Citations

Most vertebrates, including cartilaginous fishes, maintain their plasma SO42−concentration ([SO42−]) within a narrow range of 0.2–1 mM. As seawater has a [SO42−] about 40 times higher than that of the plasma, SO42−excretion is the major role of kidneys in marine teleost fishes. It has been suggested that cartilaginous fishes also excrete excess SO42−via the kidney. However, little is known about the underlying mechanisms for SO42−transport in cartilaginous fish, largely due to the extraordinarily elaborate four-loop configuration of the nephron, which consists of at least 10 morphologically distinguishable segments. In the present study, we determined cDNA sequences from the kidney of holocephalan elephant fish ( Callorhinchus milii) that encoded solute carrier family 26 member 1 (Slc26a1) and member 6 (Slc26a6), which are SO42−transporters that are expressed in mammalian and teleost kidneys. Elephant fish Slc26a1 (cmSlc26a1) and cmSlc26a6 mRNAs were coexpressed in the proximal II (PII) segment of the nephron, which comprises the second loop in the sinus zone. Functional analyses using Xenopus oocytes and the results of immunohistochemistry revealed that cmSlc26a1 is a basolaterally located electroneutral SO42−transporter, while cmSlc26a6 is an apically located, electrogenic Cl−/SO42−exchanger. In addition, we found that both cmSlc26a1 and cmSlc26a6 were abundantly expressed in the kidney of embryos; SO42−was concentrated in a bladder-like structure of elephant fish embryos. Our results demonstrated that the PII segment of the nephron contributes to the secretion of excess SO42−by the kidney of elephant fish. Possible mechanisms for SO42−secretion in the PII segment are discussed.

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.

Significance Cation/proton antiporters (CPAs) are essential to life. The sodium/proton exchanger (NHE) branch of the CPA family has been studied exhaustively and is an important drug target; however, much less is known about the recently discovered NHA branch, represented by two genes in both humans and flies. Here we show that sodium/hydrogen antiporter (NHA) function is essential to life, and that both NHAs protect against salt stress. Their transport mechanisms are radically different, however, suggesting that function cannot be inferred from structural similarity. Although NHA2 was found to be a Na + /H + exchanger as expected, NHA1 was seen to act as an electroneutral H + /Cl - cotransporter. This is an important finding for future studies of these transporters.

The mechanisms regulating proximal tubule ammonia metabolism are incompletely understood. The present study addressed the role of the proximal tubule basolateral electrogenic Na+-coupled bicarbonate cotransporter (NBCe1; Slc4a4) in renal ammonia metabolism. We used mice with heterozygous and homozygous NBCe1 gene deletion and compared these mice with their wild-type littermates. Because homozygous NBCe1 gene deletion causes 100% mortality before day 25, we studied mice at day 8 (±1 day). Both heterozygous and homozygous gene deletion caused a gene dose-related decrease in serum bicarbonate. The ability to lower urinary pH was intact, and even accentuated, with NBCe1 deletion. However, in contrast to the well-known effect of metabolic acidosis to increase urinary ammonia excretion, NBCe1 deletion caused a gene dose-related decrease in ammonia excretion. There was no identifiable change in proximal tubule structure by light microscopy. Examination of proteins involved in renal ammonia metabolism showed decreased expression of phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase, key enzymes in proximal tubule ammonia generation, and increased expression of glutamine synthetase, which recycles intrarenal ammonia and regenerates glutamine. Expression of key proteins involved in ammonia transport outside of the proximal tubule (rhesus B glycoprotein and rhesus C glycoprotein) was not significantly changed by NBCe1 deletion. We conclude from these findings that NBCe1 expression is necessary for normal proximal tubule ammonia metabolism.

Significance Endocrine control of chloride shunt conductance in Malpighian tubules of many insects is mediated by kinin diuretic peptides, but the route for chloride transport is unknown. We use Drosophila transgenics, combined with physiology, imaging, electrophysiology, and transgenic chloride reporter technology, to show that a chloride channel (CLC) encoded by ClC-a/CG31116 is uniquely localized to stellate cells and is essential for neuropeptide-stimulated, but not resting, levels of secretion. Therefore, we have resolved this issue in Drosophila ; kinin stimulates chloride flux by a transcellular route that is confined to the stellate cells.

The kidney of marine teleosts is the major site of Mg2+excretion and produces urine with a high Mg2+concentration. However, the transporters involved in Mg2+excretion are poorly understood. The cyclin M (Cnnm; also known as ancient conserved domain protein) family comprises membrane proteins homologous to the bacterial Mg2+and Co2+efflux protein, CorC. To understand the molecular mechanism of Mg2+homeostasis in marine teleosts, we analyzed the expression of the C nnm family genes in the seawater (SW) pufferfish, torafugu ( Takifugu rubripes), and the closely related euryhaline species, mefugu ( Takifugu obscurus). Database mining and phylogenetic analysis indicated that the Takifugu genome contains six members of the Cnnm family: two orthologs of Cnnm1, one of Cnnm2, one of Cnnm3, and two of Cnnm4. RT-PCR analyses indicated that Cnnm2, Cnnm3, and Cnnm4a are expressed in the kidney, whereas other members are mainly expressed in the brain. Renal expression of Cnnm3 was upregulated in SW mefugu, whereas renal expression of Cnnm2 was upregulated in freshwater (FW) mefugu. No significant difference was observed in renal expression of Cnnm4a between SW and FW mefugu. In situ hybridization and immunohistochemical analyses of the SW mefugu kidney revealed that Cnnm3 is expressed in the proximal tubule, and its product localizes to the lateral membrane. When Cnnm3 was expressed in Xenopus laevis oocytes, whole cellular Mg2+content and free intracellular Mg2+activity significantly decreased. These results suggest that Cnnm3 is involved in body fluid Mg2+homeostasis in marine teleosts.

Mutations in the electrogenic Na+/HCO3−cotransporter (NBCe1) that cause proximal renal tubular acidosis (pRTA), glaucoma, and cataracts in patients are recessive. Parents and siblings of these affected individuals seem asymptomatic although their tissues should make some mutant NBCe1 protein. Biochemical studies with AE1 and NBCe1 indicate that both, and probably all, Slc4 members form dimers. However, the physiologic implications of dimerization have not yet been fully explored. Here, human NBCe1A dimerization is demonstrated by biomolecular fluorescence complementation (BiFC). An enhanced yellow fluorescent protein (EYFP) fragment (1–158, EYFPN) or (159–238, EYFPC) was fused to the NH2or COOH terminus of NBCe1A and mix-and-matched expressed in Xenopus oocyte. The EYFP fluorescent signal was observed only when both EYFP fragments are fused to the NH2terminus of NBCe1A (EYFPN-N-NBCe1A w/ EYFPC-N-NBCe1A), and the electrophysiology data demonstrated this EYFP-NBCe1A coexpressed pair have wild-type transport function. These data suggest NBCe1A forms dimers and that NH2termini from the two monomers are in close proximity, likely pair up, to form a functional unit. To explore the physiologic significance of NBCe1 dimerization, we chose two severe NBCe1 mutations (6.6 and 20% wild-type function individually): S427L (naturally occurring) and E91R (for NH2-terminal structure studies). When we coexpressed S427L and E91R, we measured 50% wild-type function, which can only occur if the S427L-E91R heterodimer is the functional unit. We hypothesize that the dominant negative effect of heterozygous NBCe1 carrier should be obvious if the mutated residues are structurally crucial to the dimer formation. The S427L-E91R heterodimer complex allows the monomers to structurally complement each other resulting in a dimer with wild-type like function.

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.