The outer medullary collecting duct (OMCD) plays an important role in mediating transepithelial HCO transport ( ) and urinary acidification. HCO absorption by type A intercalated cells in the OMCD inner stripe (OMCDis) segment is thought to by mediated by an apical vacuolar H+-ATPase and H+-K+-ATPase coupled to a basolateral Cl−-HCO exchanger (AE1). Besides these Na+-independent transporters, previous studies have shown that OMCDis type A intercalated cells have an apical electroneutral EIPA-sensitive, DIDS-insensitive Na+-HCO cotransporter (NBC3); a basolateral Na+/H+ antiporter; and a basolateral Na+-K+-ATPase. In this study, we reexamined the Na+ dependence of transepithelial Na+ transport in the OMCDis and determined the role of apical NBC3 in intracellular (pHi) regulation in OMCDis type A intercalated cells. Control tubules absorbed HCO at a rate of ∼13 pmol · min−1 · mm−1. Lowering luminal Na+ from 140 to 40 mM decreased by ∼15% without a change in transepithelial potential (V te). Furthermore, 50 μM EIPA (lumen) also decreased by ∼13% without a change in V te. The effect of lowering luminal Na+ and adding EIPA were not additive. These results demonstrate that in the OMCDis is in part Na+ dependent. In separate experiments, the pHi recovery rate after an NH prepulse was monitored in single type A intercalated cells with confocal fluorescence microscopy. The pHi recovery rate was ∼0.21 pH/min in Na+-containing solutions and decreased to ∼0.16 pH/min with EIPA (50 μM, lumen). In tubules perfused/bathed without Na+, luminal Na+ addition resulted in a pHi recovery rate of ∼0.36 pH/min, whereas the Na+-independent recovery rate was ∼0.16 pH/min. EIPA (50 μM, lumen) decreased the Na+-dependent pHirecovery rate to ∼0.07 pH/min. The Na+-independent recovery rate was decreased to ∼0.06 pH/min by bafilomycin (10 nM, lumen) and to ∼0.10 pH/min using Schering 28080 (10 μM, lumen). These findings indicate that NBC3 contributes to pHiregulation in OMCDis type A intercalated cells and plays only a minor role in mediating in the OMCDis.
- confocal microscopy
- intracellular pH
- bicarbonate reabsorption
- outer medullary collecting duct
under baseline conditions, the outer medullary collecting duct inner stripe segment (OMCDis) has the highest rate of H+ secretion (HCO absorption) of all the collecting duct segments (16). Several pathways for transepithelial HCO absorption in the OMCDis have been identified. The rabbit OMCDis is believed to absorb HCO via an apical H+-ATPase in series with a basolateral Cl−/HCO exchanger (1, 15,21). In addition to the vacuolar H+-ATPase, a P-type, gastric-like H+-K+-ATPase also contributes to HCO absorption in the OMCDis (2, 3, 6). Previous studies by Tsuruoka et al. (23) have concluded that ∼35% of the rate of net HCO is due to the P-type H+-K+-ATPase and 65% is mediated by a vacuolar H+-ATPase. These data differ somewhat from the data of Wingo et al. (2, 3, 29), who found that ∼70% of HCO flux is sensitive to inhibition of the apical H+-K+-ATPase, and only 30% is sensitive to inhibition of the vacuolar H+-ATPase. However, both studies agreed in that the vacuolar H+-ATPase and the P-type H+-K+-ATPase are the two main apical H+-translocating systems in the OMCDis. Consistent with the importance of the apical vacuolar H+-ATPase in urinary acidification are the recent findings that patients with loss-of-function mutations in the 58- or 116-kDa subunits of the vacuolar H+-ATPase present with distal renal tubular acidosis (8, 17).
