|
- 积分
- 0
- 威望
- 0
- 包包
- 0
|
作者：Bruns A. Watts, III and David W. Good作者单位：Departments of Medicine and Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555
3 M1 q, E9 B$ n 6 q6 |- B. h3 f; g4 P* H
5 C b6 |3 A0 A/ @7 `
, T5 o' H/ U4 d: V
; }+ t+ a; ?" u9 j; G7 j% a % a, C& K8 \. \! Y8 q: K' {
O% w0 B( E" A
2 ^' C" \* |; P 6 Y! b$ v9 ]2 c
g9 B$ Z7 _; i4 {7 L9 D' ~ - E# f8 t; t* B k3 X
: Q' Z/ y: o+ ?* g6 D H' ^* z# o8 ]
$ L, T. ]3 Q; R( @/ O 【摘要】# x+ o0 X: N% E; V
Absorption of HCO 3 - in the medullary thick ascending limb (MTAL) is mediated by apical membrane Na /H exchange. The identity and function of other apical acid-base transporters in this segment have not been defined. The present study was designed to examine apical membrane HCO 3 - /OH - /H transport pathways in the rat MTAL and to determine their role in transepithelial HCO 3 - absorption. MTALs were perfused in vitro in Na - and Cl - -free solutions containing 25 mM HCO 3 -, 5% CO 2. Lumen addition of either 120 mM Cl - or 50 mM Na (50 µM EIPA present) had no effect on intracellular pH (pH i ). Lumen Cl - addition also had no effect on pH i in the presence of 145 mM Na or in the nominal absence of HCO 3 - /CO 2. Thus there was no evidence for apical Cl - /HCO 3 - (OH - ) exchange, Na -dependent Cl - /HCO 3 - exchange, or Na -HCO 3 - cotransport. In contrast, in tubules studied in Na - and Cl - -free solutions containing 25 mM HCO 3 -, 5% CO 2 and 120 mM K , removal of luminal K induced a rapid and pronounced decrease in pH i ( pH i = 0.56 ± 0.06 pH U). pH i recovered following lumen K readdition. The initial rate of net base efflux induced by lumen K removal was decreased 85% at the same pH i in the nominal absence of HCO 3 - /CO 2, indicating a dependence on HCO 3 - /CO 2 and arguing against apical K /H exchange. A combination of the apical K channel blockers quinidine (0.1 mM) and glybenclamide (0.25 mM) had no effect on the lumen K -induced pH i changes, arguing against electrically coupled K and HCO 3 - conductances. The effect of lumen K on pH i was inhibited by 1 mM H 2 DIDS. In addition, lumen addition of DIDS increased transepithelial HCO 3 - absorption from 10.7 ± 0.7 to 14.9 ± 0.7 pmol·min -1 ·mm -1 ( P < 0.001) and increased pH i slightly in MTAL studied in physiological solutions (25 mM HCO 3 - and 4 mM K ). Lumen DIDS stimulated HCO 3 - absorption in the absence and presence of furosemide. These results are consistent with an apical membrane K -dependent HCO 3 - transport pathway that mediates coupled transfer of K and HCO 3 - from cell to lumen in the MTAL. This mechanism, possibly an apical K -HCO 3 - cotransporter, functions in parallel with apical Na /H exchange and opposes transepithelial HCO 3 - absorption.
1 T9 [2 X' x) S. Q5 h. Y 【关键词】 K HCO cotransport Na /H exchange Cl /HCO exchange acidbase transport KCC2 p8 M- n$ F6 p% ^
THE MEDULLARY THICK ascending limb (MTAL) of the mammalian kidney participates in the control of acid-base balance by reabsorbing most of the filtered HCO 3 - that is not reabsorbed by the proximal tubule ( 2, 18 ). Absorption of HCO 3 - by the MTAL is regulated by a variety of important physiological factors, including ANG II, aldosterone, vasopressin, growth factors, and dietary acid or sodium intake (16-20, 38, 41). However, the transport mechanisms involved in thick ascending limb HCO 3 - absorption are incompletely understood.8 S) z7 F1 E. {6 T) t
; ^2 [6 P' v/ k9 A$ ?
As in other nephron segments, transepithelial HCO 3 - absorption in the thick ascending limb is accomplished as the combined result of secretion of protons from cell to tubule lumen across the apical membrane and transport of base equivalents (HCO 3 - ) from cell to interstitial fluid across the basolateral membrane ( 2, 18 ). In the MTAL of the rat, virtually all of the H secretion necessary for HCO 3 - absorption is mediated by the apical membrane Na /H exchanger NHE3 ( 3, 5, 21, 27, 41 ). Neither an H -ATPase nor the Na /H exchanger isoform NHE2 appears to contribute significantly to H secretion and HCO 3 - absorption in this segment ( 18, 21, 41 ). However, whether other HCO 3 - /OH - /H transport pathways are present in the apical membrane of the MTAL and influence HCO 3 - absorption is unclear. Studies using membrane vesicles prepared from digested tubule fragments suggested that the apical membrane of the rat MTAL contains a Cl - /HCO 3 - exchanger ( 13 ) and a K /H exchanger ( 4 ). Apical Cl - /HCO 3 - exchange was also postulated in the mouse cortical thick ascending limb based on the observations that transepithelial Cl - absorption was enhanced by HCO 3 - /CO 2 and inhibited by luminal addition of the disulfonic stilbene SITS ( 14 ). However, there have been no direct studies of these transport pathways in the apical membrane of intact thick ascending limbs. Also, interpretation of the vesicle studies is complicated due to significant contamination of the apical membrane preparation with basolateral membrane vesicles ( 4, 13, 29 ).
1 V# t6 u/ M) u! g1 w } z: M! P$ F' u5 v3 X
The aims of the present study were 1 ) to determine whether HCO 3 - /OH - /H transport pathways in addition to Na /H exchange are present in the apical membrane of the rat MTAL and 2 ) to define the role of these pathways in transepithelial HCO 3 - absorption. The results show that the apical membrane contains a K -dependent HCO 3 - transport pathway that opposes HCO 3 - absorption. No evidence for apical Cl - /HCO 3 - exchange or apical K /H exchange was found.4 K0 z+ }7 E a7 C6 w9 d
9 M( q' A2 f( g; \1 i8 |" F3 MMETHODS
* g/ }1 P5 \3 b; X- N5 S" U: a% A* [3 {; W( _/ W3 a0 q; O
Tubule Perfusion
9 f2 o/ a9 a) f, n T
! f# u/ j( w& j, M2 H$ c( AMTALs from male Sprague-Dawley rats (60-80 g; Taconic, Germantown, NY) were perfused in vitro as previously described ( 16, 21, 38, 39 ). The tubules were dissected from the inner stripe of the outer medulla, transferred to a bath chamber on the stage of an inverted microscope, and mounted on micropipettes for perfusion at 37°C. The composition of the perfusion and bath solutions for specific protocols is given below.+ K" D% [4 ?& X8 }. G& F
7 I4 P, n0 W, C. pMeasurement of Net HCO 3 - Absorption
9 w0 ?& _& s6 F: E* c- H$ M @2 O0 ^: L1 C) g8 k0 ]9 l* n
To measure transepithelial HCO 3 - absorption rates, tubules were perfused and bathed in control solution that contained (in mM) 146 Na , 4 K , 122 Cl -, 25 HCO 3 -, 2.0 Ca 2 , 1.5 Mg 2 , 2.0 phosphate, 1.2 SO 4 2-, 1.0 citrate, 2.0 lactate, and 5.5 glucose (equilibrated with 95% O 2 -5% CO 2; pH 7.45 at 37°C). Bath solutions also contained 0.2% fatty acid-free bovine albumin. Experimental agents were added to the luminal solution as described in results. DIDS and its H 2 analog (H 2 DIDS) were obtained from Molecular Probes (Eugene, OR) and dissolved directly into perfusion solutions.
