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【摘要】 Functional reconstruction of inner medullary thin limbs of Henle and collecting ducts (CDs) has enabled us to characterize distinctive three-dimensional vertical and lateral relationships between these segments. We previously reported that inner medullary descending thin limbs (DTLs) that form a bend at a distance greater than 1 mm below the inner medullary base express detectable aquaporin (AQP) 1 only along the initial 40% of the segment before the bend, whereas ClC-K1 is expressed continuously along all ascending thin limbs (ATLs), beginning with the prebend segment. We have now reconstructed individual CDs that are grouped together in single clusters at the base of the inner medulla; CDs belonging to each separate cluster coalesce into a single CD in the deep papilla. DTLs are positioned predominantly at the periphery of each individual CD cluster at all levels of the inner medulla and are absent from within the cluster. In contrast, ATLs are distributed near uniformly among the CDs and DTLs at all levels of the inner medulla. A second population of inner medullary DTLs averages 700 µm in length from base to bend and, as previously reported, expresses no detectable AQP1 and expresses ClC-K1 continuously beginning with the prebend segment. ATLs located within the interior of the CD clusters arise predominantly from these short AQP1-null inner medullary DTLs, suggesting there may be functional interdependence between IMCD1 segments and short-length inner medullary thin limbs exhibiting minimal water permeability along their descending segments. AQP1-expressing DTLs and CDs are apparently separated into two structurally distinct lateral compartments. A similar lateral compartmentation between the ATLs and CDs is not apparent. This architectural arrangement indicates that fluid and solutes may be preferentially transported transversely between multiple inner medullary compartments.
【关键词】 threedimensional reconstruction aquaporin ClCK Bcrystallin countercurrent multiplier concentrating mechanism
THE SIGNIFICANCE OF COMPARTMENTATION to the process of the renal medullary concentrating mechanism is indisputable ( 4, 10, 16 ). This has been most clearly established for separate compartments that exist along the corticopapillary (or vertical) axes of the outer medulla and inner medulla. Multiple membrane transport pathways, some of which have variable, region-specific transport kinetics, are expressed along the axis of nephron segments and vessels as they pass from outer medulla to the tip of the papilla. Distinctive and dynamic interactions take place between resulting axial compartments to develop and sustain a steep corticopapillary osmotic gradient.
Compartmentation arising across the lateral plane throughout the medulla is less well accepted (the lateral plane defined as the plane perpendicular to the plane that runs parallel to the corticopapillary axis). The outer medullary blood vessels, nephrons, and collecting ducts (CDs) have been clearly shown to exhibit distinct separation of structures within the lateral plane ( 6 ). In this region, vascular bundles can be considered as central axes around which CDs, descending thin limbs (DTLs), and thick ascending limbs (TALs) are positioned in distinct, spatially organized patterns ( 7 ). The vascular bundles originate in the outer stripe, and a distinctive arrangement between vessels and nephrons is expressed through the inner stripe. This organization likely plays an essential role in enabling vasculature to efficiently recycle and redistribute solutes and water so as to sustain the axial osmotic gradient of the renal medulla ( 11, 13 ).
The lateral architectural arrangement that is so clearly defined in the inner stripe collapses, however, near the base of the inner medulla. Vascular bundles appear to dissipate as descending vasa recta and ascending vasa recta diverge from each other at deeper levels of the inner medulla. CDs and thin limbs of Henle have been considered to exhibit no distinct spatial organization with respect to each other, particularly to the extent that such organization might play a significant role in the concentrating mechanism. However, there have been indications that a distinct inner medullary tubule and blood vessel architectural organization may exist. CDs form extensive arborizations during development and a distinct, if not well-defined, CD branching pattern exists in the mature kidney ( 11 ). Lemley and Kriz ( 10 ) reported that inner medullary descending vasa recta in general are more distant from CDs than are ascending vasa recta and that descending vasa recta tend to be clustered together, whereas ascending vasa recta are more uniformly distributed. Other studies have reported that DTLs in general tend to be more distant from CDs, whereas ascending thin limbs (ATLs) tend to be positioned more closely to CDs ( 1, 7 ).
