Romero, Michael F.

Mutations in the 2Cl ‐ /H + transporter CLC‐5 cause Dent Disease Type 1 (DD1) and lead to progressive renal failure by age 20‐40. A major characteristic of DD1 is the renal mishandling of Ca 2+ that increases urinary Ca 2+ excretion, calcium oxalate (CaOx) kidney stones, and kidney calcification. Interestingly, none of the DD1 symptoms include or relate to Cl ‐ homeostasis despite impaired transport function. Thus, how does a Cl ‐ transporter cause Ca 2+ dysregulation? Our laboratory has identified Drosophila Clc‐c to be the homolog of CLC‐5 with conserved amino acids at DD1 mutation sites. We hypothesize that Clc‐c shares functional similarities in Cl ‐ transport and Ca 2+ homeostasis. To test our hypothesis, we used electrophysiology to measure ion transport, and then assessed CaOx crystal formation in adult fly renal tubules (Malpighian tubules, MTs) of Drosophila . Voltage clamp experiments with Xenopus oocytes show that Clc‐c is electrogenic with the same outward‐rectifying current in Cl ‐ solutions as observed with human CLC‐5. Next, knockdown flies (Clc‐c‐KD) were generated by crossing Uro‐Gal4 (MT driver) with UAS‐Clc‐c‐RNAi flies, resulting in 50% Clc‐c mRNA expression in the MTs. Crystals in adult MT (7 days after eclosure) were present in 100% of Clc‐c‐KD compared to 71% of control flies. The average number of crystals in adult tubule pairs was significantly greater in Clc‐c‐KD flies (15±4 crystals per pair) than control flies (5±2 crystals, P= 0.02). When fed a diet with 2mM NaOx ClcC‐KD for 4 days, Clc‐c‐KD flies had a significantly greater number of crystals in each tubule (24±5 crystals) than controls (4±2 crystals, P= 0.005). However, the KD flies did not produce more crystals than control flies when fed a supraphysiological concentration of sodium oxalate (20 mM NaOx diet) for 1 or 4 days and in ex vivo assays of dissected MTs submerged in solution containing 10 mM NaOx for 1 hour. In conclusion, Drosophila Clc‐c has both identical sequences at DD1 mutation sites and similar biophysical functions to human CLC‐5. Furthermore, Clc‐c‐KD increases incidence of CaOx crystals which corresponds to the increased risks for kidney stones in DD1 patients. With this fly‐DD1 model, we will be able to assess the relationship of Clc‐c Cl ‐ transport to renal Ca deposits. Future experiments will evaluate renal pathophysiology of DD1 mutations and their relationship to Ca 2+ transport in Clc‐c transgenic flies.

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 .

Electrical slow wave activity, generated by interstitial cells of Cajal (ICC), is essential for efficient gastrointestinal (GI) transit. Electrophysiology of slow waves has been extensively studied; inclusion of bicarbonate (HCO 3 − ) in extracellular solutions is required for recording normal slow wave activity. HCO 3 − transport contributes to regulation of intracellular pH, which has multiple consequences for normal cellular function. Previous transcriptional profiling found that the Na + / HCO 3 − cotransporter (NBCe1/Slc4a4) is enriched in pacemaker myenteric ICC (ICC‐MY) compared to non pacemaker ICC in the deep muscular plexus (ICC‐DMP) of the small intestine. Our aim was to determine the distribution and identity of Slc4a4/NBCe1 variants in ICC in the GI tract. NBCe1 immunoreactivity (IR) was detected by fluorescence labeling of parafomaldehyde‐fixed cross sections and whole mounts of adult mouse small intestine, colon and stomach using primary antibodies directed against a 329 amino acid sequence in the N terminus of the NBCe1 protein. Labeling of Kit receptor tyrosine kinase with a goat anti‐Kit polyclonal antibody was used to detect ICC. Slc4a4 knockout (KO) mice were bred from heterozygous founders. Slc4a4 fl/fl mice were bred with ETV1 CreERT2/− mice to allow targeted, conditional KO of Slc4a4 in ICC in response to tamoxifen treatment (130 mg/Kg). mRNA transcripts for Slc4a4 in Kit expressing cells were isolated from Kit‐ribotag mice, which were generated by breeding Kit creERT2 with Rpl22 tm1.1Psam mice. The abundance of specific Slc4a4 transcripts was measured by quantitative real time PCR (qRT‐PCR). Green fluorescent protein (GFP)‐labeled ICC were dissociated from the small intestine of neonatal Kit copGFP mice and RNA expression levels were determined using the Single Cell‐to‐CT™ qRT‐PCR Kit from Ambion. NBCe1‐IR was absent in all cells in Slc4a4‐KO tissues but co‐localized with Kit in ICC‐MY, but not in ICC‐DMP, in small intestine of wild type mice. In the proximal and distal colon, pacemaker submucosal ICC (ICC‐SMP) but not intramuscular ICC (ICC‐IM) or ICC‐MY showed NBCe1‐IR. In the corpus and antrum of the stomach NBCe1‐IR was detected in ICC‐MY but not in ICC‐IM. NBCe1‐IR did not co‐localize with Kit in the gastric fundus. mRNA for variant C, but not variants A or B of Slc4a4, was detected in small intestinal ICC‐MY. Reduced NBCe1‐IR was restricted to small intestinal ICC‐MY of Slc4a4 conditional KO mice. NBCe1 is enriched in subtypes of ICC responsible for pacemaker activity in all regions of the GI tract indicating a role for this transporter in slow wave generation. The presence of the C variant of Slc4a4 in ICC‐MY suggests dynamic regulation of NBCe1 activity in ICC by the I P 3 R b inding protein released with i nositol 1, 4, 5‐ t risphosphate (IRBIT). Support or Funding Information Supported by NIH R01 DK57061. 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 .

Marine teleost fish precipitate divalent cations as carbonate deposits in their intestine to minimize the potential for excessive calcium entry and to stimulate water absorption by removing osmolyte ions. This carbonate deposit formation therefore plays an important role in osmoregulation in the seawater environment and also requires controlled secretion of HCO 3 − to match the amount of calcium entering the intestinal lumen. Despite this physiological importance, the process of carbonate precipitate formation has not been characterized at the molecular level. We first isolated 24 candidate cDNA clones representing all possible membrane proteins capable of transporting HCO 3 − from the euryhaline pufferfish, mefugu Takifugu obscurus based on the DNA sequence information on the T. rubripes genome, then performed Northern blot analyses to select clones highly expressed in the intestine of seawater mefugu, and finally determined the cellular locations by immunohistochemistry. Based on these observations, we proposed a model for HCO 3 − excretion across the intestinal epithelium of marine teleost fish that includes a basolateral NBCe1 (Na + /bicarbonate cotransporter) and apical Slc26a6 (Cl − /HCO 3 − exchangers).