HCO3 Reabsorption Research Articles

READINESS OF INTERPROFESSIONAL EDUCATION IN COMMUNITY MEDICINE: MEDICAL AND DENTISTRY STUDENTS

Systemic lupus erythematosus (SLE) is an autoimmune disease affecting several body systems with a variety of clinical manifestations, course of disease, and prognosis. Its clinical manifestations can involve almost all organ systems including musculoskeletal, skin and mucosa, kidney, neuropsychiatry, lung, heart, blood vessels, gastrointestinal, ocular, obstetric, endocrine, and haematology. SLE can occur with a variety of degrees of tubular abnormalities. Tubular inflammation, tubular atrophy, interstitial inflammation and fibrosis are reported in 50-70% of patients with SLE. We have reported a case of a 27-year-old female with renal tubular acidosis (RTA) type 1 who has systemic lupus erythematosus. RTA is a group of abnormalities of the renal function, characterized by renal impairment in the reabsorption of HCO3- and excreting acid (H+). The administration of corticosteroids and immunosuppressants, correction of metabolic acidosis with sodium bicarbonate, and potassium supplementation are the main modalities of therapy in patients with SLE with RTA type-1

Potential Novel Role of Membrane-Associated Carbonic Anhydrases in the Kidney.

Carbonic anhydrases (CAs), because they catalyze the interconversion of carbon dioxide (CO2) and water into bicarbonate (HCO3-) and protons (H+), thereby influencing pH, are near the core of virtually all physiological processes in the body. In the kidneys, soluble and membrane-associated CAs and their synergy with acid-base transporters play important roles in urinary acid secretion, the largest component of which is the reabsorption of HCO3- in specific nephron segments. Among these transporters are the Na+-coupled HCO3- transporters (NCBTs) and the Cl--HCO3- exchangers (AEs)-members of the "solute-linked carrier" 4 (SLC4) family. All of these transporters have traditionally been regarded as "HCO3-" transporters. However, recently our group has demonstrated that two of the NCBTs carry CO32- rather than HCO3- and has hypothesized that all NCBTs follow suit. In this review, we examine current knowledge on the role of CAs and "HCO3-" transporters of the SLC4 family in renal acid-base physiology and discuss how our recent findings impact renal acid secretion, including HCO3- reabsorption. Traditionally, investigators have associated CAs with producing or consuming solutes (CO2, HCO3-, and H+) and thus ensuring their efficient transport across cell membranes. In the case of CO32- transport by NCBTs, however, we hypothesize that the role of membrane-associated CAs is not the appreciable production or consumption of substrates but the minimization of pH changes in nanodomains near the membrane.

Adhesion‐GPCR Gpr116 (ADGRF5) is a Regulator of Urine Acidification

G‐protein coupled receptors (GPCR) are a large and diverse family of integral membrane proteins that recognize a tremendous assortment of extracellular molecules including neurotransmitters, hormones, light and odors. They are a common target of pharmaceutical drug development, and uncovering the function of novel GPCRs in the kidney represents a wealth of untapped therapeutic potential. We previously performed an mRNA screen for novel GPCRs in the kidney to identify promising yet overlooked renal GPCRs. This screen revealed that Gpr116, an adhesion‐class GPCR, is highly expressed in the kidney. To understand the role of Gpr116 in renal physiology, we have taken a multidisciplinary approach. Using transfected HEK293 cells expressing cloned Gpr116, we have validated a monoclonal antibody for Gpr116. In murine kidney, this antibody indicates localization of Gpr116 to intercalated cells (ICs) in the collecting duct of mouse kidneys. This staining is absent in kidneys from targeted knockout (KO) of Gpr116 in renal tubules (Gpr116flox/flox, ksp‐Cre). Additionally, kidney‐specific KO animals have significantly (p<0.02) reduced urine pH (5.66±0.29, n=9) compared to wild‐type (WT; 5.96±0.23, n=15) littermates. Challenging the animals with an acid load (280mM NH4Cl added to drinking water) significantly acidifies WT urine (5.71±0.18, n=14, p<0.03) but does not significantly reduce KO urine pH (5.55±0.20, n=9), suggesting the loss of Gpr116 from α‐ICs is sufficient to maximally acidify urine. KO mice do not have significantly more ICs compared to WT. Interestingly, the loss of acid in the urine is accompanied by a small, but significant (p=0.001), increase in blood pH, and a small, but significant (p<0.05), decrease in pCO2 compared to WT. We conclude that loss of Gpr116 from the nephron causes a primary loss of acid in the urine which results in a mild metabolic alkalosis (“renal tubular alkalosis”) due to reabsorption of HCO3− by α‐ICs. This study establishes a physiologic role of the previously understudied Gpr116 in the murine kidney and demonstrates the scientific potential of future investigations into novel GPCRs.Support or Funding InformationResearch was funded by NIH F32DK116499 (N.A.Z.) and NIH R01DK107726 (J.L.P.).