Previous studies have concluded that net transepithelial HCO transport ( ) in the OMCDis is Na+ independent (11,20). Furthermore, this segment has not been previously reported to actively transport Na+ (19). Interestingly, OMCDis type A intercalated cells have Na+-dependent transporters, including a basolateral Na+-K+-ATPase (14) and a basolateral Na+/H+ exchanger (4, 7, 27,28), but lack an apical Na+/H+ exchanger (4, 7). We have recently immunolocalized the electroneutral Na+-HCO cotransporter NBC3 (SLC4A7) to the apical membrane of these cells (10,13). Consistent with these immunolocalization studies, rabbit OMCDis type A intercalated cells were shown to have an apical Na+- and HCO -dependent transport process with the known functional characteristics and inhibitor profile of NBC3, i.e, sensitivity to EIPA and lack of inhibition by DIDS (12, 13). As a result of these findings, the present studies were undertaken to reexamine the Na+ dependence of in the OMCDis segment. A Nanoflo microfluorimeter, which provided better sensitivity and reproducibility to small changes in collected fluid HCO concentration than the Picapnotherm used in the earlier studies (24, 26), was used to measure transepithelial HCO absorption. Additional studies were designed to determine the contribution of the apical vacuolar H+-ATPase, H+-K+- ATPase, and NBC3 to recovery of intracellular pH (pHi) after intracellular acid loading.
MATERIALS AND METHODS
In vitro microperfusion.
Kidneys from New Zealand White rabbits were removed, and 1- to 2-mm coronal slices were made and transferred to chilled dissection medium containing (in mM) 145 NaCl, 2.5 K2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 d-glucose, 1 trisodium citrate, 4 sodium lactate, and 6 l-alanine, pH 7.4 (290 ± 2 mosmol/kgH2O). From the corticomedullary rays, OMCDis segments were isolated under a dissecting microscope with sharpened forceps. Attention was given to obtaining the ducts from deep within the OMCDis (below the termination of straight proximal tubule segments and adjacent to the medullary thick ascending limbs of Henle's loop). To maximize the reproducibility of this isolation in such a heterogeneous epithelium, relatively short segments (0.5–0.7 mm) were obtained. In vitro microperfusion was performed by the method of Burg and Green (5), as previously described (23). An isolated OMCDiswas rapidly transferred to a 1.2-ml temperature- and environmentally controlled chamber mounted on an inverted microscope and perfused and bathed at 37°C with a solution containing (in mM) 120 NaCl, 25 NaHCO3, 2.5 K2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 d-glucose, 1 trisodium citrate, 4 sodium lactate, and 6 l-alanine, pH 7.4 (290 ± 2 mosmol/kgH2O) and gassed with 94% O2-6% CO2. The specimen chamber was continuously suffused with 94% O2-6% CO2 to maintain pH at 7.4. The collecting end of the segment was sealed into a holding pipette using Sylgard 184 (Dow Corning, Midland, MI). The length of each segment was measured using an eyepiece micrometer. Twelve-nanoliter samples of tubular fluid were collected under water-saturated mineral oil by timed filling of a calibrated volumetric pipette. Collections during each period were made in triplicate. Transepithelial voltage (V te) was measured using the perfusion pipette as an electrode, with the bath serving as the reference electrode. The voltage difference between calomel cells connected via 1 M KCl-agar bridges was measured with a high-impedance electrometer (Duo 773, WPI, Sarasota, FL).
The concentration of total CO2 (assumed to be equal to that of HCO ) in perfusate (C0) and collected fluid (CL) was measured in a continuous-flow microfluorimeter (Nanoflo, WPI, Sarasota, FL) (24, 26). Because there is no net water absorption in the OMCDis (6), was calculated as (C0 /CL) × (VL /L), whereVL was the rate of collection of tubular fluid (∼1.5 nl/min), L was the tubular length (in mm), andJ was in picomoles per minute per millimeter tubular length. When > 0, there was net HCO absorption equivalent to net H+ secretion. The coefficient of variation of the Nanoflo microfluorimeter was <0.5%. This level of sensitivity allowed us to reliably detect HCO differences of 1 mM between perfused and collected fluids. In practice, at a perfusion rate of 1.5 nl/min, there was generally a difference of 3–4 mM between C0 and CL values. For experiments in which luminal Na+ was reduced, NaCl was substituted withN-methyl d-glucamine chloride.
