The AE4 transporter mediates kidney acid-base sensing.

The kidney plays a key role in the correction of systemic acid-base imbalances. Central for this regulation are the intercalated cells in the distal nephron, which secrete acid or base into the urine. How these cells sense acid-base disturbances is a long-standing question. Intercalated cells exclusively express the Na+-dependent Cl-/HCO3- exchanger AE4 (Slc4a9). Here we show that AE4-deficient mice exhibit a major dysregulation of acid-base balance. By combining molecular, imaging, biochemical and integrative approaches, we demonstrate that AE4-deficient mice are unable to sense and appropriately correct metabolic alkalosis and acidosis. Mechanistically, a lack of adaptive base secretion via the Cl-/HCO3- exchanger pendrin (Slc26a4) is the key cellular cause of this derailment. Our findings identify AE4 as an essential part of the renal sensing mechanism for changes in acid-base status.

Hydrochlorothiazide and acute urinary acidification: The "voltage hypothesis" of ENaC-dependent H+ secretion refuted.

AIM: The "voltage hypothesis" of H+ secretion states that urinary acidification following increased Na+ delivery to the collecting duct (CD) is ENaC dependent leading to transepithelial voltage-dependent increase in H+ secretion. We recently showed that furosemide acidifies the urine independently of ENaC activity. If the voltage hypothesis holds, hydrochlorothiazide (HCT) must acidify the urine. We here tested the acute effect of HCT on urine pH under normal and high ENaC expression. METHODS: Mice subjected to a control or a low-Na+ diet were anesthetized and infused (0.5 mL h-1 ) with saline. Catheterization of the urinary bladder allowed real-time measurement of diuresis and urine pH. Mice received either HCT (1 mg mL-1 ) or vehicle. Urinary Na+ and K+ excretions were determined by flame photometry. ENaC expression levels were measured by semi-quantitative Western blotting. RESULTS: (1) HCT increased diuresis and natriuresis in both diet groups. (2) K+ excretion rates increased after HCT administration from 18.6 ± 1.3 to 31.7 ± 2.5 µmol h-1 in the control diet group and from 23.0 ± 1.3 to 48.7 ± 3.0 µmol h-1 in the low-Na+ diet group. (3) Mice fed a low-Na+ diet showed a marked upregulation of ENaC. (4) Importantly, no acute changes in urine pH were observed after the administration of HCT in either group. CONCLUSION: Acute administration of HCT has no effect on urine pH. Similarly, substantial functional and molecular upregulation of ENaC did not cause HCT to acutely change urine pH. Thus, an increased Na+ load to the CD does not alter urine pH. This supports our previous finding and likely falsifies the voltage hypothesis of H+ secretion.

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

AIM: 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. METHODS: Mice were fed low (0.03%), control (0.2%) or high (2%) Na(+) diets for 25 days to preserve or promote Na(+) loss and thus ENaC activity. Once a week, the mice received either K(+) -containing gavage or a control gavage. Following the gavage treatment, the mice were placed in metabolic cages and urine was collected in real time. ENaC dependence of kaliuresis was assessed by benzamil injections prior to gavage. RESULTS: We confirmed that dietary Na(+) content is inversely related to plasma aldosterone, NCC phosphorylation and ENaC cleavage products. The novel findings were as follows: (i) acute K(+) feeding caused NCC dephosphorylation in all dietary groups; (ii) under all dietary conditions, K(+) loading induced natriuresis; (iii) high Na(+) diet markedly reduced the K(+) excretion following K(+) gavage; (iv) benzamil injection prior to K(+) loading increased natriuresis, decreased kaliuresis and eliminated the differences between the dietary groups. CONCLUSION: These data indicate that acute K(+) -induced kaliuresis is ENaC dependent. Maximal K(+) excretion rates are attenuated when ENaC is physiologically down-regulated or pharmacologically blocked. NCC is dephosphorylated following acute K(+) loading under all dietary Na(+) regimens. This leads to natriuresis, even in severely Na(+) -restricted animals.

P2X receptors trigger intracellular alkalization in isolated perfused mouse medullary thick ascending limb.

