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. 2016 Jul 1;311(1):R66-78.
doi: 10.1152/ajpregu.00477.2015. Epub 2016 Apr 27.

Sulfate transporters involved in sulfate secretion in the kidney are localized in the renal proximal tubule II of the elephant fish (Callorhinchus milii)

Kumi Hasegawa  1 , Akira Kato  2 , Taro Watanabe  3 , Wataru Takagi  4 , Michael F Romero  5 , Justin D Bell  6 , Tes Toop  7 , John A Donald  7 , Susumu Hyodo  3
Affiliations

Sulfate transporters involved in sulfate secretion in the kidney are localized in the renal proximal tubule II of the elephant fish (Callorhinchus milii)

Kumi Hasegawa et al. Am J Physiol Regul Integr Comp Physiol. .

Abstract

Most vertebrates, including cartilaginous fishes, maintain their plasma SO4 (2-) concentration ([SO4 (2-)]) within a narrow range of 0.2-1 mM. As seawater has a [SO4 (2-)] about 40 times higher than that of the plasma, SO4 (2-) excretion is the major role of kidneys in marine teleost fishes. It has been suggested that cartilaginous fishes also excrete excess SO4 (2-) via the kidney. However, little is known about the underlying mechanisms for SO4 (2-) transport in cartilaginous fish, largely due to the extraordinarily elaborate four-loop configuration of the nephron, which consists of at least 10 morphologically distinguishable segments. In the present study, we determined cDNA sequences from the kidney of holocephalan elephant fish (Callorhinchus milii) that encoded solute carrier family 26 member 1 (Slc26a1) and member 6 (Slc26a6), which are SO4 (2-) transporters that are expressed in mammalian and teleost kidneys. Elephant fish Slc26a1 (cmSlc26a1) and cmSlc26a6 mRNAs were coexpressed in the proximal II (PII) segment of the nephron, which comprises the second loop in the sinus zone. Functional analyses using Xenopus oocytes and the results of immunohistochemistry revealed that cmSlc26a1 is a basolaterally located electroneutral SO4 (2-) transporter, while cmSlc26a6 is an apically located, electrogenic Cl(-)/SO4 (2-) exchanger. In addition, we found that both cmSlc26a1 and cmSlc26a6 were abundantly expressed in the kidney of embryos; SO4 (2-) was concentrated in a bladder-like structure of elephant fish embryos. Our results demonstrated that the PII segment of the nephron contributes to the secretion of excess SO4 (2-) by the kidney of elephant fish. Possible mechanisms for SO4 (2-) secretion in the PII segment are discussed.

Keywords: cartilaginous fish; elephant fish; kidney; secretion; sulfate transporter.