Proximal renal tubular acidosis with and without Fanconi syndrome.

Proximal renal tubular acidosis (RTA) is caused by a defect in bicarbonate (HCO3−) reabsorption in the kidney proximal convoluted tubule. It usually manifests as normal anion-gap metabolic acidosis due to HCO3− wastage. In a normal kidney, the thick ascending limb of Henle’s loop and more distal nephron segments reclaim all of the HCO3− not absorbed by the proximal tubule. Bicarbonate wastage seen in type II RTA indicates that the proximal tubular defect is severe enough to overwhelm the capacity for HCO3− reabsorption beyond the proximal tubule. Proximal RTA can occur as an isolated syndrome or with other impairments in proximal tubular functions under the spectrum of Fanconi syndrome. Fanconi syndrome, which is characterized by a defect in proximal tubular reabsorption of glucose, amino acids, uric acid, phosphate, and HCO3−, can occur due to inherited or acquired causes. Primary inherited Fanconi syndrome is caused by a mutation in the sodium-phosphate cotransporter (NaPi-II) in the proximal tubule. Recent studies have identified new causes of Fanconi syndrome due to mutations in the EHHADH and the HNF4A genes. Fanconi syndrome can also be one of many manifestations of various inherited systemic diseases, such as cystinosis. Many of the acquired causes of Fanconi syndrome with or without proximal RTA are drug-induced, with the list of causative agents increasing as newer drugs are introduced for clinical use, mainly in the oncology field.

Pathophysiology of Renal Tubular Acidosis: Core Curriculum2016.

Metabolic acidosis results from either the gain of an acid or the loss of a base. The former is due to exogenous or endogenous acid loads resulting in anion gap metabolic acidosis. The latter is due to the loss of a base from either the gastrointestinal or genitourinary tract, producing nonanion gap or hyperchloremic metabolic acidosis. Renal tubular acidosis (RTA) arises from the kidney’s inability to excrete enough acid or retain enough bicarbonate (HCO3 - ), resulting in a clinical syndrome characterized by nongap metabolic acidosis, hyperchloremia, and impaired urinary acidification. In this Core Curriculum, we briefly summarize the role of the kidney in acid-base homeostasis and discuss clinical presentations, diagnoses, and treatments of RTA. OVERVIEW OF RENAL ACID-BASE HOMEOSTASIS Total-Body Acid-Base Homeostasis Metabolism of food particles generates both volatile carbonic acid, which is excreted by the lung, and fixed acid, which is generated primarily from metabolism of proteins. On a Western diet containing high levels of animal protein, an adult generates 15,000 mEq of volatile acid derived from fat and carbohydrate combustion and w1 mEq of fixed acid per kilogram of body weight from metabolism of proteins. This latter is initially neutralized by the body buffers, including HCO3 - , and then excreted by the kidney. The kidney is therefore responsible for the regeneration of lost buffers through excretion of 1 mEq of hydrogen ion (H 1 ) per kilogram of body by the reabsorption of all filtered HCO3 - and the regeneration of HCO3 - lost through metabolism of food particles. Both processes involve secretion of H 1 , initially to reabsorb filtered HCO3 - and then to generate new HCO3 - . The secreted H 1 would either combine with filtered HCO3 - , resulting in HCO3 reabsorption, or combine with urinary buffers in the tubular fluid to be excreted as titratable acids and ammonium ion (NH4 1 ), thus leading to HCO3 regeneration. Net acid excretion (NAE) by the kidney equals the sum of titratable acids and ammonium amounts minus the amount of HCO3 - :

Role of Receptor Protein Tyrosine Phosphatase γ in Sensing Extracellular CO2 and HCO3.

