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【摘要】 In the medullary thick ascending limb (MTAL) of rat kidney, inhibiting basolateral Na + /H + exchange with either amiloride or nerve growth factor (NGF) results secondarily in inhibition of apical Na + /H + exchange, thereby decreasing transepithelial HCO 3 - absorption. To assess the possible role of the Na + /H + exchanger NHE1 in this regulatory process, MTALs from wild-type and NHE1 knockout (NHE1 -/- ) mice were studied using in vitro microperfusion. The rate of HCO 3 - absorption was decreased 60% in NHE1 -/- MTALs (15.4 ± 0.5 pmol·min -1 ·mm -1 wild-type vs. 6.0 ± 0.5 pmol·min -1 ·mm -1 NHE1 -/- ). Transepithelial voltage, an index of the NaCl absorption rate, did not differ in wild-type and NHE1 -/- MTALs. Basolateral addition of 10 µM amiloride or 0.7 nM NGF decreased HCO 3 - absorption by 45-49% in wild-type MTALs but had no effect on HCO 3 - absorption in NHE1 -/- MTALs. Inhibition of HCO 3 - absorption by vasopressin and stimulation by hyposmolality, both of which regulate MTAL HCO 3 - absorption through primary effects on apical Na + /H + exchange, were similar in wild-type and NHE1 -/- MTALs. Thus the regulatory defect in NHE1 -/- MTALs is specific for factors (bath amiloride and NGF) shown previously to inhibit HCO 3 - absorption through primary effects on basolateral Na + /H + exchange. These findings demonstrate a novel role for NHE1 in transepithelial HCO 3 - absorption in the MTAL, in which basolateral NHE1 controls the activity of apical NHE3. Paradoxically, a reduction in NHE1-mediated H + extrusion across the basolateral membrane leads to a decrease in apical Na + /H + exchange activity that reduces HCO 3 - absorption.
【关键词】 growth factors sodium hydrogen exchanger type
NA + / H + EXCHANGERS (NHE) mediate the electroneutral exchange of extracellular Na + for intracellular H + across plasma membranes. At least eight NHE isoforms encoded by different genes have been identified in plasma membranes and intracellular membranes of mammalian cells ( 12, 18, 26, 28 ). NHE1 is expressed in all tissues and plays a central role in housekeeping functions such as regulation of intracellular pH (pH i ) and cell volume ( 12, 28, 31, 36 ). Increases in NHE1 activity also are involved in other important cellular processes, including proliferation, hypertrophy, adhesion, and migration ( 31, 36 ). In the kidney, NHE1 has been localized to the basolateral membrane of virtually all nephron segments where it is believed to participate in defense of pH i and cell volume ( 6, 21, 30 ). NHE1 has been ascribed no role in transepithelial H + secretion or HCO 3 - absorption by renal tubules. In contrast, NHE3 is localized specifically in the apical membrane of renal and intestinal epithelial cells, where it mediates transepithelial absorption of NaHCO 3 and/or NaCl ( 2, 3, 7, 12, 14, 23, 28, 32, 37 ).
The medullary thick ascending limb (MTAL) of the mammalian kidney participates in acid-base regulation by reabsorbing most of the filtered HCO 3 - not reabsorbed by the proximal tubule ( 2, 14 ). As in other nephron segments, HCO 3 - absorption in the MTAL is achieved as a two-step process involving secretion of H + across the apical membrane and transport of HCO 3 - across the basolateral membrane into the interstitium and then blood ( 2, 14 ). 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, 7, 14, 17, 39 ). The mechanism of basolateral HCO 3 - efflux in the MTAL is incompletely understood but may involve Cl - /HCO 3 - exchange ( 1, 10 ).
