in the distal nephrons of euryhaline and seawater pufferfishes. Am J Physiol Regul Integr Comp Physiol 300: R284 -R297, 2011. First published November 17, 2010 doi:10.1152/ajpregu.00725.2009.-The process of NaCl reabsorption in the distal nephron allows freshwater fishes to excrete hypotonic urine and seawater fishes to excrete urine containing high concentrations of divalent ions; the relevant transporters, however, have not yet been identified. In the mammalian distal nephron, NaCl absorption is mediated by Na ϩ -K ϩ -Cl Ϫ cotransporter 2 (NKCC2, Slc12a1) in the thick ascending limb, Na ϩ -Cl Ϫ cotransporter (NCC, Slc12a3) in the distal convoluted tubule, and epithelial sodium channel (ENaC) in the collecting duct. In this study, we compared the expression profiles of these proteins in the kidneys of euryhaline and seawater pufferfishes. Mining the fugu genome identified one NKCC2 gene and one NCC gene, but no ENaC gene. RT-PCR and in situ hybridization analyses demonstrated that NKCC2 was highly expressed in the distal tubules and NCC was highly expressed in the collecting ducts of euryhaline pufferfish (mefugu, Takifugu obscurus). On the other hand, the kidney of seawater pufferfish (torafugu, Takifugu rubripes), which lacked distal tubules, expressed very low levels of NCC, and, in the collecting ducts, high levels of NKCC2. Acclimation of mefugu to seawater resulted in a 2.7ϫ decrease in NCC expression, whereas NKCC2 expression was not markedly affected. Additionally, internalization of NCC from the apical surface of the collecting ducts was observed. These results suggest that NaCl reabsorption in the distal nephron of the fish kidney is mediated by NCC and NKCC2 in freshwater and by NKCC2 in seawater. distal tubule; collecting duct; NKCC2; NCC; fish kidney; dilute urine FRESHWATER FISHES LIVE IN hypo-osmotic environments, resulting in significant influx of water into the body, mainly across the gills. To maintain body fluid homeostasis, freshwater fishes import ions from surrounding water through the gills (13,22) or from the diet, and excrete excess water in hypotonic urine (ϳ10 mM Na ϩ and Cl Ϫ ) through the kidneys (30). To increase urine volume (i.e., water excretion), freshwater fishes secrete NaCl in the proximal tubule and draw interstitial water into the tubular lumen (9); this proximally introduced NaCl should be reabsorbed in the distal nephron. Reabsorption of Na ϩ and Cl Ϫ ions from urine under water-impermeable conditions is, therefore, essential for net water excretion and the survival of fishes in freshwater. Na ϩ and Cl Ϫ reabsorption is also important for seawater fishes. Unlike freshwater fishes, however, seawater fishes experience passive water efflux. To balance water loss, seawater fishes absorb water and ions through the intestine by drinking large amounts of seawater (17). Surplus ions are excreted mainly through the gills (13, 22), although divalent ions (Mg 2ϩ and SO 4 2Ϫ ) are excreted through isotonic urine (4, 30). To produce urine with high concentrations of Mg 2ϩ (ϳ140 mM) ...

The second most abundant cation in seawater (SW), Mg²⁺, is present at concentrations of ~53 mM. Marine teleosts maintain plasma Mg²⁺ concentration at 1-2 mM by excreting Mg²⁺ into the urine. Urine Mg²⁺ concentrations of SW teleosts exceed 70 mM, most of which is secreted by the renal tubular epithelial cells. However, molecular mechanisms of the Mg²⁺ secretion have yet to be clarified. To identify transporters involved in Mg²⁺ secretion, we analyzed the expression of fish homologs of the Slc41 Mg²⁺ transporter family in various tissues of SW pufferfish torafugu (Takifugu rubripes) and its closely related euryhaline species mefugu (Takifugu obscurus). Takifugu genome contained five members of Slc41 genes, and only Slc41a1 was highly expressed in the kidney. Renal expression of Slc41a1 was markedly elevated when mefugu were transferred from fresh water (FW) to SW. In situ hybridization analysis and immunohistochemistry at the light and electron microscopic levels revealed that Slc41a1 is localized to vacuoles in the apical cytoplasm of the proximal tubules. These results suggest that pufferfish Slc41a1 is a Mg²⁺ transporter involved in renal tubular transepithelial Mg²⁺ secretion by mediating Mg²⁺ transport from the cytosol to the vacuolar lumen, and support the hypothesis that Mg²⁺ secretion is mediated by exocytosis of Mg²⁺-rich vacuoles to the lumen.

