Citations

The gas in the swimbladder of teleost fish is composed primarily of O 2 , and the swimbladder volume is regulated by O 2 transfer between the luminal space of the swimbladder and the blood. In the swimbladder, lactic acid generation by anaerobic glycolysis in the gas gland epithelial cells and its recycling through the rete mirabile bundles of countercurrent capillaries are essential for local blood acidification and oxygen liberation from hemoglobin by the “Root effect.” To clarify molecules that are involved in the blood acidification and visualize the route of lactic acid movement, we analyzed the expression of 16 members of the H + /monocarboxylate transporter (MCT) family in the fugu genome and found that only MCT1b and MCT4b are highly expressed in the fugu swimbladder. Immunohistochemistry demonstrated that (i) MCT4b expresses in gas gland cells together with the glycolytic enzyme GAPDH at high level and mediate lactic acid secretion by gas gland cells, and (ii) MCT1b expresses in arterial, but not venous, capillary endothelial cells in rete mirabile and mediates recycling of lactic acid in the rete mirabile by solute‐specific transcellular transport. Electrophysiological analyses demonstrated that MCT1b is a high‐affinity lactate transporter whereas MCT4b is a low‐affinity/high‐conductance lactate transporter. These results clarified the mechanism of the blood acidification in the swimbladder by spatially organized two lactic acid transporters MCT4b and MCT1b.

Background Kidney stones (nephrolithiasis) are an expensive and painful health problem with a complex etiology with ~70% of stones composed of calcium oxalate (CaOx). The Slc26 gene family contains several proteins that transport oxalate in the gut and kidney. Slc26a6 and its homologs exchange Cl − for oxalate (ox 2− ), as well as SO 4 2− , HCO 3 − and formate. Slc26a6 −/− mice have hyperoxaluria and CaOx stones illustrating a physiologic collaboration between the gut and kidney. We developed a Drosophila genetic model for 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, renal tubule) were dissected from wild type and dPrestin knockdown mutants and soaked in ox 2− solutions. CaOx stones were visualized by 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 − /ox 2− exchange by native dPrestin. (Whole body) Wild type and dPrestin knockdown flies were fed ox 2− enriched food, and flies imaged (CaOx quantified) by micro computed tomography (MicroCT). Results Exposing dPrestin‐knockdowns to bath ox 2− elicited almost no CaOx crystals. However, exposing wt‐MT to ox 2− elicited many CaOx crystals in 20 min. Increased CaOx crystal formation was also coupled with an eYFP intensity increase, matching dPrestin Cl − /ox 2− exchange characteristics. MicroCT showed more CaOx crystals in flies that fed on ox 2− 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. [DK83007]

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.

Background Kidney 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 (S 2 O 3 ) and tannic acid (TA) are implicated to inhibit CaOx crystals in supersaturated urine. Here, we report transporter, renal tubule and whole body effects of S 2 O 3 and TA on crystal formation. Methods S 2 O 3 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 S 2 O 3 or TA. MT CaOx crystals were viewed by DIC, or whole flies by microCT. Results dPrestin transports S 2 O 3 but not TA, yet both inhibited Ox transport (K i (S 2 O 3 )~10mM; K i (TA)~100μM). S 2 O 3 competitively inhibits while TA blocks dPrestin. MT treated with S 2 O 3 or TA ↓ crystal formation compared to MT soaked in Ox only. Flies fed with S 2 O 3 or TA also showed ↓ CaOx in MTs. Conclusions S 2 O 3 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]

The ten‐member SLC26 gene family encodes anion transporters with a diversity of substrates and transport modes, functioning as electrogenic exchangers, anion sensors, and/or anion channels. Slc26a6 is perhaps the most versatile paralog, mediating electrogenic exchange of multiple divalent and monovalent anions. We have generated a cysteineless Slc26a6 for structure‐function studies, fully substituting 8 cysteines with threonine, serine, and alanine, as dictated by the functional data for single and compound mutants. Although absolute transport rates are reduced in the cysteineless Slc26a6 protein it exhibits equivalent anion specificity and electrophysiology (chloride‐bicarbonate exchange) to that of wild‐type. On Western blots, the oligomerization of wild‐type Slc26a6 is inhibited by dithiothreitol, suggesting a role for inter‐cysteine disulfide bonds; oligomerization is absent in the cysteineless protein. Co‐immunoprecipitation of FLAG‐ and MYC‐tagged Slc26a6 is easily detectable for wild‐type proteins but absent in the epitope‐tagged cysteineless proteins. The equivalent data for single and compound mutants implicate C‐terminal cytoplasmic cysteines in oligomerization of the transporter protein. In summary, the eight cysteines in Slc26a6 are dispensable for transport activity but appear to play a role in oligomerization of the transport protein.