Recently, we identified a novel role for basolateral membrane Na + /H + exchange in transepithelial HCO 3 - absorption in the rat MTAL. Inhibiting basolateral Na + /H + exchange with either amiloride or nerve growth factor (NGF) results secondarily in inhibition of apical Na + /H + exchange, thereby decreasing HCO 3 - absorption ( 16, 38 ). The control of HCO 3 - absorption in the MTAL thus involves cross talk between the basolateral and apical membrane Na + /H + exchangers. However, the molecular identity of the basolateral exchanger responsible for this regulation has been unclear. NHE1 is expressed in the basolateral membrane of the thick ascending limb ( 4, 6, 21 ), and the sensitivity of HCO 3 - absorption to inhibition by bath amiloride and EIPA is consistent with the inhibitor sensitivities of NHE1 ( 16 ). However, there is no direct evidence that NHE1 influences transcellular acid-base transport in any segment of the nephron. In addition, uncertainty arises from the fact that basolateral Na + /H + exchange in the MTAL that regulates HCO 3 - absorption is 1 ) inhibited by NGF, an effect opposite to the virtually universal stimulation of NHE1 by growth factors in other cell types ( 19, 25, 28, 36 ), and 2 ) inhibited by the ERK signaling pathway ( 40 ), which activates NHE1 in other tissues ( 12, 31 ). These findings suggest that the regulation of HCO 3 - absorption may involve a basolateral Na + /H + exchanger other than NHE1. NHE4 has been localized in the MTAL, where it may be expressed along with NHE1 in the basolateral membrane ( 11 ). In the present study, we used MTALs from NHE1 knockout mice to examine specifically the role of NHE1 in transepithelial HCO 3 - absorption. The results show that the presence of NHE1 markedly enhances transepithelial HCO 3 - absorption in the MTAL and that NHE1 is the basolateral exchanger through which amiloride and NGF, acting from the basolateral side of the tubule, inhibit apical Na + /H + exchange and HCO 3 - absorption.
METHODS
NHE1 knockout mice. NHE1 null mutant mice (NHE1 -/- ) were generated by gene targeting as described ( 5 ). All mice were genotyped by PCR analysis of tail DNA. The homozygous NHE1 -/- mutants exhibit ataxia and growth retardation, and approximately two-thirds of the mutants die from epileptic-like seizures shortly before or after weaning ( 5 ). Experiments were performed using age-matched wild-type (129SvJ/Black Swiss) and NHE1 -/- mice between 6 and 12 wk old. Body weight was 24 ± 2 g ( n = 9) for wild-type mice and 17 ± 2 g ( n = 9) for NHE1 -/- mice ( P < 0.01). Kidney weight expressed as percent body weight did not differ significantly in wild-type and NHE1 -/- mice (0.80 ± 0.04% wild-type vs. 0.83 ± 0.07% NHE1 -/- ). Within the wild-type and NHE1 -/- groups, no differences in the basal HCO 3 - absorption rate or in the regulation of HCO 3 - absorption were observed in MTALs from male and female mice; thus the results were combined. The mice were kept in microisolator cages and received standard rodent chow (National Institutes of Health 31 diet; Ziegler) and distilled water up to the time of experiments.
Tubule perfusion and measurement of net HCO 3 - absorption. MTALs from wild-type and NHE1 -/- mice were isolated and perfused in vitro using methods identical to those described previously for MTALs from rats ( 13, 39 ). The tubules were dissected from the inner stripe of the outer medulla at 15°C in control solution, transferred to a bath chamber on the stage of an inverted microscope, and mounted on concentric glass pipettes for perfusion at 37°C. 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). Experimental agents were added to the bath solution as described in RESULTS. For experiments in Fig. 5, a hyposmotic solution was prepared by removing 25 mM NaCl from the control solution ( 39 ). All bath solutions also contained 0.2% fatty acid-free bovine albumin. Solutions containing amiloride, NGF, and AVP were prepared as described ( 13, 16, 38 ). The length of the perfused tubule segments ranged from 0.41 to 0.51 mm and did not differ in wild-type and NHE1 -/- mice. There was no difference in the size (inner and outer tubule diameters) or general appearance of MTALs from wild-type and NHE1 -/- mice in the perfusion microscope. In some tubules, the transepithelial voltage was measured using calomel cells connected to lumen and bath solutions by NaCl-agar bridges ( 13 ).
Fig. 5. Effects of hyposmolality (Hypo) on HCO 3 - absorption. MTAL from WT ( A ) and NHE1 -/- ( B ) mice were studied in control solution (295 mosmol/kgH 2 O), and then hyposmolality was produced in lumen and bath by removal of 25 mM NaCl (245 mosmol/kgH 2 O) ( 39 ). J HCO 3 -, data points, lines, and P values are as in Fig. 4. Mean values are given in the text.