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Figures

Fig. 1.
Fig. 1.
Schematic drawing showing the four-loop nephron of cartilaginous fish (based on and modified from Ref. 22). The encircled numbers represent the number of loops. The representative segments that are common to both elasmobranchs and elephant fish nephrons are shown: PIa, proximal tubule Ia; PII, proximal tubule II; EDT, early distal tubule; LDT, late distal tubule; CT, collecting tubule; CV, central vessel; PS, peritubular sheath; RC, renal corpuscle.
Fig. 2.
Fig. 2.
Molecular phylogeny of vertebrate Slc26 family proteins. The elephant fish (holocephalan) sequences identified in this study are shown in bold letters with selected vertebrates (mammals and teleosts) indicated in normal font. The accession numbers of genes and mRNAs used in the analysis are listed in Table 2. Numbers at branch nodes represent Bayesian posterior probabilities.
Fig. 3.
Fig. 3.
Primary structure of elephant fish (cm) Slc26a1 and Slc26a6 cloned from the kidney. The deduced amino acid sequences are aligned with those of human (hs) Slc26a1 and Slc26a6, respectively. The accession numbers of the genes are listed in Table 2. For simplicity and to reduce alignment gaps, other Slc26 sequences, such as teleost Slc26a1 and Slc26a6, have not been included in the alignment; these show considerable similarity to mammalian Slc26a1 and Slc26a6, respectively (25, 26, 31, 39). Gray shading indicates identical amino acid residues between cmSlc26a1 (cm1) and hsSlc26a1 (hs1) and between cmSlc26a6 (cm6) and hsSlc26a6 (hs6), respectively. Identical amino acid residues between cmSlc26a1 and cmSlc26a6 are indicated by asterisks. Hyphens denote gaps introduced for the alignment. The hash mark (#) represents the glutamate residue that is important for the Slc26 transporter properties (40). Wavy lines indicate the three amino acid residues that interact with the PDZ domain. TM, transmembrane domain; STAS, a sulfate transporter and Ant-sigma factor antagonist domain. Note that the Slc26 family has been considered to have 12 TMs (indicated by horizontal solid bars), while the rat Slc26a5 was recently reported to have 14 TMs; additional putative TMs are indicated by open horizontal bars.
Fig. 4.
Fig. 4.
Tissue distribution of mRNAs encoding cmSlc26a1 (A) and cmSlc26a6 (B) in adult elephant fish. Data are presented as means ± SE. The values were normalized against those of cmACTB mRNA. n = 4 (two males and two females) for each tissue analysis, except for uterus which was n = 2.
Fig. 5.
Fig. 5.
Functional characterization of cmSlc26a1 and cmSlc26a6 using [35S]SO42−. A: [35S]SO42− uptake mediated by water-injected or cmSlc26a6 cRNA-injected oocytes was examined in Na+ buffer or K+ buffer in the presence (solid bars) or absence (open bars) of 100 mM Cl−. DIDS was used as a general anion exchanger inhibitor. a,b,cValues not sharing an identical letter are significantly different (P < 0.05). B: [35S]SO42− uptake mediated by water-injected or cmSlc26a1 cRNA-injected X. laevis oocytes. a,bValues not sharing the identical letter are significantly different (P < 0.05).
Fig. 6.
Fig. 6.
Functional characterization of cmSlc26a1 and cmSlc26a6 using Cl−-selective microelectrodes. Representative traces are shown for intracellular Cl− activity (aCli) and membrane potential (Vm) of oocytes expressing either cmSlc26a1 and cmSlc26a6, and control oocytes. In the continuous presence of 5 mM SO42−, Cl−/SO42− exchanging activity was monitored as changes in aCli and Vm after extracellular Cl− was removed (0 Cl−) and replaced by gluconate.
Fig. 7.
Fig. 7.
Kidney sections subjected to either hematoxylin-and-eosin (HE) staining (A–C) or in situ hybridization with cRNA probes for cmSlc26a1 (D–F), cmSlc26a6 (G–I), or cmNKCC2 (J–L). A, D, G, and J are low-power micrographs. The kidney lobule is separated into two zones, a sinus zone (SZ) and a bundle zone (BZ). Signals for cmSlc26a1 and cmSlc26a6 mRNAs were detected only in the SZ, while cmNKCC2 mRNA was expressed in tubules in both the sinus and bundle zones. B, E, H, and K are magnified views of the SZ. In the SZ, two major nephron segments are identifiable: a proximal II (PII; arrows) segment and a late distal tubule (LDT; arrowheads). The signals for cmSlc26a1 and cmSlc26a6 mRNAs were colocalized in the PII segments, while cmNKCC2 mRNA was expressed in the LDT. C, F, I, and L are magnified views of the BZ. In the BZ, a cross-sectional view of the five tubular segments can be identified (C, inset). cmNKCC2 mRNA was expressed in the early distal tubule, while cmSlc26a1 and cmSlc26a6 mRNAs were not expressed in the bundle zone (F, I). M: schematic representation of the elephant fish nephron showing the localization of cmNKCC2 mRNA (modified on the basis of Ref. 22). The encircled numbers represent the number of loops. CV, central vessel; RC, renal corpuscle; PS, peritubular sheath. Scale bars: 500 μm (A, D, G, J) and 100 μm (B, C, E, F, H, I, K, L).
Fig. 8.
Fig. 8.
Immunohistochemical localization of the cmSlc26a1 and cmSlc26a6 in the sinus zone of elephant fish kidney. Immunoreactive signals to cmSlc26a1 were detected in the basolateral membrane of PII cells (arrows), while signals to cmSlc26a6 were detected in the apical membrane of PII cells (C). Preabsorption of antibodies with the antigen peptides resulted in disappearance of the immunoreactive signals on the membranes (B, D). Sections are counterstained with hematoxylin. Scale bars: 50 μm.
Fig. 9.
Fig. 9.
Embryonic tissue distribution of mRNAs. RNAs encoding cmSlc26a1 (A) and cmSlc26a6 (B) were detected in stage 36 embryos. The mRNA values were normalized against those of cmEF1α mRNA. Data are presented as means ± SE; n = 6.
Fig. 10.
Fig. 10.
Schematic diagrams showing the elephant fish nephron and PII sulfate secretion. Localization of Slc26a1, Slc26a6, NKA, and NKCC2 is indicated in the diagram of elephant fish nephron. The upper left corner provides a hypothetical model for epithelial SO42− secretion in the PII segment. cmSlc26a6 is an electrogenic Cl−/SO42− exchanger localized on the apical membrane. The negative membrane potential and high concentration of Cl− in the filtered urine most probably drives SO42− secretion into the filtrate via Slc26a6. The intracellular SO42− is supplied via basolaterally located cmSlc26a1 (electroneutral SO42− transporter) from the blood sinus. The dashed line means that we have no direct evidence for SO42−/anion exchange or SO42−-cation cotransport for cmSlc26a1. The encircled numbers represent the number of loops. AM, apical membrane; BLM, basolateral membrane; CV, central vessel; NKA, Na+-K+-ATPase; RC, renal corpuscle; PS, peritubular sheath, Slc, solute carrier family.
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