Urinary acid-base excretion deciphers high acid load from colonic bicarbonate loss in intestinal failure patients with ileocolonic anastomosis - Guidance for composition of parenteral support.

BACKGROUND & AIMS: Acid-base disturbances are common in short bowel (SB) patients due to increased intestinal bicarbonate loss. However, the resulting systemic acid load has not been quantified. Base excess is used to monitor metabolic acid-base disturbances but inadequately reflects the acid load. Our aim was to investigate the systemic acid/base load in SB-patients to obtain quantitative estimates to guide the composition of parenteral support. METHODS: We calculated total acid load in SB patients by summing 24-h urinary net acid excretion (NAE) and the provision of base equivalents in parenteral support. We then compared differences among anatomical SB-types: jejunostomy (SB-J), jejunocolostomy (SB-JC), and jejunoileostomy (SB-JIC). 47 urine samples from 34 SB patients were analyzed for bicarbonate (HCO3-), ammonium (NH4+), and titratable acid (TA) concentrations. NAE was calculated as (TA + NH4+) - HCO3-. Mixed-effects repeated-measures models were used to statistically examine differences between SB-types and associations with parenteral nutrition and NAE. A healthy cohort served as control. RESULTS: In comparison to SB-J, SB-JC patients had a 4.1 mmoL/l lower base excess (95% CI: -6.3 to -1.8) and an 84.5 mmol/day higher total acid load (CI: 41.3 to 127.7). There were no significant differences between SB-JIC and SB-J regarding base excess, NAE, or total acid load. Higher amounts of infused acetate, sodium, and chloride, but not the acetate/chloride ratio, were associated with lower NAE and higher base excess. CONCLUSIONS: Due to increased colonic bicarbonate loss, patients with SB-JC have a ∼4.4-fold higher acid load than healthy controls. The ion transport mechanisms mediating this bicarbonate loss from the remaining colon need further experimental investigation. NAE could be a useful tool to adjust base infusion in SB.

Challenged Urine Bicarbonate Excretion as a Measure of Cystic Fibrosis Transmembrane Conductance Regulator Function in Cystic Fibrosis.

BACKGROUND: In cystic fibrosis (CF), renal base excretion is impaired. Accordingly, challenged urine bicarbonate excretion may be an in vivo biomarker of cystic fibrosis transmembrane conductance regulator (CFTR) function. OBJECTIVE: To evaluate the association between challenged bicarbonate excretion and clinical characteristics at baseline, quantify the CFTR modulator drug elexacaftor/tezacaftor/ivacaftor-induced changes of challenged bicarbonate excretion after 6 months of treatment, and characterize the intraindividual variation in healthy adults. DESIGN: Prospective observational study. SETTING: Cystic fibrosis clinic, Aarhus University Hospital, Denmark. PATIENTS: Fifty adult patients with CF starting CFTR modulator therapy with elexacaftor/tezacaftor/ivacaftor between May 2020 and June 2021. MEASUREMENTS: Quantification of urine bicarbonate excretion after an acute oral sodium bicarbonate challenge before and 6 months after elexacaftor/tezacaftor/ivacaftor treatment. RESULTS: At baseline, challenged urine bicarbonate excretion was associated with several CF disease characteristics. Bicarbonate excretion was higher in patients with residual function mutations. A higher bicarbonate excretion was associated with better lung function, pancreatic sufficiency, and lower relative risk for chronic pseudomonas infections. Elexacaftor/tezacaftor/ivacaftor treatment increased bicarbonate excretion by 3.9 mmol/3 h (95% CI, 1.6 to 6.1 mmol/3 h), reaching about 70% of that seen in healthy control participants. In healthy control participants, individual bicarbonate excretion at each visit correlated with the individual mean bicarbonate excretion. The median coefficient of variation was 31%. LIMITATION: Single-center study without a placebo-controlled group. CONCLUSION: Although further studies are needed to address the performance and sensitivity of this approach, this early-stage evaluation shows that challenged urine bicarbonate excretion may offer a new, simple, and safe quantification of CFTR function and the extent of its pharmacologic improvement. Elexacaftor/tezacaftor/ivacaftor partially restores renal CFTR function in patients with CF, likely resulting in decreased risk for electrolyte disorders and metabolic alkalosis. PRIMARY FUNDING SOURCE: Innovation Fund Denmark.

Dietary K+ acts as a genuine diuretic.

