Masayuki Iwano

Reduced renal α-Klotho expression in CKD patients and its effect on renal phosphate handling and vitamin D metabolism.

Renal α-Klotho (α-KL) plays a fundamental role as a co-receptor for fibroblast growth factor 23 (FGF23), a phosphaturic hormone and regulator of 1,25(OH)2 vitamin D3 (1,25VitD3). Disruption of FGF23-α-KL signaling is thought to be an early hallmark of chronic kidney disease (CKD) involving reduced renal α-KL expression and a reciprocal rise in serum FGF23. It remains unclear, however, whether the rise in FGF23 is related to the loss of renal α-KL. We evaluated α-KL expression in renal biopsy samples and measured levels of several parameters of mineral metabolism, as well as soluble α-KL (sKL), in serum and urinary samples from CKD patients (nu200a=u200a236). We found that although renal α-KL levels were significantly reduced and serum FGF23 levels were significantly elevated in early and intermediate CKD, serum phosphate levels remained within the normal range. Multiple regression analysis showed that the increases in FGF23 were significantly associated with reduced renal function and elevated serum phosphate, but were not associated with loss of renal α-KL. Moreover, despite falling renal α-KL levels, the increase in FGF23 enhanced urinary fractional excretion of phosphate and reduced serum 1,25VitD3 levels in early and intermediate CKD, though not in advanced CKD. Serum sKL levels also fell significantly over the course of CKD, and renal α-KL was a significant independent determinant of sKL. These results demonstrate that FGF23 levels rise to compensate for renal failure-related phosphate retention in early and intermediate CKD. This enables FGF23-α-KL signaling and a neutral phosphate balance to be maintained despite the reduction in α-KL. In advanced CKD, however, renal α-KL declines further. This disrupts FGF23 signaling, and serum phosphate levels significantly increase, stimulating greater FGF23 secretion. Our results also suggest the serum sKL concentration may be a useful marker of renal α-KL expression levels.

Clinical impact of albuminuria and glomerular filtration rate on renal and cardiovascular events, and all-cause mortality in Japanese patients with type 2 diabetes

BackgroundThe number of patients suffering from diabetic nephropathy resulting in end-stage kidney disease is increasing worldwide. In clinical settings, there are limited data regarding the impact of the urinary albumin-to-creatinine ratio (UACR) and reduced estimated glomerular filtration rate (eGFR) on renal and cardiovascular outcomes and all-cause mortality.MethodsWe performed a historical cohort study of 4328 Japanese participants with type 2 diabetes from 10 centers. Risks for renal events (requirement for dialysis or transplantation, or half reduction in eGFR), cardiovascular events (cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke), and all-cause mortality were assessed according to UACR and eGFR levels.ResultsDuring follow-up (median 7.0xa0years, interquartile range 3.0–8.0xa0years), 419 renal events, 605 cardiovascular events and 236 deaths occurred. The UACR levels increased the risk and the adjusted hazard ratios for these three events. In addition to the effects of UACR levels, eGFR stages significantly increased the adjusted hazard ratios for renal events and all-cause mortality, especially in patients with macroalbuminuria. Diabetic nephropathy score, based on the prognostic factors, well predicted incidence rates per 1000 patient/year for each event.ConclusionsIncreased UACR levels were closely related to the increase in risks for renal, cardiovascular events and all-cause mortality in Japanese patients with type 2 diabetes, whereas the association between high levels of UACR and reduced eGFR was a strong predictor for renal events.

Suppressed soluble Fms–like tyrosine kinase-1 production aggravates atherosclerosis in chronic kidney disease

Patients with chronic kidney disease (CKD) die of cardiovascular diseases for unknown reasons. Blood vessel formation in plaques and its relationship with plaque stability could be involved with signaling through the Flt-1 receptor and its ligands, vascular endothelial growth factor, and the closely related placental growth factor (PlGF). Flt-1 also exists as a circulating regulatory splice variant short-inhibitory form (sFlt-1) that serves as a decoy receptor, thereby inactivating PlGF. Heparin releases sFlt-1 by displacing the sFlt-1 heparin-binding site from heparin sulfate proteoglycans. Heparin could provide diagnostic inference or could also induce an antiangiogenic state. In the present study, postheparin sFlt-1 levels were lower in CKD patients than in control subjects. More importantly, sFlt-1 levels were inversely related to atherosclerosis in CKD patients, and this correlation was more robust after heparin injection, as verified by subsequent cardiovascular events. Knockout of apolipoprotein E (ApoE) and/or sFlt-1 showed that the absence of sFlt-1 worsened atherogenesis in ApoE-deficient mice. Thus, the relationship between atherosclerosis and PlGF signaling, as regulated by sFlt-1, underscores the underappreciated role of heparin in sFlt-1 release. These clinical and experimental data suggest that novel avenues into CKD-dependent atherosclerosis and its detection are warranted.

