Mads V. Sorensen

Colonic potassium handling

Homeostatic control of plasma K+ is a necessary physiological function. The daily dietary K+ intake of ~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.

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 KCa1.1 (BK) channels. Here we investigate if aldosterone stimulates colonic K+ secretion via BK channels. Luminal Ba2+ 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 Ba2+ and IBTX‐sensitive short‐circuit current in distal mouse colonic mucosa. This effect was absent in BK α‐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 α‐ and β2‐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.

Distal colonic Na(+) absorption inhibited by luminal P2Y(2) receptors.

Luminal P2 receptors are ubiquitously expressed in transporting epithelia. In steroid-sensitive epithelia (e.g., lung, distal nephron) epithelial Na+ channel (ENaC)-mediated Na+ absorption is inhibited via luminal P2 receptors. In distal mouse colon, we have identified that both, a luminal P2Y2 and a luminal P2Y4 receptor, stimulate K+ secretion. In this study, we investigate the effect of luminal adenosine triphosphate/uridine triphosphate (ATP/UTP) on electrogenic Na+ absorption in distal colonic mucosa of mice treated on a low Na+ diet for more than 2xa0weeks. Transepithelial electrical parameters were recorded in an Ussing chamber. Baseline parameters: transepithelial voltage (Vte): −13.7u2009±u20091.9xa0mV (lumen negative), transepithelial resistance (Rte): 24.1u2009±u20091.8xa0Ω cm2, equivalent short circuit current (Isc): −563.9u2009±u200963.8xa0μA/cm2 (nu2009=u200921). Amiloride completely inhibited Isc to −0.5u2009±u20098.5xa0μA/cm2. Luminal ATP induced a slowly on-setting and persistent inhibition of the amiloride-sensitive Isc by 160.7u2009±u200929.7xa0μA/cm2 (nu2009=u200912, NMRI mice). Luminal ATP and UTP were almost equipotent with IC50 values of 10xa0μM and 3xa0μM respectively. In P2Y2 knock-out (KO) mice, the effect of luminal UTP on amiloride-sensitve Na+ absorption was absent. In contrast, in P2Y4 KO mice the inhibitory effect of luminal UTP on Na+ absorption remained present. Semiquantitative polymerase chain reaction did not indicate regulation of the P2Y receptors under low Na+ diet, but it revealed a pronounced axial expression of both receptors with highest abundance in surface epithelia. Thus, luminal P2Y2 and P2Y4 receptors and ENaC channels co-localize in surface epithelium. Intriguingly, only the stimulation of the P2Y2 receptor mediates inhibition of electrogenic Na+ absorption.

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.

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, Ca2+‐activated KCa1.1 (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 Ca2+‐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 α‐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.

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 Ca2+-activated K+ channel, KCa1.1 or big potassium (BK) channel, as the only relevant K+ secretory pathway in mouse distal colon. The exact localisation of KCa1.1 channels along the crypt axis is, however, still controversial. The aim of this project was to further define the localisation of the KCa1.1 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 KCa1.1 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 KCa1.1 α-subunit were, however, under these circumstances substantially augmented (KCa1.1 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 KCa1.1-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.

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 β2-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, β2-independent mechanism. The phenotype of KCNMB2 mice was similar but milder than the phenotype of KCNMA1(-/-) mice.

Impaired aldosterone responsiveness in corticosteroid binding globulin deficient mice

Aim:u2002 Corticosteroid binding globulin (CBG) is the high affinity plasma carrier protein for cortisol. It keeps the steroids inactive, prevents them from degradation and defines the amount of free hormone acting on target tissues. Previous findings have shown insufficient responsiveness of corticosterone in peripheral tissues in CBG−/− mice despite elevated free plasma corticosterone. In the large intestine, glucocorticoids synergistically enhance the pro‐absorptive effects of aldosterone. We therefore hypothesized that CBG−/− mice have reduced responsiveness to aldosterone.

Na(+) dependence of K(+) -induced natriuresis, kaliuresis and Na(+) /Cl(-) cotransporter dephosphorylation.

High dietary K+ intake is associated with protection against hypertension. In mammals, acute K+ intake induces natriuresis and kaliuresis, associated with a marked dephosphorylation of the renal Na+/Cl− cotransporter (NCC). It has been suggested that reduced activity of NCC increases the driving force for more distal tubular epithelial Na+ channel (ENaC)‐dependent K+ secretion. This study investigated the ENaC dependence of urinary K+ and Na+ excretion following acute K+ loading.

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 β 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‐β2‐subunit (KCNMB2−/−), which apparently is the only β‐subunit of the colonic enterocyte KCa1.1 channel. We can report that: (1) Neither KCa1.1 α‐ nor the remaining β‐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 β2‐subunit of KCa1.1 channel complex despite no additional compensatory induction of KCa1.1 β‐subunits.