The protocol for study of transepithelial HCO 3 - absorption was as described ( 13, 16, 39 ). The tubules were equilibrated for 20-30 min at 37°C in the initial perfusion and bath solutions, and the lumen flow rate (normalized per unit tubule length) was adjusted to 1.6 to 2.0 nl·min -1 ·mm -1. One to three 10-min tubule fluid samples were then collected for each period (initial, experimental, recovery). The tubules were allowed to reequilibrate for 5-10 min after a change in composition of the bath and/or lumen solutions. The absolute rate of HCO 3 - absorption ( J HCO 3 -, pmol·min -1 ·mm -1 ) was calculated from the lumen flow rate and the difference between total CO 2 concentrations measured in perfused and collected fluids ( 13 ). An average HCO 3 - absorption rate was calculated for each period studied in a given tubule. When control measurements were made before and after an experimental maneuver, the values were averaged. Single-tubule values are presented in the figures. Mean values ± SE ( n = number of tubules) are presented in the text. Differences between means were evaluated using the paired or unpaired t -test, or analysis of variance with a Newman-Keuls multiple range test, as appropriate. P < 0.05 was considered statistically significant.
Immunoblot analysis. Immunoblotting of NHE1 was carried out on the inner stripe of the outer medulla, the region of the kidney highly enriched in MTALs. Inner stripe tissue dissected from wild-type and NHE1 -/- kidneys was homogenized and solubilized in Laemmli sample buffer. Samples of equal protein content (50 µg/lane) were separated by SDS-PAGE using 8% gels and transferred to PVDF membranes as described ( 40 ). The blots were blocked with 10% nonfat milk in Tris-buffered saline and incubated overnight at 4°C with monoclonal anti-NHE1 antibody (clone 4E9, 1:1,000; Chemicon). After being washed in TBS + 0.1% Tween 20, horseradish peroxidase-conjugated goat anti-mouse secondary antibody was applied and immunoreactive bands were detected using enhanced chemiluminescence (Amersham). Identical gels were run in parallel and stained with Coomassie blue to verify protein loading.
RESULTS
NHE1 protein expression. Previous reports demonstrated that only defective NHE1 transcripts are expressed and that NHE1 protein and transport activity are absent in tissues from the NHE1 -/- mice ( 5, 29 ). Immunoblot analysis in Fig. 1 confirms that NHE1 protein is undetectable in the inner stripe of the outer medulla of kidneys from NHE1 -/- mice.
Fig. 1. Na + /H + exchanger isoform 1 (NHE1) protein expression in inner stripe of the outer medulla of wild-type and NHE1 -/- mice. Immunoblots of inner stripe extracts were probed with monoclonal antibody against NHE1 (MAb 4E9). Each lane contains a sample from a different mouse. Parallel gels were subjected to Coomassie blue staining to verify protein loading.
Transepithelial HCO 3 - absorption is impaired in MTAL from NHE1 - / - mice. Absolute rates of HCO 3 - absorption by MTAL from wild-type and NHE1 -/- mice are shown in Fig. 2 A. Tubules from wild-type mice absorbed HCO 3 - at 15.4 ± 0.5 pmol·min -1 ·mm -1 ( n = 9), a value similar to basal absorption rates measured under the same conditions in MTALs from rats ( 16, 38 ). In MTAL from NHE1 -/- mice, the HCO 3 - absorption rate was reduced by 60% vs. that of wild-type, to 6.0 ± 0.5 pmol·min -1 ·mm -1 ( n = 9; P < 0.001). The transepithelial voltage, which is unrelated to NaHCO 3 absorption ( 14 ) but is an accurate index of the rate of NaCl absorption in the mouse MTAL ( 20 ), did not differ significantly between tubules from wild-type and NHE1 -/- mice (7.2 ± 0.1 mV wild-type vs. 7.3 ± 0.2 mV NHE1 -/-; Fig. 2 B ). These results support an important role for NHE1 in transepithelial HCO 3 - absorption in the MTAL.
Fig. 2. Transepithelial HCO 3 - absorption rate ( A ) and transepithelial voltage ( V TE; B ) in medullary thick ascending limbs (MTAL) from wild-type (WT) and NHE1 -/- mice. Data points are average values for single tubules. V TE data are stable values measured over a 20- to 30-min period following an initial 15- to 20-min equilibration period. P values compare WT vs. NHE1 -/- (unpaired t -test). J HCO 3 -, absolute rate of HCO 3 - absorption. Mean values are given in the text. NS, not significant.
