Rossano, Adam J.

Nephrolithiasis is a painful and costly healthcare complication. The most common kidney stones are composed of calcium oxalate and thus renal handling of oxalate is an important facet of understanding the pathogenesis of nephrolithiasis. Recently, the Drosophila melanogaster Malpighian tubule (MT) has emerged as a robust model of trans‐epithelial ion transport and nephrolithiasis as MTs readily form luminal calcium‐oxalate crystals in the presence of oxalate. Drosophila Prestin (dPrestin, Slc26a6) transports oxalate across the apical surface of the MT into the lumen but a full model of the trans‐epithelial movement of oxalate in the Drosophila MT has been lacking as the basolateral oxalate transporter has remain uncharacterized. The objective of this work was to identify and characterize the Drosophila basolateral oxalate transporter through ex vivo real‐time quantification of intracellular pH (pH i ). pH i was monitored by using the GAL4/UAS system to selectively express pHerry, a pseudo‐ratiometric genetically‐encoded pH indictor (GEpHI) in the cytosol of the principal cells of the MT. Calibration of pHerry in extracted adult MTs yielded a p K a of 7.24. Basolateral perfusion of MTs in CO 2 /HCO 3 − ‐buffered solution produced a large acidification followed by rapid recovery in all three functionally distinct segments of the anterior MT (initial, transitional, and main). Recovery was likely due to HCO 3 − loading by HCO 3 − transporters and this process could be interrupted by basolateral application of 1mM oxalate (in the initial and transitional segments) or 1mM SO 4 2− (in all three segments). The response was most robust in the transitional segment and oxalate induced a more rapid acidification than SO 4 2− ( dpH i / dt = −0.60 ± 0.09 pH units min −1 compared to −0.06 ± 0.01). A putative ortholog of the mammalian renal basolateral oxalate transporter (SAT 1; Slc26a1) was identified through sequence homology. Tissue specific knock‐down of this protein with interference RNA (RNAi) reduced the rate of acid‐loading in the transitional segment of the MT with regard to oxalate but not SO 4 2− ( dpH i / dt = −0.34 ± 0.06 pH units min −1 ; p = 0.03 compared to controls, and −0.06 ± 0.01; p = 0.94 compared to controls for oxalate and SO 4 2− respectively). These data indicate this protein is likely the Drosophila basolateral MT oxalate transporter and the basolateral movement of oxalate is functionally coupled to movement of acid equivalents, potentially as oxalate/HCO 3 − exchange, oxalate/OH − exchange, or oxalate/H + co‐transport. Support or Funding Information Funding: T32‐DK007013 to AJR, GM075148 (JBA), DK092408 and DK100227 to MFR.

The electrogenic Na + /HCO 3 − cotransporter (NBCe1/SLC4A4) maintains systemic acid/base balance by reabsorbing HCO 3 − in the renal proximal tubule but also contributes to intracellular pH (pH i ) regulation in the eye, nervous system, pancreas, and intestine. Human recessive mutations lead to diverse phenotypes (glaucoma, proximal renal tubular acidosis, poor dentition) and mouse models suggest some organ manifestations are partially penetrant. The objective of this study was to develop a full spectrum genetically‐encoded live‐imaging toolkit which could be adapted to assay NBCe1‐mediated pH i regulation across a variety of cell types to further characterize how NBCe1 recessive mutations and polymorphisms functionally interact with each other and wild‐type (WT) NBCe1 to produce alterations in cellular acid/base physiology. WT human NBCe1 (WT‐NBCe1) and a recently characterized human recessive mutation (Mut‐NBCe1) were tagged with GFP. Each construct was co‐expressed via transfection in human immortalized trabecular meshwork cells (TM5) with “pHire”, a red‐shifted (556/630nm ex/em) pH‐sensitive variant of mApple, to assay NBCe1 function through real‐time quantification of pH i via widefield epifluorescent live‐imaging. pHire successfully reported pH i of TM5 cells in response to H + loading with NH 4 Cl pulses. Calibration of pHire revealed an in vitro p K a of 7.44, a 5‐fold linear fluorescence response from pH 6.5 to 8.0, and stability following acid loads (pH 5.0) and long imaging protocols (30 min). pH i of TM5 cells fell in response to CO 2 /HCO 3 − — buffered solution and displayed a modest Na + ‐dependent recovery ( dpH i / dt = 0.02 ± 0.01 pH units min −1 in CO 2 /HCO 3 − , −0.06 ± 0.02 in Na + ‐free), consistent with intrinsic TM5‐cell NBCe1 activity. Transfection of WT‐NBCe1 did not consistently alter HCO 3 − recovery but accelerated pH i decrease in Na + ‐free solution by 10‐fold (1017 ± 3%) relative to neighboring untransfected cells. Transfection with E91R‐NBCe1 failed to similarly potentiate acidification upon Na + withdrawal (117 ± 81%), and transfection with Mut‐NBCe1 suppressed Na + ‐dependent acidification to 72 ± 28% of untransfected cells. These data suggest that although both Mut‐NBCe1 and E91R‐NBCe1 are functionally “transporter‐null” they possess different capacities to impair endogenous WT‐NBCe1, possibly through dimerization and localization deficits. Such differences could manifest discretely in specific cell types and may underlie the partial penetrance of some NBCe1‐associated phenotypes. Support or Funding Information Funding: T32‐DK007013 to AJR, DK092408, DK100227 and R25‐DK101405 to MFR.

