Citations

The SLC26A9 protein mediates Cl‐ transport as a Cl‐ channel and a Cl‐/HCO3 exchanger in the several epithelial cells, e.g., gut, lung, kidney. Topology analysis from BicA, a member of the SulP/SLC26A family, suggests that Slc26 proteins consists of 12 predicted transmembrane regions. However, the topology model of human SLC26 transporters has not yet been directly investigated. In this study, 12 HA‐inserted SLC26A9 mutants were constructed and expressed in Xenopus oocytes. Chemiluminescence was used to determine which HA‐tags were accessible from the extracellular surface. 157‐160 and 352‐353 sites of human SLC26A9 were identified as extracellular sites. Interestingly, the SLC26A9 HA (159‐160) mutant has ~40% Cl‐ transport activity, but this decreased function could be rescued by coexpression with another low function cSNP (T127N‐SLC26A9). These results revealed that the topology of TM region of SLC26A9 may different from that of BicA and also indicate that human SLC26A9 functions as dimer. Grant Funding Source : Supported by NIH EY 017732 (MFR)

In the renal proximal tubule (PT), Na + /K + ‐ATPase (NKA) is exclusively located in the basolateral domain. Through its classic ATP‐dependent ion‐pumping function, NKA generates the Na + gradient that drives apical Na + reabsorption, mostly through Na + /H + exchanger (NHE3). Accordingly, activation of NKA‐mediated ion transport decreases natriuresis through activation of basolateral (NKA) and apical Na + reabsorption (NHE3). In contrast, pharmacological evidence suggests that activation of the more recently discovered NKA signaling function triggers a cellular redistribution of NKA and NHE3 that decreases transcellular Na + flux in cultured PT cells. To obtain genetic evidence of this NKA/Src mechanism in the PT and asses its physiological importance, we used a knockdown and rescue approach in pig renal epithelial cells (LLC‐PK1). Additionally, we genetically targeted NKA α1 in the mouse PT by crossing mice expressing a sodium glucose co‐transporter 2 promoter driven Cre transgene with Floxed NKA α1 mice (PTα1 ‐/‐ ). Knockdown of 90% of NKA α1 in PT LLC‐PK1 cells increased transepithelial 22 Na flux by 2‐fold, activated NHE3 (50% decrease in inhibitory phosphorylation), and increased basolateral Na + /HCO 3 ‐ cotransporter (NBCe1A) protein content. In the PTα1 ‐/‐ mouse (4‐month males and females in a 1:1 ratio), 70% decrease in PT NKA α1 expression decreased urine output (0.51±0.14 vs 1.57±0.21 mL/24h in PTα1 +/+ , p<0.001, n=16) and absolute Na + excretion (0.14±0.05 vs 0.36±0.05 mmol/24h in PTα1 +/+ , p<0.05, n=8) by 65%, without histological or functional evidence of renal injury. Those changes were driven by increased PT Na + reabsorption, as indicated by a 65% decrease in lithium clearance (4‐month males, 1344±220 vs 3932±697 mL/24h in PTα1 +/+ , p<0.001, n=12) with unchanged GFR. This hyper‐reabsorptive phenotype of PTα1 ‐/‐ mice was coupled to increased membrane abundance of NHE3 and NBCe1A, and rescued upon crossing with floxed NHE3 mice, consistent a NKA/NHE3‐dependent mechanism. A dismantlement of caveolar NKA/Src receptor complex and intracellular redistribution of pY418Src occurred in knockdown NKA α1 PT cells, and was also observed in the PTα1 ‐/‐ hypomorphic mouse. Rescue of PT cells with wild‐type but not Src signaling‐null NKA α1 restored NHE3 and NBCe1A to basal levels, indicative of a role for NKA/Src receptor function in the tonic inhibition of Na + transporters in the PT. Hence, NKA signaling exerts a tonic inhibition on Na + reabsorption by regulating key apical and basolateral Na + transporters. This action, which is lifted upon NKA genetic suppression in cells and in vivo , tonically counteracts NKA's ATP‐driven function of basolateral Na + reabsorption. Strikingly, NKA/Src signaling is not only physiologically relevant, it is functionally dominant over NKA ion‐pumping in the control of PT reabsorption. NKA signaling therefore provides a long sought‐after mechanism for the natriuretic action of endogenous NKA ligands such as cardiotonic steroids.

