Shinya Kaname

siRNA-Based Therapy Ameliorates Glomerulonephritis

RNA interference by short interfering RNAs (siRNAs) holds promise as a therapeutic strategy, but use of siRNAs in vivo remains limited. Here, we developed a system to target delivery of siRNAs to glomeruli via poly(ethylene glycol)-poly(l-lysine)-based vehicles. The siRNA/nanocarrier complex was approximately 10 to 20 nm in diameter, a size that would allow it to move across the fenestrated endothelium to access to the mesangium. After intraperitoneal injection of fluorescence-labeled siRNA/nanocarrier complexes, we detected siRNAs in the blood circulation for a prolonged time. Repeated intraperitoneal administration of a mitogen-activated protein kinase 1 (MAPK1) siRNA/nanocarrier complex suppressed glomerular MAPK1 mRNA and protein expression in a mouse model of glomerulonephritis; this improved kidney function, reduced proteinuria, and ameliorated glomerular sclerosis. Furthermore, this therapy reduced the expression of the profibrotic markers TGF-beta1, plasminogen activator inhibitor-1, and fibronectin. In conclusion, we successfully silenced intraglomerular genes with siRNA using nanocarriers. This technique could aid the investigation of molecular mechanisms of renal disease and has potential as a molecular therapy of glomerular diseases.

Methylglyoxal induces peritoneal thickening by mesenchymal-like mesothelial cells in rats

BACKGROUND The epithelial-to-mesenchymal transition (EMT) of mesothelial cells was observed in patients on peritoneal dialysis and may be involved in peritoneal thickening. Conventional peritoneal dialysis fluids (PDFs) that contain glucose degradation products (GDPs), such as methylglyoxal (MGO) and formaldehyde (FA), are bioincompatible. The aim of this study is to analyse the participation of EMT in peritoneal thickening induced by GDPs in rats. METHODS Rat mesothelial cells were cultured with various GDPs, and the gene expression of Snail was analysed by polymerase chain reaction (PCR). Sprague-Dawley rats were administered intraperitoneally 20 mM MGO/PDFs, 20 mM FA/PDFs or 0.1% chlorhexidine gluconate (CHX)/15% ethanol/saline every day for 21 days. On Day 22, the expression of transforming growth factor-beta (TGF-beta), collagen 1, matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), Snail and receptor for advanced glycation end-products (RAGE) was analysed by PCR, enzyme-linked immunoassay or immunohistological staining. RESULTS In cell-culture experiments, the expression of Snail was enhanced by MGO, but not FA. In rats treated with 20 mM MGO, peritoneal fibrous thickening with the proliferation of mesenchymal-like mesothelial cells was observed. The expression of TGF-beta, collagen 1, MMP-2, VEGF, Snail and RAGE increased significantly (P < 0.01). In FA- or CHX-treated rats, the peritoneum was thickened with sparse collagen fibres, but mesenchymal-like mesothelial cells were not observed. CONCLUSIONS MGO induced peritoneal fibrous thickening with the proliferation of mesenchymal-like mesothelial cells in vivo. These cells may be transdifferentiated from mesothelial cells by EMT via Snail and play an important role in peritoneal fibrous thickening.

Mac-1 (CD11b/CD18) Links Inflammation and Thrombosis After Glomerular Injury

Background— Inflammation and thrombosis coexist in several disorders. Although it is recognized that leukocytes may induce a procoagulant state at sites of inflammation, the critical molecular determinants of this process remain largely unknown. Methods and Results— To examine mechanisms of inflammation-induced thrombosis, we developed a murine model of thrombotic glomerulonephritis (TGN), a known cause of acute renal failure in patients. This model, induced by lipopolysaccharide and antibody to the glomerular basement membrane, led to rapid glomerular neutrophil recruitment, thrombotic glomerular lesions with endothelial cell injury, and renal dysfunction. In mice immunodepleted of neutrophils or lacking the leukocyte-specific integrin Mac-1, neutrophil recruitment, endothelial injury, glomerular thrombosis, and acute renal failure were markedly attenuated despite the robust generation of renal cytokines. Neutrophil elastase is a likely effector of Mac-1 because its activity was reduced in Mac-1–deficient mice and the phenotype in mice deficient in Mac-1 or neutrophil elastase was similar. Platelets accumulated in glomerular capillaries within 4 hours of TGN before evidence of thrombosis. Platelet immunodepletion before TGN markedly exacerbated hematuria (hemorrhage), inflammation, and injury, whereas thrombocytopenic Mac-1–deficient mice remained resistant to disease, indicating that initial glomerular platelet deposition protects the vessel wall from neutrophil-mediated sequelae. The subsequent thrombosis relied on the interaction of Mac-1 on recruited neutrophils with glycoprotein Ibα on platelets as antibody-mediated disruption of this interaction attenuated TGN without affecting renal neutrophil accumulation. Conclusions— These observations establish Mac-1 on neutrophils as a critical molecular link between inflammation and thrombosis and suggest it as an attractive target for antithrombotic therapy.

