Freshwater fishes absorb Na+ from their dilute environment using ion-transporting cells. In larval zebrafish (Danio rerio), Na+ uptake is coordinated by (1) Na+/H+ exchanger 3b (Nhe3b) and (2) H+-ATPase-powered electrogenic uptake in H+-ATPase-rich (HR) cells and by (3) Na+-Cl--cotransporter (Ncc) expressed in NCC cells. The present study aimed to better understand the roles of these three proteins in Na+ uptake by larval zebrafish under 'normal' (800 µmol l-1) and 'low' (10 µmol l-1) Na+ conditions. We hypothesized that Na+ uptake would be reduced by CRISPR/Cas9 knockout (KO) of slc9a3.2 (encoding Nhe3b), particularly in low Na+ where Nhe3b is believed to play a dominant role. Contrary to this hypothes... More
Freshwater fishes absorb Na+ from their dilute environment using ion-transporting cells. In larval zebrafish (Danio rerio), Na+ uptake is coordinated by (1) Na+/H+ exchanger 3b (Nhe3b) and (2) H+-ATPase-powered electrogenic uptake in H+-ATPase-rich (HR) cells and by (3) Na+-Cl--cotransporter (Ncc) expressed in NCC cells. The present study aimed to better understand the roles of these three proteins in Na+ uptake by larval zebrafish under 'normal' (800 µmol l-1) and 'low' (10 µmol l-1) Na+ conditions. We hypothesized that Na+ uptake would be reduced by CRISPR/Cas9 knockout (KO) of slc9a3.2 (encoding Nhe3b), particularly in low Na+ where Nhe3b is believed to play a dominant role. Contrary to this hypothesis, Na+ uptake was sustained in nhe3b KO larvae under both Na+ conditions, which led to the exploration of whether compensatory regulation of H+-ATPase or Ncc was responsible for maintaining Na+ uptake in nhe3b KO larvae. mRNA expression of the genes encoding H+-ATPase and Ncc was not altered in nhe3b KO larvae. Moreover, morpholino knockdown of H+-ATPase, which significantly reduced H+ flux by HR cells, did not reduce Na+ uptake in nhe3b KO larvae, nor did rearing larvae in chloride-free conditions, thereby eliminating any driving force for Na+-Cl--cotransport via Ncc. Finally, simultaneously treating nhe3b KO larvae with H+-ATPase morpholino and chloride-free conditions did not reduce Na+ uptake under normal or low Na+ These findings highlight the flexibility of the Na+ uptake system and demonstrate that Nhe3b is expendable to Na+ uptake in zebrafish and that our understanding of Na+ uptake mechanisms in this species is incomplete.,© 2020. Published by The Company of Biologists Ltd.