Effects of bath amiloride and NGF. In the MTAL of the rat, addition of amiloride or NGF to the bath (basolateral) solution decreases HCO 3 - absorption through inhibition of basolateral membrane Na + /H + exchange ( 16, 38 ). To determine whether this regulation depends on NHE1, we examined the effects of amiloride and NGF on tubules from wild-type and NHE1-deficient mice. In wild-type MTALs, adding 10 µM amiloride to the bath decreased HCO 3 - absorption by 45% (from 15.2 ± 1.0 to 8.3 ± 1.7 pmol·min -1 ·mm -1; P < 0.025), and adding 0.7 nM NGF to the bath decreased HCO 3 - absorption by 49% (from 14.0 ± 1.1 to 7.1 ± 1.1 pmol·min -1 ·mm -1; P < 0.025; Fig. 3 A ). These effects were reversible and are similar to the inhibition observed previously in MTALs from rats ( 16, 38 ). In sharp contrast, addition of either 10 µM amiloride or 0.7 nM NGF to the bath had no effect on HCO 3 - absorption in MTAL from NHE1 -/- mice ( Fig. 3 B ). These results identify a key role for NHE1 in the regulation of transepithelial HCO 3 - absorption and support the hypothesis that NHE1 is the basolateral Na + /H + exchanger that mediates inhibition of HCO 3 - absorption by bath amiloride and NGF.
Fig. 3. Effects of bath amiloride (Amil) and nerve growth factor (NGF) on HCO 3 - absorption. MTALs from WT ( A ) and NHE1 -/- ( B ) mice were studied in control (cont) solution, and then 10 µM Amil and 0.7 nM NGF were added to and removed from the bath solution. Data points are average values for single tubules. Lines connect paired measurements made in the same tubule. P values are for experimental period (Amil or NGF) vs. pre- and postcontrol (ANOVA). J HCO 3 - is as in Fig. 2 A. Mean values are given in the text.
Effects of vasopressin and hyposmolality. To assess the specificity of the defect in the regulation of HCO 3 - absorption in MTALs from NHE1 -/- mice, we examined the effects of vasopressin and hyposmolality. These factors were studied because in the rat MTAL, AVP inhibits and hyposmolality stimulates HCO 3 - absorption through primary effects on the apical membrane NHE3 Na + /H + exchanger ( 8, 13, 14, 39 ). The effects of these stimuli on mouse MTALs are shown in Figs. 4 and 5. Addition of 10 -10 M AVP to the bath decreased HCO 3 - absorption by 45% (from 15.6 ± 0.6 to 8.6 ± 1.1 pmol·min -1 ·mm -1; P < 0.001) in MTAL from wild-type mice ( Fig. 4 A ) and by 48% (from 6.7 ± 0.4 to 3.5 ± 0.5 pmol·min -1 ·mm -1; P < 0.05) in MTAL from NHE1 -/- mice ( Fig. 4 B ). Hyposmolality increased HCO 3 - absorption from 14.1 ± 0.9 to 18.4 ± 1.0 pmol·min -1 ·mm -1 ( P < 0.001) in MTAL from wild-type mice ( Fig. 5 A ) and from 5.4 ± 0.9 to 9.6 ± 1.2 pmol·min -1 ·mm -1 ( P < 0.025) in MTAL from NHE1 -/- mice ( Fig. 5 B ). These effects are similar to those observed in MTALs from rats ( 13, 39 ). These results demonstrate that the regulation of HCO 3 - absorption by AVP and hyposmolality is preserved in MTALs lacking NHE1. Thus the defect in HCO 3 - transport regulation in the NHE1 -/- tubules is specific for factors (bath amiloride and NGF) shown previously to regulate apical Na + /H + exchange and HCO 3 - absorption through primary effects on basolateral membrane Na + /H + exchange ( 16, 38 ).
Fig. 4. Effects of AVP on HCO 3 - absorption. MTAL from WT ( A ) and NHE1 -/- ( B ) mice were studied in control solution, and then 10 -10 M AVP was added to and removed from the bath solution. J HCO 3 -, data points, and lines are as in Fig. 3. Mean values are given in the text. P values are for paired t -test.
