Architecture of vasa recta in the renal inner medulla of the desert rodent Dipodomys merriami: potential impact on the urine concentrating mechanism

Abstract

We hypothesize that the inner medulla of the kangaroo rat Dipodomys merriami, a desert rodent that concentrates its urine to over 6,000 mosmol/kg H₂O, provides unique examples of architectural features necessary for production of highly concentrated urine. To investigate this architecture, inner medullary vascular segments in the outer inner medulla were assessed with immunofluorescence and digital reconstructions from tissue sections. Descending vasa recta (DVR) expressing the urea transporter UT-B and the water channel aquaporin 1 lie at the periphery of groups of collecting ducts (CDs) that coalesce in their descent through the inner medulla. Ascending vasa recta (AVR) lie inside and outside groups of CDs. DVR peel away from vascular bundles at a uniform rate as they descend the inner medulla, and feed into networks of AVR that are associated with organized clusters of CDs. These AVR form interstitial nodal spaces, with each space composed of a single CD, two AVR, and one or more ascending thin limbs or prebend segments, an architecture that may lead to solute compartmentation and fluid fluxes essential to the urine concentrating mechanism. Although we have identified several apparent differences, the tubulovascular architecture of the kangaroo rat inner medulla is remarkably similar to that of the Munich Wistar rat at the level of our analyses. More detailed studies are required for identifying interspecies functional differences.

during the past two decades the three-dimensional architecture of the rat renal medulla has become increasingly pertinent to development of advanced computational models of medullary function (5, 12, 13, 15, 37, 38). Recent advances have been made in understanding the geometrical relationships of collecting ducts (CDs) and nephrons of the outer medulla and inner medulla (28, 40–42). However, much remains to be learned about compartmentation arising from dynamic flow of fluid and solutes between these structures; compartmentation that is essential for understanding the role of the renal medulla in processes of urine concentration and sodium and water balance (6, 23). For each of these processes, blood flow through medullary vasculature is highly integrated with epithelial fluid and solute fluxes, and more complete understanding of vessel architecture and connectivity between descending vasa recta (DVR) and ascending vasa recta (AVR) will more clearly define this compartmentation. The classical studies of the kangaroo rat (33) and other desert species demonstrating tolerance to low water intake underscore the potential insights these species could offer in understanding vascular compartmentation (1, 2) and its potential role in the urine concentrating mechanism.

Studies conducted chiefly in laboratory rats (Sprague-Dawley and Munich-Wistar strains) have shown that inner medullary vasculature consists of two types (23). These include vessels with a continuous endothelium (DVR) and vessels with a discontinuous (fenestrated) endothelium, most of which are AVR, which are highly permeable to fluid and solutes (19). A type of AVR commonly referred to as “interconnecting” or “communicating” capillaries exhibits no known structural or functional distinctions but is considered to be spatially resolved from other AVR (9, 23). DVR and some AVR exhibit little or no branching, whereas interconnecting capillaries generally exhibit repeated branching and form sparse networks (20, 23). DVR carry blood in a descending direction and AVR and interconnecting capillaries carry blood in an ascending direction. However, functional (43) and structural (39) studies with the laboratory rat have indicated that 10–15% of DVR have terminal fenestrated segments that carry blood in a descending direction.

In the rat kidney, DVR grouped in vascular bundles enter the inner medulla, and at various levels give rise to interconnecting capillaries, which form networks that route blood flow along the corticopapillary axis. Some DVR, albeit relatively few, descend to the tip of the papilla before breaking up into capillary networks (2, 20, 30). At various axial levels, interconnecting capillaries join AVR that are grouped with DVR in vascular bundles. These AVR then ascend along the corticopapillary axis, exiting the inner medulla into the outer medulla.

DVR and some AVR participate in countercurrent solute exchange, a process considered important for preservation of the inner medullary interstitial solute gradient. The parallel expression of fluid and solute channels and transporters in adjacent vessels and tubules would define, in part, the exchange processes that take place. One important goal in understanding renal medullary function is to clearly define the limits to which vascular networks, perhaps variably, lead to homogeneous or heterogeneous interstitial compartmental composition. The present studies with the kangaroo rat inner medulla provide a framework for understanding the extent to which fluid and solutes are distributed and exchanged among medullary compartments by way of vascular pathways in species capable of producing highly concentrated urine.