K+ balance in mammals relies on regulated renal K+ excretion matching unregulated fluctuating K+ intake. Upon a K+ rich meal, rapid and powerful K+ excretion is needed. Renal K+ secretion is stimulated by the increased tubular flow. We speculated that high K+ intake acutely increases urinary flow to stimulate K+ excretion. METHODS: Mice were K+ challenged through diets or gavage. Post K+ loading urinary output, osmolarity, [K+ ]u , [Na+ ]u , plasma osmolarity, [copeptin]p , [K+ ]p , and [Na+ ]p were measured. To locate the mechanism of K+ -induced diuresis in the glomerular/tubular system we measured creatinine excretion and assessed functional transport in isolated perfused TALs and CDs during an acute [K+ ]bl switch from 3.6 to 6.5 mM. Molecular adaptations of transport proteins involved in water reabsorption were investigated by immunoblotting. RESULTS: (1) Mice switched from a 1% to 2% K+ diet increased diuresis within 12 hours and reciprocally reduced diuresis when switched from 1% to 0.01% K+ diet. (2) A single K+ gavage load, corresponding to 25%-50% of daily K+ intake, induced 100% increase in diuresis within 30 minutes. This occurred despite augmented plasma osmolarity and AVP synthesis. (3) K+ gavage did not change GFR. (4) In isolated perfused TALs, shifting [K+ ]bl from 3.6 to 6.5 mM did not affect AVP-induced NaCl transport. (5) In sharp contrast, in isolated perfused CDs, shifting [K+ ]bl from 3.6 to 6.5 mM markedly reduced CD AVP sensitivity, ie inhibited water absorption. CONCLUSION: Dietary K+ loading induces a rapidly on-setting diuresis. The mechanism of K+ -induced diuresis involves desensitization of the CD to AVP.

Benzamil-mediated urine alkalization is caused by the inhibition of H+-K+-ATPases.

Epithelial Na+ channel (ENaC) blockers elicit acute and substantial increases of urinary pH. The underlying mechanism remains to be understood. Here, we evaluated if benzamil-induced urine alkalization is mediated by an acute reduction in H+ secretion via renal H+-K+-ATPases (HKAs). Experiments were performed in vivo on HKA double-knockout and wild-type mice. Alterations in dietary K+ intake were used to change renal HKA and ENaC activity. The acute effects of benzamil (0.2 µg/g body wt, sufficient to block ENaC) on urine flow rate and urinary electrolyte and acid excretion were monitored in anesthetized, bladder-catheterized animals. We observed that benzamil acutely increased urinary pH (ΔpH: 0.33 ± 0.07) and reduced NH4+ and titratable acid excretion and that these effects were distinctly enhanced in animals fed a low-K+ diet (ΔpH: 0.74 ± 0.12), a condition when ENaC activity is low. In contrast, benzamil did not affect urine acid excretion in animals kept on a high-K+ diet (i.e., during high ENaC activity). Thus, urine alkalization appeared completely uncoupled from ENaC function. The absence of benzamil-induced urinary alkalization in HKA double-knockout mice confirmed the direct involvement of these enzymes. The inhibitory effect of benzamil was also shown in vitro for the pig α1-isoform of HKA. These results suggest a revised explanation of the benzamil effect on renal acid-base excretion. Considering the conditions used here, we suggest that it is caused by a direct inhibition of HKAs in the collecting duct and not by inhibition of the ENaC function.NEW & NOTEWORTHY Bolus application of epithelial Na+ channel (EnaC) blockers causes marked and acute increases of urine pH. Here, we provide evidence that the underlying mechanism involves direct inhibition of the H+-K+ pump in the collecting duct. This could provide a fundamental revision of the previously assumed mechanism that suggested a key role of ENaC inhibition in this response.

NBCn1 Increases NH4 + Reabsorption Across Thick Ascending Limbs, the Capacity for Urinary NH4 + Excretion, and Early Recovery from Metabolic Acidosis.