AIMS: Extracellular ATP is an important regulator of renal tubular transport. Recently, we found that basolateral ATP markedly inhibits Na(+) and Cl(-) absorption in mouse medullary thick ascending limb (mTAL) via a P2X receptor. The underlying mechanism that mediates this ATP-dependent transport inhibition in mTAL is, however, unclear. The renal outer medullary K(+) channel (ROMK) is sensitive to intracellular pH where a reduction leads to closing of ROMK. We speculated that P2X receptor stimulation in the TAL could lead to changes in pHi , leading to a reduction in NaCl transport. METHODS: To test this hypothesis, we measured pHi in single perfused mouse mTALs using the fluorescent ratiometric dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethylester. RESULTS: Interestingly, basolateral ATP (100 µm) caused a prominent, reversible intracellular alkalization of mTAL, with an average pHi increase of 0.14 ± 0.02 (n = 14). This was completely abolished by the P2X receptor antagonist periodate-oxidized ATP (50 µm). The P2X receptor-mediated intracellular alkalization required the activity of the apical Na(+) /H(+) exchanger (NHE3). Typically, Gq -coupled receptors cause a significant acidification of tubular epithelial cells, which was confirmed in this study, by P2Y2 and Ca(2+) sensing receptor stimulation. CONCLUSION: This study reports that stimulation of basolateral P2X receptors causes a substantial intracellular alkalization in the isolated perfused mouse mTAL. This intracellular alkalization is mediated through an increased apical NHE3 activity, similar to what we previously observed when tubular transport is inhibited with furosemide. This increased NHE3 activity causes H(+) secretion in the mTAL and provides further support that the TAL is a site of urinary acidification.

P2X receptor stimulation amplifies complement-induced haemolysis.

Activation of the complement system evokes cell damage by insertion of membrane attack complexes, which constitute the basis of the pathogenesis of various haemolytic disorders. Recently, we found that haemolysis caused by other types of membrane pore-forming proteins such as α-haemolysin (HlyA) from Escherichia coli, α-toxin from Staphylococcus aureus and leukotoxin from Aggregatibacter actinomycetemcomitans inflict their cytotoxic effects through P2 receptor activation. Here we show that similar to haemolysis induced by HlyA, leukotoxin and α-toxin, complement-induced haemolysis is amplified through ATP release and subsequent P2 receptor activation. Similar results were found both in murine, sensitised ovine and human erythrocytes, with either human plasma or guinea pig serum as complement donors. Non-selective P2 antagonists (PPADS and suramin) concentration-dependently inhibited complement-induced haemolysis. More specific P2 receptor antagonists imply that P2X1 and P2X7 are the main receptors involved in this response. Moreover, complement activation produces a sustained increase in [Ca(2+)]i, which initially triggers significant erythrocyte shrinkage, most likely mediated by KCa3.1-dependent K(+) efflux. These results indicate that complement, similar to HlyA and α-toxin, requires purinergic signalling for full haemolysis and that activation of erythrocyte volume regulation protracts the process. This finding points to several new pathways to interfere with haemolytic diseases and implies that P2 receptor antagonists potentially can be used to prevent intravascular haemolysis.

UTP reduces infarct size and improves mice heart function after myocardial infarct via P2Y2 receptor.

Pyrimidine nucleotides are signaling molecules, which activate G protein-coupled membrane receptors of the P2Y family. P2Y(2) and P2Y(4) receptors are part of the P2Y family, which is composed of 8 subtypes that have been cloned and functionally defined. We have previously found that uridine-5'-triphosphate (UTP) reduces infarct size and improves cardiac function following myocardial infarct (MI). The aim of the present study was to determine the role of P2Y(2) receptor in cardiac protection following MI using knockout (KO) mice, in vivo and wild type (WT) for controls. In both experimental groups used (WT and P2Y(2)(-/-) receptor KO mice) there were 3 subgroups: sham, MI, and MI+UTP. 24h post MI we performed echocardiography and measured infarct size using triphenyl tetrazolium chloride (TTC) staining on all mice. Fractional shortening (FS) was higher in WT UTP-treated mice than the MI group (44.7±4.08% vs. 33.5±2.7% respectively, p<0.001). However, the FS of P2Y(2)(-/-) receptor KO mice were not affected by UTP treatment (34.7±5.3% vs. 35.9±2.9%). Similar results were obtained with TTC and hematoxylin and eosin stainings. Moreover, troponin T measurements demonstrated reduced myocardial damage in WT mice pretreated with UTP vs. untreated mice (8.8±4.6 vs. 12±3.1 p<0.05). In contrast, P2Y(2)(-/-) receptor KO mice pretreated with UTP did not demonstrate reduced myocardial damage. These results indicate that the P2Y(2) receptor mediates UTP cardioprotection, in vivo.

Extracellular nucleotide derivatives protect cardiomyocytes against hypoxic stress.

