Holmes, Heather L.

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.

NBCe1 is an electrogenic Na + bicarbonate cotransporter expressed as three isoforms. NBCe1A is mainly found in the kidney (100% activity), NBCe1B is a general isoform (~25% activity), and NBCe1C is found in the nervous system (20% activity). This project was to characterize mice with loss of NBCe1 in the kidney and urogenital tract and to test isoform activity ± IRBIT. Previous work has shown that NBCe1‐B is activated when IRBIT is present and interacts with the N‐terminus (Nt). Even though NBCe1C shares this Nt, the effects of IRBIT on human NBCe1C are unknown. Interstitial Cells of Cajal (ICC)pacemaker cells in the gastrointestinal tract ( c‐kit + ) have NBCe1C, IRBIT and require bicarbonate for normal gut activity. Developmentally similar ICC cells are found in the urogenital tract; however, NBCe1C locations within the kidney and urogenital tract are unknown. Using c‐kit ‐copGFP and NBCe1A‐knockout mice, kidneys, ureters, prostate, bladder, and urethra were dissected and analyzed via direct imaging, sectioning, and immunofluorescence. Oocytes injected with NBCe1C ± IRBIT were voltage clamped to determine the cotransporter's activity. “Strings” of c‐kit + ‐cells were found in the copGFP & nbce1A kidneys. In nbce1 (+/−) ‐(Het) mice (Figure), prostate ducts appeared enlarged, and in nbce1A − / − kidney, c‐kit protein was broadly expressed, showing cluster colocalization with NBCe1C. NBCe1C is not obviously expressed in WT or copGFP mice. Electrophysiology data showed that IRBIT activates NBCe1C by ~10‐fold. As c‐kit is also a marker of stem cells, NBCe1C/c‐kit association and c‐kit increase in nbce1A − / − mice, could suggest that NBCe1A loss elicits kidney repair itself (proliferation) and causes expression of other NBCe1‐isoforms. Support or Funding Information R25‐DK101405, R01‐DK057061, U54‐DK100227 (Mayo) and U54‐DK104310 (UW‐Madison) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Dysregulation of glucose homeostasis in Type 2 diabetes mellitus (T2DM) is attributed to pancreatic β‐cell failure in maintaining adequate glucose‐stimulated insulin secretion (GSIS) to compensate for diminished insulin action. Recent single cell transcriptomics studies of human pancreatic islets have identified Slc4a4 (encoding an electrogenic Na + ‐nHCO 3 − cotransporter, NBCe1) as a gene repressed in healthy non‐diabetic β‐cells, but expressed in human T2DM β‐cells. However the role of Slc4a4 /NBCe1 in pancreatic β‐cell function remains unknown. Thus, in the current study we set out to test the hypothesis that mis‐expression of Slc4a4 in β‐cells contributes to loss of GSIS and impaired glucose homeostasis in T2DM. Subsequently, we first assessed Slc4a4 mRNA expression in isolated human islets (Control vs. T2DM) by qPCR and quantified NBCe1 protein expression by immunofluorescence in autopsy‐derived human pancreas tissue (Control vs. T2DM). We observed a 4‐fold induction in Slc4a4 mRNA expression in T2DM islets compared to non‐diabetic controls ( p =0.004 vs. Control) and significantly increased NBCe1 co‐localization with insulin‐positive β‐cells of T2DM patients compared to controls ( p <0.001 vs. Control). In addition, we also demonstrated that chronic inhibition of NBCe1 activity using the channel specific inhibitor (S0859) significantly enhanced GSIS in islets isolated from T2DM patients (~1.5‐fold increase vs. untreated, p =0.006). To delineate the role NBCe1 plays on β‐cell function in vivo , we generated β‐cell specific knockout ( β‐Slc4a4 −/− ) mice by crossing Slc4a4 fl/fl (G. Shull, UC) and Ins2‐Cre mouse models. We confirmed >50% reduction in Slc4a4 mRNA expression in β‐Slc4a4 −/− islets and complete ablation of NBCe1 protein expression. In order to model the environmental stress that leads to metabolic defects common to T2DM, we exposed β‐Slc4a4 −/− mice and control littermates to either standard chow diet or 60% high fat diet (HFD) for 8 weeks ad libitum . Under chow‐fed conditions, β‐Slc4a4 −/− mice displayed normal glucose tolerance ( p >0.05 vs. β‐Slc4a4 +/+ Chow). Interestingly, β‐Slc4a4 −/− mice exposed to HFD exhibited enhanced glucose tolerance and GSIS ( p <0.05 vs. β‐Slc4a4 +/+ HFD) and thus were protected from development of T2DM phenotype characteristics of HFD treatment. Finally, to understand the mechanism by which Slc4a4 deletion enhanced β‐cell function, we performed RNA‐sequencing on islets isolated from β‐Slc4a4 −/− and β‐Slc4a4 +/+ mice exposed to chow diet or HFD. Notably, gene‐set enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) defined biological pathways revealed that Slc4a4 deletion enriched genes associated with the cell cycle ( p <0.001 vs. β‐Slc4a4 +/+ Chow; p <0.001 vs. β‐Slc4a4 +/+ HFD), while suppressing genes associated with apoptosis ( p =0.002 vs. β‐Slc4a4 +/+ Chow; p =0.004 vs. β‐Slc4a4 +/+ HFD) in the presence of both chow diet and HFD. These result suggest that mis‐expression of Slc4a4 plays a novel role in the induction of β‐cell failure in T2DM. Future studies will delineate exact molecular mechanisms underlying NBCe1‐mediated regulation of β‐cell function in health and under conditions of metabolic stress. Support or Funding Information Mayo Clinic Center of Biomedical Discovery This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Mutations in the electrogenic Na + /HCO 3 − cotransporter (NBCe1) result in proximal renal tubular acidosis, glaucoma and cataracts in patients and are only recessive. Parents and siblings of these affected individuals are asymptomatic though their tissues should make some of the 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. For our studies we chose two severe NBCe1 mutations (<10% WT function individually): S427L (naturally occurring) and E91R (for N‐terminal structure studies). When we co‐expressed S427L and E91R, we measured ~55% wild‐type function which can only occur if the S427L‐E91R heterodimer is the functional unit. To further investigate this functional dimerization, we have engineered concatemeric hkNBCe1 constructs. When the hkNBCe1 tandem‐dimer (WT‐WT) cRNA was expressed in Xenopus oocytes, the electrophysiology data demonstrate the WT‐WT homodimer functions as a WT hkNBCe1. The electrophysiology data also showed that the WT‐S427L or WT‐E91R heterodimers functioned very similarly to WT hkNBCe1. Intriguingly, we observed ~50% WT function in both the S427L‐E91R and E91R‐S427L heterodimers constructs. These data illuminate why the "NBCe1 phenotype" only occurs recessively. An eYFP fragment (1‐158, YFP N ) was fused to the N‐ or C‐terminus of hkNBCe1 and another eYFP fragment (159‐238, YFP C ) was fused to N‐ or C‐terminus of hkNBCe1. The electrophysiology data demonstrate the eYFP‐hkNBCe1 fusion proteins have WT hkNBCe1 like function. The hkNBCe1 dimerization can also be visualized by biomolecular fluorescence complementation (BiFC) method. We plan to further use these molecular tools to probe the nature of the hkNBCe1 dimer interactions. Supported by EY017732.

