1Max Planck Institute for Molecular Physiology, D-44229 Dortmund; 3MariCal, Incorporated, Portland 04101; 4Children's Hospital, Boston, Massachusetts 02115; 5Yale University School of Medicine, New Haven, Connecticut 06520; 6Department of Nephrology, Hannover Medical School, 30625 Hannover, Germany; and 2Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672
Submitted 26 February 2002 ; accepted in final form 13 May 2003
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ABSTRACT |
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renal handling of magnesium; transmembrane receptor protein; immunohistochemistry
Previous work by our group has focused on a detailed characterization of physiologically relevant characteristics of the elasmobranch kidney (12, 18, 23, 25). The excretory portion of dogfish kidney consists of multiple, metamerically formed lobules that grow together during organogenesis (19). Each lobule possesses its own vasculature where renal arteries supply perfusion to glomeruli. The multiple efferent arterioles merge with the sinusoid capillaries of the renal portal system, which, in turn, are drained via the cardinal veins to the heart. The tubules from each lobule are drained by a single large collecting duct. The lobules are separated into a mesial zone and a zone of lateral bundles. Each nephron is composed of tubular segments that travel in both zones, forming two hairpin loops in the bundles and two extended convolutions in mesial tissue. A schematic drawing of the anatomic organization of a single dogfish kidney nephron is shown in Fig. 1.
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Individual nephrons in dogfish kidney possess well-developed proximal and distal segments similar to those described for teleost kidney (21). The proximal tubule is subdivided into two major portions that are designated PI and PII (21). The epithelial cells of PI are endowed with an elaborate apical tubulovesicular apparatus and an extended lysosomal compartment that are similar to those possessed by the entire proximal tubule of mammals (segments S1-S3). By contrast, PII epithelial cells, which apparently have no counterpart in the mammalian kidney, possess an apical compartment filled with clear smooth vesicles that contain high concentrations of Mg2+ (26). Although present evidence is very limited, data suggest that the PII segment of marine elasmobranchs may engage in net reabsorption of fluid (36), although the PII segment is capable of fluid and salt secretion when proximal tubules from teleosts and elasmobranches are incubated in vitro (5). In dogfish, the luminal contents of these proximal tubule segments are delivered to the early distal tubule (EDT; the homologue of the thick ascending limb of Henle's loop) and a late distal tubule (LDT; the homologue of the distal convoluted tubule in mammals) and, finally, to the collecting tubule (CT)/collecting duct (CD).
Because Mg2+ uptake in dogfish will vary when they migrate between seawater of different salinities as well as during periods of excessive feeding, Mg2+ excretion has to be balanced with alterations in Mg2+ uptake to maintain overall Mg2+ balance. However, the mechanisms that control either Mg2+ secretion in the PII segment or possible reabsorption in the distal tubule segments of elasmobranch kidney are unknown. In this regard, it has been suggested on the basis of histological evidence using 26Mg2+ that renal Mg2+ excretion in the euryhaline marine teleost, the killifish (Fundulus heteroclitus), is the result of a two-step process where proximal tubule cells secrete Mg2+ and Mg2+ reabsorption occurs in the CD/CT system (9).