Measurement of pHi.
The dissected OMCDis was cannulated and perfused in a temperature-controlled chamber mounted on a Zeiss Axiovert inverted microscope, which was coupled to a MRC-1000 (Bio-Rad) confocal scanning unit equipped with a krypton-argon laser (13, 31). pHi was measured using carboxyseminapthorhodofluor (SNARF)-1 acetate ester (10 μM) in single type A intercalated cells as previously described (13). For studies involving the quantitation of the apical Na+-dependent, Cl−-independent H+-base flux, tubules were dissected in the following Na+-free, Cl−-free HEPES-buffered solution: (in mM) 140 tetramethylammonium hydroxide, 140 gluconic acid lactone, 2.5 K2HPO4, 7 calcium gluconate, 2 magnesium gluconate; and 5 HEPES, pH 7.4 (bubbled with 100% O2). The tubules were perfused and bathed initially after dye loading in the following HCO -buffered Na+- and Cl−-free solution: (in mM) 115 tetramethylammonium hydroxide, 115 gluconic acid lactone, 2.5 K2HPO4, 7 calcium gluconate, 2 magnesium gluconate, and 25 tetramethylammonium bicarbonate, pH 7.4 (bubbled with 6% CO2-94% O2). The tubules were then exposed on the basolateral side for 5 min to a 30 mM NH -containing solution containing (in mM) 85 tetramethylammonium hydroxide, 84 gluconic acid lactone, 2.5 K2HPO4, 7 calcium gluconate, 2 magnesium gluconate, 25 tetramethylammonium bicarbonate, and 30 ammonium hydroxide, pH 7.4 (bubbled with 6% CO2-94% O2). pHi was decreased by the rapid removal of NH . The tubules were then perfused in an Na+-containing solution comprising (in mM) 115 sodium gluconate, 2.5 K2HPO4, 7 calcium gluconate, 2 magnesium gluconate, and 25 NaHCO3, pH 7.4, bubbled with 6% CO2-94% O2, and the rate of recovery of pHi was quantified. These experiments were performed in the presence or absence of EIPA (50 μM, lumen). In separate studies, the tubules were dissected, perfused, and bathed in Na+-free and Cl−-containing solutions. For these experiments, the tubules were dissected and subsequently loaded with SNARF-1 in a solution containing (in mM) 140 tetramethylammonium chloride, 2.5 K2HPO4, 1 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4, bubbled with 100% O2. The tubules were perfused and bathed initially after dye loading in the following HCO -buffered solution: (in mM) 115 tetramethylammonium chloride, 2.5 K2HPO4, 1 CaCl2, 1 MgCl2, and 25 tetramethylammonium bicarbonate, pH 7.4, bubbled with 6% CO2-94% O2. The tubules were then exposed on the basolateral side for 5 min to a 30 mM NH -containing solution: (in mM) 85 tetramethylammonium chloride, 2.5 K2HPO4, 1 CaCl2, 1 MgCl2, 25 tetramethylammonium bicarbonate, and 30 ammonium chloride, pH 7.4, bubbled with 6% CO2-94% O2. pHi was decreased by the rapid removal of NH . The tubules were then perfused in the following Na+-free, Cl−-containing solution comprising (in mM) 115 tetramethylammonium chloride, 2.5 K2HPO4, 1 CaCl2, 1 MgCl2, and 25 tetramethylammonium bicarbonate, pH 7.4, bubbled with 6% CO2-94% O2, and the rate of recovery of pHi was quantitated. These experiments were performed in the presence or absence of bafilomycin and 3-cyanomehtyl-2-methyl-8-(phenylmethoxy)imidazo[1,2-α]pyridine (Schering 28080; inhibitor of H+-K+-ATPase; 10 μM, lumen).
