1 National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; and 2 Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521
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ABSTRACT |
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To examine the role of aquaporin-1
(AQP1) in near-isosmolar fluid reabsorption in the proximal tubule, we
compared osmolalities in micropuncture samples of late proximal tubular
fluid and plasma in wild-type (+/+) and AQP1-knockout (/
)
mice. Compared with matched wild-type mice, the
/
animals
produce a relatively hypotonic urine (607 ± 42 vs. 1,856 ± 101 mosmol/kgH2O) and have a higher plasma osmolality under
micropuncture conditions (346 ± 11 vs. 318 ± 5 mosmol/kgH2O; P < 0.05).
Measurements of tubular fluid osmolality were done in three groups of
mice, +/+,
/
, and hydrated
/
mice in which
plasma osmolality was reduced to 323 ± 1 mosmol/kgH2O. Late proximal tubular fluid osmolalities were 309 ± 5 (+/+, n = 21), 309 ± 4 (
/
, n = 24), and 284 ± 3 mosmol/kgH2O (hydrated
/
, n = 19). Tubular fluid chloride concentration averaged 152 ± 1 (+/+), 154 ± 1 (
/
), and 140 ± 1 mM (hydrated
/
).
Transtubular osmotic gradients in untreated and hydrated AQP1
/
mice were 39 ± 4 (n = 25) and 39 ± 3 mosmol/kgH2O (n = 19), values significantly higher
than in +/+ mice (12 ± 2 mosmol/kgH2O; n = 24;
both P < 0.001). AQP1 deficiency in mice generates marked
luminal hypotonicity in proximal tubules, resulting from the retrieval
of a hypertonic absorbate and indicating that near-isosmolar fluid
absorption requires functional AQP1.
water transport; kidney; micropuncture
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INTRODUCTION |
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A MAJOR FUNCTION OF THE MAMMALIAN proximal tubule is the near-isosmolar reabsorption of water and electrolytes. Aquaporin-1 (AQP1), a water channel abundantly expressed in the apical and basolateral membranes of proximal tubule cells, has been proposed to account for the high water permeability of the proximal tubule epithelium that is required for tight coupling of water and solute fluxes (9, 12). In fact, transepithelial osmotic water permeability in proximal tubules of AQP1-knockout mice is reduced to 20% of that in wild-type mice, indicating that the high water permeability in proximal tubules of wild-type mice largely reflects the presence of AQP1 and that the high transepithelial water flux occurs mainly through a transcellular pathway (8, 14). It was also found that the rate of net fluid reabsorption in proximal tubules of AQP1-knockout mice was reduced to only ~50% of control as determined in both isolated perfused tubules and in vivo micropuncture experiments (14). Without measurements of the transtubular osmotic driving force, it was not possible to reconcile the fivefold reduction in water permeability with the twofold reduction in fluid absorption.
The purpose of the present study was to test the prediction that AQP1 deficiency results in marked luminal hypotonicity generated by active NaCl transport in the proximal tubule. Our results in AQP1-knockout mice confirm the existence of a markedly augmented transtubular osmotic gradient, indicating that near-isosmolar reabsorption depends on AQP1. The increased osmotic gradient provides a driving force for water reabsorption through pathways other than AQP1 channels.
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METHODS |
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Experiments were performed in wild-type and AQP1-deficient mice
generated by targeted gene disruption (8). Measurements were done in
litter-matched, 2- to 3-mo-old mice produced by intercrossing of AQP1
heterozygotes in a CD1 genetic background. Successful experiments were
performed in four wild-type (3 males, 1 female) and eight AQP1-knockout
mice (7 males, 1 female) maintained on standard rodent chow and tap
water. Mice were anesthetized with thiobutabarbital (inactin, 100 mg/kg
ip) and ketamine (100 mg/kg im) as described previously (14). Body
temperature was maintained at 38°C by placing the animals on an
operating table with a servo-controlled heating plate. The trachea was
cannulated, and 100% oxygen was blown toward the tracheal tube
throughout the experiment. The femoral artery was cannulated with
polyethylene tubing (~300-µm outer diameter) for blood pressure
measurement and blood sample withdrawal. The jugular vein was
cannulated for continuous maintenance infusion of 2.25 g/dl BSA in
saline at a rate of 0.35 ml/h (0.8-1.4 ml · h1 · 100 g body wt
1). An initial urine sample
was collected by bladder puncture; subsequently, urine was collected by
using a bladder catheter. The left kidney was approached from a flank
incision, freed of adherent fat and connective tissue, placed in a
lucite cup, and covered with mineral oil.