AbstractBackgroundNormal gastrointestinal motility depends on electrical slow‐wave activity generated by interstitial cells of Cajal (ICC) in the tunica muscularis of the gastrointestinal tract. A requirement for HCO3− in extracellular solutions used to record slow waves indicates a role for HCO3− transport in ICC pacemaking. The Slc4a4 gene transcript encoding the electrogenic Na+/HCO3− cotransporter, NBCe1, is enriched in mouse small intestinal myenteric region ICC (ICC‐MY) that generate slow waves. This study aimed to determine how extracellular HCO3− concentrations affect electrical activity in mouse small intestine and to determine the contribution of NBCe1 activity to these effects.MethodsImmunohistochemistry and sharp electrode electrical recordings were used.Key ResultsThe NBCe1 immunoreactivity was localized to ICC‐MY of the tunica muscularis. In sharp electrode electrical recordings, removal of HCO3‐ from extracellular solutions caused significant, reversible, depolarization of the smooth muscle and a reduction in slow‐wave amplitude and frequency. In 100 mM HCO3−, the muscle hyperpolarized and slow wave amplitude and frequency increased. The effects of replacing extracellular Na+ with Li+, an ion that does not support NBCe1 activity, were similar to, but larger than, the effects of removing HCO3‐. There were no additional changes to electrical activity when HCO3‐ was removed from Li+ containing solutions. The Na+/HCO3− cotransport inhibitor, S‐0859 (30µM) significantly reduced the effect of removing HCO3− on electrical activity.Conclusions & InferencesThese studies demonstrate a major role for Na+/HCO3− cotransport by NBCe1 in electrical activity of mouse small intestine and indicated that regulation of intracellular acid:base homeostasis contributes to generation of normal pacemaker activity in the gastrointestinal tract.

Background Human kidney stones form via repeated events of mineral precipitation, partial dissolution, and reprecipitation, which are directly analogous to similar processes in other natural and manmade environments, where resident microbiomes strongly influence biomineralization. High-resolution microscopy and high-fidelity metagenomic (microscopy-to-omics) analyses, applicable to all forms of biomineralization, have been applied to assemble definitive evidence of in vivo microbiome entombment during urolithiasis. Methods Stone fragments were collected from a randomly chosen cohort of 20 patients using standard percutaneous nephrolithotomy (PCNL). Fourier transform infrared (FTIR) spectroscopy indicated that 18 of these patients were calcium oxalate (CaOx) stone formers, whereas one patient formed each formed brushite and struvite stones. This apportionment is consistent with global stone mineralogy distributions. Stone fragments from seven of these 20 patients (five CaOx, one brushite, and one struvite) were thin sectioned and analyzed using brightfield (BF), polarization (POL), confocal, super-resolution autofluorescence (SRAF), and Raman techniques. DNA from remaining fragments, grouped according to each of the 20 patients, were analyzed with amplicon sequencing of 16S rRNA gene sequences (V1–V3, V3–V5) and internal transcribed spacer (ITS1, ITS2) regions. Results Bulk-entombed DNA was sequenced from stone fragments in 11 of the 18 patients who formed CaOx stones, and the patients who formed brushite and struvite stones. These analyses confirmed the presence of an entombed low-diversity community of bacteria and fungi, including Actinobacteria, Bacteroidetes, Firmicutes , Proteobacteria , and Aspergillus niger . Bacterial cells approximately 1 μm in diameter were also optically observed to be entombed and well preserved in amorphous hydroxyapatite spherules and fans of needle-like crystals of brushite and struvite. Conclusions These results indicate a microbiome is entombed during in vivo CaOx stone formation. Similar processes are implied for brushite and struvite stones. This evidence lays the groundwork for future in vitro and in vivo experimentation to determine how the microbiome may actively and/or passively influence kidney stone biomineralization.

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.

Significance The tiny insect renal (Malpighian) tubule can transport fluid at unparalleled speed, suggesting unique specializations. Here we show that strategic allocation of major intrinsic proteins (MIPs) to specific cells within the tubule allows the separation of metabolically intense active cation transport from passive chloride and water conductance. This specialized renal architecture is general to at least many higher insects, providing a clue to the unique success of the class Insecta in colonizing an extraordinary range of ecological niches.

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-, β-, 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.