Confocal images were acquired from the bottom of the tubules with a Zeiss ×40 plan-apochromat objective (numerical aperture 1.2) at a zoom factor of 2–3. A laser line at 568 nm was used to excite the fluorescence of SNARF-1. Pairs of images at the emission wavelengths of 580 and 620 nm were acquired simultaneously on two separate photomultiplier detectors at 0.2–0.5 Hz and stored digitally. An emission ratio of 580/620 nm was used to monitor the changes in pHi (13). Analysis of pHitransients was obtained retrospectively from stored image pairs using TSCM software (Bio-Rad), as previously described (13, 30). Fluorescence ratios from each image pair were corrected by subtracting the dark current and background from each image at each wavelength. The dark current and background fluorescence were <5% of the total signal after dye loading. The fluorescence ratios were converted to pHi from the calibration parameters, which were obtained from the same cell at the end of the experiment using the high-K+-nigericin technique (13). The pHi recovery rate was then estimated using linear regression analysis of the time course of pHi recovery.
Schering 28080 was a gift of Dr. James Kaminski at Schering (Kenilworth, NJ). All other chemicals were from Sigma (St. Louis, MO).
Results are reported as means ± SE. In the transepithelial transport studies, a paired Student's t-test was used to compare group means. In the experiments in which pHi was measured, an unpaired Student's t-test was used in comparing group means.
Effects of luminal Na+reduction and EIPA on .
The effect of luminal Na+ reduction on andV te is shown in Fig.1. After the reduction of luminal Na+ from 140 to 40 mM, fell significantly from 13.0 ± 0.47 to 11.0 ± 0.32 pmol · min−1 · mm−1(n = 3), P < 0.05 (Fig.1 A), without a change in V te (Fig.1 B). EIPA (50 μM, lumen) decreased significantly from 13.5 ± 0.32 to 11.8 ± 0.40 pmol · min−1 · mm−1(n = 6), P < 0.01 (Fig.1 C), without a change in V te (Fig.1 D). The effects of lowering luminal Na+ and adding EIPA were not additive (Fig. 1, A and C). These results demonstrate that a small component of in the OMCDis is dependent on luminal Na+ and is EIPA inhibitable.
pHi recovery after intracellular acidification of type A intercalated cells.
The rate of increase in pHi was measured in type A intercalated cells after intracellular acid loading to assess the contribution of apical NBC3, vacuolar H+-ATPase, and H+-K+-ATPase to pHi regulation. To determine the role of apical NBC3, which has been previously immunolocalized to the apical membrane of type A intercalated cells, in mediating pHi recovery, we monitored the rate of EIPA-inhibitable, HCO - and Na+-dependent H+/base transport as previously described (13). The tubules were perfused and bathed in Na+- and Cl−-free solutions. After intracellular acid loading, luminal Na+ addition induced a rapid increase in pHi (Fig.2 A). The mean pHirecovery rate after luminal Na+ addition was 0.36 ± 0.02 pH/min (32 cells/3 OMCDis). These experiments confirmed that pHi recovery in these cells is in part luminal Na+ dependent. As shown in Fig.2 B, in the presence of 50 μM EIPA (lumen), the pHi recovery rate decreased to 0.07 ± 0.01 pH/min,P < 0.001 (22 cells/3 OMCDis). EIPA (50 μm, basolateral) had no effect on the rate of pHirecovery (0.33 ± 0.05 pH/min, P = not significant, 23 cells/3 OMCDis).