Seawater (SW) contains ∼10 mM Ca(2+), yet marine fish must drink seawater as their major water source. Thus marine teleosts fish need to excrete Ca(2+) to maintain whole body Ca(2+) homeostasis. In the intestine, seawater Ca(2+) interreacts with epithelial-secreted HCO(3)(-) by the intestinal epithelium, and the resulting CaCO(3) precipitates, which is rectally excreted. Recently the transporters involved in intestinal HCO(3)(-) secretion were identified. Ca(2+) is also excreted by the kidney, but the protein(s) involved in renal Ca(2+) excretion have not been identified. Here we identified a candidate transporter by using SW pufferfish torafugu (Takifugu rubripes) and its closely related euryhaline species mefugu (Takifugu obscurus), which are becoming useful animal models for studying molecular mechanisms of seawater adaptation. RT-PCR analyses of Na(+)/Ca(2+) exchanger (NCX) family members in various torafugu tissues demonstrated that only NCX2a is highly expressed in the kidney. Renal expression of NCX2a was markedly elevated when mefugu were transferred from freshwater to seawater. In situ hybridization and immunohistochemical analyses indicated that NCX2a is expressed in the proximal tubule at the apical membrane. NCX2a, expressed in Xenopus oocytes, conferred [Ca(2+)](out)- and Na(+)-dependent currents. These results suggest that NCX2a mediates renal Ca(2+) secretion at the apical membrane of renal proximal tubules and has an important role in whole body Ca(2+) homeostasis of marine teleosts.

The kidney of marine teleosts is the major site of Mg 2ϩ excretion and produces urine with a high Mg 2ϩ concentration. However, the transporters involved in Mg 2ϩ excretion are poorly understood. The cyclin M (Cnnm; also known as ancient conserved domain protein) family comprises membrane proteins homologous to the bacterial Mg 2ϩ and Co 2ϩ efflux protein, CorC. To understand the molecular mechanism of Mg 2ϩ homeostasis in marine teleosts, we analyzed the expression of the Cnnm family genes in the seawater (SW) pufferfish, torafugu (Takifugu rubripes), and the closely related euryhaline species, mefugu (Takifugu obscurus). Database mining and phylogenetic analysis indicated that the Takifugu genome contains six members of the Cnnm family: two orthologs of Cnnm1, one of Cnnm2, one of Cnnm3, and two of Cnnm4. RT-PCR analyses indicated that Cnnm2, Cnnm3, and Cnnm4a are expressed in the kidney, whereas other members are mainly expressed in the brain. Renal expression of Cnnm3 was upregulated in SW mefugu, whereas renal expression of Cnnm2 was upregulated in freshwater (FW) mefugu. No significant difference was observed in renal expression of Cnnm4a between SW and FW mefugu. In situ hybridization and immunohistochemical analyses of the SW mefugu kidney revealed that Cnnm3 is expressed in the proximal tubule, and its product localizes to the lateral membrane. When Cnnm3 was expressed in Xenopus laevis oocytes, whole cellular Mg 2ϩ content and free intracellular Mg 2ϩ activity significantly decreased. These results suggest that Cnnm3 is involved in body fluid Mg 2ϩ homeostasis in marine teleosts.Cnnm3; proximal tubule; marine teleost; magnesium homeostasis; lateral membrane SEAWATER (SW) CONTAINS ϳ53 mM Mg 2ϩ , a concentration ϳ50 times higher than that in the plasma of marine teleosts. The bladder urine Mg 2ϩ concentration of marine teleosts is 57-167 mM (4,5,29,36), and urine-to-plasma ratios of Mg 2ϩ can exceed 100. Renal tubular fluid secretion accounts for much or all initial urine production in glomerular or aglomerular marine teleosts, and the renal proximal tubule is the major site of fluid secretion and Mg 2ϩ excretion (4,5,18,33 , which is reduced to ϳ2.9 mol·kg Ϫ1 ·h Ϫ1 in final urine, indicating net Mg 2ϩ reabsorption in the renal tubule (5).Proximal tubules isolated from the winter flounder (Pseudopleuronectes americanus) generate a Mg 2ϩ concentration of 27 mM in the tubule lumen when the medium contains 1 mM Mg 2ϩ (3,11). Mg 2ϩ is secreted from the peritubular bath into the tubule lumen against electrochemical potentials; therefore, the mechanism of active Mg 2ϩ transport must be powerful (5). In the urine of marine teleosts, urinary Mg 2ϩ and Na ϩ concentrations are negatively correlated, suggesting that Mg 2ϩ excretion is coupled with Na ϩ reabsorption (32). X-ray microanalysis of freeze-dried cryosections of the kidneys of the dogfish shark (Squalus acanthias) demonstrated the presence of small apical vacuoles containing high concentrations of Mg (over 200 mM) in the proximal tubular cells (17). Ion m...