Renal histological heterogeneity and functional progress in normoalbuminuric and microalbuminuric Japanese patients with type 2 diabetes.

Background and objectives Renal histological injury patterns in type 2 diabetes are heterogeneous. We compared renal histological injury patterns using renal biopsy findings with renal function and followed up renal functional changes in normoalbuminuric and microalbuminuric patients with type 2 diabetes to determine whether renal function progresses according to injury patterns. Design, setting, participants, and measurements We examined 111 patients with type 2 diabetes with percutaneous renal biopsy (78 men, 52±11u2005years old, 59 normoalbuminuria, 52 microalbuminuria) and followed up 37 cases for 11u2005years. Light microscopy of tissues revealed renal injury patterns as: category I (CI), normal or near-normal structure; category II (CII), typical diabetic glomerulopathy; category III (CIII), atypical (disproportionately severe tubulointerstitial/vascular damage with no/mild glomerulopathy). Results There were 29 CI, 62 CII, and 20 CIII patients. CII patients had a higher frequency of chronic kidney disease (CKD) G3-4, while the injury pattern distribution was not different among the albuminuria stages. The mean glomerular volume and volume fraction of cortical interstitium were larger than those of controls. The arteriolar hyalinosis index was larger in CII and CIII, while the percent global glomerular sclerosis was larger in CKD G3-4 compared with CKD G1-2. Renal function at follow-up was decreased in CII and CIII compared with the baseline estimated glomerular filtration rate (eGFR), while the GFR decline rate was faster in CII. Conclusions In normoalbuminuric and microalbuminuric patients with type 2 diabetes, loss of GFR could indicate typical diabetic glomerulosclerosis and a high frequency of global glomerular sclerosis. Urinary biomarkers identifying histological patterns of renal injury are necessary because GFR decline rates differed according to histological injury patterns.

Telmisartan activates endogenous peroxisome proliferator-activated receptor-δ and may have anti-fibrotic effects in human mesangial cells

Telmisartan, an angiotensin II receptor type 1 blocker (ARB), was recently reported to promote lipolysis in mice by acting as a peroxisome proliferator-activated receptor (PPAR)-δ activator, although in clinical studies, it has also been recognized to activate PPAR-γ as a major cause of its pleiotropic actions. The aim of this study was to investigate whether telmisartan activates endogenous PPAR-δ and thereby exerts anti-fibrotic effects in human mesangial cells (HMC). Immunohistochemical analysis of human renal biopsy specimens revealed that PPAR-δ protein was detected in the HMC of glomeruli with moderately proliferative changes. In the HMC, both GW0742, an authentic PPAR-δ agonist, and telmisartan enhanced PPAR response element (PPRE)-luciferase activity dose dependently, and these increases were blunted by GSK0660, a specific PPAR-δ antagonist, but not by GW9662, a PPAR-γ antagonist. Telmisartan also upregulated the expression of PPAR-δ target genes related to fatty acid oxidation; that is, heart type-fatty acid-binding protein and uncoupling protein-2. These effects were inhibited by both PPAR-δ antagonism and PPAR-δ gene silencing. Transforming growth factor-β1 (TGF-β1) increased the expression of plasminogen activator inhibitor-1 (PAI-1), TGF-β1 and collagen IV. The PAI-1 expression was mediated, at least in part by the phosphorylation of extracellular signal-regulated kinases (ERKs). Telmisartan suppressed TGF-β1-stimulated PAI-1 and collagen IV expression and ERK phosphorylation, and these effects were weakened by PPAR-δ antagonism, whereas eprosartan, a non-PPAR activating ARB, did not affect TGF-β1-stimulated PAI-1 expression. These results indicate that in HMC telmisartan activates endogenous PPAR-δ and may prevent TGF-β1-induced fibrotic changes by reducing ERK phosphorylation in a PPAR-δ-dependent manner, and thus, might be useful for treating hypertensive patients with renal and metabolic disorders.

