Aberrant glycosylation and localization of polycystin-1 cause polycystic kidney in an AQP11 knockout model.

We previously reported that disruption of the aquaporin-11 (AQP11) gene in mice resulted in cystogenesis in the kidney. In this study, we aimed to clarify the mechanism of cystogenesis in AQP11(-/-) mice. To enable the analyses of AQP11 at the protein level in vivo, AQP11 BAC transgenic mice (Tg(AQP11)) that express 3×HA-tagged AQP11 protein were generated. This AQP11 localized to the endoplasmic reticulum (ER) of proximal tubule cells in Tg(AQP11) mice and rescued renal cystogenesis in AQP11(-/-) mice. Therefore, we hypothesized that the absence of AQP11 in the ER could result in impaired quality control and aberrant trafficking of polycystin-1 (PC-1) and polycystin-2 (PC-2). Compared with kidneys of wild-type mice, AQP11(-/-) kidneys exhibited increased protein expression levels of PC-1 and decreased protein expression levels of PC-2. Moreover, PC-1 isolated from AQP11(-/-) mice displayed an altered electrophoretic mobility caused by impaired N-glycosylation processing, and density gradient centrifugation of kidney homogenate and in vivo protein biotinylation revealed impaired membrane trafficking of PC-1 in these mice. Finally, we showed that the Pkd1(+/-) background increased the severity of cystogenesis in AQP11(-/-) mouse kidneys, indicating that PC-1 is involved in the mechanism of cystogenesis in AQP11(-/-) mice. Additionally, the primary cilia of proximal tubules were elongated in AQP11(-/-) mice. Taken together, these data show that impaired glycosylation processing and aberrant membrane trafficking of PC-1 in AQP11(-/-) mice could be a key mechanism of cystogenesis in AQP11(-/-) mice.

Baseline characteristics and prevalence of cardiovascular disease in newly visiting or referred chronic kidney disease patients to nephrology centers in Japan: a prospective cohort study.

BACKGROUND: About 39,000 patients were newly prescribed renal replacement therapy in Japan in 2011, resulting in a total of more than 300,000 patients being treated with dialysis. This high prevalence of treated end stage kidney disease (ESKD) patients is an emergent problem that requires immediate attention. We launched a prospective cohort study to evaluate population specific characteristics of the progression of chronic kidney disease (CKD). In this report, we describe the baseline characteristics and risk factors for cardiovascular disease (CVD) prevalence among this cohort. METHODS: New patients from 16 nephrology centers who were older than 20 years of age and who visited or were referred for the treatment of CKD stage 2-5, but were not on dialysis therapy, were recruited in this study. At enrollment, medical history, lifestyle behaviors, functional status and current medications were recorded, and blood and urine samples were collected. Estimated glomerular filtration rate (eGFR) was calculated by a modified three-variable equation. RESULTS: We enrolled 1138 patients, 69.6% of whom were male, with a mean age of 68 years. Compared with Western cohorts, patients in this study had a lower body mass index (BMI) and higher proteinuria. The prevalence of CVD was 26.8%, which was lower than that in Western cohorts but higher than that in the general Japanese population. Multivariate analysis demonstrated the following association with CVD prevalence: hypertension (adjusted odds ratio (aOR) 3.57; 95% confidence interval (CI) 1.82-7.02); diabetes (aOR 2.45; 95% CI 1.86-3.23); hemoglobin level less than 11 g/dl (aOR 1.61; 95% CI 1.21-2.15); receiving anti-hypertensive agents (aOR 3.54; 95% CI 2.27-5.53); and statin therapy (aOR 2.73; 95% CI 2.04-3.66). The combination of decreased eGFR and increased proteinuria was also associated with a higher prevalence of CVD. CONCLUSIONS: The participants in this cohort had a lower BMI, higher proteinuria and lower prevalence of CVD compared with Western cohorts. Lower eGFR and high proteinuria were associated with CVD prevalence. Prospective follow up of these study patients will contribute to establishment of individual population-based treatment of CKD.

Enhancement of pairing interaction and magnetic fluctuations toward a band insulator in an electron-doped Li(x)ZrNCl Superconductor.