When OMCDis segments were perfused and bathed in a Na+-free, Cl−-containing solution, the spontaneous pHi recovery rate after acid loading was due to the apical vacuolar H+-ATPase and H+-K+-ATPase. The Na+-independent pHi recovery rate (Fig.3 A) was 0.16 ± 0.02 pH/min (28 cells/4 OMCDis). As shown in Fig. 3 B, the pHi recovery rate was reduced to 0.06 ± 0.006 pH/min (P < 0.001, 42 cells/4 OMCDis) by luminal bafilomycin (10 nM) and to 0.10 ± 0.01 pH/min (Fig.3 C) (P < 0.001, 35 cells/4 OMCDis) by luminal Schering 28080 (10 μM; see also Fig.4).
These observations indicate that apical Na+-dependent and -independent transport processes contributed to the pHirecovery after acid loading in OMCDis type A intercalated cells. Luminal Na+ addition to tubules perfused and bathed in the absence of Na+ may have overestimated the contribution of luminal NBC3 to pHi recovery. Therefore, in separate experiments the tubules were continuously perfused and bathed in Na+-containing solutions. After NH removal, pHi initially decreased and spontaneously recovered at a rate of 0.21 ± 0.02 pH/min (25 cells/2 OMCDis). Luminal EIPA (50 μM) decreased the pHi recovery rate to 0.15 ± 0.02 pH/min (P < 0.001, 24 cells/2 OMCDis), which is comparable to the Na+-independent pHi recovery rate of 0.16 pH/min.
These studies are the first to examine the contribution of apical NBC3, H+-ATPase, and H+-K+-ATPase in type A intercalated cells to pHi regulation and in the OMCDis. After acute intracellular acidification, all three transporters contributed to pHi regulation. Using a new continuous-flow microfluorimeter, we were able to demonstrate that ∼15% of HCO transport in the OMCDiswas luminal Na+ dependent and EIPA inhibitable. These results contrast with the present view that luminal H+/base transport in the OMCDis type is entirely Na+independent.
Type A intercalated cells in the OMCDis have several potent apical and basolateral H+/base transport processes. In addition to apical NBC3 (10, 13), a vacuolar H+-ATPase and H+-K+-ATPase (1-3, 6, 7, 15, 16, 27), these cells have a basolateral Na+/H+ antiporter (4, 7, 27,28) and chloride/base exchanger (1, 15, 16, 21, 28) that contribute to pHi regulation. Therefore, type A intercalated cells in the OMCDis have two Na+-dependent H+/base transport processes, an apical electroneutral Na+-HCO cotransporter and a basolateral Na+/H+exchanger, which contribute to pHi regulation after acute acid loading. In the present study, we have focused on documenting the relative contribution of the apical transporters to pHiregulation after an intracellular acid load. It has generally been assumed that the same H+/base transport processes that contribute to pHi regulation also play an important role in in OMCDis type A intercalated cells. The results of this study demonstrate that this simple paradigm may not apply uniformly. The contribution of apical NBC3 and the H+-ATPase and H+-K+-ATPase to these processes may vary at different pHi values. From these results, it would be predicted that in patients with loss-of-function mutations in the apical H+-ATPase (8), pHiregulation might not be impaired to the same extent as the decrement in . It should be noted, however, that interstitial Na+ concentration rises sharply in the outer medulla (25). Indeed, it has been predicted that passive Na+ secretion may occur in the OMCDis (18). Furthermore, systemic acid-base changes may alter luminal, cytoplasmic, and interstitial pH in the outer medulla. The extent to which changes in luminal, cytoplasmic, and interstitial Na+ and pH modulate apical and basolateral Na+-dependent and -independent H+/base flux requires further study.
