The European Eel NCCβ Gene Encodes a Thiazide-Resistant Na-Cl Cotransporter

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Journal of the American Society of Nephrology




Background: Thiazides are among the most frequently prescribed drugs in the world for their antihypertensive effect through inhibition of the renal Na+:Cl- cotransporter, NCC, which in mammals is encode by one gene (SLC12A3). In contrast, two NCC genes are expressed in the European eel (Anguila anguila): NCCa and NCCb. Flounder and mammalian NCC exhibit different affinity for thiazides and we have previously reported a single amino acid residue (C575 in rat NCC) responsible for this difference (Castañeda-Bueno et al AJP Renal 2011), but nothing is known regarding residues conferring specificity for thiazide diuretics in NCC.

Methods: To study the functional properties of NCCb, a full length cDNA encoding NCCb from eel intestine was constructed by RT-PCR, inserted into pgh19 vector and fully sequenced. Functional expression was assessed by 22Na+ uptake using Xenopus laevis oocytes microinjected with NCCb cRNA. Rat and flounder NCC cRNA were used as controls.

Results: NCCb encodes a 1043 amino acid residues exhibiting a 55, 55, and 43 degree of identity with flounder, rat and human NCC, respectively. It is expressed along the eel gastrointestinal tract and absent in the kidney. NCCb cRNA injection into oocytes induced the appearance of a Na+ uptake pathway that is Cl- dependent, K+ independent, but is not sensitive to a variety of thiazide type diuretics (up to 1 mM concentration), to furosemide, DIDS or DIOA. A small 30% inhbition was observed with acetazolamide (100 mM). No 86Rb+ uptake was induced by NCCb. The Cl- transport kinetics revealed a Km for Cl- about 13 mM (previously observed for flounder NCC was 15 mM and rat NCC 2 mM). Interestingly, in contrast to flounder and mammalian NCC, eel NCCb is not activated by intracellular chloride depletion or co-injection with WNK informs.

Conclusions: NCCb encodes for an electroneutral Na+:Cl- cotransporter that is not sensitive to thiazide-type diuretics, with different regulatory properties when compared with mammalian NCC. Structure function studies of NCCb could lead to define the amino acid residues responsible for thiazide specificity in the NaCl cotransporter.