Am J Physiol Renal Physiol 293: F696-F704, 2007.
First published July 3, 2007; doi:10.1152/ajprenal.00231.2007
0363-6127/07 $8.00
Three-dimensional architecture of collecting ducts, loops of Henle, and blood vessels in the renal papilla
Thomas L. Pannabecker and
William H. Dantzler
University of Arizona Health Sciences Center, Department of Physiology, Tucson, Arizona
Submitted 17 May 2007
; accepted in final form 28 June 2007
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ABSTRACT
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Three-dimensional architecture of vasculature and nephrons in rat renal papilla was assessed by digital reconstruction. Descending vasa recta (DVR), ascending vasa recta (AVR), descending thin limbs (DTLs), ascending thin limbs (ATLs), and collecting ducts (CDs) were identified with antibodies against segment-specific proteins. DTLs are distributed nonuniformly in transverse sections of papilla, but lateral compartmentation between DTLs and CD clusters that occurs in outer IM makes no contribution to concentrating mechanism in papilla. ATLs are distributed nearly uniformly throughout IM. Vasa recta within 
2 mm of the papilla tip are primarily fenestrated vessels; therefore, AVR and DVR can only be determined by blood flow direction. CDs within 500 µm of the papilla tip have nearly 100% greater circumference than CDs within first 1–2 mm below the IM base. Return of water to general circulation from deep papillary CDs appears to be facilitated by a 150% increase in the number of AVR closely abutting these CDs. Consequently, average fractional CD surface area abutting AVR is 0.61, about the same as that (0.54) for smaller CDs that lie near the IM base. Interstitial nodal compartments, bounded by CDs, ATLs, and AVR, surround CDs along the axis of the IM. Fewer ATLs exist in the final 1 mm, as there are fewer loops and the number of these nodal arrangements is therefore reduced. However, tips of many of those loops reaching this area have bends with 50–100% greater transverse lengths than bends of loops near the IM base. This may be significant for solute movement out of loop bends.
three-dimensional reconstruction; aquaporin; ClC-K; 
B crystallin; countercurrent multiplier; concentrating mechanism
COLLECTING DUCTS AND DESCENDING thin limbs of Henle vary in their water or solute transport properties along the corticopapillary axis (2, 3, 19, 20). To better understand how variations in tubule function and architecture generate and sustain an inner medullary osmotic gradient (9, 13, 23), we have prepared three-dimensional functional reconstructions of tubules and vessels from the renal papilla.
We previously reported that coalescing inner medullary (IM) collecting duct (CD) clusters form organizing motifs around which loops of Henle and vasa recta are arranged in regular arrays along the corticopapillary axis from the base to the papilla tip (17, 18). Descending thin limbs (DTLs) that form a bend at a distance greater than 1 mm below the inner medullary (IM) base express detectable aquaporin-1 (AQP1) only along the initial 40% of the segment before the bend, whereas the rat kidney-specific chloride channel (ClC-K1) is expressed continuously along all ascending thin limbs (ATLs) beginning with the prebend segment (16). DTLs are positioned predominantly at the periphery of each individual CD cluster at all levels of the IM and are absent from within the cluster (17). In contrast, ATLs are distributed nearly uniformly among the CDs and DTLs at all levels of the IM. A second population of IM DTLs averages 700 µm in length from base to bend and, as previously reported, expresses no detectable AQP1 but nonetheless 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 IM DTLs, suggesting there may be functional interactions between IMCD1 segments and short-length IM loops that exhibit minimal osmotic water permeability along their descending segments. DTLs and CDs are therefore 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 IM compartments.
DVR, like DTLs, are positioned predominantly at the periphery of each individual CD cluster at all levels of the IM and are absent from within the cluster (18). AVR, like ATLs, are distributed nearly uniformly among the CDs and DTLs at all levels of the IM. DVR and AVR outside CD clusters appear to be sufficiently juxtaposed to permit good countercurrent exchange. Within CD clusters, about four AVR closely abut each CD, surrounding it in a highly symmetrical fashion. These AVR seem ideally placed to effectively move excess water reabsorbed from the CDs out of the IM. Therefore, there appears to be a separation of function between those AVR outside the CD clusters and those inside the clusters. In addition, AVR abutting each CD and ATLs within CD clusters form repeating nodal interstitial spaces, or microdomains, bordered by a CD on one side, one or more ATLs on the opposite side, and one AVR on each of the other two sides. These relationships may be highly significant for both establishing and maintaining the IM osmotic gradient.
