Masato Kasahara

Urinary neutrophil gelatinase-associated lipocalin levels reflect damage to glomeruli, proximal tubules, and distal nephrons.

Urinary neutrophil gelatinase-associated lipocalin (Ngal or lipocalin 2) is a very early and sensitive biomarker of kidney injury. Here we determined the origin and time course of Ngal appearance in several experimental and clinically relevant renal diseases. Urinary Ngal levels were found to be markedly increased in lipoatrophic- and streptozotocin-induced mouse models of diabetic nephropathy. In the latter mice, the angiotensin receptor blocker candesartan dramatically decreased urinary Ngal excretion. The reabsorption of Ngal by the proximal tubule was severely reduced in streptozotocin-induced diabetic mice, but upregulation of its mRNA and protein in the kidney was negligible, compared to those of control mice, suggesting that increased urinary Ngal was mainly due to impaired renal reabsorption. In the mouse model of unilateral ureteral obstruction, Ngal protein synthesis was dramatically increased in the dilated thick ascending limb of Henle and N was found in the urine present in the swollen pelvis of the ligated kidney. Five patients with nephrotic syndrome or interstitial nephritis had markedly elevated urinary Ngal levels at presentation, but these decreased in response to treatment. Our study shows that the urinary Ngal level may be useful for monitoring the status and treatment of diverse renal diseases reflecting defects in glomerular filtration barrier, proximal tubule reabsorption, and distal nephrons.

Growth-Dependent Induction of Angiotensin II Type 2 Receptor in Rat Mesangial Cells

Angiotensin II acts on at least two receptor subtypes, AT1 and AT2. Although the physiological role of the AT2 receptor is still poorly defined, it may be implicated in inhibition of cell growth, vasorelaxation, and apoptosis. In the present study, to investigate the role of the AT2 receptor in the kidney and its implication in hypertensive states, we examined its expression using cultured mesangial cells (MC) from normotensive Wistar-Kyoto rats (WKY) and from stroke-prone spontaneously hypertensive rats (SHRSP). Receptor binding assays were performed using a nonselective ligand, [Sar1,Ile8]angiotensin II, or AT2-selective CGP42112A. Binding assays revealed that MC from WKY exhibited both AT1 and AT2 receptors, the ratio of which was confluence-dependent. In contrast, MC from SHRSP, whose proliferation activity was much higher than those from WKY, showed only the AT1 subtype. In receptor binding and Northern blot analyses, expression of the AT2 receptor of WKY-MC was low in the growing state but significantly induced upon confluence to become abundant in the post-confluent state, whereas that of SHRSP-MC was undetectable in either state. Gene expressions of AT1A and AT1B receptors were not significantly altered in either strain during the time in culture. These results indicate that the mesangial AT2-receptor expression is growth-dependent and suggest a role in the inhibition of MC growth in WKY. Much lower expression of the AT2 receptor in MC from SHRSP may suggest involvement in their higher proliferation activity and possibly in consequent renal disorders.

Impact of Rituximab Desensitization on Blood‐Type‐Incompatible Adult Living Donor Liver Transplantation: A Japanese Multicenter Study

We evaluated the effects of rituximab prophylaxis on outcomes of ABO‐blood‐type‐incompatible living donor liver transplantation (ABO‐I LDLT) in 381 adult patients in the Japanese registry of ABO‐I LDLT. Patients underwent dual or triple immunosuppression with or without B cell desensitization therapies such as plasmapheresis, splenectomy, local infusion, intravenous immunoglobulin and rituximab. Era before 2005, intensive care unit‐bound status, high Model for End‐Stage Liver Disease score and absence of rituximab prophylaxis were significant risk factors for overall survival and antibody‐mediated rejection (AMR) in the univariate analysis. After adjustment for era effects in the multivariate analysis, only absence of rituximab prophylaxis was a significant risk factor for AMR, and there were no significant risk factors for survival. Rituximab prophylaxis significantly decreased the incidence of AMR, especially hepatic necrosis (p < 0.001). In the rituximab group, other B cell desensitization therapies had no add‐on effects. Multiple or large rituximab doses significantly increased the incidence of infection, and early administration had no advantage. In conclusion, outcomes in adult ABO‐I LDLT have significantly improved in the latest era coincident with the introduction of rituximab.

