Tetsu Akimoto

Characteristics of urinary and serum soluble Klotho protein in patients with different degrees of chronic kidney disease.

BackgroundKlotho is a single-pass transmembrane protein, which appears to be implicated in aging. The purpose of the present study was to characterize the relationship between the soluble Klotho level and renal function in patients with various degrees of chronic kidney disease (CKD).MethodsThe levels of soluble Klotho in the serum and urine obtained from one hundred thirty-one CKD patients were determined by a sandwich enzyme-linked immunosorbent assay system.ResultsThe amount of urinary excreted Klotho during the 24 hr period ranged from 1.6 to 5178 ng/day (median 427 ng/day; interquartile range [IR] 56.8-1293.1), and the serum Klotho concentration ranged from 163.9 to 2123.7 pg/ml (median 759.7 pg/ml; IR 579.5-1069.1). The estimated glomerular filtration rate (eGFR) was significantly correlated with the log-transformed values of the amount of 24 hr urinary excreted Klotho (r = 0.407, p < 0.01) and the serum Klotho levels (r = 0.232, p < 0.01). However, a stepwise multiple regression analysis identified eGFR to be a variable independently associated only with the log-transformed value of the amount of 24-hr urinary excreted Klotho but not with the log-transformed serum Klotho concentration. Despite the strong correlation between random urine protein-to-creatinine ratio and the 24 hr urinary protein excretion (r = 0.834, p < 0.01), a moderate linear association was observed between the log-transformed value of the amount of 24 hr urinary excreted Klotho and that of the urinary Klotho-to-creatinine ratio (Klotho/Cr) in random urine specimens (r = 0.726, p < 0.01).ConclusionsThe amount of urinary Klotho, rather than the serum Klotho levels, should be linked to the magnitude of the functioning nephrons in CKD patients. The use of random urine Klotho/Cr as a surrogate for the amount of 24-hr urinary excreted Klotho needs to be evaluated more carefully.

AVP inhibits LPS- and IL-1β-stimulated NO and cGMP via V1 receptor in cultured rat mesangial cells

The present study examined how arginine vasopressin (AVP) affects nitric oxide (NO) metabolism in cultured rat glomerular mesangial cells (GMC). GMC were incubated with test agents and nitrite, and intracellular cGMP content, inducible nitric oxide synthase (iNOS) mRNA, and iNOS protein were analyzed by the Griess method, enzyme immunoassay, and Northern and Western blotting, respectively. AVP inhibited lipopolysaccharide (LPS)- and interleukin-1β (IL-1β)-induced nitrite production in a dose- and time-dependent manner, with concomitant changes in cGMP content, iNOS mRNA, and iNOS protein. This inhibition by AVP was reversed by V1- but not by oxytocin-receptor antagonist. Inhibition by AVP was also reproduced on LPS and interferon-γ (IFN-γ). Protein kinase C (PKC) inhibitors reversed AVP inhibition, whereas PKC activator inhibited nitrite production. Although dexamethasone and pyrrolidinedithiocarbamate (PDTC), inhibitors of nuclear factor-κB, inhibited nitrite production, further inhibition by AVP was not observed. AVP did not show further inhibition of nitrite production with actinomycin D, an inhibitor of transcription, or cycloheximide, an inhibitor of protein synthesis. In conclusion, AVP inhibits LPS- and IL-1β-induced NO production through a V1 receptor. The inhibitory action of AVP involves both the activation of PKC and the transcription of iNOS mRNA in cultured rat GMC.