METHODS

Animals.

Dipodomys merriami (kangaroo rat) (∼30–50 g) were obtained by live trapping at the Santa Rita Experimental Range, located ∼10 miles east of Green Valley, AZ. Analyses were conducted with both male and female animals; no gender-related differences have been observed. Kangaroo rats are housed individually in cages in the University of Arizona Health Sciences animal facility for periods up to 4 mo. Bedding is Sakrete Play Sand, obtained from local commercial suppliers, and is autoclaved before use. Animals are provided with 15 g Kay-Tee wild birdseed daily and no fluids, as in captivity they do not drink free water, and they are also provided with one small leaf of spinach three times each week. Room temperature is 21–22°C and relative humidity is 30–70%. Young male Munich-Wistar rats (average wt, 90 g) were purchased from Harlan (Indianapolis, IN) and provided with rat chow and water ad lib. Animals were euthanized with CO₂. All experiments were conducted in accordance with Guide for the Care and Use of Laboratory Animals (Washington, DC: National Academy, 1996) and approved by the University of Arizona Institutional Animal Care and Use Committee.

Tissue preparation and immunohistochemistry.

Kidneys were prepared for immunohistochemistry as described previously (36). Briefly, kidneys are perfused through the aorta with phosphate-buffered saline (PBS) (pH 7.4) for 5 min, followed by periodate-lysine-paraformaldehyde (PLP) (0.01 M, 0.075 M, 2%) in PBS (pH 7.4) for 5 min; some kidneys were fixed without perfusion. The medulla is dissected free, immersed in PLP fixative for 3 h at 4°C, washed in PBS, and dehydrated through an ethanol series. Each medulla is trimmed to a size that measures ∼2,000 × 1,400 μm in the transverse dimensions near the outer medullary-inner medullary boundary. Tissue is then immersed in a solution of Spurr epoxy resin (Ted Pella) and ethanol (1:1) for 16 h (room temperature), then in 100% Spurr for 48 h (4°C), and finally embedded in 100% Spurr (12 h at 60°C). Serial transverse sections of the inner medulla were cut, beginning either at the outer medullary-inner medullary (OM-IM) boundary, or at the papilla tip. Tissue embedded in Spurrs resin was cut into 1-μm thick sections with a Leica EM UC6 or Leica Ultracut UCT ultramicrotome.

Orthologous proteins in kangaroo rat were labeled using affinity-purified polyclonal or monoclonal antibodies against the COOH-terminal region of human water channel aquaporin 1 (AQP1, mouse monoclonal, Abcam no. 9566) to label descending thin limbs of Henle, rat kidney-specific chloride channel to label ascending thin limbs of Henle (ClC-K, rabbit host, Alomone no. ACL-004), and human water channel aquaporin 2 to label collecting ducts (AQP2, goat host, Santa Cruz no. 9882). DVR were labeled with a polyclonal antibody raised in rabbits against rat urea transporter B (UT-B; diluted 1:200, provided by Jeff Sands and Janet Klein, Emory University). All tubules and blood vessels were labeled nonselectively with fluorescein-conjugated wheat germ agglutinin (Vector Laboratories, no. FL-1021). Secondary antibodies conjugated to fluorescent probes (Invitrogen/Molecular Probes or Jackson ImmunoResearch) were applied as described previously (36). Sections were mounted with Dako fluorescent mounting medium (Carpinteria, CA) and were viewed with epifluorescence microscopy (Applied Precision, DeltaVision).

Quantification of loops and vessels.

Segment-specific antibodies for the AQP1-null descending thin limb (DTL) and AVR of kangaroo rat are not available. Consequently, AVR were identified in transverse sections from nonreconstructed tissue by two methods. For the first method, AVR were identified simply on the basis of morphological criteria (wall thickness and vessel diameter) (23, 36) and absence of UT-B expression. For the second method, the total number of thin limb segments was determined from ClC-K expression, estimated prebend density (0.9%) (36), and the 1:1 ratio of DTLs and ascending thin limbs (ATLs), and the number of DVR were subtracted from the total number of tubular and vascular structures to obtain the number of AVR. Variation in the number of AVR determined with the two methods was no more than 3%.

