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Metabolic syndrome (MetS) is associated with chronic kidney disease and proteinuria. Previously, we reported that a synthetic serine protease inhibitor, camostat mesilate (CM), mitigated hypertension and proteinuria in rodent disease models. The present study evaluated the anti-hypertensive and anti-proteinuric effects of CM in MetS model rats (SHR/ND mcr-cp). Rats were divided into normal salt-fed (NS), high salt-fed (HS), HS and CM-treated (CM), and HS and hydralazine-treated (Hyd) groups. Rats were sacrificed after four weeks of treatment. Severe hypertension and proteinuria were observed in the HS group. Although CM and Hyd equally alleviated hypertension, CM suppressed proteinuria and glomerular sclerosis more efficiently than Hyd. The HS group revealed a decrease in podocyte number and podocyte-specific molecules, together with an increase in glomerular apoptotic cells and apoptosis-related proteins in the kidney. These changes were significantly attenuated by CM, but not by Hyd. Furthermore, CM ameliorated the apoptotic signals in murine cultured podocytes stimulated with the high glucose and aldosterone medium. In conclusion, CM could exert renoprotective effects in MetS model rats, together with the inhibition of podocyte apoptosis. Our study suggests that serine protease inhibition may become a new therapeutic strategy against MetS-related hypertension and renal injuries.

Background Patients undergoing hemodialysis treatment have a poor prognosis, as many develop premature aging. Systemic inflammatory conditions often underlie premature aging phenotypes in uremic patients. We investigated whether angiopoietin-like protein 2 (ANGPTL 2), a factor that accelerates the progression of aging-related and noninfectious inflammatory diseases, was associated with increased mortality risk in hemodialysis patients. Methods We conducted a multicenter prospective cohort study of 412 patients receiving maintenance hemodialysis and evaluated the relationship between circulating ANGPTL2 levels and the risk for all-cause mortality. Circulating ANGPTL2 levels were log-transformed to correct for skewed distribution and analyzed as a continuous variable. Results Of 412 patients, 395 were included for statistical analysis. Time-to-event data analysis showed high circulating ANGPTL2 levels were associated with an increased risk for all-cause mortality after adjustment for age, sex, hemodialysis vintage, nutritional status, metabolic parameters and circulating high-sensitivity C-reactive protein levels {hazard ratio [HR] 2.04 [95% confidence interval (CI) 1.10–3.77]}. High circulating ANGPTL2 levels were also strongly associated with an increased mortality risk, particularly in patients with a relatively benign prognostic profile [HR 3.06 (95% CI 1.86–5.03)]. Furthermore, the relationship between circulating ANGPTL2 levels and mortality risk was particularly strong in patients showing few aging-related phenotypes, such as younger patients [HR 7.99 (95% CI 3.55–18.01)], patients with a short hemodialysis vintage [HR 3.99 (95% CI 2.85–5.58)] and nondiabetic patients [HR 5.15 (95% CI 3.19–8.32)]. Conclusion We conclude that circulating ANGPTL2 levels are positively associated with mortality risk in patients receiving maintenance hemodialysis and that ANGPTL2 could be a unique marker for the progression of premature aging and subsequent mortality risk in uremic patients, except those with significant aging-related phenotypes.

Emerging evidence has suggested that aldosterone has direct deleterious effects on the kidney independently of its hemodynamic effects. However, the detailed mechanisms of these direct effects remain to be elucidated. We have previously reported that camostat mesilate (CM), a synthetic serine protease inhibitor, attenuated kidney injuries in Dahl salt-sensitive rats, remnant kidney rats, and unilateral ureteral obstruction rats, suggesting that some serine proteases would be involved in the pathogenesis of kidney injuries. The current study was conducted to investigate the roles of serine proteases and the beneficial effects of CM in aldosterone-related kidney injuries. We observed a serine protease that was activated by aldosterone/salt in rat kidney lysate, and identified it as plasmin with liquid chromatography-tandem mass spectrometry. Plasmin increased pro-fibrotic and inflammatory gene expressions in rat renal fibroblast cells. CM inhibited the protease activity of plasmin and suppressed cell injury markers induced by plasmin in the fibroblast cells. Furthermore, CM ameliorated glomerulosclerosis and interstitial fibrosis in the kidney of aldosterone/salt-treated rats. Our findings indicate that plasmin has important roles in kidney injuries that are induced by aldosterone/salt, and that serine protease inhibitor could provide a new strategy for the treatment of aldosterone-associated kidney diseases in humans.

A 28-year-old woman with systemic lupus erythematosus was referred to our hospital due to nephrotic-level proteinuria despite approximately 1 year of treatment with 50 to 60 mg/d of prednisolone and 100 to 150 mg/d of cyclosporine with methylprednisolone pulse therapy. Kidney biopsy showed diffuse global lupus nephritis (World Health Organization class 4-G A/C) with many intraglomerular foam cells containing cholesterol crystals. Surprisingly, proteinuria diminished after only 5 low-density lipoprotein (LDL) cholesterol apheresis sessions. This case demonstrated the potential of LDL apheresis to exhibit a remarkable effect on not only focal segmental glomerulosclerosis, but also other types of nephritis, particularly nephritis with intraglomerular foam cells.