DISCUSSION
Previous studies demonstrated that basolateral membrane Na + /H + exchange plays an important role in transepithelial HCO 3 - absorption in the rat MTAL. This occurs through a unique and paradoxical mechanism whereby a primary decrease in basolateral Na + /H + exchange results secondarily in a decrease in apical Na + /H + exchange and HCO 3 - absorption ( 16, 38 ). The reduction in apical Na + /H + exchange that is directly responsible for decreased HCO 3 - absorption is induced by inhibiting basolateral Na + /H + exchange with either amiloride or NGF. The results of the present study show that MTALs lacking NHE1 have a markedly reduced basal rate of HCO 3 - absorption and, unlike wild-type MTALs, they exhibit no change in HCO 3 - absorption in response to amiloride or NGF applied to the basolateral side of the tubule. These studies demonstrate that NHE1 is the basolateral transporter that affects the apical NHE3 Na + /H + exchanger and HCO 3 - absorption in the MTAL ( Fig. 6 ), thereby providing the first direct evidence of a regulatory role for NHE1 in transepithelial transport in the kidney.
Fig. 6. Simplified model for NHE1-mediated regulation of HCO 3 - absorption in the MTAL. HCO 3 - absorption is mediated by the apical NHE3 Na + /H + exchanger; the basolateral HCO 3 - efflux mechanisms have not yet been established. NaCl absorption is mediated by the apical Na + -K + -2Cl - cotransporter NKCC2; other transporters involved in NaCl absorption (apical ROMK K + channel; basolateral CLC-KB Cl - channel) are omitted for simplicity. Basolateral NHE1 enhances NHE3-mediated HCO 3 - absorption, as shown by experiments in which eliminating NHE1 activity by inhibition with amiloride or NGF, or by gene targeting, decreases apical NHE3 activity and HCO 3 - absorption.
Decreasing basolateral Na + /H + exchange in the MTAL by three independent maneuvers, i.e., pharmacological inhibition with amiloride or EIPA, addition of NGF, or genetic ablation of NHE1 ( Figs. 2 and 3 ), decreases luminal H + secretion and HCO 3 - absorption. Importantly, eliminating basolateral Na + /H + exchange activity by any one of these maneuvers prevents further inhibition of HCO 3 - absorption by the others ( Fig. 3 B ) ( 38 ), indicating that the three conditions inhibit HCO 3 - absorption through a common mechanism, namely, by decreasing or eliminating the activity of NHE1. Together, these studies provide strong evidence that basolateral NHE1 controls apical Na + /H + exchange activity and HCO 3 - absorption in the MTAL. The finding that NHE1 activity on the basolateral membrane enhances luminal H + secretion and HCO 3 - absorption is surprising because it is widely believed that basolateral Na + /H + exchange reduces the efficiency of transcellular HCO 3 - absorption by decreasing net basolateral base efflux and/or by increasing cell [Na + ] or pH i and secondarily inhibiting apical Na + /H + exchange ( 9, 24, 25, 30 ). Our results show clearly that the role of basolateral NHE1 in transcellular acid-base transport is more complex than this, and that, paradoxically, the dominant effect of NHE1 activity in the MTAL is to enhance HCO 3 - absorption by increasing apical Na + /H + exchange ( Fig. 2 ) ( 16 ). The effect of basolateral NHE1 to increase apical Na + /H + exchange cannot be explained by a change in the net driving force for the apical exchanger and thus appears to be mediated through an as yet unidentified signal transduction mechanism.
Several lines of evidence indicate that the reduced rate of basal HCO 3 - absorption and the absence of regulation by bath amiloride or NGF in the NHE1 -/- tubules are attributable to the lack of NHE1 and not to undetected tubule damage or to a generalized functional or metabolic defect. These are: 1 ) the basal rate of HCO 3 - absorption in the NHE1-deficient tubules is stable for several hours and is similar in magnitude to that of wild-type tubules studied in the presence of bath amiloride; 2 ) the transepithelial voltage, an accurate indicator of apical Na + -K + -2Cl - cotransport-mediated NaCl absorption in the mouse MTAL ( 20 ), does not differ in MTAL from wild-type and NHE1 knockout mice; and 3 ) HCO 3 - absorption in tubules lacking NHE1 undergoes normal regulation in response to AVP and hyposmolality, indicating that the defect in transport regulation is specific for factors (bath amiloride and NGF) that alter HCO 3 - absorption through effects on basolateral Na + /H + exchange ( 16, 38 ). In the MTAL, AVP decreases HCO 3 - absorption by inhibiting the apical NHE3 Na + /H + exchanger via cAMP ( 8, 13, 14 ); hyposmolality increases HCO 3 - absorption by stimulating apical NHE3 via phosphatidylinositol 3-kinase ( 15 ). The results of the present study show that regulation of NHE3 via these signaling pathways is intact in MTALs lacking NHE1. Thus the control of apical NHE3 activity may be dependent on, or occur independently of NHE1, depending on the physiological stimulus. The absence of an effect of NHE1 ablation on the transepithelial voltage suggests that the transport pathways involved in NaCl absorption, which are distinct from those involved in NaHCO 3 absorption (see Fig. 6 ), may be unaffected by the mechanisms underlying NHE1-mediated regulation of NHE3 and HCO 3 - absorption.
