Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio)

Abstract

The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), a member of the SLC12 family, is mainly expressed in the apical membrane of the mammalian distal convoluted tubule (DCT) cells, is responsible for cotransporting Na(+) and Cl(-) from the lumen into DCT cells and plays a major role in the mammalian renal NaCl reabsorption. The NCC has also been reported in fish, but the functional role in fish ion regulation is yet unclear. The present study used zebrafish as an in vivo model to test the hypothesis of whether the NCC plays a role in Na(+) and/or Cl(-) uptake mechanisms. Four NCCs were cloned, and only one of them, zebrafish (z) slc12a10.2 was found to predominately and specifically be expressed in gills. Double in situ hybridization/immunocytochemistry in zebrafish skin/gills demonstrated that the specific expression of zslc12a10.2 mRNA in a novel group of ionocytes differed from those of the previously-reported H(+)-ATPase-rich (HR) cells and Na(+)-K(+)-ATPase-rich (NaR) cells. Gill mRNA expression of zslc12a10.2 was induced by a low-Cl environment that stimulated fish Cl(-) influx, while a low-Na environment suppressed this expression. Incubation with metolazone, a specific inhibitor of the NCC, impaired both Na(+) and Cl(-) influx in 5-day postfertilization (dpf) zebrafish embryos. Translational knockdown of zslc12a10.2 with a specific morpholino caused significant decreases in both Cl(-) influx and Cl(-) content of 5-dpf zebrafish embryos, suggesting that the operation of zNCC-like 2 results in a net uptake of Cl(-) in zebrafish. On the contrary, zslc12a10.2 morphants showed increased Na(+) influx and content that resulted from upregulation of mRNA expressions of Na(+)-H(+) exchanger 3b and carbonic anhydrase 15a in HR cells. These results for the first time provide in vivo molecular physiological evidence for the possible role of the NCC in the Cl(-) uptake mechanism in zebrafish skin/gills.

Ion uptake and acid secretion in zebrafish (Danio rerio)

“…Double ISH and ICC experiments demonstrated specific expression of zNCCg mRNA in a novel type of ionocyte, NCC cells, which differ from the previously reported HR and NaR cells (Fig. 2C,D), and loss-of-function experiments with zNCCg morpholinos revealed significant decreases in both Cl -influx and Cl -content in the morphants (Wang et al, 2009). These results indicate the function of NCC cells, in which zNCCg may be the major player in the apical Cl --uptake pathway, although ICC localization of the zNCCg protein in zebrafish NCC cells remains to be confirmed.…”

Differential expression of Na+-Cl−cotransporter and Na+-K+-Cl−cotransporter 2 in the distal nephrons of euryhaline and seawater pufferfishes

“…2). The kidney expression of NCC is weak in winter flounder (seawater fish) (16), but strong in European eel (euryhaline fish) and zebrafish (freshwater fish) (11,54). These results suggest that NCC in the kidney is essential for fish to live in freshwater but not seawater, and there is a relationship between renal expression of NCC and the adaptability of pufferfish to freshwater.…”

“…The gill is another important osmoregulatory tissue in teleosts. Recently, a fish-specific isoform of NCC was identified that is specifically expressed in the mitochondrion-rich cells of the gill epithelium and appears to be involved in Na ϩ and Cl Ϫ uptake in certain freshwater fish species (21,24,54). No gene encoding this fish-specific NCC was identified in the mefugu genome (Fig.…”

A novel function of calcitonin gene-related peptide in body fluid Cl−homeostasis

“…and Cl 2 uptake function [6,8], resulted in the upregulation of cgrp mRNA expression (figure 1b); in addition, CGRP loss-offunction stimulated the expression of Cl 2 transporters (figure 2e) and Cl 2 influx ( figure 5). As the use of CALC morpholinos does not allow us to distinguish between the effects of CT and CGRP loss-of-function, we also performed specific CGRP gain-of-function experiments: cgrp overexpression suppressed both the expression of ncc2b mRNA (figure 4) and Cl 2 influx ( figure 5).…”

“…These findings inspired us to test the hypothesis that CGRP is involved in the control of fish body fluid Cl 2 homeostasis. The expression and function of Cl 2 transporters have been well studied in zebrafish, because of the comprehensive genetic databases and well-developed molecular physiological approaches available for this organism [6,8]. In zebrafish embryos, the skin ionocytes are responsible for the majority of ion regulation until 7-14 days post-fertilization (dpf ), at which time the gills are fully developed and functional [24].…”