Effects of icodextrin on glycemic and lipid profiles in diabetic patients undergoing peritoneal dialysis.

Aim: Icodextrin reduces glucose absorption from the peritoneal dialysate. We conducted this prospective, open-labeled, multicenter study to determine the effects of icodextrin on glycemic and lipid parameters in diabetic patients undergoing continuous ambulatory peritoneal dialysis (PD) or automated PD. Methods: Patients were recruited from 15 institutions in Japan, and a total of 51 patients (15 women and 36 men, mean age: 59 ± 10 years, median duration of PD: 13 months) were enrolled. The patients were administered an overnight or daytime dwell of 1.5 or 2.0 l of 7.5% icodextrin-containing solution. At baseline and 3, 6, 9 and 12 months after the start of icodextrin, nonfasting blood was drawn for measurement of glycated hemoglobin (HbA1C) and serum lipids. Results: During icodextrin treatment, there was no change in overall HbA1C levels compared to baseline values; however, for those with baseline HbA1C ≧6.5% (n = 22), significant decreases in HbA1C were observed. Mean total/LDL cholesterol and triglycerides were decreased significantly during icodextrin treatment, with greater decreases for patients with baseline total cholesterol ≧220 mg/dl, LDL cholesterol ≧120 mg/dl or triglycerides ≧150 mg/dl. HDL cholesterol did not differ at any time point; however, values for patients with baseline HDL cholesterol <40 mg/dl tended to increase with marginal significance. Conclusions: In the current study, switching from glucose-containing dialysis solution to icodextrin resulted in improved lipid profiles and possibly a favorable metabolic profile, particularly in patients with poor glycemic control. These hypotheses remain to be proven in controlled clinical trials.

Natriuretic Peptide Receptor Guanylyl Cyclase-A Protects Podocytes from Aldosterone-Induced Glomerular Injury

Natriuretic peptides produced by the heart in response to cardiac overload exert cardioprotective and renoprotective effects by eliciting natriuresis, reducing BP, and inhibiting cell proliferation and fibrosis. These peptides also antagonize the renin-angiotensin-aldosterone system, but whether this mechanism contributes to their renoprotective effect is unknown. Here, we examined the kidneys of mice lacking the guanylyl cyclase-A (GC-A) receptor for natriuretic peptides under conditions of high aldosterone and high dietary salt. After 4 weeks of administering aldosterone and a high-salt diet, GC-A knockout mice, but not wild-type mice, exhibited accelerated hypertension with massive proteinuria. Aldosterone-infused GC-A knockout mice had marked mesangial expansion, segmental sclerosis, severe podocyte injury, and increased oxidative stress. Reducing the BP with hydralazine failed to lessen such changes; in contrast, blockade of the renin-angiotensin-aldosterone system markedly reduced albuminuria, ameliorated podocyte injury, and reduced oxidative stress. Furthermore, treatment with the antioxidant tempol significantly reduced albuminuria and abrogated the histologic changes. In cultured podocytes, natriuretic peptides inhibited aldosterone-induced mitogen-activated protein kinase phosphorylation. Taken together, these results suggest that renoprotective properties of the endogenous natriuretic peptide/GC-A system may result from the local inhibition of the renin-angiotensin-aldosterone system and oxidative stress in podocytes.

MicroRNA-26a inhibits TGF-β-induced extracellular matrix protein expression in podocytes by targeting CTGF and is downregulated in diabetic nephropathy

