Angiotensin II Stimulates Vacuolar H+-ATPase Activity in Renal Acid-Secretory Intercalated Cells from the Outer Medullary Collecting Duct
Florina Rothenberger,
Ana Velic,
Paul A. Stehberger,
Jana Kovacikova and
Carsten A. Wagner
Institute of Physiology and Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland
Correspondence: Dr. Carsten A. Wagner, Institute of Physiology and Centre for Integrative Human Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. Phone: +41-1-63-50659; Fax: +41-1-63-56814; E-mail: wagnerca{at}access.unizh.ch
Received for publication July 18, 2006.
Accepted for publication April 10, 2007.
Final urinary acidification is mediated by the action of vacuolarH+-ATPases expressed in acid-secretory type A intercalated cells(A-IC) in the collecting duct. Angiotensin II (AngII) has profoundeffects on renal acid-base transport in the proximal tubule,distal tubule, and collecting duct. This study investigatedthe effects on vacuolar H+-ATPase activity in A-IC in freshlyisolated mouse outer medullary collecting ducts. AngII (10 nM)stimulated concanamycin-sensitive vacuolar H+-ATPase activityin A-IC in freshly isolated mouse outer medullary collectingducts via AT1 receptors, which were also detected immunohistochemicallyin A-IC. AngII increased intracellular Ca2+ levels transiently.Chelation of intracellular Ca2+ with BAPTA and depletion ofendoplasmic reticulum Ca2+ stores prevented the stimulatoryeffect on H+-ATPase activity. The effect of AngII on H+-ATPaseactivity was abolished by inhibitors of small G proteins andphospholipase C, by blockers of Ca2+-dependent and -independentisoforms of protein kinase C and extracellular signal–regulatedkinase 1/2. Disruption of the microtubular network and cleavageof cellubrevin attenuated the stimulation. Finally, AngII failedto stimulate residual vacuolar H+-ATPase activity in A-IC frommice that were deficient for the B1 subunit of the vacuolarH+-ATPase. Thus, AngII presents a potent stimulus for vacuolarH+-ATPase activity in outer medullary collecting duct IC andrequires trafficking of stimulatory proteins or vacuolar H+-ATPases.The B1 subunit is indispensable for the stimulation by AngII,and its importance for stimulation of vacuolar H+-ATPase activitymay contribute to the inappropriate urinary acidification thatis seen in patients who have distal renal tubular acidosis andmutations in this subunit.
The kidney plays a central role in controlling systemic acid-basehomeostasis by reabsorbing bicarbonate, synthesis and excretionof ammonia, and regulated secretion of acid or base equivalents.1,2The importance of these processes is underlined by both geneticand acquired defects in proteins that are involved in protonand bicarbonate transport, such as the vacuolar H+-ATPase subunitsa4 (ATP6V0A4) and B1 (ATP6V1B1) and the Cl–/HCO3–exchanger AE1 (SLC4A1).3 Acidification of the urine occurs inseveral distal nephron segments, but final urinary acidificationoccurs along the cortical and outer medullary collecting duct(OMCD), largely through the action of vacuolar H+-ATPases inacid-secretory type A intercalated cells (A-IC).2,4
Final urinary acidification and H+-ATPase activity in the collectingduct are controlled by several factors, including endothelin,angiotensin II (AngII), and aldosterone.1,2,4,5 The mechanism(s)through which AngII stimulates net acid secretion in the medullarycollecting duct has not been fully explored. AngII, however,has been shown to stimulate acid excretion and bicarbonate reabsorptionin the proximal tubule by acting on Na+/H+ exchangers, Na+/bicarbonateco-transporters, and H+-ATPases.6–9 In in vivo experiments,AngII stimulates also final urinary acidification.10–13Thus, AngII may stimulate H+-ATPases in the collecting duct.
