Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona 85724-5051
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ABSTRACT |
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Amino acids are apparently recycled between loops of Henle and vasa recta in the rat papilla in vivo. To examine more closely papillary amino acid transport, we measured transepithelial fluxes of L-[14C]alanine and [14C]taurine in thin limbs of Henle's loops isolated from rat papilla and perfused in vitro. In descending thin limbs (DTL) in vitro, unidirectional bath-to-lumen fluxes tended to exceed unidirectional lumen-to-bath fluxes for both radiolabeled amino acids, although the difference was statistically significant only for taurine. In ascending thin limbs (ATL) in vitro, unidirectional lumen-to-bath fluxes tended to exceed unidirectional bath-to-lumen fluxes, although the difference was again statistically significant only for taurine. These results are compatible with apparent directional movements of amino acids in vivo. However, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from the ATL in vivo but not compatible with apparent movement from vasa recta to DTL in vivo. There was no evidence of net active transepithelial transport when concentrations of radiolabeled amino acids were matched on both sides of perfused tubule segments. These data suggest that regulation of amino acid movement in vivo may involve the vasa recta, not the DTL of Henle's loops. The data also suggest that transepithelial movement of amino acids in thin limbs of Henle's loop may occur via a paracellular route.
L-alanine; taurine; descending thin limb; ascending thin limb; isolated tubules
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INTRODUCTION |
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AMINO ACIDS FILTERED at mammalian renal glomeruli are almost completely reabsorbed along the proximal tubules (10). However, small amounts do remain at the end of the accessible portion of the proximal tubule and these appear to be transported by more distal portions of the nephrons. In fact, for a number of acidic, basic, and neutral amino acids (including the sulfur-containing amino acid, taurine), our previous free-flow micropunctures and continuous microinfusions of ascending loops of Henle in the exposed papilla of the rat kidney indicate that reabsorption can occur beyond the tip of the loop of juxtamedullary nephrons, probably from ascending thin limbs (ATL) (5-8, 11). This reabsorption is generally not saturable, inhibitable (with the possible exception of taurine) (11), or stereospecific (5-8). However, it is not a simple nonspecific leak, because it does not occur for mannitol (6). In addition, our microinfusions of ascending vasa recta indicate that amino acids can leave the ascending vasa recta and enter ipsilateral papillary tubular structures, probably descending thin limbs (DTL) of Henle's loops, as intact amino acids without first entering the systemic circulation (6-8). This movement from vasa recta to ipsilateral tubules is inhibitable (6-8). It is also stereospecific for some (e.g., glutamate) but not other (e.g., alanine) amino acids (6-8). These observations, together with our micropuncture findings of high fractional deliveries of endogenous amino acids to the tips of Henle's loops (sometimes >1.0), higher amino acid concentrations in the ascending than in the descending vasa recta at the same level of the papilla, and high mean amino acid concentrations in the vasa recta (5), suggest that amino acids can be recycled among the loops and the vasa recta in the papilla (5-8). For some amino acids, such as taurine, which can apparently serve as an osmolyte in the renal medulla (9), this recycling may be important in maintaining appropriate medullary concentrations for this function.
Despite this body of in vivo data, the exact sites or characteristics of amino acid movements in the thin limbs of Henle's loops, if indeed they occurred in this region, were not known. To examine these questions more directly than was possible in vivo, we undertook the task of isolating and perfusing in vitro DTL and ATL from the renal papilla of rats of the same strain as those used in the in vivo studies. For this initial study, we characterized the thin limbs and examined the movements of the neutral amino acid, L-alanine, and the sulfur-containing amino acid, taurine. The unidirectional fluxes were generally compatible with the in vivo observations, but inhibition data were not.
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METHODS |
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Animals and dissection of tubules.
Male Munich-Wistar rats (80 g average body wt) were treated with
furosemide (1.5 mg/100 g body wt) by peritoneal injection to induce
diuresis and consequently to lower the medullary osmolality (2).
Animals were anesthetized with pentobarbital sodium (0.2 ml/100 g body
wt). Their kidneys were removed and bathed on ice in 280 mM sucrose-10
mM HEPES buffer, brought to pH 7.4 with Tris base and gassed with 100%
O2. The medullary portions were dissected from the kidneys,
and individual bundles were teased out along the length of the inner
medulla under a stereomicroscope. Segments of thin limbs were dissected
without the aid of enzymatic agents using reflected light from below
the dissection dish. We identified dissected segments by cell type
using an inverted microscope with Nomarski differential-interference
contrast optics at ×400 magnification (2). The DTL with their
cells with nuclei protruding into the lumen differ markedly in
appearance from the ATL with their large, round, flat nuclei (Fig.
1). The upper portion of the DTL within the
inner stripe of the outer medulla has a greater outer diameter and a
more tortuous lumen than the inner medullary portion as the cells
increase in size toward the proximal S3 segment of the outer stripe.