RATIONALE: Extracellular nucleotides have widespread effects and various cell responses. Whereas the effect of a purine nucleotide (ATP) and a pyrimidine nucleotide (UTP) on myocardial infarction has been examined, the role of different purine and pyrimidine nucleotides and nucleosides in cardioprotection against hypoxic stress has not been reported. OBJECTIVE: To investigate the role of purine and pyrimidine nucleotides and nucleosides in protective effects in cardiomyocytes subjected to hypoxia. METHODS AND RESULTS: Rat cultured cardiomyocytes were treated with various extracellular nucleotides and nucleosides, before or during hypoxic stress. The results revealed that GTP or CTP exhibit cardioprotective ability, as revealed by lactate dehydrogenase (LDH) release, by propidium iodide (PI) staining, by cell morphology, and by preserved mitochondrial activity. Pretreatment with various P2 antagonists (suramin, RB-2, or PPADS) did not abolish the cardioprotective effect of the nucleotides. Moreover, P2Y2 -/- , P2Y4 -/-, and P2Y2 -/-/P2Y4 -/- receptor knockouts mouse cardiomyocytes were significantly protected against hypoxic stress when treated with UTP. These results indicate that the protective effect is not mediated via those receptors. We found that a wide variety of triphosphate and diphosphate nucleotides (TTP, ITP, deoxyGTP, and GDP), provided significant cardioprotective effect. GMP, guanosine, and ribose phosphate provided no cardioprotective effect. Moreover, we observed that tri/di-phosphate alone assures cardioprotection. Treatment with extracellular nucleotides, or with tri/di-phosphate, administered under normoxic conditions or during hypoxic conditions, led to a decrease in reactive oxygen species production. CONCLUSIONS: Extracellular tri/di-phosphates are apparently the molecule responsible for cardioprotection against hypoxic damage, probably by preventing free radicals formation.

Impaired aldosterone responsiveness in corticosteroid binding globulin deficient mice.

AIM: 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. METHODS: We used CBG⁻(/)⁻ and CBG(+/+) mice to investigate distal colonic electrogenic Na(+) absorption. An Ussing chamber was used to quantify amiloride-sensitive Na(+) transport in distal colonic mucosa (ΔI(sc) (amil)) as a measure of the physiological effect of aldosterone. RESULTS: No differences were observed in ΔI(sc) (amil) or aldosterone levels in animals on control diet. When Na(+) restricted, CBG(+/+) mice responded with a marked up-regulation of ΔI(sc) (amil) (25-fold). In CBG⁻(/)⁻ mice this up-regulation was greatly attenuated as seen in a markedly reduced amiloride-sensitive short circuit current (reduced by ∼50%), a reduced ability to lower faecal Na(+) excretion and a significantly attenuated up-regulation of the ENaC channel γ-subunit. Diet-induced increases of total plasma aldosterone were similar in both genotypes, but CBG⁻(/)⁻ mice had an increased free plasma aldosterone fraction. SUMMARY: This study defines the functional hyporesponsiveness and aldosterone resistance in distal colon of CBG⁻(/)⁻ mice. This resistance occurs despite sufficient free corticosterone plasma level. Thus, steroid actions require an intrinsic but unknown function of CBG, which allows the sufficient supply of the hormone/s to the target tissue.

Released nucleotides amplify the cilium-dependent, flow-induced [Ca2+]i response in MDCK cells.

AIM: Changes in perfusate flow produce increases in [Ca(2+)](i) in renal epithelial cells. Cultured renal epithelia require primary cilia to sense subtle changes in flow. In perfused kidney tubules this flow response is caused by nucleotide signalling via P2Y(2) receptors. It is, however, not known whether nucleotides are released by mechanical stress applied to renal primary cilia. Here we investigate whether nucleotides are released during the cilium-dependent flow response and contribute to the flow-induced, cilium-dependent [Ca(2+)](i) signal. METHODS: MDCK cells loaded with Fluo-4-AM were observed at 37 degrees C in semi-open single or closed-double perfusion chambers. RESULTS: Our data suggest a purinergic component of the cilium-dependent flow-response: (1) ATP scavengers and P2 receptor antagonists reduced (55%) the cilium-dependent flow-response; (2) ATP added at subthreshold concentration sensitized the renal epithelia to flow changes; (3) increases in fluid flow transiently enhanced the ATP concentration in the superfusate (measured by biosensor-cells). To test if nucleotides were released in sufficient quantities to stimulate renal epithelia we used non-confluent MDCK cells without cilia as reporter cells. We confirmed that non-confluent cells do not respond to changes in fluid flow. Placing confluent, ciliated cells upstream in the in-flow path of the non-confluent cells made them responsive to fluid flow changes. This phenomenon was not observed if either non-confluent or de-ciliated confluent cells were placed upstream. The [Ca(2+)](i)-response in the non-confluent cells with ciliated cells upstream was abolished by apyrase and suramin. CONCLUSION: This suggests that subtle flow changes sensed by the primary cilium induces nucleotide release, which amplifies the epithelial [Ca(2+)](i)-response.