The effect of erythropoietin on interleukin-1β mediated increase in nitric oxide synthesis in vascular smooth muscle cells

OBJECTIVE Recently, we observed that recombinant human erythropoietin (rHuEPO) inhibits the interleukin (IL)-1beta induced nitric oxide (NO) production and inducible NO synthase (iNOS) expression in cultured rat vascular smooth muscle cells (VSMC). The mechanisms of these inhibitory effects of rHuEPO were evaluated. METHODS Reverse transcription-polymerase chain reaction (RT-PCR) was performed to identify a specific erythropoietin receptor (EpoR). Tyrosine phosphorylation of phospholipase C (PLC) was analyzed by combination of immunoprecipitation and Western blotting. Protein kinase C (PKC) activities were analyzed by phosphorylation assay of myelin basic protein (MBP4-14). VSMC were incubated with test agents for 24 h and nitrite as a stable NO metabolite was measured. iNOS mRNA and protein expression was analyzed by Northern and Western blotting, respectively. RESULTS RT-PCR analysis revealed that EpoR m-RNA was expressed; furthermore, it might be alternatively spliced in VSMC. rHuEPO induced tyrosine phosphorylation of PLC-gamma1 and activation of PKC. rHuEPO inhibited not only IL-1beta induced nitrite production, but also the expression of iNOS mRNA and protein. These inhibitory effects of rHuEPO were reversed in the presence of PKC inhibitors, calphostin C (1 pmol/l) or staurosporine (10 nmol/l). PKC activation by phorbol myristate acetate inhibited nitrite production. The inhibitory effect of rHuEPO on IL-1beta induced nitrite production was also eliminated in PKC depleted cells or in the existence of anti-EpoR antibody. CONCLUSION rHuEPO inhibits IL-1beta induced NO production by suppressing iNOS mRNA and protein expressions through EpoR, and the PLC-gamma1 and PKC pathway may be involved.

Clinical Features of Malignant Hypertension with Thrombotic Microangiopathy

Thrombocytopenia, microangiopathic hemolytic anemia, and elevated serum lactate dehydrogenase (LDH) clinically characterize thrombotic microangiopathy (TMA), which is frequently recognized among patients with malignant hypertension (MH). Sixteen consecutive patients with MH were retrospectively investigated over a 7-year period and clinical features of the subjects with TMA were evaluated. We confirmed TMA relevant to MH by the normalization of the platelet count and LDH after adequate blood pressure (BP) control was achieved. Thrombotic microangiopathy was found in 7 (44%) of 16 patients. All 7 patients had an elevated plasma renin activity (PRA). Although no significant differences were observed in PRA, the patients with TMA had a significantly higher plasma aldosterone (ALDO) (median: 403 pg/ml; IR: 305 to 568) in comparison to those without TMA (median: 220 pg/ml; IR: 147 to 287; p = 0.013). Overall, ALDO correlated with LDH (r = 0.634, p = 0.0095). However, no significant association was observed between PRA and LDH (r = 0.336, p = 0.2263). The median platelet count nadir of the patients with TMA was 8.4 × 104 per μl (IR: 7.15 to 9.95). Thrombocytopenia and elevated LDH were normalized, along with a gradual improvement of BP within an average of 5 days and 21.7 days, respectively. These results suggest that ALDO, but not PRA, may act as a potent indicator of the magnitude of vascular and organ damage related to TMA among patients with malignant hypertension (MH).

Microscopic Hematuria and Diabetic Glomerulosclerosis – Clinicopathological Analysis of Type 2 Diabetic Patients Associated with Overt Proteinuria

Background/Aims: The information available concerning the qualitative and quantitative clinical variables in cases with pathologically defined diabetic glomerulosclerosis (DGs) has been insufficient so far. In addition, the prevalence and composition of nondiabetic renal disease (NDRD) among proteinuric diabetics still remain to be delineated. Methods: The glomerular pathology, clinical correlates, and the prevalence of NDRD were retrospectively analyzed in 50 type 2 proteinuric diabetics who underwent a renal biopsy between 1990 and 2006. The patients were divided into two groups according to clinical and pathological features. Thereafter, the diagnostic contribution of the laboratory and clinical variables that were significant between the two groups were determined by logistic regression analysis. Results: There were 34 cases with pure DGs and 15 cases (30%) had NDRD with or without DGs. Although the difference in the prevalence of microscopic hematuria between these two groups was significant, it was no longer statistically significant when the patients were limited to nephrotic cases. We identified 14 hematuric cases with pathologically defined DGs, and they all had a significantly lower renal function than nonhematuric patients with DGs. The prevalence of nephrotic syndrome and retinopathy were significantly higher in the cases with hematuric DGs than in the cases with nonhematuric DGs. Based on a logistic regression analysis, the presence of nephrotic syndrome and known duration of diabetes were identified to be significant predictors for hematuria with DGs. Conclusions: Our observations suggest that the presence of hematuria may be a common feature for DGs with nephrotic syndrome.