: i. L1 [. Y5 L7 I0 d' d" B- N$ X; j) W0 u, t
The protocol for study of transepithelial HCO 3 - absorption was as described ( 16, 21 ). The tubules were equilibrated for 20-30 min at 37°C in the initial perfusion and bath solutions, and the luminal flow rate (normalized per unit tubule length) was adjusted to 1.6-1.9 nl·min -1 ·mm -1. One to three 10-min tubule fluid samples were then collected for each period (initial, experimental, and recovery). The tubules were allowed to reequilibrate for 10-15 min after an experimental agent was added to or removed from the luminal solution. The absolute rate of HCO 3 - absorption ( J HCO 3 -; pmol·min -1 ·mm -1 ) was calculated from the luminal flow rate and the difference between total CO 2 concentrations measured in perfused and collected fluids ( 16 ). Single tubule values are presented in the figures. Control measurements made at the beginning and end of an experiment (initial and recovery periods) were combined to obtain a mean control transport rate for each tubule. The individual tubule values were averaged to obtain the group means ± SE presented in the text ( n = number of tubules).
( ?( K/ l, b' @5 K
2 d4 o9 S3 e0 u' tMeasurement of Intracellular pH and Calculation of Equivalent Net Base Flux) b$ V. I! ]3 A* ]- [5 q! ]* G
8 T, ~, J8 _2 _+ LIntracellular pH (pH i ) was measured in isolated, perfused MTALs by use of the pH-sensitive dye BCECF and a computer-controlled spectrofluorometer (CM-X, SPEX Industries) coupled to the perfusion apparatus, as described elsewhere ( 39, 40 ). The tubules were perfused in the same manner used for HCO 3 - transport experiments except that the lumen and bath solutions were delivered via rapid flow systems that permit complete exchange of the solutions in less than 2 s ( 39 ). Intracellular dye was excited alternately at 500- and 440-nm wavelengths, and emission was monitored at 530 nm using a photon counter. Intracellular dye was calibrated using high K -nigericin standards at the end of each experiment to convert fluorescence excitation ratios (F500/F440) to pH i values, as previously described ( 39, 40 ). Initial rates of net base flux (pmol·min -1 ·mm -1 ) were calculated as dpH i /d t x x V, where dpH i /d t is the initial slope of the pH i record vs. time (pH U/min) measured over the first 4 s following an experimental maneuver, is the intracellular buffering power (mmol·l -1 ·pH U -1, see below), and V is cell volume per millimeter of tubule length (nl/mm). For experiments using HCO 3 - /CO 2 -free solutions, intrinsic buffering power ( i ) was measured as a function of pH i as described ( 40 ). For experiments with HCO 3 - /CO 2 -containing solutions, total buffering power ( T ) was the sum of i and the HCO 3 - /CO 2 -buffering power (computed as 2.3 [HCO 3 - ] i ) ( 38 ). V was determined from inner and outer tubule diameters as described ( 38, 40 ).% \0 s2 c3 r3 J( B$ E" h
0 r8 o8 w3 H& k. ?
Two main solutions were used for pH i experiments. To examine K -dependent transport pathways, tubules were perfused and bathed in K solution that contained (in mM) 120 K , 25 N -methyl- D -glucammonium (NMDG ), 137 gluconate, 25 HCO 3 -, 7 Ca 2 , and 1.5 Mg 2 . To examine Na - and Cl - -dependent pathways, tubules were perfused and bathed in Na - and Cl - -free solution that contained (in mM) 72 K , 75 NMDG , 139 gluconate, 25 HCO 3 -, 7 Ca 2 , and 1.5 Mg 2 . All HCO 3 - -containing solutions were gassed with 95% O 2 -5% CO 2. In some experiments, variants of these solutions were prepared that were buffered with 25 mM HEPES and were nominally HCO 3 - /CO 2 free ( Figs. 1 B and 2 D ). The HCO 3 - -free solutions were gassed with 100% O 2 and titrated to pH 7.4. Tubules were equilibrated in HEPES-buffered solutions for 40 min before measurements were taken. In individual protocols, NMDG replaced K or Na , and gluconate replaced Cl - (see RESULTS ). The total calcium concentration of gluconate solutions was adjusted so that Ca 2 activity matched that of Cl - -containing solutions ( 40 ). All bath solutions also contained 5 mM glucose. Furosemide or EIPA was present in the luminal solution in some experiments (see RESULTS ). H 2 DIDS was used in pH i experiments because of its insensitivity to light. Quinidine and glybenclamide were prepared as stock solutions in ethanol and diluted into luminal solutions to final concentrations given in RESULTS.