Regulation of blood pH-critical for virtually every facet of life-requires that the renal proximal tubule (PT) adjust its rate of H(+) secretion (nearly the same as the rate of HCO3 (-) reabsorption, JHCO3 ) in response to changes in blood [CO2] and [HCO3 (-)]. Yet CO2/HCO3 (-) sensing mechanisms remain poorly characterized. Because receptor tyrosine kinase inhibitors render JHCO3 in the PT insensitive to changes in CO2 concentration, we hypothesized that the structural features of receptor protein tyrosine phosphatase-γ (RPTPγ) that are consistent with binding of extracellular CO2 or HCO3 (-) facilitate monitoring of blood CO2/HCO3 (-) concentrations. We now report that PTs express RPTPγ on blood-facing membranes. Moreover, RPTPγ deletion in mice eliminated the CO2 and HCO3 (-) sensitivities of JHCO3 as well as the normal defense of blood pH during whole-body acidosis. Thus, RPTPγ appears to be a novel extracellular CO2/HCO3 (-) sensor critical for pH homeostasis.

A Novel Missense Mutation In The NBCe1 Gene ( SLC4A4 ) And A Novel Mode Of Inheritance For Proximal Renal Tubular Acidosis (pRTA)

The production and reabsorption of HCO3- by the nephron is critical for maintaining the body's pH buffering system. NBCe1-A, an electrogenic Na+/HCO3- cotransporter in the basolateral membrane of the renal proximal tubule, plays a key role in this process. Individuals that are homozygous for defects in NBCe1-A exhibit pRTA: a recessively-inherited plasma HCO3− deficiency that is associated with low blood pH and ocular defects. Here we present the first case of compound-heterozygous inheritance of pRTA. The patient has metabolic acidosis ([HCO3−] = 13 mM, pH = 7.26), is blind, and has inherited two heterozygous NBCe1-A mutations (R510H and Q913R), one from each parent. R510H has been previously linked to pRTA in homozygous form. Q913R is entirely novel. Biotinylation and voltage-clamp studies of these mutants in Xenopus oocytes, together with confocal microscopy of GFP-tagged mutants in HEK cells, indicate that failure of the mutant transporters to accumulate in the plasma membrane, not per-molecule HCO3− transport defects, underlie the disease in this case. However, Q913R manifests an unusual HCO3−-independent anion conductance that could in itself be pathogenic. Funded by UB:SUNY and NNSFC. Co-first authors: Myers and Yuan. Co-senior authors: Xia and Parker.

Regulation of glomerulotubular balance. III. Implication of cytosolic calcium in flow-dependent proximal tubule transport.

In the proximal tubule, axial flow (drag on brush-border microvilli) stimulates Na(+) and HCO3 (-) reabsorption by modulating both Na/H exchanger 3 (NHE3) and H-ATPase activity, a process critical to glomerulotubular balance. We have also demonstrated that blocking the angiotensin II receptor decreases baseline transport, but preserves the flow effect; dopamine leaves baseline fluxes intact, but abrogates the flow effect. In the current work, we provide evidence implicating cytosolic calcium in flow-dependent transport. Mouse proximal tubules were microperfused in vitro at perfusion rates of 5 and 20 nl/min, and reabsorption of fluid (Jv) and HCO3 (-) (JHCO3) were measured. We examined the effect of high luminal Ca(2+) (5 mM), 0 mM Ca(2+), the Ca(2+) chelator BAPTA-AM, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB), and the Ca-ATPase inhibitor thapsigargin. In control tubules, increasing perfusion rate from 5 to 20 nl/min increased Jv by 62% and JHCO3 by 104%. With respect to Na(+) reabsorption, high luminal Ca(2+) decreased transport at low flow, but preserved the flow-induced increase; low luminal Ca(2+) had little impact; both BAPTA and 2-APB had no effect on baseline flux, but abrogated the flow effect; thapsigargin decreased baseline flow, leaving the flow effect intact. With respect to HCO3 (-) reabsorption, high luminal Ca(2+) decreased transport at low flow and mildly diminished the flow-induced increase; low luminal Ca(2+) had little impact; both BAPTA and 2-APB had no effect on baseline flux, but abrogated the flow effect. These data implicate IP3 receptor-mediated intracellular Ca(2+) signaling as a critical step in transduction of microvillous drag to modulate Na(+) and HCO3 (-) transport.