A potential impact of medullary three-dimensional lateral organization has been assessed from both functional modeling and structural points of view (1, 2, 17-20, and Layton AT and Layton HE, unpublished observations). These studies have provided insights into features and characteristics of medullary structure and function that might potentially enhance or impede the efficiency of the concentrating mechanism as we know it. Overall, they have provided no convincing evidence that the lateral relationships of the inner medullary components are structurally organized to the extent that they might promote or enable lateral compartmentation within the inner medulla.
We recently described a method for producing three-dimensional functional reconstructions that qualitatively depict the expression of proteins along the axis of inner medullary thin limbs of Henle and CDs ( 12 ). By combining immunocytochemistry and semiautomated image acquisition techniques with graphical, volumetric modeling software, we have compiled multiple, serial tissue sections into three-dimensional surface and volumetric representations of inner medullary thin limbs of Henle's loops and CDs. Antibodies that specifically label the well-defined water channels aquaporin-1 (AQP1) and AQP2 and the inner medullary chloride channel ClC-K1 enabled us to identify DTLs, CDs, and ATLs, respectively. Similarly, a specific antibody against the heat shock-related protein B-crystallin enabled us to identify portions of tubules not labeled by the other antibodies.
In earlier investigations, it became apparent to us that DTLs were organized in a less uniform pattern compared with the pattern of ATL distribution in transverse sections of the inner medulla. These patterns are seen continuously in transverse sections along the axis of the inner medulla from the base to the papilla. Three-dimensional reconstructions show that inner medullary DTLs are in fact arranged in a highly organized fashion relative to CDs, and this arrangement is distinct from that of the ATLs. The lateral, spatially distinct architecture of water-permeable or water-impermeable DTL segments, and chloride-permeable ATL segments, and their relationships to the CDs and vasculature may facilitate formation of previously unrecognized inner medullary compartments that could have important implications for the process of the concentrating mechanism.
METHODS
Animals. Young male Munich-Wistar rats (average wt 90 g) were purchased from Harlan (Indianapolis, IN). The animals were anesthetized with pentobarbital sodium (0.2 ml/100 g body wt).
Tissue preparation. Seven kidneys from seven male Munich-Wistar rats were prepared for immunocytochemistry by retrograde perfusion through the aorta with PBS (pH 7.4) for 5 min followed by periodate-lysine-paraformaldehyde (0.01 M, 0.075 M, 2%) in PBS (pH 7.4) for 5 min before removal from the animal. The whole medulla was dissected free, then immersed in fixative for 3 h at 4°C, washed in PBS, dehydrated through an ethanol series, and embedded in Spurr's epoxy resin (Ted Pella). Serial 1-µm transverse sections were cut exhaustively from each medulla beginning near the base of the inner medulla and continuing in a papillary direction. The boundary between the inner medulla and outer medulla was identified on the basis of structural criteria ( 5 ). Every fifth section was placed onto a glass microscope slide for immunocytochemistry (4 sections/slide).
Immunocytochemistry. Indirect immunocytochemistry was conducted as previously described ( 12 ) using affinity-purified polyclonal antibodies against the COOH-terminal regions of the human water channel AQP1 (diluted 1:200, raised in chickens, provided by John Regan and W. Daniel Stamer, University of Arizona), the rat kidney-specific chloride channel (ClC-K, diluted 1:200, raised in rabbit; Chemicon), the human water channel AQP2 (diluted 1:200, raised in goat; Santa Cruz), and a bovine monoclonal antibody raised against purified B-crystallin (diluted 1:50, raised in mouse; Stressgen). AQP1 and AQP2 antibodies serve as markers for DTLs and CDs, respectively, the ClC-K antibody serves as a marker for ATLs, and B-crystallin serves as a common marker for all tubules. All sections from each inner medulla were labeled either with antibodies raised against AQP1 and ClC-K or with antibodies raised against AQP1, AQP2, ClC-K1, and B-crystallin.