2 H3 B% |( E$ E0 h' y1 U
" |2 p |0 T- h* X1 \Fig. 1. Apical K -dependent net base flux in medullary thick ascending limb (MTAL). MTALs were perfused and bathed in 120 mM K solution (see METHODS ), and intracellular pH (pH i ) was monitored as described ( 39, 40 ). A : tracing shows response of pH i to lumen K removal and readdition [K replaced with N -methyl- D -glucammonium (NMDG )]. This K replacement protocol was repeated in 25 mM HEPES-buffered solution that was nominally HCO 3 - /CO 2 free ( B ), in the presence of 1 mM luminal H 2 DIDS ( C ), and in the presence of 0.1 mM quinidine (Quin) plus 0.25 mM glybenclamide (Glyben) in the luminal fluid ( D ). E : summary of results in A - D. Initial rates of net base efflux induced by luminal K removal were determined from the initial rate of pH i decrease as described in METHODS. Values are means ± SE for 4 to 5 tubules in each condition. * P ( }4 o i% w$ P" [
1 o1 l8 O) [# M4 K
Fig. 2. Effects of lumen Na and Cl - addition on pH i. MTALs were perfused and bathed in Na - and Cl - -free solution containing 25 mM HCO 3 - and 5% CO 2 (see METHODS ), and pH i was monitored as described ( 39, 40 ). Tracings show the response of pH i to lumen addition of 50 mM Na (Na replaced NMDG ; A ) or 120 mM Cl - (Cl - replaced gluconate; B ). The Cl - addition protocol was repeated in the presence of 145 mM Na ( C ) and in 25 mM HEPES-buffered solution that was nominally HCO 3 - /CO 2 free ( D ). A and C : luminal solutions contained 50 µM EIPA. Tracings are representative of at least 3 experiments of each type.1 ^. Y0 B: M# \0 z3 q% F
/ z; {6 w3 W/ }6 \3 FAnalysis
& _# v% S. V+ r9 B& h
# f- K6 D4 B7 {# H! \) W/ R( SResults are presented as means ± SE. Differences between means were evaluated using the paired Student's t -test or analysis of variance with the Newman-Keuls multiple range test, as appropriate. P
+ [5 ^5 L; L, W4 A! o- }: E/ H9 E) E* o1 J Y% `
RESULTS9 B7 q7 D: p% }6 A& e; A. |4 a
8 @- F- Y9 S4 S$ J6 m! i1 A0 x, z
Evidence For Apical K -Dependent HCO 3 - Transport
- N* Y* z$ A8 N3 ]5 M
( E/ n1 G' o; _K -dependent equivalent net base flux. To determine whether the MTAL expresses an apical K -dependent acid-base transport pathway, pH i was monitored in response to luminal K removal and readdition. Tubules were perfused and bathed in 120 mM K solution that contained 25 mM HCO 3 -, 5% CO 2, and no Na or Cl - (see METHODS ). The luminal solutions also contained 10 -4 M furosemide. Initial pH i was 7.46 ± 0.02 ( n = 4). Removal of luminal K (K replaced with NMDG ) induced a rapid decrease in pH i ( Fig. 1 A ). The initial rate of net base efflux was 23.2 ± 2.7 pmol·min -1 ·mm -1 ( n = 4; Fig. 1 E ). The cell effect was reversed when K was readded to the tubule lumen ( Fig. 1 A ). The cell acidification induced by lumen K removal was unaffected by the presence of 50 µM lumen EIPA (initial rate of net base efflux = 21.5 ± 1.4 pmol·min -1 ·mm -1, n = 3; data not shown).5 Q8 b5 |( U- i9 k# {0 ?
4 F) Y+ w0 P) v" c9 D
Dependence on HCO 3 - /CO 2. To test whether the K -induced equivalent net base flux was dependent on the presence of HCO 3 - /CO 2, the K removal protocol in Fig. 1 A was repeated using HEPES-buffered solutions that were nominally HCO 3 - /CO 2 free. Initial pH i in the HCO 3 - -free solution (7.42 ± 0.02; n = 5) was similar to that in HCO 3 - -containing solution ( Fig. 1, A and B ). In the absence of HCO 3 - /CO 2, the pH i decrease in response to lumen K removal was nearly eliminated and the initial rate of K -dependent net base efflux was reduced by 85% ( Fig. 1, B and E ). These results argue against the presence of an apical K /H exchanger (or K -OH - cotransporter) and are consistent with the coupled transport of K and HCO 3 - across the apical membrane.1 B k; I7 P, A. U. ?- V4 ]
- L& i+ W/ k/ \+ w, J
Effect of H 2 DIDS. In the presence of 25 mM HCO 3 - and 5% CO 2, 1 mM luminal H 2 DIDS inhibited the pH i decrease induced by luminal K removal, reducing the initial rate of net base efflux by 60% ( Fig. 1, C and E ). These results are consistent with the previous finding that DIDS inhibited a HCO 3 - -dependent K flux in rat MTAL suspensions ( 28 ) (see DISCUSSION ).9 }& |% _) b* K T$ T* r
! F: f, N* R' ~ w, A
Effects of K channel inhibitors. To test for the possible involvement of electrically coupled K - and HCO 3 - -conductive pathways, we examined the effects of the K channel blockers quinidine and glybenclamide. These inhibitors block the activity of both the intermediate- and low-conductance K channels in the apical membrane of the MTAL ( 6, 37 ) and prevent changes in apical membrane voltage induced by changing the luminal K concentration ( 6, 22, 23 ). In MTAL studied in the 120 mM K solution containing HCO 3 - /CO 2, the pH i decrease and the initial rate of net base efflux induced by lumen K removal were unaffected by the combination of 0.1 mM quinidine and 0.25 mM glybenclamide in the luminal fluid ( Fig. 1, D and E ). These results argue against the presence of electrically coupled K and HCO 3 - fluxes and identify an apical K -dependent HCO 3 - transport pathway that operates independently of apical K channels.! x. z0 j; [( a/ P* |0 g- v
$ O2 f7 Q# c3 d6 a5 w) @
Evidence Against Na - and Cl - -Dependent Apical Transport Pathways* v0 X4 d. f: C4 V5 s m$ @0 t
4 |0 m% o# ~0 N$ s
To test for the presence of Na - or Cl - -dependent acid-base transport pathways, MTALs were perfused and bathed in Na - and Cl - -free solution (see METHODS ), and pH i was monitored in response to luminal Na or Cl - addition. In Na addition experiments or when Cl - was added in the presence of Na , 50 µM EIPA was present in the lumen to block apical Na /H exchange ( 40, 41 ). In the presence of 25 mM HCO 3 - and 5% CO 2, addition of either 50 mM Na (Na replaced NMDG ; Fig. 2 A ) or 120 mM Cl - (Cl - replaced gluconate; Fig. 2 B ) to the lumen had no effect on pH i. Luminal Cl - addition also did not affect pH i in the presence of HCO 3 - /CO 2 and 145 mM Na ( Fig. 2 C ) or in HCO 3 - /CO 2 -free solution buffered with 25 mM HEPES ( Fig. 2 D ). In Cl - addition protocols, results were similar in the absence or presence of 10 -4 M luminal furosemide. The lack of pH i response to lumen Na or Cl - addition is not the result of a detrimental effect of the experimental conditions on the tubules because 1 ) removing 72 mM K from the lumen using the Na - and Cl - -free solution induces a rapid decrease in pH i with an initial rate of net base efflux (19.5 ± 1.9 pmol·min -1 ·mm -1; n = 3) similar to that observed ( Fig. 1 ) using the K solution; 2 ) if the order of experimental conditions in Fig. 2 A is reversed so that tubules are begun in the 50 mM Na solution, removing lumen Na has no effect on pH i (data not shown); and 3 ) lumen Na addition induces a rapid pH i increase in the absence of luminal EIPA using similar solutions ( method 2 in Ref. 40). These experiments provide no evidence that Na -HCO 3 - (OH - ) cotransport, Cl - /HCO 3 - (OH - ) exchange, or Na -dependent Cl - /HCO 3 - exchange contributes significantly to the acid-base flux across the apical membrane.- x# j; n5 l; J6 M2 ^
