Boric acid is a vital micronutrient in animals; however, excess amounts are toxic to them. Little is known about whole-body boric acid homeostasis in animals. Seawater (SW) contains 0.4 mM boric acid, and since marine fish drink SW, their urinary system was used here as a model of the boric acid excretion system. We determined that the bladder urine of a euryhaline pufferfish (river pufferfish, Takifugu obscurus) acclimated to fresh water and SW contained 0.020 and 19 mM of boric acid, respectively (a 950-fold difference), indicating the presence of a powerful excretory renal system for boric acid. Slc4a11 is a potential animal homolog of the plant boron transporter BOR1; however, mammalian Slc4a11 mediates H+ (OH-) conductance but does not transport boric acid. We found that renal expression of the pufferfish paralog of Slc4a11, Slc4a11A, was markedly induced after transfer from fresh water to SW, and Slc4a11A was localized to the apical membrane of kidney tubules. When pufferfish Slc4a11A was expressed in Xenopus oocytes, exposure to media containing boric acid and a voltage clamp elicited whole-cell outward currents, a marked increase in pHi, and increased boron content. In addition, the activity of Slc4a11A was independent of extracellular Na+. These results indicate that pufferfish Slc4a11A is an electrogenic boric acid transporter that functions as a B(OH)4- uniporter, B(OH)3-OH- cotransporter, or B(OH)3/H+ exchanger. These observations suggest that Slc4a11A is involved in the kidney tubular secretion of boric acid in SW fish, probably induced by the negative membrane potential and low pH of urine.

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.

Nephrolithiasis is one of the most common urinary tract disorders, with the majority of kidney stones composed of calcium oxalate (CaOx). Given its prevalence (US occurrence 10%), it is still poorly understood, lacking progress in identifying new therapies because of its complex etiology. Drosophila melanogaster (fruitfly) is a recently developed model of CaOx nephrolithiasis. Effects of sulfate and thiosulfate on crystal formation were investigated using the Drosophila model, as well as electrophysiological effects on both Drosophila (Slc26a5/6; dPrestin) and mouse (mSlc26a6) oxalate transporters utilizing the Xenopus laevis oocyte heterologous expression system. Results indicate that both transport thiosulfate with a much higher affinity than sulfate Additionally, both compounds were effective at decreasing CaOx crystallization when added to the diet. However, these results were not observed when compounds were applied to Malpighian tubules ex vivo. Neither compound affected CaOx crystallization in dPrestin knockdown animals, indicating a role for principal cell-specific dPrestin in luminal oxalate transport. Furthermore, thiosulfate has a higher affinity for dPrestin and mSlc26a6 compared with oxalate These data indicate that thiosulfate's ability to act as a competitive inhibitor of oxalate via dPrestin, can explain the decrease in CaOx crystallization seen in the presence of thiosulfate, but not sulfate. Overall, our findings predict that thiosulfate or oxalate-mimics may be effective as therapeutic competitive inhibitors of CaOx crystallization.

Institute of Molecular, Cell & Systems Biology, College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK; 1 Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, 200 First St., SW, Rochester, MN 55905, USA Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan 3 Global Therapeutics Research Parasitology, VMRD, Zoetis Inc., 333 Portage Street, Kalamazoo, MI 49007 USA

The cation/proton antiporter (CPA) family includes the well-known sodium/proton exchanger (NHE; SLC9A) family of Na(+)/H(+) exchangers, and the more recently discovered and less well understood CPA2s (SLC9B), found widely in living organisms. In Drosophila, as in humans, they are represented by two genes, Nha1 (Slc9b1) and Nha2 (Slc9b2), which are enriched and functionally significant in renal tubules. The importance of their role in organismal survival has not been investigated in animals, however. Here we show that single RNAi knockdowns of either Nha1 or Nha2 reduce survival and in combination are lethal. Knockdown of either gene alone results in up-regulation of the other, suggesting functional complementation of the two genes. Under salt stress, knockdown of either gene decreases survival, demonstrating a key role for the CPA2 family in ion homeostasis. This is specific to Na(+) stress; survival on K(+) intoxication is not affected by sodium/hydrogen antiporter (NHA) knockdown. A direct functional assay in Xenopus oocytes shows that Nha2 acts as a Na(+)/H(+) exchanger. In contrast, Nha1 expressed in Xenopus oocytes shows strong Cl(-) conductance and acts as a H(+)-Cl(-) cotransporter. The activity of Nha1 is inhibited by chloride-binding competitors 4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene and 4,4'-dibenzamido-2,2'-stilbenedisulphonate. Salt stress induces a massive up-regulation of NHA gene expression not in the major osmoregulatory tissues of the alimentary canal, but in the crop, cuticle, and associated tissues. Thus, it is necessary to revise the classical view of the coordination of different tissues in the coordination of the response to osmoregulatory stress.