Molecular cloning and characterization of the calcium/polyvalent
cation-sensing protein (CaR) in the nephron segments of mammals have opened a
new window to an understanding of renal handling of Mg2+
(14,
15). Antibody and cDNA probes
derived from the sequence of CaRs cloned from mammals have been utilized
extensively to identify patterns of cell-specific CaR expression in multiple
mammalian tissues (10,
31,
33). These studies have
suggested that CaRs possess the ability to "sense" local
concentrations of divalent cations and regulate transepithelial ion transport
in response to such changes. CaRs are localized to specific cell types in
multiple tissues, where they serve as key integrators of divalent mineral ion
homeostasis in terrestrial mammals
(7,
10,
14,
15,
31,
33). To determine whether CaRs
serve similar roles in elasmobranch and teleost fish, we isolated a 4.16-kb
shark kidney CaR (SKCaR) cDNA from a dogfish kidney cDNA library
(29). SKCaR is a 1,027-amino
acid (AA) protein possessing overall 74% AA identity with rat kidney CaR
(29). The shark kidney
contains two major SKCaR poly A+ transcripts of 7 and 4 kb
that are similar to those in the mammalian kidney
(33,
34). A combination of RNA
blotting and immunocytochemistry reveals significant SKCaR expression in other
shark organs besides the kidney, including rectal gland, stomach, intestine,
gill, olfactory lamellae, brain, and ovary
(29). Functional expression of
SKCaR protein in human embryonic kidney cells shows that it possesses
half-maximal activation (EC50) values for Ca2+ and
Mg2+ of
7.5 and 30 mM, respectively, under mammalian
physiological ionic conditions
(3,
29). These data suggest that
SKCaR likely serves as a Mg2+ receptor in the shark kidney. In this
study, we hypothesized that the SKCaR protein might possess a highly specific
pattern of cellular expression, possibly reflecting its role as an ion sensor
in the shark kidney. Using SKCaR-specific antiserum, we report here that SKCaR
exhibits a highly specific subsegmental nephron distribution in the shark
kidney that is compatible with a role as a principal regulatory sensor for
Mg2+ homeostasis in elasmobranch kidney.
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MATERIALS AND METHODS |
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Tissue preparation. After anesthesia with tricaine (MS 222,
Sigma), dogfish were perfused via the heart and truncus arteriosus with
heparinized dogfish Ringer solution (in mM: 280 NaCl, 6 KCl, 3
MgCl2, 5 CaCl2, 0.5 Na2HPO4, 1.0
NaH2PO4, 330 urea, 5 glucose, 72 TMAO, and 8
NaHCO3 in 1 liter). The measured osmolality of this solution was
1,000 ± 30 mosM. Approximately 400-500 ml were perfused (5-10 min)
at a temperature of 0-4°C and a pressure of 120 cmH2O. Without
a change in pressure and flow, the fixation fluid was added and perfused for
5-10 min.
The fixation fluid contained 2% formaldehyde freshly prepared from
paraformaldehyde, 0.05% glutaraldehyde, and 0.5% picric acid in Sorensen's
phosphate buffer, pH 7.4. Sucrose was added to the buffer vehicle (Sorensen's
plus 150 mM NaCl) to obtain a blood hyposmotic value of 800 mosM. After
fixation, small tissue pieces were excised from the organ and thoroughly
rinsed in Sorenson's buffer plus 150 mM NaCl, which was adjusted with sucrose
to 850 mosM.
Small pieces of tissue were processed by four different methods: samples were 1) postfixed with 1% OsO4 in Sorensen's buffer, dehydrated via ethanol and acetone, and embedded in Spurr's medium; 2) embedded in OCT compound, shock-frozen in melting isopentane, and stored in liquid nitrogen; 3) dehydrated via ethanol and xylene and embedded in Paraplast (56°C); and 4) dehydrated via ethanol and embedded in LR-White resin.
Histology. Sections (0.5 µm) and thin sections (60 nm) were obtained from tissue blocks (Spurr's embedding medium) and viewed with a light microscope after being stained with toluidine blue or with an electron microscope (Zeiss EM 902 or Philipps EM 301) after being stained with uranyl acetate and lead citrate. The nomenclature of renal structures, i.e., nephron segments, blood vessels, and interstitial cells, was used in accordance with previous results with spiny dogfish and other marine elasmobranch fish (12, 18, 20, 25). In addition to structures, which are generally characteristic of the elasmobranch kidney, spiny dogfish feature a specific subdivision of the second segment of the proximal tubule PII (PIIa and PIIb) (12).
Antibody preparation. A 17-mer peptide (ARSRNSADGRSGDDLP+C for COOH-terminal conjugation), corresponding to 965-980 of the putative 1,027-AA SKCaR polypeptide, was synthesized by standard automated solid-phase techniques, conjugated to keyhole limpet hemocyanin via a cysteine sulfhydryl linkage, and used to immunize rabbits as reported previously (29). Test bleedings were screened by immunoblots against shark kidney tissue.
