Loss of ezrin expression reduced the susceptibility to the glomerular injury in mice.

Ezrin is highly expressed in glomerular podocytes and is reported to form a multi-protein complex with scaffold protein Na+/H+ exchanger regulatory factor 2 (NHERF2) and podocalyxin, a major sialoprotein. Podocalyxin-knockout mice died within 24 h of birth with anuric renal failure, whereas NHERF2-knockout mice show no apparent changes in the glomerular functions. However, the physiological roles of ezrin in glomerular podocytes remain unclear. Here, we investigated the importance of ezrin in the regulation of glomerular podocyte function using ezrin-knockdown mice (Vil2 kd/kd ). The Vil2 kd/kd mice did not exhibit apparent glomerular dysfunction, morphological defects or abnormal localisation of podocalyxin and NHERF2 in podocytes. Thus, we investigated the influence of ezrin defects on Rho-GTPase activity, as ezrin interacts with the Rho-GTPase dissociation inhibitor (Rho-GDI), which plays a key role in the regulation of podocyte actin organisation. In Vil2 kd/kd glomeruli, Rac1 activity was significantly reduced compared to wildtype (WT) glomeruli at baseline. Furthermore, Vil2 kd/kd mice showed reduced susceptibility to glomerular injury. In WT glomeruli, Rac1 activity was enhanced in nephrotic conditions, but remained at baseline levels in Vil2 kd/kd glomeruli, suggesting that loss of ezrin protects podocytes from injury-induced morphological changes by suppressing Rac1 activation.

Internalization of NKCC2 is impaired in thick ascending limb of Henle in moesin knockout mice.

Moesin is expressed in several types of cells including epithelial and endothelial cells. Several groups reported that moesin plays important roles in the regulation of the cellular motility, and the process of internalization of membrane proteins. However, the physiological roles of moesin in the kidney still remain unclear. Herein, we examined the physiological function of moesin in the kidney using moesin knockout (Msn -/y ) mice. There was no obvious abnormality in the renal morphology of Msn -/y mice. However, we found that Msn -/y mice exhibited mild hyperchloremia, and reduced glomerular filtration rate compared to wild type (WT) mice. Absolute electrolytes excretions of NaCl in Msn -/y mice were not significantly changed compared to WT mice. In the renal medulla, moesin was detected in thick ascending limb of Henle (TALH) as previously reported. To determine the physiological function of moesin in TALH, we examined the expression and subcellular localization of NKCC2 in Msn -/y mice. Interestingly, apical surface expression level, but not total expression of NKCC2 was increased in Msn -/y mice. Subcellular fractionation of renal medulla lysate and internalization assay using tubular suspension showed that the process of NKCC2 endocytosis is impaired. Since the distribution of NKCC2 in lipid raft fractions was decreased in Msn -/y mice, moesin may regulate the NKCC2 distribution to microdomain. These results suggest that moesin regulates the internalization of NKCC2. Furthermore, euhydration by water loading caused hyponatremina in Msn -/y mice, suggesting that dysfunction of moesin is associated with the nephrogenic syndrome of inappropriate antidiuresis (NSIAD).

Clarithromycin Dose-Dependently Stabilizes Rat Peritoneal Mast Cells.

BACKGROUND: Macrolides, such as clarithromycin, have antiallergic properties. Since exocytosis in mast cells is detected electrophysiologically via changes in membrane capacitance (Cm), the absence of such changes due to the drug indicates its mast cell-stabilizing effect. METHODS: Employing the whole-cell patch clamp technique in rat peritoneal mast cells, we examined the effects of clarithromycin on Cm during exocytosis. Using a water-soluble fluorescent dye, we also examined its effect on deformation of the plasma membrane. RESULTS: Clarithromycin (10 and 100 µM) significantly inhibited degranulation from mast cells and almost totally suppressed the GTP-x03B3;-S-induced increase in Cm. It washed out the trapping of the dye on the surface of mast cells. CONCLUSIONS: This study provides for the first time electrophysiological evidence that clarithromycin dose-dependently inhibits the process of exocytosis. The mast cell-stabilizing action of clarithromycin may be attributable to its counteractive effect on plasma membrane deformation induced by exocytosis.

