Proliferation of acid-secretory cells in the kidney during adaptive remodelling of the collecting duct

The renal collecting duct adapts to changes in acid-base metabolism by remodelling and altering the relative number of acid or alkali secreting cells, a phenomenon termed plasticity. Acid secretory A intercalated cells (A-IC) express apical H(+)-ATPases and basolateral bicarbonate exchanger AE1 whereas bicarbonate secretory B intercalated cells (B-IC) express basolateral (and apical) H(+)-ATPases and the apical bicarbonate exchanger pendrin. Intercalated cells were thought to be terminally differentiated and unable to proliferate. However, a recent report in mouse kidney suggested that intercalated cells may proliferate and that this process is in part dependent on GDF-15. Here we extend these observations to rat kidney and provide a detailed analysis of regional differences and demonstrate that differentiated A-IC proliferate massively during adaptation to systemic acidosis. We used markers of proliferation (PCNA, Ki67, BrdU incorporation) and cell-specific markers for A-IC (AE1) and B-IC (pendrin). Induction of remodelling in rats with metabolic acidosis (with NH(4)Cl for 12 hrs, 4 and 7 days) or treatment with acetazolamide for 10 days resulted in a larger fraction of AE1 positive cells in the cortical collecting duct. A large number of AE1 expressing A-IC was labelled with proliferative markers in the cortical and outer medullary collecting duct whereas no labeling was found in B-IC. In addition, chronic acidosis also increased the rate of proliferation of principal collecting duct cells. The fact that both NH(4)Cl as well as acetazolamide stimulated proliferation suggests that systemic but not urinary pH triggers this response. Thus, during chronic acidosis proliferation of AE1 containing acid-secretory cells occurs and may contribute to the remodelling of the collecting duct or replace A-IC due to a shortened life span under these conditions.     

The B1 subunit of the H+ATPase is a PDZ domain-binding protein. Colocalization with NHE-RF in renal B-intercalated cells

The 56-kDa B1 subunit of the vacuolar H(+)ATPase has a C-terminal DTAL amino acid motif typical of PDZ-binding proteins that associate with the PDZ protein, NHE-RF (Na(+)/H(+) exchanger regulatory factor). This B1 isoform is amplified in renal intercalated cells, which play a role in distal urinary acid-base transport. In contrast, proximal tubules express the B2 isoform that lacks the C-terminal PDZ-binding motif. Both the B1 56-kDa subunit and the 31-kDa (E) subunit of the H(+)ATPase are pulled down by glutathione S-transferase NHE-RF bound to GSH-Sepharose beads. These subunits associate in vivo as part of the cytoplasmic V1 portion of the H(+)ATPase, and the E subunit was co-immunoprecipitated from rat kidney cytosol with NHE-RF antibodies. The interaction of H(+)ATPase subunits with NHE-RF was inhibited by a peptide derived from the C terminus of the B1 but not the B2 isoform. NHE-RF colocalized with H(+)ATPase in either the apical or the basolateral region of B-type intercalated cells, whereas NHE-RF staining was undetectable in A-intercalated cells. In proximal tubules, NHE-RF was located in the apical brush border. In contrast, H(+)ATPase was concentrated in a distinct membrane domain at the base of the brush border, from which NHE-RF was absent, consistent with the expression of the truncated B2 subunit isoform in this tubule segment. The colocalization of NHE-RF and H(+)ATPase in B- but not A-intercalated cells suggests a role in generating, maintaining, or modulating the variable H(+)ATPase polarity that characterizes the B-cell phenotype.     

Expression of the 56-kDa B2 subunit isoform of the vacuolar H(+)-ATPase in proton-secreting cells of the kidney and epididymis