Slow spontaneous [Ca2+] i oscillations reflect nucleotide release from renal epithelia.

Renal epithelia can be provoked mechanically to release nucleotides, which subsequently increases the intracellular Ca(2+) concentration [Ca(2+)](i) through activation of purinergic (P2) receptors. Cultured cells often show spontaneous [Ca(2+)](i) oscillations, a feature suggested to involve nucleotide signalling. In this study, fluo-4 loaded Madin-Darby canine kidney (MDCK) cells are used as a model for quantification and characterisation of spontaneous [Ca(2+)](i) increases in renal epithelia. Spontaneous [Ca(2+)](i) increases occurred randomly as single cell events. During an observation period of 1 min, 10.9 +/- 6.7% (n = 23) of the cells showed spontaneous [Ca(2+)](i) increases. Spontaneous adenosine triphosphate (ATP) release from MDCK cells was detected directly by luciferin/luciferase. Scavenging of ATP by apyrase or hexokinase markedly reduced the [Ca(2+)](i) oscillatory activity, whereas inhibition of ecto-ATPases (ARL67156) enhanced the [Ca(2+)](i) oscillatory activity. The association between spontaneous [Ca(2+)](i) increases and nucleotide signalling was further tested in 132-1N1 cells lacking P2 receptors. These cells hardly showed any spontaneous [Ca(2+)](i) increases. Transfection with either hP2Y(6) or hP2Y(2) receptors revealed a striking degree of oscillations. Similar spontaneous [Ca(2+)](i) increases were observed in freshly isolated, perfused mouse medullary thick ascending limb (mTAL). The oscillatory activity was reduced by basolateral apyrase and substantially lower in mTAL from P2Y(2) knock out mice (0.050 +/- 0.020 events per second, n = 8) compared to the wild type (0.147 +/- 0.018 events per second, n = 9). These findings indicate that renal epithelia spontaneously release nucleotides leading to P2-receptor-dependent [Ca(2+)](i) oscillations. Thus, tonic nucleotide release is likely to modify steady state renal function.

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 P2Y(2) and a luminal P2Y(4) 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 2 weeks. Transepithelial electrical parameters were recorded in an Ussing chamber. Baseline parameters: transepithelial voltage (V (te)): -13.7 +/- 1.9 mV (lumen negative), transepithelial resistance (R (te)): 24.1 +/- 1.8 Omega cm(2), equivalent short circuit current (I (sc)): -563.9 +/- 63.8 microA/cm(2) (n = 21). Amiloride completely inhibited I (sc) to -0.5 +/- 8.5 microA/cm(2). Luminal ATP induced a slowly on-setting and persistent inhibition of the amiloride-sensitive I (sc) by 160.7 +/- 29.7 microA/cm(2) (n = 12, NMRI mice). Luminal ATP and UTP were almost equipotent with IC(50) values of 10 microM and 3 microM respectively. In P2Y(2) knock-out (KO) mice, the effect of luminal UTP on amiloride-sensitve Na(+) absorption was absent. In contrast, in P2Y(4) 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 P2Y(2) and P2Y(4) receptors and ENaC channels co-localize in surface epithelium. Intriguingly, only the stimulation of the P2Y(2) receptor mediates inhibition of electrogenic Na(+) absorption.

Role of cholinergic-activated KCa1.1 (BK), KCa3.1 (SK4) and KV7.1 (KCNQ1) channels in mouse colonic Cl- secretion.

