Na⁺-d-glucose cotransporter in the kidney ofSqualus acanthias: molecular identification and intrarenal distribution

Abstract

Using primers against conserved regions of mammalian Na ϩ -D-glucose cotransporters (SGLT), a cDNA was cloned from the kidney of spiny dogfish shark (Squalus acanthias). On the basis of comparison of amino acid sequence, membrane topology, and putative glycosylation and phosphorylation sites, the cDNA could be shown to belong to the family of sglt genes. Indeed, Na ϩ -dependent D-glucose uptake could be demonstrated after expression of the gene in Xenopus laevis oocytes. In a dendrogram, the SGLT from shark kidney has a high homology to the mammalian SGLT2. Computer analysis revealed that the elasmobranch protein is most similar to the mammalian proteins in the transmembrane regions and contains already all the amino acids identified to be functionally important, suggesting early conservation during evolution. Extramembraneous loops show larger variations. This holds especially for loop 13, which has been implied as a phlorizin-binding domain. Antibodies were generated and the intrarenal distribution of the SGLT was studied in cryosections. In parallel, the nephron segments were identified by lectins. Positive immunoreactions were found in the proximal tubule in the early parts PIa and PIb and the late segment PIIb. The large PIIa segment of the proximal tubule showed no reaction. In contrast to the mammalian kidney also the late distal tubule, the collecting tubule, and the collecting duct showed immunoreactivity. The molecular information confirms previous vesicle studies in which a low affinity SGLT with a low stoichiometry has been observed and supports the notion of a similarity of the shark kidney SGLT to the mammalian SGLT2. Despite its presence in the late parts of the nephron, the absence of SGLT in the major part of the proximal tubule, the relatively low affinity, and in particular the low stoichiometry might explain the lack of a Tm for D-glucose in the shark kidney.sodium-glucose cotransporter; intrarenal localization; cloning; fish; shark; elasmobranch ION GRADIENT-COUPLED TRANSPORT systems, in which one solute is accumulated in a secondary active mode against its own electrochemical gradient by employing the energy residing in the gradient of Na ϩ or protons, are widely distributed in nature. In the animal kingdom such transport systems mediate the uptake of nutrients in and the excretion of waste products out of the cells. In the kidney, they are involved in the reabsorption of organic and inorganic solutes such as sugars, amino acids, calcium, and phosphate. In mammalians usually the Na ϩ -Dglucose cotransporter (SGLT)-2 is involved in bulk D-glucose reabsorption with low affinity and high capacity in the early proximal tubule, whereas SGLT1 in the late part reduces intraluminal D-glucose to very low levels with a limited capacity (30). In fish, Na ϩ -dependent D-glucose transport activity has been found in brush-border membrane fractions derived from hagfish, flounder, trout, shark, and skate kidneys (6,8,9,16). The vesicle studies suggested that in the shark kidney an SGLT2 transporter is oper...

Morphological and functional characteristics of the kidney of cartilaginous fishes: with special reference to urea reabsorption

“…However, different from the mammalian kidney, no staining was detected in the middle part of proximal segment (proximal segment II), which is the major and most abundant segment of the proximal tubule in elasmobranch nephrons. A most probable explanation of this difference is that the proximal tubule II has secretory but not reabsorptive function (1). Indeed, we recently found that sulfate transporters are abundantly expressed in the proximal tubule II (Hasegawa K, Kato A, Hyodo S, unpublished data).…”

“…Na ϩ -dependent glucose transport activity was found in the little skate and spiny dogfish (62). Later, Na ϩ -D-glucose cotransporter was cloned from the kidney of spiny dogfish (1). Molecular phylogenetic analysis revealed that the SGLT obtained from the shark kidney is homologous to SGLT2.…”

“…One possible explanation could be that the proximal segments are not sufficient for efficient reabsorption of glucose. Alternatively, the shark SGLT2 may have a different function other than glucose transport, such as water and urea transports (1). The expression studies in Xenopus oocyte showed that mammalian SGLT1 also behave as Na ϩ uniporter, water channel, urea channel, and cotransporter of both water and urea (73,77,127).…”

Morphological and molecular investigations of the holocephalan elephant fish nephron: the existence of a countercurrent-like configuration and two separate diluting segments in the distal tubule

“…The complicated structure of the elasmobranch nephron has made it difficult to evaluate single nephron function by traditional physiological techniques. As a replacement for the absence of physiological evidence, mapping of membrane transporters has become a compelling technique for revealing the function of the elasmobranch nephron in recent years (Swenson et al 1994;Biemesderfer et al 1996;Hyodo et al 2004;Althoff et al 2006;Kakumura et al 2009;Yamaguchi et al 2009;Nakada et al 2010;Cutler et al 2012a, b;Li et al 2013). The distribution patterns of various transporting proteins along the nephron segments provide valuable information on the movement of small molecules, such as water, ions and urea, across the renal tubular epithelium.…”

Rhesus Glycoprotein P2 (Rhp2) Is a Novel Member of the Rh Family of Ammonia Transporters Highly Expressed in Shark Kidney

“…In situ hybridization and immunohistochemistry showed that Rhp2 was basolaterally localized in the renal tubule cells of the second and fourth loops in the sinus zone. Although several ion transporters have been localized to a specific segment of renal tubules in elasmobranch fish (21,(41)(42)(43)(44)(45), the functional significance of ammonia transport in the second and fourth loops is poorly understood. From its cellular and subcellular location, we assumed that Rhp2 facilitates ammonia transport across the basolateral membranes of renal tubule cells, but concerning the direction of the transport, we cannot conclude whether it mediates ammonia reabsorption or secretion in vivo.…”