Seawater fish use an electrogenic boric acid transporter, Slc4a11A, for boric acid excretion by the kidney.

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.

The role of Na+-coupled bicarbonate transporters (NCBT) in health and disease.

Cellular and organism survival depends upon the regulation of pH, which is regulated by highly specialized cell membrane transporters, the solute carriers (SLC) (For a comprehensive list of the solute carrier family members, see: https://www.bioparadigms.org/slc/ ). The SLC4 family of bicarbonate (HCO3-) transporters consists of ten members, sorted by their coupling to either sodium (NBCe1, NBCe2, NBCn1, NBCn2, NDCBE), chloride (AE1, AE2, AE3), or borate (BTR1). The ionic coupling of SLC4A9 (AE4) remains controversial. These SLC4 bicarbonate transporters may be controlled by cellular ionic gradients, cellular membrane voltage, and signaling molecules to maintain critical cellular and systemic pH (acid-base) balance. There are profound consequences when blood pH deviates even a small amount outside the normal range (7.35-7.45). Chiefly, Na+-coupled bicarbonate transporters (NCBT) control intracellular pH in nearly every living cell, maintaining the biological pH required for life. Additionally, NCBTs have important roles to regulate cell volume and maintain salt balance as well as absorption and secretion of acid-base equivalents. Due to their varied tissue expression, NCBTs have roles in pathophysiology, which become apparent in physiologic responses when their expression is reduced or genetically deleted. Variations in physiological pH are seen in a wide variety of conditions, from canonically acid-base related conditions to pathologies not necessarily associated with acid-base dysfunction such as cancer, glaucoma, or various neurological diseases. The membranous location of the SLC4 transporters as well as recent advances in discovering their structural biology makes them accessible and attractive as a druggable target in a disease context. The role of sodium-coupled bicarbonate transporters in such a large array of conditions illustrates the potential of treating a wide range of disease states by modifying function of these transporters, whether that be through inhibition or enhancement.

Na/K-ATPase signaling tonically inhibits sodium reabsorption in the renal proximal tubule.

Through its classic ATP-dependent ion-pumping function, basolateral Na/K-ATPase (NKA) generates the Na+ gradient that drives apical Na+ reabsorption in the renal proximal tubule (RPT), primarily through the Na+ /H+ exchanger (NHE3). Accordingly, activation of NKA-mediated ion transport decreases natriuresis through activation of basolateral (NKA) and apical (NHE3) Na+ reabsorption. In contrast, activation of the more recently discovered NKA signaling function triggers cellular redistribution of RPT NKA and NHE3 and decreases Na+ reabsorption. We used gene targeting to test the respective contributions of NKA signaling and ion pumping to the overall regulation of RPT Na+ reabsorption. Knockdown of RPT NKA in cells and mice increased membrane NHE3 and Na+ /HCO3 - cotransporter (NBCe1A). Urine output and absolute Na+ excretion decreased by 65%, driven by increased RPT Na+ reabsorption (as indicated by decreased lithium clearance and unchanged glomerular filtration rate), and accompanied by elevated blood pressure. This hyper reabsorptive phenotype was rescued upon crossing with RPT NHE3-/- mice, confirming the importance of NKA/NHE3 coupling. Hence, NKA signaling exerts a tonic inhibition on Na+ reabsorption by regulating key apical and basolateral Na+ transporters. This action, lifted upon NKA genetic suppression, tonically counteracts NKA's ATP-driven function of basolateral Na+ reabsorption. Strikingly, NKA signaling is not only physiologically relevant but it also appears to be functionally dominant over NKA ion pumping in the control of RPT reabsorption.

Function and regulation of the insect NaCCC2 sodium transport proteins.