Within the inner medulla, the narrowing tubule diameter and the
increasing density of cells per unit tubule length distinguish the
lower (DTLL) from the middle (DTLM) portion of
the DTL (Fig. 1). For purposes of perfusion, we initially characterized
the DTLM as being from the upper half and the
DTLL as being from the lower half of the inner medulla down
to the prebend region of the tubule (Fig. 1). In later experiments, we
stopped separating the DTL into DTLM and DTLL
portions for perfusion. However, most of each segment that we used for
perfusion consisted of the DTLM portion.
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Perfusion and bathing solution. The Ringer solution used for perfusing and bathing the tubules was that initially used by Chou and Knepper (2) in perfusion of thin limbs from chinchilla kidneys. It contained (in mM) 118 NaCl, 25 NaHCO3, 2.5 K2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 glucose, and 5 urea. The osmolality was about 290 mosmol/kgH2O, the pH was adjusted to 7.4, and the solution was continuously gassed with 95% O2-5% CO2 to maintain a stable pH and adequate oxygenation. When a high concentration of unlabeled amino acid was added to the perfusate or bathing medium, an equimolar concentration of sucrose was added to the other solution to keep the osmolalities and ionic strengths on the opposite sides of the epithelium equal.
Perfusion of tubules. We perfused the dissected tubules in vitro by a technique essentially the same as that originally described by Burg et al. (1) and modified for use in our laboratory (3, 4). Briefly, each tubule was transferred to a special temperature-controlled Lucite bathing chamber with a glass bottom. The ends of the tubule were held in glass micropipettes and the tubule was perfused through a micropipette with its tip (~7 µm diameter) centered in the tubule lumen. The perfusion rate was normally 15-20 nl/min. The temperature of the bathing chamber was maintained at 37°C during perfusion. To check for leaks and to be certain that there was no net fluid movement across the perfused epithelium, [3H]polyethylene glycol (PEG) (mol wt ~ 4,000) was added to the perfusate or bathing medium as appropriate. Tubules with a PEG leak >0.15% per minute or apparent net transepithelial fluid movement were discarded. The average rate of PEG leak across the epithelium for the experiments used was 0.09 ± 0.03% per minute.
Measurement of unidirectional transepithelial fluxes of amino
acids.
Because there was no net transepithelial fluid movement in these
perfused tubules, we determined the lumen-to-bath (reabsorptive) flux
of 14C-labeled amino acids (Jlb,
fmol · min1 · mm
tubule length
1) from the disappearance of
radiolabeled amino acid from the lumen using the following relationship
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(1) |
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(2) |
Determination of radioactivity. The activity of the 14C was determined by counting in a liquid scintillation system. The scintillation fluid was Ecolite (ICN Biomedicals).
Chemicals. L-[14C]alanine (sp act 163.85 mCi/mmol), [14C]taurine (sp act 108.5 mCi/mmol), and [3H]PEG (sp act 1.51 mCi/g) were obtained from Du Pont-NEN. All other chemicals were purchased from standard sources and were of the highest purity available.
Statistical analysis. Results are summarized as means ± SE. The n value is the number of tubules. Each tubule came from a different animal. In unpaired experiments, three to five flux measurements were made in each tubule, and the mean value for these measurements was used as the value for that tubule. Unpaired comparisons of fluxes between two groups of tubules were assessed with Student's t-test. For paired analyses of unidirectional fluxes in single tubules, three to five measurements of one unidirectional flux were made, and the mean value was determined followed by three to five measurements of the other unidirectional flux for which a second mean value was determined. The paired means were then compared for a series of tubules. Similarly, in inhibition studies, three to five control fluxes and three to five experimental fluxes were made in a single tubule, and the means were compared. The significance of the difference between paired means was assessed with a two-tailed t-test for paired observations. Differences for both paired and unpaired analyses were considered statistically significant when P < 0.05.
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RESULTS |
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Unidirectional transepithelial L-alanine fluxes.
Initially, we measured either Jbl or
Jlb for
L-[14C]alanine (8 µM) in separate
segments of DTL and ATL of Henle's loop. In this initial series of
experiments, we also divided the DTL into DTLM and
DTLL portions, as indicated in METHODS. The
results are shown in Table 2. In the
DTLM segments, Jbl (~125
fmol · min1 · mm
1)
was significantly greater than Jlb (~45
fmol · min
1 · mm
1).
However, in the DTLL segments, Jbl and
Jlb were essentially the same (~45
fmol · min
1 · mm
1).
In the ATL segments, Jlb (~70
fmol · min
1 · mm
1)
was significantly greater than Jbl (~20
fmol · min
1 · mm
1).