Anti-Allergic Drugs Tranilast and Ketotifen Dose-Dependently Exert Mast Cell-Stabilizing Properties.

BACKGROUND: Anti-allergic drugs, such as tranilast and ketotifen, inhibit the release of chemokines from mast cells. However, we know little about their direct effects on the exocytotic process of mast cells. Since exocytosis in mast cells can be monitored electrophysiologically by changes in the whole-cell membrane capacitance (Cm), the absence of such changes by these drugs indicates their mast cell-stabilizing properties. METHODS: Employing the standard patch-clamp whole-cell recording technique in rat peritoneal mast cells, we examined the effects of tranilast and ketotifen on the Cm during exocytosis. Using confocal imaging of a water-soluble fluorescent dye, lucifer yellow, we also examined their effects on the deformation of the plasma membrane. RESULTS: Relatively lower concentrations of tranilast (100, 250 µM) and ketotifen (1, 10 µM) did not significantly affect the GTP-x03B3;-S-induced increase in the Cm. However, higher concentrations of tranilast (500 µM, 1 mM) and ketotifen (50, 100 µM) almost totally suppressed the increase in the Cm, and washed out the trapping of the dye on the surface of the mast cells. Compared to tranilast, ketotifen required much lower doses to similarly inhibit the degranulation of mast cells or the increase in the Cm. CONCLUSIONS: This study provides electrophysiological evidence for the first time that tranilast and ketotifen dose-dependently inhibit the process of exocytosis, and that ketotifen is more potent than tranilast in stabilizing mast cells. The mast cell-stabilizing properties of these drugs may be attributed to their ability to counteract the plasma membrane deformation in degranulating mast cells.

Chlorpromazine-induced changes in membrane micro-architecture inhibit thrombopoiesis in rat megakaryocytes.

Chlorpromazine often causes severe and persistent thrombocytopenia. Several clinical studies have suggested the presence of an as-yet-unknown mechanism in this drug-induced thrombocytopenia, by which the platelet production from megakaryocytes may directly be affected. As we previously demonstrated in rat peritoneal mast cells or adipocytes, chlorpromazine is amphiphilic and preferentially partitioned into the lipid bilayers of the plasma membrane. Therefore, it can induce some structural changes in the megakaryocyte membrane surface and thus affect the process of thrombopoiesis. In the present study, employing the standard patch-clamp whole-cell recording technique, we examined the effects of chlorpromazine on the membrane capacitance and Kv1.3-channel currents in rat megakaryocytes. By electron microscopic imaging of the cellular surface, we also examined the effects of chlorpromazine on the membrane micro-architecture of megakaryocytes. Chlorpromazine markedly decreased the membrane capacitance of megakaryocytes, indicating the decreased number of invaginated plasma membranes, which was not detected by the fluorescent imaging techniques. As shown by electron microscopy, chlorpromazine actually changed the membrane micro-architecture of megakaryocytes, and was likely to halt the process of pro-platelet formation in the cells. This drug persistently decreased the membrane capacitance and almost totally and irreversibly inhibited the Kv1.3-channel currents in megakaryocytes. This study demonstrated for the first time that chlorpromazine is likely to inhibit the process of thrombopoiesis persistently in megakaryocytes, as detected by the long-lasting decrease in the membrane capacitance and the irreversible suppression of the Kv1.3-channel currents. Chlorpromazine-induced changes in the membrane micro-architecture are thought to be responsible for its persistent effects.

Salicylate inhibits thrombopoiesis in rat megakaryocytes by changing the membrane micro-architecture.