NaCCC2 transport proteins, including those from Drosophila melanogaster (Ncc83) and Aedes aegypti (aeCCC2), are an insect-specific clade with sequence similarity to Na+-K+-2Cl- cotransporters. Whereas the Na+-K+-2Cl- cotransporters and other cation-chloride cotransporters are electroneutral, recent work indicates that Ncc83 and aeCCC2 carry charge across membranes. Here, we further characterize the regulation and transport properties of Ncc83 and aeCCC2 expressed in Xenopus oocytes. In cation uptake experiments, Li+ was used as a tracer for Na+ and Rb+ was used as a tracer for K+. Li+ uptake of oocytes expressing either aeCCC2 or Ncc83 was greater than uptake in water-injected controls, activated by hypotonic swelling, and not inhibited by ouabain or ethyl cinnamate. Rb+ uptake of oocytes expressing either aeCCC2 or Ncc83 was not different than water injected controls. In oocytes expressing either aeCCC2 or Ncc83, Li+ uptake plateaued with increasing Li+ concentrations, with apparent Km values in the range of 10 to 20 mM. Following exposure to ouabain, intracellular [Na+] was greater in oocytes expressing aeCCC2 than in controls. Elevating intracellular cAMP (via 8-bromo-cAMP) in Ncc83 oocytes significantly stimulated both Li+ uptake and membrane conductances. Elevating intracellular cAMP in aeCCC2 oocytes did not affect Li+ uptake, but stimulated membrane conductances. Overall, these results confirm that the NaCCC2s resemble other cation-chloride cotransporters in their regulation and some transport properties. However, unlike other cation-chloride cotransporters, they carry charge across membranes.

Specialized stellate cells offer a privileged route for rapid water flux in Drosophila renal tubule.

Insects are highly successful, in part through an excellent ability to osmoregulate. The renal (Malpighian) tubules can secrete fluid faster on a per-cell basis than any other epithelium, but the route for these remarkable water fluxes has not been established. In Drosophila melanogaster, we show that 4 genes of the major intrinsic protein family are expressed at a very high level in the fly renal tissue: the aquaporins (AQPs) Drip and Prip and the aquaglyceroporins Eglp2 and Eglp4 As predicted from their structure, and by their transport function by expressing these proteins in Xenopus oocytes, Drip, Prip, and Eglp2 show significant and specific water permeability, whereas Eglp2 and Eglp4 show very high permeability to glycerol and urea. Knockdowns of any of these genes result in impaired hormone-induced fluid secretion. The Drosophila tubule has 2 main secretory cell types: active cation-transporting principal cells, wherein the aquaglyceroporins localize to opposite plasma membranes, and small stellate cells, the site of the chloride shunt conductance, with these AQPs localizing to opposite plasma membranes. This suggests a model in which osmotically obliged water flows through the stellate cells. Consistent with this model, fluorescently labeled dextran, an in vivo marker of membrane water permeability, is trapped in the basal infoldings of the stellate cells after kinin diuretic peptide stimulation, confirming that these cells provide the major route for transepithelial water flux. The spatial segregation of these components of epithelial water transport may help to explain the unique success of the higher insects in regulating their internal environments.

Cl- and H+ coupling properties and subcellular localizations of wildtype and disease-associated variants of the voltage-gated Cl-/H+ exchanger ClC-5.

Dent disease 1 (DD1) is caused by mutations in the CLCN5 gene encoding a voltage-gated electrogenic nCl-/H+ exchanger ClC-5. Using ion-selective microelectrodes and Xenopus oocytes, here we studied Cl-/H+ coupling properties of WT ClC-5 and four DD1-associated variants (S244L, R345W, Q629*, and T657S), along with trafficking and localization of ClC-5. WT ClC-5 had a 2Cl-/H+ exchange ratio at a Vh of +40 mV with a [Cl-]out of 104 mm, but the transport direction did not reverse with a [Cl-]out of 5 mm, indicating that ClC-5-mediated exchange of two Cl- out for one H+ in is not permissible. We hypothesized that ClC-5 and H+-ATPase are functionally coupled during H+-ATPase-mediated endosomal acidification, crucial for ClC-5 activation by depolarizing endosomes. ClC-5 transport that provides three net negative charges appeared self-inhibitory because of ClC-5's voltage-gated properties, but shunt conductance facilitated further H+-ATPase-mediated endosomal acidification. Thus, an on-and-off "burst" of ClC-5 activity was crucial for preventing Cl- exit from endosomes. The subcellular distribution of the ClC-5:S244L variant was comparable with that of WT ClC-5, but the variant had a much slower Cl- and H+ transport and displayed an altered stoichiometry of 1.6:1. The ClC-5:R345W variant exhibited slightly higher Cl-/H+ transport than ClC-5:S244L, but co-localized with early endosomes, suggesting decreased ClC-5:R345W membrane trafficking is perhaps in a fully functional form. The truncated ClC-5:Q629* variant displayed the lowest Cl-/H+ exchange and was retained in the endoplasmic reticulum and cis-Golgi, but not in early endosomes, suggesting the nonsense mutation affects ClC-5 maturation and trafficking.

