Cloning, function, and localization of human, canine, and Drosophila ZIP10 (SLC39A10), a Zn²⁺ transporter

Abstract

Zinc (Zn²⁺) is the second most abundant trace element, but is considered a micronutrient, as it is a cofactor for many enzymes and transcription factors. Whereas Zn²⁺ deficiency can cause cognitive immune or metabolic dysfunction and infertility, excess Zn²⁺ is nephrotoxic. As for other ions and solutes, Zn²⁺ is moved into and out of cells by specific membrane transporters: ZnT, Zip, and NRAMP/DMT proteins. ZIP10 is reported to be localized at the apical membrane of renal proximal tubules in rats, where it is believed to play a role in Zn²⁺ import. Renal regulation of Zn²⁺ is of particular interest in light of growing evidence that Zn²⁺ may play a role in kidney stone formation. The objective of this study was to show that ZIP10 homologs transport Zn²⁺, as well as ZIP10, kidney localization across species. We cloned ZIP10 from dog, human, and Drosophila (CG10006), tested clones for Zn²⁺ uptake in Xenopus oocytes and localized the protein in renal structures. CG10006, rather than foi (fear-of-intimacy, CG6817) is the primary ZIP10 homolog found in Drosophila Malpighian tubules. The ZIP10 antibody recognizes recombinant dog, human, and Drosophila ZIP10 proteins. Immunohistochemistry reveals that ZIP10 in higher mammals is found not only in the proximal tubule, but also in the collecting duct system. These ZIP10 proteins show Zn²⁺ transport. Together, these studies reveal ZIP10 kidney localization, a role in renal Zn²⁺ transport, and indicates that CG10006 is a Drosophila homolog of ZIP10.

INTRODUCTION

Zn²⁺ is the second most biologically abundant trace element, following iron, is redox neutral, and is an essential nutrient for nearly all organisms. The physiological importance of Zn²⁺ homeostasis is illustrated by its wide range of functions in the immune, endocrine, reproductive, skeletal, and neuronal systems and also due to the deleterious consequences of inherited diseases and severe zinc deficiencies (16, 19). Nonetheless, whereas Zn²⁺ has low toxicity (mM range), excess Zn²⁺ can be deleterious, e.g., causing inadequate copper absorption secondarily associated with sideroblastic anemia (2). Approximately 90% of zinc is stored in skeletal muscle and bone, with 5% in the liver and integument, and the remaining 2–3% in other tissues (16). Intestinal Zn²⁺ absorption is strictly regulated, increasing when dietary Zn²⁺ is limited, and decreasing via gastrointestinal secretion and renal excretion when in excess. As with all solutes requiring homeostasis, physiological Zn²⁺ levels are tightly controlled by specific, membrane-localized, Zn²⁺ import and export proteins.

Eukaryotic Zn²⁺ transporters are classified into two major families (19): Slc30 [zinc transporter (ZnT)] family (12) and Slc39 [Zrt-, Irt-like protein (Zip)] family (13). Many studies indicate that the ZnT transporter family acts to decrease intracellular Zn²⁺ levels by transporting Zn²⁺ from the cytosol to the lumen of endosomes, vesicles, or secretory granules, then subsequently to the extracellular space, i.e., ZnT proteins are viewed as Zn²⁺ export transporters. By contrast, the Zip transporter family proteins are thought to increase intracellular, cytosolic Zn²⁺ levels by transporting Zn²⁺ either from the extracellular space or organellar lumen into the cytosol, i.e., Zip proteins are viewed as import transporters. In human and mammalian genomes, 14 Zip transporter family members and 10 ZnT family members have been identified (20). Zn²⁺ can also be transported by macrophages and epithelia using the H⁺-coupled divalent metal transporter DMT1 [natural resistance-associated macrophage protein 2 (NRAMP2)] (22).

ZIP10 is regulated by external Zn²⁺ depletion or replenishment (15), cytokine signaling via the JAK/STAT pathway (23), and the metal-regulatory transcription factor-1 (20). ZIP10 cloning and some functionality were reported initially in rat (15) and then mouse (5, 29). In these initial studies, investigators reasoned that rat ZIP10 can import Zn²⁺ into proximal tubule cells based on Zn²⁺ uptake by LLC-PK1 transfected with rat ZIP10. Pawan and coworkers (26) showed that ZIP10-mediated Zn²⁺ uptake in rat renal and intestinal cells is regulated by thyroid hormones controlling overall cellular Zn²⁺ homeostasis. ZIP10 upregulation augments intracellular Zn²⁺ concentrations, a required cofactor for enzymes and transcription factors related to cell proliferation, and could serve as a reparative response mechanism to kidney injury.

Additionally, Pal and colleagues (25) reported a significant increase in ZIP10 expression in a highly aggressive renal cell carcinoma, revealing ZIP10 quantification as an indicator of tumor aggressiveness. Our laboratory’s own preliminary work found in canine genomewide association study that ZIP10 may be associated with calcium oxalate nephrolithiasis (8, 31). To better understand where and how ZIP10 might be associated with normal renal physiology and renal disease states, we sought to further elucidate the localization and functional details of ZIP10 in the kidney.