AIM: Colonic crypts are the site of Cl- secretion. Basolateral K+ channels provide the driving force for luminal cystic fibrosis transmembrane regulator-mediated Cl- exit. Relevant colonic epithelial K+ channels are the intermediate conductance Ca2+-activated K(Ca)3.1 (SK4) channel and the cAMP-activated K(V)7.1 (KCNQ1) channel. In addition, big conductance Ca2+-activated K(Ca)1.1 (BK) channels may play a role in Ca2+-activated Cl- secretion. Here we use K(Ca)1.1 and K(Ca)3.1 knock-out mice, and the K(V)7.1 channel inhibitor 293B (10 microm) to investigate the role of K(Ca)1.1, K(Ca)3.1 and K(V)7.1 channels in cholinergic-stimulated Cl- secretion. METHODS: A Ussing chamber was used to quantify agonist-stimulated increases in short circuit current (Isc) in distal colon. Chloride secretion was activated by bl. forskolin (FSK, 2 microm) followed by bl. carbachol (CCH, 100 microm). Luminal Ba2+ (5 mm) was used to inhibit K(Ca)1.1 channels. RESULTS: K(Ca)1.1 WT and KO mice displayed identical FSK and CCH-stimulated Isc changes, indicating that K(Ca)1.1 channels are not involved in FSK- and cholinergic-stimulated Cl- secretion. CCH-stimulated DeltaIsc was significantly reduced in K(Ca)3.1 KO mice, underscoring the known relevance of this channel in the activation of Cl- secretion by an intracellular Ca2+ increasing agonist. The residual CCH effect observed in K(Ca)3.1 KO mice suggests that yet another K+ channel is driving the CCH-stimulated Cl- secretion. In the presence of the specific K(V)7.1 channel blocker 293B, the residual CCH effect was abolished. CONCLUSIONS: This demonstrates that both K(Ca)3.1 and K(V)7.1 channels are activated by cholinergic agonists and drive Cl- secretion. In contrast, K(Ca)1.1 channels are not involved in stimulated electrogenic Cl- secretion.

K+ secretion activated by luminal P2Y2 and P2Y4 receptors in mouse colon.

Extracellular nucleotides are important regulators of epithelial ion transport, frequently exerting their action from the luminal side. Luminal P2Y receptors have previously been identified in rat distal colonic mucosa. Their activation by UTP and ATP stimulates K+ secretion. The aim of this study was to clarify which of the P2Y receptor subtypes are responsible for the stimulated K+ secretion. To this end P2Y2 and P2Y4 knock-out mice were used to measure distal colonic ion transport in an Ussing chamber. In mouse (NMRI) distal colonic mucosa, luminal UTP and ATP with similar potency induced a rapid and transient increase of the transepithelial voltage (V(te)) (UTP: from -0.81 +/- 0.23 to 3.11 +/- 0.61 mV, n = 24), an increase of equivalent short circuit current (I(sc)) by 166.9 +/- 22.8 microA cm(-2) and a decrease of transepithelial resistance (R(te)) from 29.4 +/- 2.4 to 23.5 +/- 2.0 Omega cm2. This effect was completely inhibited by luminal Ba2+ (5 mm, n = 5) and iberiotoxin (240 nm, n = 6), indicating UTP/ATP-stimulated K+ secretion. RT-PCR analysis of isolated colonic crypts revealed P2Y2, P2Y4 and P2Y6 specific transcripts. The luminal UTP-stimulated K+ secretion was still present in P2Y2 receptor knock-out mice, but significantly reduced (DeltaV(te): 0.83 +/- 0.26 mV) compared to wild-type littermates (DeltaV(te): 2.08 +/- 0.52 mV, n = 9). In P2Y4 receptor knock-out mice the UTP-induced K+ secretion was similarly reduced. Luminal UTP-stimulated K+ secretion was completely absent in P2Y2/P2Y4 double receptor KO mice. Basolateral UTP showed no effect. In summary, these results indicate that both the P2Y2 and P2Y4 receptors are present in the luminal membrane of mouse distal colonic mucosa, and stimulation of these receptors leads to K+ secretion.

Transepithelial pressure pulses induce nucleotide release in polarized MDCK cells.

The release of nucleotides is involved in mechanosensation in various epithelial cells. Intriguingly, kidney epithelial cells are absolutely dependent on the primary cilium to sense changes in apical laminar flow. During fluid passage, the renal epithelial cells are subjected to various mechanical stimuli in addition to changes in the laminar flow rate. In the distal part of the collecting duct, the epithelial cells are exposed to pressure changes and possibly distension during papillary contractions. The aim of the present study was to determine whether nucleotide release contributes to mechanosensation in kidney epithelial cells, thereby establishing whether pressure changes are sufficient to produce nucleotide-mediated responses. Madin-Darby canine kidney (MDCK) cells grown on permeable supports were mounted in a closed double perfusion chamber on an inverted microscope. The intracellular Ca(2+) concentration ([Ca(2+)](i)) was monitored with the Ca(2+)-sensitive fluorescence probe fluo 4. Transepithelial pressure pulses of 30-80 mm Hg produced a transient increase in [Ca(2+)](i) of MDCK cells. This response is independent of the primary cilium, since it is readily observed in immature cells that do not yet express primary cilia. The amplitudes of the pressure-induced Ca(2+) transients varied with the applied chamber pressure in a quantity-dependent manner. The ATPase apyrase and the P2Y antagonist suramin significantly reduced the pressure-induced Ca(2+) transients. Applying apyrase or suramin to both sides of the preparation simultaneously nearly abolished the pressure-induced Ca(2+) response. In conclusion, these observations suggest that rapid pressure changes induce both apical and basolateral nucleotide release that contribute to mechanosensation in kidney epithelial cells.