These observations (Jbl greater than
Jlb suggesting secretion in the DTL; and
Jlb greater than Jbl suggesting
reabsorption in the ATL) are in accord with what appeared to be the
dominant direction of the L-alanine fluxes in our in vivo
microinfusion studies (7).
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Unidirectional transepithelial taurine fluxes.
In the case of taurine, we measured both Jbl and
Jlb for [14C]taurine in
single perfused segments of DTL and ATL. The concentration of
[14C]taurine varied from 20 to 40 µM in
different experiments. However, in each individual experiment, the same
solution containing the same concentration of
[14C]taurine was used for measuring both
Jbl and Jlb. As in the case of
the alanine fluxes, the sequence in which Jbl and
Jlb were measured was alternated from tubule to
tubule in these experiments. The results are shown in Table
4. The average values for both unidirectional fluxes for [14C]taurine (Table
4) were significantly larger than those for L-[14C]alanine (Table 2). This was
not simply the result of the higher concentration of taurine than
L-alanine used in these flux measurements, because it held even when the concentrations of
[14C]taurine and
L-[14C]alanine were the same (~20
µM). In the case of the unidirectional [14C]taurine fluxes measured in the same
tubule, Jbl was significantly greater than
Jlb in the DTL and Jlb was
significantly greater than Jbl in the ATL (Table
4). These data suggesting secretion in the DTL and reabsorption in the
ATL are in agreement with what appeared to be the dominant direction of
the taurine fluxes in our in vivo microinfusion studies (7, 11).
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Attempts to inhibit unidirectional fluxes. We examined possible inhibition of the unidirectional fluxes of L-[14C]alanine by adding unlabeled L-alanine to the bathing medium or perfusion solution. The addition of 10 or 50 mM unlabeled L-alanine to bathing medium containing 20 µM L-[14C]alanine actually tended to increase, not decrease, Jbl of L-[14C]alanine in both the DTL and ATL, but the change was not statistically significant (0.10 < P < 0.20). In four experiments, we also examined the effect of adding 50 mM L-phenylalanine to the bath containing 20 µM L-[14C]alanine on Jbl of L-[14C]alanine in the DTL. In proximal tubules, L-phenylalanine inhibits reabsorption of numerous neutral amino acids (10). Moreover, it inhibits the transfer of radiolabeled L-alanine from ascending vasa recta to ipsilateral tubular structures in vivo (7). However, L-phenylalanine also had no significant effect on Jbl of L-[14C]alanine (0.20 < P < 0.30). The addition of 10 mM unlabeled L-alanine to perfusion medium containing 20 µM L-[14C]alanine also had no significant effect on Jlb of L-[14C]alanine in either the DTL (0.50 < P < 0.60) or the ATL (0.30 < P < 0.40).
We also attempted to inhibit the unidirectional fluxes of [14C]taurine by adding unlabeled taurine to the bathing medium or perfusion solution. The addition of 2 mM unlabeled taurine to bathing medium containing 20 µM [14C]taurine had no significant effect on Jbl of [14C]taurine in either the DTL (0.20 < P < 0.30) or the ATL (0.70 < P < 0.80). Similarly the addition of 2 mM unlabeled taurine to perfusion medium containing 20 µM [14C]taurine had no significant effect on Jlb of [14C]taurine in either the DTL (0.10 < P < 0.20) or the ATL (0.40 < P < 0.50). In two experiments, we even examined the effect of 100 mM unlabeled taurine on Jbl and Jlb of [14C]taurine in one DTL and one ATL, but there was no inhibition (data not shown).Direct measurements of net transepithelial fluxes.
In a series of experiments, we attempted to measure net movement of
L-[14C]alanine and
[14C]taurine directly by matching isotope
concentrations in the initial perfusate and bathing medium and
measuring the change in concentration of radiolabeled substrate between
the initial perfusate and the collected fluid. Unfortunately, it proved
extremely difficult, even using the same original solution, to be
absolutely certain that the concentration of radiolabeled substrate in
initial perfusate and bathing medium were identical. Therefore, the
data were limited in reliability by scatter in the apparent fluxes.
Nevertheless, in the case of
L-[14C]alanine, the apparent flux
did not differ from zero in four DTL (4.30 ± 1.82 fmol · min1 · mm
1;
0.10 < P < 0.20) and four ATL (3.52 ± 2.05 fmol · min
1 · mm
1;
0.20 < P < 0.30). Similarly, in the case of
[14C]taurine, the apparent flux did not differ
from zero in six DTL (3.42 ± 20.0 fmol · min
1 · mm
1;
0.80 < P < 0.90) and six ATL (20.8 ± 13.5 fmol · min
1 · mm
1;
0.20 < P < 0.30).