Expression of the regulated isoform of the electrogenic Na+/HCO3- cotransporter, NBCe1, is enriched in pacemaker interstitial cells of Cajal.

Interstitial cells of Cajal (ICCs) generate electrical slow waves, which are required for normal gastrointestinal motility. The mechanisms for generation of normal pacemaking are not fully understood. Normal gastrointestinal contractility- and electrical slow-wave activity depend on the presence of extracellular HCO3-. Previous transcriptional analysis identified enrichment of mRNA encoding the electrogenic Na+/HCO3- cotransporter (NBCe1) gene (Slc4a4) in pacemaker myenteric ICCs in mouse small intestine. We aimed to determine the distribution of NBCe1 protein in ICCs of the mouse gastrointestinal tract and to identify the transcripts of the Slc4a4 gene in mouse and human small intestinal tunica muscularis. We determined the distribution of NBCe1 immunoreactivity (NBCe1-IR) by immunofluorescent labeling in mouse and human tissues. In mice, NBCe1-IR was restricted to Kit-positive myenteric ICCs of the stomach and small intestine and submuscular ICCs of the large intestine, that is, the slow wave generating subset of ICCs. Other subtypes of ICCs were NBCe1-negative. Quantitative real-time PCR identified >500-fold enrichment of Slc4a4-207 and Slc4a4-208 transcripts ["IP3-receptor-binding protein released by IP3" (IRBIT)-regulated isoforms] in Kit-expressing cells isolated from KitcreERT2/+, Rpl22tm1.1Psam/Sj mice and from single GFP-positive ICCs from Kittm1Rosay mice. Human jejunal tunica muscularis ICCs were also NBCe1-positive, and SLC4A4-201 and SLC4A4-204 RNAs were >300-fold enriched relative to SLC4A4-202. In summary, NBCe1 protein expressed in ICCs with electrical pacemaker function is encoded by Slc4a4 gene transcripts that generate IRBIT-regulated isoforms of NBCe1. In conclusion, Na+/HCO3- cotransport through NBCe1 contributes to the generation of pacemaker activity in subsets of ICCs.NEW & NOTEWORTHY In this study, we show that the electrogenic Na+/HCO3- cotransporter, NBCe1/Slc4a4, is expressed in subtypes of interstitial cells of Cajal (ICCs) responsible for electrical slow wave generation throughout the mouse gastrointestinal tract and is absent in other types of ICCs. The transcripts of Slc4a4 expressed in mouse ICCs and human gastrointestinal smooth muscle are the regulated isoforms. This indicates a key role for HCO3- transport in generation of gastrointestinal motility patterns.

NBCe1-A is required for the renal ammonia and K+ response to hypokalemia.

Hypokalemia increases ammonia excretion and decreases K+ excretion. The present study examined the role of the proximal tubule protein NBCe1-A in these responses. We studied mice with Na+-bicarbonate cotransporter electrogenic, isoform 1, splice variant A (NBCe1-A) deletion [knockout (KO) mice] and their wild-type (WT) littermates were provided either K+ control or K+-free diet. We also used tissue sections to determine the effect of extracellular ammonia on NaCl cotransporter (NCC) phosphorylation. The K+-free diet significantly increased proximal tubule NBCe1-A and ammonia excretion in WT mice, and NBCe1-A deletion blunted the ammonia excretion response. NBCe1-A deletion inhibited the ammoniagenic/ammonia recycling enzyme response in the cortical proximal tubule (PT), where NBCe1-A is present in WT mice. In the outer medulla, where NBCe1-A is not present, the PT ammonia metabolism response was accentuated by NBCe1-A deletion. KO mice developed more severe hypokalemia and had greater urinary K+ excretion during the K+-free diet than did WT mice. This was associated with blunting of the hypokalemia-induced change in NCC phosphorylation. NBCe1-A KO mice have systemic metabolic acidosis, but experimentally induced metabolic acidosis did not alter NCC phosphorylation. Although KO mice have impaired ammonia metabolism, experiments in tissue sections showed that lack of ammonia does impair NCC phosphorylation. Finally, urinary aldosterone was greater in KO mice than in WT mice, but neither expression of epithelial Na+ channel α-, ß-, and γ-subunits nor of H+-K+-ATPase α1- or α2-subunits correlated with changes in urinary K+. We conclude that NBCe1-A is critical for the effect of diet-induced hypokalemia to increase cortical proximal tubule ammonia generation and for the expected decrease in urinary K+ excretion.