Sodium coupled bicarbonate transporters in the kidney, an update.

Recently five genes have been cloned, which code for sodium dependent bicarbonate transport proteins. These genes belong to the SLC4A gene family. This short review summarizes our knowledge of these gene products with respect to their renal distribution and function. The best characterized members are the SLC4A4 and SLC4A7. SLC4A4 codes for an electrogenic Na(+), HCO(3) (-)-cotransporter (NBCe1), which is present in the basolateral membranes of proximal tubules and is responsible for the bicarbonate efflux here, and thus about 80% of the renal bicarbonate reabsorption. SLC4A7 codes for an electroneutral NBC (called NBC3 and NBCn1), which is present basolaterally in the thick ascending limb and the distal part of the collecting ducts and in intercalated cells (either apically or basolaterally) in the connecting and collecting tubules. In the thick ascending limb NBCn1 may be important for NH(4) (+) reabsorption. SLCA5 codes for an electrogenic NBC (called NBC4 and NBCe2), which based on RT-PCR is located to the kidney but the exact localization awaits a good antibody. This is also the case for the SLC4A8 and SLC4A10 gene products, which are sodium dependent Cl(-), HCO(3) (-) exchangers. The recent development in this field substantially increases our understanding of the complex renal regulation of acid base status.

Expression and regulation of the Na+-K+-2Cl- cotransporter NKCC1 in the normal and CFTR-deficient murine colon.

Defective regulation and/or reduced expression of the Na+-K+-2Cl- cotransporter NKCC1 may contribute to the severe secretory defect that is observed in cystic fibrosis, but data concerning the expression and function of NKCC1 in cystic fibrosis transmembrane conductance regulator (CFTR)-deficient cells are equivocal. We therefore investigated NKCC1 mRNA expression, Na+-K+-2Cl- cotransport activity and regulation by cAMP in crypts isolated from the proximal colon of CFTR-containing (CFTR (+/+)) and CFTR-deficient (CFTR (-/-)) mice. mRNA expression levels were determined by semiquantitative PCR, transport rates were measured fluorometrically in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetomethylester (BCECF)-loaded crypts, cytoplasmic volume changes were assessed by confocal microscopy, and [Cl-]i changes were examined by N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) quenching. NKCC1 mRNA expression levels were not significantly reduced in CFTR (-/-) crypts compared to controls. Azosemide-sensitive NH4+ influx (used as a measure of Na+-K+-2Cl- cotransport) was 2.23 +/- 0.72 vs. 1.56 +/- 0.16 mM min-1, and increased by 63.6 % in (+/+) and 87.3 % in (-/-) crypts upon stimulation for 5 min with forskolin. After 20 min of stimulation with forskolin, the Na+-K+-2Cl- cotransport rates in (-/-) and (+/+) crypts were identical. Crypt cross-sectional area and [Cl-]i decreased only in (+/+) crypts upon stimulation. In conclusion, normal NKCC1 expression levels, somewhat reduced Na+-K+-2Cl- cotransport rates, but preserved activation by cAMP were found in colonic crypts from CFTR (-/-) mice, ruling out a severe dysfunction of the Na+-K+-2Cl- cotransporter in the CF intestine. Furthermore, these studies establish the existence of a direct, cell-volume- and [Cl-]i-independent activation of colonic NKCC1 by an increase in intracellular cAMP.

P2Y(2) receptor-mediated inhibition of amiloride-sensitive short circuit current in M-1 mouse cortical collecting duct cells.