BACKGROUND/AIMS: Salicylate causes drug-induced immune thrombocytopenia. However, some clinical studies indicate the presence of additional mechanisms in the drug-induced thrombocytopenia, by which the platelet production from megakaryocytes may directly be affected. Since salicylate is amphiphilic and preferentially partitioned into the lipid bilayers of the plasma membrane, it can induce some structural changes in the megakaryocyte membrane surface and thus affect the process of thrombopoiesis. METHODS: Employing the standard patch-clamp whole-cell recording technique, we examined the effects of salicylate on the membrane capacitance in rat megakaryocytes. Taking electron microscopic imaging of the cellular surface, we also examined the effects of salicylate on the membrane micro-architecture of megakaryocytes. RESULTS: Salicylate significantly decreased the membrane capacitance of megakaryocytes, indicating the decreased number of invaginated plasma membranes, which was not detected by the fluorescent imaging technique. As shown by electron microscopy, salicylate actually halted the process of pro-platelet formation in megakaryocytes. CONCLUSION: This study demonstrated for the first time that salicylate inhibits the process of thrombopoiesis in megakaryocytes, as detected by the decrease in the membrane capacitance. Salicylate-induced changes in the membrane micro-architecture are thought to be responsible for its effects.

Mast cell involvement in the progression of peritoneal fibrosis in rats with chronic renal failure.

AIM: Peritoneal fibrosis is a serious complication in patients with end stage renal disease (ESRD), especially those undergoing long-term peritoneal dialysis therapy. Since the peritoneum is a major site of mast cell accumulation, and since mast cells are known to facilitate the progression of organ fibrosis, they would also contribute to the pathogenesis of peritoneal fibrosis. The aim of this study was to reveal the involvement of mast cells in the progression of peritoneal fibrosis in chronic renal failure. METHODS: Using a rat model with chronic renal failure (CRF) resulting from 5/6 nephrectomy, we examined the histopathological features of the rat peritoneum and compared them to those of age-matched sham-operated rat peritoneum. By treating the CRF rats with a potent mast cell stabilizer, tranilast, we also examined the involvement of mast cells in the progression of peritoneal fibrosis. RESULTS: The CRF rat peritoneum was characterized by the wide staining of collagen III and an increased number of myofibroblasts, indicating the progression of fibrosis. Compared to sham-operated rat peritoneum, the number of toluidine blue-stained mast cells was significantly higher in the fibrotic peritoneum of CRF rats. The mRNA expression of fibroblast-activating factors and stem cell factor was significantly higher in peritoneal mast cells obtained from CRF rats than in those obtained from sham-operated rats. Treatment with tranilast significantly suppressed the progression of peritoneal fibrosis in CRF rats. CONCLUSIONS: This study demonstrated for the first time that the number of mast cells was significantly increased in the fibrotic peritoneum of CRF rats. The proliferation of mast cells and their increased activity in the peritoneum were thought to be responsible for the progression of peritoneal fibrosis.

Benidipine suppresses in situ proliferation of leukocytes and slows the progression of renal fibrosis in rat kidneys with advanced chronic renal failure.

BACKGROUND/AIMS: Leukocytes, such as lymphocytes and macrophages, predominantly express delayed rectifier K(+) channels (Kv1.3) in their plasma membranes. In our previous study, the overexpression of these channels in leukocytes was strongly associated with their proliferation in kidneys and the progression of renal fibrosis in advanced-stage chronic renal failure (CRF). Since benidipine, a long-acting 1,4-dihydropyridine Ca(2+) channel blocker, is also highly potent as a Kv1.3 channel inhibitor, it could exert therapeutic efficacy in advanced CRF. METHODS: Male Sprague-Dawley rats that underwent 5/6 nephrectomy followed by a 14-week recovery period were used as the model of advanced CRF. Benidipine hydrochloride (5 mg/kg) was started at 8 weeks after nephrectomy and orally administered daily for 6 weeks. The histopathological features of the kidneys were examined in vehicle-treated and benidipine-treated CRF rat kidneys. Cellular proliferation of leukocytes and the cortical expression of proinflammatory cytokines were also examined. RESULTS: In CRF rat kidneys, Kv1.3 channels began to be overexpressed in leukocytes as early as 8 weeks after nephrectomy. In the cortical interstitium of benidipine-treated CRF rat kidneys, both immunohistochemistry and real-time PCR demonstrated decreased expression of fibrotic markers. Benidipine treatment significantly reduced the number of proliferating leukocytes within the cortical interstitium and decreased the expression of cell cycle markers and proinflammatory cytokines. CONCLUSION: This study demonstrated for the first time that benidipine slowed the progression of renal fibrosis in rat kidneys with advanced CRF. Kv1.3 channels overexpressed in leukocytes were thought to be the most likely therapeutic targets of benidipine in decreasing the number of proliferating leukocytes and repressing the production of inflammatory cytokines.