Targeted renal knockdown of Na+/H+ exchanger regulatory factor Sip1 produces uric acid nephrolithiasis in Drosophila.

Nephrolithiasis is one of the most common kidney diseases, with poorly understood pathophysiology, but experimental study has been hindered by lack of experimentally tractable models. Drosophila melanogaster is a useful model organism for renal diseases because of genetic and functional similarities of Malpighian (renal) tubules with the human kidney. Here, we demonstrated function of the sex-determining region Y protein-interacting protein-1 (Sip1) gene, an ortholog of human Na+/H+ exchanger regulatory factor (NHERF1), in Drosophila Malpighian tubules and its impact on nephrolithiasis. Abundant birefringent calculi were observed in Sip1 mutant flies, and the phenotype was also observed in renal stellate cell-specific RNA interference Sip1 knockdown in otherwise normal flies, confirming a renal etiology. This phenotype was abolished in rosy mutant flies (which model human xanthinuria) and by the xanthine oxidase inhibitor allopurinol, suggesting that the calculi were of uric acid. This was confirmed by direct biochemical assay for urate. Stones rapidly dissolved when the tubule was bathed in alkaline media, suggesting that Sip1 knockdown was acidifying the tubule. SIP1 was shown to collocate with Na+/H+ exchanger isoform 2 (NHE2) and with moesin in stellate cells. Knockdown of NHE2 specifically to the stellate cells also increased renal uric acid stone formation, and so a model was developed in which SIP1 normally regulates NHE2 activity and luminal pH, ultimately leading to uric acid stone formation. Drosophila renal tubules may thus offer a useful model for urate nephrolithiasis.

Regulation of renal NaDC1 expression and citrate excretion by NBCe1-A.

Citrate is critical for acid-base homeostasis and to prevent calcium nephrolithiasis. Both metabolic acidosis and hypokalemia decrease citrate excretion and increase expression of Na+-dicarboxylate cotransporter 1 (NaDC1; SLC13A2), the primary protein involved in citrate reabsorption. However, the mechanisms transducing extracellular signals and mediating these responses are incompletely understood. The purpose of the present study was to determine the role of the Na+-coupled electrogenic bicarbonate cotransporter (NBCe1) A variant (NBCe1-A) in citrate metabolism under basal conditions and in response to acid loading and hypokalemia. NBCe1-A deletion increased citrate excretion and decreased NaDC1 expression in the proximal convoluted tubules (PCT) and proximal straight tubules (PST) in the medullary ray (PST-MR) but not in the PST in the outer medulla (PST-OM). Acid loading wild-type (WT) mice decreased citrate excretion. NaDC1 expression increased only in the PCT and PST-MR and not in the PST-MR. In NBCe1-A knockout (KO) mice, the acid loading change in citrate excretion was unaffected, changes in PCT NaDC1 expression were blocked, and there was an adaptive increase in PST-MR. Hypokalemia in WT mice decreased citrate excretion; NaDC1 expression increased only in the PCT and PST-MR. NBCe1-A KO blocked both the citrate and NaDC1 changes. We conclude that 1) adaptive changes in NaDC1 expression in response to metabolic acidosis and hypokalemia occur specifically in the PCT and PST-MR, i.e., in cortical proximal tubule segments; 2) NBCe1-A is necessary for normal basal, metabolic acidosis and hypokalemia-stimulated citrate metabolism and does so by regulating NaDC1 expression in cortical proximal tubule segments; and 3) adaptive increases in PST-OM NaDC1 expression occur in NBCe1-A KO mice in response to acid loading that do not occur in WT mice.