Image analysis.

Separate sets of digitized, serial images were generated by capturing immunofluorescence from each tissue section. Continuous surface and volume representations for each vessel were constructed as described previously from serial sections no greater than 3 to 5 μm apart (36) with Amira visualization and volume modeling software (Mercury, Chelmsford, MA). Reconstructions were created for vessels of a single inner medulla. Quantitative analyses were carried out on two-dimensional images using Photoshop (Adobe) and the Image Processing Toolkit (Reindeer Graphics).

Electron microscopy.

Kidneys were prepared for electron microscopy by retrograde perfusion through the aorta with 0.08 M cacodylate buffer, pH 7.2, containing 0.6% NaCl and 3% dextran (mean molecular mass 38 kDa) (3). This was followed by perfusion with the same buffer containing 3% glutaraldehyde. The kidney was removed and the whole medulla was dissected free. The outer medulla was discarded and the inner medulla was cut into five sections transverse to the corticopapillary axis. These were immersed in fixative for 18 h at 4°C, washed in buffer, postfixed with 1% osmium tetroxide in 0.08 M cacodylate buffer at 4°C, dehydrated through a graded series of ethanol solutions, and embedded in epoxy resin. Grids were stained with uranyl acetate and lead citrate. Thin sections were cut on a Leica Ultracut microtome (Leica, Deerfield, IL) and examined and photographed using a Philips CM-12S electron microscope (Philips Electronic Instruments, Mahwah, NJ).

Statistical analyses.

Data combined from three or more samples are reported as means and SE (N = number of replicates). The statistical significance of differences between means was determined with one-way ANOVA and Scheffé's post hoc test (P < 0.05).

RESULTS

Architecture of DVR in the kangaroo rat inner medulla.

The inner medullary DVR were identified with immunohistochemistry using antibodies labeling the urea transporter UT-B. UT-B is coexpressed with the water channel AQP1 in most, but not all, DVR (Fig. 1). AQP1 expression levels in DVR are substantially lower than those seen in DTLs. AQP1 is also expressed in the initial ∼60–70% of the inner medullary DTL segment of long-looped nephrons, as shown in an earlier study of kangaroo rat loop of Henle and CD architecture (36). AQP1-positive DVR and DTLs can be readily differentiated from each other on the basis of wall thickness and vessel diameter as observed in transverse sections (Fig. 1).

Fig. 1.

Coimmunolocalization of aquaporin 1 (AQP1) and urea transporter B (UT-B) in descending vasa recta (DVR) in a single transverse section from kangaroo rat inner medulla, ∼1,000 μm below the outer medulla. Descending thin limbs (DTLs) are strongly labeled for AQP1, whereas DVR are weakly labeled. AQP1 and UT-B are coexpressed in most DVR. *DVR. **One UT-B-positive DVR expresses no detectable AQP1. Scale bars, 25 μm.

The UT-B-positive DVR and AQP1-positive DTLs of the kangaroo rat are spatially separate from groups of CDs and are distributed in a nonuniform pattern, as shown in a transverse section from about 900 μm below the outer medulla (Fig. 2A). A comparable pattern occurs in the Munich-Wistar rat at the same level (Fig. 2B). Although general spatial characteristics are comparable for the two species, as previously reported, the number density of AQP1-positive DTLs is greater in the kangaroo rat (Fig. 2A) than in the Munich-Wistar rat (Fig. 2B) due to AQP1 expression in a significantly longer proportion of the kangaroo rat DTL (36). CDs coalesce as they descend the corticopapillary axis, thereby forming distinct clusters; however, as delineation of coalescing clusters requires full reconstruction through the deep inner medulla, clusters cannot be determined in this image (36). In the Munich-Wistar rat (29) (Fig. 2B) and also in the kangaroo rat (36), this spatial separation defines two interstitial regions in the transverse dimension: the intercluster and intracluster regions. The space encompassed by the CDs and surrounding interstitium and associated nephrons and blood vessels is referred to as the intracluster region. The intercluster region encompasses the interstitial space that separates adjacent groups of coalescing CDs.

Fig. 2.

