40 research outputs found
Pathogenesis of nephrogenic diabetes insipidus by aquaporin-2 C-terminus mutations
Pathogenesis of nephrogenic diabetes insipidus by aquaporin-2 C-terminus mutations.BackgroundWe previously reported three aquaporin-2 (AQP2) gene mutations known to cause autosomal-dominant nephrogenic diabetes insipidus (NDI) (Am J Hum Genet 69:738, 2001). The mutations were found in the C-terminus of AQP2 (721delG, 763 to 772del, and 812 to 818del). The wild-type AQP2 is a 271 amino acid protein, whereas these mutant genes were predicted to encode 330 to 333 amino acid proteins due to the frameshift mutations leading to the creation of a new stop codon 180 nucleotides downstream. The Xenopus oocyte expression study suggested that the trafficking of the mutant AQP2s was impaired.MethodsTo determine the cellular pathogenesis of these NDI-causing mutations in mammalian epithelial cells, Madin-Darby canine kidney (MDCK) cells were stably transfected with the wild-type AQP2, or the 763 to 772del mutant AQP2, or both. Cells were grown on the membrane support to examine the localization of AQP2 proteins by immunofluorescence microscopy.ResultsConfocal immunofluorescence microscopy showed that the wild-type AQP2 was expressed in the apical region, whereas the mutant AQP2 was apparently located at the basolateral region. Furthermore, the wild-type and mutant AQP2s were colocalized at the basolateral region when they were cotransfected, suggesting the formation of mixed oligomers and thereby mistargeting.ConclusionMixed oligomers of the wild-type and the 763 to 772del mutant AQP2s are mistargeted to the basolateral membrane due to the dominant-negative effect of the mutant. This defect is very likely to explain the pathogenesis of autosomal-dominant NDI. The mistargeting of the apical membrane protein to the basolateral membrane is a novel molecular pathogenesis of congenital NDI
Primary cilia disappear in rat podocytes during glomerular development
Most tubular epithelial cell types express primary cilia, and mutations of primary-cilium-associated proteins are well known to cause several kinds of cystic renal disease. However, until now, it has been unclear whether mammalian podocytes express primary cilia in vivo. In this study, we determined whether primary cilia are present in the podocytes of rat immature and mature glomeruli by means of transmission electron microscopy of serial ultrathin sections. In immature glomeruli of fetal rats, podocytes express the primary cilia with high percentages at the S-shaped body (88 ± 5%, n = 3), capillary loop (95 ± 4%, n =  4), and maturing glomerulus (76 ± 13%, n = 5) stages. The percentage of ciliated podocytes was significantly lower at the maturing glomerulus stage than at the former two stages. In mature glomeruli of adult rats, ciliated podocytes were not found at all (0 ± 0%, n = 11). These findings indicate that the primary cilia gradually disappear in rat podocytes during glomerular development. Since glomerular filtration rate increases during development, the primary cilia on the podocytes are subjected to a stronger bending force. Thus, the disappearance of the primary cilia presumably prevents the entry of excessive calcium-ions via the cilium-associated polycystin complexes and the disturbance of intracellular signaling cascades in mature podocytes
Glomerular Endothelial Cells Form Diaphragms during Development and Pathologic Conditions
Unlike most fenestrated capillary endothelial cells, adult glomerular endothelial cells (GEnC) are generally thought to lack diaphragms at their fenestrae, but this remains controversial. In this study, morphologic and immunocytochemical analyses demonstrated that, except for a small fraction, GEnC of adult rats lacked diaphragmed fenestrae, which contain the transmembrane glycoprotein PV-1. In contrast, the GEnC in embryonic rats exhibited diaphragmed fenestrae and expressed PV-1 protein. The luminal surface of the fenestral diaphragm possesses a high density of anionic sites, thereby compensating for the functional immaturity of the embryonic glomerular filtration barrier. In addition, GEnC with diaphragmed fenestrae and PV-1 expression were significantly increased in adult rats with Thy-1.1 nephritis, presumably reflecting a process of restorative remodeling of the glomerular capillary tuft after injury; therefore, the reappearance of PV-1 expression and diaphragmed fenestrae may serve as a marker of glomerular capillary remodeling
Inhibition of connexin43 dephosphorylation is involved in protective effects of diltiazem on cardiac function during hypoxic injury
Background: Connexin43 (Cx43), a gap
junction protein, mediates cell-cell communication via
electrical and chemical coupling. Ischemic stress of the
cardiac muscle interrupts intercellular communication by
changing the distribution and phosphorylation status of
Cx43. This may be a factor contributing to reentrant
arrhythmia. The calcium channel blocker diltiazem is
known for its protective and anti-arrhythmogenic effect
in ischemic heart disease. In this study, we assess the
effect of diltiazem pretreatment upon ischemia-induced
phosphorylation change of Cx43
Methods: Langendorff preparations of isolated
Wistar rat hearts were performed. After stabilization,
hearts were treated with (D+) or without diltiazem (D-),
then subjected to hypoxia-reoxygenation. After
perfusion, the left ventricle was prepared for
immunocytochemistry and immunoblot analysis.
Results: During perfusion, left ventricular function
was better in the D+ group than the D- group.
Immunostaining of the heart indicated that
dephosphorylated Cx43 (dpCx43) signal was increased
after hypoxic perfusion, and this finding was confirmed
by immunoblot data. The quantitative area analysis of
dpCx43 using the immunohistochemical approach
showed that the dpCx43-positive area was enlarged, as
the hypoxic perfusion time was longer, and it was
reduced by pretreatment of diltiazem. There was a
negative correlation between the dpCx43 area and
%RPP (rate-pressure product), calculated by heart rate
and contraction force.
Conclusions: Pretreatment of diltiazem could protect
the heart against hypoxia-reoxygenation injury by attenuation of dephosphorylation of Cx43. The antiarrhythmic
mechanism of diltiazem may include the
preservation of phosphorylation status of Cx43 after
hypoxia-reoxygenation injury