The Impact of Nephrectomy and Renal Transplantation on Serum Levels of Soluble Klotho Protein

BACKGROUND Klotho, a single-pass transmembrane protein primarily expressed in the kidneys, parathyroid glands, and choroid plexus of the brain, has a short cytoplasmic tail and a long extracellular domain, which can be cleaved and released as a soluble form. However, information regarding the origins and kinetics of soluble serum Klotho remains poorly understood. We evaluated serial changes in serum Klotho levels among living donors before and after retroperitoneoscopic nephrectomy as well as in their renal transplant recipients. METHODS The levels of soluble Klotho in serum obtained from 10 living donors and their renal transplant recipients were determined using a sandwich enzyme-linked immunosorbent assay system. RESULTS Serum soluble Klotho was detectable in all subjects. The baseline serum Klotho concentrations in the living donors ranged from 726.4 to 1417.1 pg/mL (median, 909.8 pg/mL; interquartile ranges [IR], 754.8-1132.4), whereas that in the concomitant renal transplant recipients ranged from 397.5 to 1047.2 pg/mL (median, 613.0 pg/mL; IR, 445.9-750.8; P = .003). The levels of soluble serum Klotho measured 5 days after retroperitoneoscopic nephrectomy (median, 619.0 pg/mL; IR, 544.6-688.5; P = .001) were significantly lower than the baseline values. Among the renal transplant recipients, no significant changes in serum Klotho levels were observed during the observation period. CONCLUSION Our data regarding soluble serum Klotho levels obtained from living donors support the idea that the kidneys are a major source of soluble serum Klotho in human subjects without a deterioration of renal function. In recipients, concomitant acute kidney injuries and immunosuppressive protocols might modulate the release of soluble Klotho from the grafts into the circulation.

Febuxostat for Hyperuricemia in Patients with Advanced Chronic Kidney Disease

Febuxostat is a nonpurine xanthine oxidase (XO) inhibitor, which recently received marketing approval. However, information regarding the experience with this agent among advanced chronic kidney disease (CKD) patients is limited. In the current study, we investigated the effects of oral febuxostat in patients with advanced CKD with asymptomatic hyperuricemia. We demonstrated, for the first time, that not only the serum levels of uric acid (UA) but also those of 8-hydroxydeoxyguanosine, an oxidative stress marker, were significantly reduced after six months of febuxostat treatment, with no adverse events. These results encouraged us to pursue further investigations regarding the clinical impact of lowering the serum UA levels with febuxostat in advanced CKD patients in terms of concomitantly reducing oxidative stress via the blockade of XO. More detailed studies with a larger number of subjects and assessments of the effects of multiple factors affecting hyperuricemia, such as age, sex, and dietary habits, would shed light on the therapeutic challenges of treating asymptomatic hyperuricemia in patients with various stages of CKD.

Ascites from patients with encapsulating peritoneal sclerosis augments NIH/3T3 fibroblast proliferation.