Vacuolar H+-ATPases are composed of at least 13 subunits inmammals of which multiple isoforms exist.4,14 The B subunitbelongs to the cytosolic V1 domain and is essential for vacuolarH+-ATPase function in yeast.4,14 In mammals, two isoforms ofthe B subunit, B1 (ATP6V1B1) and B2 (ATP6V1B2), have been identified.H+-ATPases that contain the B1 subunit were detected in theinner ear, in lung and epididymis, and in the kidney. In kidney,expression of the B1 subunit is restricted to IC in the latedistal tubule, connecting segment, and cortical and medullarycollecting ducts. In these cells, the B1 subunit is found inthe plasma membrane and seems to be involved in acid extrusion.4,14Mutations in the B1 subunit in human cause distal renal tubularacidosis with sensorineural deafness.15 This type of metabolicacidosis is thought to be caused by the partial or completelack of proton secretion in the collecting duct. Accordingly,a mouse model that is deficient for the B1 subunit is not ableto acidify urine appropriately and remove an acid load.16 H+-ATPaseactivity in IC from B1-deficient mice is attenuated.16 The B2subunit, in contrast, is found in various tissues and can bedetected both in the plasma membrane and in intracellular organellesand may serve a housekeeping function.4 In mouse and rat kidney,B2 is also found in IC with some labeling of the apical plasmamembrane, suggesting that B2 may take part in acid excretion.17The exact function of the B1 and B2 isoforms, however, has notbeen fully understood to date.
Here we investigated the stimulatory effect of AngII H+-ATPaseactivity in A-IC of mouse OMCD. Moreover, we tested whetherthe B1 subunit is important for the hormonal stimulation thatwas observed with AngII. We present evidence for a specificfunction of the B1 subunit in the stimulation of H+-ATPase activityby AngII.
Stimulation of H+-ATPase Activity in Mouse OMCD A-IC by AngII
In single mouse OMCD A-IC, the mean initial intracellular pH(pHi) was acidified to 6.35 ± 0.012 using the NH4Cl prepulsein the absence of extracellular Na+. Intracellular pHi recoveredslowly (alkalinized) during the subsequent superfusion withNa+-free solution with a rate of 0.032 ± 0.002 unitspH/min. Upon re-addition of Na+, pHi increased rapidly to theinitial value mediated by Na+/H+ exchange as described previously(Table 1).18,19
Table 1. Summary of pHi measurements in single intercalated cells in mouse OMCD fragmentsa
Preincubation of mouse OMCD with 10 nM AngII for 10 min increasedthe Na+-independent alkalinization rate two- to three-fold to0.080 ± 0.005 units pH/min. Addition of 1 nM AngII inthe same experimental series did not significantly increasethe alkalization rate (0.035 ± 0.002 units pH/min; Figure 1A).High supraphysiologic concentrations of 100 nM AngII resultedin a small stimulation (0.047 ± 0.005 units pH/min).The enhanced rate of pHi recovery in the absence of sodium wasmediated by vacuolar H+-ATPase activity. OMCD were incubatedfor 10 min before experiments with the specific H+-ATPase inhibitorconcanamycin (200 nM).20 In the presence of concanamycin, therate of intracellular alkalinization was reduced 60 to 70% incontrol OMCD, and no stimulation was observed after incubationwith AngII (Figure 1B). Inhibition of H+/K+-ATPases with SCH28080(100 µM) reduced the rate of pHi recovery only by approximately10 to 15%, similar in extent to the residual pHi recovery ratein the presence of concanamycin. Thus, AngII stimulates onlyvacuolar H+-ATPase activity in A-IC of isolated mouse OMCD.
Figure 1. Angiotensin II (AngII) stimulates vacuolar H+-ATPase activity in freshly isolated mouse outer medullary collecting duct (OMCD) intercalated cells (IC). (A) Original tracings of intracellular pH measurements in single OMCD IC in the absence or presence of 10 nM AngII. (B) AngII (10 nM) stimulated Na+-independent pHi recovery of OMCD IC. OMCD were preincubated for 10 min with AngII, and AngII was present in all Na+-free solutions. AngII at 1 nM did not cause any significant stimulation, whereas AngII at 10 nM increased pHi recovery. (C) The major part of the Na+-independent pHi recovery is due to the activity of vacuolar H+-ATPases and can be blocked by the specific H+-ATPase inhibitor concanamycin (concan.; 200 nM), whereas the H+/K+-ATPase inhibitor SCH28080 (100 µM) reduced pHi recovery only by 10 to 15%, similar in extent to the residual pHi recovery rate in the presence of concanamycin. The values of control and AngII-treated OMCD are shown again for comparison. *Significantly different from control; #significantly different from 10 nM AngII.