Objective: This study evaluated the non-inferiority of renoprotection afforded by benidipine versus hydrochlorothiazide in hypertensive patients with chronic kidney disease (CKD). Methods: In this prospective, multicenter, open-labeled, randomized trial, the antialbuminuric effects of benidipine and hydrochlorothiazide were examined in renin-angiotensin system (RAS) inhibitor-treated patients with blood pressure (BP) readings of ≥ 130/80 mmHg and ≤ 180/110 mmHg, a urinary albumin to creatinine ratio (UACR) of ≥ 300 mg/g, and an estimated glomerular filtration rate (eGFR) of ≥ 30 ml/min/1.73m2. Patients received benidipine (n = 176, final dose: 4.8 mg/day) or hydrochlorothiazide (n = 170, 8.2 mg/day) for 12 months. Results: Benidipine and hydrochlorothiazide exerted similar BP- and eGFR-decreasing actions. The UACR values for benidipine and hydrochlorothiazide were 930.8 (95% confidence interval: 826.1, 1048.7) and 883.1 (781.7, 997.7) mg/g at baseline, respectively. These values were reduced to 790.0 (668.1, 934.2) and 448.5 (372.9, 539.4) mg/g at last observation carried forward (LOCF) visits. The non-inferiority of benidipine versus hydrochlorothiazide was not demonstrated (benidipine/hydrochlorothiazide ratio of LOCF value adjusted for baseline: 1.67 (1.40, 1.99)). Conclusions: The present study failed to demonstrate the non-inferiority of the antialbuminuric effect of benidipine relative to that of hydrochlorothiazide in RAS inhibitor-treated hypertensive patients with macroalbuminuria.

Nephrosclerosis/ischemic nephropathy (NS/IN) ranks third among renal diseases requiring dialysis in Japan. Although it is an important renal disease in terms of frequency, its prevalence, new incidence, and risk factors are not fully elucidated. We analyzed the prevalence, incidence, concurrent diseases, and risk factors of NS/IN by using data from specific health checkups of Kumamoto citizens between 2008 and 2010. Although the prevalence of NS/IN was 1−2 % in people in their 40s, it increased sharply with age, reaching 17.6 % in people aged 70–74 years. The incidence of new NS/IN was 0.4−0.5 % per year. In multivariate logistic regression analysis, factors such as age, male gender, body mass index (BMI), hyperuricemia, hypertension, and dyslipidemia correlated with NS/IN. When risk factors associated with NS/IN progress were evaluated by multivariate logistic regression analysis, four factors—male gender, hypertension, BMI, and current smoking—significantly correlated. The analysis of Kumamoto citizens aged 40–74 years receiving specific health checkups showed that in addition to hypertension and age that were considered important, male gender and obesity are also risk factors for NS/IS independent from hypertension.

The number of the chronic renal failure (CRF) patients is increasing explosively. Hypertension, proteinuria, inflammation, fibrosis, and oxidative stress are intertwined in a complicated manner that leads to the progression of CRF. However, the therapeutic strategies to delay its progression are limited. Since serine proteases are involved in many processes that contribute to these risk factors, we investigated the effects of a synthetic serine protease inhibitor, camostat mesilate (CM), on the progression of CRF in 5/6 nephrectomized (Nx) rats. Eighteen male Sprague-Dawley rats were divided into three groups: a sham-operated group ( n = 6), a vehicle-treated Nx group ( n = 6), and a CM-treated Nx group ( n = 6). Following the 9-wk study period, both proteinuria and serum creatinine levels were substantially increased in the vehicle-treated Nx group, and treatment with CM significantly reduced proteinuria and serum creatinine levels. The levels of podocyte-associated proteins in glomeruli, such as nephrin and synaptopodin, were markedly decreased by 5/6 nephrectomy, and this was significantly ameliorated by CM. CM also suppressed the levels of inflammatory and fibrotic marker mRNAs including transforming growth factor-β1, TNF-α, collagen types I, III, and IV, and reduced glomerulosclerosis, glomerular hypertrophy, and interstitial fibrosis in histological studies. Furthermore, CM decreased the expression of NADPH oxidase component mRNAs, as well as reactive oxygen species generation and advanced oxidative protein product levels. Our present results strongly suggest the possibility that CM could be a useful therapeutic agent against the progression of CRF.

Aldosterone plays an important role in the regulation of blood pressure by modulating the activity of the epithelial sodium channel (ENaC) that consists of α-, β-, and γ-subunits. Aldosterone induces a molecular weight shift of γENaC from 85 to 70 kDa that is necessary for the channel activation. In vitro experiments demonstrated that a dual cleavage mechanism is responsible for this shift. It has been postulated that furin executes the primary cleavage in the Golgi and that the second cleavage is provided by other serine proteases such as prostasin or plasmin at the plasma membrane. However, the in vivo contribution of serine proteases to this cleavage remains unclear. To address this issue, we administered the synthetic serine protease inhibitor camostat mesilate (CM) to aldosterone-infused rats. CM decreased the abundance of the 70-kDa form of ENaC and led to a new 75-kDa form with a concomitant increase in the urinary Na-to-K ratio. Because CM inhibits the protease activity of serine proteases such as prostasin and plasmin, but not furin, our findings strongly indicate that CM inhibited the second cleavage of γENaC and subsequently suppressed ENaC activity. The results of our current studies also suggest the possibility that the synthetic serine protease inhibitor CM might represent a new strategy for the treatment of salt-sensitive hypertension in humans.