Anti-albuminuric effect of the aldosterone blocker eplerenone in non-diabetic hypertensive patients with albuminuria: a double-blind, randomised, placebo-controlled trial

BACKGROUND Renin-angiotensin system inhibitors have renoprotective effects in patients with chronic kidney disease, but most patients treated with these drugs have residual urinary albumin excretion. Some small clinical studies show that mineralocorticoid receptor blockade reduces albuminuria. Our study aimed to examine the beneficial effects of addition of a selective aldosterone antagonist, eplerenone, to renin-angiotensin system inhibitors in hypertensive patients with non-diabetic chronic kidney disease. METHODS In this double-blind, randomised, placebo-controlled trial, we enrolled hypertensive patients, aged 20–79 years, with albuminuria (urinary albumin-to-creatinine ratio [UACR] in the first morning void urine of 30–599 mg/g), an estimated glomerular filtration rate of 50 mL/min per 1·73 m2 or more, and who had received an angiotensin-converting enzyme inhibitor, an angiotensin receptor blocker, or both, for at least 8 weeks. Participants were from 59 clinics and hospitals in Japan. Eligible patients were randomly assigned (1:1), stratified by baseline characteristics, to either low-dose eplerenone (50 mg/day) or placebo, with continuation of standard antihypertensive treatment to attain therapeutic goals (<130/80 mm Hg) for 52 weeks. We assessed efficacy in all patients who received allocated treatment, provided a baseline and post-treatment urine sample, and remained in follow-up. We assessed safety in all patients who received allocated treatment. The primary efficacy measure was percent change in UACR in the first morning void urine at week 52 from baseline. The trial is registered at the clinical trials registry of University Hospital Medical Information Network (UMIN), trial identification number UMIN000001803. FINDINGS Between April 1, 2009, and March 31, 2012, we randomly allocated 170 patients to the eplerenone group and 166 patients to the placebo group. In the primary efficacy analysis, mean percent change in UACR from baseline was −17·3% (95% CI −33·65 to −0·94) for 158 patients in the eplerenone group compared with 10·3% (−6·75 to 22·3) for 146 patients in the placebo group (absolute difference −27·6% [–51·15 to −3·96]; p=0·0222). In the safety analyses, 53 (31%) of 169 patients in the eplerenone group had adverse events (five serious), as did 49 (30%) of 163 in the placebo group (seven serious). Although mean serum potassium concentration was higher in the eplerenone group than the placebo group, severe hyperkalaemia (>5·5 mmol/L) was not recorded in either group. INTERPRETATION Addition of low-dose eplerenone to renin-angiotensin system inhibitors might have renoprotective effects through reduction of albuminuria in hypertensive patients with non-diabetic chronic kidney disease, without serious safety concerns. FUNDING Pfizer.

Diagnostic criteria for atypical hemolytic uremic syndrome proposed by the Joint Committee of the Japanese Society of Nephrology and the Japan Pediatric Society.

Atypical hemolytic uremic syndrome (aHUS) is rare and comprises the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. Recently, abnormalities in the mechanisms underlying complement regulation have been focused upon as causes of aHUS. The prognosis for patients who present with aHUS is very poor, with the first aHUS attack being associated with a mortality rate of approximately 25%, and with approximately 50% of cases resulting in end‐stage renal disease requiring dialysis. If treatment is delayed, there is a high risk of this syndrome progressing to renal failure. Therefore, we have developed diagnostic criteria for aHUS to enable its early diagnosis and to facilitate the timely initiation of appropriate treatment. We hope these diagnostic criteria will be disseminated to as many clinicians as possible and that they will be used widely.

Clinical guides for atypical hemolytic uremic syndrome in Japan

Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. In 2013, we developed diagnostic criteria to enable early diagnosis and timely initiation of appropriate treatment for aHUS. Recent clinical and molecular findings have resulted in several proposed classifications and definitions of thrombotic microangiopathy and aHUS. Based on recent advances in this field and the emerging international consensus to exclude secondary TMAs from the definition of aHUS, we have redefined aHUS and proposed diagnostic algorithms, differential diagnosis, and therapeutic strategies for aHUS.

Effects of nocturnal oxygen therapy on sleep apnea syndrome in peritoneal dialysis patients.