Transport proteins involved in Zn²⁺ movement within the renal tubular system, with the exception of rat, have not been well localized or characterized. In this study, we report the cloning, function, and renal localization of SLC39A10 (ZIP10) from three species: human, dog, and fly. ⁶³Zn²⁺ uptake studies indicate that fly, dog, and human clones transport Zn²⁺, making them functional homologs of mouse and rat ZIP10. Currently, both dogs and flies are used as translatable models of calcium oxalate nephrolithiasis. The present results will assist in explaining the role of Zn²⁺ and Zn²⁺ transport in the kidney.

MATERIALS AND METHODS

Animals and tissues.

Flies (Drosophila melanogaster) were kept on standard medium or dietary salt substitution in vials at 22°C, 12:12-h photoperiod, and 40% relative humidity. Wild-type (Oregon R) and flies expressing the ZIP10 (CG10006) RNAi were used for cloning and RNAi-mediated experiments. Human cortical/medullary tissue was collected from non-neoplastic kidney tissue of patients distal to tumor by at least 2 cm. The Institutional Review Board for Human Research (Mayo Clinic College of Medicine, Rochester, MN) approved these studies. Dog cortex/medullary kidney tissues were collected from control animals used in collaborative studies being performed within the Division of Cardiovascular Research. Addendums to animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) (Mayo Clinic College of Medicine, Rochester, MN). Xenopus care and oocyte harvest were also IACUC approved. Both protocols are in accordance with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals.”

Human, dog, and fly Slc39a10 (ZIP10) cloning.

Full-length cDNAs of human and dog kidney Slc39a10, as well as whole fly body, were amplified and inserted into the pGEMHE Xenopus laevis expression vector (21). All clones were obtained via PCR primer design to the predicted starts and stops from the genomic DNA of the respective organisms. The resulting plasmids were linearized with NotI restriction enzyme and transcribed into cRNA in vitro using T7 RNA polymerase and mMESSAGE mMACHINE kits (Ambion, Austin, TX).

Oocyte isolation and injection.

Xenopus laevis defolliculated oocytes were prepared as described previously (28) and injected with 50 nl of water (control) or human, dog, or fly cRNA at a concentration of 0.5 µg/µl (12.5 ng/oocyte) using a Nanoject-II injector (Drummond Scientific, Broomall, PA). Uptake and electrophysiology experiments were performed 2–4 days after injection.

⁶³Zn uptake studies.

⁶³Zn citrate in >99% radiochemical and radioisotopic purities was produced using a low-energy cyclotron, as previously described (6). The carrier medium for ⁶³Zn citrate was 2 ml sterile 4% sodium citrate. The ⁶³Zn citrate (~10.5 mCi) solution was diluted to 10 ml with 300 µl of stock solution defined below before addition to oocytes. Stock solutions were ND90 or ND96 (90 or 96 mM NaCl, respectively, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, pH 7.5 or 8.5) with isoosmotic ion replacements (choline chloride for 0 Na, gluconate for 0 Cl, bicarbonate for ${\text{HCO}}_3^{-}$ ND90, and 90 mM KCl for K⁺ ND90). Oocytes were preincubated with ND90, pH 7.5, for 20–30 min, followed by 30-min uptake in the above solutions containing ⁶³Zn-zinc citrate. The cells were then washed in ice-cold ND90, pH 7.5, containing 1 mM nonradioactive ZnCl₂ to remove any nonspecific binding. ⁶³Zn uptake in each oocyte was determined by measurement of ⁶³Zn radioactivity using a gamma-counter, corrected for isotopic decay, and expressed as counts/min (CPM). Zinc uptake (nanomoles per hour per oocyte) was calculated as ⁶³Zn uptake (CPM)/⁶³Zn administered (CPM). Uptake experiments were performed in duplicate with 10 oocytes in each experimental group for a total of 20 oocytes/group. Uptake data were log-transformed for statistical analysis to compare uptake between species and across solutions within each species. Statistical significance was determined by ANOVA, followed by Tukey’s test for pairwise comparisons implemented with R software for statistical computing (http://www.R-project.org/), and an adjusted P value < 0.05 was considered significant.

Electrophysiology and two-electrode voltage clamp.

Electrophysiology protocols were performed as previously reported (30).

ZIP10 immunolocalization in oocytes.

Oocytes were injected with either ZIP10 cRNA from all three species or water controls, as described above. Immunolocalization was done as described previously (3). Sections were incubated with rabbit polyclonal IgG anti-ZIP10 (no. 6099; primary; ProSci, Poway, CA) at 4°C overnight and allowed to incubate at room temperature (RT) with goat-anti-rabbit-AF568 (secondary). Cell nuclei were then stained with DAPI, and ZIP10 surface staining was visualized via fluorescent microscopy with AF568 (red) and DAPI (blue) filters.

Cell type-specific knockdown of fly ZIP10.