Abstract:  Encapsulating peritoneal sclerosis (EPS) remains one of the major causes of dropout in continuous ambulatory peritoneal dialysis by reducing ultrafiltration capacity. To demonstrate whether ascites from patients with EPS (EPS ascites) has fibroblast proliferation activity, we used NIH/3T3 fibroblasts to examine the effects of EPS ascites on fibroblast proliferation activity by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Encapsulating peritoneal sclerosis ascites dose‐dependently augmented NIH/3T3 fibroblast proliferation. The protein kinase C inhibitors and the tyrosine kinase inhibitors partially inhibited the stimulatory effects of EPS ascites on fibroblast proliferation activity. In EPS ascites, levels of interleukin (IL)‐1β, IL‐6, IL‐8, transforming growth factor (TGF)‐β1, hepatocyte growth factor (HGF), and platelet‐derived growth factor (PDGF)‐AB were elevated. The treatment with IL‐1β, HGF, TGF‐β1, and PDGF‐AB alone or in combination at similar concentrations to those in EPS ascites exhibited small but significant fibroblast proliferation activities. We conclude that EPS ascites stimulate NIH/3T3 fibroblast proliferation via protein kinase C and tyrosine kinase. The elevated cytokine and growth factors partly contribute to the EPS ascites‐induced fibroblast proliferation.

Effects of Raloxifene on Bone Metabolism in Hemodialysis Patients With Type 2 Diabetes

Background Osteoporosis and chronic kidney disease are common conditions in older adults, and often occur concurrently. Bone disease is caused by increased bone turnover accompanying secondary hyperparathyroidism, and by factors such as bone metabolic disorder accompanying kidney disease and postmenopausal or age-related osteoporosis, even in hemodialysis patients. Raloxifene is commonly used for the treatment of postmenopausal osteoporosis in the general population, and may be a treatment option for osteoporosis in hemodialysis patients. However, the effects of raloxifene in hemodialysis patients with type 2 diabetes have not been examined in detail. Objectives This study was performed to investigate the effects of raloxifene on bone turnover markers and bone density in postmenopausal women with type 2 diabetes mellitus who were undergoing hemodialysis in Japan. Patients and Methods The subjects were 60 female patients on maintenance hemodialysis (non-diabetic, n=30; diabetic, n=30). Raloxifene hydrochloride (60 mg) was administered to 14 diabetic patients and 14 non-diabetic patients for one year, and these patients were compared with control groups (no raloxifene) of 16 diabetic patients and 16 non-diabetic patients. Serum levels of N-terminal cross-linking telopeptide of type I collagen (NTx), bone alkaline phosphatase, and intact parathyroid hormone (iPTH) were measured, and bone density was determined by quantitative heel ultrasound at the speed of sound (SOS) in the calcaneus during this period. Results There were no significant differences in the levels of bone turnover markers except for iPTH after treatment of diabetic and non-diabetic patients with raloxifene for one year. SOS increased after treatment with raloxifene, but was significantly decreased in the control groups. Treatment with raloxifene resulted in a significant decrease in NTx and a significant increase in SOS in both diabetic and non-diabetic patients. There were no significant differences between the diabetic and non-diabetic patients who received raloxifene. Conclusions Treatment with raloxifene can suppress reduction in bone density in postmenopausal women with type 2 diabetes who are undergoing hemodialysis.

Central venous stenosis among hemodialysis patients is often not associated with previous central venous catheters.

It is widely assumed that central venous stenosis (CVS) is most commonly associated with previous central venous catheterization among the chronic hemodialysis (HD) patients. We evaluated the validity of this assumption in this retrospective study. The clinical records from 2,856 consecutive HD patients with vascular access failure during a 5-year period were reviewed, and a total of 26 patients with symptomatic CVS were identified. Combined with radiological findings, their clinical characteristics were examined. Only seven patients had a history of internal jugular dialysis catheterization. Diagnostic multidetector row computed tomography angiography showed that 7 of the 19 patients with no history of catheterization had left innominate vein stenosis due to extrinsic compression between the sternum and arch vessels. These patients had a shorter period from the time of creation of the vascular access to the initial referral (9.2 ± 7.6 months) than the rest of the patients (35.5 ± 18.6 months, p = 0.0017). Our findings suggest that cases without a history of central venous catheterization may not be rare among the HD patients with symptomatic CVS. However, those still need to be confirm by larger prospective studies of overall chronic HD patients with symptomatic CVS.