Slc 26 gene family encodes multifunctional anion transport proteins: sensor, exchangers and channels. Structurally, Slc26 members have 10–14 predicted transmembrane spans followed by a cytoplasmic domain. This carboxy‐terminus contains a STAS (sulfate transporter anti‐sigma) domain whose function we have studied in mouse Slc26a9 (Chang et al , JBC 284: 28306, 2009). Mutations in SLC26 proteins lead to a number of diseases including diastrophic dysplasia, congenital chloride diarrhea, deafness and potentially Ca‐oxalate, kidney stones. While several groups have addressed the exchanger vs. channel nature of Slc26 proteins, elements within each Slc26 protein contributing to (or controlling) the anion transport character have not been probed. Interestingly, mapping the STAS domain conserved motifs reveals a hypervariable region. This hypervariable region ranges between 30 and 130 amino acids; no similarities exist between different Slc26 members. We made a SLC26A9 construct (Δloop), lacking this STAS subregion of SLC26A9. We expressed Δloop in Xenopus oocytes and monitored transport using ion selective microelectrodes and voltage clamping. SLC26A9 Δloop showed >5‐fold increased Cl − currents compared with the wild type SLC26A9 without obvious change in Cl − ‐HCO 3 − exchange activity. We also made several SLC26A9 mutants to remove putative phosphorylation sites located in this loop: T620A, S621A, S623A, T626A and S628A(replacing T or S with A). All of these mutants show decreased (10–50%) Cl − currents compared with the wild type SLC26A9. These results suggest this variable loop contains a channel‐inhibitory motif and also phosphorylation sites maintaining basic SLC26A9 function. We hypothesize that this variable loop is a regulatory element collaborating with conserved STAS domain regions and that this structural interaction may confer specific transport modes for SLC26A9 and possibly other SLC26 proteins.

Malaria transmission requires Plasmodium invasion of the Anopheles mosquito midgut. This critical first step involves adhesion to chondroitin sulfate (ChSO 4 )[Dinglasan et al, PNAS 2007], a midgut epithelial ligand. Sulfate (SO 4 2− ) for ChSO 4 biosynthesis is supplied by midgut SO 4 2− uptake. Slc26 proteins transport many anions: Cl − , HCO 3 − , oxalate (ox 2− ), and SO 4 2− . Recently, we found that Drosophila Slc26a5 is an electrogenic Cl − /SO 4 2− , Cl − /ox 2− and Cl − / n HCO 3 − exchanger [Hirata et al, JASN 2009], and SO 4 2− transport exceeded ox 2− transport. Therefore we searched EST and genomic databases to identify two Slc26a5 orthologues (AgSlc26a5A and ‐ B). We expressed AgSlc26a5A and B in Xenopus oocytes to evaluate transport using microelectrodes. AgSlc26a5A is found in the salivary glands and gastric caeca. However, AgSlc26a5A did not obviously transport Cl − , HCO 3 − , SO 4 2− or ox 2− . On the other hand, AgSlc26a5B has transport properties similar to mouse Slc26a6 and Drosophila Slc26a5. That is, AgSlc26a5B mediates electrogenic Cl − /SO 4 2− , Cl − /ox 2− and Cl − / n HCO 3 − exchange. And, AgSlc26a5B is enriched in the hindgut (including Malpighian tubules) and the posterior midgut. These results suggest that AgSlc26a5B can alkalinize the Anopheles midgut and is also a candidate for SO 4 2− supplier to midgut cell. Thus, we hypothesize that control of AgSlc26a5B activity could control parasite ( Plasmodium ) invasion.