We have used a method of three-dimensional tissue reconstruction to investigate architecture of the terminal two millimeters of the rat IM (papilla). This architecture is contrasted with that of the outer IM, which we define as the first 3–3.5 mm of the IM that lie below the border of the outer medulla (OM) and IM. The results indicate that in this papillary region, ATLs and AVR are arranged in a relatively uniform fashion as occurs in the outer IM. However, the pattern of DTLs ringing CD clusters that is so apparent in the outer IM disappears in the papilla as clusters reach convergence. Nearly all vessels are fenestrated, and urea transporter UT-B expression is absent. Interstitial nodal spaces are reduced in number and appear more dilated compared with the outer IM, where loops of Henle are more abundant and closely packed. Approximately 30% of papillary ATLs exhibit wide transverse extensions that closely appose CDs in the deep papilla (within 500 µm from the papilla tip). This unique architecture may be significant for renal papillary function.
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METHODS
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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). All experiments were conducted in accordance with The Guide for the Care and Use of Laboratory Animals (Washington, DC: National Academy Press, 1996) and approved by the Institutional Animal Care and Use Committee.
Tissue preparation for immunohistochemistry.
Kidneys 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, the OM was discarded, and the IM was immersed in fixative for 3 h at 4°C and washed in PBS. Tissue was then prepared for either cryosectioning or epoxy sectioning for immunohistochemistry. For cryosectioning, tissue was immersed in 30% sucrose in PBS for 2 h and then frozen in plastic forms containing Tissue-Tek Optimal Cutting Temperature Compound (Sakura-Finetek, Torrance, CA) that were placed on the surface of an isopentane bath cooled with liquid nitrogen. For epoxy sectioning, tissue was dehydrated through an ethanol series and embedded in Spurr's epoxy resin (Pella). Serial transverse sections were cut at a thickness of 5 (cryosections) or 1 µm (epoxy) either exhaustively or partially from medullas beginning near the base of the IM and continuing in a papillary direction, or from medullas beginning near the papillary tip and continuing in a cortical direction. To reconstruct uninterrupted segments of either nephrons or vessels from the complete kidney, two sets of 1-µm serial sections were prepared from epoxy-embedded tissue. The initial 1-µm sections for each set were offset from each other by 2 µm, and each set had 5-µm steps between sections. Consecutive sections were placed onto glass microscope slides for immunocytochemistry (4 sections/slide). The boundary between the IM and OM was identified on the basis of structural criteria (10).
Immunocytochemistry.
Generally, two sets of serial sections, as described above, were prepared for each kidney, one set labeled for nephrons and CDs, and one set labeled for vasa recta and CDs. Nephron segments were labeled by indirect immunocytochemistry as described previously (16–18) 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, or mice, Serotec); rat ClC-K (diluted 1:200, raised in rabbits, Chemicon); the human water channel AQP2 (diluted 1:200, raised in goats, Santa Cruz); and a bovine monoclonal antibody raised against purified B-crystallin (diluted 1:50, raised in mice, Stressgen). AQP1 and AQP2 antibodies serve as markers for DTLs and collecting ducts, respectively, the ClC-K antibody serves as a marker for ATLs, and B-crystallin serves as a common marker for all tubules (16).
The second set of sections was labeled for vasa recta and CDs. AVR and capillaries were labeled with a polyclonal antibody raised in chickens against rat PV-1, a plasmalemmal vesicle protein formerly known as gp68 (22) (diluted 1:500, provided by Dr. Radu Stan, Dartmouth College). PV-1 is a component of the fenestral diaphragm, whose physiological function is presently poorly understood. In the rat IM, only AVR and capillaries are fenestrated and these all are believed to have diaphragms. DVR were labeled with a polyclonal antibody raised in rabbits against rat UT-B (diluted 1:200, provided by Dr. Jeff Sands, Emory University). Collecting ducts were labeled for AQP2 as described above.
