Epithelial Na+ Channel Activation after Bile Duct Ligation with Mineralocorticoid Receptor Blockade.

BACKGROUND: Sodium and fluid retention in liver disease are classically thought to result from reduced effective circulating volume and stimulation of the renin-angiotensin-aldosterone system (RAAS). However, evidence of fluid retention in patients without RAAS activation suggests the involvement of additional mechanisms. In vitro, bile acids activate the epithelial Na+ channel (ENaC) found in the aldosterone-sensitive distal nephron. If this occurs in vivo, ENaC may become activated in liver disease even with antagonism of aldosterone signaling. METHODS: To test this, we performed bile duct ligation to induce liver disease and increase circulating bile acids in mice given spironolactone to antagonize aldosterone signaling. We analyzed effects on blood, urine and body composition. We also determined the effects of taurocholic acid, a primary conjugated bile acid elevated in liver disease, on ion fluxes in microperfused rabbit collecting ducts. RESULTS: Bile duct ligation increased benzamil-sensitive natriuresis compared to sham, indicating ENaC activation. These effects were not explained by effects on ENaC expression, cleavage, or localization. Bile duct ligated mice also gained significantly more fluid than sham-operated animals. Blocking ENaC reversed fluid gains in bile duct ligated mice but had no effect in shams. In dissected collecting ducts from rabbits, which express ENaC, taurocholic acid stimulated net Na+ absorption. CONCLUSIONS: Our results provide experimental evidence for a novel aldosterone-independent mechanism for sodium and fluid retention in liver disease.

Long-term outcome of childhood-onset complicated nephrotic syndrome after a multicenter, double-blind, randomized, placebo-controlled trial of rituximab.

BACKGROUND: Although rituximab effectively prevents relapses of complicated frequently relapsing nephrotic syndrome (FRNS) and steroid-dependent nephrotic syndrome (SDNS), data of long-term outcomes and safety are limited. METHODS: Fifty-one patients (age, 3-38 years) with childhood-onset complicated FRNS or SDNS, who received rituximab in investigator-initiated multicenter prospective trials were enrolled. Rituximab was administered at 375 mg/m2 once weekly for 4 weeks, and immunosuppressive agents were discontinued according to the study protocol. We investigated relapses, re-administration of immunosuppressive agents, additional rituximab treatment, body height, renal function, and late adverse events during the observation period. RESULTS: Forty-eight patients (94%) developed relapses during the observation period (median, 59 months) and the 50% relapse-free survival was 261 days. Thirty patients (59%) developed SDNS, 44 (86%) required re-administration of immunosuppressive agents, and 22 (43%) received additional rituximab treatment. All patients who were receiving immunosuppressive agents at rituximab treatment required either immunosuppressive agents or additional rituximab treatment. On the contrary, 5 of the 13 patients without immunosuppressive agents at rituximab treatment required neither immunosuppressive agents nor additional rituximab treatment and 3 of them did not develop relapse during observation period. Growth failure due to steroid toxicity did not progress and none of the patients developed chronic renal insufficiency. None of the patients suffered from rituximab-related late adverse events. CONCLUSIONS: As most patients suffer from relapses after B-cell recovery, long-term immunosuppressive agents or additional rituximab treatment is necessary. However, some patients who can discontinue immunosuppressive agents before rituximab treatment may achieve long-term remission after rituximab treatment without immunosuppressive agents.

Extrapontine myelinolysis associated with severe hypernatremia in infancy.

Extrapontine myelinolysis (EPM) is an uncommon disorder in children, with few pediatric cases reported to date. We report the first case of an infant with EPM without central pontine myelinolysis (CPM) presenting with severe hypernatremia. On admission, the infant had impaired consciousness, mild dehydration, and severe hypernatremia (190 mmol/L). The following day, the patient developed abnormal involuntary movements. Brain magnetic resonance imaging (MRI) confirmed EPM without CPM. He recovered without sequelae, and clinical examinations were within normal limits approximately 6 months after discharge. Brain MRI at 1 year after onset showed complete disappearance of the previous EPM regions. To the best of our knowledge, this represents the youngest patient with EPM without CPM presenting with severe hypernatremia. Given that treatment for osmotic demyelination syndrome (ODS) is yet to be established, preventing the development of ODS is crucial.