The data in this study differ somewhat from those of Weiner et al. (27, 28), who concluded that in OMCDis type A intercalated cells, pHi recovery from an acid load is mediated entirely by the apical H+-ATPase and H+-K+-ATPase and basolateral Na+/H+ exchange. The contribution of apical Na+-dependent H+/base transport processes to pHi regulation was not addressed, because previous functional studies had failed to detect an apical Na+/H+ exchange process in these cells (4, 7). It was assumed that there were no other apical Na+-dependent H+/base transport processes that contributed to pHi regulation. NBC3 was subsequently detected on the apical membrane of type A intercalated cells in the OMCDis (10, 13). In the study by Weiner et al. (27), in HEPES-buffered solutions the rate of basolateral Na+-dependant pHi recovery in type A intercalated cells was ∼0.38 pH/min, which approximated the rate of apical Na+-dependent pHi recovery measured in the present study using HCO -buffered solutions. Although not strictly comparable because of differences in methodology (e.g., minimum pHi achieved, composition of solutions), given the larger buffer capacity in HCO -buffered solutions, the data suggest that apical Na+-dependent base transport is potentially a potent mechanism in type A intercalated cells responsible for pHi recovery after acid loading. Although not the specific goal of the present study, it would be of interest to address the relative contribution of apical and basolateral Na+-dependent H+/base transport processes when studied under identical conditions.
We have shown that NBC3, an electroneutral EIPA-sensitive Na+-HCO cotransporter is immunolocalized to the apical membrane of type A intercalated in the OMCDis(10, 13). Furthermore, functional studies have demonstrated that these cells have an apical Na+- and HCO -dependent H+/base transport process that has the same functional properties as NBC3 and contributes to pHi regulation after changes in luminal Na+(12, 13). Our present data show that NBC3, in addition to the vacuolar H+-ATPase and H+-K+-ATPase, contributes to pHirecovery after acid loading. We have previously shown than NBC3 is also immunolocalized to the basolateral membrane of type B intercalated cells (10). The role NBC3 plays in mediating basolateral HCO transport in this cell type remains to be determined.
In a previous study, it was shown that ∼65% of in the OMCDis was mediated by the apical vacuolar H+-ATPase and ∼35% by a P-type H+-K+-ATPase (23). The findings in the present study indicate that ∼15% of in the OMCDis is luminal Na+ dependent. Previous studies have concluded that in this segment is Na+ independent (11, 20). In the present study, we were able to reproducibly demonstrate a component of HCO absorption in OMCDis that was luminal Na+ dependent. We are unable to explain why other investigators had not previously observed a dependence on luminal Na+. A possibility is that the more sensitive Nanoflo microfluorimeter used in the present study provided better sensitivity and reproducibility to small changes in collected fluid HCO concentration than the Picapnotherm used in the earlier studies (24, 26).
In addition to demonstrating that transepithelial HCO absorption is in part dependent on luminal Na+, we have also shown that luminal EIPA inhibits transepithelial HCO absorption in the OMCDis and that the effects of luminal Na+reduction and EIPA addition are not additive. There are no previous studies of the effect of luminal EIPA on in the OMCDis. Stone et al. (20) examined the effect of amiloride (10−5 M) in OMCDis and failed to detect a decrease in transepithelial HCO absorption. Higher doses of amiloride were not used in this study. Previous studies have suggested that, based on a model of transport in the turtle bladder, luminal amiloride indirectly decreases electrogenic H+ secretion in type A intercalated cells by blocking principal cell apical Na+ (ENaC) channels (22). The results of the present study suggest that an additional potential mechanism involves direct inhibition of apical NBC3-mediated NaHCO3 absorption in type A intercalated cells.
This work was supported by National Institutes of Health Grants HL-59156 (K.-P. Yip); DK-50603 (G. J. Schwartz), and DK-58563, the Max Factor Family Foundation, the Richard and Hinda Rosenthal Foundation, and the Fredericka Taubitz Foundation (I. Kurtz).
Address for reprint requests and other correspondence: I. Kurtz, UCLA Division of Nephrology, 10833 Le Conte Ave., Rm. 7–155 Factor Bldg., Los Angeles, CA 90095-1689 (E-mail:).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 16, 2002;10.1152/ajprenal.00241.2001
- Copyright © 2002 the American Physiological Society