore, the formula and the nomogram should be applied in treating nondiabetic patients; a separate formula may be needed for patients with diabetic nephropathy.

Comparative effects of cyclosporine A and FK-506 on endothelin secretion by a cultured renal cell line, LLC-PK1.

Cyclosporine A (CSA) stimulated endothelin secretion by a cultured renal epithelial cell line, LLC-PK1. A less nephrotoxic immunosuppressant (FK-506) did not affect endothelin secretion. Putative endothelin converting enzyme inhibitors or specific receptor antagonists may therefore prevent CSA nephrotoxicity.

Endothelin-1 receptors in rat renal glomeruli

Summary Renal glomeruli form a dynamic structure capable of regulating the glomerular filtration rate (GFR). To explore possible regulating effects of endothelin-1 (ET-1) upon glomerular functions, the presence of specific ET-1 receptors and the biological actions of ET-1 were studied in isolated rat renal glomeruli. The specific binding of [125I]ET-1 to glomeruli was time and temperature dependent. Scatchard analysis of the binding data indicated the presence of a single class of high-affinity binding sites with the apparent dissociation constant of 8.3 nM and the maximal binding capacity of 1.5 pmol/mg protein. ET-1 stimulated prostaglandin (PG) E2 production in glomeruli with the time course and the concentration dependency similar to those found in [125I]ET-1 binding to glomeruli. These results indicate the presence of high-affinity binding sites for ET-1 linked to PGE2 production in glomeruli, suggesting a possible role of ET-1 in the regulation of glomerular function.

Signal transduction mechanism of interleukin 6 in cultured rat mesangial cells

Interleukin 6 (IL‐6) is one of the potent autocrine growth factors for mesangial cells. We investigated the signal transduction mechanism or IL‐6 in cultured rat mesangial cells. IL‐6 induced a transient increase of inositol 1,4,5‐trisphosphate (Ins 1,4,5‐P3) followed by a transient and sustained increase of intracellular calcium concentration, suggesting that IL‐6 stimulates phosphoinositide turnover. IL‐6 also stimulated prostaglandin E2 (PGE2) production. The IL‐6‐concentration dependency in PGE2 production was similar to that in Ins 1,4,5‐P3 production. We concluded that the action of IL‐6 on mesangial cells is exerted at least partially through the enhancement of phosphoinositide turnover and PGE2 production.

Comparison of Methylprednisolone Plus Prednisolone with Prednisolone Alone as Initial Treatment in Adult-Onset Minimal Change Disease: A Retrospective Cohort Study

BACKGROUND AND OBJECTIVES Previous studies suggested that intravenous methylprednisolone possibly accelerates remission of proteinuria in adult-onset minimal change disease; its impact on relapse of proteinuria is unknown. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This multicenter retrospective cohort study included 125 adult-onset minimal change disease patients diagnosed by kidney biopsy between 2000 and 2009 and treated initially with corticosteroid in five nephrology centers in Japan participating in the Study of Outcomes and Practice Patterns of Minimal Change Disease. Times to first remission and first relapse of proteinuria after initiating the first immunosuppressive therapy were compared between 65 patients with initial use of intravenous methylprednisolone followed by prednisolone and 60 patients with initial use of prednisolone alone using multivariate Cox proportional hazards models. After calculating the probability of receiving methylprednisolone and prednisolone using a logistic regression model (propensity score), the results were ascertained using propensity score-matched and -stratified models. RESULTS During the median 3.6 years of observation (interquartile range=2.0-6.9), all 65 patients in the methylprednisolone and prednisolone group achieved remission within 11 (8-20) days of the corticosteroid initiation, whereas in the prednisolone group, 58 of 60 patients (96.7%) achieved remission within 19 (12-37) days (P<0.001). After achieving first remission, 32 (49.2%) patients in the methylprednisolone and prednisolone group and 43 (74.1%) patients in the prednisolone group developed at least one relapse. Multivariate Cox proportional hazards models revealed that methylprednisolone and prednisolone use was significantly associated with early remission (multivariate-adjusted hazard ratio, 1.56; 95% confidence interval, 1.06 to 2.30) and lower incidence of relapse (0.50; 95% confidence interval, 0.29 to 0.85) compared with prednisolone use alone. These results were ascertained in propensity score-based models. No significant difference was observed in incidence of adverse events, including infection, aseptic osteonecrosis, cataract, diabetes, and gastrointestinal bleeding. CONCLUSIONS Initial use of methylprednisolone was associated with earlier remission and lower incidence of relapse in adult-onset minimal change disease patients. Efficacy of methylprednisolone should be evaluated in randomized controlled trials.