We previously reported that a deficiency in the vasopressin V1a receptor (V1aR) results in type 4 renal tubular acidosis, which suggests that vasopressin exerts direct effects on the physiological actions of aldosterone. We investigated the role of vasopressin for nucleocytoplasmic transport of mineralocorticoid receptor (MR) in the intercalated cells. Vasopressin V1aR-deficient (V1aR−/−) mice showed largely decreased expression of MR and 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) in the medulla of the kidney, which was partially ameliorated by fludrocortisone treatment. The incubation of IN-IC cells, an intercalated cell line established from temperature-sensitive SV40 large T antigen-expressing rats, with aldosterone or vasopressin increased the nuclear-to-cytoplasmic ratio of the MR from 11.2 to 47.2% and from 18.7 to 61.2%, respectively, in 30 min without any changes in MR expression from the whole cell extract. The immunohistochemistry analysis of the IN-IC cells revealed the nuclear accumulation of MRs after a 30-min incubation with aldosterone or vasopressin. These effects were accompanied by an increase in regulator of chromosome condensation-1 (RCC-1) due to aldosterone and a decrease in Ran GTPase-activating protein 1 (Ran Gap1) due to vasopressin. RNA interference against V1aR abolished the nuclear accumulation of MR induced by aldosterone or vasopressin. Vasopressin increased PKCα and -β1 expression, and aldosterone increased PKCδ and -ζ expression, but these effects were abolished with a V1aR knockdown. These results suggest that vasopressin directly regulates the nucleocytoplasmic transport of MRs via the V1aR in the intercalated cells of the collecting ducts.

Although the involvement of cytomegalovirus (CMV) infections in the development of thrombotic microangiopathy (TMA) in HIV patients and transplant recipients has been reported, it is still controversial whether CMV itself can cause TMA. We report herein a rare case with rapid improvement of TMA by ganciclovir treatment in a patient who is neither HIV-positive nor a transplant recipient, suggesting a pathogenic role for CMV in TMA.

Proteases are involved in numerous essential biological processes including blood clotting, controlled cell death, and tissue differentiation. Prostasin, a glycosylphosphatidylinositol-anchored serine protease, has been identified as a potential regulator of the epithelial sodium channel (ENaC) function in the kidney, lung, and airways. ENaC is composed of three homologous subunits α, β, and, γ. The dual cleavage of α subunit by furin and γ subunit by prostasin and furin releases inhibitory segments from ENaC, leading to the channel activation. Protease nexin-1, an endogenous prostasin inhibitor, inhibits ENaC activity through the suppression of prostasin activity, strongly suggesting the possibility that a coordinated regulation of serine proteases and serine protease inhibitors plays a key role in the sodium handling in the kidney. Camostat mesilate (CM), a synthetic serine protease inhibitor, reduced prostasin activity and subsequently decreased ENaC current. Oral administration of CM to Dahl salt-sensitive rats resulted in a significant decrease in blood pressure with an elevation of the urinary sodium/potassium ratio. These findings suggest that synthetic serine protease inhibitors such as CM might represent a new class of antihypertensive drugs in patients with salt-sensitive hypertension.

AIM Intravenous vitamin D therapy is an established treatment for secondary hyperparathyroidism (SHPT). However, no protocols have been established for maintenance therapy with intravenous or oral vitamin D after control of intact parathyroid hormone (iPTH) within the target range. METHODS Step I. For patients with SHPT (200 ≤ iPTH ≤ 500 pg/ml), a dose of 2.5 mg maxacalcitol (OCT) was administered intravenously three times a week with oral sevelamer hydrochloride; the dose was increased to a 10 µg maximum three times a week to control iPTH to < 150 pg/ml. Step II. When iPTH reached the target level, patients were assigned to Group A (oral alfacalcidol 1.0 µg/day) or B (oral alfacalcidol 0.25 µg/ day). Serum iPTH, calcium, and inorganic phosphorus were measured each month for 6 months. Maintenance rates for the target iPTH levels were evaluated, < 150 pg/ml at Step I and < 200 pg/ml at Step II. RESULTS iPTH decreased to < 150 pg/ml by OCT in 24 of 35 patients (68.6%). During the 24-week observation period, iPTH was controlled for 83.3% patients in Group A vs. 36.4% for Group B (p < 0.05). No dropouts due to hypercalcemia or hyperphosphatemia occurred. CONCLUSION OCT dose titration was effective for SHPT. A higher daily dose of oral alfacalcidol (1.0 µg) appears to be more effective than a lower dose (0.25 µg) as maintenance therapy after iPTH control.

Sodium balance, extracellular fluid volume, and ultimately blood pressure are maintained by precise regulation of the activity of epithelial sodium channels (ENaC). Multiple mechanisms such as hormones, intracellular factors, and other regulatory factors contribute to regulation of ENaC activity. Prostasin, a glycosylphosphatidylinositol-anchored serine protease, has been identified as an activator of ENaC that increases its open probability. Furin cleaves αENaC at two sites and γENaC at one site at the Golgi. Prostasin cleaves γENaC at one site that is distinct from the furin site at the plasma membrane. Dual cleavage of α- and γ-subunit releases inhibitory segments from ENaC, leading to channel activation. Protease nexin-1 (PN-1), an endogenous prostasin inhibitor, inhibits ENaC activity through suppression of prostasin activity. Aldosterone and transforming growth factor-β1 reciprocally regulate expression of prostasin, PN-1, and ENaC in renal epithelial cell, resulting in sodium retention or natriuresis, respectively. These findings strongly suggest the possibility that coordinated regulation of serine protease, serpin, and ENaC expression plays a key role in sodium handling in the kidney.