Glucocorticoid mediates the transcription of OAT-PG, a kidney-specific prostaglandin transporter.

OAT-PG is a kidney-specific prostaglandin transporter and exclusively expressed at the basolateral membrane of proximal tubules in rodent kidneys. We previously reported that OAT-PG was dominantly expressed in the male kidney similar to the other SLC22 family proteins as organic anion transporter (OAT) 1 and OAT3. Recently, Wegner et al. revealed that a transcription factor, B-cell CLL/lymphoma 6 (BCL6), is associated with the male-dominant expressions of OAT1 and OAT3 in the rat kidney. Here, we performed the luciferase assay to investigate whether OAT-PG is also transcriptionally regulated by BCL6. However, the promoter activity of OAT-PG was not directly affected by BCL6 overexpression nor the testosterone treatment, suggesting that different regulatory mechanisms underlie the male-dominant transcriptional regulation of OAT-PG compared to those of OAT1 and OAT3. We newly found that adrenalectomy (Adx) of male rat caused a significant reduction of OAT-PG expression without any significant changes in the OAT1 and OAT3 expressions, and it was recovered by the dexamethasone administration. Furthermore, the renocortical PGE2 concentration was markedly increased in Adx male rat, concomitant with the downregulation of OAT-PG, and it was reduced to the basal level by dexamethasone treatment. In the luciferase assay, dexamethasone stimulated OAT-PG promoter activity but not OAT1. The luciferase activity responsiveness to dexamethasone was significantly reduced by the deletion of glucocorticoid response elements in the OAT-PG promoter region. These results suggest that glucocorticoid plays an important role in the regulation of the renocortical PGE2 concentration by the transcriptional regulation of OAT-PG in the rat kidney.

Decreased expression of a novel prostaglandin transporter, OAT-PG, facilitates renocortical PGE2 accumulation during rat pregnancy.

BACKGROUND: Prostaglandin (PG)-specific organic anion transporter (OAT-PG) is a recently identified renal transporter involved in the local clearance of prostaglandin E2 (PGE2). Since the renal biosynthesis of PGE2 is not increased during pregnancy, this transporter expression would affect the gestational changes in the renal PGE2 content. METHODS: Kidneys from rats at different gestational stages were used to examine gestational changes in the renocortical PGE2 concentration. The renal expression of OAT-PG and the enzymes for PGE2 synthesis was also examined sequentially, together with the gestational changes in renal renin production. RESULTS: The renocortical PGE2 concentration was significantly increased during midterm to late pregnancy, with a maximum increase of 47.6 ± 11.5% from the virgin value. Although the expression of the enzymes, such as cyclooxygenases and PG synthases, was not increased, that of OAT-PG was significantly decreased throughout pregnancy, inversely correlating with changes in the renocortical PGE2 concentration. Renal renin production was significantly increased during pregnancy. CONCLUSION: This study demonstrated for the first time that the tissue PGE2 concentration was increased in pregnant rat kidneys, which may be associated with the gestational rise in glomerular filtration rate. The decreased expression of OAT-PG was thought to be responsible for the increased tissue PGE2 content.

Ezrin, a membrane cytoskeletal cross-linker, is essential for the regulation of phosphate and calcium homeostasis.

Ezrin cross-links plasma membrane proteins with the actin cytoskeleton. In the kidney, ezrin mainly localizes at the brush border membrane of proximal tubules with the scaffolding protein, Na(+)/H(+) exchanger regulatory factor (NHERF) 1. NHERF1 interacts with the sodium/phosphate cotransporter, Npt2a. Defects in NHERF1 or Npt2a in mice cause hypophosphatemia. Here we studied the physiological role of ezrin in renal phosphate reabsorption using ezrin knockdown mice (Vil2). These mice exhibit hypophosphatemia, hypocalcemia, and osteomalacia. The reduced plasma phosphate concentrations were ascribed to defects in urinary phosphate reabsorption. Immunofluorescence and immunoblotting indicated a marked reduction in renal Npt2a and NHERF1 expression at the apical membrane of proximal tubules in the knockdown mice. On the other hand, urinary loss of calcium was not found in Vil2 mice. Plasma concentrations of 1,25-dihydroxyvitamin D were elevated following reduced plasma phosphate levels, and mRNA of the vitamin D-dependent TRPV6 calcium channel were significantly increased in the duodenum of knockdown mice. Expression of TRPV6 at the apical membrane, however, was significantly decreased. Furthermore, tibial bone mineral density was significantly lower in both the adult and young Vil2 mice. These results suggest that ezrin is required for the regulation of systemic phosphate and calcium homeostasis in vivo.

