Citations

Mutations in the electrogenic Na+/ nHCO3− 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 (zNBCe1) 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 (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 ( aNai), we examined the function of zNBCe1 expressed in Xenopus oocytes. Zebrafish NBCe1 shared transport properties with mammalian NBCe1s, demonstrating electrogenic Na+ and HCO3− 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+ and HCO3−. Thus zebrafish promise to be useful model system for studies of NBCe1 physiology.

Abstract Vitamin D and vitamin D metabolites such as 25-hydroxyvitamin D and 1α,25-dihydroxyvitamin D [1α,25(OH)2D3] circulate in the serum of fish. The receptor for 1α,25(OH)2D3 (VDR) has previously been cloned from fish intestine, and ligand binding assays have shown the presence of the VDR in the gills, intestine, and liver of fish. Using immunohistochemical methods with specific antibodies against the VDR, we now report that the VDR is widely expressed in tissues of the adult male and female zebrafish, Danio rerio, specifically in epithelial cells of gills, tubular cells of the kidney, and absorptive cells in the intestine. Additionally, the VDR is expressed in the skin, the olfactory organ, the retina, brain, and spinal cord. Sertoli cells of the testis, oocytes, acinar cells of the pancreas, hepatocytes, and bile duct epithelial cells express substantial amounts of the receptor. Osteoblast-like cells and chondrocytes also express VDR. Preimmune serum and antiserum preadsorbed with Danio VDR protein fails to detect VDR in the same tissues. The VDR is also present in the developing eye, brain, and otic vesicle of 48- and 96-h postfertilization zebrafish embryos. Parenteral administration of 1α,25(OH)2D3 increases concentrations of VDR in intestinal epithelial cells but not in epithelial cells of the gills. Lithocholic acid, however, does not alter concentrations of VDR after parenteral administration. The data suggest that VDR is widely distributed in tissues of the zebrafish, D. rerio, and is likely to play important roles in epithelial transport, bone, and endocrine function. Furthermore, concentrations of the receptor seem to be regulated by its ligand, 1α,25-dihydroxyvitamin D but not by lithocholic acid. Zebrafish may serve as a useful model in which to assess the function of the VDR in diverse tissues.