A case of infant botulism infection due to consumption of untreated well-water.

A 7-week-old boy with flaccid paralysis was diagnosed with infant botulism caused by Clostridium botulinum toxin type A. In this case of infant botulism, untreated well-water was identified as a potential source of this infection.

Development of enzyme-linked immunosorbent assays for urinary thiazide-sensitive Na-Cl cotransporter measurement.

The Na-Cl cotransporter (NCC) in the distal convoluted tubules in kidney is known to be excreted in urine. However, its clinical significance has not been established because of the lack of quantitative data on urinary NCC. We developed highly sensitive enzyme-linked immunosorbent assays (ELISAs) for urinary total NCC (tNCC) and its active form, phosphorylated NCC (pNCC). We first measured the excretion of tNCC and pT55-NCC in urinary exosomes in pseudohypoaldosteronism type II (PHAII) patients since PHAII is caused by NCC activation. Highly increased excretion of tNCC and pNCC was observed in PHAII patients. In contrast, the levels of tNCC and pNCC in the urine of patients with Gitelman's syndrome were not detectable or very low, indicating that both assays could specifically detect the changes in urinary NCC excretion caused by the changes of NCC activity in the kidney. Then, to test whether these assays could be feasible for a more general patient population, we measured tNCC and pNCC in the urine of outpatients with different clinical backgrounds. Although urinary protein levels >30 mg/dl interfered with our ELISA, we could measure urinary pNCC in all patients without proteinuria. Thus we established highly sensitive and quantitative assays for urinary NCC, which could be valuable tools for estimating NCC activity in vivo.

Functional hearing loss and developmental imbalances.

OBJECTIVE: Functional hearing loss (FHL) is a disorder in which there are abnormal values on a hearing test, despite the absence of organic abnormalities in the peripheral and central auditory pathways. Here, we examined the developmental characteristics of FHL and the importance of intervention by analyzing the clinical characteristics of children with this disorder. METHODS: We retrospectively examined 16 patients assessed under a diagnosis of FHL. After interventions such as psychological counseling by our pediatrics and psychiatry departments, we compared the clinical profiles of patients in which hearing was "improved/normalized" and "unimproved". RESULTS: Fourteen patients visited a pediatrician and two chose not to do so. A discrepancy between the maximum and minimum values of the four index scores was observed in all patients in which WISC-IV (the fourth version of the Wechsler Intelligence Scale for Children) was performed (n = 12). The discrepancy between the verbal comprehension index (VCI) and perceptual reasoning index (PRI) was significantly greater in "unimproved" patients than in "improved/normalized" patients. Hearing improved, or was normalized, after intervention in six of 16 patients. CONCLUSIONS: Developmental imbalances were suspected in all 12 children who visited a pediatrician and completed the WISC-IV. Cooperation with pediatricians, psychiatrists, and other health professionals is desirable in supporting patients diagnosed with FHL.

Myelin oligodendrocyte glycoprotein antibody-associated disease in a patient with symptoms of aseptic meningitis who achieved spontaneous remission: A case report and review of the literature.