Aims/hypothesisThe accumulation of extracellular matrix (ECM) is a characteristic of diabetic nephropathy, and is partially caused by profibrotic proteins TGF-β and connective tissue growth factor (CTGF). We aimed to identify microRNAs (miRNAs) targeting CTGF on podocytes in diabetic nephropathy.MethodsWe investigated miRNAs targeting CTGF on podocytes with miRNA array analysis and identified a candidate miRNA, miR-26a. Using overexpression and silencing of miR-26a in cultured podocytes, we examined changes of ECM and its host genes. We further investigated glomerular miR-26a expression in humans and in mouse models of diabetic nephropathy.ResultsmiR-26a, which was downregulated by TGF-β1, was expressed in glomerular cells including podocytes and in tubules by in situ hybridisation. Glomerular miR-26a expression was downregulated by 70% in streptozotocin-induced diabetic mice. Transfection of miR-26a mimics in cultured human podocytes decreased the CTGF protein level by 50%, and directly inhibited CTGF expression in podocytes, as demonstrated by a reporter assay with the 3′-untranslated region of the CTGF gene. This effect was abolished by a mutant plasmid. miR-26a mimics also inhibited TGF-β1-induced collagen expression, SMAD-binding activity and expression of its host genes CTDSP2 and CTDSPL. Knockdown of CTDSP2 and CTDSPL increased collagen expression in TGF-β-stimulated podocytes, suggesting that host genes also regulate TGF-β/SMAD signalling. Finally, we observed a positive correlation between microdissected glomerular miR-26a expression levels and estimated GFR in patients with diabetic nephropathy.Conclusions/interpretationThe downregulation of miR-26a is involved in the progression of diabetic nephropathy both in humans and in mice through enhanced TGF-β/CTGF signalling.

Pleiotrophin triggers inflammation and increased peritoneal permeability leading to peritoneal fibrosis

Long-term peritoneal dialysis induces peritoneal fibrosis with submesothelial fibrotic tissue. Although angiogenesis and inflammatory mediators are involved in peritoneal fibrosis, precise molecular mechanisms are undefined. To study this, we used microarray analysis and compared gene expression profiles of the peritoneum in control and chlorhexidine gluconate (CG)-induced peritoneal fibrosis mice. One of the 43 highly upregulated genes was pleiotrophin, a midkine family member, the expression of which was also upregulated by the solution used to treat mice by peritoneal dialysis. This growth factor was found in fibroblasts and mesothelial cells within the underlying submesothelial compact zones of mice, and in human peritoneal biopsy samples and peritoneal dialysate effluent. Recombinant pleiotrophin stimulated mitogenesis and migration of mouse mesothelial cells in culture. We found that in wild-type mice, CG treatment increased peritoneal permeability (measured by equilibration), increased mRNA expression of TGF-β1, connective tissue growth factor and fibronectin, TNF-α and IL-1β expression, and resulted in infiltration of CD3-positive T cells, and caused a high number of Ki-67-positive proliferating cells. All of these parameters were decreased in peritoneal tissues of CG-treated pleiotrophin-knockout mice. Thus, an upregulation of pleiotrophin appears to play a role in fibrosis and inflammation during peritoneal injury.

Intra-abdominal fat area is a predictor for new onset of individual components of metabolic syndrome: MEtabolic syndRome and abdominaL ObesiTy (MERLOT study)

Objective: To investigate the significance of intra-abdominal fat area (IAFA) on new onset of individual components of the metabolic syndrome: high blood pressure, dyslipidemia, or hyperglycemia. Methods: We conducted a longitudinal study using checkup data of a hospital from 1994 to 2010. Of 25,255 subjects, we examined 1,380 Japanese, who underwent computed tomography to measure IAFA and had no metabolic syndrome components at baseline. Results: During 3.6 years of the mean follow-up period, one of metabolic syndrome components occurred in 752 subjects. Of three components, high blood pressure was more prevalent. The multiple Cox regression analysis disclosed that IAFA is significantly associated with onset of metabolic syndrome components (HR: 1.05 per 10 cm2, 95%CI: 1.03–1.07). This finding was independent of BMI, and significant even in non-obese individuals with body mass index <25 kg/m2. Conclusions: MERLOT study demonstrates that IAFA is an independent predictor for new onset of individual components of the metabolic syndrome, even in non-obese healthy Japanese.

