Itsuro Kazama

Leucine-Rich Repeat-Containing G Protein-Coupled Receptor-4 (LGR4, Gpr48) Is Essential for Renal Development in Mice

Leucine-rich repeat-containing G protein-coupled receptor (LGR)-4 is a G protein-coupled receptor (GPCR) with a seven-transmembrane domain structure. LGRs are evolutionally and structurally phylogenetic, classified into three subgroups and are members of the so-called orphan receptors whose ligands have yet to be identified. We generated knockout mice lacking Lgr4(Gpr48) by targeted deletion of part of exon 18, which codes for the transmembrane and signal-transducing domains of the receptor. Lgr4 null mice were born at much less than the 25% expected frequency from crosses of Lgr4 heterozygous mice (Lgr4+/–). Lgr4 null mice that survived in utero died shortly after birth in almost all cases. We observed striking renal hypoplasia in the null mice, accompanied by elevated concentration of plasma creatinine. Histological analysis of the P0 null mouse kidney showed a notable decrease in the total number and density of the glomerulus. Thus, the function of Lgr4 is essential to regulate renal development in the mouse. This study suggests that the Lgr4 gene is a new and important member of LGRs involved in a group of genes responsible for hereditary disease in the kidney.

Angiotensin converting enzyme I/D polymorphism and hypertension : the Ohasama study

Objective Angiotensin-converting enzyme (ACE) I/D polymorphism in intron 16 of the ACE gene was analyzed in a general Japanese population in relation to self-blood pressure (BP) measurement at home (home BP) and ambulatory BP monitoring (ABPM) to determine the association between genetic variants of this polymorphism and hypertension. Design A cross-sectional study. Methods and results We genotyped the ACE I/D polymorphism in 1245 subjects with home BP and 803 subjects with ABPM in Ohasama, a rural community in Japan. All the subjects were 40 years of age and over, and gave written informed consent for the present genetic analysis. Hypertensive subjects were defined as those receiving antihypertensive drugs and those who had a home BP higher than 135 mmHg in systole and/or higher than 85 mmHg in diastole. The frequencies of the II, ID, and DD genotypes in these Japanese subjects were 0.45, 0.45, and 0.10, indicating a lower frequency of the D allele (0.33) than in Caucasians. There was no significant difference of BP level, prevalence of hypertension or nocturnal decline in BP among the genotypes. There were no differences in the prevalence of previous cardiovascular disease, age, body mass index, male gender, smoking, or biochemical and hormonal parameters among the three genotypes. Conclusion The present results indicate the absence of direct effects of the ACE D-allele on BP level, prevalence of hypertension, prevalence of cardiovascular disease, and circadian BP variation. We conclude there is little association between ACE I/D polymorphism and hypertension in the general Japanese population.

Suppressive effects of nonsteroidal anti-inflammatory drugs diclofenac sodium, salicylate and indomethacin on delayed rectifier K+-channel currents in murine thymocytes

Lymphocytes predominantly express delayed rectifier K+-channels (Kv1.3) in their plasma membranes, and the channels play crucial roles in the lymphocyte activation and proliferation. Since nonsteroidal anti-inflammatory drugs (NSAIDs), the most commonly used analgesic and antipyretic drugs, exert immunomodulatory effects, they would affect the channel currents in lymphocytes. In the present study, employing the standard patch-clamp whole-cell recording technique, we examined the effects of diclofenac sodium, salicylate and indomethacin on the channel currents in murine thymocytes and the membrane capacitance. Diclofenac sodium and salicylate significantly suppressed the pulse-end currents of the channel. However, indomethacin suppressed both the peak and the pulse-end currents with a significant increase in the membrane capacitance. This study demonstrated for the first time that NSAIDs, such as diclofenac sodium, salicylate and indomethacin, exert inhibitory effects on thymocyte Kv1.3-channel currents. The slow inactivation pattern induced by indomethacin was thought to be associated with microscopic changes in the plasma membrane surface detected by the increase in the membrane capacitance.