: Y/ j; z0 \' u& w3 E. X! F
Role in Transepithelial HCO 3 - Absorption
2 j) j e. z- w4 A1 n9 N% `9 ~) D" L8 ]/ m: q `6 D* z% N) ^
Effect of luminal DIDS on HCO 3 - absorption. The results in Fig. 1 are consistent with the presence of a DIDS-sensitive K -dependent HCO 3 - transporter in the apical membrane of the MTAL. Under physiological conditions, the cell-to-lumen K concentration difference would be expected to drive the coupled transport of K and HCO 3 - from cell to tubule lumen, resulting in luminal HCO 3 - addition that would detract from net HCO 3 - absorption [the apical [HCO 3 - ] gradient is small compared with the [K ] gradient, based on measurements of pH i ( 21, 22 )]. To test this, we examined the effect on HCO 3 - absorption of inhibiting apical K -dependent HCO 3 - transport with DIDS. In MTAL studied in control solution (see METHODS ), adding 1 mM DIDS or 1 mM H 2 DIDS to the lumen increased HCO 3 - absorption by 39%, from 10.7 ± 0.7 to 14.9 ± 0.7 pmol·min -1 ·mm -1 ( P % J; J$ J! l9 K7 P6 u* x
" u6 o. B% P% Q0 x
Fig. 3. Luminal DIDS increases HCO 3 - absorption. MTALs were studied in control solution (see METHODS ) in the absence ( A ) or presence ( B ) of 10 -4 M luminal furosemide (Furos) and then 1 mM DIDS or 1 mM H 2 DIDS was added to the luminal solution. Data points are average values for single tubules. Lines connect paired measurements made in the same tubule. P values are for paired t -test. J HCO 3 -, absolute rate of HCO 3 - absorption. Mean values are given in RESULTS.$ _3 i. a0 M, {( f( o% n
/ K* S5 s* p7 a7 Z* M e0 r
Effect of luminal EIPA in the presence of DIDS. In the MTAL, the H secretion necessary for HCO 3 - absorption is mediated by apical membrane Na /H exchange ( 18, 21, 41 ). In MTAL perfused with 1 mM DIDS, addition of 50 µM EIPA to the lumen virtually abolished HCO 3 - absorption (14.9 ± 0.3 pmol·min -1 ·mm -1 DIDS vs. 1.9 ± 0.2 pmol·min -1 ·mm -1 DIDS EIPA, n = 3; P
1 r* }8 F$ H S! P7 n( v, @+ E7 N( e! G. X8 }
Fig. 4. Effect on HCO 3 - absorption of adding 50 µM EIPA to the lumen in the presence of 1 mM luminal DIDS. Data points, lines, P value, and J HCO 3 - are as in Fig. 3. Mean values are given in RESULTS.
! g& [" |' u0 W z
$ u! ?& {' z" b1 n! HEffect of luminal DIDS on steady-state pH i. If an apical K -dependent HCO 3 - transport mechanism mediates transfer of HCO 3 - from cell to lumen, then inhibiting this pathway with H 2 DIDS may cause intracellular alkalinization. Steady-state pH i was monitored in tubules perfused and bathed in the control solution used to study HCO 3 - absorption. The luminal solution also contained 10 -4 M furosemide. Addition of 1 mM H 2 DIDS to the lumen induced a small, reversible increase in pH i ( pH i = 0.10 ± 0.02 pH U; P
& ]4 O1 B; r, k- X) P, q5 \8 {$ ~0 o1 x8 f9 l6 w5 F( b( i9 n
Fig. 5. Luminal H 2 DIDS increases steady-state pH i. MTALs were studied in control (Cont) solution used to measure HCO 3 - absorption (see METHODS ), and pH i was monitored as described ( 38, 40 ). A : tracing shows the effect of adding 1 mM H 2 DIDS to the tubule lumen. B : values show pH i ± SE for 3 experiments similar to A. Pre- and post-H 2 DIDS control values were averaged and compared with pH i in the presence of H 2 DIDS in the same tubule. * P 0 c9 `7 p$ x8 c& ]: Z5 z
% e# Y4 j( M9 x$ k% o xDISCUSSION4 `: ?4 N/ a# b: i1 r* i
) c0 u# I$ N# s7 {
K -Dependent HCO 3 - Transport in the Apical Membrane7 f; j Q! [7 l& c$ m; j
! t9 t" w. w+ A, V+ |* t
Transepithelial absorption of HCO 3 - by the MTAL depends on proton secretion mediated by apical membrane Na /H exchange (NHE3) ( 3, 5, 18, 21, 41 ). The identity and function of other apical acid-base transporters in this segment are poorly defined. In the present study, we found that the MTAL contains an apical K -dependent base transport pathway that is dependent on HCO 3 - /CO 2, inhibited by DIDS, and independent of apical K channels. Adding DIDS to the tubule lumen to inhibit this pathway increases net HCO 3 - absorption and steady-state pH i. These results provide functional evidence for an apical membrane K -dependent HCO 3 - transporter that mediates the coupled transfer of K and HCO 3 - from cell to tubule lumen. This transporter functions in parallel with apical membrane Na /H exchange and opposes transepithelial HCO 3 - absorption.
& y( a- O. [ f- p' s+ \6 z& f. x6 Y
Evidence for coupled transport of K and HCO 3 - has been presented previously in two systems: suspensions of rat MTAL and squid giant axon. In alkali-loaded MTALs in suspension, a HCO 3 - efflux pathway was described that was dependent on cell K , independent of Na and Cl -, and inhibited by DIDS ( 28 ). Also, K efflux from the cells was enhanced by a HCO 3 - gradient ( 28 ). In a detailed series of studies examining pH i regulation in the squid giant axon, a pathway for coupled transport of K and HCO 3 - was identified that was best explained by a K -HCO 3 - cotransporter ( 24, 25, 42 ). In both the MTAL suspensions and squid giant axon, the K -coupled HCO 3 - flux was unrelated to membrane voltage and could not be explained by K , H , or HCO 3 - conductances ( 24, 25, 28 ). Consistent with these findings, our results show that the K -induced HCO 3 - flux in the apical membrane of the MTAL did not differ when the luminal K concentration was changed in the absence or presence of K channel blockers, conditions associated with marked differences in the apical membrane voltage ( 6, 22, 23, 37 ). Thus these studies suggest that the putative K -HCO 3 - cotransporter(s) may be electroneutral. K -HCO 3 - cotransport in the squid axon was not inhibited by DIDS, suggesting a transport pathway different from that in the MTAL ( 25 ). Studies using MTAL suspensions cannot localize transporters to the apical or basolateral membrane. However, it was presumed that K -coupled HCO 3 - transport was basolateral where it would mediate HCO 3 - efflux for HCO 3 - absorption ( 28 ). Our results show, however, that K -dependent HCO 3 - transport is apically located, where it detracts from net HCO 3 - absorption (see below). Whether the basolateral membrane may also contain a K -dependent HCO 3 - transport mechanism is unclear ( 8, 28, 29 ).
