Romero, Michael F.

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).

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.

Background Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a leading cause of end‐stage renal disease in adults affecting 1 in 800 people in the US. Knocking down bicaudal‐C (BicC) in zebrafish and Xenopus has been reported as a method used to model the cystic changes in ADPKD. Gamberi and colleagues [JASN 25: 538A, 2014] recently presented a similar knockdown in Drosophila melanogaster resulting in cystic Malpighian Tubules (MTs) homologous to the human kidney. Additionally, calcium oxalate (CaOx) kidney stones affect ~1 in 10 people in the US, with the incidence increasing ~2‐fold in PKD patients. Methods Our group has investigated the PKD phenotype in Drosophila using a Gal4/UAS‐RNAi system to knockdown the expression of BicC in MTs. We initiated CaOx crystal formation by feeding Na‐oxalate or adding to tubule bathing solution [Hirata, AJP 2012]. Results Penetrance of Uro, CapaR, and c724 drivers in the MTs were compared using UAS‐YFP, resulting in Uro::BicC expression in ~50% of cells with CapaR::BicC approaching 100% and c724 expression specific to MT stellate cells only. MT morphology was analyzed: controls [WT (0%); parental (18%)] occurrence v BicC knockdowns [Uro::BicC (25%); CapaR::BicC (15%); c724::BicC (6.3%)]. Functional tests using Ramsay secretion assays reveal a slower secretion rate in Uro::BicC, CapaR::BicC, and c724::BicC knockdowns compared to WT MTs. In CapaR::BicC knockdowns, slower secretion rates are observed even after stimulation with Drosophila ‐kinin. Since there is an increased risk for ADPKD‐related kidney stones, MT calcium oxalate (CaOx) crystallization experiments were performed to quantify crystal formation in BicC knockdown vs WT MTs. While crystals are present in all oxalate exposed MTs, BicC knockdowns using Uro::BicC and c724::BicC form more CaOx crystals than CapaR::BicC and WT. However, crystals formed by Uro::BicC and c724::BicC knockdowns have a significantly smaller total crystal area and average area/individual crystal compared to both CapaR::BicC and WT. Conclusions These results suggest that BicC knockdown parallels kidney stone increases associated with human PKD, but does not yet consistently mimic morphologic ADPKD‐induced cystic phenotypes usually observed in PKD models. Support or Funding Information DK90278, DK101405, DK101405

Dent disease 1, an X‐linked inherited kidney disease caused by mutations in the CLCN5 gene, is characterized by low molecular weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets, and progressive renal failure. CLCN5 encodes the CLC‐5 protein found at the apical membrane in the kidney proximal tubule as well as endosome membranes of other tubule segments. Approximately 80 human mutations of the CLCN5 gene have been identified consisting of nonsense, missense, splice site, insertion and deletion mutations. However, the genotype and phenotype correlation are poor, and the mechanisms of CLC‐5 dysfunction that cause disease are still unclear. Moreover, there has been a lack of systematic study of CLC‐5 mutational effects on Cl − /H + exchanger activity. To address this need we used ion selective microelectrodes to monitor intracellular pH (pH i ) and intracellular Cl − concentration ([Cl i ]) of Xenopus oocytes expressing WT and mutant CLC‐5 while clamping at +40 mV. Common and novel mutations were identified in Dent patients by collaborators in the Rare Kidney Stone Consortium. The apparent H + :Cl − coupling ratio through the CLC‐5 protein was calculated using the Gibbs‐free energy equation. Cellular localization and protein trafficking of WT and mutant CLC‐5 in renal epithelial cells were also examined by immunofluorescence microscopy using eGFP/HA double‐tagged CLC‐5 and organelle‐specific markers and quantified by flow cytometry. In the presence of 104 mM Cl − (ND96), the pH i and [Cl − ] i of the CLC‐5 WT increased 340 ± 59 ×10 −4 pH unit/min and 2.8 ± 0.5 mM/min, respectively. T657S increased at a similar rate as WT, while there was a significant decrease in the rate of pH i and [Cl − ] i change for S244L, R345W, and Q629X compared to WT. Decreasing extracellular chloride to 5 mM decreased WT ClC‐5 pH i and [Cl − ] i by reversing transport: −7 ± 65 ×10 −4 pH unit/min and −0.2 ± 0.2 mM/min, respectively. Using these measurements, the stoichiometry of WT CLC‐5 was calculated to be 1.80 ± 0.02 (nCl − :H + ) in ND96, similar to the stoichiometry estimated for T657S. However, the stoichiometry was significantly altered for S244L, R345W, and Q629X. WT ClC‐5 acts as Cl − /H + exchanger at +40mV with 104 mM Cl − in the extracellular solution. Lowering extracellular Cl − reverses the transport direction, and the ClC‐5 exchanger functions at a significantly slower rate. Although S244L has normal surface expression, S244L and R345W mutations have significantly decreased pH and Cl − exchange rates in response to solution manipulations, suggesting these mutations have an altered ion exchange coupling ratio (stoichiometry). Interestingly, while R345W was retained in the ER suggesting defective protein maturation, R345W surface localization was rescued by lowering the incubation temperature from 37 to 30°C. Q629X was retained in the ER, not localized in early endosomes, and also has abolished Cl − /H + exchange activity, suggesting that Q629X affects protein maturation and trafficking while T657S is a benign, non‐disease causing mutation. These data suggest studying CLC‐5 mutational effects on both transport function and cellular localization is essential to better understand divergent phenotypes in Dent disease. Support or Funding Information NIH DK101405, U54KD083908, U54DK100227, Mayo O'Brien Urology Research Center, Mayo Clinic Rare Kidney Stone Consortium