Significance of combined cyclosporine−prednisolone therapy and cyclosporine blood concentration monitoring for idiopathic membranous nephropathy with steroid-resistant nephrotic syndrome: a randomized controlled multicenter trial

BackgroundCombined treatment with cyclosporine microemulsion preconcentrate (CyA MEPC) and steroids has been widely used for idiopathic membranous nephropathy (IMN) associated with steroid-resistant nephrotic syndrome (SRNS). Recent studies have shown that once-a-day and preprandial administration of CyA MEPC is more advantageous than the conventional twice-a-day administration in achieving the target blood CyA concentration at 2xa0h post dose (C2). We designed a randomized trial to compare these administrations.MethodsIMN patients with SRNS (age 16–75xa0years) were divided prospectively and randomly into 2 groups. In group 1 (nxa0=xa023), 2–3xa0mg/kg body weight (BW) CyA MEPC was given orally once a day before breakfast. In group 2 (nxa0=xa025), 1.5xa0mg/kg BW CyA MEPC was given twice a day before meals. CyAxa0+xa0prednisolone was continued for 48xa0weeks.ResultsGroup 1 showed a significantly higher cumulative complete remission (CR) rate (pxa0=xa00.0282), but not when incomplete remission 1 (ICR1; urine protein 0.3–1.0xa0g/day) was added (pxa0=xa00.314). Because a C2 of 600xa0ng/mL was determined as the best cut-off point, groups 1 and 2 were further divided into subgroups A (C2xa0≥600xa0ng/mL) and B (C2xa0<600xa0ng/mL). Groups 1A and 2A revealed significantly higher cumulative remission (CRxa0+xa0ICR1) (pxa0=xa00.0069) and CR-alone (pxa0=xa00.0028) rates. On the other hand, 3 patients with high CyA levels (C2xa0>900xa0ng/mL) in Group 1A were withdrawn from the study because of complications.ConclusionCyAxa0+xa0prednisolone treatment is effective for IMN with associated SRNS at a C2 of ≥600xa0ng/mL. To achieve remission, preprandial once-a-day administration of CyA at 2–3xa0mg/kg BW may be the most appropriate option. However, we should adjust the dosage of CyA by therapeutic drug monitoring to avoid complications.

Renal resistive index correlates with peritubular capillary loss and arteriosclerosis in biopsy tissues from patients with chronic kidney disease.

BackgroundThe renal resistive index (RI) is a Doppler-derived measure that reportedly correlates with renal histological changes and renal disease severity and outcome. The aim of this study was to investigate the factors related to the RI elevation in chronic kidney disease (CKD).MethodsUsing Doppler ultrasonography, RIs were determined in 30 patients with CKD, after which they were correlated with interstitial fibrosis, arteriosclerosis, arteriolosclerosis and peritubular capillary (PTC) density. PTC-positive areas were determined based on CD34 immunostaining. Interstitial fibrosis was detected with Masson trichrome staining. All histological markers were assessed using quantitative and semi-quantitative analyses and evaluated statistically using Pearson correlation tests, unpaired t tests and stepwise multiple regression analysis.ResultsRI correlated positively with age (rxa0=xa00.603, pxa0=xa00.0004), systolic blood pressure (rxa0=xa00.775, pxa0<xa00.0001), diastolic blood pressure (rxa0=xa00.575, pxa0=xa00.001), interstitial fibrosis (rxa0=xa00.381, pxa0=xa00.038) and arteriosclerosis (rxa0=xa00.520, pxa0=xa00.003), and negatively with creatinine clearance (rxa0=xa0−0.471, pxa0=xa00.009) and CD34+ (PTC) areas (rxa0=xa0−0.437, pxa0=xa00.016). Patients with hypertension or diabetes mellitus showed higher RIs (pxa0<xa00.05) than those without the ailments. Multivariate analysis showed PTC and arteriosclerosis to be independent variables correlating with RI (r2xa0=xa00.321, pxa0<xa00.05).ConclusionsTo our knowledge, this is the first report of using RI measurements to evaluate peritubular capillary loss. Our findings indicate that increases in RI are associated with both arteriosclerosis and loss of PTCs.

Renal redox dysregulation in AKI: application for oxidative stress marker of AKI

Oxidative stress is a major determinant of acute kidney injury (AKI); however, the effects of an AKI on renal redox system are unclear, and few existing AKI markers are suitable for evaluating oxidative stress. We measured urinary levels of the redox-regulatory protein thioredoxin 1 (TRX1) in patients with various kinds of kidney disease and in mice with renal ischemia-reperfusion injury. Urinary TRX1 levels were markedly higher in patients with AKI than in those with chronic kidney disease or in healthy subjects. In a receiver operating characteristic curve analysis to differentiate between AKI and other renal diseases, the area under the curve for urinary TRX1 was 0.94 (95% confidence interval, 0.90-0.98), and the sensitivity and specificity were 0.88 and 0.88, respectively, at the optimal cutoff value of 43.0 μg/g creatinine. Immunostaining revealed TRX1 to be diffusely distributed in the tubules of normal kidneys, but to be shifted to the brush borders or urinary lumen in injured tubules in both mice and humans with AKI. Urinary TRX1 in AKI was predominantly in the oxidized form. In cultured human proximal tubular epithelial cells, hydrogen peroxide specifically and dose dependently increased TRX1 levels in the culture supernatant, while reducing intracellular levels. These findings suggest that urinary TRX1 is an oxidative stress-specific biomarker useful for distinguishing AKI from chronic kidney disease and healthy kidneys.