" W& }6 N9 `- z: j) T
" e% b# _# _. Q4 BA transport protein that mediates coupled transport of K and HCO 3 - has not been molecularly identified, but some possibilities can be considered. First, K could replace Na on a Na -HCO 3 - cotransporter. This possibility is unlikely because 1 ) we found no evidence for Na -dependent HCO 3 - transport in the apical membrane of the MTAL and 2 ) members of the Na -HCO 3 - cotransporter gene family (NBC) do not have significant affinity for K ( 32, 33 ). Second, HCO 3 - could replace Cl - on a K -Cl - cotransporter. Consistent with this possibility, three of the four known members of the K -Cl - cotransporter family are present in the kidney (KCC1, 3, and 4) ( 11, 31, 35 ), and KCC1 and KCC4 are inhibited by DIDS ( 30 ). KCC4 has been localized to the basolateral membrane of the MTAL, where it may contribute to transcellular Cl - absorption ( 22, 31, 35 ). However, evidence for KCC expression in the apical membrane of kidney cells has not been presented. Also, to our knowledge, it has not been established whether KCCs can transport HCO 3 -. Thus the possible role of K -Cl - cotransporters in mediating K -dependent HCO 3 - flux requires further study. Highly selective inhibitors of K -Cl - cotransporters have not yet been identified. A third possibility is that K -HCO 3 - cotransport may be mediated by a novel transporter distinct from the cation chloride cotransporter ( 11 ) or Na -dependent bicarbonate cotransporter ( 10, 32 ) gene families. Our data suggest that the MTAL is a viable model to investigate this hypothesis." S% f. J8 Q: h
0 {. ~; M; Y' ~9 _: M( C, }Absence of Other Apical HCO 3 - /OH - /H Transport Pathways( w8 t; ~0 C: W ^
% R) ~' u( l- {" u
We found no evidence that transport pathways other than Na /H exchange ( 21, 40, 41 ) and K -dependent HCO 3 - transport contribute significantly to the acid-base flux across the apical membrane of the MTAL. Using three different protocols involving luminal Cl - addition, we found no evidence for a measurable component of apical Cl - /HCO 3 - (OH - ) exchange in the rat MTAL. Based on the finding that 50% of net Cl - absorption was dependent on HCO 3 - /CO 2, it was proposed that apical Cl - /HCO 3 - exchange functioned in parallel with apical Na /H exchange to mediate a portion of NaCl absorption in the mouse cortical thick ascending limb ( 14 ). However, in a separate study of the same segment, no dependence of NaCl absorption on HCO 3 - /CO 2 was found ( 12 ). Although the explanation for the differing results in the mouse cortical TAL is unclear, it is conceivable that HCO 3 - /CO 2 could influence NaCl absorption under certain conditions in this segment through cellular mechanisms other than apical Cl - /HCO 3 - exchange. In the MTAL, the Cl - /HCO 3 - exchanger AE2 has been localized to the basolateral membrane, where it likely mediates HCO 3 - efflux for HCO 3 - absorption ( 1, 8, 34 ). The Na -HCO 3 - cotransporter NBCnl, which is electroneutral and insensitive to amiloride or EIPA ( 8, 10, 26 ), also has been localized to the basolateral membrane of the MTAL ( 8, 26, 36 ). We found no evidence for Na -HCO 3 - cotransport activity in the apical membrane, consistent with the absence of apical NBC staining in the immunolocalization studies. Last, our findings that the apical K -dependent net base flux in the MTAL is eliminated in the absence of HCO 3 - /CO 2 and is inhibited by DIDS argue against the involvement of an apical K /H exchanger (or an H -K -ATPase). It is possible that the K /H exchange activity reported previously in apical membrane vesicles from MTAL suspensions reflects transport activity present in basolateral membranes or contaminating membranes from other cell types or intracellular organelles ( 4, 13, 29 ). Alternatively, K /H exchange observed in membrane vesicles may not be functionally active in intact MTALs. Our finding that the lumen K -induced pH i change is not affected by luminal EIPA indicates that transport of K on the apical Na /H exchanger does not contribute significantly to the apical acid-base flux.7 K2 h5 l! }$ }$ U6 E) q
/ ]# n/ @9 [' s* n9 F' }2 o0 W# m8 _
Role of Apical K -Dependent HCO 3 - Transport in HCO 3 - Absorption' f6 O2 t+ y% }& C$ B, m; _
" C8 x% E) y9 @; g, E
Under physiological conditions, a K -HCO 3 - cotransporter in the apical membrane should mediate the net transfer of K and HCO 3 - into the tubule lumen, driven by the large cell-to- lumen K concentration difference ( 22 ). This HCO 3 - transport would be expected to diminish net HCO 3 - absorption. Consistent with this prediction, luminal DIDS significantly increased HCO 3 - absorption in the MTAL. Although DIDS is a nonselective inhibitor of anion transport pathways, several lines of evidence support the view that DIDS increased HCO 3 - absorption through inhibition of apical K -dependent HCO 3 - transport: 1 ) DIDS caused a sizable inhibition of the K -induced apical net base flux; 2 ) no evidence for Na - or Cl - -dependent HCO 3 - /OH - transport pathways was found in the apical membrane; 3 ) DIDS increased HCO 3 - absorption in the presence of luminal furosemide, ruling out an indirect effect on Na -K -2Cl - cotransport; 4 ) DIDS increased HCO 3 - absorption when Cl - and HCO 3 - were the only luminal anions, ruling out potential effects on organic anion or divalent anion transporters; and 5 ) DIDS has no direct effect on the activity of the apical Na /H exchanger NHE3 ( 9 ). In the presence of DIDS, HCO 3 - absorption was eliminated by luminal EIPA, consistent with mediation of the HCO 3 - absorption by apical Na /H exchange. It appears, therefore, that the HCO 3 - absorption rate increases with DIDS because transport of HCO 3 - into the lumen via apical K -HCO 3 - cotransport is inhibited and no longer opposes HCO 3 - absorption mediated by apical Na /H exchange. We cannot be certain from our data whether the apical Na /H exchange rate is increased, decreased, or unchanged when K -HCO 3 - cotransport is inhibited. However, if it is decreased, then this decrease must be quantitatively less than the decrease in K -HCO 3 - cotransport to result in an increase in net HCO 3 - absorption. An important area for future work will be to examine coupling between K -dependent HCO 3 - transport and Na /H exchange in the apical membrane. Based on the small effect of luminal DIDS on pH i, and the relative insensitivity of apical Na /H exchange to physiological changes in pH i ( 21, 40 ), it is unlikely that K -HCO 3 - cotransport influences apical Na /H exchange activity through effects on pH i.% e. r- H& J/ ^5 [7 P$ k
) k) O* K" H: A0 ~Our studies provide the first evidence that K -dependent HCO 3 - transport plays a role in transepithelial acid-base transport and suggest that the control of HCO 3 - absorption in the MTAL can be achieved through regulation of apical Na /H exchange, apical K -dependent HCO 3 - transport, or both processes. Factors that regulate K -HCO 3 - cotransport currently are unknown. Acute changes in extracellular K concentration do not appear to be a major influence on MTAL HCO 3 - absorption, because increasing K concentration in both luminal and bath solutions did not affect HCO 3 - absorption in vitro ( 15 ). This likely is because changing extracellular K concentration has multiple effects that are not observed with luminal DIDS, such as changes in intracellular K or Cl - activity or membrane voltage that may influence basolateral HCO 3 - efflux. A broad range of physiological factors and signal transduction pathways has been identified that regulates MTAL HCO 3 - absorption through effects on apical Na /H exchange activity ( 7, 18, 19, 21, 27, 38, 40, 41 ). Future studies aimed at identifying hormones and other factors that modulate K -dependent HCO 3 - transport will be necessary to understand the role of this pathway in acid-base regulation. One interesting possibility is that downregulation of apical K -HCO 3 - cotransport activity could result in an increase in MTAL HCO 3 - absorption that contributes to metabolic alkalosis in chronic K depletion.