This was performed as described previously (11, 18). To specifically knock down fly ZIP10, we used the CapaR-GAL4 driver (32), whereby the promoter of the tubule principal cell-specific gene neuropeptide Capa receptor drives GAL4 expression and crossed it to a CG10006 fly line (101031: Vienna Drosophila Resource Center), possessing a transposable element directed against fly ZIP10.

Dog, fly, human, and mouse Slc39a10 (ZIP10) renal immunofluorescence.

Malpighian tubules (MT) from female wild-type (Oregon R), as well as RNAi-mediated ZIP10 knockdown flies were dissected and transferred immediately to poly-l-lysine-coated slides. Tubules were fixed in 4% paraformaldehyde/0.1% phosphate buffer for 1 h. Tubules were then incubated with anti-ZIP10 used above at 4°C overnight. Tubules were incubated for 3 h at RT with goat-anti-rabbit-AF568. Tubules were stained with DAPI and visualized via fluorescent microscopy. The same ZIP10 antibody was used in mammalian tissues. Human, dog, and mouse cortical tissue samples were trimmed and fixed in 4% paraformaldehyde/PBS for 1 h at 4°C. Tissue was placed in 10% (10 min), 16% (1 h), and 18% (1 h) sucrose/PBS solution. Immediately, tissue was placed in 20% sucrose/PBS overnight. The next day, tissue was flash frozen and embedded in OCT, and cryosections (10 µm) were prepared. For immunofluorescence, slides were allowed to rehydrate in PBS followed by blocking buffer incubation [10% donkey serum/1% BSA/PBST for ZIP10 and aquaporin-2 (AQP-2); 10% BlokHen (Aves Laboratories, Tigard, OR)/1% BSA/PBST for monocarboxylate transporter-1 (MCT-1)]. Slides were incubated overnight (4°C) with their primary antibodies/blocking buffer: ZIP10, MCT-1, or AQP-2 (chicken polyclonal IgY anti-MCT-1, Chemicon, Billerica, MA; goat polyclonal IgG anti-AQP-2, Novus Biologics, Littleton, CO). Slides were allowed to incubate with their secondary antibodies (Jackson ImmunoResearch, West Grove, PA): Cy3 (donkey-anti-rabbit), donkey-anti-chicken-AF647 (MCT-1), or donkey-anti-goat-AF647 (AQP-2) for 1 h at RT and were incubated with DAPI. ZIP10 fluorescence was visualized using Cy3 (red), and Alexa 647 for MCT-1 (green) and AQP-2 (yellow). For LTA (lotus tetragonolobus agglutinin; Vector Laboratories), we used a fluorescein-labeled version and incubated with slides during the secondary antibody application.

Western blotting.

Kidneys from human, dog, and mouse were collected as described above and placed in ice-cold homogenization buffer containing 250 mM sucrose, 20 mM HEPES (pH 7.4 with HCl), 100 mM NaCl, and 2 mM sodium EDTA, and were homogenized using a PowerGen 125 (Fisher Scientific). The homogenate was centrifuged [15 min at 1,150 g, 5424 R centrifuge (Eppendorf)] at 4°C. The pellet (P1) containing debris and nuclei was discarded, and the previous step was repeated a second time. The resultant supernatant (S1) was centrifuged [30 min at 20,000 g, 5424 R centrifuge (Eppendorf)] at 4°C, and the supernatant (S2) was discarded. The resulting microsomal pellet (P2) containing plasma and organellar membranes was resuspended in homogenization buffer, assayed for protein content (Bradford assay), and stored at −20°C. Protein (0.6 µg) was loaded into each well, and Western blotting was performed using a WES Simple Western automated immunoblot system (ProteinSample, San Jose, CA), according to the manufacturer’s instructions. The same rabbit polyclonal IgG anti-ZIP10 antibody (as above) utilized in the immunolocalization studies mentioned above was utilized to perform Western blots.

RESULTS

Protein comparison of human (h), dog (d), mouse (m), and Drosophila (fly, CG10006) Slc39a10(ZIP10).

To highlight SLC39A10 (i.e., ZIP10) as a gene of interest in kidney stone disease (8, 22, 31), we cloned the ZIP10-cDNA for dog (KY094513) and human ZIP10 (NM_001127257) (Fig. 1A). Two genes, foi/CG6817 and dZip71B/CG10006, were identified as the closest Drosophila orthologs (34) (http://flybase.org/reports/FBgn0036461.html) (Fig. 1B). Pileup analysis (Fig. 1B) shows that there are multiple blocks of identity between the human, dog, and mouse ZIP10 cDNAs and CG10006. Divergence analysis indicates that CG10006 is ~30% identical to the three mammalian cDNAs (Fig. 1B). However, data available in FlyAtlas (http://flyatlas.org/atlas.cgi) revealed that CG6817 (i.e., fear of intimacy, foi) has low expression in MTs (fly renal structures): larval MT (95 ± 1), and adult MT (95 ± 1). Whereas, CG10006 is expressed almost exclusively in the tubules (Fig. 1C) at high levels: larval MT (3,219 ± 48) and adult MT (902 ± 145). Thus CG10006 was better suited for evaluation of renal Zn²⁺ handling in flies.

