Ryoichi Imamura

Analytic validation of the enzyme multiplied immunoassay technique for the determination of mycophenolic acid in plasma from renal transplant recipients compared with a high-performance liquid chromatographic assay

The analysis of mycophenolic acid (MPA) has proved a valuable adjunct to the clinical care of organ transplant recipients. The analytic validation of the enzyme multiplied immunoassay technique (EMIT) for the determination of MPA in plasma is described. The EMIT MPA standard curve was 0 to 15.0 &mgr;g/mL, and curve storage was maintained for 4 weeks. The MPA EMIT assay proved reliable and reproducible, as shown by the intra-assay and interassay coefficients of variation (1.58–3.68% and 1.23–7.57%, respectively). Excellent linear correlation (r = 0.999) was observed for dilution linearity. The sensitivity of the assay was 0.01 &mgr;g/mL. Recoveries of 99.4% to 104.2% were obtained by spiking aliquots of three controls of known MPA concentrations with MPA. No interference was observed for endogenous substances and coadministered immunosuppressant drugs, and no cross-reactivity from the major metabolite MPA glucuronide was found. The high-performance liquid chromatography (HPLC) assay used protein precipitation and C18 ion-pair chromatography with ultraviolet detection at 304 nm. Plasma concentrations of MPA were measured using EMIT and HPLC. A linear relationship was observed between EMIT and HPLC (EMIT = 1.091 × HPLC - 0.089;r 2 = 0.990, n = 129). These results indicate that EMIT is a simple, rapid, and sensitive assay method for the measurement of MPA in plasma.

Treatment Adherence in Renal Transplant Recipients: A Questionnaire Survey on Immunosuppressants

INTRODUCTION To achieve a high graft survival rate, patient adherence to immunosuppressive therapy is critical. It is extremely difficult to establish the actual adherence status of transplant recipients; only a few surveys on the issue have been performed in Japan. METHODS We conducted a questionnaire survey mainly on treatment adherence to calcineurin inhibitors among renal transplant recipients. RESULTS The survey demonstrated some degree of nonadherence in a relatively high percentage of the patients. The adherence rate was significantly lower for the evening than the morning dose (McNemar test, P < .001). It significantly decreased with time following transplantation for both the morning and the evening doses (logistic regression analysis, P = .025 and <.001, respectively). CONCLUSIONS Immunosuppressive treatment places a substantial burden on patients, some of whom cannot continue regular treatment at specified time points due to daily life restrictions after they have returned to work.

Carbamylated Erythropoietin Improves Angiogenesis and Protects the Kidneys from Ischemia-Reperfusion Injury:

Several studies have shown that erythropoietin (EPO) can protect the kidneys from ischemia-reperfusion injury and can raise the hemoglobin (Hb) concentration. Recently, the EPO molecule modified by carbamylation (CEPO) has been identified and was demonstrated to be able to protect several organs without increasing the Hb concentration. We hypothesized that treatment with CEPO would protect the kidneys, partly due to the increased peritubular capillaries. The therapeutic effect of CEPO was evaluated using an endothelial tube formation assay in vitro, and a rat ischemia-reperfusion injury model in vivo. EPO treatment showed the tendency of increased tube formation, while CEPO treatment induced more capillary-like formation than EPO. Ischemia-reperfusion-induced kidneys exhibited characteristic nuclei of apoptosis in tubular epithelial cells with decreased peritubular capillaries, while EPO treatment inhibited tubular apoptosis with preserved endothelial cells. Moreover, CEPO-treated kidneys showed minimal tubular apoptosis with increased peritubular capillary endothelial cells. In conclusion, we identified a new therapeutic approach using CEPO to protect kidneys from ischemia-reperfusion injury by promoting angiogenesis.

Risk factors for malignancy in Japanese renal transplant recipients

Among recipients of renal transplants, the incidences of renal cancer and gastrointestinal cancer are higher and that of skin cancer is much lower in Japan than in Europe and North America.

Nonerythropoietic derivative of erythropoietin protects against tubulointerstitial injury in a unilateral ureteral obstruction model

BACKGROUND Erythropoietin (EPO), a member of the cytokine type I superfamily, acts to increase circulating erythrocytes primarily by preventing apoptosis of erythroid progenitors, is known to protect tissues and can raise haemoglobin (Hb) concentrations. Recently, a second receptor for EPO comprising the EPO receptor and beta-common receptor has been reported to mediate EPO-induced tissue protection. EPO modified by carbamylation (CEPO) only signals through this second receptor. Accordingly, we hypothesized that treatment with CEPO, which would not increase Hb concentrations, would protect against tubular damage and thereby inhibit tubulointerstitial injuries. METHODS We evaluated therapeutic effects of CEPO using a rat unilateral ureteral obstruction model. RESULTS CEPO decreased tubular apoptosis and alpha-smooth muscle actin (alphaSMA) expression in the absence of polycythaemia, while the untreated obstructed kidneys exhibited increased tubular apoptosis with expanded (alphaSMA) expression. While EPO treatment similarly inhibited tubular apoptosis and alphaSMA expression, EPO treatment increased Hb concentrations and induced a wedge-shaped infarction. CONCLUSION We established a therapeutic approach using CEPO to protect against tubulointerstitial injury. The therapeutic value of this approach warrants further attention and preclinical studies.