The proximal tubule electrogenic, sodium‐coupled bicarbonate transporter, NBCe1, has a critical role in two proximal tubule acid‐base functions, bicarbonate reabsorption and ammonia metabolism. A third component of acid‐base homeostasis involves organic anion metabolism. The purpose of the current study was to determine NBCe1's role in organic anion metabolism by comparing mice with NBCe1 deletion to wild‐type (WT) littermates. Because NBCe1 KO causes early post‐natal mortality, we studied mice at day 8±1. Also, because NBCe1 deletion causes metabolic acidosis, we compared effects of NBCe1 deletion to that of experimental metabolic acidosis in adult WT mice. Both NBCe1 knockout (KO) and acidosis decreased citrate excretion, but their effect on proximal tubule citrate transporters differed. NaDC‐1 is the primary apical citrate transporter, and NBCe1 KO decreased NaDC‐1 mRNA expression and it decreased proximal tubule apical immunolabel. In contrast, acidosis in WT mice increased NaDC‐1 mRNA increased and proximal tubule apical immunolabel. The basolateral citrate transporter is NaDC‐3. NBCe1 deletion decreased NaDC‐3 expression whereas metabolic acidosis increased expression. A second key urinary organic anion is 2‐oxoglutarate. NBCe1 KO increased 2‐oxoglutarate excretion ~15‐fold, whereas metabolic acidosis decreased excretion by ~85%. NBCe1 deletion did not alter expression of the enzyme involved in 2‐oxoglutarate generation (Glud1), whereas metabolic acidosis increased it. There were no substantial changes in expression of the 2‐oxoglutarate dehydrogenase complex components, Ogdh, Dlst or Dld, with either NBCe1 KO or acidosis. Expression of the putative apical 2‐oxoglutarate transporter, OAT10, was decreased similarly by NBCe1 KO and by acidosis. Thus, effects of NBCe1 KO on 2‐oxoglutarate excretion did not correlate with changes in proteins involved in production, metabolism, basolateral transport (NaDC‐3) or its putative apical transporter (OAT10). 2‐oxoglutarate is proposed to stimulate urine alkalinization by activating the Oxgr1 receptor in pendrin‐positive intercalated cells. However, NBCe1 KO causes a urine pH of ~4.2 despite increased 2‐oxoglutarate excretion. Pendrin, the primary mechanism of collecting duct bicarbonate secretion, exhibited significantly decreased expression with NBCe1 KO. Expression of the 2‐oxoglutarate receptor, Oxgr1, was unaffected by NBCe1 KO, whereas it was decreased by metabolic acidosis. In summary: 1) NBCe1 is necessary for normal expression of the proximal tubule citrate transporters, NaDC‐1 and NaDC‐3; 2) NBCe1 is necessary for normal 2‐oxoglutarate metabolism through mechanisms independent of expression of known transport and metabolic pathways; and, 3) the lack of 2‐oxoglutarate urinary alkalinization in NBCe1 KO mice likely results from decreased expression of its target transporter, pendrin. Thus, NBCe1 expression is necessary for normal renal metabolism of the key organic acids, citrate and 2‐oxoglutarate. We conclude that NBCe1 expression is necessary for normal regulation of each of the three major components of proximal tubule acid‐base homeostasis, filtered bicarbonate reabsorption, ammonia metabolism and organic anion transport. Support or Funding Information Funds from the NIH (R01–DK045788 to IDW, R01‐EY017732 to MFR and R01‐DK095879 to KHS) and from the Department of Veterans Affairs (1I01BX000818 to IDW) supported these studies.