Acute inhibition of NHE1 with amiloride or NGF and chronic loss of function of NHE1 by gene ablation result in similar decreases in basal HCO 3 - absorption ( Figs. 2 and 3 A ). This supports the view that the reduced rate of HCO 3 - absorption in MTAL from the NHE1 knockout mice is due simply to the loss of the regulatory influence of NHE1 on NHE3 and not to a chronic adaptation in the apical exchanger. Direct studies of NHE3 activity and apical NHE3 protein level in the NHE1 -/- tubules will be required to address this issue. At present, we do not know whether other basolateral NHE isoforms, such as NHE4, may be expressed in MTALs from the knockout mice and provide partial compensation for the absence of NHE1. If so, they do not replace the functions of NHE1 to enhance basal HCO 3 - absorption or to mediate regulation of HCO 3 - absorption by bath amiloride or NGF.
One of the most prominent features of NHE1 is that its activity is stimulated by growth factors. This stimulation has been a virtually universal finding, occurring in a wide variety of cell types and with virtually all mitogens ( 19, 25, 31, 36 ). Our finding that NGF inhibits HCO 3 - absorption in the MTAL through inhibition of NHE1 ( Fig. 3 ) is thus noteworthy, because it provides the first direct evidence for inhibition of NHE1 by a growth factor. In addition, NGF decreases basolateral NHE1 activity through activation of the ERK signaling pathway ( 40 ). This finding also is in distinct contrast to regulation in other cell types, where the ERK pathway is a key mediator of NHE1 activation ( 12, 31 ).
Although the mouse MTAL has both apical and basolateral Na + /H + exchange activity, the ability of this segment to absorb HCO 3 - has been unclear ( 14, 35, 41 ). Our results demonstrate directly that the mouse MTAL absorbs HCO 3 - in vitro at rates similar to those observed in MTALs from rats ( 13, 38, 39 ). In addition, factors that regulate HCO 3 - absorption in the rat MTAL have similar effects in the mouse MTAL, including the regulation by bath amiloride and NGF mediated through basolateral Na + /H + exchange. The marked reduction in basal HCO 3 - absorption in the MTAL of NHE1 -/- mice is not associated with a measurable change in blood acid-base values ( 5 ). This likely reflects compensatory upregulation of H + secretion and HCO 3 - absorption in the collecting ducts or other nephron segments, similar to the compensatory mechanisms that maintain renal HCO 3 - absorption and acid excretion in NHE3-deficient mice ( 33 ).
Our finding that basolateral NHE1 enhances apical Na + /H + exchange in the MTAL may be relevant to other epithelia that express NHE1 and NHE3. A loss of cross talk between basolateral NHE1 and apical Na + /H + exchange has been suggested recently as a possible explanation for decreased NaCl absorption by parotid gland duct cells from NHE1 knockout mice ( 29 ). Changes in NHE1 activity could regulate NHE3-mediated NaCl or NaHCO 3 transport in other epithelia, such as the renal proximal tubule, intestine, and bile duct. A functional link between NHE1 and NHE3 also could be important in pathophysiological conditions. NHE1 activity is increased in a variety of cells and tissues in patients with essential hypertension and in hypertensive animal models ( 27, 34 ). Both NHE1 and NHE3 activities are increased in proximal tubule cells of the spontaneously hypertensive rat, a model of genetic hypertension ( 27 ). Also, transgenic mice overexpressing NHE1 have salt-sensitive hypertension in association with increased renal sodium retention ( 22 ). Our results raise the possibility that an increase in basolateral NHE1 activity leading secondarily to increased apical Na + /H + exchange could promote renal sodium retention and thus contribute to the pathogenesis of salt-sensitive hypertension.