B1 and B2 are two highly homologous isoforms of the vacuolar H⁺-ATPase (V-ATPase) 56-kDa B subunit. We investigated whether the B2 subunit is expressed alongside B1 in proton-secreting cells of the rodent kidney collecting duct (intercalated cells, IC) and epididymis (clear cells) by using antibodies against distinct COOH-terminal peptides from the two B isoforms. B2 was detected not only in the kidney proximal tubule, thick ascending limb, distal convoluted tubule, and connecting segment but also in A- and B-type IC of collecting ducts (CD) in both rat and mouse. B2 had a predominant cytoplasmic localization in most IC but was clearly located in a tighter apical band together with the V-ATPase 31-kDa E subunit in some A-IC, especially in the medulla. Apical membrane staining was confirmed by immunogold electron microscopy. B2 was very weakly expressed on the basolateral membranes of B-IC in control kidney CD, but some connecting segment B-IC had more distinct basolateral staining. In response to chronic carbonic anhydrase inhibition by acetazolamide, many A-IC showed a strong apical membrane localization of B2, where it colocalized with E and B1. In rat and mouse epididymis, B2 isoform expression was detected in clear cells, where it was concentrated in subapical vesicles. Unlike B1, B2 did not colocalize with the E subunit in the apical microvilli. These findings indicate that in addition to its role in the acidification of intracellular organelles, the B2 isoform could also contribute to transepithelial proton secretion and the maintenance of acid-base homeostasis. vacuolar H⁺-ATPase B subunit; intercalated cells; clear cells; urogenital tract; immunofluorescence     

Defective kidney anion-exchanger 1 (AE1, Band 3) trafficking in dominant distal renal tubular acidosis (dRTA)

dRTA (distal renal tubular acidosis) results from the failure of the a-intercalated cells in the distal tubule of the nephron to acidify the urine. A truncated form of AE1 (anion-exchanger 1; Band 3), kAE1 (kidney isoform of AE1), is located in the basolateral membrane of the intercalated cell. Mutations in the AE1 gene cause autosomal dominant and recessive forms of dRTA. All the dominant dRTA mutations investigated cause aberrant trafficking of kAE1, resulting in its intracellular retention or mistargeting to the apical plasma membrane. Therefore the intracellular retention of hetero-oligomers containing wild-type and dRTA mutants, or the mistargeted protein in the apical membrane neutralizing acid secretion, explains dominant dRTA. The kAE1 (Arg(901)-->stop) mutant has been studied in more detail, since the mistargeting kAE1 (Arg(901)-->stop) from the basolateral to the apical membrane is consistent with the removal of a basolateral localization signal. The C-terminal amino acids deleted by the Arg(901)-->stop mutation, contain a tyrosine motif and a type II PDZ interaction domain. The tyrosine residue (Tyr(904)), but not the PDZ domain, is critical for basolateral localization. In the absence of the N-terminus of kAE1, the C-terminus was not sufficient to localize kAE1 to the basolateral membrane. This suggests that a determinant within the kAE1 N-terminus co-operates with the C-terminus for kAE1 basolateral localization. Interestingly, Tyr(359), in the N-terminal domain, and Tyr(904) in the C-terminus of AE1 are phosphorylated in red blood cells. A potential scheme is suggested where successive phosphorylation of these residues is necessary for correct localization and recycling of kAE1 to the basolateral membrane.     

AQP5 is expressed in type-B intercalated cells in the collecting duct system of the rat, mouse and human kidney

We screened human kidney-derived multipotent CD133+/CD24+ ARPCs for the possible expression of all 13 aquaporin isoforms cloned in humans. Interestingly, we found that ARPCs expressed both AQP5 mRNA and mature protein. This novel finding prompted us to investigate the presence of AQP5 in situ in kidney. We report here the novel finding that AQP5 is expressed in human, rat and mouse kidney at the apical membrane of type-B intercalated cells. AQP5 is expressed in the renal cortex and completely absent from the medulla. Immunocytochemical analysis using segment- and cell type-specific markers unambiguously indicated that AQP5 is expressed throughout the collecting system at the apical membrane of type-B intercalated cells, where it co-localizes with pendrin. No basolateral AQPs were detected in type-B intercalated cells, suggesting that AQP5 is unlikely to be involved in the net trans-epithelial water reabsorption occurring in the distal tubule. An intriguing hypothesis is that AQP5 may serve an osmosensor for the composition of the fluid coming from the thick ascending limb. Future studies will unravel the physiological role of AQP5 in the kidney.     