The doping dependence of specific heat and magnetic susceptibility has been investigated for Li(x)ZrNCl superconductors derived from a band insulator. As the carrier concentration is decreased, the anisotropy of superconducting gap changes from highly anisotropic to almost isotropic. It was also found that, upon reducing carrier density, the superconducting coupling strength and the magnetic susceptibility are concomitantly enhanced in parallel with T(c), while the density of states at the Fermi level is kept almost constant. Theoretical calculations taking into account the on-site Coulomb interaction reproduced the experimental results, suggesting a possible pairing mediated by magnetic fluctuations, even in the doped band insulators.

Disruption of aquaporin-11 produces polycystic kidneys following vacuolization of the proximal tubule.

Aquaporin-11 (AQP11) has been identified with unusual pore-forming NPA (asparagine-proline-alanine) boxes, but its function is unknown. We investigated its potential contribution to the kidney. Immunohistochemistry revealed that AQP11 was localized intracellularly in the proximal tubule. When AQP11 was transfected in CHO-K1 cells, it was localized in intracellular organelles. AQP11-null mice were generated; these mice exhibited vacuolization and cyst formation of the proximal tubule. AQP11-null mice were born normally but died before weaning due to advanced renal failure with polycystic kidneys, in which cysts occupied the whole cortex. Remarkably, cyst epithelia contained vacuoles. These vacuoles were present in the proximal tubules of newborn mice. In 3-week-old mice, these tubules contained multiple cysts. Primary cultured cells of the proximal tubule revealed an endosomal acidification defect in AQP11-null mice. These data demonstrate that AQP11 is essential for the proximal tubular function. AQP11-null mice are a novel model for polycystic kidney diseases and will provide a new mechanism for cystogenesis.

Disease-causing mutant WNK4 increases paracellular chloride permeability and phosphorylates claudins.

Mutations in the WNK4 gene cause pseudohypoaldosteronism type II (PHAII), an autosomal-dominant disorder of hyperkalemia and hypertension. The target molecules of this putative kinase and the molecular mechanisms by which the mutations cause the phenotypes are currently unknown. Although recent reports found that expression of WNK4 in Xenopus oocytes causes inhibition of the thiazide-sensitive NaCl cotransporter and the renal K channel ROMK, there may be additional targets of WNK4. For example, an increase in paracellular chloride permeability has been postulated to be a mediator of PHAII pathogenesis, a possibility supported by the localization of WNK4 at tight junctions in vivo. To determine the validity of this hypothesis, we measured transepithelial Na and Cl permeability in Madin-Darby canine kidney II cells stably expressing wild-type or a pathogenic mutant of WNK4. We found that transepithelial paracellular Cl permeability was increased in cells expressing a disease-causing mutant WNK4 (D564A) but that Na permeability was decreased slightly. Furthermore, WNK4 bound and phosphorylated claudins 1-4, major tight-junction membrane proteins known to be involved in the regulation of paracellular ion permeability. The increases in phosphorylation of claudins were greater in cells expressing the mutant WNK4 than in cells expressing wild-type protein. These results clearly indicate that the pathogenic WNK4 mutant possesses a gain-of-function activity and that the claudins may be important molecular targets of WNK4 kinase. The increased paracellular "chloride shunt" caused by the mutant WNK4 could be the pathogenic mechanism of PHAII.

Expression of CLC-KB gene promoter in the mouse cochlea.

SUMMARY: We have investigated the expression of chloride channels by examining the cochlea of mice harboring the enhanced green fluorescence protein (EGFP) gene driven by an 11 kbp human CLC-KB gene promoter. CLC-KB was seen not only on the stria vascularis but on spiral ligament and limbal fibrocytes, interdental cells and satellite cells of spiral ganglion neurons that are known to possess both Na,K-ATPase and the Na-K-Cl co-transporter (NKCC). These results suggest that some fibrocytes possessing both the CLC-KB and the NKCC may be involved in the regulation of cell volume, transport and recycling of Cl- such as is seen in the stria vascularis. Moreover, these fibrocytes may recycle Cl- through CLC that accompany Na+ and K+ into the cell via NKCC.

Human CLC-KB gene promoter drives the EGFP expression in the specific distal nephron segments and inner ear.