TASK-2 K₂p K⁺ channel: thoughts about gating and its fitness to physiological function.

TASK-2 (K2P5) was one of the earliest members of the K2P two-pore, four transmembrane domain K(+) channels to be identified. TASK-2 gating is controlled by changes in both extra- and intracellular pH through separate sensors: arginine 224 and lysine 245, located at the extra- and intracellular ends of transmembrane domain 4. TASK-2 is inhibited by a direct effect of CO2 and is regulated by and interacts with G protein subunits. TASK-2 takes part in regulatory adjustments and is a mediator in the chemoreception process in neurons of the retrotrapezoid nucleus where its pHi sensitivity could be important in regulating excitability and therefore signalling of the O2/CO2 status. Extracellular pH increases brought about by HCO3 (-) efflux from proximal tubule epithelial cells have been proposed to couple to TASK-2 activation to maintain electrochemical gradients favourable to HCO3 (-) reabsorption. We demonstrate that, as suspected previously, TASK-2 is expressed at the basolateral membrane of the same proximal tubule cells that express apical membrane Na(+)-H(+)-exchanger NHE-3 and basolateral membrane Na(+)-HCO3 (-) cotransporter NBCe1-A, the main components of the HCO3 (-) transport machinery. We also discuss critically the mechanism by which TASK-2 is modulated and impacts the process of HCO3 (-) reclaim by the proximal tubule epithelium, concluding that more than a mere shift in extracellular pH is probably involved.

A mathematical model of rat proximal tubule and loop of Henle (1137.9)

PT and LOH function are coupled, with proximal transport determining loop fluid composition, and loop transport modulating glomerular filtration via tubuloglomerular feedback (TGF). To examine this interaction, we began with published models of superficial rat proximal convoluted tubule (PCT) (including flow‐dependent transport in a compliant tubule), and rat thick ascending Henle limb (AHL). This PCT was scaled down to represent outer medullary proximal straight tubule (PST), which was connected to thick AHL via a short descending limb. Superficial PT was scaled up to a juxtamedullary nephron, and connected to thick AHL via outer and inner medullary descending limbs, and inner medullary thin AHL. Medullary interstitial solute concentrations were specified. End‐tubule hydrostatic pressure was required to conform to a distal nephron flow resistance. The TGF signal was represented as a linear function of end‐AHL cytosolic Cl concentration. These two distal conditions required iterative solution of the model. Calculations revealed Na, K, and HCO3 reabsorption in all segments, with thin limb transport driven by the increases in lumen concentrations produced by osmotic water reabsorption. PST was a critical locus for NH4 secretion, due to high interstitial NH4, and cellular uptake via the Na,K‐ATPase. Late AHL cytosolic Cl was a sigmoidal function of flow, and appears suitable to represent the TGF signal.Grant Funding Source: Supported by NIH RO1‐DK29857

Effects of Atrial Natriuretic Peptide on Bicarbonate Transport in Long- and Short-Looped Medullary Thick Ascending Limbs of Rats

Atrial natriuretic peptide (ANP) is known to influence NaCl transport in the medullary thick ascending limbs (MAL), where the largest NaCl reabsorption occurs among distal nephron segments in response to arginine vasopressin (AVP). In the present study, we investigated the effect of ANP on bicarbonate (HCO3 −) transport in the MAL using an isolated tubule perfusion technique. The HCO3 − concentration was measured using free-flow ultramicro-fluorometer. We first observed basal HCO3 − reabsorption in both long- and short-looped MALs (lMALs, and sMALs, respectively). AVP inhibited HCO3 − reabsorption in both lMALs and sMALs, whereas ANP did not change HCO3 − transport. However, in the presence of AVP, ANP restored the HCO3 − reabsorption inhibited by AVP both in lMAL and sMAL. The effects of ANP on HCO3 − transport was mimicked by cyclic GMP. The mRNA expression level of the vasopressin V2 receptor in lMALs was significantly higher than in sMALs, whereas expression of the V1a receptor was unchanged. In summary, AVP inhibits HCO3 − transport, and ANP counteracts the action of AVP on HCO3 − transport both in lMALs and sMALs.