BACKGROUND: The electroneutral Na+/HCO3 - cotransporter NBCn1 (Slc4a7) is expressed in basolateral membranes of renal medullary thick ascending limbs (mTALs). However, direct evidence that NBCn1 contributes to acid-base handling in mTALs, urinary net acid excretion, and systemic acid-base homeostasis has been lacking. METHODS: Metabolic acidosis was induced in wild-type and NBCn1 knockout mice. Fluorescence-based intracellular pH recordings were performed and NH4 + transport measured in isolated perfused mTALs. Quantitative RT-PCR and immunoblotting were used to evaluate NBCn1 expression. Tissue [NH4 +] was measured in renal biopsies, NH4 + excretion and titratable acid quantified in spot urine, and arterial blood gasses evaluated in normoventilated mice. RESULTS: Basolateral Na+/HCO3 - cotransport activity was similar in isolated perfused mTALs from wild-type and NBCn1 knockout mice under control conditions. During metabolic acidosis, basolateral Na+/HCO3 - cotransport activity increased four-fold in mTALs from wild-type mice, but remained unchanged in mTALs from NBCn1 knockout mice. Correspondingly, NBCn1 protein expression in wild-type mice increased ten-fold in the inner stripe of renal outer medulla during metabolic acidosis. During systemic acid loading, knockout of NBCn1 inhibited the net NH4 + reabsorption across mTALs by approximately 60%, abolished the renal corticomedullary NH4 + gradient, reduced the capacity for urinary NH4 + excretion by approximately 50%, and delayed recovery of arterial blood pH and standard [HCO3 -] from their initial decline. CONCLUSIONS: During metabolic acidosis, NBCn1 is required for the upregulated basolateral HCO3 - uptake and transepithelial NH4 + reabsorption in mTALs, renal medullary NH4 + accumulation, urinary NH4 + excretion, and early recovery of arterial blood pH and standard [HCO3 -]. These findings support that NBCn1 facilitates urinary net acid excretion by neutralizing intracellular H+ released during NH4 + reabsorption across mTALs.

Impaired renal HCO3- secretion in CFTR deficient mice causes metabolic alkalosis during chronic base-loading.

AIM: Cystic fibrosis patients have an increased risk of developing metabolic alkalosis presumably as a result of altered renal HCO3- handling. In this study, we directly assess the kidneys' ability to compensate for a chronic base-load in the absence of functional CFTR. METHODS: Comprehensive urine and blood acid-base analyses were done in anaesthetized WT mice or mice lacking either CFTR or pendrin, with or without 7 days of oral NaHCO3 loading. The in vivo experiments were complemented by a combination of immunoblotting and experiments with perfused isolated mouse cortical collecting ducts (CCD). RESULTS: Base-loaded WT mice maintained acid-base homeostasis by elevating urinary pH and HCO3- excretion and decreasing urinary net acid excretion. In contrast, pendrin KO mice and CFTR KO mice were unable to increase urinary pH and HCO3- excretion and unable to decrease urinary net acid excretion sufficiently and thus developed metabolic alkalosis in response to the same base-load. The expression of pendrin was increased in response to the base-load in WT mice with a paralleled increased pendrin function in the perfused CCD. In CFTR KO mice, 7 days of base-loading did not upregulate pendrin expression and apical Cl- /HCO3- exchange function was strongly blunted in the CCD. CONCLUSION: CFTR KO mice develop metabolic alkalosis during a chronic base-load because they are unable to sufficiently elevate renal HCO3- excretion. This can be explained by markedly reduced pendrin function in the absence of CFTR.

Impaired Renal HCO3- Excretion in Cystic Fibrosis.

BACKGROUND: Patients with cystic fibrosis (CF) do not respond with increased urinary HCO3- excretion after stimulation with secretin and often present with metabolic alkalosis. METHODS: By combining RT-PCR, immunohistochemistry, isolated tubule perfusion, in vitro cell studies, and in vivo studies in different mouse models, we elucidated the mechanism of secretin-induced urinary HCO3- excretion. For CF patients and CF mice, we developed a HCO3- drinking test to assess the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in urinary HCO3-excretion and applied it in the patients before and after treatment with the novel CFTR modulator drug, lumacaftor-ivacaftor. RESULTS: ß-Intercalated cells express basolateral secretin receptors and apical CFTR and pendrin. In vivo application of secretin induced a marked urinary alkalization, an effect absent in mice lacking pendrin or CFTR. In perfused cortical collecting ducts, secretin stimulated pendrin-dependent Cl-/HCO3- exchange. In collecting ducts in CFTR knockout mice, baseline pendrin activity was significantly lower and not responsive to secretin. Notably, patients with CF (F508del/F508del) and CF mice showed a greatly attenuated or absent urinary HCO3--excreting ability. In patients, treatment with the CFTR modulator drug lumacaftor-ivacaftor increased the renal ability to excrete HCO3-. CONCLUSIONS: These results define the mechanism of secretin-induced urinary HCO3- excretion, explain metabolic alkalosis in patients with CF, and suggest feasibility of an in vivo human CF urine test to validate drug efficacy.