In summary, our data show that the basolateral Na + /H + exchanger NHE1 is a major determinant of transepithelial HCO 3 - absorption in the MTAL. The basal rate of HCO 3 - absorption is markedly reduced in MTALs from NHE1 knockout mice, and regulation of HCO 3 - absorption by factors that act via primary effects on basolateral Na + /H + exchange ( 16, 38 ) is eliminated. These studies demonstrate that basolateral NHE1 enhances HCO 3 - absorption by increasing apical NHE3 Na + /H + exchanger activity, an effect opposite to that predicted on the basis of its effects on the net driving force for apical Na + /H + exchange. This raises the possibility that NHE1 might regulate transepithelial absorptive processes in other tissues as well.
GRANTS
This work was supported by National Institutes of Health Grants DK-38217 (to D. Good) and DK-50594 (to G. Shull) and by a grant from The John Sealy Memorial Endowment Fund for Biomedical Research (to D. Good).
【参考文献】
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.
Alpern RJ. Renal acidification mechanisms. In: The Kidney, edited by Brenner BM. Philadelphia: Saunders, Vol. 1, 2000, p. 455-519.
Amemiya 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.
Attmane-Elakeb A, Chambrey R, Tsimaratos M, Leviel F, Blanchard A, Warnock DG, Paillard M, and Podevin RA. Isolation and characterization of luminal and basolateral plasma membrane vesicles from the medullary thick ascending loop of Henle. Kidney Int 50: 1051-1057, 1996.
Bell SM, Schreiner CM, Schultheis PJ, Miller ML, Evans RL, Vorhees CV, Shull GE, and Scott WJ. Targeted disruption of the murine Nhe1 locus induces ataxia, growth retardation, and seizures. Am J Physiol Cell Physiol 276: C788-C795, 1999.
Biemesderfer D, Reilly RF, Exner M, Igarashi P, and Aronson PS. Immunocytochemical characterization of Na + -H + exchanger isoform NHE-1 in rabbit kidney. Am J Physiol Renal Fluid Electrolyte Physiol 263: F833-F840, 1992.
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.
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.
Boron WF and Boulpaep EL. Intracellular pH regulation in the renal proximal tubule of the salamander. Na-H exchange. J Gen Physiol 81: 29-52, 1983.
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.
Chambrey R, St. John PL, Eladari D, Quentin F, Warnock DG, Abrahamson DR, Podevin RE, and Paillard M. Localization and functional characterization of Na + /H + exchanger isoform NHE4 in rat thick limbs. Am J Physiol Renal Physiol 281: F707-F717, 2001.
Counillon L and Pouyssegur J. The expanding family of eucaryotic Na + /H + exchangers. J Biol Chem 275: 1-4, 2000.
Good 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.
Good DW. The thick ascending limb as a site of renal bicarbonate reabsorption. Semin Nephrol 13: 225-235, 1993.
Good DW, Di Mari J, and Watts BA III. Hyposmolality stimulates Na + /H + exchange and HCO 3 - absorption in renal thick ascending limb via PI3-kinase. Am J Physiol Cell Physiol 279: C1443-C1454, 2000.
Good DW, George T, and Watts BA III. Basolateral membrane Na + /H + exchange enhances HCO 3 - absorption in rat medullary thick ascending limb: evidence for functional coupling between basolateral and apical membrane Na + /H + exchangers. Proc Natl Acad Sci USA 92: 12525-12529, 1995.
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.
Goyal S, Vanden Heuvel G, and Aronson PS. Renal expression of novel Na + /H + exchanger isoform NHE8. Am J Physiol Renal Physiol 284: F467-F473, 2003.
Grinstein S, Rotin D, and Mason MJ. Na + /H + exchange and growth factor-induced cytosolic pH changes. Role in cellular proliferation. Biochim Biophys Acta 988: 73-97, 1989.
Hebert SC, Culpepper RM, and Andreoli TE. NaCl transport in mouse medullary thick ascending limbs. I. Functional nephron heterogeneity and ADH-stimulated NaCl cotransport. Am J Physiol Renal Fluid Electrolyte Physiol 241: F412-F431, 1981.
Krapf R and Solioz M. Na + /H + antiporter mRNA expression in single nephron segments of rat kidney cortex. J Clin Invest 88: 783-788, 1991.