AT1 Receptor Is Necessary for the Stimulatory Effect of AngII
AngII mainly signals through two main receptor subtypes, AT1and AT2.21 Both subtypes of receptors were previously identifiedin the OMCD.22 Saralasin (1 µM) and losartan (1 µM),two AT1 receptor antagonists, prevented completely the stimulatoryeffect of AngII (Figure 2), whereas PD123.319, an inhibitorof AT2 receptors, partially reduced the stimulatory effect ofAngII (Figure 2), indicating that the stimulation of H+-ATPaseactivity by AngII is mainly mediated via AT1 receptors.
Figure 2. AT1 receptors are necessary for the stimulation of vacuolar H+-ATPase activity by AngII. OMCD were incubated with blockers for AngII type 1 or 2 receptors in the absence and presence of AngII (10 nM). Blocking of AT1 receptors with saralasin (1 µM) or losartan (1 µM) prevented the AngII effect. However, blocking of the type 2 receptor AT2 with PD123.319 (1 µM) partially reduced the stimulatory effect. The values of control and AngII-treated OMCD are shown again for comparison. *Significantly different from control; #significantly different from the 10 nM AngII activity.
Furthermore, immunohistochemistry with an affinity-purifiedantibody against the AT1 receptor showed in the OMCD stronglabeling of both principal cells and IC at the luminal pole.The basolateral membrane showed a weaker labeling. IC were identifiedas being negative for the aquaporin-2 water channel presentonly in principal/segment-specific cells (Figure 3). The antibodyalso stained arterial smooth muscle cells and apical and basolateralmembranes of proximal tubules as described previously.22,23
Figure 3. Localization of AT1 receptors in OMCD IC. Mouse kidneys were stained with antibodies against the AT1 receptor (red, C and F) and the principal cell–specific aquaporin-2 (AQP-2) water channel (green, B and E). Overlays are shown in A and D. AT1 staining was seen in cells that were positive and negative for AQP-2, demonstrating expression in principal cells and IC (IC marked with *). In IC, AT1 staining was mostly associated with the apical pole (arrowhead). Magnifications: x650 in A through C; x800 in D through F.
Role of Small G Proteins, Phospholipase C, and Intracellular Ca2+
AT1 receptors couple intracellularly mainly via pertussis toxin(PTX)-sensitive small G proteins to phospholipase C, releasingintracellular Ca2+ and activating the protein kinase C (PKC)pathway.21,24 PTX (200 ng/ml), an inhibitor of Gi and Go smallG proteins, had no effect alone but completely blocked the stimulatoryeffect of AngII (Figure 4A). Inhibition of phospholipase C activitywith U73122 (10 µM) also impeded the stimulation by AngII.U73122 alone reduced the rate of alkalinization slightly butsignificantly (Figure 4A).
Figure 4. The stimulatory effect of AngII is mediated via small G proteins and phospholipase C (PLC) and requires intracellular Ca2+. (A) The stimulatory effect of angiotensin is mediated via pertussis toxin (PTX)-sensitive (200 ng/ml) small G protein (Go or Gi). U73122 (10 µM), an inhibitor of PLC activity, prevented the stimulation of vacuolar H+-ATPase activity induced by AngII. (B) Superfusion of IC with 10 nM AngII induced a transient rise in intracellular calcium. ATP (100 µM) was used a positive control (original tracing, left). Summary of data from 10 independent experiments showing the increase in intracellular calcium concentrations (right). (C) Chelation of intracellular Ca2+ with BAPTA-AM (50 µM) prevented the vacuolar H+-ATPase stimulation. Depletion of endoplasmic reticulum Ca2+ stores with thapsigargin (1 µM) had a similar effect. The values of control and AngII-treated OMCD are shown again for comparison. *Significantly different from control; #significantly different from 10 nM AngII.
Intracellular calcium measurements demonstrated that AngII induceda rapid and transient increase in [Ca2+]i that was smaller thanan ATP-induced (100 µM) rise in the same cells (Figure 4B).Chelation of intracellular Ca2+ by preincubation with 1,2-bis(2-amino-5-fluoro-phenoxy)ethane-N,N,N'N'-tetraaceticacid tetrakis (acetoxymethyl)ester (BAPTA-AM) (50 µM)abolished the stimulation of H+-ATPases by AngII. For examinationof whether release of Ca2+ from endoplasmic reticulum (ER) storeswas involved in this process, ER Ca2+ stores were depleted withthapsigargin (1 nM), which resulted in a significant inhibitionof the AngII effect (Figure 4C). Thus, intracellular Ca2+ playsan important role in the stimulation of H+-ATPases by AngII.