Stimulatory effect of thrombin on endothelin-1 production in isolated glomeruli and cultured mesangial cells of rats.

To determine whether endothelin-1 (ET-1) is released in close proximity to its binding sites on glomerular mesangial cells, and also to elucidate the regulatory factors responsible for its release, we measured immunoreactive ET-1 (ir-ET-1) in the incubation media of isolated glomeruli and cultured mesangial cells of rats using enzyme immunoassay under both basal and thrombin-stimulated conditions. ir-ET-1 was released time-dependently, from isolated glomeruli and cultured mesangial cells. Thrombin (2 U/ml) stimulated the release from both preparations. The release of immunoreactive big endothelin-1 (ir-big ET-1), which was assayed by using enzyme immunoassay, was also time-dependent, and the release was increased by thrombin (2 U/ml; at 4 h, 8 h, and 24 h) in cultured mesangial cells. The releases of big ET-1 and its converted product ET-1 from mesangial cells, an established target for ET-1, suggests that ET-1 acts as an autocrine factor for the cells.

A case of MPO-ANCA-positive polyarteritis nodosa complicated by exudative otitis media, mononeuritis multiplex, and acute renal failure

In December 2008, a 69-year-old Japanese woman was admitted to the Department of Otorhinolaryngology because of hearing impairment due to bilateral exudative otitis media, and was discharged without complete recovery despite conventional treatment. Two weeks later, she was readmitted for worsened deafness, numbness, gait disturbance, and general fatigue. She was referred to our department for general investigation. On admission, laboratory examination revealed severe inflammatory signs and active nephritic urinary sediments. Cranial computed tomography (CT) revealed progressive exudative otitis media and sinusitis. Initially, Wegener’s granulomatosis was suspected. Nasal cavity biopsy, however, showed no granuloma formation or vasculitis. Serology revealed high titer of myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA), suggestive of microscopic polyangitis (MPA). However, contrast CT identified stenosis of a celiac artery, and renal biopsy showed tubulointerstitial changes with minor glomerular abnormalities. Therefore, polyarteritis nodosa (PAN) was suspected and treatment with intravenous methylprednisolone was initiated. However, a lacunar infarct developed followed by cerebral hemorrhage, and the patient died 19 days after readmission. Autopsy revealed fibrinoid necrosis, neutrophilic infiltration, and giant cell reaction in small to medium-sized arteries in multiple organs. These findings led to diagnosis of systemic vasculitis anatomically compatible with PAN. This was a rare case of a patient with MPO-ANCA-positive PAN who may have developed bilateral exudative otitis media and hearing loss as the initial manifestation of PAN.

Regulation of MAP-Kinase Activation by 8-Iso-Prostaglandin F2a in Cultured Rat Aortic Smooth Muscle Cells

8-iso-prostaglandin F2α (8-iso-PGF2α), an F2-isoprostane, is produced in vivo by a cyclooxygenase-independent, free radical-catalyzed mechanism involving peroxidation of arachidonic acid1. This isoprostane is a highly potent renal vasoconstrictor2 and stimulates proliferation of cultured rat aortic smooth muscle cells (AoSMC) through enhancement of phosphoinositide turnover3. AoSMC have distinct F2-isoprostane binding sites, although these putative receptors bear homology to the thromboxane A2 (TxA2) receptor4. In this study, we report the activation of mitogen activated protein kinases (MAP-kinases)5 by 8-iso-PGF2α in AoSMC and its regulating mechanism involving protein kinase C (PKC) and pertussis toxin (PT)-sensitive GTP binding proteins.