Intact colonic KCa1.1 channel activity in KCNMB2 knockout mice.

Mammalian potassium homeostasis results from tightly regulated renal and colonic excretion, which balances the unregulated dietary K+ intake. Colonic K+ secretion follows the pump-leak model, in which the large conductance Ca2+-activated K+ channel (KCa1.1) is well established as the sole, but highly regulated apical K+ conductance. The physiological importance of auxiliary ß and γ subunits of the pore-forming α-subunit of the KCa1.1 channel is not yet fully established. This study investigates colonic K+ secretion in a global knockout mouse of the KCa1.1-ß2-subunit (KCNMB2-/-), which apparently is the only ß-subunit of the colonic enterocyte KCa1.1 channel. We can report that: (1) Neither KCa1.1 α- nor the remaining ß-subunits were compensatory transcriptional regulated in colonic epithelia of KCNMB2-/- mice. (2) Colonic epithelia from KCNMB2-/- mice displayed equal basal and ATP-induced KCa1.1-mediated K+ conductance as compared to KCNMB2+/+ (3) K+ secretion was increased in KCNMB2-/- epithelia compared to wild-type epithelia from animals fed an aldosterone-inducing diet. (4) Importantly, the apical K+ conductance was abolished by the specific blocker of KCa1.1 channel iberiotoxin in both KCNMB2+/+ and KCNMB2-/- mice. Recently a novel family of auxiliary γ-subunits of the KCa1.1 channel has been described. (5) We detected the γ1-subunit (LRRC26) mRNA in colonic epithelia. To investigate the physiological role of the γ1-subunit of KCa1.1 channels in colonic K+ secretion, we acquired an LRRC26 knockout mouse. (6) Unexpectedly, LRRC26 mice had a perinatal lethal phenotype, thus preventing functional measurements. On this basis we conclude that colonic K+ secretion is intact or even increased in mice lacking the ß2-subunit of KCa1.1 channel complex despite no additional compensatory induction of KCa1.1 ß-subunits.

Hyperaldosteronism after decreased renal K+ excretion in KCNMB2 knockout mice.

The kidney is the primary organ ensuring K(+) homeostasis. K(+) is secreted into the urine in the distal tubule by two mechanisms: by the renal outer medullary K(+) channel (Kir1.1) and by the Ca(2+)-activated K(+) channel (KCa1.1). Here, we report a novel knockout mouse of the ß2-subunit of the KCa1.1 channel (KCNMB2), which displays hyperaldosteronism after decreased renal K(+) excretion. KCNMB2(-/-) mice displayed hyperaldosteronism, normal plasma K(+) concentration, and produced dilute urine with decreased K(+) concentration. The normokalemia indicated that hyperaldosteronism did not result from primary aldosteronism. Activation of the renin-angiotensin-aldosterone system was also ruled out as renal renin mRNA expression was reduced in KCNMB2(-/-) mice. Renal K(+) excretion rates were similar in the two genotypes; however, KCNMB2(-/-) mice required elevated plasma aldosterone to achieve K(+) balance. Blockade of the mineralocorticoid receptor with eplerenone triggered mild hyperkalemia and unmasked reduced renal K(+) excretion in KCNMB2(-/-) mice. Knockout mice for the α-subunit of the KCa1.1 channel (KCNMA1(-/-) mice) have hyperaldosteronism, are hypertensive, and lack flow-induced K(+) secretion. KCNMB2(-/-) mice share the phenotypic traits of normokalemia and hyperaldosteronism with KCNMA1(-/-) mice but were normotensive and displayed intact flow-induced K(+) secretion. Despite elevated plasma aldosterone, KNCMB2(-/-) mice did not display salt-sensitive hypertension and were able to decrease plasma aldosterone on a high-Na(+) diet, although plasma aldosterone remained elevated in KCNMB2(-/-) mice. In summary, KCNMB2(-/-) mice have a reduced ability to excrete K(+) into the urine but achieve K(+) balance through an aldosterone-mediated, ß2-independent mechanism. The phenotype of KCNMB2 mice was similar but milder than the phenotype of KCNMA1(-/-) mice.