Intracellular pH regulation is vital for normal cellular function and is controlled by membrane H + and HCO 3 ‑ transporters. Most pH reporters have green fluorescence (i.e., BCECF, pHlorin) making simultaneous Ca 2+ , Cl ‐ , or other sensing (i.e., green fluorescent sensors) or GFP‐tags impossible. We previously reported a red‐fluorescent pH sensor ( pHire ) with an alkaline‐shifted pKa that increases fluorescence with increasing intracellular pH. pHire was transiently transfect into TM5 cells, but these fibroblast‐like cells did not address continuous expression in epithelial or excitable cells. Our objective was to evaluate pHire responses in multiple cell‐types and show that it can be used for fluorescent multiplexing. We selected pHire‐expressing rat pancreatic β‐cells (INS1 pHire ) and porcine kidney epithelial cells (LLCPK1 pHire ), and then FACS sorted to keep the top 10‐20% fluorescent cells. To test pHire’s usefulness for intact animals, we made transgenic, UAS‐pHire Drosophila , allowing pHire expression in specific tissues via genetic crossing with promoter‐GAL4 lines. By using perfusion chambers, we monitored cellular pHire‐responses to acid‐loading (NH 4 Cl pulse) and CO 2 /HCO 3 ‑ addition. Stable selection of pHire cell lines ensures single cells as well as contiguous cell layers for acid‐base transporter evaluation. Addition of NH 4 Cl quickly alkalinizes all pHire cells. Different acid‐recovery rates were found in individual cells with LLCPK1 pHire cells recovering faster than INS1 pHire cells. CO 2 /HCO 3 ‑ addition acidified both mammalian cell‐types. LLCPK1 pHire cells typically show a Na + dependent recovery (consistent with electrogenic Na + bicarbonate cotransporter, NBCe1, activity) that is absent from INS1 pHire cells. Infection of INS1 pHire cells with a NBCe1A‐GFP AAV allowed clear demonstration of NBCe1 activity. To test pHire’s stability in living animals, we next generated a UAS‐pHire transgenic Drosophila line, allowing pHire expression in specific tissues via genetic crossing with various promoter‐GAL4 lines. We tested renal epithelial (principal cells: Uro‐GAL4, CapaR‐GAL4; stellate cells: c724‐GAL4) and CNS (elav‐GAL4) expression. As observed in mammalian cells, NH 4 Cl acid‐loading showed robust and more uniform non‐HCO 3 ‑ recovery. The pH‐calibration curves for pHire in Drosophila cells were similar those obtained in mammalian cells (pKa>7.4). Thus, pHire characteristics are not changed by continuous expression in whole organisms. This study shows that the pHire sensor can be constitutively expressed into multiple cell lines or whole animals (flies) with an ideal pH range of pH 6 to 9. pHire may also be multiplexed with GFP labels and biosensors to allow real‐time, pH measurements with other ions of interest at a cellular scale. Finally, our data demonstrate that genetically encoded pHire is a reliable and useful tool for studying acid‐base transporters in a variety of cell‐types.

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. AbstractNeuronal 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.