Selective Assembly of V-ATPase Subunit Isoforms in Mouse Kidney

The kidney plays vital roles in acid–base homeostasis, and the reabsorption of water, ions, and proteins. These processes are achieved through acidification of urine and endosomes of proximal tubule epithelial cells. Multisubunit vacuolar-type proton ATPase (V-ATPase) is one of the major acidification-machinery proteins that localizes to the apical or basolateral plasma membranes of intercalated cells in collecting ducts and the endosomal region at the base of brush border microvilli in proximal tubules. Multiple subunit isoforms of V-ATPase, which are expressed in kidney, have been identified. One obvious question is whether the pumps at different locations in the kidney have their own unique subunit identities. We have used a combination of methods to study this enzyme in kidney including immunocytochemical staining and immunoprecipitation analyses. The subunit isoforms of V-ATPase exhibited selective association/assembly in kidney: kidney-specific isoforms predominantly formed the intercalated cell proton pump, whereas the pump located in the brush border comprised ubiquitously expressed counterparts.     

Immunofluorescent localization of the Rab-GAP protein TBC1D4 (AS160) in mouse kidney

TBC1D4 (or AS160) was identified as a Rab-GTPase activating protein (Rab-GAP) that controls insulin-dependent trafficking of the glucose transporter GLUT4 in skeletal muscle cells and in adipocytes. Recent in vitro cell culture studies suggest that TBC1D4 may also regulate the intracellular trafficking of kidney proteins such as the vasopressin-dependent water channel AQP2, the aldosterone-regulated epithelial sodium channel ENaC, and the Na(+)-K(+)-ATPase. To study the possible role of TBC1D4 in the kidney in vivo, we raised a rabbit polyclonal antibody against TBC1D4 to be used for immunoblotting and immunohistochemical studies. In immunoblots on mouse kidney homogenates, the antibody recognizes specific bands at the expected size of 160 kDa and at lower molecular weights, which are absent in kidneys of TBC1D4 deficient mice. Using a variety of nephron-segment-specific marker proteins, immunohistochemistry reveals TBC1D4 in the cytoplasm of the parietal epithelial cells of Bowman's capsule, the thin and thick limbs of Henle's loop, the distal convoluted tubule, the connecting tubule, and the collecting duct. In the latter, both principal as well as intercalated cells are TBC1D4-positive. Thus, with the exception of the proximal tubule, TBC1D4 is highly expressed along the nephron and the collecting duct, where it may interfere with the intracellular trafficking of many renal transport proteins including AQP2, ENaC and Na(+)-K(+)-ATPase. Hence, TBC1D4 may play an important role for the control of renal ion and water handling and hence for the control of extracellular fluid homeostasis.     

Angiotensin II stimulates H?-ATPase activity in intercalated cells from isolated mouse connecting tubules and cortical collecting ducts

Intercalated cells in the collecting duct system express V-type H(+)-ATPases which participate in acid extrusion, bicarbonate secretion, and chloride absorption depending on the specific subtype. The activity of H(+)-ATPases is regulated by acid-base status and several hormones, including angiotensin II and aldosterone. Angiotensin II stimulates chloride absorption mediated by pendrin in type B intercalated cells and this process is energized by the activity of H(+)-ATPases. Moreover, angiotensin II stimulates bicarbonate secretion by the connecting tubule (CNT) and early cortical collecting duct (CCD). In the present study we examined the effect of angiotensin II (10 nM) on H(+)-ATPase activity and localization in isolated mouse connecting tubules and cortical collecting ducts. Angiotensin II stimulated Na(+)-independent intracellular pH recovery about 2-3 fold, and this was abolished by the specific H(+)-ATPase inhibitor concanamycin. The effect of angiotensin II was mediated through type 1 angiotensin II receptors (AT(1)-receptors) because it could be blocked by saralasin. Stimulation of H(+)-ATPase activity required an intact microtubular network--it was completely inhibited by colchicine. Immunocytochemistry of isolated CNT/CCDs incubated in vitro with angiotensin II suggests enhanced membrane associated staining of H(+)-ATPases in pendrin expressing intercalated cells. In summary, angiotensin II stimulates H(+)-ATPases in CNT/CCD intercalated cells, and may contribute to the regulation of chloride absorption and bicarbonate secretion in this nephron segment.     