Image analysis. Separate stacks of digitized, serial images were generated by capturing AQP1, AQP2, ClC-K1, or B-crystallin immunofluorescence from each tissue section. Final images measured 900 x 1,600 pixels (608 x 1,081 µm within the central region of the inner medulla) and encompassed a vertical depth of up to 3.3 mm beginning at the base of the inner medulla. Continuous surface and volume representations for each tubule were constructed as described previously ( 12 ) with Amira 2.3 visualization and volume modeling software (Indeed-Visual Concepts; Berlin-Dahlem). In the reconstructed three-dimensional surface representations, the tubule positions and lengths are drawn to scale in the x -, y -, and z -axes. Diameters of reconstructed tubules are approximately those that exist at the base of the inner medulla in vivo.
Pattern differentiation of DTL and ATL distributions in two-dimensional images was determined by comparing the mean and variance of distances between nearest neighbors using the method of Schwarz and Exner ( 15 ). The mean and variance of nearest neighbor distances for all DTLs and for all ATLs in individual images were determined using PhotoShop (Adobe) and the Image Processing Toolkit (Reindeer Graphics). The expected mean and variance of nearest-neighbor distances were calculated from the observed feature density (no. of features/unit area), assuming a random distribution as represented by a Poisson distribution ( 15 ). The ratio of the observed mean nearest-neighbor distance to the expected distance ( Q ) and the ratio of the corresponding observed mean variance to the expected variance (R) will each be equal to nearly one for features that are randomly distributed ( 15 ). By contrast, for a uniform pattern of distribution Q 1 and R << 1 and for a clustered pattern Q < 1 and R < 1 ( 15 ).
RESULTS
Patterns of CD, DTL, and ATL lateral distribution near the base of the inner medulla. A representative immunohistochemical transverse section from the rat renal inner medulla showing the CDs, DTLs, ATLs, or all tubules collectively, is shown in Fig. 1. This section is from the inner core region of the inner medulla and is positioned 40 µm below the base of the inner medulla. Individual CDs ( Fig. 1, shown in blue in A-D ) tend to be grouped or bundled together, and there are interbundle spaces devoid of CDs. DTLs and ATLs ( Fig. 1 ), vasculature (not shown), and interstitum occupy the interbundle spaces. The AQP1-expressing DTLs ( Fig. 1 B, white tubules) encircle CD clusters as asymmetric rings in a reticulated pattern or network. The ATLs ( Fig. 1C, white tubules) are more uniformly spaced with an apparently more uniform density. For the remainder of this paper, we define "uniform" patterns of ATL distribution to also imply a relatively uniform density. All tubules are shown collectively in Fig. 1 D (white tubules).
Fig. 1. Representative example of the collecting ducts (CDs; A ), descending thin limbs (DTLs; B ), ascending thin limbs (ATLs; C ), and all tubules collectively ( D ) from a single transverse section (900 x 1,600 pixels) of the rat renal inner medulla. CDs are shown in blue in all panels; other segments are shown in white. This section is from the inner core region of the inner medulla and is from a depth of 40 µm below the base of the inner medulla. ATLs are arranged in a relatively uniform pattern. DTLs are arranged in a reticulated pattern. CDs tend to be grouped together as separate bundles, and there are interbundle spaces devoid of CDs. Scale bars, 100 µm.
We have compared distribution patterns for AQP1-expressing DTLs and ATLs in transverse sections from the inner medullas of kidneys from more than 15 rats, and they all exhibit reticulated patterns or uniform patterns, respectively, as is apparent in Fig. 1. The distinct two-dimensional patterns of AQP1-expressing DTL and ATL distribution extend across the entire inner medulla, as shown in Fig. 2. One of the most apparent differences in distribution is the lower number density of AQP1-expressing DTLs compared with ATLs. Although the actual number of DTLs and ATLs is equal, not all DTLs express AQP1, whereas all ATLs express ClC-K1, thereby leading to this disparity. The density of AQP1-expressing DTLs and ATLs was determined for kidneys from seven of these animals by counting all tubules expressing AQP1 or ClC-K1 in a single transverse section from each kidney. For these seven kidneys, the density of AQP1-expressing DTLs was 639.2 ± 64.5 tubules/mm 2 and density of ATLs was 1,433.2 ± 157.3 tubules/mm 2 (means ± SE; significantly different at P < 0.05 Student's paired t -test).