Role of the AT 1A receptor in the HCO 3 ‐induced inhibition of HCO 3 reabsorption by mouse proximal tubules

Previous works showed that the proximal tubule (PT) senses the changes of basolateral (BL) [CO2] on HCO3 reabsorption (JHCO3) requires an active apical AT1 receptor. I hypothesized that an active apical AT1 receptor is critical for the PT HCO3 sensitivity. In isolated perfused wild type (WT) mouse PT, I increased [HCO3]BL from 0, 22 to 44 mM (fixed [CO2]BL = 5%, pHBL = 7.4), caused the JHCO3 fall from 235 ± 13 (n = 6), 144 ± 7 (baseline, n = 30) to 58 ± 13 pmol mm−1 min−1 (n = 6, p < 0.01). Luminal 10−8 M candesartan (AT1 inhibitor) reduced the baseline JHCO3 by ~51% (71 ± 8, n = 12, p < 0.01), and the PT response on JHCO3 to 0 mM [HCO3]BL (90 ± 9, n = 6) or 44 mM [HCO3]BL (70 ± 12, n = 6) was eliminated. Whereas, luminal 10−7 M PD123319 (AT2 inhibitor) did not affect the PT HCO3 sensitivity. Added BL 240 nM lisinopril (ACE inhibitor) to block the endogenous ANG II generation and thereby secretion to the PT lumen, the data was identical to the candesartan data above. Finally, isolated perfused the AT1A-null mouse PT, the baseline JHCO3 fell by ~23% (110 ± 9, n = 12, p = 0.01), the PT response to 0 mM [HCO3]BL (113 ± 11, n = 6) was eliminated, and the JHCO3 fell to pedestal in response of 44 mM [HCO3]BL (48 ± 9, n = 6, p = 0.82). Thus, the work is consistent with the idea that the PT HCO3 sensitivity on JHCO3 requires an active apical AT1 receptor. High [HCO3]BL is a more powerful modulator ruling out the PT adaptation mechanism during the AT1A signal-transduction cascade is frozen.

Luminal Alkalinization Attenuates Proteinuria-Induced Oxidative Damage in Proximal Tubular Cells

A highly acidic environment surrounds proximal tubular cells as a result of their reabsorption of HCO(3)(-). It is unclear whether this luminal acidity affects proteinuria-induced progression of tubular cell damage. Here, we investigated the contribution of luminal acidity to superoxide (O(2)(·-)) production induced by oleic acid-bound albumin (OA-Alb) in proximal tubular cells. Acidic media significantly enhanced OA-Alb-induced O(2)(·-) production in the HK-2 proximal tubular cell line. Simultaneous treatment with both OA-Alb and acidic media led to phosphorylation of the intracellular pH sensor Pyk2. Highly phosphorylated Pyk2 associated with activation of Rac1, an essential subcomponent of NAD(P)H oxidase. Furthermore, knockdown of Pyk2 with siRNA attenuated the O(2)(·-) production induced by cotreatment with OA-Alb and acid. To assess whether luminal alkalinization abrogates proteinuria-induced tubular damage, we studied a mouse model of protein-overload nephropathy. NaHCO(3) feeding selectively alkalinized the urine and dramatically attenuated the accumulation of O(2)(·-)-induced DNA damage and proximal tubular injury. Overall, these observations suggest that luminal acidity aggravates proteinuria-induced tubular damage and that modulation of this acidic environment may hold potential as a therapeutic target for proteinuric kidney disease.