For selected experiments, SKCaR immune antiserum was affinity purified as described previously (30). Purified immune IgG fractions were absorbed to a column containing covalently attached peptide conjugated via a 5-thio-2-nitro-benzoic acid-thiol-agarose linkage. After extensive washing, the purified anti-SKCaR antiserum was eluted at pH 2.5, followed by rapid titration to pH 8.0. Both raw and affinity-purified antisera were stored at -80°C in multiple aliquots.
Immunoblot analyses. To provide further evidence for the specificity of the antiserum for SKCaR in addition to comparisons of immunoblots and immunocytochemical sections exposed to immune antiserum vs. preimmune serum as previously reported (29), immunoblots containing kidney membranes were probed using affinity-purified antibodies before and after their preincubation with a 100-fold molar excess of competing peptide (30). In all blots, we routinely observed that the dye front of all lanes containing shark tissue fractions displayed reactivity (see Fig. 2) that was not ablated by preincubation with an appropriate peptide. This apparent reactivity of the dye front was due to binding of secondary antiserum because dye front reactivity was present without the addition of primary anti-SKCaR antiserum (either affinity purified, immune, or preimmune) but not when secondary antiserum was omitted (data not shown).
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Immunohistochemistry. Cryosections (4 µm) and Paraplast sections (2-4 µm) were treated with 0.1% H2O2 and used for indirect immunolabeling. After the blocking of unspecific binding with a mixture of 0.2% coldwater fish gelatin, 2% BSA, and 2% fetal calf serum, sections were incubated with primary antiserum (SKCaR antibody; see above), and specific antibody binding sites were revealed with the immunoperoxidase technique, involving biotin-streptavidin amplification (ABC Elite Kit, Vector Laboratories, Burlingame, CA) with methyl green as a counterstain. Routine controls were performed 1) with the omission of primary antiserum and 2) with incubation with preimmune serum. Permanent mounts (Gelmount) were viewed and photographed with an Axiophot light microscope (Carl Zeiss, Göttingen, Germany).
Immunoelectron microscopy. Thin sections (60 nm) were obtained from LR-White blocks with an ultramicrotome (Ultracut E, Leica) and incubated with anti-SKCaR antibody, followed by anti-rabbit IgG-colloidal gold conjugate (10-nm gold particles, Aurion-Gent). The sections were counterstained with uranyl acetate and viewed with Zeiss EM 902 and Philipps 301 electron microscopes.
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RESULTS |
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When immunoblots containing crude membranes isolated from dogfish kidney were probed with affinity-purified anti-SKCaR antibody, prominent bands of 240, 140, and 91 kDa were present (Fig. 2C). These bands were completely ablated after preincubation with an excess of competing peptide. These bands display molecular masses similar to those reported previously for SKCaR protein expressed in human embryonic kidney cells (29) as well as anti-CaR-reactive proteins present in a variety of mammalian tissues (33, 34).
Anti-SKCaR labeling of specific nephron subsegments. Anti-SKCaR antibody labeled specific nephron subsegments in both mesial tissue and lateral bundle zones of dogfish kidney (Fig. 3). The PIa segment in the lateral bundle zone displayed apical SKCaR staining within the region of its brush border (Fig. 3 and see Fig. 6). The PIb segment, which displays multiple bands within mesial tissue near the glomeruli, showed SKCaR-specific staining only at the base of the microvilli of a few cells (Fig. 3).
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The PIIa and PIIb segments of the proximal tubule present in mesial tissue displayed markedly different patterns of anti-SKCaR staining characteristics (Figs. 3, 4, 5): PIIa cells exhibited no SKCaR staining. In contrast, PIIb cells of all 14 animals studied displayed specific SKCaR immunoreactivity that was observed at their apical membranes (brush border). Interestingly, the intensity of SKCaR labeling of PIIb apical membrane varied greatly among various individual animals studied despite the fact that consistent SKCaR labeling was observed in most other shark nephron segments (see below).