Objective. To assess retrospectively whether continuously high serum alanine aminotransferase (ALAT) levels (80 IU) in the first three successive years after the diagnosis of liver cirrhosis (LC) are predictive of a subsequent high incidence of hepatocellular carcinoma (HCC) in patients with Child Stage A hepatitis C virus (HCV)-LC. Material and methods. The study comprised 132 HCV-LC (Child Stage A) patients who had not received interferon therapy but had been treated with anti-inflammatory agents. At the end of a 3-year follow-up after the diagnosis of LC, the patients were subdivided into three groups according to their serum ALAT levels and the subsequent incidence of HCC was assessed. Results. The cumulative incidence of HCC starting from 3 years after the diagnosis of LC in the continuously high ALAT group (annual average over 3 years alwaysor =80 IU; n=41; Group A) was markedly higher than that in the continuously low ALAT group (always80 IU; n=48; Group B) (p0.005) during an observation period of 7.9+/-3.7 years. The incidence of HCC in Group A was 11.8%/year. The odds ratios of developing HCC in Group A and Group C (mixed high and low ALAT levels; n=43) were 5.1-fold and 1.5-fold that of Group B, respectively. A multivariate analysis revealed that the ALAT group was independently associated with HCC development. Conclusions. Continuously high ALAT levels for three successive years following the diagnosis of LC can be predictive of a very high incidence of HCC in Child A HCV-LC patients. Prospective trials using therapeutic approaches aimed at decreasing ALAT levels are necessary in order to confirm a positive impact of ALAT reduction on the incidence of HCC in patients with HCV-LC.

The epithelial sodium channel (ENaC) plays an important role in the regulation of blood pressure by modulating Na reabsorption in the kidney. Dahl salt-sensitive rats on high-salt diet develop severe hypertension, and high-salt diet has been reported to stimulate ENaC mRNA expression in the kidney abnormally in Dahl salt-sensitive rats despite a suppressed plasma aldosterone concentration (PAC).We investigated the effect of high-salt diet on ENaC protein expression in Dahl salt-resistant and Dahl salt-sensitive rats, and examined the effect of amiloride (5 mg/kg per day) and eplerenone (0.125% diet) on blood pressure and renal injury in Dahl salt-sensitive rats.Dahl salt-sensitive rats developed hypertension and renal damage following 4 weeks of treatment with high-salt diet. Although PAC and kidney aldosterone content were all suppressed by the high-salt diet in Dahl salt-sensitive rats, both beta and gammaENaC mRNA expression and protein abundance were significantly increased. The molecular weight shift of gammaENaC from 85 to 70 kDa, an indication of ENaC activation, was clearly increased in Dahl salt-sensitive rats on high-salt diet compared with the low-salt group or Dahl salt-resistant rats on high-salt diet. Four weeks of treatment with amiloride, but not eplerenone, significantly ameliorated hypertension and kidney injury in Dahl salt-sensitive rats fed high-salt diet, suggesting aberrant aldosterone-independent activation of ENaC.These results suggest that inappropriate expression and activation of ENaC could be one of the underlying mechanisms by which Dahl salt-sensitive rats develop salt-sensitive hypertension and organ damage, and indicate a therapeutic benefit of amiloride in salt-sensitive hypertension where ENaC is excessively activated.

Background Estimation of glomerular filtration rate (GFR) is limited by differences in creatinine generation among ethnicities. Our previously reported GFR-estimating equations for Japanese had limitations because all participants had a GFR less than 90 mL/min/1.73 m2 and serum creatinine was assayed in different laboratories. Study Design Diagnostic test study using a prospective cross-sectional design. New equations were developed in 413 participants and validated in 350 participants. All samples were assayed in a central laboratory. Setting & Participants Hospitalized Japanese patients in 80 medical centers. Patients had not participated in the previous study. Reference Test Measured GFR (mGFR) computed from inulin clearance. Index Test Estimated GFR (eGFR) by using the modified isotope dilution mass spectrometry (IDMS)-traceable 4-variable Modification of Diet in Renal Disease (MDRD) Study equation using the previous Japanese Society of Nephrology Chronic Kidney Disease Initiative (JSN-CKDI) coefficient of 0.741 (equation 1), the previous JSN-CKDI equation (equation 2), and new equations derived in the development data set: modified MDRD Study using a new Japanese coefficient (equation 3), and a 3-variable Japanese equation (equation 4). Measurements Performance of equations was assessed by means of bias (eGFR − mGFR), accuracy (percentage of estimates within 15% or 30% of mGFR), root mean squared error, and correlation coefficient. Results In the development data set, the new Japanese coefficient was 0.808 (95% confidence interval, 0.728 to 0.829) for the IDMS–MDRD Study equation (equation 3), and the 3-variable Japanese equation (equation 4) was eGFR (mL/min/1.73 m2) = 194 × Serum creatinine−1.094 × Age−0.287 × 0.739 (if female). In the validation data set, bias was −1.3 ± 19.4 versus −5.9 ± 19.0 mL/min/1.73 m2 (P = 0.002), and accuracy within 30% of mGFR was 73% versus 72% (P = 0.6) for equation 3 versus equation 1 and −2.1 ± 19.0 versus −7.9 ± 18.7 mL/min/1.73 m2 (P Limitation Most study participants had chronic kidney disease, and some may have had changing GFRs. Conclusion The new Japanese coefficient for the modified IDMS–MDRD Study equation and the new Japanese equation are more accurate for the Japanese population than the previously reported equations.

Renal aquaporin-2 (AQP2) expression plays a key role in urine concentration. However, it is not known whether metabolic acidosis affects urine-concentrating ability through AQP2 expression in the kidney and urine. We examined urinary excretion and renal expression of AQP2 in control and acidosis rats, using RT-competitive PCR, immunoblot and immunocytochemistry. Urinary excretion of AQP2 is decreased by 92% even with the increase in AQP2 mRNA and protein expressions in the collecting ducts by metabolic acidosis in rats. Urine osmolality in control rats was 1670+/-198 mOsm per kg H(2)O, and immunocytochemistry revealed the presence of AQP2 in the apical plasma membrane of the principal cells in the collecting ducts. Urine osmolality in acidosis rats was lower than that in control (1397+/-243 mOsm per kg H(2)O), and immunocytochemistry showed the diffuse presence of AQP2 in the cytoplasm of the principal cells. Differential centrifugation-coupled immunoblot showed a significant decrease in the ratio of AQP2 in plasma membrane-enriched fraction to that in intracellular vesicle-enriched fraction by metabolic acidosis. In summary, AQP2 translocation is largely decreased by metabolic acidosis even with increased expression in the collecting ducts. A disorder of AQP2 translocation in the collecting ducts with acidosis may be responsible for the diuresis in patients with chronic renal failure.

