Holmes, Heather L.

SLC26A9 is a Cl - channel and electrogenic Cl - -HCO 3 ‑ exchanger found in several human epithelia, including the lung, intestine / GI tract, and kidney. SLC26 proteins are defined by a C-terminal, cytoplasmic sulfate transporter / anti-σ-domain (STAS). In mammals, several STAS domains have protein-protein interactions with the regulatory-region of CFTR (R-CFTR), the Cl - channel associated with cystic fibrosis. However, the functional consequences, i.e., regulatory effects (up/downregulation), of STAS-to-R-CFTR binding seem to differ by SLC26 member. Our prior work showed that the SLC26A9:CFTR interaction decreases SLC26A9 channel and exchange activity. If SLC26A9-STAS was replaced with SLC26A6-STAS, no inhibition of SLC26A9 conductance occurred when interacting with R-CFTR (PMID 19643730). Complete deletion of SLC26A9-STAS removed Cl - conductance. Thus, we hypothesized that a disordered loop within STAS is the likely interaction area, and therefore deleted this STAS-disordered loop (ΔLoop) to test this region’s functionality. We used a voltage clamp to measure Cl - currents (IV; conductance G calculated) in Xenopus laevis oocytes (n=10 / construct) injected with cRNA of ΔLoop and subcomponent areas of SLC26A9. Each construct contained a 3xFLAG tag such that stained oocytes from these constructs allowed determination of membrane expression. ΔLoop has three subregions: K/R rich, Middle, S/T rich. Both ΔLoop and ΔLoop(K/R) increase Cl - currents (and conductance) by 5x greater comparted to wild-type SLC26A9. Five amino acid extension of ΔLoop (N- or C-terminal) produces Cl - currents equivalent to ΔLoop deletion. However, 10 amino acid extension of ΔLoop (N- or C- direction) produces currents equivalent to WT-SLC26A9. Membrane localization indicates that ΔLoop and regional deletion constructs have similar membrane abundance to WT, while boundary deletions inhibit channel trafficking to the membrane. As ΔLoop(S/T) slightly increased conductance, the ordered S/T-rich motif [T 620 SVSYITFS 628 ] was examined for possible phosphorylation effects by serial alanine substitution. Each alanine substitution in the S/T-rich motif decrease SLC26A9 Cl - conductance vs WT-SLC26A9. However, for S623A the conductance decrease was due to minimal plasma membrane expression rather than apparent change of channel activity. Together these data suggest that the disordered loop is multifaceted and can maintain low conductance (autoinhibition) or by potential phosphorylation. As cystic fibrosis (CF) results from loss-of-function, trafficking or degradation mutations, the robust Cl - conductance from elimination of SLC26A9 autoinhibitory, disordered loop, presents an opportunity to supplement or replace Cl - conductance in CF tissues and potentially patients. R25-DK101405 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Key Points Robotic ultrasound performed favorably compared with magnetic resonance imaging for evaluating total kidney volume. Collagen evaluation by two novel methods of picrosirius red imaging were more informative than the standard method by brightfield imaging. Findings can improve research by increasing speed and access to total kidney volume determination and sensitivity of collagen assessment. Background 3D imaging and histology are critical tools for assessing polycystic kidney disease (PKD) in patients and animal models. Magnetic resonance (MR) imaging provides micron resolution but is time consuming and expensive, and access to equipment and expertise is limited. Robotic ultrasound (US) imaging has lower spatial resolution but is faster, more cost-effective, and accessible. Similarly, picrosirius red (PSR) staining and brightfield microscopy are commonly used to assess fibrosis; however, alternative methods have been shown in non-kidney tissues to provide greater sensitivity and more detailed structural characterization. Methods In this study, we evaluated the utility of robotic US and alternative methods of quantifying PSR staining for PKD research. We compared longitudinal total kidney volume measurements using US and MR imaging. We additionally compared PSR imaging and quantification using standard brightfield microscopy with that by circularly polarized light with hue analysis and fluorescence imaging analyzed using curvelet transform fiber extraction software for automatic detection of individual collagen fibers. Results Increased total kidney volume was detected by US in Pkd1 RC/RC versus wild-type (WT) at time points spanning from early to established disease. US interobserver variability was greater but allowed scanning in 2–5 minutes/mouse, whereas MR imaging required 20–30 minutes/mouse. While no change in fibrotic index was detected in this cohort of relatively mild disease using brightfield microscopy, polarized light showed fibers skewed thinner in Pkd1 RC/RC versus WT. Fluorescence imaging showed a higher density of collagen fibers in Pkd1 RC/RC versus WT, and fibers were thinner and curvier with no change in length. In addition, fiber density was higher in both glomeruli and tubules in Pkd1 RC/RC , and glomeruli had a higher fiber density than tubules in Pkd1 RC/RC and trended higher in WT. Conclusions These studies show robotic US is a rigorous imaging tool for preclinical PKD research. In addition, they demonstrate the increased sensitivity of polarized and fluorescence analysis of PSR-stained collagen.