Image analysis.
Separate stacks of digitized, serial images were generated by capturing UT-B, PV-1, AQP1, AQP2, ClC-K1, or B-crystallin immunofluorescence from each tissue section. Continuous surface and volume representations for each vessel and tubule were constructed as described previously (16–18) with Amira visualization and volume modeling software (Mercury, Chelmsford, MA). Continuous three-dimensional surface views of each vessel or tubule were created from serial sections no greater than 5 µm apart. In the reconstructed three-dimensional surface representations, the vessel and tubule positions and lengths are drawn to scale in the x-, y-, and z-axes. Diameters of reconstructed vessels and tubules are, unless otherwise stated in the figure legend, approximately those which exist near the base of the IM in vivo. Three-dimensional reconstructions and two-dimensional images are typical images derived from a minimum of three kidneys. A random number generator (Microsoft Excel) was used to select tubules for dimensional analyses. Where appropriate, means ± SE with number of replicates are given in RESULTS.
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RESULTS
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Axial distribution of CDs and loops of Henle in the papilla.
Clusters of CDs arise in the IM through the process of branching morphogenesis, beginning in early embryogenesis. During this process, a single CD exists initially, arising from the ureteric bud, and subsequently branches iteratively in an organized fashion, resulting in the mature CD system of nearly 7,000 CDs in the rat renal IM (4, 15, 21). The first several generations of the ureteric bud undergo transformation to form the renal pelvis, ureter, and ducts of Bellini at the papilla tip (1).
For an analysis of renal physiological processes such as the concentrating mechanism, we consider the mature collecting system as a group of coalescing CDs that carry fluid and solutes along the corticopapillary axis toward their termination at the ducts of Bellini. Single clusters that consist of as many as 6–12 individual CDs at the base of the IM (OM-IM border) descend parallel to the IM axis and coalesce into one single CD at variable depths below the base (17). Each of these single CDs combines with an adjacent CD and continues descending deeper, thereby forming a group of CD clusters that eventually coalesce into a single CD within an 2-mm region above the papilla tip. These may or may not join other CDs yet again, and eventually merge with the papillary epithelium to form the ducts of Bellini.
For the IM, the expression of AQP1 in DTLs and expression of UT-B in DVR occur at the base and continue through the initial 3 mm of the IM (Fig. 1). Little or no expression of these two transporters is observed within the terminal 2 mm of the papilla. In contrast, expression of AQP2 in CDs is continuous from base to papilla tip. One defining architectural feature of the primary CD clusters is the existence of AQP1-positive DTLs and UT-B-positive DVR positioned circumferentially along the outer edge of their perimeters at the level of the IM base (17, 18). Figure 2 shows this arrangement of DTLs in a transverse section near the base of the IM. Individual CD clusters are positioned (although not shown in the figure) within the areas encircled by DTLs. In contrast, ATLs are distributed nearly uniformly throughout the IM (as seen in transverse sections) and are positioned both within and around the periphery of CD clusters (Fig. 2). This concept of CD clusters encircled with DTLs becomes less distinct in the deep papilla. As shown in a transverse section 500 µm above the papilla tip, the DTLs are arranged in a nonuniform fashion transversely across the papilla (Fig. 3A). Descending CD segments that arise from the primary CD clusters near the base of the IM have coalesced to the point where there remain a total of just 13 individual CD clusters (Fig. 3B). At this level, each cluster consists of a mean of 8.8 CD segments (range 3–26). The DTLs belonging to the deepest loops descend adjacent to single CDs but are not arranged in an organized fashion around the periphery of single CD clusters (Fig. 3B) as occurs near the base of the IM (Fig. 2). Rather, they tend to intermingle equally within each CD cluster as well as along the cluster periphery. ATLs and prebend segments, in contrast, are positioned in a relatively uniform fashion transversely across the papilla (Fig. 3C), similar to the pattern of ATL and prebend distribution that occurs near the base of the IM (Fig. 2).