Immunolocalization of AQP2, AQP1, and UT-B in the renal inner medulla in transverse sections located ∼900 μm below the outer medulla. A: kangaroo rat; B: Munich-Wistar rat. DVR and DTLs of both species are arranged nonuniformly around groups of collecting ducts (CDs). Both images are overlays of 2 sections no more than 3 μm apart. Scale bars, 100 μm.

The intracluster and intercluster regions associated with an assumed CD cluster are outlined in Fig. 3A. The intracluster region is defined by an irregular polygon that connects the perimeters of all CDs. The intercluster boundary is defined by Euclidean distance map analysis (9, 29, 31) and is represented by a contour line (white line in Fig. 3A) that corresponds to the linear array of points that are most distant from any CD. AQP1-positive DTLs and UT-B-positive DVR tend to lie within the intercluster region (Fig. 3A). ATLs and AVR are distributed near uniformly across the intracluster and intercluster regions (36).

Fig. 3.

A: immunolocalization of AQP2, AQP1, and UT-B in a transverse section from kangaroo rat inner medulla, ∼100 μm below the outer medulla. The white line represents a Euclidean Distance Map boundary surrounding an assumed CD cluster. The red line represents an irregular polygon that encompasses CDs that form an assumed CD cluster. The intracluster region lies within the red line. The intercluster region lies between the red and white lines. See results for additional details. The three vascular bundles that are circled were reconstructed and are shown in B. *Reconstructed CD in Fig. 6. B: reconstruction of 9 CDs and associated DVR and AQP1-positive DTLs, extending from near the outer medullary-inner medullary (OM-IM) boundary to about 500 μm below the outer medulla. Tubule and vessel diameters are not to scale. Animal gender was female. Scale bars 50 μm.

To evaluate the three-dimensional vascular architecture, we traced blood vessels through serial transverse sections (5 μm apart) of the inner medulla from a single kangaroo rat. All UT-B-positive DVR associated with a group of CDs near the OM-IM boundary (0.057 mm² total area) were traced beginning near the OM-IM boundary and continuing at least as far as their first branch point or thru the first 1,750 μm below the outer medulla. As they descend, the DVR fan out from each other but remain compartmentalized within the intercluster region. DVR associated with three vascular bundles were reconstructed, continuing from near the OM-IM boundary to 500 μm below the OM-IM boundary (Fig. 3). The UT-B-positive DVR maintain their separation from CDs as they descend along the medullary axis (Fig. 3), as do AQP-1-positive DTLs (36). The lengths of UT-B-positive DVR within the three bundles can be fit with linear trend lines, which suggests that the UT-B-positive segment lengths for DVR within each bundle decline at a nearly linear rate for the interval examined (0 to 1,750 μm below the OM-IM boundary) (Fig. 4).

Fig. 4.

Lengths of UT-B expression in DVR from three reconstructed inner medullary vascular bundles. DVR (circled in Fig. 3B) were traced through the first 1,750 μm below the OM-IM boundary. Each DVR was assigned a serial ID number (abscissa) and this was correlated with its length of inner medullary UT-B expression (ordinate). Boxed points represent DVR that express UT-B to levels deeper than 1,750 μm. Linear trend lines for each bundle (1, 2, or 3) were calculated from lengths <1,750 μm [N = 5 DVR for each bundle; correlation coefficients (R²) for bundles 1, 2, and 3 are 0.94, 0.98, and 0.84].

UT-B-positive DVR branch once or twice near their termini. Beyond this point, vessel branching becomes more frequent, possibly signaling onset of a capillary network. UTB-positive DVR generally continue their descent as UT-B-negative segments for variable distances. No UT-B-positive DVR directly join AVR in a simple hairpin type loop configuration, at least through the first 1,750 μm below the OM-IM boundary.

The UT-B-positive DVR number density was determined at 500-μm intervals beginning at the OM-IM boundary and continuing to a depth of 2,000 μm below the outer medulla (Fig. 5). The number density of UT-B-positive DVR declines at progressively deeper levels along the corticopapillary axis, reaching statistical significance at and below 1,500 μm below the outer medulla.

Fig. 5.