Free-flow micropuncture was performed as previously described (14). Briefly, end-proximal segments were identified by injecting a bolus of artificial tubular fluid stained with FD&C green from a 3- to 4-µm tip pipette connected to a pressure manometer. After identification of an end-proximal segment, the pipette was withdrawn and the tubule was observed for signs of leakage. Timed fluid collections in four to seven nephrons per experiment were made with oil-filled pipettes over 3-5 min. Immediately after micropuncture, a blood sample was collected from the femoral artery in a heparinized tube. Tubular fluid volumes were determined from column length in a constant-bore capillary. Chloride concentration in tubular fluid was measured by electrometric titration (11). Osmolalities in plasma and late proximal tubular fluid were determined by freezing point depression using a nanoliter osmometer (Clifton Technical Physics, Hartford, NY) with an eight-hole cooling plate. All tubular samples were measured against two plasma samples in opposite positions in the plate and a 300- mosmol/kgH2O standard. Plasma osmolality was also determined from freezing point depression in a 50-µl sample by using a macroosmometer (Roebling).
In three experiments in male AQP1 /
mice, plasma
osmolality was reduced by the intravenous administration of the
V2-receptor agonist
1-deamino-[8-D-arginine]-vasopressin (dDAVP; 1 ng) and infusion of a 0.3% NaCl-0.8% glucose solution at a rate of
1.6 ml/h. After 45 min of infusion, a blood sample was taken and
micropuncture was performed as described above. Another blood sample
was taken at the end of the experiment. For every tubular fluid
collection, the time was noted and the corresponding plasma osmolality
was determined from interpolation of the plasma values, assuming a linear change in plasma osmolality between the two time points.
Statistical significance of differences between AQP1 +/+ and AQP1
/
mice was assessed by unpaired t-test.
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RESULTS |
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Body and kidney weights averaged 39 ± 2 g and 292 ± 37 mg
in wild-type (n = 4) and 33 ± 2 g and 243 ± 16 mg in AQP1
/
mice (n = 5), respectively. Mean arterial blood
pressure was 115 ± 4 mmHg in wild-type and 109 ± 5 mmHg in AQP1
/
mice. As reported previously (8), all AQP1
/
mice had a substantially lower urine osmolality than
AQP1 +/+ mice (mean values: 607 ± 42 vs. 1,856 ± 101 mosmol/kgH2O; P < 0.001). Under micropuncture
conditions, plasma osmolality was significantly higher in AQP1
/
than +/+ mice whether determined by microosmometry (346 ± 11 vs. 316 ± 5 mosmol/kgH2O; P < 0.05) or
by the standard macroscopic method (346 ± 11 vs. 323 ± 3 mosmol/kgH2O; P < 0.05). Plasma Cl concentrations were 121 ± 5 mM in wild-type (n = 4) and 137 ± 6 mM in AQP1
/
mice (n = 3). Plasma osmolalities in six
wild-type and six AQP1
/
mice taken directly from their
cages averaged 330 ± 1.5 and 322 ± 1 mosmol/kgH2O,
respectively, indicating that the elevated plasma osmolality in the
experimental AQP1
/
mice was a result of the surgical intervention.