Underestimation of urinary biomarker-to-creatinine ratio resulting from age-related gain in muscle mass in rats.

Recent efforts have been made to identify useful urinary biomarkers of nephrotoxicity. Furthermore, the application of urine to the other toxicities as new biomarker source has been recently expanded. Meanwhile, correction of urinary biomarker concentrations according to fluctuations in urine flow rate is required for adequate interpretation of the alteration. The urinary biomarker-to-creatinine ratio (UBCR) is widely used because of the convenience, while the urinary biomarker-excretion rate is regarded as the gold standard corrective method. Because creatinine is a catabolite in energy production in muscles, we hypothesized that altered muscle mass could affect creatinine kinetics, ultimately affecting UBCR. However, no study has examined this hypothesis. In this study, we examined the influence of muscle mass gain on UBCR, using male Sprague-Dawley rats during the growth phase, 6-12-week old. Both plasma creatinine and excretion of urinary creatinine (Ucr excretion) showed increases with muscle mass gain in rats, in which the alterations of UBCR were lowered. The renal mRNA level of the organic cation transporter-2 (Oct2), a creatinine transporter, showed an age-related increase, whereas the mRNA level of multidrug and toxin extrusions-1 (Mate1) remained constant. Multiple regression analysis showed that the increase in creatinine clearance highly contributed to the age-related increase in Ucr excretion compared to the mRNA levels of Oct2 and Mate1. This suggested that the age-related increase in Ucr excretion may be attributable to the increased transglomerular passage of creatinine. In conclusion, the results suggest that muscle mass gain can affect creatinine kinetics, leading to underestimation of UBCR. Therefore, it is important to understand the characteristics of the corrective method when using urinary biomarker, the failure of which can result in an incorrect diagnosis.

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) Ca(2+) 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.

Biomarker panel of cardiac and skeletal muscle troponins, fatty acid binding protein 3 and myosin light chain 3 for the accurate diagnosis of cardiotoxicity and musculoskeletal toxicity in rats.

Cardiotoxicity and musculoskeletal toxicity can be life-threatening, and thus have strong impact on both the development and marketing of drugs. Because the conventional biomarkers such as aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase (CK) have low detection power, there has been increasing interest in developing biomarkers with higher detection power. The current study examined the usefulness of several promising biomarkers, cardiac and skeletal muscle troponins (cTnI, cTnT and sTnI), fatty acid binding protein 3 (FABP3) and myosin light chain 3 (MYL3), and compared the obtained data to AST, LDH and CK in rat models treated with various myotoxic and non-myotoxic compounds (isoproterenol, metaproterenol, doxorubicin, mitoxantrone, allylamine, cyclosporine A, cyclophosphamide, aminoglutethimide, acetaminophen, methapyrilene, allylalcohol and α-naphthylisothiocyanate). These promising biomarkers were found to be superior to the conventional biomarkers, as they had a specific and abundant distribution within the heart and/or skeletal muscles; exhibited a positive correlation between the amplitude of increases and the degree of pathological alterations; had higher diagnostic accuracy for detecting pathological alterations; and had the additive effect of improving the diagnostic accuracy of conventional biomarkers. However, these promising biomarkers have several drawbacks including a rapid clearance, the fact that they are affected by renal dysfunction, and different reactivity to the mode of action of individual myotoxicants. In conclusion, the promising biomarkers cTnI, cTnT, FABP3, MYL3, and sTnI demonstrated sensitivity and specificity for cardiac and skeletal myotoxicity that was superior to those of conventional biomarkers, while we should pay attention to the drawbacks of these biomarkers when used in toxicity studies.