Fig. 1.

Slc39a10 sequence analyses. A: sequence pileup of human SLC39A10 (H; NM_001127257), dog slc39a10 (D; KY094513), mouse (M; NP_76624), and Drosophila (CG10006; dZip71B, fly ZIP10). Human, dog, mouse, and Drosophila ZIP10 cDNAs were amplified from kidney (human, dog, and mouse) or whole body (fly) by RT-PCR using gene-specific primers based on 5′ and 3′ expressed sequence tag primers. Black shading indicates identical amino acids in all four (human, dog, mouse, and fly) gene products, whereas gray shading indicates similar functional groups. B: identity and divergence analysis of ZIP10 clones. C: distribution of CG10006 mRNA in larval (left) and adult (right) Drosophila. Data are mined from FlyAtlas.org, an Affymetrix microarray-derived expression atlas of Drosophila (4).

⁶³Zinc transport by human, dog, and fly Slc39a10 (ZIP10).

Xenopus oocytes were injected with SLC39A10 cRNAs (copy RNAs) from each species, using water as a control. Three days after cRNA injection, we performed 30 min ⁶³Zn²⁺ uptake incubations. All three clones showed a significant increase in ⁶³Zn²⁺ uptake compared with water-injected controls (Fig. 2A), with fly ZIP10 (CG10006) showing an eightfold increase, hZIP10 showing a fivefold increase, and dZIP10 showing a sevenfold increase (in nmol·h⁻¹·oocyte⁻¹: 0.47 water, 3.81 fly ZIP10, 2.47 hZIP10, 3.04 dZIP10). Thus fly ZIP10, hZIP10, and dZIP10 all transport Zn²⁺. Interestingly, fly ZIP10 transported significantly more Zn²⁺ than human ZIP10 (Fig. 2A).

Fig. 2.

⁶³Zn²⁺ uptake by ZIP10 clones in Xenopus laevis oocytes expressing recombinant human, dog, or Drosophila ZIP10. A: Xenopus laevis oocytes injected with cRNA coding for either human, dog, or fly (Drosophila) ZIP10 (Slc39a10) and water controls were used for ⁶³Zn²⁺ uptake. The data from the pH 7.5 uptake solution is shown. All species had significant uptake (P < 0.001) compared with water, and no interspecies differences were detected (P > 0.05), as determined by ANOVA with Tukey’s post hoc test. B: the same four groups of oocytes were placed in six different solutions with varying isoosmotic ion replacements, and ⁶³Zn²⁺ uptake was measured in nanomoles per hour per oocyte; n = 10 oocytes per solution done in two replicates with a total n = 120 oocytes per species. Log-scaled data are shown. Xenopus laevis oocytes injected with cRNA coding for either human, dog, or fly (Drosophila) ZIP10 (Slc39a10) were placed in either pH 7.5 ND90/96 (black symbols), pH 8.5 ND90/96, pH 8.5 ${\text{HCO}}_3^{-}$, pH 7.5 0 mM Na⁺, pH 7.5 0 mM Cl⁻, or pH 7.5 KCl (high potassium), and ⁶³Zn²⁺ uptake was measured in nanomoles per hour per oocyte; n = 10 oocytes per solution done in two replicates. Log-scaled data are shown. ⁶³Zn²⁺ uptake did not differ significantly between solutions for any species (P > 0.05), as determined by ANOVA with Tukey’s post hoc test.

Since hZIP2 and ZIP8 were previously reported as a Zn²⁺-${\text{HCO}}_3^{-}$ cotransporter (9), we tested if ZIP10 might be pH or ${\text{HCO}}_3^{-}$ dependent. Starting solution was a pH 7.5 NaCl-ringer (see materials and methods). Adjusting solution pH to 8.5 did not change uptake for any clone. Since we did not want to bubble our ⁶³Zn²⁺ solutions with CO₂ (to maintain pH 7.5), we replaced NaCl and KCl with NaHCO₃ and KHCO₃. This resulted in a solution pH of ~8.5 (so the non-${\text{HCO}}_3^{-}$ solution was 8.5). This pH 8.5-${\text{HCO}}_3^{-}$solution did not significantly alter uptake for any clone (Fig. 2B). Combined, these data indicate that high pH does not affect transport for ZIP10. These data do not support that ZIP10 operates as a Zn²⁺-${\text{HCO}}_3^{-}$ cotransporter.

To further determine the ionic coupling of ZIP10, we performed ion replacements during ⁶³Zn²⁺ uptake. Replacement of Na⁺ with choline or Cl⁻ with gluconate did not change uptake (Fig. 2B). Depolarization (pH 7.5, KCl) also did not alter ⁶³Zn²⁺ uptake.

Intracellular pH and cellular currents.