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

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.

NBCe1-A Regulates Proximal Tubule Ammonia Metabolism under Basal Conditions and in Response to Metabolic Acidosis.

Renal ammonia metabolism is the primary mechanism through which the kidneys maintain acid-base homeostasis, but the molecular mechanisms regulating renal ammonia generation are unclear. In these studies, we evaluated the role of the proximal tubule basolateral plasma membrane electrogenic sodium bicarbonate cotransporter 1 variant A (NBCe1-A) in this process. Deletion of the NBCe1-A gene caused severe spontaneous metabolic acidosis in mice. Despite this metabolic acidosis, which normally causes a dramatic increase in ammonia excretion, absolute urinary ammonia concentration was unaltered. Additionally, NBCe1-A deletion almost completely blocked the ability to increase ammonia excretion after exogenous acid loading. Under basal conditions and during acid loading, urine pH was more acidic in mice with NBCe1-A deletion than in wild-type controls, indicating that the abnormal ammonia excretion was not caused by a primary failure of urine acidification. Instead, NBCe1-A deletion altered the expression levels of multiple enzymes involved in proximal tubule ammonia generation, including phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and glutamine synthetase, under basal conditions and after exogenous acid loading. Deletion of NBCe1-A did not impair expression of key proteins involved in collecting duct ammonia secretion. These studies demonstrate that the integral membrane protein NBCe1-A has a critical role in basal and acidosis-stimulated ammonia metabolism through the regulation of proximal tubule ammonia-metabolizing enzymes.

Reassessment of the Transport Mechanism of the Human Zinc Transporter SLC39A2.

The human zinc transporter SLC39A2, also known as ZIP2, was shown to mediate zinc transport that could be inhibited at pH <7.0 and stimulated by HCO3-, suggesting a Zn2+/HCO3- cotransport mechanism [Gaither, L. A., and Eide, D. J. (2000) J. Biol. Chem. 275, 5560-5564]. In contrast, recent experiments in our laboratory indicated that the functional activity of ZIP2 increases at acidic pH [Franz, M. C., et al. (2014) J. Biomol. Screening 19, 909-916]. The study presented here was therefore designed to reexamine the findings about the pH dependence and to extend the functional characterization of ZIP2. Our current results show that ZIP2-mediated transport is modulated by extracellular pH but independent of the H+ driving force. Also, in our experiments, ZIP2-mediated transport is not modulated by extracellular HCO3-. Moreover, a high extracellular [K+], which induces depolarization, inhibited ZIP2-mediated transport, indicating that the transport mechanism is voltage-dependent. We also show that ZIP2 mediates the uptake of Cd2+ ( Km ∼ 1.57 µM) in a pH-dependent manner ( KH+ ∼ 66 nM). Cd2+ transport is inhibited by extracellular [Zn2+] (IC50 ∼ 0.32 µM), [Cu2+] (IC50 ∼ 1.81 µM), and to a lesser extent [Co2+], but not by [Mn2+] or [Ba2+]. Fe2+ is not transported by ZIP2. Accordingly, the substrate selectivity of ZIP2 decreases in the following order: Zn2+ > Cd2+ ≥ Cu2+ > Co2+. Altogether, we propose that ZIP2 is a facilitated divalent metal ion transporter that can be modulated by extracellular pH and membrane potential. Given that ZIP2 expression has been reported in acidic environments [Desouki, M. M., et al. (2007) Mol. Cancer 6, 37; Inoue, Y., et al. (2014) J. Biol. Chem. 289, 21451-21462; Tao, Y. T., et al. (2013) Mol. Biol. Rep. 40, 4979-4984], we suggest that the herein described H+-mediated regulatory mechanism might be important for determining the velocity and direction of the transport process.

Expression of the B splice variant of NBCe1 (SLC4A4) in the mouse kidney.