Zebrafish Na+/H+exchanger 3b (zNHE3b) is highly expressed in the apical membrane of ionocytes where Na+is absorbed from ion-poor fresh water against a concentration gradient. Much in vivo data indicated that zNHE3b is involved in Na+absorption but not leakage. However, zNHE3b-mediated Na+absorption has not been thermodynamically explained, and zNHE3b activity has not been measured. To address this issue, we overexpressed zNHE3b in Xenopus oocytes and characterized its activity by electrophysiology. Exposure of zNHE3b oocytes to Na+-free media resulted in significant decrease in intracellular pH (pHi) and intracellular Na+activity ( aNai). aNaiincreased significantly when the cytoplasm was acidified by media containing CO2-HCO3−or butyrate. Activity of zNHE3b was inhibited by amiloride or 5-ethylisopropyl amiloride (EIPA). Although the activity was accompanied by a large hyperpolarization of ∼50 mV, voltage-clamp experiments showed that Na+/H+exchange activity of zNHE3b is electroneutral. Exposure of zNHE3b oocytes to medium containing NH3/NH4+resulted in significant decreases in pHiand aNaiand significant increase in intracellular NH4+activity, indicating that zNHE3b mediates the Na+/NH4+exchange. In low-Na+(0.5 mM) media, zNHE3b oocytes maintained aNaiof 1.3 mM, and Na+-influx was observed when pHiwas decreased by media containing CO2-HCO3−or butyrate. These results provide thermodynamic evidence that zNHE3b mediates Na+absorption from ion-poor fresh water by its Na+/H+and Na+/NH4+exchange activities.

Background: Zn is implicated as having a role in the formation of calcium oxalate (CaOx) kidney stones. Our studies indicate that Slc39a10 (Zip10) may be important in this process. Zip10 has identifiable sequences in human, dog and fly (Drosophila). As we are interested in a translational model of kidney stones, we examined Zip10 from all 3 species.

Na /H Na NH 4 (cid:2) exchange activities of zebrafish NHE3b expressed in Xenopus oocytes. Am Physiol 2014. Na (cid:2) /H (cid:2) exchanger 3b (zNHE3b) is highly expressed in the apical membrane of ionocytes where Na (cid:2) is absorbed from ion-poor fresh water against a concentration gradient. Much in vivo data indicated that zNHE3b is involved in Na (cid:2) absorption but not leakage. However, zNHE3b-mediated Na (cid:2) absorption has not been thermodynamically explained, and zNHE3b activity has not been measured. To address this issue, we overexpressed zNHE3b in Xenopus oocytes and characterized its activity by electrophysiology. Exposure of zNHE3b oocytes to Na (cid:2) -free media resulted in significant decrease in intracellular pH (pH i ) and intracellular Na (cid:2) activity ( a Na i ). a Na i increased significantly when the cytoplasm was acidified by media containing CO 2 -HCO 3 (cid:3) or butyrate. Activity of zNHE3b was inhibited by amiloride or 5-ethylisopropyl amiloride (EIPA). Although the activity was accompanied by a large hyperpolarization of (cid:4) 50 mV, voltage-clamp experiments showed that Na (cid:2) /H (cid:2) exchange activity of zNHE3b is electroneutral. Exposure of zNHE3b oocytes to medium containing NH 3 /NH 4 (cid:2) resulted in significant decreases in pH i and a Na i and significant increase in intracellular NH 4 (cid:2) activity, indicating that zNHE3b mediates the Na (cid:2) /NH 4 (cid:2) exchange. In low-Na (cid:2) (0.5 mM) media, zNHE3b oocytes maintained a Na i of 1.3 mM, and Na (cid:2) -influx was observed when pH i was decreased by media containing CO 2 - HCO 3 (cid:3) or butyrate. These results provide thermodynamic evidence that zNHE3b mediates Na (cid:2) absorption from ion-poor fresh water by its Na (cid:2) /H (cid:2) and Na (cid:2) /NH 4 (cid:2) exchange activities. Quantitative compared between and control and the statistical significances P were calculated by unpaired two-sided Student’s t -test Prism

