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

FAT is a component of glomerular slit diaphragms

FAT is a component of glomerular slit diaphragms.BackgroundSlit diaphragms are intercellular junctions of podocytes of the renal glomerulus. The molecular composition of slit diaphragms is still elusive. Slit diaphragms are characterized by the presence of a wide intercellular space. The morphological feature is shared by desmosomes and adherens junctions, which contain members of the cadherin superfamily. Thus, we have hypothesized that some components of slit diaphragms belong to the cadherin superfamily. Consequently, we have isolated cDNA encoding FAT from reverse-transcribed (RT) glomerular cDNA by homology polymerase chain reaction (PCR) using primers based on conserved sequences in cadherin molecules. FAT is a novel member of the cadherin superfamily with 34 tandem cadherin-like extracellular repeats, and it closely resembles the Drosophila tumor suppressor fat.MethodsExpression of FAT was examined in glomeruli of the adult rat kidney by the ribonuclease protection assay and in situ hybridization. To localize the FAT protein in podocytes minutely, we prepared affinity-purified antibody against FAT by immunizing rabbits against an oligopeptide corresponding to the C-terminal 20 amino acids.ResultsExpression of FAT mRNA was detected in total RNA from glomeruli. In situ hybridization revealed significant signals in podocytes. Western blot analysis using solubilized glomeruli showed a single band, in which the molecular weight was more than 500 kD. Immunostaining of cultured epithelial cells from rat kidney (NRK52E) revealed FAT accumulation in cell–cell contact sites. In the glomerulus, FAT staining was observed distinctly along glomerular capillary walls. Double-label immunostaining using monoclonal antibody against slit diaphragms (mAb 5-1-6) showed identical localization of anti-FAT antibody and mAb 5-1-6. Furthermore, the double-label immunogold technique with ultrathin cryosections demonstrated that gold particles for FAT cytoplasmic domain were located at the base of slit diaphragms labeled by mAb 5-1-6 and that the cytoplasmic domain of FAT colocalized with ZO-1, a cytoplasmic component associated with slit diaphragms.ConclusionThe molecular structure of FAT and its colocalization with 5-1-6 antigen and ZO-1 indicate that FAT is a component of slit diaphragms

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

Deletion of Rac1GTPase in the Myeloid Lineage Protects against Inflammation-Mediated Kidney Injury in Mice.

Macrophage-mediated inflammation has been implicated in various kidney diseases. We previously reported that Rac1, a Rho family small GTP-binding protein, was overactivated in several chronic kidney disease models, and that Rac1 inhibitors ameliorated renal injury, in part via inhibition of inflammation, but the detailed mechanisms have not been clarified. In the present study, we examined whether Rac1 in macrophages effects cytokine production and the inflammatory mechanisms contributing to kidney derangement. Myeloid-selective Rac1 flox control (M-Rac1 FC) and knockout (M-Rac1 KO) mice were generated using the cre-loxP system. Renal function under basal conditions did not differ between M-Rac1 FC and KO mice. Accordingly, lipopolysaccharide (LPS)-evoked kidney injury model was created. LPS elevated blood urea nitrogen and serum creatinine, enhanced expressions of kidney injury biomarkers, Kim-1 and Ngal, and promoted tubular injury in M-Rac1 FC mice. By contrast, deletion of myeloid Rac1 almost completely prevented the LPS-mediated renal impairment. LPS triggered a marked induction of macrophage-derived inflammatory cytokines, IL-6 and TNFα, in M-Rac1 FC mice, which was accompanied by Rac1 activation, stimulation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and reactive oxygen species overproduction. These changes were inhibited in M-Rac1 KO mice. LPS evoked F4/80-positive macrophages accumulation in the kidney, which was not affected by myeloid Rac1 deficiency. We further tested the role of Rac1 signaling in cytokine production using macrophage cell line, RAW264.7. Exposure to LPS increased IL-6 and TNFα mRNA expression. The LPS-driven cytokine induction was dose-dependently blocked by the Rac1 inhibitor EHT1864, NADPH oxidase inhibitor diphenyleneiodonium, and NF-κB inhibitor BAY11-7082. In conclusion, genetic ablation of Rac1 in the myeloid lineage protected against LPS-induced renal inflammation and injury, by suppressing macrophage-derived cytokines, IL-6 and TNFα, without blocking recruitment. Our data suggest that Rac1 in macrophage is a novel target for the treatment of kidney disease through inhibition of cytokine production

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