Prostasin, a membrane-bound serine protease, is known to increase the activity of the epithelial sodium channel (ENaC). Gene expression of prostasin was shown to be regulated by aldosterone, which increases the rate of sodium reabsorption through ENaC. To clarify the physiological relationships among prostasin, aldosterone and ENaC, we developed a specific radioimmunoassay (RIA) for human prostasin. Prostasin levels in urine were determined in 26 normotensive and 121 hypertensive subjects. Aldosterone content in urine and plasma, urinary Na/K ratio and other clinical parameters were also measured. We observed a highly significant correlation between prostasin and aldosterone concentration in urine (correlation coefficient: 0.673, P

白血球除去療法 (LCAP) は，体外循環下に患者末梢血から白血球を除去する治療法であり，潰瘍性大腸炎（UC）の重要な治療法の一つである．LCAP施行の際には，メシル酸ナファモスタット（NM）が抗凝固剤として選択される．これはUC患者における消化管出血のリスク軽減のためや，セルソーバEX® を構成する陰性荷電膜と血液の接触によるブラジキニン（BK）の産生を抑制し，BKに誘発される血圧低下などのアナフィラキシー様症状を抑制するためである．しかしNMに対しアレルギーを有する患者にヘパリンを抗凝固剤として使用すると，産生されたBKによる症状のためLCAPを中止せざるを得ないことがある．今回，私たちは初めてNMアレルギーの患者に対し，NMの代わりにメシル酸ガベキサート（GM）を使用することで，LCAP施行中のBK産生抑制およびBK関連症状の消失を認めたため報告する．BKによる症状のためLCAPを中止せざるを得なかったNM不耐性患者において，GMはLCAPの継続を可能とし，UCの改善に貢献するものと思われる．

Arginine vasopressin (AVP) and hypertonicity in the renal medulla play a major role in the urine concentration mechanism. Previously, we showed that rat vasopressin V2 receptor (rV2R) promoter activity was increased by vasopressin V2R stimulation and decreased by vasopressin V1a receptor (V1aR) stimulation in a LLC-PK1 cell line stably expressing rat V1aR (LLC-PK1/rV1aR). In the present study, we investigated the effects of hypertonicity on the rV2R promoter activity and on the suppression of rV2R promoter activity by V1aR stimulation in LLC-PK1/rV1aR cells. rV2R promoter activity was increased in NaCl- or mannitol-induced hypertonicity. The hypertonicity-responsive site in the rV2R promoter region was limited to 10 bp, including the Sp1 motif. The increase of V2R promoter activity by hypertonicity was significantly inhibited by a JNK inhibitor (SP600125) and PKA inhibitor (H89). In contrast, rV2R promoter activity was remarkably suppressed by V1aR stimulation in the hypertonic condition rather than in the isotonic condition. The AVP-stimulated intracellular Ca2+ concentration was increased in the hypertonic condition, suggesting the functional activation of V1aR by hypertonicity. In conclusion, 1) V2R promoter activity is increased by hypertonicity via the JNK and PKA pathways, 2) suppression of V2R expression by the V1aR-Ca2+ pathway is enhanced by hypertonicity, and 3) hypertonicity enhances the V1aR-Ca2+ pathway. The counteractivity of V2R and V1aR could be required to maintain minimum urine volume in the dehydrated state.

Intravenous iron administration (IVIR) is effective for correcting anemia in hemodialysis (HD) patients, but it also enhances the generation of hydroxyl radicals. Previously we demonstrated that IVIR increases oxidized serum albumin levels in HD patients. However, the effect of IVIR frequencies on the oxidative stress has never been studied before. Therefore, we compared the two IVIR schedules recommended by the Japanese Society for Dialysis Therapy guideline 2004 by measuring oxidized albumin in chronic HD patients.Twenty-two HD patients were divided into two IVIR protocol groups (group I: 40 mg of iron 3 times a week for 4 weeks, group II: 40 mg of iron once a week for 3 months). These protocols differ in IVIR frequency, but receive the same amount of iron (total 520 mg). We compared these two regimens by determining the levels of hemoglobin, serum ferritin, advanced oxidation protein products (AOPP), and oxidized albumin at 0, 4, 8, 12, 16, and 20 weeks.Both patient groups resulted in a similar and significant increase in hemoglobin levels, whereas group I markedly induced AOPP and oxidation of serum albumin than group II at 4 weeks (P0.05). AOPP and oxidation of serum albumin was also gradually declined by 20 weeks, while the oxidized albumin and AOPP in group II was not significantly changed during the entire experimental period. Transferrin saturation and serum ferritin levels were also increased in group I compared with group II at 4 weeks (P0.001). In addition, we found a strong positive correlation between oxidized albumin and serum ferritin levels (r=0.615, P0.05), suggesting the possibility that the accumulation of iron stores has a causative role in the progression of oxidative stress in HD patients treated with IVIR.The results of this study indicate that lower frequency IVIR protocol is recommended to reduce IVIR-induced oxidative stress in HD patients.