BACKGROUND: A few case reports have described patients with myelin oligodendrocyte glycoprotein antibody (MOG-Ab)-associated demyelinating syndrome who presented with symptoms of aseptic meningitis. All such patients required immunotherapy. We report a patient with MOG-Ab-associated disorder (MOGAD) who presented with symptoms of aseptic meningitis and improved without treatment. CASE: A 13-year-old girl presented with fever, headache, decreased appetite, and neck stiffness. Cerebrospinal fluid (CSF) analysis revealed pleocytosis and magnetic resonance imaging (MRI) showed leptomeningeal enhancement. The patient was diagnosed with aseptic meningitis at admission. However, there were no signs of recovery 4 days after admission (i.e., 8 days after disease onset). Therefore, we performed extensive investigations to identify the cause of the underlying infection and inflammation. On day 14 after admission, the serum MOG-Ab test performed at admission came back positive (1:128) and she was diagnosed with MOGAD. She was discharged on day 18 after admission, because her symptoms, CSF pleocytosis, and MRI findings had improved. About 6 weeks after discharge, MRI revealed hyperintensity without gadolinium enhancement. However, her serum MOG-Ab test was negative. We did follow-ups for 11 months but found no new neurological symptoms. DISCUSSION AND CONCLUSION: To the best of our knowledge, this is the first ever report of a pediatric patient with MOGAD experiencing spontaneous remission with no demyelinating symptoms during an extended follow-up period.

Molecular pathogenesis of pseudohypoaldosteronism type II: generation and analysis of a Wnk4(D561A/+) knockin mouse model.

WNK1 and WNK4 mutations have been reported to cause pseudohypoaldosteronism type II (PHAII), an autosomal-dominant disorder characterized by hyperkalemia and hypertension. To elucidate the molecular pathophysiology of PHAII, we generated Wnk4(D561A/+) knockin mice presenting the phenotypes of PHAII. The knockin mice showed increased apical expression of phosphorylated Na-Cl cotransporter (NCC) in the distal convoluted tubules. Increased phosphorylation of the kinases OSR1 and SPAK was also observed in the knockin mice. Apical localization of the ROMK potassium channel and transepithelial Cl(-) permeability in the cortical collecting ducts were not affected in the knockin mice, whereas activity of epithelial Na(+) channels (ENaC) was increased. This increase, however, was not evident after hydrochlorothiazide treatment, suggesting that the regulation of ENaC was not a genetic but a secondary effect. Thus, the pathogenesis of PHAII caused by a missense mutation of WNK4 was identified to be increased function of NCC through activation of the OSR1/SPAK-NCC phosphorylation cascade.

Generation and analyses of R8L barttin knockin mouse.

Barttin, a gene product of BSND, is one of four genes responsible for Bartter syndrome. Coexpression of barttin with ClC-K chloride channels dramatically induces the expression of ClC-K current via insertion of ClC-K-barttin complexes into plasma membranes. We previously showed that stably expressed R8L barttin, a disease-causing missense mutant, is retained in the endoplasmic reticulum (ER) of Madin-Darby canine kidney (MDCK) cells, with the barttin ß-subunit remaining bound to ClC-K α-subunits (Hayama A, Rai T, Sasaki S, Uchida S. Histochem Cell Biol 119: 485-493, 2003). However, transient expression of R8L barttin in MDCK cells was reported to impair ClC-K channel function without affecting its subcellular localization. To investigate the pathogenesis in vivo, we generated a knockin mouse model of Bartter syndrome that carries the R8L mutation. These mice display disease-like phenotypes (hypokalemia, metabolic alkalosis, and decreased NaCl reabsorption in distal tubules) under a low-salt diet. Immunofluorescence and immunoelectron microscopy revealed that the plasma membrane localization of both R8L barttin and the ClC-K channel was impaired in these mice, and transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) as well as thiazide-sensitive chloride clearance were significantly reduced. This reduction in transepithelial chloride transport in tAL, which is totally dependent on ClC-K1/barttin, correlated well with the reduction in the amount of R8L barttin localized to plasma membranes. These results suggest that the major cause of Bartter syndrome type IV caused by R8L barttin mutation is its aberrant intracellular localization.

Calcium and calcimimetics regulate paracellular Na+ transport in the thin ascending limb of Henle's loop in mouse kidney.