As reported earlier, free-flow collections in the last surface loop of
the proximal tubule revealed a modest increase in late proximal tubular
flow rate in AQP1 /
mice compared with wild-type animals
(6.9 ± 0.5 vs. 4.8 ± 0.4 nl/min; n = 17 and 21 nephrons, respectively; P < 0.001). Tubular fluid osmolality at the end of the proximal tubule was identical in AQP1
/
and
wild-type mice (309 ± 5.5 vs. 309 ± 4.5 mosmol/kgH2O;
n = 21 and 24, respectively; not significant). However, the
tubular fluid-to-plasma osmolality ratio (TF/Posm ) was
significantly higher in wild-type than in AQP1
/
mice
(0.98 ± 0.02 vs. 0.89 ± 0.01), and the transtubular osmotic
difference was significantly augmented in the AQP1-knockout animals
(
39 ± 4 vs.
11.5 ± 3 mosmol/kgH2O;
n = 21 and 24, respectively; P < 0.001) (Fig.
1). In a subset of nephrons, Cl
concentrations were measured together with osmolality and also found to
be similar between AQP1 +/+ and
/
mice (152 ± 1 vs. 154 ± 1 mM; n = 11; not significant).
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In three additional AQP1 /
mice, infusion of hypotonic
fluid (0.3% NaCl-0.8% glucose at 1.6 ml/h) plus administration of dDAVP (1 ng) established a plasma osmolality of 323 ± 2 mosmol/kgH2O and a Cl concentration of 127 ± 1 mM, values
not different from those found in wild-type animals (323 ± 3 mosmol/kgH2O, and 121 ± 4.5 mM). Under these conditions,
osmolality and chloride concentration in late proximal tubular fluid
averaged 284 ± 3 mosmol/kgH2O (n = 19) and 140 ± 1.1 mM (n = 15), respectively, significantly reduced compared with AQP1 +/+ mice (P < 0.001 for both osmolality
and Cl concentration). The TF/Posm of 0.88 ± 0.01 and
transtubular osmotic difference of
39 ± 3 mosmol/kgH2O in these hydrated mice were similar than those
observed in AQP1
/
mice without hydration (Fig. 1).
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DISCUSSION |
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The main result of the present study is the observation that, in mice
without functional AQP1 water channels, the osmolality of tubular fluid
at the end of the proximal convoluted tubule is markedly reduced
compared with plasma osmolality. The increased luminal hypotonicity in
AQP1-deficient mice provides direct evidence for the notion that water
channels are required for fluid absorption in the proximal tubule to
operate near isotonicity. Using single nephron filtration and proximal
absorption rates of 9.6 and 4.7 nl/min in wild-type and 8.1 and 2.1 nl/min in AQP1 /
mice, respectively, as reported
previously (14), one can calculate by mass balance that mean
absorbate tonicity was ~330 mosmol/kgH2O in wild-type mice and ~450 mosmol/kgH2O in AQP1-knockout
mice. Thus the proximal tubule has the capacity to generate a
remarkably hypertonic absorbate when epithelial water permeability is limited.
A small but significant reduction in luminal tonicity compared with plasma was found in wild-type mice. The issue of the precise level of luminal tonicity in previous studies in rat and rabbit proximal tubules has been somewhat controversial (13). Nevertheless, there is wide agreement that a macroscopic reduction in luminal tonicity provides a major driving force for water absorption and that the transtubular osmotic gradient is small and difficult to establish experimentally because of the high osmotic water permeability of the proximal tubule (1, 4, 15). The degree of luminal hypotonicity in wild-type mice observed in the present study is very similar to that reported by Liu et al. (5) in micropuncture studies in rats in which luminal tonicity at the end of the proximal tubule was reduced 7.5 mosmol/kgH2O below that of plasma. In addition to analytic problems, using the tonicity of systemic plasma as a reference point may further soften the validity of measurements of transtubular gradients. Differences may exist between systemic plasma and the interstitial fluid compartment surrounding proximal tubules, and the luminal tonicity may fall across the glomerular capillaries by Donnan distribution effects (5).
In contrast to the marginal luminal hypotonicity in wild-type mice, we
found a marked augmentation in the transtubular osmotic gradient at the
end of the proximal tubules in AQP1-deficient mice. The magnitude of
this gradient was independent of the level of systemic plasma tonicity.