Effects of dietary cation-anion difference on ruminal metabolism and blood acid-base regulation in dairy cows receiving 2 contrasting levels of concentrate in diets

Dietary cation-anion difference [DCAD=Na+K − Cl in mEq/kg of dry matter (DM)] increases DM intake (DMI) in cows fed diets containing rapidly degraded starch. Increased DMI of diets containing rapidly degraded starch could potentially exacerbate subacute acidosis. The objective of this study was to determine metabolic effects of increasing DCAD in low and high starch diets. Six cannulated Holstein cows were blocked into 2 groups of 3 cows and assigned to two 3 × 3 Latin squares in a split-plot design. Each group received a level of concentrate at either 20 or 40% on a DM basis. The diet containing 20% concentrate supplied 4% rapidly degraded starch, whereas the diet containing 40% concentrate supplied 22% rapidly degraded starch. Diets in each square were formulated to provide a DCAD of 0, 150, or 300 mEq/kg of DM. The 3 values were obtained by manipulating Na and Cl contents. Increasing the proportion of rapidly degraded starch decreased rumen pH and the acetate to propionate ratio but did not affect digestibility, blood acid-base status, pH of urine, and strong ion excretion. Increasing DCAD increased DMI, the effect being higher when the cows were fed the 40% concentrate diet. Increasing DCAD did not affect mean ruminal pH, molar proportion of VFA, and fiber digestibility; reduced the range of rumen pH decrease during the meal in cows fed the 40% concentrate diet; and strongly increased blood pH and blood HCO3 concentration. Increasing DCAD increased urine pH and modified the urinary excretion of minerals. With low DCAD, 70% of Cl and only 16% of Na were excreted in urine whereas with high DCAD, 33% of Cl and 53% of Na were excreted. These results suggest that DMI of cows fed diets rich in rapidly degraded starch and low DCAD was limited to maintain the blood pH in a physiological range. Increasing DCAD allowed the cows to increase DMI because of the ability of positive DCAD to maintain blood acid-base status. A localized rumen buffering effect could not be excluded and could be linked with a higher amount of HCO3 recycled into the rumen. Main mechanisms involved in regulating blood pH might be renal excretion of protons and strong ions and renal HCO3 reabsorption.

Separate Gating Mechanisms Mediate the Regulation of K2P Potassium Channel TASK-2 by Intra- and Extracellular pH

TASK-2 (KCNK5 or K(2P)5.1) is a background K(+) channel that is opened by extracellular alkalinization and plays a role in renal bicarbonate reabsorption and central chemoreception. Here, we demonstrate that in addition to its regulation by extracellular protons (pH(o)) TASK-2 is gated open by intracellular alkalinization. The following pieces of evidence suggest that the gating process controlled by intracellular pH (pH(i)) is independent from that under the command of pH(o). It was not possible to overcome closure by extracellular acidification by means of intracellular alkalinization. The mutant TASK-2-R224A that lacks sensitivity to pH(o) had normal pH(i)-dependent gating. Increasing extracellular K(+) concentration acid shifts pH(o) activity curve of TASK-2 yet did not affect pH(i) gating of TASK-2. pH(o) modulation of TASK-2 is voltage-dependent, whereas pH(i) gating was not altered by membrane potential. These results suggest that pH(o), which controls a selectivity filter external gate, and pH(i) act at different gating processes to open and close TASK-2 channels. We speculate that pH(i) regulates an inner gate. We demonstrate that neutralization of a lysine residue (Lys(245)) located at the C-terminal end of transmembrane domain 4 by mutation to alanine abolishes gating by pH(i). We postulate that this lysine acts as an intracellular pH sensor as its mutation to histidine acid-shifts the pH(i)-dependence curve of TASK-2 as expected from its lower pK(a). We conclude that intracellular pH, together with pH(o), is a critical determinant of TASK-2 activity and therefore of its physiological function.