The effect of Ca(2+) and calcimimetics on NaCl transport was investigated in the in vitro isolated microperfused mouse thin ascending limb of Henle's loop. In the presence of a transmural NaCl gradient, the transepithelial diffusional potential was 13.7 +/- 0.4 mV (n = 17). When the Ca(2+) in the bath was increased from 1.5 to 4.5 mM at 37 degrees C, the relative permeability of Na(+) to Cl(-) (P (Na) /P (Cl)) estimated from the diffusional voltage deflection due to the transepithelial NaCl gradient (V (d)) changed from 0.371 +/- 0.017 to 0.341 +/- 0.015 (n = 10, P < 0.0001). When the Ca(2+) in the lumen was increased from 1.5 to 4.5 mM, the P (Na) /P (Cl) decreased from 0.349 +/- 0.013 to 0.330 +/- 0.013 (n = 5, P < 0.002). The addition of 0.1 mM neomycin and 0.2 mM gentamicin to the bath or lumen also decreased the P (Na) /P (Cl). The same effect on P (Na) /P (Cl) of Ca(2+) and calcimimetics occurred in ClC-K1 (kidney-specific chloride channel) knockout mice. The addition of 300 mug/ml protamine to the bath strongly inhibited changes to P (Na) /P (Cl) induced by basolateral Ca(2+). These data indicate that ambient Ca(2+) and calcimimetics inhibit Na(+) transport in the thin ascending limb, which is known to occur via the paracellular shunt pathway. Our observations strongly suggest that Ca(2+) is involved in the regulation of paracellular Na(+) permeability in the thin ascending limbs.

Calcium-sensing receptor stimulates luminal K+-dependent H+ excretion in medullary thick ascending limbs of Henle's loop of mouse kidney.

The calcium-sensing receptor (CaSR) is known well as a sensor of extracellular calcium for regulating parathyroid hormone secretion. CaSR is located along all nephron segments in the kidney. While hypercalcemia strongly enhances urinary acidification, the relationship between CaSR and acid-base metabolism in the kidney is still uncertain. In the present study, we examined whether CaSR activation caused acid secretion in the medullary thick ascending limb (mTAL), which is one of the major nephron segments involved in both mineral and acid-base regulation. The effects of a potent calcimimetic neomycin (Neo) on intracellular pH (pHi) were analyzed in the in vitro miroperfused mouse mTALs. The mTALs were incubated with 2,7-bis-(2-carboxyethyl)-5(6)-carboxyfluoresceine-acetoxymethylester (BCECF-AM) for microfluorescent pHi measurements. In HCO(3)(-)/CO(2)-buffered solution, the steady-state pHi was 7.17 +/- 0.01 (n = 19). Basolateral Neo at 0.4 mM in basolateral side significantly alkalinized the mTAL cells to 7.28 +/- 0.02 (n = 19), while Neo in the lumen had no effect on pHi. Neo in the basolateral side alkalinized the mTALs in the absence of ambient Na(+) and the presence of H(+)-ATPase inhibitor bafilomycin in the lumen, indicating that the effect of Neo is unrelated to Na(+)-dependent acid-base transporters such as Na(+)-H(+) exchangers and Na(+)-HCO(3)(-) cotransporter, or to luminal H(+)-ATPase. In contrast, the effect of Neo on pHi was inhibited by K(+) removal or treatment with specific H(+)-K(+)-ATPase (HKa) inhibitors, ouabain and Sch-28080, in the lumen. Our results suggest that hypercalcemia induces urinary acidification partly by stimulating luminal K(+)-dependent H(+)-excretion via CaSR in mouse mTALs.

AKAPs-PKA disruptors increase AQP2 activity independently of vasopressin in a model of nephrogenic diabetes insipidus.