AngII Stimulates through Ca2+-Dependent and -Independent PKC
The inhibition of PKC activity with chelerythrine (1 µM)prevented completely the effect of AngII. Several isoforms ofPKC have recently been described in mouse OMCD IC by immunohistochemistry,including the classic Ca2+-dependent isoforms PKC- and PKC-1and the novel Ca2+-independent PKC- and PKC- isoforms.25–27On the basis of the observation that intracellular Ca2+ playsa critical role, we tested the involvement of Ca2+-dependentand -independent PKC isoforms. Incubation with Gö 6976(1 µM), an inhibitor of Ca2+-dependent PKC subtypes PKC-and PKC-1, reduced the stimulation of H+-ATPases partially.Also Ro-31-2880 (1 µM), an inhibitor of Ca2+-independentPKC- and PKC- isoforms, attenuated the stimulatory effect partially(Figure 5). Using Gö 6976 and Ro-31-2880 each at a concentrationof 10 µM, the stimulation that was caused by AngII wascompletely abolished. This may indicate that Gö 6976 andRo-31-2880 used at higher concentrations may be less isoformspecific. Moreover, the effect of AngII was completely inhibitedwhen the activation of extracellular signal–regulatedkinase 1/2 (ERK1/2) was prevented using PD098059 (20 µM)(Figure 6). Preincubation with PD098059 alone had no effecton H+-ATPase activity.
Figure 5. Different protein kinase C (PKC) isoforms are involved in the pathway activated by AngII. (A) Inhibition of PKC with chelerythrine (1 µM) completely prevented vacuolar H+-ATPase stimulation. (B and C) Inhibition of classic Ca2+-dependent and novel independent PKC isoforms PKC- and PKC-1 with Gö 6976 (1 µM) and PKC- and PKC- with Ro-31-2880 (1 µM), respectively, partially reduced stimulation. Higher concentrations of Gö 6976 and Ro-31-2880 (10 µM) completely blocked stimulation. *Significantly different from control; #significantly different from 10 nM AngII.
Figure 6. The stimulatory effect of AngII is mediated via extracellular signal–regulated kinase 1/2 (ERK1/2). Incubation of OMCD with ERK1/2 inhibitor PD098059 (20 µM) attenuated AngII-induced stimulation. *Significantly different from control.
Microtubular Network and SNARE Protein Are Involved in the Stimulatory Effect of AngII
Stimulation of H+-ATPase activity in various cells involvescolchicine-sensitive trafficking and fusion of vesicles withthe membrane depending on soluble not attachment receptor (SNARE)proteins and cellubrevin.7,19,28–33 Disruption of themicrotubular network with colchicine (preincubation for 10 min,10 µM) reduced the basal rate of alkalinization significantlypossibly by inhibition of recycling of H+-ATPases. Colchicinecompletely abolished the effect of AngII (Figure 7). In addition,cleaving of cellubrevin, part of the membrane vesicle fusioncomplex, with tetanus toxin (50 nM) had no effect on basal activitybut prevented the stimulation by AngII. Thus, the effect ofAngII requires trafficking and SNARE-dependent fusion of vesiclesthat carry H+-ATPases or some of their subunits or possibleaccessory or regulatory proteins.
Figure 7. The stimulatory effect of AngII depends on an intact microtubular network and SNARE protein complexes. Disruption of the microtubular network by preincubation for 10 min with colchicine (Colch., 10 µM) or cleavage of cellubrevin, part of the SNARE protein complex, with tetanus toxin (50 nM) before exposure to AngII completely prevented the stimulatory effect. Colchicine alone reduced basal activity significantly. *Significantly different from control; #significantly different from 10 nM AngII.
AngII Stimulates H+-ATPases via PI3 Kinases
Phosphatidylinositol-3 kinases (PI3-K) have been implicatedin the assembly, trafficking, and stimulation of vacuolar H+-ATPasesby glucose,34 and AT1 receptor–mediated and PI3-K–dependentstimulation of Na+/H+ exchanger NHE3 activity was described.35The PI3-K inhibitor wortmannin (1 µM) prevented the stimulationof the intracellular alkalinization rate induced by AngII, withoutaffecting basal activity (Figure 8).