UNLABELLED Sleep apnea syndrome (SAS) is common in patients with end-stage renal disease (ESRD). Although the treatment of choice is continuous positive airway pressure (CPAP) particularly for obstructive SAS, long-term compliance is not satisfactory. We investigated the effectiveness of nocturnal oxygen therapy on sleep apnea and autonomic nervous dysfunction in peritoneal dialysis (PD) patients with SAS. METHODS 40 patients on PD in our outpatient clinic were screened for SAS by pulse oximetry. We set the indication for nocturnal oxygen therapy at 4% oxygen desaturation index (4% ODI; defined as the number of falls of oxygen saturation > or = 4% per hour) > 5 or average nocturnal saturation < 95%. For SAS patients, 2 l per minute of oxygen was given during sleep and polysomnography was performed before and 1 month after oxygen administration. The heart rate variability was analyzed to assess autonomic nervous activity. RESULTS 23 patients fulfilled the indication for oxygen therapy and 11 patients agreed to participate in the study. After oxygen therapy, the apnea-hypopnea index (AHI) and the frequencies of hypopnea and central apnea were significantly decreased (AHI: from 31.1 +/- 8.8 to 12.7 +/- 8.5, p < 0.01; hypopnea: from 19.5 +/- 4.3 to 3.5 +/- 3.2, p < 0.01; central apnea: from 4.0 +/- 4.0 to 0.8 +/- 1.2, p < 0.05), whereas that of obstructive apnea was not changed. An analysis of heart rate variability showed that oxygen therapy did not alter autonomic activity after 1 month of oxygen therapy. CONCLUSIONS Nocturnal oxygen therapy decreases hypopnea and central apnea in PD patients with SAS. Nocturnal oxygen therapy may be useful for the treatment of SAS in PD patients, particularly when central apnea and hypopnea are predominant.

Guidelines for the management and investigation of hemolytic uremic syndrome.

The first guidelines for the diagnosis and treatment of HUS following the Shiga toxin producing Escherichia coli (STEC) infection was published by The Japanese Society of Pediatric Nephrology (JSPN) in 2000. Since then, there has been considerable advancement in the understanding and treatment of acute encephalopathy one of the most serious complications in HUS. Furthermore, the etiology, conditions and treatments of atypical HUS have been elucidated. Therefore, a set of comprehensive guidelines for HUS that reflects recent clinical evidence is necessary. The aim of this set of guidelines is to provide a support, tool for daily medical practice and to contribute to the standardization and accessibility of HUS-related medical care, as well as to improve level of safety for HUS patients.

Immunopathologic co-localization of MPO, IgG, and C3 in glomeruli in human MPO-ANCA-associated glomerulonephritis.

Myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA)- associated glomerulonephritis (GN) is characterized by pauci-immune necrotizing glomerulonephritis(NGN). Although it has been thought that MPO-ANCA is involved in the pathogenesis of capillary injuries in NGN via activation of neutrophils, recent studies suggest a possible role of other factors such as immunoglobulins precipitated on the glomeruli. Here we performed a pathological study investigating a relationship of deposition of MPO, IgG, complements with regard to MPO-positive cells and glomerular capillaries in human MPO-ANCA-associated GN. Renal specimen including 317 glomeruli obtained from 20 patients with MPOANCA- associated GN were analyzed. All of the specimens showed significant focal segmental deposition of IgG. There was a significant glomerular infiltration of MPO-positive cells along with deposition of extracellular MPO in the active lesions of segmental and global NCG, with CD34 staining being decreased in the adjacent areas. IgG deposits were almost colocalized with C3 and partly with MPO, which are also associated with a decrease in CD34 staining, suggesting that immune complex formation and the resultant capillary injuries. Actually occurred, the colocalization of MPO, IgG and C3 was seen only in the glomerular lesions with low severity and activity. These results suggest that not only MPO itself released from the neutrophils but also immune complexes composed of MPO and anti-MPO antibody may play some pathogenetic roles for the glomerular injuries especially in the early phase of human MPO-ANCA-associated GN.

Laboratory and imaging features of kidney involvement in autoimmune pancreatitis: incidence, correlation, and steroid therapy response.

AIM Autoimmune pancreatitis (AIP) is a rare subtype of chronic pancreatitis. AIP has been suggested to be complicated by tubulointerstitial nephritis or glomerulonephritis, implying that the kidney is involved as a phenotype of IgG4-positive multi-organ lymphoproliferative syndrome; however, the clinical significance of this novel entity is not well-defined. METHODS We conducted a retrospective cohort analysis of 47 (male, 39; female, 8) AIP patients. RESULTS The patients (mean age, 70.3 +/- 9.5 years) had a mean observation period of 4.1 years. Before treatment, renal dysfunction with an eGFR of 30 and 15 ml/min/1.73 m2 developed only in 10.6% (5/47) and 2.1% (1/47) of the patients, respectively. Nevertheless, urinary N-acetyl-beta-D-glucosaminidase and alpha1-microglobulin levels were elevated in 78.6% (11/14) and 30.8% (4/13) of the patients, respectively. Renal involvement in contrast-enhanced CT imaging was present in 18.2% (8/44) of the patients and was associated with proteinuria (p = 0.04) and a decrease in eGFR (p < 0.01). Furthermore, a follow-up CT study (mean, 545 days) revealed improved kidney lesions in 80.0% (4/5) of the patients after oral corticosteroid administration. In contrast, first-time kidney involvements appeared newly in 3.6% (1/28) of the patients after steroid therapy for nonrenal AIP symptoms, and in 14.3% (1/7) of the patients under no specific therapy (p = 0.02). CONCLUSION Although severe renal failure develops rarely in AIP patients, renal abnormalities have been significantly detected by biochemical and radiological tests. Oral corticosteroid administration, even when not targeting symptomatic nephropathy, can treat and prevent kidney involvements in AIP.