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DISCUSSION |
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In the present study, we were able to isolate papillary thin limbs of
Henle's loops from the kidneys of Munich-Wistar rats without the aid
of enzymatic agents and to perfuse segments of these thin limbs in
vitro. Therefore, we were able to examine amino acid transport in vitro
in tubule segments of the same type and from the same strain of rats as
we had used for our micropuncture and continuous microinfusion studies
in vivo (5-8, 11). In these initial in vitro studies, we chose to
examine the transport of L-alanine, a prevalent neutral
amino acid, and taurine, a neutral -amino (sulfonic) acid that can
exist in very high concentrations in the papillary vasa recta (5) and
that may be important as a cellular osmolyte in the renal medulla (9).
For both radiolabeled amino acids, there was a tendency for the flux into the DTL to dominate and for the flux out of the ATL to dominate. These observations are in agreement with the in vivo data that indicate that radiolabeled amino acid continuously microinfused into papillary ATL is reabsorbed to a significant extent and that radiolabeled amino acid continuously microinfused into papillary ascending vasa recta appears to enter the ipsilateral DTL (although this tubule site of entry was not shown directly in vivo) (6-8, 11). However, when the unidirectional fluxes were examined in the same tubule segments so that a paired analysis could be performed, the domination of Jbl in the DTL and Jlb in the ATL, although appearing to hold for both amino acids, was statistically significant only for taurine. Statistical significance only reached the 10% level for L-alanine. It is possible that the lack of statistical significance reflected variability in the tubule segments dissected (e.g., deep versus superficial) since only superficial segments were microinfused in vivo. Such variability may not have shown up to the same extent for taurine where the unidirectional fluxes and the differences between them were much larger than for L-alanine.
In the present study with perfused thin limbs, we were unable to saturate or inhibit any of the unidirectional fluxes of the radiolabeled form of the amino acids with high concentrations of the unlabeled form of the amino acids. Although we had no reason to expect that active net transepithelial transport of these amino acids could occur in the thin limbs of Henle's loop, especially in view of the failure of inhibition of the unidirectional fluxes, we attempted to examine this possibility directly by looking for net transepithelial transport when the concentrations of radiolabeled amino acid in the bathing medium and initial perfusate were identical. Unfortunately, it is extraordinarily difficult to be certain that these low concentrations of radiolabeled amino acids are exactly matched, and this leads to considerable variability in the results. Nevertheless, even taking the scatter into account, we found no evidence of active net transepithelial transport.
The failure to saturate or inhibit Jlb in the ATL in vitro is in agreement with the in vivo microinfusions of the ATL for alanine and most other amino acids (6, 7). However, reabsorption of radiolabeled taurine during in vivo microinfusions of the ATL is saturable and inhibitable, although this may only reflect the inhibition that clearly occurs in the ascending thick limb of Henle's loop (11).
The lack of saturation or inhibition of Jbl in the DTL does not fit with the less direct observations for amino acids during in vivo microinfusions of the ascending vasa recta (6-8). For almost every radiolabeled amino acid examined during these in vivo studies (including both L-alanine and taurine), movement from the vasa recta into ipsilateral tubular structures could be inhibited (6-8). Although we could be certain of the transfer of intact radiolabeled amino acids from vasa recta to ipsilateral tubular structures and of the inhibition of such transfer in these in vivo studies, we could not be certain of the exact site in the tubules where transfer occurred (6-8). Indeed, it is possible that the inhibition observed in vivo could have involved the exit step from the ascending vasa recta. We formerly thought that saturable, inhibitable carrier-mediated transport was more likely to occur in the epithelia of the thin limbs than in the vasa recta (6-8). However, in view of the present results, the involvement of the vasa recta needs to be reconsidered.
In summary, the unidirectional fluxes for radiolabeled L-alanine and taurine observed in isolated thin limbs of Henle's loop in vitro reveal a pattern similar but certainly not exactly equivalent to that observed in vivo. The general lack of inhibition of amino acid fluxes during the current in vitro studies suggests that the transepithelial movement of these amino acids may occur via a paracellular route. However, in the case of taurine, the paired unidirectional fluxes indicate that this would have to be a rectifying pathway, and it is difficult to imagine such rectification in the absence of active transport. In view of our inability to be certain of the exact site of tubular transfer during the in vivo studies, the present in vitro results could be compatible with the in vivo observations, especially if the vasa recta provide a controlling barrier to transport.
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ACKNOWLEDGEMENTS |
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We thank Diane Abbott for assistance with data analysis.
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FOOTNOTES |
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This study was supported in part by National Institutes of Health Grant DK-16294 and Training Grants HL-07249, NS-07309, and GM-08400, and by Grant ES-06694 to the Southwest Environmental Health Sciences Center.
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: W. H. Dantzler, Department of Physiology, College of Medicine, University of Arizona, Tucson, AZ 85724-5051 (E-mail: dantzler{at}u.arizona.edu).
Received 23 December 1998; accepted in final form 30 March 1999.
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