Figure 8. The stimulation with AngII requires activation of phosphatidylinositol-3 (PI-3) kinases. The PI3 kinase inhibitor wortmannin (Wort.; 1 µM) prevented the stimulation of vacuolar H+-ATPase activity. *Significantly different from control; #significantly different from 10 nM AngII.
B1 Subunit of the Vacuolar H+-ATPase Is Required for Stimulation
H+-ATPases contain either the B1 or the B2 isoform, formingpart of the cytosolic V1 domain.4 The B1 subunit is specificallyexpressed in IC.36,37 It has been speculated that the B subunitmay be involved in cell-specific subcellular localization, trafficking,or enzyme regulation.37 Hence, we used mice that were deficientfor the B1 subunit (Atp6v1b1–/–) and tested whether(1) OMCD IC express H+-ATPase activity and (2) activity canbe stimulated by AngII.
The total rate of pHi alkalinization was similar in OMCD ICfrom B1-deficient mice (0.032 ± 0.002 units pH/min) ascompared with wild-type mice (Figure 1). Inhibition of H+-ATPaseswith concanamycin (200 nM) decreased the rate of alkalinizationsignificantly (0.027 ± 0.001 units pH/min), demonstratingthat a vacuolar H+-ATPase is functional in the plasma membraneof IC despite the lack of the B1 subunit (Figure 9A). However,the concanamycin-sensitive rate of alkalinization was lowerthan that observed in wild-type mice (compare with Figure 1).AngII (10 nM) had no effect on the rate of alkalinization inthe absence or presence of concanamycin. To rule out that AT1receptors were absent or unresponsive in the IC of B1-deficientmice, we measured intracellular calcium in response to AngIIand ATP as detailed previously. Both AngII and ATP eliciteda rise in intracellular calcium similar to what had been observedin wild-type OMCD (Figure 9B). Furthermore, AT1 receptor localizationwas not altered as assessed by immunohistochemistry (Figure 9C).Hence, vacuolar H+-ATPases that lack the B1 subunit cannot bestimulated by AngII.
Figure 9. The B1 subunit of the vacuolar H+-ATPase is necessary for its stimulation with AngII. (A) Preincubation of OMCD from B1-deficient mice with AngII (10 nM) did not show any significant stimulatory effect on the rate of intracellular alkalinization. Concanamycin (200 nM), a specific inhibitor of vacuolar H+-ATPases, reduced the rate of intracellular alkalinization in the absence and presence of AngII to the same extent, demonstrating that AngII did not stimulate proton pumps that lacked the B1 subunit. (B) The presence of functional AngII receptors in OMCD IC from Atp6v1b1-deficient mice was confirmed by measurement of intracellular Ca2+ changes in response to AngII (10 nM) and ATP (100 µM) as a positive control. Intracellular Ca2+ increased in OMCD IC to a similar extent as shown in Figure 4 (n = 12 cells, four OMCD). (C) Staining for the AT1 receptor (red) and the AQP-2 water channel (green) in the OMCD of B1-deficient mice. *Significantly different from control; #significantly different from 10 nM AngII.
Here we describe two related findings: The stimulation of H+-ATPaseactivity in acid-secretory IC by AngII and that stimulationrequires the presence of the B1 isoform. The stimulatory effectof AngII on several renal acid-base transport systems has beenextensively documented.6,7,9,38,39 These processes are mostlymediated via AT1 receptors. Here we show that AngII stimulatesalso vacuolar H+-ATPase activity in the acid-secretory IC ofthe OMCD. Both pharmacologic evidence and immunohistochemistrydemonstrate that AT1 receptors are present and involved. However,our preparation does not allow distinguishing between the stimulationof basolateral and/or luminal receptors. Urinary AngII concentrationsin the OMCD are in the range of 10 to 30 nM,40,41 originatingmainly from AngII synthesis along the nephron.40,42 During metabolicacidosis, an increase in AngII has been reported, and the abilityof the kidney to adapt to changes in acid-base status is influencedby AngII.10,12,13,43,44 The less stimulatory effect of 100 nMAngII is consistent with high concentrations of AngII even inhibitingbicarbonate reabsorptive mechanisms, an effect that may occurat least in part via stimulation of AT2 receptors.45,46
Some reports indicated that AngII may inhibit urinary acidification,bicarbonate reabsorption, or H+-ATPase activity in the rabbitinitial cortical collecting duct and the rat OMCD and innermedullary collecting duct, respectively.47–50 However,these studies are in contrast to a large number of observationsdemonstrating that AngII stimulates final urinary acidificationin these nephron segments and that blockade of the angiotensinsystem reduces urinary acidification.5,10–12,39,44,51,52We cannot exclude that the discrepancies may be either due todifferent species used or because we used superfusion of OMCDin contrast to perfused nephron segments, which may alter theaccess of AngII and other inhibitors to the luminal membrane.