Fig. 2. Representative example of the DTLs ( A ) and ATLs ( B ) from a single nearly complete transverse section of the rat renal inner medulla. The inner medulla is enclosed within the white lines in each panel. Portions of the outer medulla lie outside of the white lines. This section lies near the base of the inner medulla. Scale bar, 500 µm.
The consistency of a reticulated DTL distribution pattern and a relatively uniform ATL pattern among different kidneys was assessed by comparing the mean and variation of distances between nearest neighbors in transverse sections (see METHODS ). For the seven images from the seven kidneys, the area (means ± SE) was 1.01 ± 0.15 mm 2 and mean feature counts (means ± SE) were 616 ± 75 (DTL) and 1,370 ± 160 (ATL). DTL values for Q and R were 1.14 ± 0.02 and 0.60 ± 0.04 (means ± SE, n = 7). ATL values for Q and R were 1.42 ± 0.01 and 0.39 ± 0.02 (means ± SE, n = 7). All values are significantly different from each other (ANOVA with Tukey's post hoc test; P < 0.05). The DTL pattern is significantly less uniform than the ATL pattern, since Q DTL is significantly less than Q ATL and since R DTL is significantly greater than R ATL. Neither DTL nor ATL arrangements exhibit a random ( Q 1, R 1) or a clustered ( Q < 1, R < 1) pattern.
Three-dimensional lateral relationships of inner medullary CDs and thin limbs. Figure 3 shows the relationships between three CD clusters and DTLs in the central core region of the inner medulla. The section shown in Fig. 3 A lies at the base of the inner medulla, and the section shown in Fig. 3 B lies 150 µm below this section in a papillary direction. Each of the three CD clusters coalesced into a single CD at nearly 3 mm below the inner medullary base along the vertical axis [2.98 (red), 2.91(green), and 3.15 (blue) mm]. In the rat, all of the CDs coalesce into several ducts of Bellini at the tip of the papilla, so at some point deeper in the inner medulla (beyond our reconstruction) these three CDs will join other CDs.
Fig. 3. Spatial relationships between DTLs (white) and 3 separate CD clusters in the central core region of the inner medulla. The CDs associated with each cluster are the same color. CDs not associated with these 3 clusters are shown in pale blue. The section shown in A lies at the base of the inner medulla; the section shown in B lies 150 µm below the section shown in A in a papillary direction. Scale bars, 100 µm.
It is apparent in transverse sections, such as Fig. 3 A-B, that numerous CDs are enclosed by each asymmetric DTL ring. Furthermore, each linear array of DTLs is associated with two or more "ring" configurations. A comparison of the general lateral relationships between DTLs and CDs in the two images indicates a remarkable tendency for these segments to retain their relative lateral positions along the vertical axis. These observations led us to further examine the three-dimensional structural relationships between thin limbs and CDs. For this purpose, the three separate inner medullary CD clusters shown in Fig. 3 and their neighboring DTLs and ATLs were reconstructed in their entirety. The relationships of the reconstructed DTLs or ATLs to the CDs for one of these clusters (the red CD cluster shown in Fig. 3 ), at the base of the inner medulla, are shown in Figs. 4 A and 5 A. Figures 4 A and 5 A each depict a region that encompasses 150 µm, beginning at the inner medullary base and continuing vertically in a papillary direction. The DTLs are spatially separate from the CDs in the horizontal plane, and this spatial separation continues along the entire axial length of the CDs ( Fig. 4, A-F ). In contrast, the ATLs are distributed relatively uniformly within and around the CDs, and this pattern also continues from the base of the inner medulla to the papilla ( Fig. 5, A-F ).