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.

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.

Polycystic kidney disease (PKD) is an inherited disorder characterized by renal cyst formation and enlargement of the kidney. PKD severity can be staged noninvasively by measuring total kidney volume (TKV), a promising biomarker that has recently received regulatory qualification. In preclinical mouse models, where the disease is studied and potential therapeutics are evaluated, the most popular noninvasive method of measuring TKV is magnetic resonance imaging (MRI). Although MRI provides excellent 3D resolution and contrast, these systems are expensive to operate, have long acquisition times, and, consequently, are not heavily used in preclinical PKD research. In this study, a new imaging instrument, based on robotic ultrasound (US), was evaluated as a complementary approach for assessing PKD in rodent models. The objective was to determine the extent to which TKV measurements on the robotic US scanner correlated with both in vivo and ex vivo reference standards (MRI and Vernier calipers, respectively). A cross-sectional study design was implemented that included both PKD-affected mice and healthy wild types, spanning sex and age for a wide range of kidney volumes. It was found that US-derived TKV measurements and kidney lengths were strongly associated with both in vivo MRI and ex vivo Vernier caliper measurements ( R 2 =0.94 and 0.90, respectively). In addition to measuring TKV, renal vascular density was assessed using acoustic angiography (AA), a novel contrast-enhanced US methodology. AA image intensity, indicative of volumetric vascularity, was seen to have a strong negative correlation with TKV ( R 2 =0.82), suggesting impaired renal vascular function in mice with larger kidneys. These studies demonstrate that robotic US can provide a rapid and accurate approach for noninvasively evaluating PKD in rodent models.