Extracellular nucleotides modulate renal ion transport. Our previous results in M-1 cortical collecting duct cells indicate that luminal and basolateral ATP via P2Y2 receptors stimulate luminal Ca2+-activated Cl- channels and inhibit Na+ transport. Here we address the mechanism of ATP-mediated inhibition of Na+ transport. M-1 cells had a transepithelial voltage (V(te)) of -31.4 +/- 1.3 mV and a transepithelial resistance (R(te)) of 1151 +/- 28 Omegacm(2). The amiloride-sensitive short circuit current (I(sc)) was -28.0 +/- 1.1 microA/cm2. The ATP-mediated activation of Cl- channels was inhibited when cytosolic Ca2+ increases were blocked with cyclopiazonic acid (CPA). Without CPA the ATP-induced [Ca2+](i) increase was paralleled by a rapid and transient R(te) decrease (297 +/- 51 Omegacm(2)). In the presence of CPA, basolateral ATP led to an R(te) increase by 144 +/- 17 Omegacm(2) and decreased V(te) from -31 +/- 2.6 to -26.6 +/- 2.5 mV. Isc dropped from -28.6 +/- 2.4 to -21.6 +/- 1.9 microA/cm2. Similar effects were observed with luminal ATP. In the presence of amiloride, ATP was without effect. This reflects ATP-mediated inhibition of Na+ absorption. Lowering [Ca2+](i) by removal of extracellular Ca2+ did not alter the ATP effect. PKC inhibition or activation were without effect. Na+ absorption was activated by pH(i) alkalinization and inhibited by pH(i) acidification. ATP slightly acidified M-1 cells by 0.05 +/- 0.005 pH units, quantitatively not explaining the ATP-induced effect. In summary this indicates that extracellular ATP via luminal and basolateral P2Y2 receptors inhibits Na+ absorption. This effect is not mediated via [Ca2+](i), does not involve PKC and is to a small part mediated via intracellular acidification.

PKA site mutations of ROMK2 channels shift the pH dependence to more alkaline values.

Close similarity between the rat native low-conductance K(+) channel in the apical membrane of renal cortical collecting duct principal cells and the cloned rat ROMK channel strongly suggest that the two are identical. Prominent features of ROMK regulation are a steep pH dependence and activation by protein kinase A (PKA)-dependent phosphorylation. In this study, we investigated the pH dependence of cloned renal K(+) channel (ROMK2), wild-type (R2-WT), and PKA site mutant channels (R2-S25A, R2-S200A, and R2-S294A). Ba(2+)-sensitive outward whole cell currents (holding voltage -50 mV) were measured in two-electrode voltage-clamp experiments in Xenopus laevis oocytes expressing either R2-WT or mutant channels. Intracellular pH (pH(i)) was measured with pH-sensitive microelectrodes in a different group of oocytes from the same batch on the same day. Resting pH(i) of R2-WT and PKA site mutants was the same: 7.32 +/- 0.02 (n = 22). The oocytes were acidified by adding 3 mM Na butyrate with external pH (pH(o)) adjusted to 7.4, 6.9, 6.4, or 5.4. At pH(o) 7.4, butyrate led to a rapid (tau: 163 +/- 14 s, where tau means time constant, n = 4) and stable acidification of the oocytes (DeltapH(i) 0.13 +/- 0. 02 pH units, where Delta means change, n = 12). Intracellular acidification reversibly inhibited ROMK2-dependent whole cell current. The effective acidic dissociation constant (pK(a)) value of R2-WT was 6.92 +/- 0.03 (n = 8). Similarly, the effective pK(a) value of the N-terminal PKA site mutant R2-S25A was 6.99 +/- 0.02 (n = 6). The effective pK(a) values of the two COOH-terminal PKA site mutant channels, however, were significantly shifted to alkaline values; i.e., 7.15 +/- 0.06 (n = 5) for R2-S200A and 7.16 +/- 0.03 (n = 8) for R2-S294A. The apparent DeltapH shift between the R2-WT and the R2-S294A mutant was 0.24 pH units. In excised inside-out patches, alkaline pH 8.5 activated R2-S294A channel current by 32 +/- 6.7%, whereas in R2-WT channel patches alkalinzation only marginally increased current by 6.5 +/- 1% (n = 5). These results suggest that channel phosphorylation may substantially influence the pH sensitivity of ROMK2 channel. Our data are consistent with the hypothesis that in the native channel PKA activation involves a shift of the pK(a) value of ROMK channels to more acidic values, thus relieving a H(+)-mediated inhibition of ROMK channels.

In vitro peritonitis: basic inflammatory reactions in a two-chamber coculture model of human peritoneum.