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Fig. 1. Longitudinal cryosection from rat inner medulla (IM; base of the IM is near top edge of image, and papilla tip is near bottom edge of image). Descending thin limbs (DTLs)/aquaporin-1 (AQP1)/red; descending vasa recta (DVR)/urea transporter (UT-B)/green; collecting ducts (CDs)/AQP2/blue. Scale bar = 500 µm.
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Fig. 2. Transverse sections from rat IM (1,000 µm below IM base). A: DTLs/AQP1/red. B: ascending thin limbs (ATLs) and prebends/kidney-specific chloride channel (ClC-K1)/green. Scale bars = 100 µm.
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Axial distribution of vasa recta and capillaries in the papilla.
All IM vasa recta descend from the OM (DVR), subsequently forming fenestrated vessels that continue in a descending (DVR), ascending (AVR), or transverse direction (18). The dense and confluent lateral meshwork of fenestrated vessels that is arranged around CDs continues axially as the CDs descend to the tip of the papilla. UT-B-positive vessels are nearly undetectable within the terminal 2 mm of the papilla (Fig. 1). We previously reported that relatively short terminal portions of all UT-B-positive DVR are fenestrated to variable extents (18). Because we have observed no fenestrated vessels that become both PV-1 negative and UT-B negative at higher levels, we tentatively conclude there are no nonfenestrated, UT-B-negative vessels in the IM. Therefore, most vessels in the lower 2 mm of the papilla are fenestrated vasa recta or capillaries, with a significant proportion of these continuing in a descending direction. A representative immunohistochemical transverse section from the rat renal papilla showing the CDs and AVR is shown in Fig. 4. The fraction of total volume (volume fraction) occupied by AVR at 4–5 mm below the IM base is 22.5 ± 4.0% (n = 3).
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Fig. 4. Transverse section through the papilla showing distribution of CD/AQP2 (blue) and ascending vasa recta (AVR)/plasmalemmal vesicle protein (PV-1; red) 4 mm below the base of the IM. Inset: magnification of area outlined by white box. Scale bars = 200 µm. Inset: 50 µm.
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Interstitial nodal spaces in the deep papilla.
Underlying the confluent vascular network is the symmetrical arrangement of AVR abutting each CD from the IM base to the papilla tip (Fig. 5, A–C). Near the base of the IM, there are on average about four AVR associated with each CD (18). This ratio of about four AVR to each CD remains relatively constant through the first 4 mm of the IM (Fig. 5D). At a depth of 3 mm below the IM base, the mean CD circumference has increased slightly compared with the circumference at the base (Fig. 5E). This increase has been shown to result from increased cell diameter, with little or no change in lumen diameter along the axis (6). A more marked increase in circumference occurs within the deepest 0.5–1 mm, a region where CDs begin to coalesce into the 10–15 CDs that form the ducts of Bellini. In this region of the papilla tip, the AVR:CD ratio increases markedly to a mean of 10 in parallel with an increase in the CD circumference (Fig. 5D). The mean fractional CD surface area abutting AVR is 0.62 ± 0.01 (means ± SE; n = 3). This compares with a mean fractional surface area of 0.54 for CD abutting AVR within 1 mm below the base of the IM (18). Because of near-parallel increases in axial CD circumference and number of abutting AVR, the mean surface contact area between CD and AVR remains nearly constant at 50–60% along the entire axis of the IM (Fig. 5F).
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Fig. 5. Transverse sections showing patterns formed by CD/AQP2 (blue) and AVR/PV-1 (red) across the central core region of the IM. A–C: sections from 3,000, 4,500, and 5,000 µm below the inner medullary base, respectively. The number of AVR abutting each CD ranges 4–5 through most of the papilla (A). In the final 0.5 mm, the number of abutting AVR increases sharply (B and C) to 6–10 in the terminal 0.5 mm (D). The average individual CD circumference in the papilla is significantly larger compared with the CD circumference at the IM base, with marked increase in the final 0.5–1.0 mm (E). As a result of these changes, the average absolute surface area per CD that abuts AVR increases markedly in the final 0.5–1.0 mm, but the percentage of surface area remains constant (F). D–F: mean values. Scale bars = 50 µm.