The solute carrier 26 (Slc26) transporters are anion transporters with diverse substrate specificity. They are expressed mainly in epithelia and play a central role in secretion and absorption of anions such as chloride, oxalate (ox 2− ), sulfate (SO 4 2− ), bicarbonate (HCO 3 − ), iodide, formate and hydroxyl. In mammals the Slc26a6 protein is expressed in kidney and gut epithelia, and mediates electrogenic Cl − /ox 2− , Cl − / n HCO 3 − , Cl − /SO 4 2− exchange. Recently, chicken and zebrafish prestin (Slc26a5) were shown to mediate electrogenic Cl − /ox 2− and Cl − /SO 4 2− exchange rather than being an auditory sensor. We have cloned and characterized the Slc26a5 (fPrestin) transporter from the euryhaline pufferfish Takifugu obscurus (mefugu). This fPrestin mRNA is expressed ubiquitously unlike fSlc26a6A‐C which is found in kidney and gut. While the fSlc26a6A‐C mRNAs are increased in response to a freshwater to saltwater transition, fPrestin expression is unchanged. We next expressed fSlc26a5 in Xenopus oocytes and evaluated transport using microelectrodes. These experiments reveal that fPrestin mediates electrogenic Cl − /ox 2− , and Cl − /SO 4 2− as previously shown for chicken and zebrafish Slc26a5. Furthermore, fPrestin also can mediate electrogenic Cl − / n HCO 3 − exchange. These transport features are quite similar to mammalian Slc26a6 which has been implicated in renal Ca‐oxalate, stones.

The Na + /monocarboxylate transporters (SMCT1 and SMCT2) are expressed several organ between them intestine and kidney, suggesting that its main physiological function are the absorption of fuel molecules involved in cellular metabolism and have only one ‐COOH group in its chemical structure; (lactate, propionate). Drugs with similar chemical properties also are translocated by SMCTs (B3 vitamin, pyrazinoate). However some propionate‐derivative NSAIDs (ibuprofen) reduce the activity of mammalian SMCT1. Using 22 Na + uptake in Xenopus oocytes injected with zebrafish SMCT’s (zSMCT1, zSMCT2) or human SMCT1 cRNA, we evaluated the effect of aspirin and salicylate on the function SMCT’s. Our results indicate a 22 Na + uptake that is aspirin concentration dependent in all SMCT’s. Similar affinity by aspirin was observed between hSMCT1 (K m ~2.4mM) and zSMCT1 (K m ~2.5 mM) in contrast to a lower affinity of zSMCT2 (K m ~5mM). Also the difference of substrate affinity between zSMCT2 and hSMCT1 was evident by salicylate ( hSMCT1 K m ~4mM vs zSMCT1 K m ~30 mM). Interestingly, although zSMCT1 and hSMCT1 shares a similar affinity and transport capacity for several substrates, the salicylate transport by zSMCT1 was not detected and only reduced a small percentage propionate transport induced by zSMCT1 (~10%) in contrast of ibuprofen which drops to 80%. The clear difference of salicylate transporter between SMCT1 could be explained by changes in the structure of proteins because the amino acids sequences are approximately 50% different, so the salicylate could be candidate for studies of structural‐function between SMCT1. Grant Funding Source : CONACYT48709MJ(CP)

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. Methods: Zip10 cDNAs were cloned from human & dog (kidney) and fly (whole fly) using gene specific primers and RT‐PCR. Clones were put into a Xenopus oocyte expression vector so that Zip10 function could be measured. Three days post‐injection, oocytes were pre‐incubated in ND96, followed by 30 min in ND96 + 10 μM ZnCl + the PET isotope 63 Zn, with varying pHs and ion concentrations. 63 Zn uptake was measured in each oocyte using a gamma counter. Zip10 oocytes were also checked using electrophysiology (ion selective electrodes & voltage clamping). Results: Zip10 mRNA & protein are found in gut and kidney. In mouse kidney, Zip10 antibodies stained the apical membrane of proximal tubules. All clones transported 63 Zn (nmol/oocyte/hr): hZip10 = 70±8; cZip10= 44 ±7; dZip10= 64 ±8; water = 0.4 ±0.1. These clones seem to be most active at pH 7.5 as uptakes decreased at 5.5 and 8.5. Replacement of either all Cl ‐ (with HCO 3 舉 ) or all Na + (with choline) reduced Zn‐uptake. Electrophysiology revealed no evidence of I or V changes with addition of Zn (electroneutral). Conclusions: Our results indicated that all three Zip10 clones transport Zn 2+ . Understanding the detailed mechanisms of these Zn transporters in oocytes presents the opportunity to better use our Drosophila model in the study of CaOx stone formation and absorption. Grant Funding Source : Supported by DK83007, DK92408