Sodium-coupled bicarbonate transporters are critical for renal electrolyte transport. The electrogenic, sodium-coupled bicarbonate cotransporter, isoform 1 (NBCe1), encoded by the SLC4A4 geneencoded by the SLC4A4 gene has five multiple splice variants; the A splice variant, NBCe1-A, is the primary basolateral bicarbonate transporter in the proximal convoluted tubule. This study's purpose was to determine if there is expression of additional NBCe1 splice variants in the mouse kidney, their cellular distribution, and their regulation by metabolic acidosis. In wild-type mice, an antibody reactive only to NBCe1-A showed basolateral immunolabel only in cortical proximal tubule (PT) segments, whereas an antibody reactive to all NBCe1 splice variants (pan-NBCe1) showed basolateral immunolabel in PT segments in both the cortex and outer medulla. In mice with NBCe1-A deletion, the pan-NBCe1 antibody showed basolateral PT immunolabel in both the renal cortex and outer stripe of the outer medulla, and immunoblot analysis showed expression of a ~121-kDa protein. RT-PCR of mRNA from NBCe1-A knockout mice directed at splice variant-specific regions showed expression of only NBCe1-B mRNA. In wild-type kidney, RT-PCR confirmed expression of mRNA for the NBCe1-B splice variant and absence of mRNA for the C, D, and E splice variants. Finally, exogenous acid loading increased expression in the proximal straight tubule in the outer stripe of the outer medulla. These studies demonstrate that the NBCe1-B splice variant is present in the PT, and its expression increases in response to exogenous acid loading, suggesting it participates in the PT contribution to acid-base homeostasis.

Prostatic collagen architecture in neutered and intact canines.

BACKGROUND: Prostate stiffness and increased collagen content both associate with the presence of urinary symptoms in men but mechanisms responsible, including impact of age and androgens, are unknown. Dogs develop prostate-related urinary dysfunction similar to humans, but mechanisms are also unknown. Mice have been used to examine how prostatic collagen accumulation affects voiding but whether mouse prostatic collagen organization resembles human or dog has not been evaluated. Here, we have constructed the first comprehensive, comparative maps of collagen architecture in canine, human, and mouse prostate and test whether canine prostatic collagen content is increased by aging and reduced by castration. METHODS: Complete transverse prostate sections were stained with picrosirius red and imaged with confocal microscopy to reveal and compare collagen architecture across species. Canine prostatic collagen fiber length, diameter, and density in prostatic urethral, periurethral, peripheral, and capsular regions were quantified and compared among four experimental groups: young intact, young neutered, old intact, and old neutered dogs. RESULTS: Surprisingly, the majority of collagen was localized to the prostatic urethra in canine, human, and mouse. In canine and human, capsular regions also featured a dense collagen network but it appeared less dense than around prostatic urethra. Older, intact male canines exhibited overall denser prostate collagen fibers and had thicker capsular fibers than young, intact males. Prostatic glandular regions undergo dramatic atrophy and regression following castration, and our finding of neutered animals having increased collagen fiber density in both periurethral and peripheral regions is consistent with glandular contraction and increased proportion of stroma. CONCLUSIONS: Collagen architecture in dog appears similar to that in humans when cross sections are compared side-by-side. Canine collagen organization is affected by both age and androgen status, suggesting these factors may contribute to collagen accumulation in some males.

Transport proteins NHA1 and NHA2 are essential for survival, but have distinct transport modalities.

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.

Na+ /H+ exchange via the Drosophila vesicular glutamate transporter mediates activity-induced acid efflux from presynaptic terminals.