Maintaining pH is critical for proper cellular and organ function. To measure intracellular pH, we made a novel genetically encoded protein, pHire, that increases red fluorescence with increasing pH. Thus, pHire allows for the coupling of multiple fluorescent tags without spectral overlap. We have expressed pHire in mammalian epithelial cells, Drosophila, and mice. In this study, our goal was to characterize pHire’s spectral properties by expression in human cells (HEK293) and in E.coli. To biophysically characterize pHire, we made a maltose binding protein (MBP)-pHire fusion protein to purify from E.coli. Pelleting individual colony growths revealed pink bacterial pellets which fluoresced with mCherry filters. Rather than purifying the MBP-pHire protein, equal volumes of bacteria were permeabilized with nigericin and high K+ solution for pH 5 to 9. These aliquots were then used to gather the fluorescence excitation and emission spectra of pHire, allowing us to determine the excitation and emission peaks of pHire as a function of pH. Our results showed peaks at Ex579 nm and Em611 nm, or roughly mCherry. Pelleted bacteria could also be fixed in perfusion chambers, revealing the same calibration curve as in mammalian cells and flies. Next, stable HEK293-pHire cells were selected to assay acid-base transport using NH4Cl or CO2/HCO3− in perfusion experiments. These HEK-pHire cells have less fluorescence than MBP-pHire bacteria but show the same Ex/Em peaks. However, an in-frame ATG 5’ to pHire in a mouse (adding 23 amino acids), removed pHire pH-dependence and decreased bacterial fluorescent output. Together these results indicate that pHire is biophysically similar in cells tested but that unstructured N-terminal additions block pH-responses. Funding: R25-DK101405, R21-DK129897. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The A‐splice variant of Na + ‐dependent, electrogenic bicarbonate transporter‐1 (SLC4A4, NBCe1‐A) is generally believed to be the only SLC4A4 isoform expressed in the mammalian proximal tubule, and to have critical roles in filtered bicarbonate reabsorption, proximal tubule ammonia metabolism and proximal tubule organic anion metabolism. The purpose of the current study was to examine expression of other splice variants in the mouse kidney. We used recently generated mice with TALEN‐generated deletion of A and D NBCe1 splice‐variants, but with intact expression of the B, C and E isoforms. RT‐PCR using primers specific to the B, C and E isoforms showed mRNA of non‐A,D splice variants was expressed in the kidney of mice with intact NBCe1‐A expression and with NBCe1‐A‐specific deletion. We then used an antibody that recognizes portions of NBCe1 present in all splice isoforms and examined immunolabel in the kidney of NBCe1‐A‐deletion mice. There was basolateral immunolabel in the proximal convoluted tubule in the cortex, and lesser intensity basolateral immunolabel in the proximal tubule straight tubule in both the cortex and outer medulla. Specificity of this antibody was demonstrated by identifying that mice with deletion of all splice variants had no detectable immunolabel. An antibody specific to the A‐splice variant showed basolateral immunolabel throughout the entire proximal tubule of mice with intact NBCe1‐A expression, but no detectable immunolabel in mice with NBCe1‐A‐specific deletion. Thus, an NBCe1 splice variant other than NBCe1‐A appears to be expressed in the mouse proximal tubule. In acid‐loaded NBCe1‐A‐deletion mice, immunolabel observed using the pan‐specific anti‐NBCe1 antibody showed increased immunolabel in the proximal straight tubule, particularly in the outer medulla. These studies show that splice variants of NBCe1 other than NBCe1‐A/D are present in the mouse kidney and that their expression appears to be increased with acid‐loading. We conclude that these splice variants may have important roles in proximal tubule filtered bicarbonate reabsorption, ammonia metabolism and organic anion metabolism. Support or Funding Information The studies were supported by funds from the NIH (R01–DK045788 to IDW, R01‐DK107798 to IDW and JWV, and R01‐EY017732 to MFR) and the Department of Veterans Affairs (1I01BX000818 to IDW).

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.

Anoctamins (ANO) are activated by intracellular Ca2+ and function as phospholipid scramblases and/or Cl- channels. ANO4 is mutated in miniature schnauzers with calcium oxalate stones (CaOx), and ANO4 protein in urinary extracellular vesicles is decreased in human CaOx stone formers. In Drosophila Malpighian tubules (MT), subdued (ANO4-homolog) functions as Ca2+-activated Cl- channel, scramblase, and plays a role in host defense against gram-negative bacteria. Increasing evidence shows the participation of bacteria in urinary stone formation in humans. We used a Drosophila avatar to investigate whether subdued changes CaOx crystallization and if crystallization increases with bacterial infection.Uro:Gal4 flies were crossed with UAS:subdued-RNAi flies to knockdown (KD) subdued in MT principal cells (PC) or with w1118 flies to generate normal controls (WT). F1 generation underwent CaOx crystallization in association or not with uropathogenic E. coli (UPEC) infection. In vivo, flies were fed on diet supplemented with 20mM sodium Ox (NaOx) + UPEC:eGFP for 4 days. For ex vivo assays, dissected MTs were submerged in solution containing 10mM NaOx+ UPEC:eGFP for 90 minutes. subdued-KD in PC reduced subdued mRNA by 56% in MT. Neither crystal number nor size were changed by subdued-KD after prolonged NaOx feeding. However, when UPEC is introduced with the NaOx diet, subdued-KD flies exhibited a large bacterial presence in MT lumen and produced enlarged CaOx crystals compared to WT flies. Similar augmented presence of UPEC in subdued-KD MTs was observed during ex vivo assays. Short-term exposure to NaOx+UPEC resulted in increased number of crystals in subdued-KD MTs compared to WT MTs, but no difference was observed in crystal size.Fly cell atlas ( flycellatlas.org ) revealed that subdued is also found in MT stellate cells (SC; water and Cl- secretion). C724:Gal4>UAS:subdued-RNAi flies were used to assess the effect of subdued-KD in SC. This s ubdued-KD slightly increased bacterial infection but did change CaOx crystallization in MTs after 4 days of NaOx+UPEC feeding.The presence of subdued in PC limits CaOx crystals formation and aggregation through reducing bacterial invasion in the MT lumen. These Drosophila results suggest a role for ANO4 in bacterial-related human and dog kidney stone formation. Future experiments will determine whether other functional aspects of subdued affect CaOx crystallization. U54-DK100227, R01-DK092408, FAPESP (2022/01226-1), Mayo Foundation. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.