Number densities of inner medullary UT-B-positive DVR along the corticopapillary axis. The number density declines at a rate that is linear relative to depth below the outer medulla. Number densities were determined from random cross-sectional areas of the inner medulla. A linear trend line was fit to the mean values (y = −0.12x + 609.1; R² = 0.94) (means ± SE, N = 4 kidneys). Means sharing a common numeral are not significantly different from each other (ANOVA; P < 0.05). Animal genders were female (1) and male (2–4).

Architecture of AVR and interconnecting capillaries in the inner medulla.

As we have no marker for AVR or AQP1-null DTLs, these vessels and tubules were identified by their absence of immunolabeling for AQP1, AQP2, UT-B, or ClCK, and on the basis of wall thickness and vessel diameter and were visualized by labeling with wheat germ agglutinin (see methods). In three-dimensional reconstructions, AVR were identified as extensions of the DVR. AQP1-null DTLs were identified as extensions of the ClCK-labeled prebend segments. AVR exhibiting little or no branching lie in close association with DVR within the bundles of the intercluster region. In contrast, AVR that consist of interconnecting capillaries, relatively short transverse-running segments, or vessels that undergo repeated branching are intermixed among the CDs. About four of these branching AVR abut each CD and run parallel to it along its entire length (Figs. 6 and 7). Each AVR branches at various intervals, at which point it may pass to a neighboring CD and continue its course.

Fig. 6.

Reconstruction showing, at left, a single CD and abutting, branching AVR, and at right, UT-B-positive DVR and nonbranching ascending vasa recta (AVR) within a vascular bundle. Reconstruction extends from near the OM-IM boundary to about 750 μm below the outer medulla. The reconstructed CD is marked with an asterisk in Figs. 3A and 7. The reconstructed vascular bundle is bundle number 1 in Fig. 3A. Scale bar, 250 μm in the vertical dimension; tubule and vessel diameters are approximate.

Fig. 7.

Immunolocalization of AQP2, AQP1, ClC-K, and UT-B in inner medullary transverse sections from ∼500 μm below the outer medulla. Interstitial nodal spaces (marked with X's) are bordered by a single CD, two abutting AVR, and at least one ascending thin limb (ATL) or prebend segment. Image is an overlay of two transverse sections, ∼3 μm apart, each labeled with one of two antibody panels as described in methods. AQP1-negative descending thin limbs (DTLs) (DTL-) were identified from loop reconstructions. A reconstruction of the CD marked with an asterisk and abutting AVR is shown in Fig. 6. Animal gender was female. Scale bar, 25 μm.

AVR/DVR number density ratio in the inner medulla.

The total numbers of DVR and AVR were identified in transverse sections (0.06 to 0.11 mm² per section) at 2,000 μm below the outer medulla on the basis of segment-specific protein expression and morphological criteria, as noted above. Each section included multiple vascular bundles. The mean density of UT-B-positive DVR was 611 ± 72 DVR/mm², the mean density of AVR was 2,848 ± 253 AVR/mm², and the mean AVR-to-DVR ratio was 4.7 ± 0.4 (mean ± SE; n = 4). The AVR-to-DVR ratio determined from sections may be an overestimate to some degree as a percentage of UT-B-negative vessels are descending.

Interstitial nodal spaces in the inner medulla.

The AVR (typically about 4) that abut each CD are juxtaposed with ATL and prebend segments that lie opposite the CD to produce defined interstitial compartments. These compartments (previously referred to as interstitial nodal spaces) (26) run along the axial length of every CD and are distributed throughout the entire inner medulla (Fig. 7). Because of their positioning away from groups of CDs, the AQP1-positive DTLs and DVR rarely abut the interstitial nodal spaces. When viewed at the ultrastructural level, the capillaries (AVR) forming interstitial nodal spaces are seen to be fenestrated and to lie about 0.5 μm from CDs (Fig. 8). Interstitial nodal spaces are open spaces of varying dimensions. Interstitial cells (some lipid-laden) are sometimes seen in close association with interstitial nodal spaces (Fig. 8).

Fig. 8.

Transverse ultrathin section from midway along the corticopapillary axis showing ultrastructure of interstitial nodal spaces. A: interstitial nodal spaces are marked with an X. Fenestrated blood vessels (AVR) are marked with asterisks. B: magnification of boxed area in A. Black arrow identifies fenestrations of AVR endothelium. An interstitial cell containing lipid droplets (white arrows) lies juxtaposed with the AVR and CD. Scale bars, 10 μm (A); 500 nm (B).