AIMS Recently, we reported the effectiveness of PAC therapy, a combination therapy with prostaglandin (PG) and angiotensin-converting enzyme inhibitor (ACE-I), as a new tool for the prevention of chronic kidney disease. In the current study, we continually treated these patients with or without PG and analyzed the survival rate of renal function by Kaplan-Meier method and Cox regression analysis. MATERIAL AND METHODS 52 patients (serum creatinine 2.9 A+/- 1.9 mg/dl) were followed-up for 48 months. 26 patients continued to receive ACE-I monotherapy and the remaining 26 patients were treated by PAC therapy. Primary end-point was defined as a decrease in 1/Cr by 0.2 (dl/mg), initiation of renal replacement therapy or death. RESULTS At the end of the study, PAC therapy significantly reduced the risk for the decline in renal function compared to ACE-I monotherapy by 54%. Survival time was longer in PAC group (21.7 A+/- 2.2 and 35.1 A+/- 3.9 months, in ACE-I monotherapy and PAC therapy, p < 0.05). Cox regression analysis indicated that age, sex and blood pressure except urinary protein excretion did not relate to the risk reduction by PAC therapy. CONCLUSION PAC therapy was proved to reduce the progression of end-stage renal failure.

In the proximal tubules of rat (r) kidney, the polyspecific organic cation transporters (OCTs), rOCT1 and rOCT2, mediate the baso-lateral uptake of various organic cations, including many drugs, toxins, and endogenous compounds, and the apical type of H(+)/ organic cation antiporter, rat multidrug and toxin extrusion 1 (rMATE1), mediate the efflux of organic cations. Renal clearances of H(2) receptor antagonists, including famotidine, were reported to be decreased in patients with kidney disease. Therefore, acute kidney injury (AKI) could influence renal excretion and disposition of organic cations accompanied by the regulation of organic cation transporters. The aim of this study was to investigate the pharmacokinetic alteration of cationic drugs and the expression of tubular organic cation transporters, rOCT1, rOCT2, and rMATE1, in ischemia/reperfusion (I/R)-induced AKI rats. I/R-induced AKI increased the plasma concentration of i.v. administrated famotidine, a substrate for rOCT1 and rOCT2, or tetraethylammonium (TEA), a substrate for rOCT1, rOCT2, and rMATE1. The areas under the plasma concentration curves for famotidine and TEA were 2- and 6-fold higher in I/R rats than in sham-operated rats, respectively. The accumulation of TEA into renal slices was significantly decreased, suggesting that organic cation transport activity at the basolateral membranes was reduced in I/R rat kidney. The protein expressions of basolateral rOCT2 and luminal rMATE1 were down-regulated in I/R rat kidneys. These data suggest that the urinary secretion of cationic drugs via epithelial organic cation transporters is decreased in AKI.

The effect of acute renal failure (ARF) induced by ischemia/reperfusion (I/R) of rat kidney on the expression of organic anion transporters (OATs) was examined. The level of serum indoxyl sulfate (IS), a uremic toxin and substrate of OATs in renal tubules, shows a marked increase with the progression of ARF. However, this increase was significantly attenuated by ingestion of cobalt. The level of mRNA and protein of both rOAT1 and rOAT3 were markedly depressed in the ischemic kidney. The uptake of p-aminohippuric acid (PAH) and estrone sulfate (ES) by renal slices of ischemic rats was significantly reduced compared to control rats. Renal slices taken from ischemic rats treated with cobalt displayed significantly elevated levels of ES uptake. Cobalt intake did not affect PAH uptake, indicating the functional restoration of rOAT3 but not rOAT1. The expression of Na(+)/K(+)-ATPase was markedly depressed in the ischemic kidney, suggesting that the inward Na(+) gradient in renal tubular cells had collapsed, thereby reducing the outward gradient of alpha-ketoglutarate, a driving force of both rOATs. The decreased expression of Na(+)/K(+)-ATPase was significantly restored by cobalt treatment. Our results suggest that the downregulation of renal rOAT1 and rOAT3 could be responsible for the increase in serum IS level of ischemic rats. Cobalt treatment has a significant protective effect on ischemia-induced ARF, being accompanied by the restoration of rOAT3 and/or Na(+)/K(+)-ATPase function.

Prostasin has been shown to regulate sodium handling in the kidney. Recently, a serine protease inhibitor, protease nexin-1 (PN-1), was identified as an endogenous inhibitor for prostasin. Therefore, we hypothesized that PN-1 may regulate sodium reabsorption by reducing prostasin activity, and that expression of PN-1 was regulated by transforming growth factor-beta1 (TGF-beta1) or aldosterone, like prostasin. cRNAs for epithelial sodium channel (ENaC), prostasin, and PN-1 were expressed in Xenopus oocytes, and the amiloride-sensitive sodium currents (I(Na)) were measured. The effect of TGF-beta1 and aldosterone on the mRNA and protein abundance of PN-1 and ENaC was detected by real-time polymerase chain reaction and immunoblotting in M-1 cells. Expression of PN-1 substantially decreased prostasin-induced I(Na) by approximately 68% in oocytes. Treatment of M-1 cells with 20 ng/ml TGF-beta1 significantly increased protein expression of PN-1 by 3.8+/-0.5-fold, whereas administration of 10(-6) M aldosterone markedly decreased protein expression of PN-1 to 53.7+/-6.7%. Basolateral, but not apical, application of TGF-beta1 significantly reduced I(eq). To elucidate the involvement of PN-1 in basal ENaC activity, we silenced the expression of PN-1 by using short-interfering RNA. This increased I(eq) by 1.6+/-0.1-fold. Our study indicates that PN-1 could have a natriuretic role by inhibiting prostasin activity and suggests the possibility that aldosterone and TGF-beta reciprocally regulate the expression of PN-1 in renal epithelial cells contributing to salt retention or natriuresis, respectively by an additional mechanism. PN-1 could represent a new factor that contributes to regulation of ENaC activity in the kidney.