Voltage-dependent biphasic effects of chloroquine on delayed rectifier K+-channel currents in murine thymocytes

Lymphocytes are of rich in delayed rectifier K+-channels (Kv1.3) in their plasma membranes, and the channels play crucial roles in the lymphocyte activation and proliferation. Since chloroquine, a widely used anti-malarial drug, exerts immunosuppressive effects, it will affect the channel currents in lymphocytes. In the present study, employing the standard patch-clamp whole-cell recording technique, we examined the effects of chloroquine on the channels expressed in murine thymocytes. Published papers report that chloroquine will inhibit voltage-dependent K+-channel currents by plugging into the open-pore. We observed, indeed, that chloroquine suppressed the pulse-end currents of Kv1.3-channels at higher voltage steps. Surprisingly, however, we found that the drug enhanced the peak currents at both higher and lower voltage steps. Since chloroquine showed such biphasic effects on the thymocyte K+-channels, and since those effects were voltage dependent, we examined the effects of chloroquine on the activation and the inactivation of the channel currents. We noted that chloroquine shifted both the activation and the inactivation curves toward the hyperpolarizing potential, and that those shifts were more emphasized at lower voltage steps. We conclude that chloroquine facilitates both the activation and the inactivation of Kv1.3-channel currents in thymocytes, and that those effects are voltage dependent.

Urinary Concentration Defect and Limited Expression of Sodium Cotransporter, rBSC1, in a Rat Model of Chronic Renal Failure

Background: Renal urinary concentration is associated with enhanced expression of sodium cotransporter (rBSC1) in thick ascending limb of Henle. Overexpression of rBSC1 was reported recently in hypertrophied nephrons after unilateral nephrectomy (UNX) and in kidney isografts. Since urinary concentration defect and hypertrophy of residual nephrons are major manifestations of chronic renal failure (CRF), we investigated the rBSC1 signals for RNA and protein in a rat model of CRF. Methods: Rats underwent 5/6 nephrectomy and examined 8 weeks after operation. rBSC1 mRNA was examined by competitive PCR and in situ hybridization, and rBSC1 protein signals by western blotting and immunohistochemistry. Rats that underwent sham-operation, UNX, or 5/6 nephrectomy followed by a 3-week recovery period (acute renal failure), were used as control. Water intake was restricted for 24 h in subgroups of control and CRF rats. Results: Microscopic examination showed hypertrophy of residual nephrons in both UNX and CRF rats. Signals for rBSC1 mRNA and protein were enhanced at basal condition only in rats with UNX. Under basal conditions, CRF rats demonstrated low urinary osmolality in spite of high plasma arginine vasopressin levels. Water restriction resulted in increased signals for rBSC1 mRNA and protein and concentration of urine in sham-operated rats, but such increases were absent and urinary concentration was incomplete in CRF rats. Conclusions: Compensatory overexpression and upregulation of rBSC1 expression in response to dehydration are both absent in CRF rats. These limitations are thought to be the underlying mechanisms of urinary concentrating defect seen in CRF.

Benidipine persistently inhibits delayed rectifier K(+)-channel currents in murine thymocytes.

Lymphocytes predominantly express delayed rectifier K+-channels (Kv1.3) in their plasma membranes, and the channels play crucial roles in the lymphocyte activation and proliferation. Since 1,4-dihydropyridine (DHP) Ca2+ channel blockers (CCBs), which are highly lipophilic, exert relatively stronger immunomodulatory effects than the other types of CCBs, they would affect the Kv1.3-channel currents in lymphocytes. In the present study, employing the standard patch-clamp whole-cell recording technique in murine thymocytes, we examined the effects of benidipine, one of the most lipophilic DHPs, on the channel currents and the membrane capacitance and compared them with those of nifedipine. Both drugs significantly suppressed the peak and the pulse-end currents of the channels with significant decreases in the membrane capacitance. However, the effects of benidipine were more marked than those of nifedipine and were irreversible after the drug withdrawal. This study demonstrated for the first time that DHP CCBs, such as nifedipine and benidipine, exert inhibitory effects on thymocyte Kv1.3-channel currents. The persistent effect of benidipine was thought to be associated with its sustained accumulation in the plasma membranes as detected by the long-lasting decrease in the membrane capacitance.

Differential effects of clarithromycin and azithromycin on delayed rectifier K(+)-channel currents in murine thymocytes.

