1 Department of Physiology and Biophysics, College of Medicine, University of South Florida, Tampa, Florida 33612; 2 Department of Pharmacology, Jichi Medical School, Tochigi 329-04, Japan; 3 Departments of Pediatrics and Medicine, School of Medicine, University of Rochester, Rochester, New York 14642; and 4 Division of Nephrology, David Geffen School of Medicine, University of California, Los Angeles, California 90095
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ABSTRACT |
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The outer medullary collecting
duct (OMCD) plays an important role in mediating transepithelial
HCO-HCO
1 · mm
1. Lowering
luminal Na+ from 140 to 40 mM decreased
confocal microscopy; NBC3; intracellular pH; bicarbonate reabsorption; outer medullary collecting duct
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INTRODUCTION |
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UNDER BASELINE CONDITIONS, the outer
medullary collecting duct inner stripe segment (OMCDis) has
the highest rate of H+ secretion (HCO/HCO
Previous studies have concluded that net transepithelial
HCO
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MATERIALS AND METHODS |
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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 OMCDis was 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 (Vte) 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).
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
-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 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
-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-
]pyridine (Schering 28080; inhibitor of H+-K+-ATPase;
10 µM, lumen).
Data acquisition. 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 pHi transients 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.
Chemicals. Schering 28080 was a gift of Dr. James Kaminski at Schering (Kenilworth, NJ). All other chemicals were from Sigma (St. Louis, MO).
Statistics. 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.
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RESULTS |
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Effects of luminal Na+
reduction and EIPA on
1 · mm
1
(n = 3), P < 0.05 (Fig.
1A), without a change in Vte (Fig.
1B). EIPA (50 µM, lumen) decreased
1 · mm
1
(n = 6), P < 0.01 (Fig.
1C), without a change in Vte (Fig.
1D). 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
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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-free solutions. After
intracellular acid loading, luminal Na+ addition induced a
rapid increase in pHi (Fig.
2A). The mean pHi
recovery 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.
2B, 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 pHi
recovery (0.33 ± 0.05 pH/min, P = not significant, 23 cells/3 OMCDis).
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DISCUSSION |
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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
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
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
We have shown that NBC3, an electroneutral EIPA-sensitive
Na+-HCO
In a previous study, it was shown that ~65% of
In addition to demonstrating that transepithelial
HCO5 M) in OMCDis and failed to
detect a decrease in transepithelial HCO
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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: IKurtz{at}mednet.ucla.edu).
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
Received 1 August 2001; accepted in final form 12 July 2002.
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