Fig. 4. Three-dimensional reconstruction showing spatial relationships of DTLs (red tubules) to CDs (blue tubules) for the red CD cluster shown in Fig. 3. DTL segments that do not express aquaporin-1 (AQP1) were identified by their expression of B-crystallin and are shown in gray. DTLs are spatially separate from the CDs, and A-E show that this relationship continues along the entire axial length of the CD cluster. Axial lengths of A-E are shown in F with lowercase letters. Tubules are oriented in a corticopapillary direction, with the upper edge of the image near the base of the inner medulla. Scale bar, 500 µm.
Fig. 5. Spatial relationships between ATLs (green tubules; including prebend segments) and CDs (blue tubules) for the red CD cluster shown in Fig. 3. ATLs are distributed relatively uniformly within and around the CDs, and A-E show that this relationship continues along the entire axial length of the CD cluster. Axial lengths of A-E are shown in F with lowercase letters. Tubules are oriented in a corticopapillary direction, with the upper edge of the image near the base of the inner medulla. Scale bar, 500 µm.
For two of the three CD clusters that we reconstructed (red and blue CDs in Fig. 3 ), DTLs reside completely outside the CD clusters at the base of the inner medulla so that these CD clusters each constitute a single compartment relative to the DTLs. A linear array of DTLs does not pass across the interior of the CD clusters along their entire axial length. Several DTLs that pass partially in the blue cluster near the inner medullary base ( Fig. 3 ) have turned away from the interior of this cluster by 165 µm below the base. Therefore, the compartment occupied by the CDs remains intact and separate from the compartment occupied by DTLs along the entire axial length. The third CD cluster that we reconstructed was split by several DTLs into two CD compartments near the inner medullary base (green CDs in Fig. 3 ). These DTLs later separated from the CD, and the two CD clusters merged to form a single distinct compartment at 600 µm below the base of the inner medulla (not shown).
ATLs that lie within the CD clusters arise from short AQP1-null inner medullary long loops. In most thin limbs that we have reconstructed and that form loops below 1 mm from the inner medullary base, AQP1 is expressed continuously along the initial 40% of the inner medullary DTL segment ( 12 ). Beyond this point, AQP1 expression becomes intermittent for variable short lengths and is absent from then on (shown as gray tubules in Fig. 4 ). These AQP1-expressing DTLs form the ring-like arrangement described above and shown in Figs. 1 and 3. In the ascending segments of these loops, ClC-K1 is expressed continuously along a length of 150-200 µm before the bend (the prebend segment) and then continuously through the entire ascent to the base of the inner medulla (for this pattern in ATLs, see Fig. 5 ). As noted, these ATLs are distributed near uniformly among DTLs and CDs; however, underneath this uniform lateral arrangement lies a distinct, nonuniform pattern of distribution based on loop length. The ATLs that lie within the interior of CD clusters arise from short, AQP1-null inner medullary long loops. This population of short DTLs forms a loop within the outermost 1 mm of the inner medulla (12; average length is 699.5 ± 62.3 µm, mean ± SE; n = 58 tubules), and these tubules express no detectable AQP1.
The relationship between these short AQP1-null thin limbs and their proximity to CDs was determined as follows. ATLs that are associated with the red CD cluster shown in Fig. 3 were divided into each of three groups according to their positions relative to CDs. For this comparison, these ATL positions were determined at the base of the inner medulla. Group 1 includes those ATLs that are interposed between two CDs, which lie adjacent to the ATL on two opposite sides; group 2 includes ATLs that lie adjacent to just one CD; and group 3 includes ATLs that 0.5 tubule diameter from a CD. By these definitions, ATLs in group 1 lie within the CD cluster and ATLs in groups 2 and 3 lie at the periphery or outside of the CD clusters. The relative positions of these ATLs and CDs at the base of the inner medulla are shown in Fig. 6, A-C. The reconstructed tubules positioned along the initial 1,500 µm of the inner medullary vertical axis, beginning at the base and continuing in a papillary direction, are shown in Fig. 6, A'-C''. The mean lengths for ATLs (from base to bend) in each group are shown in Fig. 7. These data show that the ATLs that lie within the interior of the CD cluster ( group 1 ) at the base of the inner medulla arise from the shortest long loops, whereas the ATLs that lie at the periphery or outside of the CD cluster ( groups 2 and 3 ) tend to be from the longest loops.