Mechanisms of renal control of potassium homeostasis in complete aldosterone deficiency.

Aldosterone-independent mechanisms may contribute to K(+) homeostasis. We studied aldosterone synthase knockout (AS(-/-)) mice to define renal control mechanisms of K(+) homeostasis in complete aldosterone deficiency. AS(-/-) mice were normokalemic and tolerated a physiologic dietary K(+) load (2% K(+), 2 days) without signs of illness, except some degree of polyuria. With supraphysiologic K(+) intake (5% K(+)), AS(-/-) mice decompensated and became hyperkalemic. High-K(+) diets induced upregulation of the renal outer medullary K(+) channel in AS(-/-) mice, whereas upregulation of the epithelial sodium channel (ENaC) sufficient to increase the electrochemical driving force for K(+) excretion was detected only with a 2% K(+) diet. Phosphorylation of the thiazide-sensitive NaCl cotransporter was consistently lower in AS(-/-) mice than in AS(+/+) mice and was downregulated in mice of both genotypes in response to increased K(+) intake. Inhibition of the angiotensin II type 1 receptor reduced renal creatinine clearance and apical ENaC localization, and caused severe hyperkalemia in AS(-/-) mice. In contrast with the kidney, the distal colon of AS(-/-) mice did not respond to dietary K(+) loading, as indicated by Ussing-type chamber experiments. Thus, renal adaptation to a physiologic, but not supraphysiologic, K(+) load can be achieved in aldosterone deficiency by aldosterone-independent activation of the renal outer medullary K(+) channel and ENaC, to which angiotensin II may contribute. Enhanced urinary flow and reduced activity of the thiazide-sensitive NaCl cotransporter may support renal adaptation by activation of flow-dependent K(+) secretion and increased intratubular availability of Na(+) that can be reabsorbed in exchange for K(+) secreted.

Rapid dephosphorylation of the renal sodium chloride cotransporter in response to oral potassium intake in mice.

A dietary potassium load induces a rapid kaliuresis and natriuresis, which may occur even before plasma potassium and aldosterone (aldo) levels increase. Here we sought to gain insight into underlying molecular mechanisms contributing to this response. After gastric gavage of 2% potassium, the plasma potassium concentrations rose rapidly (0.25 h), followed by a significant rise of plasma aldo (0.5 h) in mice. Enhanced urinary potassium and sodium excretion was detectable as early as spot urines could be collected (about 0.5 h). The functional changes were accompanied by a rapid and sustained (0.25-6 h) dephosphorylation of the NaCl cotransporter (NCC) and a late (6 h) upregulation of proteolytically activated epithelial sodium channels. The rapid effects on NCC were independent from the coadministered anion. NCC dephosphorylation was also aldo-independent, as indicated by experiments in aldo-deficient mice. The observed urinary sodium loss relates to NCC, as it was markedly diminished in NCC-deficient mice. Thus, downregulation of NCC likely explains the natriuretic effect of an acute oral potassium load in mice. This may improve renal potassium excretion by increasing the amount of intraluminal sodium that can be exchanged against potassium in the aldo-sensitive distal nephron.

Basolateral P2X receptors mediate inhibition of NaCl transport in mouse medullary thick ascending limb (mTAL).

Extracellular nucleotides regulate epithelial transport via luminal and basolateral P2 receptors. Renal epithelia express multiple P2 receptors, which mediate significant inhibition of solute absorption. Recently, we identified several P2 receptors in the medullary thick ascending limb (mTAL) including luminal and basolateral P2Y(2) receptors (Jensen ME, Odgaard E, Christensen MH, Praetorius HA, Leipziger J. J Am Soc Nephrol 18: 2062-2070, 2007). In addition, we found evidence for a basolateral P2X receptor. Here, we investigate the effect of basolateral ATP on NaCl absorption in isolated, perfused mouse mTALs using the electrical measurement of equivalent short-circuit current (I'(sc)). Nonstimulated mTALs transported at a rate of 1,197 ± 104 µA/cm(2) (n = 10), which was completely blockable with luminal furosemide (100 µM). Basolateral ATP (100 µM) acutely (1 min) and reversibly reduced the absorptive I'(sc). After 2 min, the reduction amounted to 24.4 ± 4.0% (n = 10). The nonselective P2 receptor antagonist suramin blocked the effect. P2Y receptors were found not to be involved in this effect. The P2X receptor agonist 2-methylthio ATP mimicked the ATP effect, and the P2X receptor antagonist periodate-oxidized ATP blocked it. In P2X(7)(-/-) mice, the ATP effect remained unaltered. In contrast, in P2X(4)(-/-) mice the ATP-induced inhibition of transport was reduced. A comprehensive molecular search identified P2X(4), P2X(5), and P2X(1) receptor subunit mRNA in isolated mouse mTALs. These data define that basolateral ATP exerts a significant inhibition of Na(+) absorption in mouse mTAL. Pharmacological, molecular, and knockout mouse data identify a role for the P2X(4) receptor. We suggest that other P2X subunits like P2X(5) are part of the P2X receptor complex. These data provide the novel perspective that an ionotropic receptor and thus a nonselective cation channel causes transport inhibition in an intact renal epithelium.