Clinical characteristics and risk factors for septic shock in patients receiving emergency drainage for acute pyelonephritis with upper urinary tract calculi

BackgroundAcute pyelonephritis (APN) is a common complication of ureteral obstruction caused by urolithiasis, and it can be lethal if it progresses to septic shock. We investigated the clinical characteristics of patients undergoing emergency drainage and assessed risk factors for septic shock.MethodsA retrospective study was performed of 98 patients (101 events) requiring emergency drainage at our urology department for obstructive APN associated with upper urinary tract calculi from January 2003 to January 2011. Clinical characteristics were summarized, and risk factors for septic shock were assessed by logistic regression analysis.ResultsObjective evidence of sepsis was found in 64 (63.4%) events, and 21 events (20.8%) were categorized as septic shock. Ninety-six patients recovered, but 2 patients died of septic shock. Multivariate analysis revealed that age and the presence of paralysis were independent risk factors for septic shock.ConclusionsAPN associated with upper urinary tract calculi is a severe disease that should be treated with caution, particularly when risk factors are present.

Bcl-2 Protects Tubular Epithelial Cells From Ischemia/Reperfusion Injury by Dual Mechanisms

Ischemia/reperfusion (I/R) injury, which induces extensive loss of tubular epithelial cells, is associated with delayed graft function following kidney transplantation. Recent reports have suggested that cell death by I/R injury occurs by autophagy, a cellular degradation process responsible for the turnover of unnecessary or dysfunctional organelles and cytoplasmic proteins, as well as by apoptosis. Recently, we demonstrated that overexpression of the anti-apoptotic factor, Bcl-2, inhibited tubular apoptosis and subsequent tubulointerstitial damage after I/R injury. Autophagy is also observed in cells undergoing cell death in several diseases. Therefore, we hypothesized that increased Bcl-2 protein may protect tubular epithelial cells by suppressing autophagy and inhibiting apoptosis. In the present study, a transgenic mouse model (LC3-GFP TG) in which autophagosomes are labeled with LC3-GFP and Bcl-2/LC3-GFP double transgenic mice (Bcl-2/LC3-GFP TG) were used to examine the effect of Bcl-2 on I/R-induced autophagy. I/R injury, which is associated with marked disruption of normal tubular morphology, promoted the formation of LC3-GFP dots, representing extensively induced autophagosomes. On electron microscopy, the autophagosomes contained mitochondria in I/R-injured tubular epithelial cells. In contrast, Bcl-2 augmentation suppressed the formation of autophagosomes and there was less tubular damage. In conclusion, Bcl-2 augmentation protected renal tubular epithelial cells from I/R injury by suppressing autophagosomal degradation and inhibiting tubular apoptosis.

Intravital two-photon microscopy assessment of renal protection efficacy of siRNA for p53 in experimental rat kidney transplantation models

Renal ischemia-reperfusion (I/R) injury, which is unavoidable in renal transplantation, frequently influences both short- and long-term allograft survival. Despite decades of laboratory and clinical investigations, and the advent of renal replacement therapy, the overall mortality rate due to acute tubular injury has changed little. I/R-induced DNA damage results in p53 activation in proximal tubule cells (PTC), leading to their apoptosis. Therefore, we examined the therapeutic effect of temporary p53 inhibition in two rat renal transplantation models on structural and functional aspects of injury using intravital two-photon microscopy. Nephrectomized Sprague-Dawley rats received syngeneic left kidney transplantation either after 40 min of intentional warm ischemia or after combined 5-h cold and 30-min warm ischemia of the graft. Intravenously administrated siRNA for p53 (siP53) has previously been shown to be filtered and reabsorbed by proximal tubular epithelial cells following the warm ischemia/reperfusion injury in a renal clamp model. Here, we showed that it was also taken up by PTC following 5 h of cold ischemia. Compared to saline-treated recipients, treatment with siP53 resulted in conservation of renal function and significantly suppressed the I/R-induced increase in serum creatinine in both kidney transplantation models. Intravital two-photon microscopy revealed that siP53 significantly ameliorated structural and functional damage to the kidney assessed by quantification of tubular cast formation and the number of apoptotic and necrotic tubular cells and by evaluation of blood flow rate. In conclusion, systemic administration of siRNA for p53 is a promising new approach to protect kidneys from I/R injury in renal transplantation.

Prevalence, characteristics, and outcome of BK virus nephropathy in Japanese renal transplant patients: analysis in protocol and episode biopsies

Abstract:  Background:  BK virus nephropathy (BKN) is recognized as a cause of graft loss in renal transplant patients. This may be related to the introduction of new and potent immunosuppressive regimens. In Japan, our experience regarding its prevalence, clinical significance, and outcome is still limited. In this study, our primary purpose is to outline the prevalence, outcome, and clinical characteristics of BKN as observed at Osaka University Hospital.

Carbamylated erythropoietin ameliorates cyclosporine nephropathy without stimulating erythropoiesis.

The introduction of cyclosporine (CsA) has improved graft survival, but it causes nephropathy, which limits its clinical utility. Recently, we reported that carbamylated erythropoietin (CEPO) protected kidneys from ischemia reperfusion injury as well as EPO. To investigate the clinical applications of CEPO, we next evaluated the long-term therapeutic effect of CEPO using a CsA-induced nephropathy model. CsA caused renal dysfunction, while EPO/CEPO administration significantly improved renal function. EPO treatment significantly increased Hb concentration, while CEPO treatment neither enhanced nor reduced Hb concentration. CsA treatment induced tubular apoptosis, while EPO/CEPO administration inhibited it and increased PI3 kinase activation and Akt phosphorylation. In parallel, morphological assessment revealed that EPO/CEPO significantly reduced CsA-induced interstitial fibrosis and inhibited interstitial macrophage infiltration. In addition, real-time RT-PCR demonstrated that cortical mRNA levels of TGF-β1 and type I collagen were suppressed in the EPO/CEPO group. These results suggest a new therapeutic approach using CEPO to protect kidneys from CsA-induced nephropathy.