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Mutations in the electrogenic [Na.sup.+]/nHC[O.sup.-.sub.3] cotransporter (NBCe1, SLC4A4) cause severe proximal renal tubular acidosis, glaucoma, and cataracts in humans, indicating NBCe1 has a critical role in acid-base homeostasis and ocular fluid transport. To better understand the homeostatic roles and protein ontogeny of NBCe1, we have cloned, localized, and downregulated NBCe1 expression in zebrafish, and examined its transport characteristics when expressed in Xenopus oocytes. Zebrafish NBCe1 (zN-BCe1) is 80% identical to published mammalian NBCe1 cDNAs. Like other fish NBCe1 clones, zebrafish NBCe1 is most similar to the pancreatic form of mammalian NBC (S1c4a4-B) but appears to be the dominant isoform found in zebrafish. In situ hybridization of embryos demonstrated mRNA expression in kidney pronephros and eye by 24 h postfertilization (hpf) and gill and brain by 120 hpf. Immunohistochemical labeling demonstrated expression in adult zebrafish eye and gill. Morpholino knockdown studies demonstrated roles in eye and brain development and caused edema, indicating altered fluid and electrolyte balance. With the use of microelectrodes to measure membrane potential ([V.sub.m]), voltage clamp (VC), intracellular pH ([pH.sub.i], or intracellular [Na.sup.+] activity (a[Na.sub.i]), we examined the function of zNBCe1 expressed in Xenopus oocytes. Zebrafish NBCe1 shared transport properties with mammalian NBCe1s, demonstrating electrogenic [Na.sup.+] and HC[O.sup.-.sub.3] transport as well as similar drug sensitivity, including inhibition by 4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene and tenidap. These data indicate that NBCe1 in zebrafish shares many characteristics with mammalian NBCe1, including tissue distribution, importance in systemic water and electrolyte balance, and electrogenic transport of [Na.sup.+] and HC[O.sup.-.sub.3]. Thus zebrafish promise to be useful model system for studies of NBCel physiology. intracellular pH; acid base; membrane current; bicarbonate transport; Xenopus oocyte; teleost; Danio rerio', cellular buffering; immunohistochemistry; in situ; electrophysiology doi: 10.1152/ajpcell.00679.2008

A study was conducted on the influence of heat stress on Na+-glucose cotransport using porcine-derived renal epithelial cell line monolayers. Release of alkaline phosphatase or lactate dehydrogenase show that mild heat shock is not associated with luminal membrane surface area or cell survival. Results also show that Na+-glucose cotransport is both protected and improved through heat shock protein treatment.

The failure of the polycystins (PCs) to function in primary cilia is thought to be responsible for autosomal dominant polycystic kidney disease (ADPKD). Primary cilia integrate multiple cellular signaling pathways, including calcium, cAMP, Wnt, and Hedgehog, which control cell proliferation and differentiation. It has been proposed that mutated PCs result in reduced intracellular calcium, which in turn upregulates cAMP, protein kinase A (PKA) signaling, and subsequently other proliferative signaling pathways. However, the role of PKA in ADPKD has not been directly ascertained in vivo, although the expression of the main regulatory subunit of PKA in cilia and other compartments (PKA-RIα, encoded by PRKAR1A) is increased in a mouse model orthologous to ADPKD. Therefore, we generated a kidney-specific knockout of Prkar1a to examine the consequences of constitutive upregulation of PKA on wild-type and Pkd1 hypomorphic (Pkd1RC) backgrounds. Kidney-specific loss of Prkar1a induced renal cystic disease and markedly aggravated cystogenesis in the Pkd1RC models. In both settings, it was accompanied by upregulation of Src, Ras, MAPK/ERK, mTOR, CREB, STAT3, Pax2 and Wnt signaling. On the other hand, Gli3 repressor activity was enhanced, possibly contributing to hydronephrosis and impaired glomerulogenesis in some animals. To assess the relevance of these observations in humans we looked for and found evidence for kidney and liver cystic phenotypes in the Carney complex, a tumoral syndrome caused by mutations in PRKAR1A. These observations expand our understanding of the pathogenesis of ADPKD and demonstrate the importance of PRKAR1A highlighting PKA as a therapeutic target in ADPKD. [ABSTRACT FROM AUTHOR]