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Interstitial nodal spaces are defined as three-dimensional, partitioned regions of interstitial space, or microdomains, that are positioned alongside CDs in their descent through the IM (18). These interstitial spaces are bounded by a single CD and its two adjacent AVR (Fig. 6) and one or more ATLs (18). Interstitial nodal spaces are relatively abundant in the outer region of the IM, as shown in Fig. 6A. The number density of ATLs declines markedly along the papillary axis as loops of Henle form bends and loop back toward the OM (Fig. 6B) (6). This is paralleled by a decline in CD number density and a marked increase in the volume of interstitium (Fig. 6B) (6). In the papilla tip, the interstitial nodal spaces become increasingly dilated relative to those of the outer IM as the interstitial volume increases (Fig. 7A compared with 7B); the number of interstitial nodal spaces per unit papilla volume becomes slightly reduced (Fig. 6A compared with 6B).
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Fig. 6. Transverse sections showing interstitial nodal spaces (marked with X) formed by ATL/ClC-K1 (green), AVR/PV-1 (red), and CD/AQP2 (blue) in the central core region of the IM. A and B: sections from 1.4 and 5.0 mm below the base of the IM, respectively. Nodal spaces adjacent to CDs are numerous in the outer IM (A), becoming less numerous in the papilla tip (B). Scale bars = 50 µm.
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Fig. 7. Magnified view of interstitial nodal spaces (marked with X) formed by ATL/ClC-K1 (green), AVR/PV-1 (red), and CD/AQP2 (blue) in the central core region of the IM. Nodal spaces near the base of the IM (A) are smaller than those occurring in the deep papilla (B). A and B: sections from 1.4 and 5.0 mm below the base of the IM, respectively. Scale bars = 10 µm.
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Increased transverse length of ATLs at papillary tip.
Most of the bends of loops of Henle along the papillary axis resemble U-shaped hairpins. These narrow-bend loops exhibit very little transverse extension as the loop makes a sharp 180° turn. In contrast, in the terminal 500-µm region of the papilla tip, some, but not all, loop bends exhibit 5- to 10-fold greater transverse length than the average transverse length of narrow bends. Unlike the tight, U-shaped bends of the narrow-bend loops, these wide-bend loops exhibit boot-shaped bends that extend transversely, running perpendicular to the corticopapillary axis (Fig. 8). As indicated above, the CDs of the papillary tip exhibit increased diameters compared with CDs at higher levels. The wide transverse loops form very close associations with these large-diameter CDs, nearly encircling them before the points where the CDs merge with the papillary surface to form the ducts of Bellini (Fig. 9; Supplementary Video 1; all supplementary material is available in the online version of this article at the journal web site).
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Fig. 8. Transverse section from 70 µm above the papilla tip shows several terminal CD segments (AQP2, blue). Several of these form ducts of Bellini as they merge with the papillary surface epithelium. Some of the bends of loops of Henle (ClC-K1, green) exhibit wide transverse lengthening (arrows), and several of these wide bends lie closely adjacent to CDs. Scale bar = 100 µm.
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Fig. 9. Three-dimensional reconstruction of several papillary CDs (AQP2, blue), ATLs (ClC-K1, green), and AQP1-null DTLs (-B crystallin, yellow). Tight narrow bends of loops of Henle (3 top arrows) and wide transverse bends of loops of Henle (2 bottom arrows) are shown. Wide transverse bends of 2 loops reaching to near the tip of the papilla almost completely encompass a final CD segment (blue) before its merging with the papillary wall (surface epithelium; translucent gray) to form a duct of Bellini. Relative diameters of loops and CDs in this image nearly approximate true dimensions. Scale bar = 100 µm. See Supplemental Video 1 for additional details.