The anion exchanger pendrin (SLC26A4) and renal acid-base homeostasis

The anion exchanger pendrin (Pds, SLC26A4) transports various anions including bicarbonate, chloride and iodide. In the kidney, pendrin is exclusively expressed on the luminal pole of bicarbonate-secretory type B intercalated cells. Genetic ablation of pendrin in mice abolishes luminal chloride-bicarbonate exchanger activity from type B intercalated cells suggesting that pendrin is the apical bicarbonate extruding pathway. The renal expression of pendrin is developmentally adapted and pendrin positive cells originate from both the uretric bud and mesenchyme. In adult kidney, pendrin expression and activity is regulated by systemic acid-base status, dietary electrolyte intake (mostly chloride), and hormones such as angiotensin II and aldosterone which can affect subcellular localization, the relative number of pendrin expressing cells, and the overall abundance consistent with a role of pendrin in maintaining normal acid-base homeostasis. This review summarizes recent findings on the role and regulation of pendrin in the context of the kidneys role in acid-base homeostasis in health and disease.     

The association between familial distal renal tubular acidosis and mutations in the red cell anion exchanger (band 3, AE1) gene.

In distal renal tubular acidosis (dRTA) the tubular secretion of hydrogen ion in the distal nephron is impaired, leading to the development of metabolic acidosis, frequently accompanied by hypokalemia, nephrocalcinosis, and metabolic bone disease. The condition can be familial, when it is usually inherited as an autosomal dominant, though there is a rarer autosomal recessive form associated with nerve deafness. It has been shown that the autosomal dominant form of dRTA is associated with a defect in the anion exchanger (AE1) of the renal collecting duct intercalated cell. This transporter is a product of the same gene (AE1) as the erythrocyte anion exchanger, band 3. In this review we will look at the evidence for this association. Studies of genomic DNA from families with this disorder have shown, both by genetic linkage studies and by DNA sequencing, that affected individuals are heterozygous for mutations in the AE1 gene whilst unaffected family members have a normal band 3 sequence. Mutations have been found in the region of proposed helices 6 and 7 of the membrane domain of band 3 and involve amino acids Arg-589 and Ser-613, and in the COOH-terminal domain of band 3. Studies of red cell band 3 from these families have provided information on the effect these mutations have on the structure and function of erythrocyte band 3. Expression studies of the erythroid and kidney isoforms of the mutant AE1 proteins, in Xenopus laevis oocytes, have shown that they retained chloride transport activity, suggesting that the disease in the dRTA families is not related simply to the anion transport activity of the mutated proteins. A possible explanation for the dominant effect of these mutant AE1 proteins in the kidney cell is that these mutations affect the targeting of AE1 from the basolateral to the apical membrane of the alpha-intercalated cell.     

Decreased expression of Slc26a4 (Pendrin) and Slc26a7 in the kidneys of carbonic anhydrase II-deficient mice

BACKGROUND/AIMS: Intercalated cells (ICs) of the kidney collecting duct are rich in carbonic anhydrase II (CAII), which facilitates proton and bicarbonate transport. Bicarbonate secretion is mediated via Pendrin (Slc26a4), which is expressed on the apical membrane of B-ICs and nonA-nonB ICs in the cortical collecting ducts (CCD). Bicarbonate absorption is mediated via anion exchanger 1 (AE1-Slc4a1) in the CCD and via AE1 and possibly Slc26a7 in the OMCD. Both exchangers are expressed on the basolateral membrane of A-ICs. The aim of this study was to examine the expression of pendrin, Slc26a7, and AE1 in the kidneys of CAII-deficient (CAR2-null) mice. METHODS: For the expression studies, we used real-time RT-PCR, Northern hybridization, immunolabeling, and immunoblotting. RESULTS: Pendrin mRNA expression was reduced 63% along with decreased pendrin immunolabeling in the cortex of CAR2-null mice present predominantly in nonA-nonB ICs. Slc26a7 mRNA expression was decreases by 73% and Slc26a7 immunolabeling, present in A-ICs, severely reduced in the outer medulla of CAR2-null mice. AE1 mRNA expression was decreased to a similar degree (62%) along with reduced AE1 immunolabeling. The expression of aquaporin 2 (AQP2) water channel, exclusively present in principal cells of the collecting duct, was comparable in the wild type and CAR2-null mice. CONCLUSION: CAII deficiency results in a significant decrease in the gene and protein expression of bicarbonate transport proteins from Slc26 gene family - Slc26a4 (pendrin) and Slc26a7. These results emphasize the critical role of CAII for the maintenance of the intercalated cell phenotype.     

Unraveling trafficking of the kidney anion exchanger 1 in polarized MDCK epithelial cells.