Abstract Context: Lymphocytes predominantly express delayed rectifier K+-channels (Kv1.3) in their plasma membranes, and the channels play crucial roles in the lymphocyte activation and proliferation. Since macrolide antibiotics, such as clarithromycin and azithromycin, exert immunomodulatory effects, they would affect the Kv1.3-channel currents in lymphocytes. Objective: This study determined the physiological involvement in the mechanisms of immunomodulation by these antibiotics. Materials and methods: Employing the standard patch-clamp whole-cell recording technique in murine thymocytes, we examined the effects of 30 and 100 µM clarithromycin and azithromycin on the Kv1.3-channel currents and the membrane capacitance. Results: Clarithromycin significantly suppressed the peak currents (30 µM, 178 ± 5.6 to 111 ± 2.0 pA/pF; 100 µM, 277 ± 4.4 to 89.6 ± 10 pA/pF) and the pulse-end currents (30 µM, 47.5 ± 2.2% to 15.5 ± 3.3%; 100 µM, 48.5 ± 1.4% to 15.8 ± 1.0%) of thymocyte Kv1.3-channels without significant effects on the membrane capacitance. In contrast, azithromycin did not affect the channel currents. However, it significantly decreased the membrane capacitance (30 µM, 4.68 ± 0.14 to 3.74 ± 0.13 pF; 100 µM, 4.47 ± 0.06 to 3.37 ± 0.08 pF), indicating its accumulation in the plasma membrane. Discussion and conclusion: This study demonstrated for the first time that clarithromycin exerts inhibitory effects on thymocyte Kv1.3-channel currents, while azithromycin decreases the membrane capacitance without affecting the channel currents. These differences in the effects of the macrolide antibiotics may reflect differences in the mechanisms of immunomodulation by which they control the production of cytokines.

Adult onset Fanconi syndrome : extensive tubulo-interstitial lesions and glomerulopathy in the early stage of Chinese herbs nephropathy

A 33-year-old woman was transferred to our hospital with a 5-month history of taking Chinese herbal medicine containing aristolochic acid. She presented with metabolic acidosis, severe anemia, hypophosphatemia and uric aciduria, and generalized aminoaciduria and glycosuria, features which were consistent with the clinical manifestations of Fanconi syndrome. Renal biopsy was performed when her plasma creatinine was 0.7 mg/dl and creatinine clearance was 46 ml/min per 1.73 m2. The renal specimen showed extensive interstitial edema with focal fibrosis, tubular atrophy, and focal glomerulopathy, which suggested the presence of glomerular endothelial damage or glomerular ischemia. Although steroid therapy ameliorated the plasma electrolyte levels, renal failure progressed, and hemodialysis therapy was initiated approximately 18 months after the time of renal biopsy. This patient demonstrated the early renal lesions of Chinese herbs nephropathy in association with various clinical manifestations. The characteristic glomerulopathy found in the present patient is considered to be an additional renal lesion, leading to the renal failure.

HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes

BACKGROUND Since lymphocytes predominantly express delayed rectifier K(+)-channels (Kv1.3) that trigger lymphocyte activation, statins, which exert immunosuppressive effects, would affect the channel currents. METHODS Employing the patch-clamp technique in murine thymocytes, we examined the effects of statins on Kv1.3-channel currents and the membrane capacitance (Cm). RESULTS Pravastatin significantly suppressed the pulse-end currents of the channels. Lovastatin and simvastatin also suppressed the peak currents, significantly decreasing the Cm. CONCLUSIONS This study demonstrated for the first time that statins inhibit thymocyte Kv1.3-channels. The slow inactivation patterns induced by lovastatin and simvastatin may be associated with their accumulation in the plasma membranes.

Compensatory thrombopoietin production from the liver and bone marrow stimulates thrombopoiesis of living rat megakaryocytes in chronic renal failure.

Background/Aims: Decreased thrombopoiesis has been ascribed a role in the pathogenesis of uremic bleeding in chronic renal failure (CRF). However, serum thrombopoietin (TPO) levels are usually elevated in CRF patients, suggesting increased thrombopoiesis. The aim of this study was to determine the thrombopoietic activity in CRF. Methods: Male Sprague-Dawley rats that underwent 5/6 nephrectomy were used as the model of CRF. Age-matched sham-operated rats were used as controls. Single megakaryocytes were isolated from the rat bone marrow, and their size distribution was examined. Megakaryocyte membrane invaginations were monitored by confocal imaging of di-8-ANEPPS staining, and patch clamp whole-cell recordings of membrane capacitance. TPO gene expression was assessed in various tissues. Results: Circulating platelet counts and the number of large megakaryocytes were increased in the bone marrow of CRF rats. Massive di-8-ANEPPS staining and increased membrane capacitance in large megakaryocytes demonstrated increased membrane invaginations. Unaffected Kv1.3-channel currents per cell surface area demonstrated unaltered channel densities. TPO transcription was decreased in the renal cortex but increased in the liver and bone marrow of CRF rats. Conclusion: Increased thrombopoiesis in CRF was thought to be a reactive mechanism to platelet dysfunction. Increased TPO production from the liver and bone marrow compensated for decreased production from damaged kidneys.