The secretory KCa1.1 channel localises to crypts of distal mouse colon: functional and molecular evidence.

The colonic epithelium absorbs and secretes electrolytes and water. Ion and water absorption occurs primarily in surface cells, whereas crypt cells perform secretion. Ion transport in distal colon is regulated by aldosterone, which stimulates both Na(+) absorption and K(+) secretion. The electrogenic Na(+) absorption is mediated by epithelial Na(+) channel (ENaC) in surface cells. Previously, we identified the large conductance Ca(2+)-activated K(+) channel, K(Ca)1.1 or big potassium (BK) channel, as the only relevant K(+) secretory pathway in mouse distal colon. The exact localisation of K(Ca)1.1 channels along the crypt axis is, however, still controversial. The aim of this project was to further define the localisation of the K(Ca)1.1 channel in mouse distal colonic epithelium. Through quantification of mRNA extracted from micro-dissected surface and crypt cells, we confirmed that Na(+)/K(+)/2Cl(-) (NKCC1) is expressed primarily in the crypts and γ-ENaC primarily in the surface cells. The K(Ca)1.1 α-subunit mRNA was like NKCC1, mainly expressed in the crypts. The crypt to surface expression pattern of the channels and transporters was not altered when plasma aldosterone was elevated. The mRNA levels for NKCC1, γ-ENaC and K(Ca)1.1 α-subunit were, however, under these circumstances substantially augmented (K(Ca)1.1 α-subunit, twofold; NKCC1, twofold and ENaC, tenfold). Functionally, we show that ENaC-mediated Na(+) absorption and BK channel-mediated K(+) secretion are two independent processes. These findings show that K(Ca)1.1-mediated K(+) secretion mainly occurs in the crypts of the murine distal colon. This is in agreement with the general model of ion secretion being preferentially located to the crypt and not surface enterocytes.

Adrenaline-induced colonic K+ secretion is mediated by KCa1.1 (BK) channels.

Colonic epithelial K(+) secretion is a two-step transport process with initial K(+) uptake over the basolateral membrane followed by K(+) channel-dependent exit into the lumen. In this process the large-conductance, Ca(2+)-activated K(Ca)1.1 (BK) channel has been identified as the only apparent secretory K(+) channel in the apical membrane of the murine distal colon. The BK channel is responsible for both resting and Ca(2+)-activated colonic K(+) secretion and is up-regulated by aldosterone. Agonists (e.g. adrenaline) that elevate cAMP are potent activators of distal colonic K(+) secretion. However, the secretory K(+) channel responsible for cAMP-induced K(+) secretion remains to be defined. In this study we used the Ussing chamber to identify adrenaline-induced electrogenic K(+) secretion. We found that the adrenaline-induced electrogenic ion secretion is a compound effect dominated by anion secretion and a smaller electrically opposing K(+) secretion. Using tissue from (i) BK wildtype (BK(+/+)) and knockout (BK(/)) and (ii) cystic fibrosis transmembrane regulator (CFTR) wildtype (CFTR(+/+)) and knockout (CFTR(/)) mice we were able to isolate the adrenaline-induced K(+) secretion. We found that adrenaline-induced K(+) secretion: (1) is absent in colonic epithelia from BK(/) mice, (2) is greatly up-regulated in mice on a high K(+) diet and (3) is present as sustained positive current in colonic epithelia from CFTR(/) mice. We identified two known C-terminal BK alpha-subunit splice variants in colonic enterocytes (STREX and ZERO). Importantly, the ZERO variant known to be activated by cAMP is differentially up-regulated in enterocytes from animals on a high K(+) diet. In summary, these results strongly suggest that the adrenaline-induced distal colonic K(+) secretion is mediated by the BK channel and probably involves aldosterone-induced ZERO splice variant up-regulation.