Mutations in the electrogenic Na+/nHCO-3 cotransporter (NBCeI, SLC4A4) cause severe proximal renal tubular acidosis, glaucoma, and cataracts in humans, indicating NBCe1 has a critical role in acid-base homeostasis and ocular fluid transport. To better understand the homeostatic roles and protein ontogeny of NBCe1, we have cloned, localized, and downregulated NBCe1 expression in zebrafish, and examined its transport characteristics when expressed in Xenopus oocytes. Zebrafish NBCeI (ZNBCe1) is 80% identical to published mammalian NBCeI cDNAs. Like other fish NBCe1 clones, zebrafish NBCeI is most similar to the pancreatic form of mammalian NBC (Slc4a4-B) but appears to be the dominant isoform found in zebrafish. In situ hybridization of embryos demonstrated mRNA expression in kidney pronephros and eye by 24 h postfertilization (hpf) and gill and brain by 120 hpf. Immunohistochemical labeling demonstrated expression in adult zebrafish eye and gill. Morpholino knockdown studies demonstrated roles in eye and brain development and caused edema, indicating altered fluid and electrolyte balance. With the use of microelectrodes to measure membrane potential (Vm), voltage clamp (VC), intracellular pH (pHi), or intracellular Na+ activity (aNa1), we examined the function of zNBCe1 expressed in Xenopus oocytes. Zebrafish NBCe1 shared transport properties with mammalian NBCeIs, demonstrating electrogenic Na+ and HCO-3 transport as well as similar drug sensitivity, including inhibition by 4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene and tenidap. These data indicate that NBCeI in zebrafish shares many characteristics with mammalian NBCe1, including tissue distribution, importance in systemic water and electrolyte balance, and electrogenic transport of Na+ and HCO-3. Thus zebrafish promise to be useful model system for studies of NBCe1 physiology. [ABSTRACT FROM AUTHOR]

The SLC26 gene family encodes anion transporters with diverse functional attributes: (a) anion exchanger, (b) anion sensor, and (c) anion conductance (likely channel). We have cloned and studied Slc26a9, a paralogue expressed mostly in lung and stomach. Immunohistochemistry shows that Slc26a9 is present at apical and intracellular membranes of lung and stomach epithelia. Using expression in Xenopus laevis oocytes and ion-sensitive microelectrodes, we discovered that Slc26a9 has a novel function not found in any other Slc26 proteins: cation coupling. Intracellular pH and voltage measurements show that Slc26a9 is a nCl(-)-HCO(3)(-) exchanger, suggesting roles in gastric HCl secretion or pulmonary HCO(3)(-) secretion; Na(+) electrodes and uptakes reveal that Slc26a9 has a cation dependence. Single-channel measurements indicate that Slc26a9 displays discrete open and closed states. These experiments show that Slc26a9 has three discrete physiological modes: nCl(-)-HCO(3)(-) exchanger, Cl(-) channel, and Na(+)-anion cotransporter. Thus, the Slc26a9 transporter channel is uniquely suited for dynamic and tissue-specific physiology or regulation in epithelial tissues. [ABSTRACT FROM AUTHOR]

Neuregulin is required for proper oligodendrocyte development, but which receptors are involved and whether neuregulin promotes or inhibits maturation remain controversial. To assess the roles of the neuregulin receptor ErbB4 in oligodendrocyte development, we examined oligodendrocyte initiation and maturation in cultures derived from erbB4 knock-out mice and rat spinal cord in the presence of neutralizing erbB4 antibodies. No differences in the development of O4+ oligodendrocytes were detected in the presence or absence of erbB4 signaling. All four epidermal growth factor receptor family members were detected in the ventral neural tube at approximately the time of initial oligodendrocyte development, consistent with redundancy in neuregulin receptor signaling at the onset of oligodendrocyte development. In contrast, greater numbers of differentiated (monoclonal antibody O1+) oligodendrocytes developed in neural tube explants from erbB4-/- mice than either erbB4+/+ or erbB4+/- littermates as well as in cultures treated with anti-erbB4. These data indicate that ErbB4 is not required for oligodendrocyte development and, in fact, inhibits oligodendrocyte lineage maturation. Together with previous studies, these data suggest a model in which early oligodendrocyte lineage development is regulated by promiscuous neuregulin receptor signaling, but subsequent lineage progression occurs through a balance of receptor-specific promotion or inhibition of maturation. [ABSTRACT FROM AUTHOR]