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The EDT, which is present exclusively in the lateral bundle zone, is contiguous with the LDT, which thereafter performs numerous bands in mesial tissue. EDT cells were diffusely labeled by anti-SKCaR antiserum (Figs. 3 and 6). The LDT is present in mesial tissue, where it courses along the pathway of PIIa tubules and is frequently in close proximity to both PIIa and PIIb cells (Figs. 3, 4, 5). LDT cells in all animals examined showed sharply defined SKCaR staining at their apical cell membranes. In addition, only very weak immunostaining was observed at the LDT basolateral membrane in two animals (Fig. 4).
Electron microscopy of LDT cells revealed that they possess short, stubby microvilli with a marked asymmetry of the apical cell membrane, where the extracellular (luminally facing) side was thickened and had a fuzzy coat (glycocalyx). Immunoelectron microscopy of the apical region of LDT cells showed that anti-SKCaR immunoreactivity protein was 1) in the immediate vicinity of the cell membrane; 2) in the apical cytoplasmic region, presumably at apical vesicles; 3) associated with membrane-bound granules located in close proximity to the apical membrane; and 4) outside the cell in the glycocalyx (Fig. 7).
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Significant anti-SKCaR immunoreactivity was observed in the CT as well as in the CD (Figs. 3 and 6). The CT at the vascular field of the glomerulus was labeled by the chromogenic reaction. In CT and CD cells, SKCaR antibody binding was confined to the region of the apical cell membrane and its adjacent cytoplasmic zone, where membrane-bound granules abound.
SKCaR staining was also observed in the cytoplasm of small, round cells with large spherical nuclei that were arranged in islets in the interstitium of the lateral bundle zone (Fig. 3). These cells belong to the renal lymphomyeloid tissue that is involved in hematopoiesis of elasmobranch fish (22). Although the function of these cells is presently unknown, they may correspond to hematopoietic cells that possess CaR proteins in mammals (15).
In summary, we consistently found SKCaR labeling in nephron segments PIa, PIb, PIIb, EDT, the apical membrane of the LDT, the CT/CD system, and in a subpopulation of cells of the interstitial tissue. However, we observed that SKCaR reactivity was less pronounced in PIIb of four animals, and with the exception of two animals, the basolateral membrane of LDT was not labeled.
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DISCUSSION |
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The reason for the variability of anti-SKCaR staining that we observed in the PIIb and LDT tubular segments is not presently known. We speculate that this variability could possibly result from wild-caught animals that were in different stages of acclimatization to either captivity or conditions before being captured for study. Further studies are also required to more carefully define any physiological variables that might contribute to differences in SKCaR staining in these tubular segments.
The significance of the observed pattern of SKCaR, a Mg2+ sensor, for the handling of Mg2+ by marine fish will be discussed below, with particular reference to the elaborate organization of the elasmobranch kidney. Due to the paucity of transport studies in the elasmobranch nephron (17, 37), these specific SKCaR staining patterns provide the basis for us to propose a functional model of Mg2+ homeostasis in the elasmobranch kidney that highlights specific aspects of putative recycling of Mg2+ between Mg2+-sensing and/or -reabsorbing cells and Mg2+-secreting cells of the shark's multisegmental nephron. In turn, this model provides a series of testable hypotheses that form the basis of future experiments directed at a more detailed understanding of divalent mineral transport and recycling by the elasmobranch kidney.
Overall renal handling of Mg2+ in marine fish. Although
renal Mg2+ excretion is essential for the survival of elasmobranchs
in the marine environment because seawater contains a high concentration of
Mg2+ (50-60 mM) relative to their plasma Mg2+
(plasma
1-3 mM), functional data quantifying the specific contribution of
various nephron segments to this process of net Mg2+ excretion are
scarce (17,
37). The few measurements of
S. acanthias kidney show that urinary flow in conscious, restrained
spiny dogfish is low (
0.3
ml·h-1·kg-1), <40% of the inulin
clearance (
1 ml·h-1·kg-1)
(4,
38). In contrast,
urinary-to-plasma Mg2+ concentration ratios are >40 and indicate
net tubular secretion (8).