Overexpression of Delayed Rectifier K(+) Channels Promotes In situ Proliferation of Leukocytes in Rat Kidneys with Advanced Chronic Renal Failure.

Leukocytes, such as lymphocytes and macrophages, predominantly express delayed rectifier K(+) channels (Kv1.3), and the channels play crucial roles in the activation and proliferation of the cells. Since lymphocytes are activated in patients with end-stage renal disease (ESRD), the channels expressed in those cells would contribute to the progression of renal fibrosis in advanced-stage chronic renal failure (CRF). In the present study, using a rat model with advanced CRF that underwent 5/6 nephrectomy followed by a 14-week recovery period, we examined the histopathological features of the kidneys and the leukocyte expression of Kv1.3-channels and cell cycle markers. Age-matched sham-operated rats were used as controls. In the cortical interstitium of advanced CRF rat kidneys, leukocytes proliferated in situ and overexpressed Kv1.3 channel protein in their cytoplasm. Treatment with margatoxin, a selective Kv1.3-channel inhibitor, significantly suppressed the number of leukocytes and the progression of renal fibrosis with a significant decrease in the cortical cell cycle marker expression. This study demonstrated for the first time that the number of leukocytes was dramatically increased in rat kidneys with advanced CRF. The overexpression of Kv1.3 channels in the leukocytes was thought to contribute to the progression of renal fibrosis by stimulating cell cycling and promoting cellular proliferation.

Sex hormones induce a gender-related difference in renal expression of a novel prostaglandin transporter, OAT-PG, influencing basal PGE2 concentration.

Based on the nucleotide sequence of a mouse prostaglandin-specific transporter (mOAT-PG), we identified a rat homolog (rOAT-PG) which shares 80% identity with mOAT-PG in a deduced amino acid sequence. rOAT-PG transports PGE(2) and colocalizes with 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a metabolic enzyme for PGs, in proximal tubules, suggesting that rOAT-PG is involved in PGE(2) clearance to regulate its physiological function in the renal cortex. We found that the expression level of rOAT-PG in the renal cortex was much higher in male rats than in female rats whereas there was no gender difference in the expression level of cyclooxygenase-2, a key enzyme producing PGE(2), and 15-PGDH in the renal cortex. Tissue PGE(2) concentration in the renal cortex was lower in male rats than in female rats, suggesting that renocortical PGE(2) concentration is primarily determined by the expression level of OAT-PG, which is regulated differently between male and female rats. Castration of male rat led to a remarkable reduction in OAT-PG expression and a significant increase in renocortical PGE(2) concentration. These alterations were recovered by testosterone supplementation. These results suggest that OAT-PG is involved in local PGE(2) clearance in the renal cortex. Although the physiological importance of the gender difference in local PGE(2) clearance is still unclear, these findings might be a key to clarifying the physiological roles of PGE(2) in the kidney.

Clinical characteristics of young-onset and medical treatment-requiring hypertension identified by targeted screening in university health check-up.

Based on targeted screening for hypertension at a university health check-up, we previously reported a high incidence of white-coat hypertension and estimated prevalence of hypertension requiring medical treatments (HT) as around 0.1% in young population aged less than 30. In spite of such low prevalence, continuous screening for seven consecutive years (2003-2009) increased the number of HT students to 20 (19 males and 1 female). We presently assessed the clinical characteristics of these HTs. Renovascular hypertension was found in the only female HT and aortic valve regurgitation in two HTs. Resting 17 HTs were diagnosed as having essential hypertension (EH). A father and/or a mother had EH in 16 out of 17 EHs, and blood pressure (BP) at home was slightly elevated (135-145 mm Hg in systolic) except three obese EHs (body mass index more than 30) who demonstrated more than 160 mm Hg in systolic. Plasma aldosterone-renin ratio (ARR) of EHs did not differ from that of normal controls, and Pearson correlation coefficient (R) between ARR and systolic BP (SBP) was -0.2. Its partial correlation coefficient, however, was statistically significant (R = -0.55, P = .026) after correcting for body mass index, which was significantly correlated with both SBP (P = .006, after correcting for ARR) and ARR (P = .004, after correcting for SBP). In conclusion, most of young-onset HTs are male EHs, and aortic valve regurgitation should be carefully checked. Excess plasma renin activity would be one of additional characteristics of young-onset EH to male gender, genetic background, and increased body mass.