5 e5 j9 Q4 b5 @8 [2 Y+ S: Y/ y J! c/ d& G" z
In summary, we obtained functional evidence for a K -dependent HCO 3 - transport pathway, possibly a K -HCO 3 - cotransporter, in the apical membrane of the rat MTAL. This pathway functions in parallel with apical Na /H exchange and decreases the rate of transepithelial HCO 3 - absorption, presumably by transporting K and HCO 3 - from cell to tubule lumen. The molecular identity of the K -dependent HCO 3 - transporter and physiological factors that regulate this pathway remain to be identified.
. K" i, |% O k( y
% k) M; L: U1 D N/ I& ~. RGRANTS
0 H! x: ^5 u. L1 ^( _4 e L
! {+ |( \$ j6 \: Y1 O b; t3 gThis work was supported by National Institutes of Health Grant DK-38217.
3 l% i; L6 i# v) N9 Y! H, V! O. N! S9 M2 Z
ACKNOWLEDGMENTS
7 d5 o$ {* ~9 ~5 \6 u7 Y* V) \0 t
We thank L. Reuss for a critical reading of the manuscript.4 u* [- s( B# y! M5 l- j) ]9 G8 L
【参考文献】
c: D: q& m8 ^ C: ?1 j( q' C- c" I Alper SL, Stuart-Tilley AK, Biemesderfer D, Shmukler BE, and Brown D. Immunolocalization of AE2 anion exchanger in rat kidney. Am J Physiol Renal Physiol 273: F601-F614, 1997.3 S. N( L4 H# u7 S, `
0 I, C9 {; M$ X8 [; o7 r7 N/ }( g* |
+ q( @4 ^' Z) aAlpern RJ. Renal acidification mechanisms. In: The Kidney, edited by Brenner BM. Philadelphia, PA: Saunders, vol. I, 2000, p. 455-519.* ~( q: j( X2 H8 f: A; [# }6 Y
9 [8 O; e3 J$ ~7 s
: k: S% O. i( ?- H% E7 c4 L! |. q
) q2 y* A- V# WAmemiya M, Loffing J, Lotscher M, Kaissling B, Alpern RJ, and Moe OW. Expression of NHE-3 in the apical membrane of rat renal proximal tubule and thick ascending limb. Kidney Int 48: 1206-1215, 1995.
R$ v5 f# ]# E* x3 X
# N6 n* f& v1 f2 Z' z: M+ u: o- Z C) h3 k8 o( V
. s+ g8 G( K/ W0 j( y" KAttmane-Elakeb A, Boulanger H, Vernimmen C, and Bichara M. Apical location and inhibition by arginine vasopressin of K /H antiport of the medullary thick ascending limb of rat kidney. J Biol Chem 272: 25668-25677, 1997.
! M; m, y: O6 {" f! |
% [! j# p8 }3 R) S% ?9 M' O
; [3 i( Y& t% ^* W. ^6 j1 V4 j/ Z
Biemesderfer D, Rutherford PA, Nagy T, Pizzonia JH, Abu-Alfa AK, and Aronson PS. Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal rat kidney. Am J Physiol Renal Physiol 273: F289-F299, 1997., C0 c a& h- i; E ~0 {# H9 O+ A
2 `4 n" I8 r- S0 E5 Y% B. F* w
5 h5 [" A; Q: ^: C. k- k# z9 w: `3 k7 J
Bleich M, Schlatter E, and Greger R. The luminal K channel of the thick ascending limb of Henle's loop. Pflügers Arch 415: 449-460, 1990.1 P3 z# P" u& y1 W' I4 u
/ F6 X. _) t& s7 M
7 @8 |' F1 P4 D1 {' Z' Z
2 l* z7 M& L7 b5 ]Borensztein P, Juvin P, Vernimmen C, Poggioli J, Paillard M, and Bichara M. cAMP-dependent control of Na /H antiport by AVP, PTH, and PGE 2 in rat medullary thick ascending limb cells. Am J Physiol Renal Fluid Electrolyte Physiol 264: F354-F364, 1993.4 v8 L% t0 ?& }$ D9 O2 F
5 [/ F* k# D! ~$ ?- Z m: O6 H+ D
1 b5 D8 ]# v$ A0 o0 R; ]
4 b2 G/ Y6 r9 U% i8 [ @
Bourgeois S, Masse S, Paillard M, and Houillier P. Basolateral membrane Cl - -, Na -, and K -coupled base transport mechanisms in rat MTALH. Am J Physiol Renal Physiol 282: F655-F668, 2002.) S; W! k' v7 o: q7 Q
% V' D h% ]: K, \ ?
6 c. s. l" H6 m d% A+ }( v6 h, z. T% E* G. r* r; y* w3 j
Chambrey R, Achard JM, and Warnock DG. Heterologous expression of rat NHE4: a highly amiloride-resistant Na /H exchanger isoform. Am J Physiol Cell Physiol 272: C90-C98, 1997.4 z4 r2 {) g/ R6 o2 \2 F, ]
: g" O6 L9 y: w+ L) S, C& o
$ ?4 F+ S2 H+ ~- H$ r
( H L5 L* k" \( w0 xChoi I, Aalkjaer C, Boulpaep EL, and Boron WF. An electroneutral sodium/bicarbonate cotransporter NBCn1 and associated sodium channel. Nature 405: 571-575, 2000.