Kuro-o M, Hanaoka K, Hiroi Y, Noguchi T, Fujimori Y, Takewoki S, Hayasaka M, Katoh H, Miyagishi A, Nagai R, Yazaki Y, and Nabeshima Y. Salt-sensitive hypertension in transgenic mice overexpressing Na + -proton exchanger. Circ Res 76: 148-153, 1995.
Lorenz JN, Schultheis PJ, Traynor T, Shull GE, and Schnermann J. Micropuncture analysis of single-nephron function in NHE3-deficient mice. Am J Physiol Renal Physiol 277: F447-F453, 1999.
Mackovic-Basic M and Kurtz I. H + /base transport in proximal straight tubule and thin descending limb of Henle. Semin Nephrol 10: 122-131, 1990.
Noel J and Pouyssegur J. Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na + /H + exchanger isoforms. Am J Physiol Cell Physiol 268: C283-C296, 1995.
Numata M and Orlowski J. Molecular cloning and characterization of a novel (Na +, K + )/H + exchanger localized to the trans-Golgi network. J Biol Chem 276: 17387-17394, 2001.
Orlov S, Adragna NC, Adarichev VA, and Hamet P. Genetic and biochemical determinents of abnormal monovalent ion transport in primary hypertension. Am J Physiol Cell Physiol 276: C511-C536, 1999.
Orlowski J and Grinstein S. Na + /H + exchangers of mammalian cells. J Biol Chem 272: 22373-22376, 1997.
Park K, Evans RL, Watson GE, Nehrke K, Richardson L, Bell SM, Schultheis PJ, Hand AR, Shull GE, and Melvin JE. Defective fluid secretion and NaCl absorption in the parotid glands of Na + /H + exchanger-deficient mice. J Biol Chem 276: 27042-27050, 2001.
Preisig PA and Alpern RJ. Basolateral membrane H/HCO 3 transport in renal tubules. Kidney Int 39: 1077-1086, 1991.
Putney LK, Denker SP, and Barber DL. The changing face of the Na + /H + exchanger, NHE1: structure, regulation, and cellular actions. Annu Rev Pharmacol Toxicol 42: 527-552, 2002.
Schultheis PJ, Clarke LL, Meneton P, Miller ML, Soleimani M, Gawenis LR, Riddle TM, Duffy JJ, Doetschman T, Wang T, Giebisch G, Aronson PS, Lorenz JN, and Shull GE. Renal and intestinal absorptive defects in mice lacking the NHE3 Na + /H + exchanger. Nat Genet 19: 282-285, 1998.
Shull GE, Miller ML, and Schultheis PJ. Lessons from genetically engineered animal models. VIII. Absorption and secretion of ions in the gastrointestinal tract. Am J Physiol Gastrointest Liver Physiol 278: G185-G190, 2000.
Siffert W and Dusing R. Sodium-proton exchange and primary hypertension. An update. Hypertension 26: 649-655, 1995.
Sun AM, Saltzberg SN, Kikeri D, and Hebert SC. Mechanisms of cell volume regulation by the mouse medullary thick ascending limb of Henle. Kidney Int 38: 1019-1029, 1990.
Wakabayashi S, Shigekawa M, and Pouyssegur J. Molecular physiology of vertebrate Na + /H + exchangers. Physiol Rev 77: 51-74, 1997.
Wang T, Yang CL, Abbiati T, Schultheis PJ, Shull GE, Giebisch G, and Aronson PS. Mechanism of proximal tubule bicarbonate absorption in NHE3 null mice. Am J Physiol Renal Physiol 277: F298-F302, 1999.
Watts BA III, George T, and Good DW. Nerve growth factor inhibits HCO 3 - absorption in renal thick ascending limb through inhibition of basolateral membrane Na + /H + exchange. J Biol Chem 274: 7841-7847, 1999.
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.
Watts BA III and Good DW. Extracellular signal-regulated kinase mediates inhibition of Na + /H + exchange and HCO 3 - absorption by nerve growth factor in MTAL. Am J Physiol Renal Physiol 282: F1056-F1063, 2002.
Winters CJ, Reeves WB, and Andreoli TE. A survey of transport properties of the thick ascending limb. Semin Nephrol 11: 236-247, 1991.
作者单位：1 Departments of Medicine and 2 Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555; and 3 Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267