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Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona 85724-5051
Submitted 6 April 2004 ; accepted in final form 3 June 2004
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ABSTRACT |
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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.
three-dimensional reconstruction; aquaporin; ClC-K1; 
B-crystallin; countercurrent multiplier; concentrating mechanism
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.
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METHODS |
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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).
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RESULTS |
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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. 1B, 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. 1D (white tubules).
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1, R1) 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. 3A lies at the base of the inner medulla, and the section shown in Fig. 3B 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.
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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).
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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 lie >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.
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Ten short AQP1-null DTLs are identified in Fig. 6, A-C; most of these 10 DTLs are associated with group 1 ATLs (Fig. 6A). 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.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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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 (IMCD1 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 IMCD1 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.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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