Anoctamins typically function as Ca 2+ activated Cl − channels (CaCC) or as specific, phospholipid scramblases. GWAS of miniature schnauzers (>40% with recurrent calcium oxalate (CaOx) kidney stones) revealed a risk locus including an anoctamin 4 (ANO4) variant. ANO4 in mammals was initially reported as a phosphatidylserine (PS) scramblase. Urinary extracellular vesicles (EV) show decreased ANO4 protein in human stone formers. However, in Drosophila renal structures (Malpighian tubules, MTs), subdued (the ANO4‐homolog), functions as both a CaCC and scramblase. Since subdued also functions to defend against gram‐negative bacteria, it was knocked down to determine whether CaOx crystals were altered by bacterial infection. Subdued was knockdown (KD) in MT principle cells by crossing Uro:Gal4 flies with UAS: subdued ‐RNAi flies. F1‐flies were evaluated using CaOx crystallization in dissected MTs as well as after 4d NaOx feeding protocols. To evaluate infection, uropathic E. coli (UPEC):eGFP were fed to flies or added to NaOx solution before an overnight time‐lapse. Interestingly, the canine ANO4 variant has a 0.01 allele variant frequency at a residue conserved in humans, canines and Drosophila . Rapid crystallization experiments show that MTs with subdued‐KD have fewer CaOx crystals than control MTs. However, prolonged NaOx feeding assays showed that subdued‐KD in MTs produces enlarged CaOx crystals. These feeding assays also showed an unexpected phenotype: crystal clustering and matting structures (e.g., bacterial biofilms). These structures have similar patterning to human kidney stones [Sivaguru et al., PMID: 30213974]. Following a four day feeding of NaOx, when UPEC + NaOx is introduced overnight ( ex vivo ), a three hour bacterial event occurs, causing crystals to more tightly pack. Finally, when UPEC is introduced during a rapid crystallization experiment, the bacteria heavily localize in subdued‐KD MT lumens while control MTs have little eGFP‐fluorescence. Thus, UPEC facilitates CaOx crystal aggregates in subdued‐KD MTs while the nature of this interaction is unknown. In human kidney, ANO4 localizes in thick ascending limb (NKCC2 + ), while in murine kidney localizes more proximal in the Loop of Henle (UMOD + ). As mice are resistant to CaOx stones, these slight location differences may reveal altered TAL function by ANO4. This Drosophila avatar indicates that subdued (ANO4) normally limits CaOx crystal aggregation and limits UPEC growth. As an apparent pathogenic amino acid is conserved in mammals and flies; subdued’ s role in fly bacterial infection suggests an additional ANO4 role in human kidney stone formation. These results indicate that examining antibacterial treatments with UPEC infection may allow an antibiotic approach to more efficiently eliminate kidney stones. Future experiments determine what functional aspect of subdued alters CaOx crystallization. Moreover, this simple kidney stone avatar‐the fly – continues to provide new and mechanistic insights to CaOx stone formation. Support or Funding Information Support: Oxalosis & Hyperoxaluria Foundation, R25‐DK101405, Mayo Foundation Drosophila MT (Uro:Gal4 x UAS‐ subdued ‐RNAi) viewed with (A) DIC for CaOx birefringence, (B) fluorescence to visualize UPEC:eGFP and (C) merged to show coincidence of UPEC with CaOx crystals. The red line indicates the length of the CaOx/UPEC mass. This is not observed in control MTs. Figure 1

Dipteran Na + ‐dependent cation‐chloride cotransporters (CCCs) group into two clades. Drosophila melanogaster Ncc69 is a bona fide Na + ‐K + ‐Cl − cotransporter and has a single ortholog in Aedes aegypti (aeNKCC1). Drosophila Ncc83 has not been physiologically characterized and has two orthologs in Ae. aegypti (aeCCC2 and aeCCC3). Previous work showed that oocytes expressing aeCCC2 have higher Na + conductances than controls. Moreover, in flux assays using Li + as a Na + tracer, Li + transport by aeCCC2 was not dependent upon extracellular Cl − and not inhibited by the loop diuretics furosemide or bumetanide. These are surprising results, since previously characterized CCCs are electroneutral, Cl − ‐dependent, and sensitive to the loop diuretics. We have further characterized aeCCC2 and performed an initial analysis of Ncc83, using two‐electrode voltage clamp. Following a hypotonic pre‐incubation, changes in membrane potential upon Na + replacement were 20 ± 3 mV in Ncc83 and 26 ± 4 mV in aeCCC2, approximately two‐fold greater than before swelling. Na + ‐dependent currents in oocytes expressing aeCCC2 depended on extracellular Na + with saturable Michaelis‐Menton kinetics. The apparent affinity for Na + was 4.6 ± 0.5 mM (mean, SEM, n=5). To characterize the function of Ncc83 in Drosophila Malpighian tubules (MT), we developed procedures to assess the cation concentrations of secreted fluid in Ramsay secretion assays using cation‐exchange chromatography. The physiological cations were readily detectable in 10,000–50,000 fold dilutions of secreted fluid, and Li + and Rb + were also detectable when added to the bathing saline as tracers for Na + and K + . In w118‐control flies, fluid secreted by MT measured using these procedures had a [K + ] of 104.1 ± 8.8 mM (mean, SEM, n=7) and [Na + ] of 61.2 ± 7.8 mM. In preliminary experiments, [K + ] was 112.7 ± 1.3 mM (n=4) and [Na + ] was 48.2 ± 4.1 mM in fluid secreted by MTs of flies with MT‐specific knockdown of Ncc83 using the GAL4‐UAS RNAi system. Further work is needed to determine the physiological roles of Ncc83 and aeCCC2. Funding: American Physiological Society Research Career Enhancement Award, NIH F33 GM131599, and Kenyon College. Support or Funding Information Funding: American Physiological Society Research Career Enhancement Award, NIH F33 GM131599, and Kenyon College.