Congenital nephrogenic diabetes insipidus (NDI) is characterized by the inability of the kidney to concentrate urine. Congenital NDI is mainly caused by loss-of-function mutations in the vasopressin type 2 receptor (V2R), leading to impaired aquaporin-2 (AQP2) water channel activity. So far, treatment options of congenital NDI either by rescuing mutant V2R with chemical chaperones or by elevating cyclic adenosine monophosphate (cAMP) levels have failed to yield effective therapies. Here we show that inhibition of A-kinase anchoring proteins (AKAPs) binding to PKA increases PKA activity and activates AQP2 channels in cortical collecting duct cells. In vivo, the low molecular weight compound 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives increase AQP2 activity to the same extent as vasopressin, and increase urine osmolality in the context of V2R inhibition. We therefore suggest that FMP-API-1 may constitute a promising lead compound for the treatment of congenital NDI caused by V2R mutations.

Psychometric validation of a new measurement instrument for time-oriented patient information in electronic medical records: A questionnaire survey of physicians.

RATIONALE, AIMS, AND OBJECTIVES: Time is an important element in medical data. Physicians record and store information about patients' disease progress and treatment response in electronic medical records (EMRs). Because EMRs use timestamps, physicians can identify patterns over time regarding a patient's disease and treatment (eg, laboratory values and medications). However, analyses of physicians' use and satisfaction with EMRs have focused on functionality, storage, and system operation rather than the use of time-oriented information. This study aimed to understand physicians' needs regarding time-oriented patient information in EMRs in clinical practice. METHODS: The reliability and validity of the items in the questionnaire were evaluated in 87 physicians at a national university hospital. Internal consistency was satisfactory (Cronbach alpha coefficient, 0.87). RESULTS: Four dimensions were identified in exploratory factor analysis. Correlations between the 4 dimensions supported the construct validity of the items. Scores of time-oriented patients' medical history in the 4 dimensions showed a significant association with physician age. Based on confirmatory factor analysis, associations were significant and positive (P < .001). In terms of the needs of physicians regarding time-oriented patient information in EMRs, both time-oriented treatment results followed by time-oriented team information had significant positive associations. CONCLUSION: Our study suggests that 4 specific time-oriented patient information factors in EMRs are needed by physicians. Exploring physicians' needs regarding patient-specific time-oriented information may provide a better understanding of the barriers facing the adoption and use of EMRs (eg, decision-making and practice safety concerns) and lead to better acceptance of EMRs in physicians' clinical practices.

Wnt5a induces renal AQP2 expression by activating calcineurin signalling pathway.

Heritable nephrogenic diabetes insipidus (NDI) is characterized by defective urine concentration mechanisms in the kidney, which are mainly caused by loss-of-function mutations in the vasopressin type 2 receptor. For the treatment of heritable NDI, novel strategies that bypass the defective vasopressin type 2 receptor are required to activate the aquaporin-2 (AQP2) water channel. Here we show that Wnt5a regulates AQP2 protein expression, phosphorylation and trafficking, suggesting that Wnt5a is an endogenous ligand that can regulate AQP2 without the activation of the classic vasopressin/cAMP signalling pathway. Wnt5a successfully increases the apical membrane localization of AQP2 and urine osmolality in an NDI mouse model. We also demonstrate that calcineurin is a key regulator of Wnt5a-induced AQP2 activation without affecting intracellular cAMP level and PKA activity. The importance of calcineurin is further confirmed with its activator, arachidonic acid, which shows vasopressin-like effects underlining that calcineurin activators may be potential therapeutic targets for heritable NDI.

Reconstruction of tubular structures in three-dimensional collagen gel culture using proximal tubular epithelial cells voided in human urine.

Human proximal tubular (PT) epithelial cells were isolated from urine and monoclonally cultured as monolayers for 1 wk, after which they were subcultured between two layers of collagen gel, designated a "collagen gel sandwich." Under these culture conditions, PT cells formed three-dimensional tubular structures exhibiting distinct polarized cell morphology. Scanning and transmission electron microscopic studies showed that they bore numerous microvilli at the apical surface and that they closely contacted the collagen gel at the basal surface. These studies indicate that PT cells exfoliated in urine still exhibit the potential to proliferate and form organized structures mimicking in vivo tubules. Because of the current lack of useful culture systems for human tubular epithelial cells originating from kidney tissue, we suggest that this unique culture system using voided PT cells in urine could open up new avenues to study not only the mechanisms of morphogenesis but also the physiology of human PT cells.