Rapid Detection of the Mycobacterium tuberculosis Complex by Use of Quenching Probe PCR (geneCube)

Early detection of tuberculosis (TB) is essential for infection control. The GENECUBE (TOYOBO) is a novel fully automated gene analyzer that can amplify target DNAs within 60 min. In this study, we evaluated the ability of the GENECUBE to directly detect Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium complex (MAC) in clinical specimens. The results were then compared with those obtained using conventional culture, microscopy and the COBAS AMPLICOR (Roche). We examined a total of 516 frozen samples from 69 patients who showed culture-positive infection (73 samples; 39 MTBC, 32 MAC and 2 mixed infections), and from 354 patients who were culture-negative (443 samples). Assays using the GENECUBE had a sensitivity of 85.4% and a specificity of 99.8% for detection of MTBC, and a sensitivity of 85.3% and a specificity of 99.8% for detection of MAC. These results are similar to those obtained using the AMPLICOR system, but were obtained much more rapidly (1 h with the GENECUBE vs. 5.5 h with the AMPLICOR). The GENECUBE thus enables a significant shortening of the assay time with no loss of sensitivity or specificity.ABSTRACT Early detection of tuberculosis (TB) is essential for infection control. The geneCube (Toyobo) is a novel fully automated gene analyzer that can amplify target DNAs within 60 min. In this study, we evaluated the ability of the geneCube to directly detect Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium complex (MAC) in clinical specimens. The results were then compared with those obtained using conventional culture, microscopy, and the Cobas Amplicor assay (Roche). We examined a total of 516 frozen samples from 69 patients who showed culture-positive infection (73 samples; 39 MTBC, 32 MAC, and 2 mixed infections) and from 354 patients who were culture negative (443 samples). Assays using the geneCube had a sensitivity of 85.4% and a specificity of 99.8% for detection of MTBC and a sensitivity of 85.3% and a specificity of 99.8% for detection of MAC. These results are similar to those obtained using the Amplicor system but were obtained much more rapidly (1 h with the geneCube versus 5.5 h with the Amplicor system). The geneCube thus enables a significant shortening of the assay time with no loss of sensitivity or specificity.

Short-chain fatty acids, GPR41 and GPR43 ligands, inhibit TNF-α-induced MCP-1 expression by modulating p38 and JNK signaling pathways in human renal cortical epithelial cells

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are produced predominantly by gut microbiota fermentation of dietary fiber. SCFAs are newly identified as endogenous ligands of two orphan G protein-coupled receptors, GPR41 and GPR43, which have the potential to modulate inflammation. Therefore, GPR41 and GPR43 may mediate the link between the gut microbiome status and various disease conditions including renal inflammation. This study aimed at investigating whether SCFAs activate GPR41 and GPR43, and thereby exert anti-inflammatory effects in human renal cortical epithelial cells (HRCEs) as a main component of kidney tissue. Immunohistochemical analyses of human renal biopsy specimens revealed the expression of GPR41 and GPR43 protein in the distal renal tubules and collecting tubules. TNF-α increased the expression of monocyte chemoattractant protein-1 (MCP-1), a potential fibrotic inducer, at least partly via enhancing phosphorylation of p38 and JNK in HRCEs. SCFAs, especially propionate, attenuated TNF-α- stimulated MCP-1 expression by inhibiting the phosphorylation of p38 and JNK. This inhibitory effect was considerably attenuated by an inactivator of the Gi/o-type G protein and a Gβγ (i/o) blocker, but not by a Gα (i/o) blocker. Furthermore, SCFA-mediated inhibition of MCP-1 expression was significantly blocked by siRNA-induced gene silencing of GPR41 and GPR43. In conclusion, SCFAs lowered TNF-α-induced MCP-1 expression by reducing phosphorylation of p38 and JNK in a GPR41/43-dependent manner in HRCEs, suggesting that SCFA modification may be a new therapeutic tool for preventing progression of renal inflammation and fibrosis.