Kidney anion exchanger 1 (kAE1) is a membrane glycoprotein expressed at the basolateral membrane of type A intercalated cells in the kidney collecting tubule. Mutations occurring in the gene encoding this protein can give rise to distal renal tubular acidosis (dRTA), a disease characterized by an impaired urine acidification, nephrocalcinosis, and renal failure. Here we review how the study of dRTA mutants in polarized epithelial cells has shed light on the cellular mechanisms resulting in this renal disease.     

Knockdown of V-ATPase subunit A (atp6v1a) impairs acid secretion and ion balance in zebrafish (Danio rerio)

In the skin of zebrafish embryo, the vacuolar H(+)-ATPase (V-ATPase, H(+) pump) distributed mainly in the apical membrane of H(+)-pump-rich cells, which pump internal acid out of the embryo and function similarly to acid-secreting intercalated cells in mammalian kidney. In addition to acid excretion, the electrogenic H(+) efflux via the H(+)-ATPases in the gill apical membrane of freshwater fish was proposed to act as a driving force for Na(+) entry through the apical Na(+) channels. However, convincing molecular physiological evidence in vivo for this model is still lacking. In this study, we used morpholino-modified antisense oligonucleotides to knockdown the gene product of H(+)-ATPase subunit A (atp6v1a) and examined the phenotype of the mutants. The H(+)-ATPase knockdown embryos revealed several abnormalities, including suppression of acid-secretion from skin, growth retardation, trunk deformation, and loss of internal Ca(2+) and Na(+). This finding reveals the critical role of H(+)-ATPase in embryonic acid -secretion and ion balance, as well.     

Localization of carbonic anhydrase XII to the basolateral membrane of H+-secreting cells of mouse and rat kidney.

Membrane-associated carbonic anhydrase (CA) has a crucial role in renal HCO(3)(-) absorption. CA activity has been localized to both luminal and basolateral membranes of the tubule epithelial cells. CA XII is a transmembrane isoenzyme that has been demonstrated in the basolateral plasma membrane of human renal, intestinal, and reproductive epithelia. The present study was designed to demonstrate the distribution of CA XII expression in the rodent kidney. A new polyclonal antibody to recombinant mouse CA XII was used in both Western blotting and immunohistochemistry. Western blotting analysis revealed a 40-45-kD polypeptide in CA XII-expressing CHO cells and isolated membranes of mouse and rat kidney. Immunofluorescence staining localized CA XII in the basolateral plasma membranes of S1 and S2 proximal tubule segments. Abundant basolateral staining of CA XII was seen in a subpopulation of cells in both cortical and medullary collecting ducts. Double immunofluorescence staining identified these cells as H(+)-secreting type A intercalated cells. The localization of CA XII in the peritubular space of proximal tubules suggests that it may play a role in renal HCO(3)(-) absorption, whereas the function of CA XII in the type A intercalated cells needs further investigation.     

Pendrin as a novel target for diuretic therapy

The Cl(-)/HCO(3)(-) exchanger pendrin (SLC26A4, PDS) and the thiazide-sensitive NaCl cotransporter NCC (SLC12A3) are expressed on the apical membranes of distal nephron segments and mediate salt absorption, with pendrin working in tandem with the epithelial Na channel (ENaC) and NCC working by itself. Pendrin is expressed on the apical membrane of intercalated cells in late distal convoluted tubule (DCT), connecting tubule (CNT) and the cortical collecting duct (CCD) whereas the thiazide-sensitive NaCl cotransporter NCC is primarily detected on the apical membrane of DCT cells. Recent studies indicate that pendrin expression is increased in kidneys of NCC knockout mice, raising the possibility that pendrin and NCC can compensate for loss of the other by increasing their expression and activity. Current investigations in our laboratories demonstrate that pendrin plays an important role in compensatory salt absorption in response to the loop diuretics and the thiazide derivatives. These studies further demonstrate that whereas single deletion of pendrin or NCC does not cause salt wasting in mutant mice under baseline conditions, double knockout of pendrin and NCC causes profound polyuria and polydipsia, along with salt wasting under basal conditions. As a result, animals develop significant dehydration. We propose that pharmacologic inhibition of pendrin and NCC can provide a novel and strong diuretic regimen for patients with fluid overload, including those with congestive heart failure, nephrotic syndrome or renal failure.     