Zebrafish Na(+)/H(+) exchanger 3b (zNHE3b) is highly expressed in the apical membrane of ionocytes where Na(+) is absorbed from ion-poor fresh water against a concentration gradient. Much in vivo data indicated that zNHE3b is involved in Na(+) absorption but not leakage. However, zNHE3b-mediated Na(+) absorption has not been thermodynamically explained, and zNHE3b activity has not been measured. To address this issue, we overexpressed zNHE3b in Xenopus oocytes and characterized its activity by electrophysiology. Exposure of zNHE3b oocytes to Na(+)-free media resulted in significant decrease in intracellular pH (pH(i)) and intracellular Na(+) activity (aNa(i)). aNa(i) increased significantly when the cytoplasm was acidified by media containing CO₂-HCO₃(-) or butyrate. Activity of zNHE3b was inhibited by amiloride or 5-ethylisopropyl amiloride (EIPA). Although the activity was accompanied by a large hyperpolarization of ∼50 mV, voltage-clamp experiments showed that Na(+)/H(+) exchange activity of zNHE3b is electroneutral. Exposure of zNHE3b oocytes to medium containing NH₃/NH₄(+) resulted in significant decreases in pH(i) and aNa(i) and significant increase in intracellular NH₄(+) activity, indicating that zNHE3b mediates the Na(+)/NH₄(+) exchange. In low-Na(+) (0.5 mM) media, zNHE3b oocytes maintained aNa(i) of 1.3 mM, and Na(+)-influx was observed when pHi was decreased by media containing CO₂-HCO₃(-) or butyrate. These results provide thermodynamic evidence that zNHE3b mediates Na(+) absorption from ion-poor fresh water by its Na(+)/H(+) and Na(+)/NH₄(+) exchange activities.

Na/H and Na/NH4-exchange activities of zebrafish NHE3b expressed in Xenopus oocytes 1 2 Yusuke Ito, Akira Kato, Taku Hirata, Shigehisa Hirose, and Michael F. Romero 3 4 Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan; and 5 Department of Physiology and Biomedical Engineering, Nephrology & Hypertension and O’Brien 6 Urology Research Center, Mayo Clinic College of Medicine, Rochester, Minnesota 7 8 Running head: FUNCTIONAL CHARACTERIZATION OF ZEBRAFISH NHE3B 9 10 Kew Words: Na/H exchange, Na/NH4 exchange, NHE3, zebrafish, Xenopus oocyte 11 electrophysiology, ammonium, intracellular pH 12 13 Contact information: 14 15 Michael F. Romero, PhD 16 Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, 200 First St. SW, 17 Rochester, MN 55905. Tel.: +1-507-2848127; E-mail: romero.michael@mayo.edu 18 19 * Y. Ito and A. Kato contributed equally to this work. 20 21 22 Articles in PresS. Am J Physiol Regul Integr Comp Physiol (January 8, 2014). doi:10.1152/ajpregu.00363.2013

BackgroundKidney stones are an expensive and painful health problem with calcium oxalate (CaOx) being 70% of cases. We developed a Drosophila model of CaOx stones [Hirata et al., AJP‐Renal Physiol, 2012]. This work showed that dPrestin (Slc26a5/a6) exchanges Cl− for oxalate (Ox). Thiosulfate (S2O3) and tannic acid (TA) are implicated to inhibit CaOx crystals in supersaturated urine. Here, we report transporter, renal tubule and whole body effects of S2O3 and TA on crystal formation.MethodsS2O3 and TA transport effects were examined by voltage clamping of dPrestin‐expressing Xenopus oocytes. Cis‐inhibition of oxalate transport was also examined in (a) dissected Malpighian tubules (MT) or flies fed ox‐enriched food plus S2O3 or TA. MT CaOx crystals were viewed by DIC, or whole flies by microCT.ResultsdPrestin transports S2O3 but not TA, yet both inhibited Ox transport (Ki(S2O3)~10mM; Ki(TA)~100μM). S2O3 competitively inhibits while TA blocks dPrestin. MT treated with S2O3 or TA ↓ crystal formation compared to MT soaked in Ox only. Flies fed with S2O3 or TA also showed ↓ CaOx in MTs.ConclusionsS2O3 and TA are both inhibitors of CaOx crystallization. These experiments show that these inhibitors have distinct mechanisms of action. Our Results indicate that (a) this Drosophila model can be used as a chemical screening assay for CaOx stone prevention and (b) TA, due to low Ki, might be a reasonable therapy for certain CaOx stones patients.[DK83007, DK92408]