A 59-year-old woman with a 10-year history of hemodialysis was admitted to our hospital for painful skin ulcers on her right thigh, right calf, and left upper arm. A whole-body plain computed tomographic scan showed diffuse calcification of the uterus and marked calcification of the mitral valve. Skin biopsy specimens from the left thigh showed calcium deposition in numerous small blood vessels in the dermis and fat, leading to a diagnosis of calcific uremic arteriolopathy (CUA). Despite antibiotic therapy and aggressive wound care for 2 months, the skin ulcers enlarged and the patient's general condition worsened. Surprisingly, oral administration of etidronate disodium (200 mg/d) strikingly improved the focal infection and decreased the size of the skin ulcers within several days. She was discharged from the hospital 2 months later, when epithelialization of the ulcers was almost complete. We report a case of CUA that was improved dramatically by treatment with etidronate. Etidronate therapy should be considered for refractory CUA.

The predicted structure of the autosomal recessive polycystic kidney disease protein, fibrocystin, suggests that it may function as a receptor, but its function remains unknown. To understand its function, we searched for proteins that interact with the intracellular C-terminus of fibrocystin using the yeast two-hybrid system. From the screening, we found calcium modulating cyclophilin ligand (CAML), a protein involved in Ca2+ signaling. Immunofluorescent analysis showed that both proteins are co-localized in the apical membrane, primary cilia, and the basal body of cells derived from the distal nephron Epitope-tagged expression constructs of both proteins were co-immunoprecipitated from COS7 cells. The intracellular C-terminus of fibrocystin interacts with CAML, a protein with an intracellular distribution that is similar to that of PKD2. Fibrocystin may participate in regulation of intracellular Ca2+ in the distal nephron in a manner similar to PKD1 and PKD2 that are involved in autosomal dominant polycystic kidney disease.

Hyperosmolality in the renal medullary interstitium is generated by the renal countercurrent multiplication system, in which the medullary thick ascending limb (MAL) and the outer medullary collecting duct (OMCD) primarily participate. Since arginine vasopressin (AVP) regulates Na-K-ATPase activity directly via protein kinase A and indirectly via hyperosmolality, we investigated the acute and chronic effects of hyperosmolality on Na-K-ATPase and AVP-dependent cAMP generation in the MAL and OMCD. Microdissected MAL and OMCD from control and dehydrated rats were used for the measurement of Na-K-ATPase activity, mRNA expression of alpha-1, beta-1, and beta-2 subunits of Na-K-ATPase, and AVP-dependent cAMP generation. Na-K-ATPase activity in the MAL from dehydrated rats, as measured in isotonic medium, was higher than that of control rats. Moreover, incubation of samples in hypertonic medium (490 mOsm/kg H2O) further increased Na-K-ATPase activity. Dehydration increased alpha-1, beta-1, and beta-2 mRNA expression in the MAL without changing that in the OMCD. Western blot analysis revealed that in the outer medulla, the expression of beta-1, but not that of alpha-1 or beta-2, was stimulated by dehydration. Incubation of MAL or OMCD in hypertonic medium increased AVP-dependent cAMP generation. Higher levels of AVP-dependent cAMP were generated in the MAL from dehydrated rats than that of controls, although incubation in hypertonic medium did not lead to additional increases in AVP-dependent cAMP accumulation. In contrast, AVP-dependent cAMP generation in the OMCD was stimulated by dehydration, and was further stimulated by incubation in hypertonic medium. These findings demonstrate that Na-K-ATPase is upregulated short- and long-term hyperosmolality in the MAL, but not in OMCD.

Inhibition of prostasin expression by TGF-β1 in renal epithelial cells. Background Prostasin has been shown to be involved in the regulation of sodium handling in the kidney. TGF-β1 has been demonstrated to suppress αENaC expression and sodium uptake. Therefore, we hypothesized that TGF-β1 may regulate prostasin expression to modulate sodium reabsorption in the kidney. Methods To determine if TGF-β1 has an effect on prostasin expression, we isolated 2.9kb of the rat prostasin promoter, and measured its transcriptional activity with a luciferase assay in mouse cortical collecting duct cell line (M-1). The effect of TGF-β1 on the mRNA and protein abundance of prostasin, and amiloride-sensitive 22 Na uptake was determined. Results Treatment of M-1 cells with 20ng/mL of TGF-β1 for 24 hours significantly decreased the promoter activity by 50 ± 1%, and the inhibitory effect was dose dependent over the range of 0.1 to 20 ng/mL. We identified a 50bp region (-410 to -360) containing c-Rel–like sequence in prostasin promoter that is responsible for the TGF-β1–mediated inhibition, and found that TGF-β1 increases IκBα expression in M-1 cells. TGF-β1 reduced endogenous prostasin mRNA and protein expression in M-1 cells by 50 ± 12% and 44 ± 12%, respectively, and the amiloride-sensitive 22 Na uptake by 35.9 ± 4.8%. Conclusion Our findings indicate the possibility that TGF-β1 transcriptionally inhibits prostasin expression by the induction of IκBα and the subsequent inhibition of NF-κB/Rel activity in M-1 cells, and also suggest the possibility that TGF-β1 might inhibit sodium reabsorption through a reduction in prostasin expression and subsequent inhibition of ENaC activity.