Decrease in urinary creatinine in acute kidney injury influences diagnostic value of urinary biomarker-to-creatinine ratio in rats.

Recent research has revealed several useful urinary biomarkers of renal dysfunction such as acute kidney injury (AKI). For adequate evaluation of altered urinary biomarkers, it is necessary to consider the influence of varied urine flow rate (UFR). Calculation of the excretion rate of a urinary biomarker (UFR-correction) is the gold standard for the correction of UFR variation. An alternative method that is widely used is to calculate the ratio of the biomarker level to urinary creatinine (Ucr-correction). To date, the equivalence between these two methods has been examined only in a steady state situation such as diabetic nephropathy, and the urinary biomarkers examined have been limited to proteinuria and albuminuria. Therefore, we comprehensively addressed the relationship between Ucr-correction and UFR-correction of ten urinary biomarkers N-acetyl-ß-d-glucosaminidase (NAG), lactate dehydrogenase (LDH), total protein, albumin, kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, clusterin, ß(2)-microglobulin, cystatin-c and glutathione S-transferase-α in non-steady state situations such as AKI. All ten urinary biomarkers showed larger amplitude increases in AKI by Ucr-correction than by UFR-correction in linear regression analysis. Moreover, receiver operating characteristic curves analysis suggested that, at least for the biomarkers NAG and LDH, Ucr-correction had higher diagnostic power than UFR-correction. We observed a decrease in the Ucr excretion in AKI that was accompanied by a reduction in creatinine clearance and reduced mRNA expression of the renal organic cation transporter-2, which is known to function as a transporter for creatinine. These results may provide a mechanistic explanation for the phenomena obtained in Ucr-correction. In conclusion, while Ucr-correction could overestimate the degree of AKI, it could also provide higher diagnostic power for AKI than UFR-correction. We should take into consideration of these backgrounds when using the Ucr-correction.

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.

A novel transporter of SLC22 family specifically transports prostaglandins and co-localizes with 15-hydroxyprostaglandin dehydrogenase in renal proximal tubules.

We identified a novel prostaglandin (PG)-specific organic anion transporter (OAT) in the OAT group of the SLC22 family. The transporter designated OAT-PG from mouse kidney exhibited Na(+)-independent and saturable transport of PGE(2) when expressed in a proximal tubule cell line (S(2)). Unusual for OAT members, OAT-PG showed narrow substrate selectivity and high affinity for a specific subset of PGs, including PGE(2), PGF(2alpha), and PGD(2). Similar to PGE(2) receptor and PGT, a structurally distinct PG transporter, OAT-PG requires for its substrates an alpha-carboxyl group, with a double bond between C13 and C14 as well as a (S)-hydroxyl group at C15. Unlike the PGE(2) receptor, however, the hydroxyl group at C11 in a cyclopentane ring is not essential for OAT-PG substrates. Addition of a hydroxyl group at C19 or C20 impairs the interaction with OAT-PG, whereas an ethyl group at C20 enhances the interaction, suggesting the importance of hydrophobicity around the omega-tail tip forming a "hydrophobic core" accompanied by a negative charge, which is essential for substrates of OAT members. OAT-PG-mediated transport is concentrative in nature, although OAT-PG mediates both facilitative and exchange transport. OAT-PG is kidney-specific and localized on the basolateral membrane of proximal tubules where a PG-inactivating enzyme, 15-hydroxyprostaglandin dehydrogenase, is expressed. Because of the fact that 15-keto-PGE(2), the metabolite of PGE(2) produced by 15-hydroxyprostaglandin dehydrogenase, is not a substrate of OAT-PG, the transport-metabolism coupling would make unidirectional PGE(2) transport more efficient. By removing extracellular PGE(2), OAT-PG is proposed to be involved in the local PGE(2) clearance and metabolism for the inactivation of PG signals in the kidney cortex.