To more directly determine whether Zn²⁺ would change intracellular pH (pH_i) with and without ${\text{HCO}}_3^{-}$, we measure pH_i in ZIP10 expressing oocytes (Fig. 3, A–C). Figure 3A shows that addition of 1 mM Zn without or with 33 mM ${\text{HCO}}_3^{-}$ does not elicit a pH_i change. Figure 3, B and C, shows the experiments, except that the ZIP10 oocytes are also voltage clamped. When clamping the oocyte, Zn²⁺ also does not elicit a current or change pH_i. Finally, voltage steps with the addition of 1 mM or 5 mM Zn²⁺ does not reveal a voltage-dependent current (Fig. 3D).

Fig. 3.

Electrophysiology characterization of ZIP10 in Xenopus oocytes. Xenopus oocytes were injected with human ZIP10 cRNA. A: nonvoltage clamped experiment in which intracellular pH (pH_i) and membrane potential (V_m) were measured, and 1 mM ZnCl₂ (blue shading) was added in the absence or presence of 5% CO₂:33 mM ${\text{HCO}}_3^{-}$ (pH 7.5; tan shading). B and C: similar experiments in which pH_i is measured while the oocytes is clamped at −20 mV. D: current-voltage curves of ZIP10 oocytes with 0 mM Zn²⁺ (ND96), 1 mM Zn²⁺, and 5 mM Zn²⁺. The red-dotted circle in C indicates an air bubble in the system, which also manifests as a quick current spike. The repeat maneuver shows no pH_i or current change.

Human, dog, mouse, and Drosophila (fly) Slc39a10 (ZIP10) tissue localization.

ZIP10 has only been localized to rodent kidney (15). An anti-ZIP10 antibody was raised against an 18-amino acid synthetic peptide near the center of human ZIP10; however, the exact peptide is not revealed by the manufacturer. Thus, before staining renal tissue from other animals, we determined whether the ZIP10 antibody would recognize recombinant ZIP10 protein expressed in Xenopus oocytes (Fig. 4). Oocytes were injected with water (Fig. 4A, control), dog ZIP10 (Fig. 4B), human ZIP10 (Fig. 4C), and Drosophila CG10006 (Fig. 4D). Dog, human, and Drosophila membrane protein was recognized by the ZIP10 antibody, whereas the water-injected controls showed no ZIP10 staining (Fig. 4A), indicating that the ZIP10 antibody recognizes dog SLC39A10 protein, human SLC39A10 protein, and the Drosophila CG10006 protein. To verify that our aliquots of the commercial ZIP10-antibody recognized the correct-sized protein, we preformed Western analysis (WES) using kidney homogenates of mouse, dog, and human kidney (Fig. 5). These blots show immunoreactivity of a 94-kDa protein, which is the predicted size of ZIP10 in all three species (Fig. 5A). To normalize for protein loading, a ratio against β-actin was done (Fig. 5B).

Fig. 4.

Human, dog, and Drosophila ZIP10 expression in Xenopus oocyte plasma membrane. Xenopus laevis oocytes were injected with cRNA coding for either water (control; A), dog ZIP10 (B), human ZIP10 (C), or dZIP10 (CG10006; D). To determine whether a commercially available ZIP10 antibody would detect the expressed Zip proteins, oocytes were processed using immunohistochemistry 3–5 days after cRNA injection. Fluorescent immunohistochemistry shows recognition of recombinant protein epitopes across species (red: human, dog, and fly), but not water-injected control. DAPI denotes cell interior as counterstain (blue). Magnification is at ×20.

Fig. 5.

ZIP10 (Slc39A10) expression in normal mouse (M), dog (D), and human (H) kidney. A: immunoblot analysis of ZIP10 expressions in kidneys from normal mouse, dog, and human tissue. The apparent molecular mass for mouse, dog, and human ZIP10 (94 kDa) is the same across species and matches the reported weight recognized by the rabbit polyclonal antibody. B: graphical representation of ZIP10 protein levels normalized to β-actin loading controls.

Since ZIP10 has been previously localized in rat kidney and out ZIP10 antibody recognizes the correct protein, we initially localized ZIP10 in the mouse kidney (Fig. 6). Immunofluorescence of mouse kidney sections illustrates that the ZIP10 antibody recognizes protein at the apical membrane of the proximal tubule. MCT-1 (Slc16a1) is specific to the basolateral membrane of the proximal tubule and is colocalized with ZIP10 reactivity (Fig. 6A). AQP-2 counterstaining, collecting duct (CD) marker, revealed no colocalization with Zip10 reactivity in mouse (Fig. 6B). LTA, a proximal tubule glycocalyx marker, colocalized with Zip10 while uromodulin (UMOD), a marker of the thick ascending limb, did not (Fig. 6C). These colocalization studies clearly indicate that Zip10 is found predominantly at the apical membranes of proximal tubules in the mouse kidney. Zip10 colocalization with LTA (proximal tubule-specific marker), but not AQP-2 (collecting duct-specific marker), illustrates that Zip10 is exclusively located in the mouse proximal tubule and not in other regions of the nephron (Fig. 6D).

Fig. 6.