ROLE OF DOPAMINE IN MODULATING FLOW‐DEPENDENT SODIUM AND BICARBONATE TRANSPORT IN MOUSE PROXIMAL TUBULE

We have previously demonstrated that increases in luminal flow rate produce proportional increases in Na+ reabsorption by mouse proximal tubules. Since dopamine inhibits sodium reabsorption by stimulating NHE3 endocytosis and antagonizes the stimulation of proximal tubule transport by norepinephrine, we investigated whether increase of NHE3 endocytosis by dopamine will block flow‐stimulation of proximal tubule transport. Mouse proximal tubules were microperfused in vitro at perfusion rates of 5 and 20nl/min, and the net reabsorption of fluid (Jv) and HCO3− (JHCO3) were measured. The effect of dopamine and dopamine receptor inhibitors SCH23390 (DA1), sulpiride (DA2), 8‐Bromo‐cAMP and a cAMP‐dependent protein kinase inhibitor H89 on flow‐stimulated proximal tubule transport was examined. Increase in perfusion rate from 5 to 20nl/min significantly increased Jv from 0.88 to 1.5 nl/min/mm and JHCO3 from 61.72 to 128.53 pmole/min/mm. Luminal dopamine did not change Jv and JHCO3 at low flow rate but abolished the increments of Jv and JHCO3 induced by high flow rate. SCH23390 completely blocked, but sulpiride partially blocked the effect of dopamine. 8‐Bromo‐cAMP reduced Jv and JHCO3 at both flow rates and the effect of dopamine was blocked by H89. These results indicate that dopamine activates its receptor and inhibits flow‐stimulated proximal tubule transport by a PKA‐mediated mechanism. Recruitment of NHE3 to the apical membrane is critical for flow‐stimulated Na+ and HCO3− absorption in the kidney proximal tubule.

Angiotensin II‐induced stimulation of NHE3 is dependent on IRBIT and CaMKII

NHE3 plays an essential role in the reabsorption of NaCl, HCO3−, and fluid in the proximal tubules of the kidney. Accumulating evidence has suggested that angiotensin II (ANG II) at physiological concentrations exerts a stimulatory effect on renal reabsorption of NaCl and filtered water by targeting NHE3. However, the underlying mechanisms remain incompletely elucidated. Herein, we aimed to determine the role of IRBIT in ANG II‐induced stimulation of NHE3 and to elucidate the underlying mechanisms. In OKP cells, 1 nM ANG II stimulated NHE3 activity by ~22%. In comparison, knockdown of IRBIT resulted in ~7% increase, whereas ectopic expression of IRBIT enhanced NHE3 activity by ~33%. Furthermore, ANG II induced over 20% increase in the surface NHE3 expression in OKP cells as determined by surface biotinylation, which was confirmed by immunofluorescence labeling of NHE3 on the apical membrane. CaMKII was shown to phosphorylate IRBIT and, hence, we tested whether CaMKII is required for ANG II‐activation of NHE3. Our results showed that inhibition of CaMKII by KN‐93 or ectopic expression of the natural inhibitor protein of CaMKII resulted in significant suppression of NHE3 activation by ANG II. In conclusion, our current results demonstrate that ANG II regulates NHE3 via CaMKII‐dependent mechanism and identify IRBIT as a physiological regulator of NHE3 in renal proximal epithelial cells.

Effect of knocking out receptor protein tyrosine phosphatase γ (RPTPγ) in the CO2‐induced stimulation of HCO3 reabsorption by mouse renal proximal tubules

Previous work on isolated perfused proximal tubules (PTs) shows that: (1) raising basolateral (BL) [CO2] markedly increases HCO3 reabsorption rate (JHCO3) and (2) blocking apical AT1 receptors or knocking out AT1A receptors eliminates the JHCO3 response to [CO2]BL. We hypothesize that the “orphaned” RPTPγ, with an extracellular carbonic‐anhydrase‐like domain, is a CO2 sensor. We perfused PTs from wild‐type vs RPTPγ‐null mice, using out‐of‐equilibrium solutions to vary [CO2]BL at fixed [HCO3]BL and pHBL. In wild‐type PTs, raising [CO2]BL from 0% to 5% (“baseline”) to 20% (fixed [HCO3]BL = 22 mM, pHBL = 7.4) increased JHCO3 from 79 ± 6 (n = 14) to 136 ± 7 (n = 40) to 189 ± 4 pmol mm−1 min−1 (“pmm”, n = 14). In RPTPγ‐null PTs, baseline JHCO3 was reduced by 23% to 105 ± 7 pmm (n = 24, p = 0.002, 1‐way ANOVA), and the responses to 0% [CO2]BL (JHCO3 = 98 ± 7, n = 6) and 20% [CO2]BL (JHCO3 = 105 ± 12, n = 6) were eliminated. Finally, when we added 10−8 M candesartan (non‐peptide AT1 antagonist) to the lumen of RPTPγ‐null PTs, baseline JHCO3 was further fell by 29% to the “pedestal” level (JHCO3 = 75 ± 8 pmm, n = 18, p = 0.012, 1‐way ANOVA). Moreover, and the responses to 0% [CO2]BL (JHCO3 = 58 ± 8, n = 4) and 20% [CO2]BL (JHCO3 = 83 ± 23, n = 5) were eliminated. Thus, knocking out RPTPγ eliminates the PT response to changes in [CO2]BL, consistent with the idea that RPTPγ is a CO2 sensor, as part of a signal‐transduction cascade that also requires the apical AT1 receptor.