Introduction: Dent Disease type 1 (DD1) results from X-linked, mutations in CLC-5 (2Cl-/H+ exchanger) and results in progressive renal failure by 20-40. DD1 is characterized by kidney Ca2+ mishandling, and subsequent hypercalcuria, calcium oxalate (CaOx) kidney stones, and kidney calcification as well as proteinuria. We identified Drosophila Clc-c as the CLC-5 homolog with conserved amino acids at DD1 mutation sites. We hypothesize that Clc-c shares functional similarities in Cl- transport, Ca2+ homeostasis and protein processing. Methods and Results: By voltage clamping Xenopus oocytes expressing Clc‑c and CLC-5, we find that both are electrogenic and have the same outward-rectifying current in Cl- solutions. Cl- transport (current at +80mV) was lowered by acidity (2.37±0.55 μA, pH 6.0, P=0.06), but not by alkaline solution (4.45±0.96 μA, pH 8.5, P= 0.7) when compared to the standard solution of pH 7.5 (4.97±1.12 μA). Similarly, Cl- transport was lowered by homologous DD1 mutations S393L (0.67±0.098 μA, P<0.0001), R494W (2.9±0.46 μA, P =0.006), and Q777x (1.13±0.28 μA, P =0.0001) vs. WT (7.4±1.2 μA) as observed previously for CLC-5 DD1 mutations: S244L, R345W, and Q629x (Tang et al, Physiol Rep 4[8], 2016). To determine cell membrane and possible intracellular localization of DD1 mutations, HA-tagged Clcc and mutations (identical placement as in CLC-5-HA, Tang 2016) were made. Likewise, transgenic Drosophila expressing Clc-c-GFP were made to localize Clc-c in cell and intracellular membranes of renal tubules (Malpighian tubules, MT) in vivo (Judd-Mole, U Monash, Dissertation, 2016). RNAi MT knockdowns of Clc-c (Clc-c-KD) were made and resulted in 50% decrease in Clc-c mRNA expression. CaOx crystals in anterior MT (7 days after eclosure) were present in all Clc-c-KD and were more abundant (15±4 crystals/fly) than w1118 controls (5±2 crystals/fly, P= 0.02). MT secretions showed that Clc-c KD flies contained higher [Ca2+] while all other cations (Na+, NH4+, K+, Mg2+), and volume were the same as w1118 . Similarly, urinary protein concentration (4-6 pooled flies per n) appears elevated in DD1 flies (1.41 ± 0.14 mg/mL, n=3) vs w1118 (1.16 ± 0.08 mg/mL, n=5). Conclusion: Drosophila Clc-c has similarities to human CLC-5 including impaired function of voltage-gated Cl- transport, homologous DD1 mutations, increased CaOx crystal formation, and elevated urinary [Ca2+] and protein. Our results indicate that the Drosophila Clc-c avatar may help to provide more direct insights to the cellular and organismal pathophysiology for future investigations of Cl- transport, Ca2+ homeostasis and urinary protein loss in DD1. F32 DK128987, T32 DK007013 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.

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.

AbstractMarine teleosts ingest large amounts of seawater containing various ions, including 0.4 mM boric acid, which can accumulate at toxic levels in the body. However, the molecular mechanisms by which marine teleosts absorb and excrete boric acid are not well understood. Aquaporins (Aqps) are homologous to the nodulin‐like intrinsic protein (NIP) family of plant boric acid channels. To investigate the potential roles of Aqps on boric acid transport across the plasma membrane in marine teleosts, we analyzed the function of Aqps of Japanese pufferfish (Takifugu rubripes) expressed in Xenopus laevis oocytes. Takifugu genome database contains 16 genes encoding the aquaporin family members (aqp0a, aqp0b, aqp1aa, aqp1ab, aqp3a, aqp4a, aqp7, aqp8bb, aqp9a, aqp9b, aqp10aa, aqp10bb, aqp11a, aqp11b, aqp12, and aqp14). When T. rubripes Aqps (TrAqps) were expressed in X. laevis oocytes, a swelling assay showed that boric acid permeability was significantly increased in oocytes expressing TrAqp3a, 7, 8bb, 9a, and 9b. The influx of boric acid into these oocytes was also confirmed by elemental quantification. Electrophysiological analysis using a pH microelectrode showed that these TrAqps increase B(OH)3 permeability. These results indicate that TrAqp3a, 7, 8bb, 9a, and 9b act as boric acid transport systems, likely as channels, in marine teleosts.

Abstract Through its classic ATP‐dependent ion‐pumping function, basolateral Na/K‐ATPase (NKA) generates the Na + gradient that drives apical Na + reabsorption in the renal proximal tubule (RPT), primarily through the Na + /H + exchanger (NHE3). Accordingly, activation of NKA‐mediated ion transport decreases natriuresis through activation of basolateral (NKA) and apical (NHE3) Na + reabsorption. In contrast, activation of the more recently discovered NKA signaling function triggers cellular redistribution of RPT NKA and NHE3 and decreases Na + reabsorption. We used gene targeting to test the respective contributions of NKA signaling and ion pumping to the overall regulation of RPT Na + reabsorption. Knockdown of RPT NKA in cells and mice increased membrane NHE3 and Na + /HCO 3 − cotransporter (NBCe1A). Urine output and absolute Na + excretion decreased by 65%, driven by increased RPT Na + reabsorption (as indicated by decreased lithium clearance and unchanged glomerular filtration rate), and accompanied by elevated blood pressure. This hyper reabsorptive phenotype was rescued upon crossing with RPT NHE3 −/− mice, confirming the importance of NKA/NHE3 coupling. Hence, NKA signaling exerts a tonic inhibition on Na + reabsorption by regulating key apical and basolateral Na + transporters. This action, lifted upon NKA genetic suppression, tonically counteracts NKA's ATP‐driven function of basolateral Na + reabsorption. Strikingly, NKA signaling is not only physiologically relevant but it also appears to be functionally dominant over NKA ion pumping in the control of RPT reabsorption.