Focuses on the protein kinase C regulation of p-glycoprotein-mediated xenobiotic secretion in renal proximal tubules of the kidneys. Role of protein kinase C (PKC) in the control of p-glycoprotein; Negative correlation of glycoprotein xenobiotic secretion with PKC activity; Drug transport in renal proximal tubules.

Monolayers of the porcine-derived renal epithelial cell line, LLC-PK1, were used to characterize the effects of heat stress on Na1-glucose cotransport. Transepithelial current dependent on 5 mM glucose (IGlc), phloridzin-sensitive current (Iphz), and total transepithelial current (Itotal) were measured as indicators of Na1-glucose cotransport. Severe heat shock (SHS; 45°C for 1 h, then 37°C for measurements) decreased transepithelial electrical resistance (TER), IGlc, Iphz, and Itotal 50-70%. Mild heat shock (MHS; 42°C for 3 h, then 37°C for 12 h) induced accumulation of 72-kDa heat shock protein (HSP-72), decreased damage to TER from SHS, and prevented damage to IGlc, Iphz, and Itotal. Kinetic analysis showed that SHS damaged and MHS protected total Na1-glucose transport capacity (Vmax of IGlc). MHS alone increased TER (50%), IGlc (20%), Itotal (20%), and Vmax of IGlc (25%). On enhancement of the Na1 gradient by depletion of intracellular Na+, MHS increased IGlc 50% and had no effect on transepithelial Na+-dependent sulfate reabsorptive flux measured concurrently or in Na+- replete tissues. These effects of MHS were not reflected in effects on cell survival or luminal membrane surface area as indicated by lactate dehydrogenase or alkaline phosphatase release. In conclusion, HSP-72-inducing heat treatment both protected and enhanced Na+-glucose cotransport independently of the luminal membrane Na+ gradient and selectively with respect to effects on TER, reabsorptive sulfate transport, cell survival, and luminal membrane surface area. [ABSTRACT FROM AUTHOR]

The author reflects on the renal replacement therapy for kidney repairement including hepatocyte growth factor, cell migration, and isoforms.

While zebrafish embryos in the first five days after fertilization are clear and amenable to optical analysis, older juveniles and adults are not, due to pigmentation development and tissue growth. Thus other imaging methods are needed to image adult specimens. NMR is a versatile tool for studies of biological systems and has been successfully used for in vivo zebrafish microscopy. In this work we use NMR microscopy (MRM) for assessment of zebrafish specimens, which includes imaging of formalin fixed (FF), formalin fixed and paraffin embedded (FFPE), fresh (unfixed), and FF gadolinium doped specimens. To delineate the size and shape of various organs we concentrated on 3D MRM. We have shown that at 7 T a 3D NMR image can be obtained with isotropic resolution of 50 μm/pxl within 10 min and 25 μm/pxl within 4 h. Also, we have analyzed sources of contrast and have found that in FF specimens the best contrast is obtained by T1 weighting (3D FLASH, 3D FISP), whereas in FFPE specimens T2 weighting (3D RARE) is the best. We highlight an approach to perform segmentation of the organs in order to study morphological changes associated with mutations. The broader implication of this work is development of NMR methodology for high contrast and high resolution serial imaging and automated analysis of morphology of various zebrafish mutants. High resolution MRM was used to image formalin‐fixed and formalin‐fixed paraffin embedded zebrafish specimens to study morphology of the mutants. It is demonstrated that 3D images with resolution up to 25 μm/pixel with strong contrast (T1W, T2W, MT, DWI or their combinations) could be obtained. That enables (semi)automatic organ segmentation. [ABSTRACT FROM AUTHOR]