Transepithelial osmotic gradients of identical magnitude were found in
mice with ambient plasma tonicity and in mice in which plasma tonicity
was acutely reduced to levels found in wild-type mice. The reason for
the marked elevation in plasma tonicity in AQP1-null mice undergoing
micropuncture appears to be related to the surgical intervention
because the osmolality of plasma taken from untreated AQP1
/
mice was within the range of that from normal mice. It
is likely that the combination of a compromised concentrating mechanism
(2, 10) and hypertonic fluid absorption in the proximal tubule renders
AQP1-null mice highly dependent on drinking as the source for free
water addition. It has been shown previously that urine osmolality of
AQP1-knockout mice cannot be increased by vasopressin (8). Thus, even
if these animals have high ambient vasopressin concentrations, free water absorption along collecting ducts may be insufficient to sustain
body fluid isotonicity over a 3- to 4-h experiment. Increased luminal
hypotonicity in AQP1-deficient mice is the first experimental demonstration that near-isotonicity of proximal tubular fluid and of
proximal tubular absorbate is directly dependent on epithelial water
permeability. The serous acinus of the salivary gland is another
fluid-transporting epithelium although the direction of near-isosmotic
fluid transport is secretory rather than absorptive. It has recently
been observed that deletion of AQP5, the water channel present in the
apical membrane of acinar epithelial cells, results in a decrease in
agonist-stimulated saliva secretion and is associated with the
production of a hypertonic saliva (7). Thus the normally near-isotonic
primary saliva in the salivary gland acinus becomes hypertonic in the
AQP5-knockout mice. Like the proximal tubule, the salivary gland acinus
is able to pump Na against a considerable opposing concentration gradient.
The present results provide an explanation for our previous observation that osmotic water permeability in proximal tubules of AQP1-deficient mice was reduced by ~80%, whereas the rate of fluid reabsorption in the proximal tubule of AQP1-knockout mice was reduced by only ~50% (14). It would seem that despite the reduction in osmotic water permeability an increased osmotic driving force permits continued fluid absorption, albeit at a reduced rate. We believe that, in view of the markedly enhanced transtubular osmotic gradient alternative, nonosmotic mechanisms of fluid absorption need not be invoked in the proximal tubule of AQP1-knockout mice (6). The non-AQP1-mediated water absorption presumably utilizes the paracellular pathway and/or permeation of the lipid bilayer. The augmentation of the osmotic driving force that permits residual water flux in the absence of AQP1 provides partial compensation for the proximal absorption defect and therefore contributes to the relatively mild salt- and water-losing phenotype of AQP1 deficiency.
Although Na concentrations were not measured in these experiments one can estimate, assuming that one-half of the osmotic gradient is due to Na, that luminal Na concentration along the proximal tubule fell by ~5 mM in wild-type and by ~20 mM in AQP1- deficient mice. The attainment of a concentration gradient in the absence of transepithelial water flux is a reflection of the strength of the Na pump in relation to Na backflux (16). Previous studies in which coupling of Na and water fluxes was prevented by nonabsorbable sugars have shown that the proximal tubule can generate a limiting transtubular Na gradient of 30-35 mM before net fluxes of Na and water become zero (3). The present results suggest a similar Na concentration-lowering effect of the Na pump, considering that continued water absorption prevents the attainment of a true equilibrium state.
In summary, AQP1 deficiency in mice generates marked luminal hypotonicity in proximal tubules compatible with the retrieval of a hypertonic absorbate and indicating that near-isosmolar fluid absorption requires functional AQP1.
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ACKNOWLEDGEMENTS |
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We thank Dr. Tonghui Ma and Liman Qian for mouse generation, breeding, and genotype analysis.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-35124 and by intramural funds from the NIDDK. Dr. Vallon was a visiting scientist from the Department of Pharmacology, University of Tübingen, Germany, who was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Va 118/4-1).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Schnermann, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bldg. 10, Rm. 4 D51, 10 Center Dr., MSC 1370, Bethesda, MD 20892 (E-mail: jurgens{at}intra.niddk.nih.gov).
Received 20 January 2000; accepted in final form 29 March 2000.
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