Human atrial natriuretic peptide is a useful criterion in treatment of nocturia

BACKGROUND Nocturia is one of the major problems of elderly people. The possible causes of nocturia include irritation of lower urinary tract obstruction, disturbance of the fluid balance, and sleep disturbance. In a significant proportion of patients, the mechanism of nocturia is still unclear and a definitive method of treatment has yet to be determined. This study investigated how to treat nocturia. MATERIALS AND METHODS We analyzed 51 patients who visited Kobe City General Hospital between January 1998 and June 1999 with nocturia (three or more nocturnal voidings) and no daytime urological problems. Twenty-two of these patients were given daytime diuretic therapy (azosemide 60 mg), while 29 other patients received a minor tranquilizer. The method of treatment was selected randomly. Correlations between the plasma level of atrial natriuretic peptide (hANP) and the effect of treatment were analyzed. RESULTS Daytime diuretics decreased the nocturnal frequency of voiding in 10 out of 22 patients. The plasma hANP level at the first visit was significantly higher in the patients with improvement (p = 0.0026). Furthermore, the plasma hANP level was significantly decreased after daytime diuretic therapy in patients who showed improvement of nocturia with diuretic therapy (p = 0.0180). Minor tranquilizer administration decreased nocturia in 22 out of 29 patients. The plasma hANP level at the first visit was significantly lower in the patients who improved (p = 0.0021). CONCLUSIONS These findings suggest that patients with higher plasma hANP levels should be treated as having subclinical heart failure, while nocturia in patients with a normal plasma hANP level might be caused by sleep disturbance.Background: Nocturia is one of the major problems of elderly people. The possible causes of nocturia include irritation of lower urinary tract obstruction, disturbance of the fluid balance, and sleep disturbance. In a significant proportion of patients, the mechanism of nocturia is still unclear and a definitive method of treatment has yet to be determined. This study investigated how to treat nocturia. Materials and methods: We analyzed 51 patients who visited Kobe City General Hospital between January 1998 and June 1999 with nocturia (three or more nocturnal voidings) and no daytime urological problems. Twenty-two of these patients were given daytime diuretic therapy (azosemide 60 mg), while 29 other patients received a minor tranquilizer. The method of treatment was selected randomly. Correlations between the plasma level of atrial natriuretic peptide (hANP) and the effect of treatment were analyzed. Results: Daytime diuretics decreased the nocturnal frequency of voiding in 10 out of 22 patients. The plasma hANP level at the first visit was significantly higher in the patients with improvement (p = 0.0026). Furthermore, the plasma hANP level was significantly decreased after daytime diuretic therapy in patients who showed improvement of nocturia with diuretic therapy (p = 0.0180). Minor tranquilizer administration decreased nocturia in 22 out of 29 patients. The plasma hANP level at the first visit was significantly lower in the patients who improved (p = 0.0021). Conclusions: These findings suggest that patients with higher plasma hANP levels should be treated as having subclinical heart failure, while nocturia in patients with a normal plasma hANP level might be caused by sleep disturbance.

Adrenomedullin inhibits connective tissue growth factor expression, extracellular signal-regulated kinase activation and renal fibrosis

Systemic administration of the potent vasodilating peptide adrenomedullin reduces cardiac and renal fibrosis in hypertensive animals. Here, we investigated the effects of kidney-specific adrenomedullin gene delivery in normotensive rats after unilateral ureteral obstruction, an established model of renal tubulointerstitial fibrosis. Overexpression of exogenous adrenomedullin in the renal interstitium following ureteral obstruction significantly prevented fibrosis and proliferation of tubular and interstitial cells. In this model, there is upregulation of connective tissue growth factor (CTGF) mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation, and adrenomedullin overexpression suppressed both of these activities without altering the blood pressure. In NRK-49F renal fibroblasts, adrenomedullin reduced transforming growth factor-beta-induced CTGF and fibronectin mRNA upregulation through the cyclic AMP/protein kinase A signaling pathway, and suppressed ERK phosphorylation and cell proliferation. In the kidneys with an obstructed ureter, adrenomedullin receptor gene expression was upregulated along with cyclic AMP production in kidney slices. The latter effect was partially blocked by a neutralizing antibody to adrenomedullin, indicating that an endogenous peptide-receptor system was activated. Our results show that overexpression of exogenous adrenomedullin in the ureteral-obstructed kidney prevents tubulointerstitial fibrosis and cell proliferation through the cyclic AMP-mediated decrease of CTGF induction and ERK phosphorylation.