Role of a receptor tyrosine kinase in the HCO3‐induced inhibition of HCO3 reabsorption by rabbit S2 proximal tubule

Previous work showed that: (1) Proximal tubules (PTs) respond to increases in basolateral (BL) [CO2] or [HCO3] by, respectively, stimulating or inhibiting HCO3 reabsorption (JHCO3), suggesting the presence of CO2/HCO3 sensors near the basolateral membrane. (2) Studies with PD168393 (blocks erbB1 via alkylating a Cys in the ATP‐binding pocket) and BPIQ‐I (competes with ATP) suggest that the CO2 sensitivity of JHCO3 requires an active receptor tyrosine kinase (RTK), probably erbB1. Here, I hypothesize an active RTK also is required for the HCO3 sensitivity of JHCO3. I perfused the lumen of an isolated rabbit S2 PT with 5% CO2/22 mM HCO3 solution. Adding BL 35 nM PD168393 significantly decreased "baseline" JHCO3 (i.e. [HCO3]BL = 22 mM, [CO2]BL = 5%, pHBL = 7.4) from 69 ± 3 (n = 32) to 43 ± 4 pmol mm−1 min−1 ("pmm", n = 6, P &lt; 0.0001). Using an out‐of‐equilibrium (OOE) solution to decrease [HCO3]BL to 0 mM, I found that BL 35 nM PD168393 decreased JHCO3 from 103 ± 6 (n = 7) to 62 ± 7 pmm (n = 6, P &lt; 0.0006) at a fixed [CO2]BL of 5% and pHBL of 7.4. Using OOE solutions to increase [HCO3]BL to 44 mM, I found that BL 35 nM PD168393 had no effect on the already‐low JHCO3 (n = 7, P &lt;0.79). Thus, BL 35 nM PD168393 decreased baseline JHCO3 by 37%, reduced the JHCO3 response at 0 mM HCO3, and eliminated the response at 44 mM. An active RTK, perhaps erbB1, plays a central role in both CO2 and HCO3 signaling regulating H+ secretion by PTs. (Supported by AHA SDG 0735432N)

Structural requirements for the electrogenicity of the electrogenic Na‐HCO3 cotransporter NBCe1

Na/HCO3 cotransporters, including the electrogenic NBCe1 and the electroneutral NBCn1, belong to SLC4 family. The NBCe1‐A splicing variant plays a key role in HCO3 reabsorption by renal proximal tubules. NBCe1 and NBCn1 have 13 putative transmembrane (TM) segments and a large extracellular loop between TM5 and 6 (“the Loop”). Choi et al (J Physiol, 578:131, 2007) previously demonstrated that the electrogenecity of NBCe1‐A requires the interaction between TM1‐5 and TM6‐13, but does not require the cytoplasmic N terminus (Nt), the Loop, or the cytoplasmic C terminus (Ct). To understand better the structural basis of electrogenecity of NBCe1, we constructed a series of chimeras of human NBCe1‐A and NBCn1‐X (i.e., lacking cassette B in the Ct), starting with one consisting of Nt∼Loop of NBCe1‐A spliced to TM6∼Ct of NBCn1. We exposed Xenopus oocytes expressing chimeras or wild‐type NBCe1‐A to 1.5% CO2/10 mM HCO3 (pH 7.5), and monitored pHi and Vm. We found that this first chimera exhibits the Na conductance characteristic of NBCn1, but no HCO3 transport. Restoring TM6‐7 of NBCe1‐A restored some electrogenic HCO3 transport (i.e., CO2/HCO3 elicited a slight hyperpolarization and pHi recovery). The chimera in which we restored TM6‐9 of NBCe1‐A functions as a typical NBCe1‐A. We conclude that the putative TM6‐9 of NBCe1 (vs NBCn1) is very important, but TM10‐13 of NBCe1 is not essential, for the electrogenecity of NBCe1‐A.