We developed an in vitro model of the peritoneum by coculturing human umbilical vein endothelial cells (HUVEC) and human peritoneal mesothelial cells (HPMC) to gather information on peritoneal physiology and to closer reflect the in vivo situation in humans. HUVEC and HPMC were seeded on collagen-coated polytetraflourethylene-insert membranes of pore size 3 microm. HUVEC were grown on the bottom of the membrane and HMPC on the top. The confluent cells were monitored by measuring transepithelial resistance and by confocal microscopy. The transmigration of PMNs as an important mechanism during secondary peritonitis was studied in this two-chamber model. PMNs were isolated by density separation. After stimulation of HMPC with the complement factor 5 split product C5a (1 ng/ml) or tumor necrosis factor-alpha (TNF-alpha; 10 or 50 microg/ml) for 1 h, 1 x 10(6) PMN were given to the lower compartment. Controls were cocultured cells without stimulation. After 1, 2, and 6 h nonadherent PMNs in the upper compartment were harvested and counted, interleukin-8 was measured in each compartment, and cells on the membrane were paraffin-embedded for immunohistochemistry. Each experiment was performed four times. Cells grew to confluence within 2-5 days and were detected on their respective seeding side by CD34 and cytokeratin 18 counterstaining. Transmigration of PMNs after C5a or TNF-alpha stimulation showed a significant time-dependent increase between 1 h and 6 h (P<0.05). PMNs were found in significantly higher numbers after stimulation with either C5a or TNF-alpha at 1, 2, and 6 h than without stimulation. After stimulation of HPMC, interleukin-8 secretion to the apical compartment increased in a time-dependent fashion, resulting in a gradient between the two chambers. Linear regression analysis revealed significant correlation between transmigrated PMN and interleukin-8 in stimulated cocultures; no correlation was found in controls. This new in vitro peritoneum consisting of cocultured mesothelial and endothelial cells may allow more detailed assessment of peritoneal pathophysiology. Generation of an interleukin-8 gradient affecting the migration of PNMs across the cocultured membrane represents a parameter which may be addressed in further studies.

Cyclosporin-A-induced effects on the free Ca2+ concentration in LLC-PK1-cells and their mechanisms.

Here we have examined the effects of Cyclosporin A (CyA) on the free intracellular Ca2+ concentration ([Ca2+]i) of LLC-PK1/PKE20 cells to evaluate mechanisms of CyA nephrotoxicity using Fura-2 microspectrofluorometry or digital fluorescence video imaging. The CyA-associated changes were compared to the effects of tacrolimus (Tac), a structurally unrelated immunosuppressant with similar cellular pathways which also causes nephrotoxicity. CyA (EC50(: 1 nmol/l, n=16) and Tac (EC50: 1 nmol/l, n=5) caused a concentration-dependent increase of [Ca2+]i which was substantially attenuated by reducing the external Ca2+ concentration (10(-6) mol/l). Similarly Cyclosporin H, a non-immunosuppressive analogue of CyA, stimulated a Ca2+ influx. Nicardipine (10(-6) mol/l) reduced the CyA- and the Tac-induced Ca2+ influx to 52+/-16% (n=10) and 13+/-10% (n=13) of control respectively. Diltiazem and verapamil (10(-6) mol/l) were also effective, but flufenamate (10(-4) mol/l), Gd3+ (10(-5) mol/l) and La3+ (10(-5) mol/l) were not. In the absence of extracellular Ca2+ CyA led to a small but significant [Ca2+]i increase, indicating additional release from internal stores. Depletion of inositol-1,4,5-trisphosphate-(InsP3-) sensitive Ca2+ stores by extracellular ATP (10(4) mol/l) in low-Ca2+ solution completely suppressed the CyA-induced [Ca2+]i rise. CyA had no effect on the cellular InsP3 concentration. Furthermore, inhibition of phospholipase-Cbeta (PLCbeta) by U73122 (2x10(-5) mol/l) did not alter the CyA-stimulated [Ca2+]i rise. A direct effect of CyA on InsP3-sensitive Ca2+ stores, the InsP3 receptor, the Ca2+ content of the stores or involvement of additional stores is assumed. Incubation with CyA for 1, 12 and 24 h enhanced the rise in [Ca2+]i peak induced by ATP, arginine vasopressin (AVP) and angiotensin II. In summary, CyA stimulated a [Ca2+]i increase in LLC-PK1 cells through Ca2+ release from InsP3-sensitive stores and Ca2+ influx via a nicardipine-sensitive pathway. The CyA-mediated [Ca2+]i increase is independent of PLCbeta activity and InsP3 metabolism. CyA caused long-term enhancement of the agonist-induced rise in [Ca2+]i. The effects of CyA on Ca2+ signaling appear to be independent of its immunosuppressive action.