Colonic potassium handling.

Homeostatic control of plasma K+ is a necessary physiological function. The daily dietary K+ intake of approximately 100 mmol is excreted predominantly by the distal tubules of the kidney. About 10% of the ingested K+ is excreted via the intestine. K+ handling in both organs is specifically regulated by hormones and adapts readily to changes in dietary K+ intake, aldosterone and multiple local paracrine agonists. In chronic renal insufficiency, colonic K+ secretion is greatly enhanced and becomes an important accessory K+ excretory pathway. During severe diarrheal diseases of different causes, intestinal K+ losses caused by activated ion secretion may become life threatening. This topical review provides an update of the molecular mechanisms and the regulation of mammalian colonic K+ absorption and secretion. It is motivated by recent results, which have identified the K+ secretory ion channel in the apical membrane of distal colonic enterocytes. The directed focus therefore covers the role of the apical Ca2+ and cAMP-activated BK channel (KCa1.1) as the apparently only secretory K+ channel in the distal colon.

The essential role of luminal BK channels in distal colonic K+ secretion.

Distal colonic K(+) excretion is determined by the balance of K(+) absorption and K(+) secretion of the enterocytes. K(+) secretion occurs via active basolateral K(+) uptake mostly via the NKCC1 co-transporter followed by K(+) exit via a luminal K(+) channel. The specific focus here is directed towards the luminal secretory K(+) channel (1). Several recent observations highlight the pivotal role of the large conductance, Ca(2+)-activated K(Ca)1.1 (BK, KCNMA) channel as the only functionally relevant luminal K(+) efflux pathway in mouse distal colon (2, 3). This conclusion was based on defining results from BK knock-out mice. The following key observations were made: 1. BK channels mediate the resting distal colonic K(+) secretion (2, 4), 2. They are acutely stimulated by activation of luminal nucleotide receptor and elevations of intracellular Ca(2+) (2, 4, 5), 3. Colonic BK channels are up-regulated by increases of plasma aldosterone (3), 4. In addition, the cAMP-stimulated distal colonic K(+) secretion is apparently mediated via BK channels, 5. Finally, aldosterone was found to up-regulate specifically the ZERO (e.g. cAMP activated) C-terminal splice variant of the BK channel. In summary, we suggest that the sole exit pathway for transcellular (K+) secretion in mammalian distal colon is the BK channel, which is the target for short term intracellular Ca(2+) and cAMP activation and long term aldosterone regulation.

Aldosterone increases KCa1.1 (BK) channel-mediated colonic K+ secretion.

Mammalian K(+) homeostasis results from highly regulated renal and intestinal absorption and secretion, which balances the unregulated K(+) intake. Aldosterone is known to enhance both renal and colonic K(+) secretion. In mouse distal colon K(+) secretion occurs exclusively via luminal K(Ca)1.1 (BK) channels. Here we investigate if aldosterone stimulates colonic K(+) secretion via BK channels. Luminal Ba(2+) and iberiotoxin (IBTX)-sensitive electrogenic K(+) secretion was measured in Ussing chambers. In vivo aldosterone was augmented via a high K(+) diet. High K(+) diet led to a 2-fold increase of luminal Ba(2+) and IBTX-sensitive short-circuit current in distal mouse colonic mucosa. This effect was absent in BK alpha-subunit-deficient (BK(-/-)) mice. The resting and diet-induced K(+) secretion was stimulated by luminal ionomycin. In BK(-/-) mice luminal ionomycin did not stimulate K(+) secretion. In vitro addition of aldosterone likewise triggered a 2-fold increase in K(+) secretion, which was inhibited by the mineralocorticoid receptor antagonist spironolactone and the BK channel blocker IBTX. Semi-quantification of mRNA from colonic crypts showed up-regulation of BK alpha- and beta(2)-subunits in high K(+) diet mice. The BK channel could be detected luminally in colonic crypt cells by immunohistochemistry. The expression level of the channel in the luminal membrane was strongly up-regulated in K(+)-loaded animals. Taken together, these data strongly suggest that aldosterone-induced K(+) secretion occurs via increased expression of luminal BK channels.