Mg2+ homeostasis and functional evidence from the proximal and distal tubules. Secretion of Mg2+ occurs in isolated dogfish proximal tubules (35). In previous work, we used quantitative transmission electron microscopy to demonstrate that Mg2+ is sequestered at high concentrations within secretory apical vesicles of PII cells in European dogfish (Scyliorhinus caniculus) (26). Micropuncture studies (37) in a related elasmobranch, the little skate (Raja erinacea), show that the final urinary Mg2+ concentration is 10-fold higher compared with its plasma value. Transtubular concentration differences (TF/P) for Mg2+ reveal that large increases in TF/P Mg2+ occur in PII, suggesting that significant Mg2+ secretion occurs within this segment. Mg2+ transport in elasmobranchs also appears to be very similar to Mg2+ secretion in the proximal tubule of two other marine and euryhaline teleost fish species, the flounder Pleuronectes americanus and the killifish (F. heteroclitus) (11). Transport kinetics with isolated tubules show saturation far below plasma concentration, suggesting that Mg2+ secretion by proximal tubules cannot alone be held responsible for the tight regulation of Mg2+ concentration in the plasma of marine fish. Accordingly, Beyenbach and co-workers (5, 6) have suggested that proximal tubules in marine fish can be considered to work as devices for clearance of Mg2+ from blood akin to the clearance of plasma solutes by glomerular filtration.
Evidence for Mg2+ reabsorption in distal tubule segments of marine elasmobranchs is very limited. TF/P values obtained from micropuncture studies of proximal segments vs. CD and final urine of the little skate suggest that more distal nephron segments may be involved in both tubular reabsorption and secretion of Mg2+ (37). Although studies of isolated distal tubules of S. acanthias have revealed great similarity to the diluting segment of mammals and other vertebrates (13, 16), no studies of Mg2+ transport have been performed to quantify Mg2+ transport in this dogfish nephron segment.
Selected aspects of elasmobranch kidney morphology relevant to Mg2+ homeostasis. The morphological investigations of the kidneys of Chondrichthyes (sharks, skates, and chimeras) have uncovered an extremely complex organization (for references and reviews, see Refs. 12, 18, 20, 25, and 28). Previous comparative anatomic studies in elasmobranchs have revealed the presence of nephron segments that are homologous to corresponding segments in other vertebrates (20). Moreover, it is interesting to note that proximal and EDT segments in kidneys of both mammals and elasmobranchs are spatially separated and their renal tissue is zonated. These patterns of kidney tissue zonation are present in many other vertebrates, including agnathean lampreys, archaic fish such as Polypteridae and lungfish, and amphibian and higher vertebrates (Sauropsida and Mammalia) (21), and might have originated early in vertebrate evolution.
Thus despite the fact that mammalian and cartilaginous fish kidneys are organized differently, it is intriguing that the kidney of spiny dogfish possesses two renal zones that superficially resemble those of the mammalian renal cortex and medulla, respectively. Mesial tissue of dogfish kidney contains a close association of proximal tubules (segments PIb, PIIa, and PIIb) as well as LDT. This complement of dogfish nephron segments present in mesial tissue appears to be similar to the mammalian renal cortex with its assembly of proximal tubules, distal convoluted tubule, CT, and cortical CD. By contrast, the lateral bundles of dogfish kidney bear a similarity to the mammalian renal medulla in that the EDT and the CT/CD of dogfish kidney are specially associated like that of the thick ascending limb of Henle's loop and medullary CD of the mammalian kidney. As discussed below, these anatomic associations might be functionally relevant in renal Mg2+ handling in the elasmobranch kidney in a manner that is important, as specific nephron segments appear to do so in the mammalian kidney.
Functional considerations for SKCaR localization in individual elasmobranch nephron segments. The specific distribution pattern of SKCaR protein in individual cell types present in dogfish nephron segments suggests a unifying hypothesis whereby Mg2+ excretion in dogfish is effectively regulated by SKCaR, which senses local Mg2+ concentrations. Based on the combination of our previous research on the structural features and Mg2+ transport of elasmobranch nephron segments, SKCaR localization data presented here, and published reports of CaRs in mammalian kidneys and cells, we can speculate on the performance of the different segments in dogfish kidney as described below.