Fig. 6. Spatial relationships between DTLs (red tubules), ATLs (green tubules), and CDs (dark blue tubules) associated with the red CD cluster shown in Fig. 3. ATLs were categorized into 3 groups related to their lateral proximity to CDs, as described in RESULTS. Members of each group are shown in a transverse section located at the base of the inner medulla. A : group 1; B, group 2; C, group 3. In A-C, open red figures represent AQP1-null DTLs, solid red figures represent AQP1-expressing DTLs, white outlined figures represent ATLs not associated with the CD cluster, and light blue figures represent CDs not associated with the CD cluster. Two prebend segments from group 1 are included in A. One ATL from each of groups 2 and 3 ( B and C ) extends below the region of reconstruction, and their DTLs were therefore not reconstructed. A ', B ', and C ' show DTLs and CDs; A '', B '', and C '' show ATLs and CDs. Gray tubules in A ', B ', and C ' represent AQP1-null DTLs. Scale bars, 100 µm.
Fig. 7. Relationships between proximity of ATLs to CDs and thin limb lengths. ATLs were categorized in 3 groups defined by their lateral relationships with CDs, as described in RESULTS. Lengths of loops in each group (measured from inner medullary base to loop bend; means ± SE) are shown for ATLs of red CD cluster shown in Fig. 3 (striped bars) and lengths for all ATLs associated with the 3 CD clusters shown in Fig. 3 (open bars). Significantly different means for each category of the 2 data sets are indicated by a common symbol (ANOVA with Tukey's post hoc test; P < 0.05).
A similar comparison was made for the entire ensemble of ATLs that are associated with the three CD clusters shown in Fig. 3. For these three clusters, the ATLs that lie within the clusters ( group 1 ) at the base of the inner medulla arise from the shortest loops, whereas the ATLs that lie at the periphery or outside of the CD cluster ( groups 2 and 3 ) tend to be from the longest loops ( Fig. 7 ). In fact, no loops extending beyond 1.8 mm were located within group 1. In two additional kidneys, the lengths of ATLs that lie within two CD clusters from each kidney ( group 1 ATLs) were measured. Because these CDs have not been completely reconstructed, the clusters were identified on the basis of CD proximity to one another and by the arrangement of DTLs that encircled them. The mean lengths of the group 1 ATLs in two CD clusters from each of these two kidneys were (mean ± SE) 585.3 ± 110.4 ( n = 15) and 803.6 ± 195.8 ( n = 11); 95% confidence intervals were 356.6-814.0 and 387.7-1,219.6, respectively, for each kidney.
Ten short AQP1-null DTLs are identified in Fig. 6, A-C; most of these 10 DTLs are associated with group 1 ATLs ( Fig. 6 A ). The proximity of short-length inner medullary DTLs to the interior of the CD cluster is not so tightly correlated as with ATLs, although DTLs giving rise to group 1 ATLs appear to be positioned more closely to CD cluster interiors than do DTLs giving rise to group 3 ATLs.
DISCUSSION
CDs that form an ensemble at the base of the inner medulla coalesce as they descend the inner medulla along the corticopapillary axis and form a single CD deep in the papilla. Each ensemble of CDs exhibits a clear lateral separation from surrounding DTLs along the corticopapillary axis. Both AQP1-expressing and AQP1-null segments of each DTL lie at the periphery of the CD clusters. In contrast, ATLs do not exhibit such a lateral separation from CD clusters. The lateral ATL distribution appears more uniform than that of DTLs, and there is a clear underlying three-dimensional organization whereby ATLs arising from short-length, AQP1-null loops are positioned within CD clusters, and ATLs arising from longer-length loops are positioned at the periphery and outside of CD clusters. These general patterns of CD, DTL, and ATL distribution appear to be recapitulated throughout most of the central region of the inner medulla (a region that includes nearly all the papilla; Fig. 2 ).