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To define the architecture of the wide-bend loops, all those loops of Henle whose prebends lie entirely below the 500-µm upper limit of the papilla tip were reconstructed for one kidney, and their dimensions were measured. This reconstruction of the complete population of loops of Henle and CDs within the terminal 500 µm of the papillary tip is shown in Fig. 10 (Supplementary Video 2). By convention, the prebend segment is considered to consist of the ClC-K1-expressing segment that extends from the terminal DTL to the midpoint of the loop bend (Fig. 11, inset: segment that extends from A to the point midway between B and C). For our analysis, the total prebend segment is defined as the ClC-K1-positive segment that extends from the terminal DTL to the point where the ATL begins its axial ascent (Fig. 11, inset, segments A–C). The transverse segment (Fig. 11, inset, segments B and C) is identical to the total prebend segment with the exclusion of the axial portion of the descending side of the loop. By these definitions, the entire loop bend is considered part of the prebend segment; for wide transverse segments, inclusion of the transverse segment significantly impacts the total prebend length; for narrow loop bends, inclusion of the transverse segment is immaterial. The loops can be categorized into two distinct populations, as shown in Fig. 11: those with tight hairpin bends exhibiting transverse segment lengths <30 µm and those with transverse segment lengths ranging from 30 to >200 µm. For comparison with the transverse segments in the final 500 µm, the lengths of transverse segments lying between 800 and 4,100 µm above the tip, selected randomly from two kidneys, are also included in Fig. 11.
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Fig. 10. Three-dimensional reconstruction of all papillary CDs (AQP2, blue), ATLs (ClC-K1, green), and DTLs ( B-crystallin, yellow) from a single kidney. Papillary surface epithelium is shown in gray. A: lateral view. B: axial view from papilla tip. Scale bars = 100 µm. See Supplemental Video 2 for additional details.
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Fig. 11. Transverse length of loop bend (length of transverse segment) plotted against distance of the bend above the papilla tip. For our analysis, the papillary loop bend consists of 2 parts (see inset), the total prebend segment (segment encompassed from A to C) and the transverse segment (segment encompassed from B to C). Loops that were measured were located either in the papilla at 0–500 µm above the tip (all loops from 1 kidney) or within a central region of the IM at 800–4,100 µm above the tip (loops randomly selected from 2 kidneys).
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The mean total prebend length of narrow-bend loops within the terminal 500 µm of the papillary tip is 170 µm, and the mean length of their transverse segments is 17 µm. This total prebend length of 170 µm is similar to the mean length of 164 µm that we reported for prebend segments in the first 3 mm of the IM (16). The mean total prebend length of wide-bend loops is 217 µm; this includes their transverse segments, whose mean length is 94 µm. Using the conventional definition of a prebend segment (ClC-K1-positive segment that extends from the terminal DTL to the midpoint of the loop bend), the mean prebend lengths for both narrow and wide loops are similar, 161 µm for the narrow loops and 170 µm for the wide loops. Narrow-bend loops can therefore be considered as having dimensions comparable to those of wide-bend loops; an architectural distinction can be made, however, between narrow-bend loops, whose prebend segments run entirely parallel to the corticopapillary axis, and wide-bend loops, whose prebend segments are folded and run both parallel and perpendicular to the corticopapillary axis.
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DISCUSSION
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The sharpest rise in solute concentration and, thus, in urine concentration occurs within the renal papilla of the mammalian kidney, with the NaCl concentration nearly doubling in the final 500 µm (8). The architecture of the outer IM contrasts notably with papillary architecture, and several of the architectural features described here likely play significant roles in developing and sustaining the steep interstitial solute gradient in the deep papilla. One of these is the relationship between DTLs and CDs. The distinctive arrangement of DTLs surrounding CD clusters that is so apparent in the outer IM is clearly lacking in the papilla. One possible role for this arrangement in the outer IM, in combination with interstitial composition and organization, is the restriction of lateral solute diffusion (17). The absence of this arrangement in the papilla indicates that this specific relationship between DTLs and CD clusters makes no contribution to the concentrating mechanism in this region.