Boron is a vital micronutrient and is toxic at high concentrations, however, little is known about whole‐body boric acid homeostasis in animals. Slc4a11 was reported to function as a Na+‐coupled borate transporter in mammals, presumably for borate absorption. However, seawater (SW) contains 0.4 mM boric acid, and the bladder urine of a euryhaline pufferfish mefugu (Takifugu obscures) in SW contains ~20 mM boric acid. Therefore, mefugu kidney is a good model to study a borate efflux system. In the mefugu kidney, a paralog of Slc4a11 (Slc4a11A) was markedly induced after transfer to SW and localized to the apical membrane of renal tubules. When Xenopus oocytes expressing Slc4a11A were voltage‐clamped at a holding potential of −60 mV and exposed medium containing borate, intracellular pH was increased and an outward current (anion influx) was observed. The borate current was not altered when Na+ was replaced with other cations such as choline and Li+, but was eliminated when Na+ was replaced with borate chelating agent NMDG. The borate (boron) influx was confirmed by elemental analysis of the oocytes. These results indicate that Slc4a11A is a Na+‐independent B(OH) 4− channel which is suitable for borate secretion, and clarify at first the mechanism of the borate efflux system in animal that prevent marine fishes from the toxic effects of borate.

Purpose: The limitations imposed by human clinical studies and mammalian models of nephrolithiasis have hampered the development of effective medical treatments and preventive measures for decades. The simple but elegant Drosophila melanogaster is emerging as a powerful translational model of human disease, including nephrolithiasis. It may provide important information essential to our understanding of stone formation. We present the current state of research using D. melanogaster as a model of human nephrolithiasis.Materials and Methods: We comprehensively reviewed the English language literature using PubMed (R). When necessary, authoritative texts on relevant subtopics were consulted.Results: The genetic composition, anatomical structure and physiological function of Drosophila malpighian tubules are remarkably similar to those of the human nephron. The direct effects of dietary manipulation, environmental alteration and genetic variation on stone formation can be observed and quantified in a matter of days. Several Drosophila models of human nephrolithiasis have been developed, including genetically linked and environmentally induced stones. A model of calcium oxalate stone formation is among the most recent fly models of human nephrolithiasis.Conclusions: The ability to readily manipulate and quantify stone formation in D. melanogaster models of human nephrolithiasis presents the urological community with a unique opportunity to increase our understanding of this enigmatic disease.

Secretion of HCO(3)- at the apical side of the epithelial cells of the choroid plexus is an essential step in the formation of cerebrospinal fluid. Anion conductance with a high degree of HCO(3)- permeability has been observed and suggested to be the major pathway for HCO(3)- transport across the apical membrane. Recently, it was found that NBC (Na(+)/HCO(3)- co-transporter) 4, an electrogenic member of the NBC family, was expressed in the choroid plexus. We found that a novel variant of the NBC4 [NBC4g/Slc4a5 (solute carrier family 4, sodium bicarbonate co-transporter, member 5)] is almost exclusively expressed in the apical membrane of rat choroid plexus epithelium at exceptionally high levels. RNA interference-mediated knockdown allowed the functional demonstration that NBC4g is the major player in the HCO(3)- transport across the apical membrane of the choroid plexus epithelium. When combined with a recent observation that in choroid plexus epithelial cells electrogenic NBC operates with a stoichiometry of 3:1, the results of the present study suggest that NBC4g mediates the efflux of HCO(3)- and contributes to cerebrospinal fluid production.

The gut and Malpighian tubules of insects are the primary sites of active solute and water transport for controlling hemolymph and urine composition, pH, and osmolarity. These processes depend on ATPase (pumps), channels and solute carriers (Slc proteins). Maturation of genomic databases enables us to identify the putative molecular players for these processes. Anion transporters of the Slc4 family, AE1 and NDAE1, have been reported as HCO3- transporters, but are only part of the story. Here we report Dipteran (Drosophila melanogaster (d) and Anopheles gambiae (Ag)) anion exchangers, belonging to the Slc26 family, which are multi-functional anion exchangers. One Drosophila and two Ag homologues of mammalian Slc26a5 (Prestin) and Slc26a6 (aka, PAT1, CFEX) were identified and designated dPrestin, AgPrestinA and AgPrestinB. dPrestin and AgPrestinB show electrogenic anion exchange (Cl−/nHCO3-, Cl-/SO42- and Cl−/oxalate2−) in an oocyte expression system. Since these transporters are the only Dipteran Slc26 proteins whose transport is similar to mammalian Slc26a6, we submit that Dipteran Prestin are functional and even molecular orthologues of mammalian Slc26a6. OSR1 kinase increases dPrestin ion transport, implying another set of physiological processes controlled by WNK/SPAK signaling in epithelia. All of these mRNAs are highly expressed in the gut and Malpighian tubules. Dipteran Prestin proteins appear suited for central roles in bicarbonate, sulfate and oxalate metabolism including generating the high pH conditions measured in the Dipteran midgut lumen. Finally, we present and discuss Drosophila genetic models that integrate these processes.