Human CLC-KB has been identified as a kidney-specific member of the CLC chloride channel family, and mutations of the human CLC-KB gene are known to cause Bartter syndrome type III. A precise understanding of the localization of this channel in the human kidney is imperative to our understanding of the pathophysiology, but this has remained unclear due to the high homology between human CLC-KB and CLC-KA, another kidney-specific member of the same family. The high intraspecies homology also rules out an exact correlation of the human isoforms (CLC-KA and CLC-KB) to the mouse and rat isoforms (CLC-K1 and CLC-K2, respectively). This study created transgenic mice harboring the enhanced green fluorescence protein (EGFP) gene driven by an 11-kbp human CLC-KB gene promoter. Three transgenic lines were generated, and all of them showed EGFP fluorescence in the kidney, with an identical pattern of localization to the thick ascending limb of Henle's loop, distal tubules, connecting tubules, and intercalated cells of the collecting duct. This localization is exactly the same as that of mouse CLC-K2 identified in a previous report (Kobayashi et al. J Am Soc Neph 12: 1327-1334, 2001). EGFP fluorescence was also detected in the inner ear, more specifically in marginal cells of the stria vascularis and dark cells of the vestibular labyrinth, suggesting that human CLC-KB could play an important role in the fluid transport mechanism of the inner ear. The results (1) confirmed that CLC-KB is the true human homologue of rat and mouse CLC-K2 and (2) established that the 11-kbp human CLC-KB gene promoter is sufficient to elicit the precise expression in specific cell types of the kidney and inner ear.

CLC-3 deficiency leads to phenotypes similar to human neuronal ceroid lipofuscinosis.

BACKGROUND: CLC-3 is a member of the CLC chloride channel family and is widely expressed in mammalian tissues. To determine the physiological role of CLC-3, we generated CLC-3-deficient mice (Clcn3-/- ) by targeted gene disruption. RESULTS: Together with developmental retardation and higher mortality, the Clcn3-/- mice showed neurological manifestations such as blindness, motor coordination deficit, and spontaneous hyperlocomotion. In histological analysis, the Clcn3-/- mice showed a pattern of progressive degeneration of the retina, hippocampus and ileal mucosa, which resembled the phenotype observed in cathepsin D knockout mice. The defect of cathepsin D results in a lysosomal accumulation of ceroid lipofuscin containing the mitochondrial F1F0 ATPase subunit c. In immunohistochemistry and Western blot analysis, we found that the subunit c was heavily accumulated in the lysosome of Clcn3-/- mice. Furthermore, we detected an elevation in the endosomal pH of the Clcn3-/- mice. CONCLUSIONS: These results indicated that the neurodegeneration observed in the Clcn3-/- mice was caused by an abnormality in the machinery which degrades the cellular protein and was associated with the phenotype of neuronal ceroid lipofuscinosis (NCL). The elevated endosomal pH could be an important factor in the pathogenesis of NCL.

Intrarenal and cellular localization of CLC-K2 protein in the mouse kidney.

CLC-K2, a kidney-specific member of the CLC chloride channel family, is thought to play an important role in the transepithelial Cl(-) transport in the kidney. This consensus was first reached shortly after it was demonstrated that the mutations of the human CLCNKB gene resulted in Bartter's syndrome type III. To clarify the pathogenesis, the exact intrarenal and cellular localization of CLC-K2 by immunohistochemistry of the Clcnk1-/- mouse kidney were investigated by use of an anti-CLC-K antibody that recognized both CLC-K1 and CLC-K2. CLC-K2 is expressed in the thick ascending limb of Henle's loop and distal tubules, where it is localized to the basolateral membranes. The localization of CLC-K2 to these nephron segments strongly implies that CLC-K2 confers the basolateral chloride conductance in the thick ascending limb of Henle's loop and distal tubules, where Cl(-) is taken up by the bumetanide-sensitive Na-K-2Cl cotransporter or the thiazide-sensitive Na-Cl cotransporter at the apical membranes. CLC-K2 expression was also shown to extend into the connecting tubule in the basolateral membrane. CLC-K2 was found in basolateral membranes of the type A intercalated cells residing along the collecting duct. This localization strongly suggests that CLC-K2 confers the basolateral conductance in the type A intercalated cells where Cl(-) is taken up by the anion exchanger in exchange for HCO(3)(-) at the basolateral membranes. These aspects of CLC-K2 localization suggest that CLC-K2 is important in Cl(-) transport in the distal nephron segments.