AT1 receptors couple to phospholipase C via small G proteins;increase intracellular Ca2+ concentrations; and activate PKC,ERK1/2, and PI3-K as well as a variety of other signal pathwaysin a cell- and tissue-specific manner.21,53 Our results demonstratethat in OMCD IC, AngII signals through a distinct cascade tostimulate H+-ATPase activity involving PTX-sensitive G proteins(Go, Gi), phospholipase C, intracellular Ca2+, different PKCisoforms, ERK1/2 mitogen-activated protein kinases, and PI3-K.The data indicate that at least two isoforms of PKC may participatein the stimulatory effect of AngII: A classic Ca2+-dependentand Gö 6976-sensitive PKC- and/or PKC-1 and a novel Ca2+-independentand Ro-31-2880–sensitive PKC- and/or PKC- isoform. Immunohistochemistrydemonstrated the expression of PKC-, PKC-1, PKC-, and PKC- inIC of the collecting duct.25,27 Evidence from freshly isolatedOMCD IC as well as from cell culture models19,31,54 suggeststhat PKC has a stimulatory effect on H+-ATPase activity. Inaddition, our experiments indicate that intact ERK1/2 mitogen-activatedprotein kinases and PI3-K are required for the stimulation tooccur. PI3-K are implied in the glucose-stimulated assembly,trafficking, and stimulation of vacuolar H+-ATPases in renalcell lines derived from the proximal tubule.34 Also, AngII stimulatesNa+/H+ exchange activity in a proximal tubular cell line viaPI3-K.35
Regulation of H+-ATPase activity can occur through several distinctmechanisms,4 and trafficking of vacuolar H+-ATPases plays amajor role. Physiologic stimuli lead to increased proton secretionsuch as CO2, acidification, or hormones such as AngII or aldosterone.4,7,19,33Insertion of pumps or its subunits into the membrane requiresan intact microtubular network7,19,33,54,55 and proteins thatare involved in vesicle fusion, such as SNARE proteins.28,29,56The inhibition of H+-ATPase stimulation by colchicine and tetanustoxin suggests strongly that AngII increases proton pump activitythrough trafficking of proton pumps or some of its subunitsor accessory proteins into the membrane.
Evidence from yeast suggests that specific isoforms of somesubunits could play a role in targeting, trafficking, and adaptivechanges in the ratio between ATP hydrolysis and proton pumping.4Particularly the two isoforms of the B subunit and various isoformsof the a subunit have been implicated.4,37 No evidence for isoform-specificfunctions for mammalian subunits has so far been described eventhough the cell- and organ-specific expression of some subunitshas been noted. Organ-specific inherited diseases such as malignantinfant osteopetrosis (mutations in the a3 subunit), distal renaltubular acidosis (a4 subunit), and distal renal tubular acidosiswith sensorineural deafness (B1 subunit) underlined the conceptof isoform-specific functions of vacuolar H+-ATPases.4 Herewe find that H+-ATPases in the OMCD that lack the B1 subunitmaintain a basic level of activity but cannot be stimulatedby AngII. This activity is most likely due to substitution oflacking B1 subunits by the B2 isoform, which show enhanced luminalappearance in the B1-deficient mice.16,17 Apparently, the B1isoform performs functions that cannot be compensated by theB2 isoform, suggesting that B1 is involved in the cell-specificstimulation that could involve trafficking or assembly of pumps.This interpretation is also in line with some recent data demonstratingthat overexpression of some B1 mutations that were found inpatients impairs assembly and trafficking of proton pumps tothe apical membrane.57 The absence of a functional B1 subunitin patients and mice leads to defective urinary acidification.15,16AngII has been shown to be involved in the adaptive increasein urinary acidification during acidosis, and that vacuolarH+-ATPases in IC that lacking the B1 subunit are insensitiveto stimulation may help to explain this phenotype.