Nephron structure and immunohistochemical localization of ion pumps and aquaporins in the kidney of frogs inhabiting different environments

Amphibians inhabit areas ranging from completely aqueous to terrestrial environments and move between water and land. The kidneys of all anurans are similar at the gross morphological level: the structure of their nephrons is related to habitat. According to the observation by light and electron microscopy, the cells that make up the nephron differ among species. Immunohistochemical studies using antibodies to various ATPases showed a significant species difference depending on habitat. The immunoreactivity for Na+,K(+)-ATPase was low in the proximal tubules but high in the basolateral membranes of early distal tubules to collecting ducts in all species. In the proximal tubule, apical membranes of the cells were slightly immunoreactive to H(+)-ATPase antibody in aquatic species. In the connecting tubule and the collecting duct, the apical membrane of intercalated cells was immunoreactive in all species. In aquatic species, H+,K(+)-ATPase immunoreactivity was observed in cell along the proximal, distal tubule to the collecting duct. However, H+,K(+)-ATPase was present along the intercalated cells of the distal segments from early distal to collecting tubules in terrestrial and semi-aquatic species. In the renal corpuscle, the neck segment and the intermediate segment, immunoreactivities to ion pumps were not observed in any of the species examined. Taking together our observations, we conclude that in the aquatic species, a large volume of plasma must be filtered in a large glomerulus and the ultrafiltrate components are reabsorbed along a large and long proximal segment of the nephron. Control of tubular transport may be poorly developed when a small short distal segment of the nephron is observed. On the contrary, terrestrial species have a long and well-developed distal segment and regulation mechanisms of tubular transport may have evolved in these segments. Thus, the development of the late distal segments of the nephron is one of the important factors for the terrestrial adaptation.     

Differential renal distribution of NHERF isoforms and their colocalization with NHE3, ezrin, and ROMK

Na(+)/H(+) exchanger regulatory factor (NHERF) and NHERF2 are PDZ motif proteins that mediate the inhibitory effect of cAMP on Na(+)/H(+) exchanger 3 (NHE3) by facilitating the formation of a multiprotein signaling complex. With the use of antibodies specific for NHERF and NHERF2, immunocytochemical analysis of rat kidney was undertaken to determine the nephron distribution of both proteins and their colocalization with other transporters and with ezrin. NHERF was most abundant in apical membrane of proximal tubule cells, where it colocalized with ezrin and NHE3. NHERF2 was detected in the glomerulus and in other renal vascular structures. In addition, NHERF2 was strongly expressed in collecting duct principal cells, where it colocalized with ROMK. These results indicate a striking difference in the nephron distribution of NHERF and NHERF2 and suggests NHERF is most likely to be the relevant biological regulator of NHE3 in the proximal tubule, while NHERF2 may interact with ROMK or other targets in the collecting duct. The finding that NHERF isoforms occur in different cell types suggests that NHERF and NHERF2 may subserve different functions in the kidney.     

Pendrin immunoreactivity in the gill epithelium of a euryhaline elasmobranch

Pendrin is an anion exchanger in the cortical collecting duct of the mammalian nephron that appears to mediate apical Cl(-)/HCO3(-) exchange in bicarbonate-secreting intercalated cells. The goals of this study were to determine 1) if pendrin immunoreactivity was present in the gills of a euryhaline elasmobranch (Atlantic stingray, Dasyatis sabina), and 2) if branchial pendrin immunoreactivity was influenced by environmental salinity. Immunoblots detected pendrin immunoreactivity in Atlantic stingray gills; pendrin immunoreactivity was greatest in freshwater stingrays compared with freshwater stingrays acclimated to seawater (seawater acclimated) and marine stingrays. Using immunohistochemistry, pendrin-positive cells were detected on both gill lamellae and interlamellar regions of freshwater stingrays but were more restricted to interlamellar regions in seawater-acclimated and marine stingray gills. Pendrin immunolabeling in freshwater stingray gills was more apical, discrete, and intense compared with seawater-acclimated and marine stingrays. Regardless of salinity, pendrin immunoreactivity occurred on the apical region of cells rich with basolateral vacuolar-proton-ATPase, and not in Na(+)-K(+)-ATPase-rich cells. We suggest that a pendrin-like transporter may contribute to apical Cl(-)/HCO3(-) exchange in gills of Atlantic stingrays from both freshwater and marine environments.