The extent to which we find an absence of DVR (nonfenestrated vessels expressing UT-B) from the papilla has not been documented previously. UT-B-positive structures have previously been identified only in the outer 50% of the IM (5), and we have confirmed this observation. Knepper et al. (6) measured a volume fraction of 24% for the sum total of vasa recta 4 mm below the base of the IM; vessel identity was determined by the presence of red blood cells. The volume fraction of papillary AVR (fenestrated vessels) that we report (22.5%) is comparable, indicating that very few if any DVR (nonfenestrated vessels expressing UT-B) exist at this level. Furthermore, electron micrographs of longitudinal sections indicate nearly all vasa recta from the rat deep papilla are fenestrated (Wilhelm Kriz, personal communication). Therefore, in the papilla, a distinction between ascending and descending vessels can only be determined by direction of blood flow. Moreover, because all vessels appear to be of the same fenestrated type, it is likely that they all have similar permeabilities to solutes and water.
Although the fenestrated vessels abutting the CDs can be determined to have ascending flow by tracing them upward and can readily be called AVR, it is very difficult to know which other perpendicular vessels in the complex network of fenestrated vessels not abutting CDs have descending or ascending flow. At the very least, there appears to be no systematic arrangement of descending and ascending vessels that would readily function in countercurrent exchange. Moreover, if countercurrent exchange in the IM involves primarily nonfenestrated DVR exhibiting UT-B-facilitated urea transport lying near fenestrated AVR, then it appears that this is limited to regions outside the CD clusters in the outer IM (18).
CD surface area per unit papillary volume remains essentially unchanged from base of the IM to the tip of the papilla; in contrast, the ATL surface area per unit papillary volume declines markedly as loops decline in number with increased depth (Pannabecker and Dantzler, unpublished observations). Accordingly, the increase in volume of the interstitium as seen in Figs. 6 and 7 (6, 11) arises chiefly from reduced loop number density, not from reduced CD number density. This increased interstitial volume probably contains an abundance of hyaluronan because McPhee (14) found that almost all the hyaluronan in the IM of rats is located in the terminal 2 mm. Hyaluronan may play some role in the IM concentrating mechanism (7).
The interstitial nodal spaces that are bounded by a single CD, two adjacent AVR, and one or more ATLs decline in number along the descending axis of the papilla. In parallel with the increased interstitial fractional volume with greater depth is an apparent increase in the interstitial space included within the nodes, thereby giving the impression of nodal dilation. These spaces would presumably contain hyaluronan not present in the outer IM. Quantitative analyses of these interstitial nodal spaces will be required to precisely define their geometry along the axis of the IM and, thus, to assess their possible role in solute exchange and compartmentation (18).
The number of long loops of Henle that reach the deep papilla, as well as the density of loops per unit papillary volume, is markedly reduced compared with that of the outer IM, potentially limiting delivery of NaCl into this region. Positioning of the wide transverse loops in close association with large-diameter CDs in the deep papilla could compensate for a reduced loop population. This architecture could facilitate delivery of NaCl to the interstitium in a manner that could maximize production of the IM osmotic gradient and enhance fluid reabsorption from the CDs in the face of spatial constraints. The possible importance of solute reabsorption near loop bends deep in the IM has been emphasized by Layton and Davies (12).
The number of tight bends at the tip is about the same as the number of bends exhibiting wide transverse lengths, suggesting there may be two distinct, functional populations of long loops of Henle in this region. Although it appears possible that geometrical constraints could lead to the development of wide transverse segments, the fact that only about one-half of the loops in this region have these wide segments suggests that multiple factors underlie their existence.
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GRANTS
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This study was supported in part by National Institutes of Health (NIH) Grant DK-16294, Grant ES-06694 for the Southwest Environmental Health Sciences Center; and NIH Training Grants HL-07249 and GM-08400.
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ACKNOWLEDGMENTS
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We thank Drs. John Regan and Dan Stamer of the University of Arizona, Radu Stan of Dartmouth College, and Jeff Sands of Emory University for providing antibodies, and Marvin Landis of the University of Arizona Center for Computing and Information Technology for assistance with image analyses. We also thank the following students for assistance with three-dimensional reconstructions: Charles Davis, Tyler Davis, Christopher Galea, and Erica Montgomery.
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FOOTNOTES
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Address for reprint requests and other correspondence: T. L. Pannabecker, Univ. of Arizona Health Sciences Center, Dept. of Physiology, AHSC 4130, 1501 N. Campbell Ave., Tucson, AZ 85724-5051 (e-mail: pannabec{at}u.arizona.edu)
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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