Expression of UT-B in papillary surface epithelium.

UT-B is also expressed in the papillary surface epithelial cells, predominantly on the basolateral membrane (Fig. 9). UT-B is expressed almost continuously, but with variable levels of expression along the circumference of the papillary surface epithelium, at least in the outer inner medulla.

Fig. 9.

Immunolocalization of UT-B in DVR and papillary epithelium. Section is from 2,000 μm below the outer medulla. Boxed area in A is enlarged in B. Scale bars, 100 μm (A); 20 μm (B).

DISCUSSION

Three-dimensional functional reconstructions of inner medullary vasa recta reveal a number of spatial relationships that may be significant for the process of vascular countercurrent exchange and the urine concentrating mechanism in animals capable of producing a highly concentrated urine. Previous studies have shown that CD clusters form the organizing motif in the inner medulla of the kangaroo rat with nephrons and vessels arranged within and around them in an organized fashion (36). Comparable architecture with some variations occurs in the Munich-Wistar rat (25, 27, 28). This architecture leads to two distinct interstitial compartments in the transverse dimension, the intercluster and intracluster interstitial regions. CD clusters are discrete groups of neighboring CDs that coalesce as they descend through the inner medulla. DTLs and bundles of DVR are arrayed outside and around each CD cluster with most of them lying in the intercluster region. Relative to ATLs and AVR, these DTLs and DVR are somewhat distant from CDs.

UT-B is the only known urea transporter in DVR and is considered to provide the major pathway for urea flux across the DVR endothelium (23). Although kangaroo rat DVR express the urea transporter UT-B and the water channel AQP1 along much of their length, as previously reported for rat (22), a terminal segment with decreased or undetectable UT-B and AQP1 expression occurs in all DVR. The UT-B-negative segment does not connect directly to an AVR that ascends, unbranched, to the outer medulla, but instead connects to a UT-B-negative vascular network, a network consisting of fenestrated vessels that are closely associated with the CDs. These vessels ascend and at some point join AVR at variable distances above the DVR terminus (these distances were not determined in this study, as we have no markers for the kangaroo rat AVR) and carry plasma to the outer medulla and cortex. The decline in number density of UT-B-positive DVR at progressively deeper inner medullary levels results from loss of UT-B expression and from DVR peeling away from their vascular bundles one-by-one at all axial levels, to join the capillary networks of AVR, as is known to occur in rats and other rodents (9, 23).

Interstitial nodal spaces, formed from one CD, two AVR, and one or more ATLs or prebend segments, are extracellular compartments first identified in the Munich-Wistar rat (26, 28) that may sequester solute reabsorbed from CDs (14). The ultrastructural morphology of interstitial nodal spaces in kangaroo rat (Fig. 8), when viewed in transverse sections, appears very similar to that of Munich-Wistar rat interstitial nodal spaces (26). Ladder-like arrangements of interstitial cells along the corticopapillary axis of the rat (10, 16, 35) may further compartmentalize interstitial nodal spaces at ∼1- to 10-μm intervals. Actin filament arrangements and lipid composition of renal interstitial cells have been correlated with papillary blood flow and urine concentration and the antihypertensive lipid medullipin (10, 21). Interstitial nodal spaces are laterally separated from many of the AQP1-positive DTLs and DVR since these segments lie outside CD clusters.

Mathematical simulations support an important role for interstitial nodal spaces in generating the inner medullary osmotic gradient of the Munich-Wistar rat (11). Their existence provides compelling structural evidence for preferential NaCl reabsorption into an interstitial compartment that preferentially drives fluid reabsorption from water-permeable CDs. Interstitial nodal space architecture may also promote urea recycling within the inner medulla. One model has hypothesized existence of hypertonic absorbate (relative to CD fluid) within these interstitial nodal spaces (14) that could preferentially enter abutting AVR and be carried to higher medullary levels within the CD cluster where the local absorbate would have lower solute concentrations and lower osmolality. At these higher medullary levels, solutes may diffuse from AVR into interstitial nodal spaces and augment the local concentrating effect. Also, the AVR fluid may promote osmotic water withdrawal from the interstitial nodal spaces. In addition, diffusion of urea from the AVR into interstitial nodal spaces high in the inner medulla may help limit the diffusion of urea from CDs so that sufficient urea can be delivered by the CDs to the deeper inner medulla.