In the outer stripe of the outer medulla, a clear separation of thin limbs from blood vessels and CDs has been reported ( 7, 10 ). Below the junction of the inner and outer stripe, the short-loop DTLs (derived from superficial and midcortical glomeruli) descend within the periphery of vascular bundles, whereas the short-loop TALs lie distant from the bundles nearer to CDs. The long-loop DTLs (derived from deep midcortical and juxtamedullary glomeruli) descend distant from the vascular bundles, whereas the long-loop TALs lie nearer to, although not within, the bundles. The DTLs and TALs of both short and long loops therefore occupy different lateral compartments within the inner stripe, and this compartmentation led Kriz et al. ( 7 ) to suggest that different functional roles exist for short and long loops.
The vascular bundles disperse after they descend below the inner-outer medullary junction. Beyond this point, DTLs are thought to lie closer than ATLs to vascular bundles, and ATLs are thought to lie closer than DTLs to the CDs ( 7 ). However, more precisely defined lateral relationships for thin limbs, CDs, and blood vessels in the inner medulla have not been reported. Our studies show that, whereas the vascular bundle of the inner stripe of the outer medulla can be considered a central axis around which the tubules are regularly arranged ( 10 ), the CDs of the inner medulla can be considered a central axis around which DTLs are nonuniformly arranged and among which ATLs are near-uniformly interspersed.
Lateral bulk flow of solutes and/or water in the inner medulla is likely retarded by the interstitium. Interstitial cells are arranged in a manner that suggests a possible barrier function, and these cells lie within a matrix of viscoelastic hyaluronan gel ( 3 ). Laterally oriented capillaries are absent. These features alone are suggestive of lateral compartmentation. The lateral separation between DTLs and CDs described in this report, as well as the absence of lateral separation between ATLs and CDs, arguably could generate lateral fluid and/or solute compartmentation that is further augmented by interstitial composition.
In the passive mechanism of countercurrent multiplication, ATLs are considered to be conduits that deliver NaCl to the inner medullary interstitium ( 14 ). This process is driven by high ATL tubular fluid NaCl concentration relative to urea and putative high NaCl permeability relative to urea for ATLs. The tubular fluid NaCl concentration exceeds interstitial NaCl, which leads to NaCl diffusion into the interstitum. Assuming NaCl efflux from ATL exceeds urea influx, ATLs will deliver dilute fluid to the outer medulla. The passive mechanism of countercurrent multiplication proposes that these processes, along with CD solute and fluid reabsorption and countercurrent exchange by vasa recta, participate in generating and sustaining the steep osmotic inner medullary gradient ( 13, 14 ). An architectural arrangement that places CDs and ATLs in a contiguous assemblage that lacks DTLs might have material influence on the diffusive forces that drive solute (chiefly NaCl and/or urea) and fluid reabsorption from these segments; this influence conceivably continues at least roughly through the outer third of the inner medulla; this distance corresponds to the vertical depth occupied by short-length intracluster ATLs. CD segments of the outer third of the inner medulla (IMCD 1 segments) exhibit low urea permeability and other functional characteristics that differ from deeper CD segments ( 14 ). ATLs that arise from short long-looped, water-impermeable DTLs and that are intimately associated with IMCD 1 segments, i.e., the intracluster ATLs, may exhibit functional interdependencies with these CD segments in a manner that is related to their distinctive solute and fluid permeabilities.
GRANTS
This study was supported in part by National Institutes of Health Research Grant DK-16294; Grant ES-06694 from the Southwest Environmental Health Sciences Center; Training Grants HL-07249 and GM-08400; and National Science Foundation Grant IBN9814448.
ACKNOWLEDGMENTS
We are grateful to Diane Abbott for technical assistance. We thank Drs. John Regan and Dan Stamer of the University of Arizona for providing AQP1 antibodies and Marvin Landis of the University of Arizona Center for Computing and Information Technology for assistance with image analyses. We also thank Anita and Harold Layton of Duke University for offering insightful discussions during the course of these studies.
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作者单位：Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona 85724-5051