Immunofluorescent detection of mouse ZIP10 (Slc39a10). A: immunofluorescence of mouse kidney section costained with Zip10 (red) and monocarboxylate transporter-1 [MCT-1; green; basolateral membrane of proximal tubules (PT)]. Note there is additional apical Zip10 staining. B: immunofluorescence of a mouse kidney section costained with Zip10 (red) and aquaporin-2 [AQP-2; yellow; apical membrane of collecting duct (CD)]. DAPI denotes PT cell nuclei (blue). C: midcortical section of mouse kidney stained with Zip10 (red), LTA [lotus tetragonolobus agglutinin; green; glycocaylx of PT), and uromodulin (UMOD or Tamm Horsfall; white; thick ascending limb]. D: cortical section of mouse kidney stained with Zip10 (red) and LTA (green; glycocaylx of PT). Bars = 100 µm.

Figure 4D shows that the ZIP10 antibody recognizes the recombinant Drosophila CG10006 (fly ZIP10) protein with ZIP10 reactivity ubiquitously expressed along the MT-luminal border (Fig. 7A). To further test the ZIP10 antibody specificity, we used a MT principal cell-specific ZIP10 knockdown (CapaR-GAL4: UAS-CG10006-RNAi). Figure 7B shows no MT luminal staining in these CG10006-knockdown MTs, further indicating the ZIP10 antibody recognition of the CG10006 protein in Drosophila. Recognition of recombinant ZIP10 from all four species (Fig. 4) indicates evolutionary conservation of this protein across species. These data further indicate CG10006 to be a Drosophila homolog of human and dog ZIP10.

Fig. 7.

Immunofluorescent detection of ZIP10 in the Drosophila Malpighian tubule (MT). A: immunohistochemistry showing specific labeling of ZIP10 (red) in the MT lumen in a wild-type (WT) Oregon R female, anterior MT. B: when CG10006-RNAi is driven by CapaR-Gal4 (MT principal cells), there is no specific labeling with the ZIP10 antibody, which does recognize the Drosophila epitope (Fig. 3D). DAPI denotes principal and stellate cell nuclei (blue). Magnification is at ×20.

Knowing that the ZIP10 antibody recognizes the recombinant proteins, we sought to determine whether dog and human ZIP10 protein localization was similar to that of mouse and fly. Both dog (Fig. 8A) and human (Fig. 9A) kidney displayed ZIP10 proximal tubular apical reactivity confirmed with MCT-1 colocalization. However, dog and human nonproximal tubules appeared immunoreactive (Figs. 8A and 9A, respectively). Unfortunately, LTA does not seem to react with dog kidney glycocalyx (not shown), so we examined Na⁺-K⁺-2Cl⁻ cotransporter 2 (NKCC2) and UMOD localization with ZIP10 in cortex (Fig. 8B) and medulla (Fig. 8C). NKCC2 and UMOD colocalize, but there is little if any localization with ZIP10 in dog. Colocalization staining with AQP-2 indicates cortical CD colocalization in dog (Fig. 8, D–F) with apical ZIP10 expression. In human kidney, ZIP10 localizes with MCT-1 but not NKCC2 (Fig. 9B), LTA labels proximal tubule apical membranes and colocalizes with ZIP10 in human (Fig. 9C), but ZIP10 does not localize with UMOD (Fig. 9C). As with dog kidney, ZIP10 shows obvious colocalization with AQP-2 in human kidney, indicating robust protein expression in CD. Both Figs. 8 and 9 illustrate dog and human ZIP10 extending beyond the proximal nephron, prominently in the cortical CD (see cartoon in Fig. 10).

Fig. 8.

Immunofluorescent detection of ZIP10 (Slc39a10) in normal dog kidney. A: immunofluorescence showing specific labeling of dog ZIP10 (red) on the apical membrane of proximal tubule cells colocalized with monocarboxylate transporter-1 (MCT-1; green; basolateral membrane). B: cortical section of dog kidney costained with Zip10 (red), Na⁺-K⁺-2Cl⁻ cotransporter 2 [NKCC2; green, apical, thick ascending limb (TAL)], and uromodulin (UMOD; white; TAL). C: near-medullary section of dog kidney costained with Zip10, NKCC2, and Tamm Horsfall showing clear TAL segments. D: immunofluorescence colocalizing ZIP10 with aquaporin-2 (AQP-2; yellow) marking the apical membrane of cortical collecting duct (CCD) cells. E and F: ZIP10 and AQP-2 alone, respectively, from D. DAPI denotes cell nuclei (blue). Bar = 100 µm.

Fig. 9.

Immunofluorescent detection of ZIP10 (SLC39A10) in normal, adult human kidney. Immunofluorescent staining of normal human kidney sections is shown. The white bar in each panel is 100 µm. A: costaining of ZIP10 (red), monocarboxylate transporter-1 [MCT-1; green; proximal tubule (PT)], and DAPI. Obviously costained PTs are indicated. B: costaining using ZIP10 (red), MCT-1 (green; PT), and Na⁺-K⁺-2Cl⁻ cotransporter 2 [NKCC2; white; thick ascending limb (TAL)]. C: costaining using ZIP10 (red), lotus tetragonolobus agglutinin (LTA; green; PT), and uromodulin (UMOD; white; TAL). D: as in Fig. 8 (dog kidney) shows colocalization of ZIP10 (red) and AQP-2 (yellow; CD) in some but not all tubules. DAPI denotes cell nuclei (blue). CCD, cortical collecting duct.