Nephrolithiasis is a major public health problem with a complex and varied etiology. Most stones are composed of calcium oxalate (CaOx), with dietary excess a risk factor. Because of complexity of mammalian system, the details of stone formation remain to be understood. Here we have developed a nephrolithiasis model using the genetic model Drosophila melanogaster, which has a simple, transparent kidney tubule. Drosophilia reliably develops CaOx stones upon dietary oxalate supplementation, and the nucleation and growth of microliths can be viewed in real time. The Slc26 anion transporter dPrestin (Slc26a5/6) is strongly expressed in Drosophilia kidney, and biophysical analysis shows that it is a potent oxalate transporter. When dPrestin is knocked down by RNAi in fly kidney, formation of microliths is reduced, identifying dPrestin as a key player in oxalate excretion. CaOx stone formation is an ancient conserved process across >400 My of divergent evolution (fly and human), and from this study we can conclude that the fly is a good genetic model of nephrolithiasis.

You have accessJournal of UrologyStone Disease: Basic Research I1 Apr 20122070 DEVELOPMENT OF DROSOPHILA KIDNEY STONE MODEL AND REAL-TIME VISUALIZATION OF CALCIUM OXALATE CRYSTAL FORMATION Taku Hirata, Pablo Cabrero, Dan Bondeson, Donald Berkholz, Erik Ritman, James Thompson, Julian Dow, and Michael Romero Taku HirataTaku Hirata Rochester, MN More articles by this author , Pablo CabreroPablo Cabrero Glasgow, United Kingdom More articles by this author , Dan BondesonDan Bondeson Rochester, MN More articles by this author , Donald BerkholzDonald Berkholz Rochester, MN More articles by this author , Erik RitmanErik Ritman Rochester, MN More articles by this author , James ThompsonJames Thompson Rochester, MN More articles by this author , Julian DowJulian Dow Glasgow, United Kingdom More articles by this author , and Michael RomeroMichael Romero Rochester, MN More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.2236AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Kidney stones (nephrolithiasis) are a painful and expensive health care issue having a complex etiology. Roughly 70% of stones are composed of calcium oxalate (CaOx). The Slc26 gene family encodes several proteins that transport oxalate in the gut and kidney. Slc26a6 and its homologs exchange Cl− for oxalate (ox2−), plus SO42−, HCO3− and formate. Slc26a6 knockout mice have hyperoxaluria and CaOx stones illustrating a physiologic collaboration between the gut and kidney. Drosophila Slc26a5/6 (dPrestin) mRNA is heavily expressed in the gut and Malpighian tubules (“kidney”). We developed a Drosophila genetic model for investigating CaOx stones (dPrestin, Slc26a5/6, manipulation) allowing rapid gut, renal and whole-body physiological assessment (min/h v. mo/y). METHODS (Tissue) First, Malpighian tubules (MT) were dissected from wild type and dPrestin knockdown fly mutants and soaked in ox2− solutions. CaOx crystals were visualized as birefringence using DIC microscopy. Second, we generated Drosophila expressing eYFP in the MT, MT+gut or whole body. eYFP is a halide (Cl−) sensor, allowing quantification of Cl−/ox2− exchange by native dPrestin. (Whole body) Wild type and dPrestin knockdown flies were fed ox2− enriched food, and flies imaged (CaOx quantified) by micro computed tomography (MicroCT). RESULTS Exposing dPrestin-knockdowns to bath ox2− elicited few CaOx crystals. However, exposing wt-MT to ox2− elicited many CaOx crystals in as little as 20 min. Increased CaOx crystal formation was also coupled with an eYFP intensity increase, matching dPrestin Cl−/ox2− exchange characteristics. MicroCT showed more CaOx crystals in flies that fed on ox2− longer. CONCLUSIONS Our observations indicate that Drosophila and dPrestin manipulation can be used for rapid, detailed studies of gut, renal and whole organism oxalate metabolism & CaOx stone formation. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e835 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Taku Hirata Rochester, MN More articles by this author Pablo Cabrero Glasgow, United Kingdom More articles by this author Dan Bondeson Rochester, MN More articles by this author Donald Berkholz Rochester, MN More articles by this author Erik Ritman Rochester, MN More articles by this author James Thompson Rochester, MN More articles by this author Julian Dow Glasgow, United Kingdom More articles by this author Michael Romero Rochester, MN More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...