Katsuki Kobayashi

Disruption of Aquaporin-11 Produces Polycystic Kidneys following Vacuolization of the Proximal Tubule

ABSTRACT 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.

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.

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 Henles 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.

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

BackgroundAbout 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.MethodsNew 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.ResultsWe 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.ConclusionsThe 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.