Fig. 10.

Nephron cartoon summarizing differences between mouse and dog/human Zip10 staining. Two nephron diagrams show Zip10 reactivity: mouse (left) and dog or human (right). The thick red line indicates tubule areas where ZIP10 protein staining was found. CCD, cortical collecting duct; DT, distal tubule; IMCD, inner medullary collecting duct; TAL, thick ascending limb.

Together, our results indicate that these three mammalian species all express ZIP10, and that it is localized prominently on the apical membrane of the proximal tubule (mouse, Fig. 6A; dog, Fig. 8A; and human, Fig. 9A). Furthermore, dog (Fig. 8D) and human (Fig. 9D) ZIP10 protein is in cortical CDs, indicating that there is ZIP10 expression beyond the proximal tubule in mammals of a higher order than mice and rats, i.e., canines and primates (Fig. 10).

DISCUSSION

Zinc homeostasis is controlled by Zn²⁺ export and import proteins. These distinct transporter groups are encoded by three solute-linked carrier (Slc) gene families: Zip (Slc39; importers) (13); ZnT (Slc30; exporters) (12, 26); and DMT/NRAMP proteins (Slc11 H⁺ coupled DMTs) (22, 24). Zip transporters increase cytosolic Zn²⁺ availability by facilitating extracellular Zn²⁺ uptake, as well as vesicular Zn²⁺ release into the cytosol (13). In contrast, ZnT transporters reduce cytosolic Zn²⁺ availability by facilitating Zn²⁺ efflux into the extracellular environment or intracellular vesicles (12). DMT1/NRAMP2 is a general H⁺-coupled transition metal transporter, localized to apical epithelial membranes, and is involved in divalent metal uptake (Fe²⁺ > Zn²⁺ > Cd²⁺, Ni²⁺, Co²⁺) into cells or intracellular compartments (10, 22).

ZIP10 has been studied in various murine cell types and organ systems, including erythrocytes (29), testicles (5), liver and brain (20), the immune system (B-cell development) (23), and oocytes (17). ZIP10 is suggested to be involved in human breast cancer metastasis and invasiveness (14), as well as renal cell carcinoma aggressiveness (25). However, ZIP10 expression, function, and localization in renal tubular systems are not well understood, and ZIP10 has only been characterized in rat brush border membranes. Functional data from this system suggests rat ZIP10 mRNA expression is regulated by zinc levels, as well as functions to import Zn²⁺ across the rat renal brush border membranes (15). Kaler and Prasad (15) reported that SLC39A10 was abundantly expressed in human kidney, but did not specify precise tissue localization. While human ZIP10 has been used as a cancer marker (7), its function and localization in the human kidney have not been explored. Furthermore, no information has been previously reported for dog ZIP10.

Comparatively, two Drosophila homologs identify with mammalian ZIP10, CG10006 and CG6817 (foi). Figure 1, B and C, illustrate that foi is less divergence from mammalian and other ZIP10 proteins (34) and, based on molecular sequence distances, has been designated as Zip6 or ZIP10 (27, 34). However, foi has relatively low expression in fly renal tubules (MTs), whereas ZIP10 has moderate to high renal expression in mammals (http://www.proteinatlas.org). In contrast, CG10006 is enriched in MTs (Fig. 1C). Since our interest was in renal Zn²⁺ transport, we focused on CG10006.

Step one is to determine transporter protein localization on the tissue level, cell type, and intracellularly. This study used immunohistochemistry to investigate ZIP10 localization in mouse, dog, and human kidney, as well as Drosophila MTs. Figures 4–9 illustrate that ZIP10 from these four species are detected with the ZIP10 antibody. Not surprisingly, mouse ZIP10, like rat ZIP10 (15), is localized predominantly on the apical membrane of the proximal tubule without any detection elsewhere in the kidney (Fig. 6A). While it is not surprising that mammalian ZIP10 is found in the apical membrane of the proximal tubule, staining of dog and human kidney indicates that ZIP10 is also found in other renal cortical regions, particularly the cortical collecting duct (CCD) (Figs. 8B and 9B). Dog and human CCD ZIP10 localization could indicate a final reabsorptive process facilitating Zn²⁺ movement from the CCD lumen into the peritubular capillaries, especially as a mechanism to maintain Zn²⁺ homeostasis in Zn²⁺-deficient states.

Hypothetically, rodents should possess the same mechanism for Zn²⁺ reabsorption in distal nephron segments. Li and coworkers (33) have reported ZIP10 mRNA in mouse DCT cells. However, these cell-line mRNA results are not supported by ZIP10 immunolocalization in mouse kidney. The results here indicate that there are distinct differences in rodents vs. dog and human kidney localization, and presumably physiology. It is attractive to speculate that such a difference may contribute to rodents being very resilient to forming kidney stones. Nevertheless, this speculation may be difficult to test and requires further investigation.