Rad18 is involved in postreplication repair mainly through monoubiquitination of proliferating cell nuclear antigen (PCNA). Here we show that Rad18 protein was detected in human cells as two major bands at 75 and 85 kDa by Western blot. The bands were identified as nonubiquitinated and monoubiquitinated forms of Rad18, respectively, by mass spectrometry. Multiple ubiquitinated bands of Rad18 were detected in vitro in the presence of E1, E2 (Rad6), and methylated ubiquitin, indicating that Rad18 was monoubiquitinated at multiple sites through autoubiquitination. Rad18 self-associates, and this interaction was abolished by replacing one of the conserved cysteine residues with phenylalanine in the zinc finger domain (C207F). In the C207F mutant Rad18, monoubiquitination of Rad18 was not observed in vivo, suggesting that self-association was critical for monoubiquitination. Monoubiquitinated Rad18 was detected mainly in the cytoplasm, whereas nonubiquitinated Rad18 was detected predominantly in the nuclei. Furthermore, Rad18 was shown to be polyubiquitinated in cells treated with proteasome inhibitors. Purified Rad18 was also polyubiquitinated in an in vitro system containing E1, E2 (Rad6), and ubiquitin, and it was degraded by the addition of proteasomes. These results suggest that the amount of Rad18 in the nucleus is regulated differentially by mono- and polyubiquitination.

Background: Nephrotic syndrome is characterized by severe proteinuria and sodium and water retention. Although endothelin (ET) 1 can cause natriuresis or antinatriuresis, the role played by ET-1 in proteinuria and in sodium retention due to nephrotic syndrome remains unclear. Methods: We investigated the role played by the ET-1 system in sodium and water retention and in proteinuria in puromycin aminonucleoside induced nephrotic syndrome in rats using microdissected nephron segments, competitive polymerase chain reaction, and Western blot. Results: The expression of prepro ET-1, ET-converting enzyme 1 (ECE-1), and ET A receptor mRNAs, but not ET B receptor mRNA, in the glomeruli was increased in rats with nephrotic syndrome. The cGMP generation in the glomeruli induced by atrial natriuretic peptide and ET-1 was decreased, whereas the ET-3-induced cGMP generation was increased in rats with nephrotic syndrome. ECE-1 mRNA expression was increased not only in the glomeruli, but also in the thick ascending limbs and collecting ducts. The protein expression of ECE-1 was increased in the membrane fraction of the cortex and in the outer and the inner medulla of nephrotic rats. Blockade of ET A and B receptors by bosentan did not inhibit the occurrence of nephrotic syndrome. However, the administration of bosentan increased the urinary sodium excretion. Conclusion: These data suggest that an activated ET-1-ET A receptor pathway in glomeruli and/or an increased ECE-1 mRNA expression in distal segments may participate in sodium and water retention, but not in the occurrence of nephrotic syndrome.

Background: Na- and Cl-dependent organic solute cotransporters participate in transporting neurotransmitters in the brain and organic osmolytes in the kidney. Methods: We examined the intranephron localization and regulation of the renal osmotic-stress-induced Na-Cl organic solute cotransporter (ROSIT) mRNA expression using microdissected nephron segments from control and dehydrated rats and RT-PCR. To further know the mechanisms of gene regulation of ROSIT, microdissected proximal straight tubules (PST) were incubated in isotonic (290 mosm/kg H2O) or hyperosmotic (490– 1,090 mosm/kg H2O) solution. Results: ROSIT mRNA was expressed predominantly in PST and to a lesser extent in cortical thick ascending limbs and cortical collecting ducts in control rats, and dehydration caused an increase in the expression in whole nephron segments. ROSIT mRNA in PST was decreased with time by incubation in isotonic solution. Incubation of PST in hypertonic solution by adding NaCl increased mRNA expression as early as 15 min (1.5- and 3-fold at 15 and 30 min, respectively). This stimulating effect of NaCl was largest at 890 mosm/kg H2O. Hypertonicity by mannitol or myoinositol also increased ROSIT mRNA expression. In contrast, hyperosmolality by urea reduced ROSIT mRNA expression. GAPDH mRNA expression, an internal standard, did not change by incubation in NaCl or mannitol solution. Conclusion: In summary, ROSIT mRNA expression was most abundant in PST in control and it was stimulated in whole nephron segments by dehydration. ROSIT mRNA expression in PST was stimulated by hypertonicity but not by urea. These data suggest that ROSIT may participate in the transport of amino acid under control conditions and organic osmolytes in dehydration.

Hypocalcemia, hyperphosphatemia and resistance to the action of parathyroid hormone (PTH) are well-characterized features in advanced chronic renal failure (CRF). Their pathogenesis has been attributed to both PTH receptor (PTH-R) down-regulation and postreceptor abnormalities. In this study, we examined the renal expression of the PTH-R mRNA in CRF (5/6 nephrectomy) rats. Experiments were also performed to determine whether an acidic condition and PTH itself influence PTH-R mRNA expression. RT-competitive PCR was used to examine mRNA expression, and polyclonal antibody against PTH-R was used for Western blot. PTH-R mRNA expression was abundant in glomeruli, proximal convoluted and straight tubules (PCT, PST), small in medullary and cortical thick ascending limbs, and cortical collecting ducts and not detectable in outer and inner medullary collecting ducts. The expression was significantly decreased in PCT and PST in CRF rats. Decrease in PTH-R mRNA expression was observed 1 week after the induction of CRF. PTH-R protein was decreased at 2 (–23%) and 4 (–45%) weeks in renal cortex, but not in medulla in CRF rats. PTH-R mRNA expression in PST was decreased by low pH (7.1 or 6.7) incubation compared with that at pH 7.4. PTH(1–34) (10–9 M) increased PTH-R mRNA expression in PST from control rats by 250%. The stimulatory effect of PTH on PTH-R mRNA expression was decreased by the incubation at low pH medium. In summary, renal PTH-R is down-regulated in CRF rats. The decrease in mRNA expression in PCT and PST causes the decrease in PTH-R protein. Metabolic acidosis may participate in the down-regulation of PTH-R in early stage of CRF. This abnormality could be important in the pathogenesis of secondary hyperparathyroidism of CRF.