7 ?# G' j) |$ q- S
$ X* R& Z9 e. p, k; L' ]6 d% K C- X6 q( ]7 g9 R& Z' }7 P* N5 P9 M3 w
/ x7 y" w1 z: ~. f$ W9 lDelpire E and Mount B. Human and murine phenotypes associated with defects in cation-chloride cotransporter. Annu Rev Physiol 64: 803-843, 2002.
- ?: e# ]6 ]1 n. j- I2 }! G# @4 e, C/ o4 d# L% S# y [6 I
5 M6 j1 a7 f/ m' @. [
( T9 L0 Y: H' P
Di Stephano A, Greger R, de Rouffignac C, and Wittner M. Active NaCl transport in the cortical thick ascending limb of Henle's loop of the mouse does not require the presence of bicarbonate. Pflügers Arch 420: 290-296, 1992.! X3 a8 P3 D" P$ t+ F
" U7 M5 d! a" }$ v, G
8 ?# D* d3 Q' R* D b) N* V% j. i/ D! Q) W
Eladori D, Blanchard A, Leviel F, Paillard M, Stuart-Tilley AK, Alper SL, and Podevan RA. Functional and molecular characterization of luminal and basolateral Cl - /HCO 3 - exchangers in rat thick limbs. Am J Physiol Renal Physiol 275: F334-F342, 1998.1 A2 G5 ]* ~. D/ {6 U U4 Q
5 p) A2 S/ H2 n6 L1 I. q Q( D3 s+ _' l; M/ w- `7 o. B
5 |& U% }6 n) |- j7 VFriedman PA and Andreoli TE. CO 2 -stimulated NaCl absorption in the mouse renal cortical thick ascending limb of Henle. J Gen Physiol 80: 683-711, 1982.
1 x5 J! M, L. D6 M ^1 u
* ^- B) G. U. N& |
2 S6 M. X* ]* M& f! m9 M, j# j
\ Z+ l( K8 a9 v2 aGood DW. Active absorption of NH 4 by rat medullary thick ascending limb: inhibition by potassium. Am J Physiol Renal Fluid Electrolyte Physiol 255: F78-F87, 1988./ ?+ d8 D) m! N
& H" W( \& c# c9 G; k4 K
% T" w9 I, n3 [ z
" J! z0 f% D# I- I! Y# w' yGood DW. Inhibition of bicarbonate absorption by peptide hormones and cyclic adenosine monophosphate in rat medullary thick ascending limb. J Clin Invest 85: 1006-1013, 1990.
u$ a" I& P: V& {6 A" ~: L& _. a, j2 H. _- B3 E3 `
' h- `8 n ]* X; h; ^
. V+ C& ~1 ]. @) }" M
Good DW. Adaptation of HCO 3 - and NH 4 transport in rat MTAL: effects of chronic metabolic acidosis and Na intake. Am J Physiol Renal Fluid Electrolyte Physiol 258: F1345-F1353, 1990.! D, W9 V% {7 J7 [2 M b
3 f. _) \ m* A# u( n* x
* \: o5 N L) |; ]- z* Q5 X0 T d7 H
! Y X1 ~1 j% J$ e3 R1 J QGood DW. The thick ascending limb as a site of renal bicarbonate reabsorption. Semin Nephrol 13: 225-235, 1993.
) U: f: M2 M, h4 ~8 ?! c9 y7 ^2 U1 P' e& E6 B" X' J. T/ O
: N: B" i; j9 e: X* U0 t9 w+ s1 ]% }5 w! r1 F
Good DW, George T, and Wang DW. Angiotensin II inhibits HCO 3 - absorption via a cytochrome P450-dependent signaling pathway in rat medullary thick ascending limb. Am J Physiol Renal Physiol 276: F726-F736, 1999.
% ~. L r, C% n' o7 a( w- g2 H8 Q: m- U2 s2 }* Q; _
) o0 p8 ?$ U9 r5 e
- I' Y# d% ^+ C. d- w# d5 }6 ^6 qGood DW, George T, and Watts BA III. Aldosterone inhibits HCO 3 - absorption via a nongenomic pathway in medullary thick ascending limb. Am J Physiol Renal Physiol 283: F699-F706, 2002.
* J2 X! T1 z* S) |8 l% q+ Y! Y% q; {7 h5 G
; G/ b. J, c/ I( Z) p
* v+ j7 ?. H j# \Good DW and Watts BA III. Functional roles of apical membrane Na /H exchange in rat medullary thick ascending limb. Am J Physiol Renal Fluid Electrolyte Physiol 270: F691-F699, 1996.
. Z% v6 Q' F0 U; u8 \: r6 @8 I6 G
% T' Z2 i' }0 q' D$ b4 I' N" T- @9 w4 c: v; S' K6 r1 P8 v% n) _
Greger R. Ion transport mechanisms in thick ascending limb of Henle's loop of mammaliam nephron. Physiol Rev 65: 760-797, 1985.
3 T# l7 K" [4 T* z9 @
9 w+ j0 J' s% A% ~* j% _ F/ V% p2 I
( ]& v' z6 O* Z3 y5 s2 T9 N; O% D- V9 _
Greger R and Schlatter E. Properties of the lumen membrane of the cortical thick ascending limb of Henle's loop of rabbit kidney. Pflügers Arch 396: 315-324, 1983.