We identified AngII as a potent stimulus for H+-ATPases in OMCDA-IC. The stimulation is transmitted via a distinct signal cascadeand requires the presence of the B1 subunit of the vacuolarH+-ATPase. The lack of stimulation by AngII may explain theinappropriate adaptive urinary acidification during acidosisof patients with mutations in the B1 subunit.
Animals
C57BL-6J (Jackson Laboratory, Bar Harbor, ME) and B1-deficientmice (Atp6v1b1–/–; male 12 to 15 wk of age, 30 to35 g) were housed under standard conditions. Breeding and genotypingof Atp6v1b1–/– mice has been described previously.16All studies were approved by the Local Swiss Veterinary Authority(Veterinäramt, Zurich, Switzerland) and were accordingto Swiss Animal Welfare Laws.
Isolation of OMCD
OMCD were prepared from mouse kidney as described previously.18,19
Intracellular pH and Ca2+ Measurements
Coverslips were transferred to a thermostatically controlledperfusion chamber (approximately 3 ml/min flow rate) maintainedat 37°C on an inverted microscope (Zeiss Axiovert 200, Feldbach,Switzerland) equipped with a video imaging system (Visitron,Munich, Germany). The isolated OMCD were incubated in a HEPES-bufferedRinger's solution that contained either the pH-sensitive dyeBCECF-AM [2',7'-bis(2carboxylethyl)-5(6)-carboxyfluoresceinester; 10 µM; Molecular Probes, Eugene, OR] or the calcium-sensingdye FURA-2-AM (5 µM; Molecular Probes) for 20 min andwere washed to remove all non de-esterified dye. pHi was measuredby alternately exciting the dye with a 10-mm-diameter spot oflight at 495 and 440 nm for BCECF and 340 and 380 nm for FURA-2while monitoring the emission at 532 or 510 nm, respectively,with a video imaging system. Each experiment was calibratedfor pHi using the nigericin/high K+ method, and the obtainedratios were converted to pHi.18,19,58 FURA-2 measurements werecalibrated for [Ca2+]i with high (2 mM) and zero calcium solutionsin the presence of a calcium ionophore and [Ca2+]i calculated.59All experiments were performed in the nominal absence of bicarbonate.The initial solution was a HEPES-buffered Ringer solution (125mM NaCl, 3 mM KCl, 1 mM CaCl2, 1.2 mM MgSO4, 2 mM KH2PO4, and32.2 mM HEPES [pH 7.4]). Cells were acidified by using the NH4Cl(20 mM) prepulse technique and washed into a Na+-free solution(Na+ was replaced by equimolar concentrations of N-methyl-d-glucamine).The rate of H+-ATPase activity was determined as the concanamycin-sensitivepHi alkalinization rate in the absence of Na+. Rates were calculatedover the same range of pHi (6.55 to 6.75) for all cells studied.OMCD were used only for one single experiment (one NH4Cl prepulseas repetitive intracellular acidification altered recovery ratesin our hands). For the inhibitor studies, OMCD were preincubatedwith the inhibitors alone for 10 min, then co-incubated withBCECF for another 10 min. The inhibitors were also added toall solutions in the absence or presence of AngII, respectively.All chemicals were from Sigma (Buchs, Switzerland) and Calbiochem(Dietikon, Switzerland).
Immunohistochemistry
Immunostaining on paraformaldehyde/lysine/periodate-perfusedkidneys was carried out as described previously60 using rabbitanti-AT1 receptor (Santa Cruz Biotechnology, Santa Cruz, CA),221:200, and goat anti-human AQP-2 (Santa Cruz Biotechnology),1:200, antibodies.
Statistical Analyses
Data are presented as means ± SEM. All data were testedfor significance using the one-way ANOVA test, and only resultswith P < 0.05 were considered statistically significant.
This study was supported by a grant from the Swiss NationalResearch Foundation to C.A.W. (31-068318) and the Hartmann-MüllerStiftung (Zurich, Switzerland).
Footnotes
F.R. and A.V. contributed equally to this work and thereforeshare first authorship.
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
and PKC-
1 and the novel Ca2+-independent PKC-
and PKC-
isoforms.25–27 On the basis of the observation that intracellular Ca2+ plays a critical role, we tested the involvement of Ca2+-dependent and -independent PKC isoforms. Incubation with Gö 6976 (1 µM), an inhibitor of Ca2+-dependent PKC subtypes PKC-
