Reassessing the contribution of the Na⁺/H⁺ exchanger Nhe3b to Na⁺ uptake in zebrafish (Danio rerio) using CRISPR/Cas9 gene editing.

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.

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