+ B( }! {( q) L: X h# ^# q6 V" [# ?: S
" N$ `. y; C+ M
7 f5 G8 M& _! W: m6 O" | S! M: ~ v9 sHogan EM, Cohen MA, and Boron WF. K - and HCO 3 - -dependent acid-base transport in squid giant axons. I. Base efflux. J Gen Physiol 106: 821-844, 1995.6 m! H- V3 R T `$ ^
9 L3 h* o; K {4 C" C; t3 }+ V
# R# q5 h- \0 z3 L; F' J5 q# |' a; \. z0 O7 q! S
Hogan EM, Cohen MA, and Boron WF. K - and HCO 3 - -dependent acid-base transport in squid giant axons. II. Base influx. J Gen Physiol 106: 845-862, 1995.0 b/ ~. K- F! d' W' }
1 k. t2 [5 f1 d j! w: t& V+ x2 S% M9 l- x: F4 O! ?" G
E, ~% i$ _) S" W" wKwon TH, Fulton C, Wang W, Kurtz I, Frokiaer J, Aalkjaer C, and Nielsen S. Chronic metabolic acidosis upregulates rat kidney Na-HCO 3 - cotransporters NBCn1 and NBC3 but not NBC1. Am J Physiol Renal Physiol 282: F341-F351, 2002.. V5 J/ P/ [- H( o9 x! d+ Y
4 ]% b2 P) D" g7 q
/ T) T; E1 q% o) [4 g/ c1 x, k. p* d5 k, F/ W
Laghmani K, Borensztein P, Ambuhl P, Froissart M, Bichara M, Moe OW, Alpern RJ, and Paillard M. Chronic metabolic acidosis enhances NHE3 protein abundance and transport activity in rat thick ascending limb by increasing NHE3 mRNA. J Clin Invest 99: 24-30, 1997.' V; N& \$ n; }% h$ d/ T/ c! c, m
8 h' V) g6 y1 g5 L' t. @- x0 t
# ?5 ?& ^, {. t# h9 X
% x: v( l! G0 Q9 pLeviel F, Borensztein P, Houillier P, Paillard M, and Bichara M. Electroneutral K /HCO 3 - cotransport in cells of medullary thick ascending limb of rat kidney. J Clin Invest 90: 869-878, 1992.
, K$ q6 @+ S) w0 J) p6 ^ G7 P/ J2 n7 f& ?
$ U a0 w1 W9 X! \$ |4 p
/ |* r( L! y* W- {- GLeviel F, Eladari D, Blanchard A, Poumarat JS, Paillard M, and Podevin RE. Pathways for HCO 3 - exit across the basolateral membrane in rat thick limbs. Am J Physiol Renal Physiol 276: F847-F856, 1999.7 Q6 V m) N6 q; L- \" ~/ E P
5 z* J" d* n( W( A& w
, t. v' p1 H8 P2 `% Y% |5 F& J( O" @0 f
Mercado A, Song L, Vazquez N, Mount DB, and Gamba G. Functional comparison of the K -Cl - cotransporters KCCl and KCC4. J Biol Chem 275: 30326-30334, 2000.% U0 o7 G5 F& N9 t
% V& v% k3 C2 Q! D0 L3 }% Q; i
, {$ P, S+ l! }/ K) N2 O: O4 p2 p5 P
Mount DB, Song L, Adriana M, Gamba G, and Delpire E. Basolateral localization of renal tubular K-Cl cotransporters. J Am Soc Nephrol 11: 35A, 2000.
8 J ]* `) e$ \2 S/ [% U v
9 b# {8 ~! j" n# P1 y" o, r) m0 O9 c$ M: k/ v. f3 |1 \
8 z0 E2 N7 m) L; dRomero MF and Boron WF. Electrogenic Na /HCO 3 - cotransporters: cloning and physiology. Annu Rev Physiol 61: 699-723, 1999.
9 _" ^! q* B' {( ?3 d. W" m. P% Y# x6 [2 V' D
# W: C8 U# E$ f5 t
# m( i* V1 } jSoleimani M and Burnham CE. Physiologic and molecular aspects of the Na :HCO 3 - cotransporter in health and disease processes. Kidney Int 57: 371-384, 2000.3 X$ z; t1 g; Z. U( G- }
3 M/ ]9 | D# R, A: \' R
! }' t/ n n3 e0 \' x- }3 n1 Z; l0 @# \; _6 [" ~0 _
Sun AM. Expression of Cl - /HCO 3 - exchanger in the basolateral membrane of mouse medullary thick ascending limb. Am J Physiol Renal Physiol 274: F358-F364, 1998.
3 Q: N/ b/ z& D% i
4 _# J! `) c& G+ ]. A
& ], `! O0 C1 v% h; k) R4 s1 n L" _+ L2 ]; O7 S. _( ^
Velazquez H and Silva T. Cloning and localization of KCC4 in rabbit kidney: expression in distal convoluted tubule. Am J Physiol Renal Physiol 285: F49-F58, 2003.3 q1 _; y& M B g- [+ e
1 C: ^9 v; r0 X9 ^: Y+ J5 m
$ \! U/ h9 U7 j3 a
/ B- ], s! t% i; IVorum H, Kwon TH, Fulton C, Simonsen B, Choi I, Boron W, Maunsbach A, Nielsen S, and Aalkjaer C. Immunolocalization of electroneutral Na -HCO 3 - cotransporter in rat kidney. Am J Physiol Renal Physiol 279: F901-F909, 2000.
. U% [; |0 H7 g& X
6 @( F/ S8 ?) j. F* A6 r# m
1 j0 c' U& w7 ^; E9 `
5 D/ x& ?! ?1 O/ _8 u ^! x9 m% D' fWang WH. Two types of K channel in thick ascending limb of rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 267: F599-F605, 1994.
1 |9 _0 a$ ^3 o4 H) _
9 w0 @2 H( Z' d0 r5 f. p) @: y6 t' O% W7 y
* U& q' U' t7 g. ~ ?Watts BA III, George T, and Good DW. Nerve growth factor inhibits HCO 3 - aborption in renal thick ascending limb through inhibition of basolateral membrane Na /H exchange. J Biol Chem 274: 7841-7847, 1999.* M& Z* U A, w, u3 m, F8 `4 f% h
g* l9 N) ?1 m% {1 w
% L) ^- e( a( k7 S+ ?
4 ]' ]$ _/ O7 o, [' S3 k
Watts BA III and Good DW. Effects of ammonium on intracellular pH in rat medullary thick ascending limb: mechanisms of apical membrane NH 4 transport. J Gen Physiol 103: 917-936, 1994.
" a9 t7 @$ H1 @% C& f# P8 I; r$ O3 V8 u4 J; t
# _/ V J- {2 x; R2 c/ N0 P- c: |$ w2 A# { X1 n
Watts BA III and Good DW. Apical membrane Na /H exchange in rat medullary thick ascending limb: pH i -dependence and inhibition by hyperosmolality. J Biol Chem 269: 20250-20255, 1994.* w- o K5 A+ z- c# X. ]
) z2 s0 z& C) b# b. X7 F/ v3 O( p' J' x
: n# ^; v- j! E' j1 `; e
2 o- M7 L4 W+ u7 z4 B1 ~
Watts BA III and Good DW. Hyposmolality stimulates apical membrane Na /H exchange and HCO 3 - absorption in renal thick ascending limb. J Clin Invest 104: 1593-1602, 1999.
M3 z# F' A$ J3 L% O4 x! h) Y) O) W9 \
0 g, x2 w3 u @. m/ B) {
2 P- w' g3 A1 V+ M% l/ l/ MZhao J, Hogan EM, Bevensee MO, and Boron WF. Out-of-equilibrium CO 2 /HCO 3 - solutions and their use in characterizing a new K/HCO 3 cotransporter. Nature 374: 636-639, 1995. |
|
发表于 2009-4-22 08:13