KEY POINTS: Intracellular pH regulation is vital to neurons as nerve activity produces large and rapid acid loads in presynaptic terminals. Rapid clearance of acid loads is necessary to maintain control of neurotransmission, but neuronal acid clearance mechanisms remain poorly understood. Glutamate is loaded into synaptic vesicles via the vesicular glutamate transporter (VGLUT), a mechanism conserved across phyla, and this study reports a previously unknown role for VGLUT as an acid-extruding protein when deposited in the plasmamembrane during exocytosis. The finding was made in Drosophila (fruit fly) larval motor neurons through a combined pharamacological and genetic dissection of presynaptic pH homeostatic mechanisms. A dual role for VGLUT serves to integrate neuronal activity and pH regulation in presynaptic nerve terminals. ABSTRACT: Neuronal activity can result in transient acidification of presynaptic terminals, and such shifts in cytosolic pH (pHcyto ) probably influence mechanisms underlying forms of synaptic plasticity with a presynaptic locus. As neuronal activity drives acid loading in presynaptic terminals, we hypothesized that the same activity might drive acid efflux mechanisms to maintain pHcyto homeostasis. To better understand the integration of neuronal activity and pHcyto regulation we investigated the acid extrusion mechanisms at Drosophila glutamatergic motorneuron terminals. Expression of a fluorescent genetically encoded pH indicator, named 'pHerry', in the presynaptic cytosol revealed acid efflux following nerve activity to be greater than that predicted from measurements of the intrinsic rate of acid efflux. Analysis of activity-induced acid transients in terminals deficient in either endocytosis or exocytosis revealed an acid efflux mechanism reliant upon synaptic vesicle exocytosis. Pharmacological and genetic dissection in situ and in a heterologous expression system indicate that this acid efflux is mediated by conventional plasmamembrane acid transporters, and also by previously unrecognized intrinsic H+ /Na+ exchange via the Drosophila vesicular glutamate transporter (DVGLUT). DVGLUT functions not only as a vesicular glutamate transporter but also serves as an acid-extruding protein when deposited on the plasmamembrane.

Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis.

Epithelia frequently segregate transport processes to specific cell types, presumably for improved efficiency and control. The molecular players underlying this functional specialization are of particular interest. In Drosophila, the renal (Malpighian) tubule displays the highest per-cell transport rates known and has two main secretory cell types, principal and stellate. Electrogenic cation transport is known to reside in the principal cells, whereas stellate cells control the anion conductance, but by an as-yet-undefined route. Here, we resolve this issue by showing that a plasma membrane chloride channel, encoded by ClC-a, is exclusively expressed in the stellate cell and is required for Drosophila kinin-mediated induction of diuresis and chloride shunt conductance, evidenced by chloride ion movement through the stellate cells, leading to depolarization of the transepithelial potential. By contrast, ClC-a knockdown had no impact on resting secretion levels. Knockdown of a second CLC gene showing highly abundant expression in adult Malpighian tubules, ClC-c, did not impact depolarization of transepithelial potential after kinin stimulation. Therefore, the diuretic action of kinin in Drosophila can be explained by an increase in ClC-a-mediated chloride conductance, over and above a resting fluid transport level that relies on other (ClC-a-independent) mechanisms or routes. This key segregation of cation and anion transport could explain the extraordinary fluid transport rates displayed by some epithelia.

Insulin and SGK1 reduce the function of Na+/monocarboxylate transporter 1 (SMCT1/SLC5A8).

SMCTs move several important fuel molecules that are involved in lipid, carbohydrate, and amino acid metabolism, but their regulation has been poorly studied. Insulin controls the translocation of several solutes that are involved in energetic cellular metabolism, including glucose. We studied the effect of insulin on the function of human SMCT1 expressed in Xenopus oocytes. The addition of insulin reduced α-keto-isocaproate (KIC)-dependent 22Na+ uptake by 29%. Consistent with this result, the coinjection of SMCT1 with SGK1 cRNA decreased the KIC-dependent 22Na+ uptake by 34%. The reduction of SMCT1 activity by SGK1 depends on its kinase activity, and it was observed that the coinjection of SMCT1 with S442D-SGK1 (a constitutively active mutant) decreased the KIC-dependent 22Na+ uptake by 50%. In contrast, an SMCT1 coinjection with K127M-SGK1 (an inactive mutant) had no effect on the KIC-dependent Na+ uptake. The decreasing SMCT1 function by insulin or SGK1 was corroborated by measuring [1-14C]acetate uptake and the electric currents of SMCT1-injected oocytes. Previously, we found that SMCT2/Slc5a12-mRNA, but not SMCT1/Slc5a8-mRNA, is present in zebrafish pancreas (by in situ hybridization); however, SLC5a8 gene silencing was associated with the development of human pancreatic cancer. We confirmed that the mRNA and protein of both transporters were present in rat pancreas using RT-PCR with specific primers, Western blot analysis, and immunohistochemistry. Additionally, significant propionate-dependent 22Na+ uptake occurred in pancreatic islets and was reduced by insulin treatment. Our data indicate that human SMCT1 is regulated by insulin and SGK1 and that both SMCTs are present in the mammalian pancreas.