Effect of a blackout in pediatric patients with home medical devices during the 2011 eastern Japan earthquake.

BACKGROUND: during the eastern Japan earthquake in 2011 and the following prolonged blackout, pediatric patients with home medical devices sought electricity at the pediatric department. We retrospectively studied the effect of this earthquake and the following blackout. METHODS: we hand-reviewed pediatric admission records in Tohoku University Hospital for new inpatients attributed to the earthquake from March 11, 2011 to April 12, 2011. A survey by questionnaire regarding the situation during the earthquake was performed for parents of technology-assisted patients. RESULTS: during the study period, 24 pediatric patients were admitted to the pediatric department. Eighteen technology-assisted pediatric patients, including those with home respirators, accounted for 75% of new pediatric admissions. Patients who were admitted for electricity shortage stayed in the hospital for a mean of 11.0days (3-25days). The questionnaire survey showed that 55% of technology-assisted patients were admitted to medical centers for evacuation. The majority of patients (89%) with ventilators were eventually admitted to medical centers during the earthquake. Most of the parents of technology-assisted patients experienced a prolonged petrol shortage and difficulty in communications with medical centers. CONCLUSION: the current study suggests that technology-assisted pediatric patients with neurological disorders as the primary disease can overwhelm the capacity of hospital inpatient facilities in certain situations. Disaster preparedness should consider assuring power requirements in healthcare facilities and preparing backup power generators lasting for at least 24h for these patients. Preparing alternative measures for emergent electricity and communications could remedy serious conditions during a disaster.

Ca2+ dependence of flow-stimulated K secretion in the mammalian cortical collecting duct.

Apical low-conductance SK and high-conductance Ca(2+)-activated BK channels are present in distal nephron, including the cortical collecting duct (CCD). Flow-stimulated net K secretion (J(K)) in the CCD is 1) blocked by iberiotoxin, an inhibitor of BK but not SK channels, and 2) associated with an increase in [Ca(2+)](i), leading us to conclude that BK channels mediate flow-stimulated J(K). To examine the Ca(2+) dependence and sources of Ca(2+) contributing to flow-stimulated J(K), J(K) and net Na absorption (J(Na)) were measured at slow (approximately 1) and fast (approximately 5 nl.min(-1).mm(-1)) flow rates in rabbit CCDs microperfused in the absence of luminal Ca(2+) or after pretreatment with BAPTA-AM to chelate intracellular Ca(2+), 2-aminoethoxydiphenyl borate (2-APB), to inhibit the inositol 1,4,5-trisphosphate (IP(3)) receptor or thapsigargin to deplete internal stores. These treatments, which do not affect flow-stimulated J(Na) (Morimoto et al. Am J Physiol Renal Physiol 291: F663-F669, 2006), inhibited flow-stimulated J(K). Increases in [Ca(2+)](i) stimulate exocytosis. To test whether flow induces exocytic insertion of preformed BK channels into the apical membrane, CCDs were pretreated with 10 microM colchicine (COL) to disrupt microtubule function or 5 microg/ml brefeldin-A (BFA) to inhibit delivery of channels from the intracellular pool to the plasma membrane. Both agents inhibited flow-stimulated J(K) but not J(Na) (Morimoto et al. Am J Physiol Renal Physiol 291: F663-F669, 2006), although COL but not BFA also blocked the flow-induced [Ca(2+)](i) transient. We thus speculate that BK channel-mediated, flow-stimulated J(K) requires an increase in [Ca(2+)](i) due, in part, to luminal Ca(2+) entry and ER Ca(2+) release, microtubule integrity, and exocytic insertion of preformed channels into the apical membrane.