The SKCaR protein localized to the apical membrane of dogfish kidney proximal tubule segments PIa and PIb may be involved in the modulation of various cellular transport functions, including transepithelial fluxes of divalent cations as well as perhaps modulating proton secretion. This interesting possibility is suggested by studies of CaR function in rat proximal tubule, where it has been suggested that CaRs might regulate proton secretion and other ion fluxes (33). In this regard, elasmobranch nephrons are well known to be capable of robust proton secretion, resulting in a low pH value of 4 that is maintained throughout the renal tubule. Urinary acidification begins as early as the PIa segment in the countercurrent bundle according to intravital microscopy (27), and low pH is thought to be important in preventing stone formation within the elasmobranch nephron, especially the CT/CD system, where high concentrations of Mg2+ salts exist.
PIIa cells likely perform the primary step in Mg2+ excretion, yet they do not contain SKCaR protein. PIIa is an exceptionally long segment in mesial tissue of marine Chondrichthyes and provides a significant mass of specialized large epithelial cells. Without the presence of SKCaR, extraction of Mg2+ from the surrounding peritubular circulation may possibly be regulated by local Mg2+ availability. Alternatively, PIIa cell function could be modulated by cross talk with adjacent SKCaR-containing cells such as PIa or PIIb cells located in neighboring segments. Future experiments will be necessary to distinguish between these possibilities or possible regulation of PIIa Mg2+ secretion by autocoids.
Present studies of mammalian distal tubule have emphasized the importance of CaRs to modulate cellular transport of both divalent (Mg2+ and Ca2+) as well as monovalent (Na+) cations. Hebert (14) has summarized the salient features of the regulation of thick ascending limb function by CaRs. Bapty and co-workers (2) further defined renal Mg2+ handling by the study of cultured immortalized mouse distal convoluted tubule cells that possess an extracellular polyvalent cation-sensing mechanism responsive to Mg2+, Ca2+, and neomycin. In mammals, distal tubules reabsorb >15% of filtered Mg2+ (32). Thus these data suggest that mammalian distal convoluted tubule exhibits a renal cell type that both senses and transports Mg2+.
It is intriguing to note that the diffuse distribution of SKCaR in shark EDT cells (perhaps localized to the basolateral membrane) and apical localization in shark LDT might correspond to the presence of CaR proteins on the mammalian medullary thick ascending limb and distal convoluted tubule (33). By analogy to the function of the thick ascending limb of Henle's loop and distal convoluted tubule in rats, we suggest that EDT and LDT are very likely segments where Mg2+ is reabsorbed in dogfish. Moreover, Riccardi et al. (33) have reported the presence of apical punctuate CaR antibody staining in some type A intercalated cells in rat CD. Interestingly, dogfish LDT cells display cytoplasmic studs and apically located H+-K+-ATPase, which are characteristically found in mammalian type A intercalated cells (23).
In a manner similar to the mammalian inner medullary CD (10, 34), the apical membrane of the dogfish kidney CT/CD system stains intensely with anti-SKCaR antibody. While the role of SKCaR in dogfish CT/CD is uncertain, it may modulate NaCl-coupled water reabsorption, as has been proposed for the flounder urinary bladder (29), and prevent excessively high concentrations of Mg2+ within shark urine, as has been proposed for both flounder bladder (29) and mammalian inner medullary CD (10, 34). The overall hypothesis outlined above can be tested by multiple experimental paradigms. A combination of subcellular immunolocalization efforts together with detailed transport studies of both divalent and monovalent ions including protons will be facilitated by the labeling pattern of SKCaR and together provide a better understanding of divalent cation metabolism in elasmobranch and teleost fish as well as Mg2+ wasting and stone formation in mammals.
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DISCLOSURES |
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H. Hentschel was the recipient of a New Investigator Award of the Mount Desert Island Biological Laboratory, Salsbury Cove, ME.
A portion of the results has been presented at the 31st Annual Meeting of the American Society of Nephrology, Philadelphia, PA, 1998, and was published in abstract form (3, 24).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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