Moreover, CG10006, while only 30% identical to mammalian ZIP10, is immunologically related and found in the apical membrane of adult MTs (Fig. 7A). Although this differs from a basolateral location reported in larvae (35), this could reflect a difference in insect Zn²⁺ requirements, especially since there was significant Zn²⁺ uptake compared with hZIP10, thus suggesting a role for ZIP10 across metamorphosis. Drosophila Zip10 localization in our studies is specific to MT principal cells, as the CapaR-CG10006-RNAi removes immunoreactivity (Fig. 7B). These data indicate that CG10006 is a ZIP10 homolog in Drosophila and suggest evolutionary conservation of renal-localized Zn²⁺ transport proteins between invertebrates and vertebrates.

Our studies directly tested ZIP10 clone Zn²⁺ transport function by expressing these proteins in Xenopus oocytes. We originally tried to assay ZIP10 function using Zn-selective microelectrodes, as we have previously done for pH, Na⁺, Cl⁻, K⁺, and ${\text{NH}}_4^{+}$. As Zn²⁺ is divalent, the maximum, ideal-electrode response is 30 mV/decade Zn²⁺ concentration. Calibration of the Zn²⁺ ionophore revealed that its response was ~20 mV/decade Zn²⁺ concentration, making it very difficult to use for quantification. These experiments did reveal that addition of even 5 mM Zn²⁺ to oocyte bathing solutions did not cause voltage or current changes. Nonetheless, these experiments indicated that ZIP10 proteins are electroneutral. Using two-electrode voltage clamp with human ZIP10 resulted in no current stimulated by addition of Zn²⁺. Moreover, pH_i measurements revealed that, in the presence and absence of ${\text{HCO}}_3^{-}$, Zn²⁺ addition did not change pH_i. These results corroborate previous studies, which have shown that the similar ZIP transporter, ZIP2, also does not transport ${\text{HCO}}_3^{-}$ (36). These data indicate that Zn²⁺-${\text{HCO}}_3^{-}$ cotransport is unlikely to occur through ZIP10.

We directly assessed Zn²⁺ transport using Zn²⁺ isotopic uptake. Since ⁶³Zn²⁺ is a short-lived PET isotope (t_0.5 = 38.5 min), we could perform uptake measurements for short durations and maintain high specific activity. Our experiments clearly show that Zn²⁺ is transported by all the ZIP10 clones (Fig. 2A). Gaither and Eide (9) are the only ones to propose a transport mechanism for any of the mammalian Zip proteins. To elucidate the mechanism of ZIP10 transport, we tested the role of OH⁻ and ${\text{HCO}}_3^{-}$ on Zn²⁺ transport (Figs. 2B and 3). These experiments illustrate that neither elevated extracellular pH nor ${\text{HCO}}_3^{-}$ stimulate Zn²⁺ transport. Moreover, if pH_i is measure, Zn²⁺ does not elicit a pH_i change (Fig. 3, A–C), and voltage clamping indicates that there are no Zn²⁺ evoke currents (Fig. 3, B–D). Therefore, in contrast to Zip2, ZIP10 protein activities are not enhanced. Replacement of Na⁺ and Cl⁻ also did not affect Zn²⁺ uptake (Fig. 2B). Thus these experiments do not provide a discrete model of the mechanism of Zn²⁺ transport, but rather support the general conclusion that all ZIP10 clones transport Zn²⁺ as their substrate, regardless of species.

While it is attractive to speculate that renal ZIP10-mediated Zn²⁺ transport is identical among mammals, ZIP10 protein appears more widespread in dog and human kidney compared with rat and mouse kidney (Fig. 10). Without knowing ionic coupling or solute gradients, it is difficult to predict if proximal tubule and CCD ZIP10-mediated Zn²⁺ movements are in the same uptake or export direction. Perhaps additional distal nephron Zip transporters enable additional control of systemic Zn²⁺. This study does establish that CG10006 is the ZIP10-fly homolog, ZIP10 proteins are Zn²⁺ transporters, and that ZIP10 in human kidney is expressed beyond the proximal tubule. ZIP10’s role in the CCD remains to be elucidated.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-092408, U54-DK100227 (O’Brien Urology Research Center), and R25-DK-101405, and a grant from the Oxalosis and Hyperoxaluria Foundation. G. M. Landry was supported by Grant T32-DK-007013.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

G.M.L., T.H., J.A.T.D., and M.F.R. conceived and designed research; G.M.L., E.F., H.L.H., T.H., A.K., P.W., K.S., C.J.R.G., M.-H.C., M.K.P., H.J., A.B., M.-C.F., N.M., P.C., J.A.T.D., T.R.D., and M.F.R. performed experiments; G.M.L., E.F., M.P.A., and M.F.R. interpreted results of experiments; G.M.L., E.F., and M.F.R. prepared figures; G.M.L., P.C., J.A.T.D., and M.F.R. drafted manuscript; G.M.L. and M.F.R. edited and revised manuscript; G.M.L. and M.F.R. approved final version of manuscript; E.F. and M.F.R. analyzed data.