The AVR-to-DVR (AVR/DVR) number density ratio is important in accounting for mass balance of fluid and solute flows into and out of the inner medulla. All vessels leaving the inner medulla are generally considered to pass into the outer medulla by way of vascular bundles (23), although there is evidence that some take an alternative pathway in the Munich-Wistar rat (9). An AVR/DVR ratio of about 4.7:1, based on number densities of all UTB-positive vessels and estimated AVR in the kangaroo rat inner medulla, approximates the ratio of 4:1 (39) and 3.96:1 (7) determined at about 2,000 μm above the papillary tip of the Munich-Wistar rat, but varies from the ratio of ∼2:1 for the same region of the Sprague-Dawley rat (17). AVR/DVR ratios determined in these studies were based on immunohistochemistry or morphological criteria for the full complement of fenestrated and nonfenestrated vessels. In contrast, on the basis of functional ascending and descending blood flow, AVR/DVR ratios are about 2:1 for Munich-Wistar rat (4, 43) and hamster (18). These latter values likely reflect numbers of vessels that lie in the intercluster region that participate in countercurrent flows and they disregard flows through branching, transverse-running capillaries. The analysis of detailed architecture of unbranched descending and ascending blood vessels that may be spatially configured for countercurrent exchange in the kangaroo rat will require an antibody that is specific for AVR. Kangaroo rat inner medullary architecture supports a broad paradigm that countercurrent exchange between DVR and AVR is restricted to a compartment that is structurally distant from CDs but in relatively close proximity to AQP1-positive DTLs (39).

Expression of UT-B in the papillary surface epithelium parallels what is seen in other species (8). Urea fluxes across the rabbit papillary epithelium are relatively low, suggesting that urea flux from the pelvic space into the medullary interstitium is unlikely to contribute to the inner medullary urea gradient (32).

Perspectives and Significance

Blood flow patterns in the kangaroo rat, as indicated by vascular architecture, appear comparable to those of the laboratory rat. The presence of interstitial nodal spaces in the kangaroo rat broadens our view of their potential physiological relevance having been shown to exist in species with high and moderate urine concentrating ability (28). Although the structural elements of the interstitial nodal spaces, as previously defined for the Munich-Wistar rat, appear in the kangaroo rat, species-specific structural and functional variations (not yet determined) may occur and these potentially impact urine concentrating ability. Kangaroo rats have longer water-permeable DTLs and shorter NaCl-permeable prebend segments than those of Munich-Wistar rat (36), an architecture that potentially supports a steeper driving force for NaCl reabsorption into interstitial nodal spaces in the former species and a higher urine concentrating ability (24). Also, the proportions of ATLs and AVR that form the interstitial nodal spaces along the inner medullary axis may be relevant to understanding their fluid and solute flows within solute mixing and countercurrent systems (11). Finally, plasma vasopressin titer of kangaroo rat exceeds that of Sprague-Dawley rat in hydrated or dehydrated animals (34), so there is reason to hypothesize that vasopressin regulation of kangaroo rat tubular function differs from that of the laboratory rat.

Some of the most efficient mechanisms that have evolved to maintain fluid and solute homeostasis should be most apparent in animals that face extreme environmental conditions. Detailed analyses of tubulovascular structure and function in the desert-dwelling kangaroo rat provide insights into physiological features that may be significant in achieving fluid and solute homeostasis under extreme conditions. The distinctive radial tubulovascular organization of the renal inner medulla may be important in establishing functionally distinct compartments, and this compartmentation may be essential to the urine concentrating mechanism for species with either low or high concentrating capacity.

GRANTS

This research was supported by the National Science Foundation Grant IOS-0952885 and by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-083338.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: T.I., V.B.U., and T.L.P. performed experiments; T.I., V.B.U., and T.L.P. analyzed data; W.H.D. and T.L.P. conception and design of research; W.H.D. edited and revised manuscript; W.H.D. and T.L.P. approved final version of manuscript; T.L.P. interpreted results of experiments; T.L.P. prepared figures; T.L.P. drafted manuscript.