Articles in PresS. Am J Physiol Renal Physiol (November 28, 2012). doi:10.1152/ajprenal.00547.2012
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Axial compartmentation of descending and ascending thin limbs of Henle’s loops
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Kristen Y. Westrick, Bradley Serack, William H. Dantzler, and Thomas L. Pannabecker
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University of Arizona Health Sciences Center
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Department of Physiology, AHSC 4128
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1501 N. Campbell Avenue
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Tucson, Arizona 85724-5051
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Running Head:
Axial compartmentation of descending and ascending thin limb
Address for Correspondence:
Dr. Thomas L. Pannabecker
University of Arizona Health Sciences Center
Department of Physiology, AHSC 4128
1501 N. Campbell Avenue
Tucson, Arizona 85724-5051
Telephone:
e-mail:
(520) 626-6481
[email protected]
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Copyright © 2012 by the American Physiological Society.
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Abstract
In the inner medulla, radial organization of nephrons and blood vessels around collecting
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duct (CD) clusters leads to two lateral interstitial regions and preferential intersegmental
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fluid and solute flows. As the descending (DTLs) and ascending thin limbs (ATLs) pass
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through these regions, their transepithelial fluid and solute flows are influenced by variable
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transepithelial solute gradients and structure-to-structure interactions. The goal of this
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study was to quantify structure-to-structure interactions, so as to better understand
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compartmentation and flows of transepithelial water, NaCl and urea, and generation of the
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axial osmotic gradient. To accomplish this, we determined lateral distances of AQP1-
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positive and AQP1-negative DTLs and ATLs from their nearest CDs, so as to gauge
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interactions with intercluster and intracluster lateral regions, and interactions with
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interstitial nodal spaces (INSs). DTLs express reduced AQP1 and low transepithelial water
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permeability along their deepest segments. Deep AQP1-null segments, prebend segments,
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and ATLs lie equally near to CDs. Prebend segments and ATLs abut CDs and INSs
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throughout much of their ascent; however, the distal 30% of ATLs of longest loops lie
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distant from CDs as they approach the outer medullary boundary, and have minimal
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interaction with INSs. These relationships occur, regardless of loop length. Finally, we
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show that ascending vasa recta separate intercluster AQP1-positive DTLs from DVR,
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thereby minimizing dilution of gradients that drive solute secretion. We hypothesize that
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DTLs and ATLs enter and exit CD clusters in an orchestrated fashion that is important for
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generation of the corticopapillary solute gradient by minimizing NaCl and urea loss.
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Keywords: aquaporin, concentrating mechanism, urea transport, tubule permeability, renal
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anatomy
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Introduction
From the outer medullary-inner medullary (OM-IM) boundary to deep within the
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inner medulla (about 3.0-3.5 mm in rat kidneys), the dominating organizing structural
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elements for the arrangement of the thin limbs of Henle’s loops and of the vasa recta are
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primary clusters of CDs (27, 28, 30). This organization produces two clearly distinct lateral
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regions, the intracluster and the intercluster regions (Fig. 1). The intracluster region
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includes the space occupied by CDs of each cluster and the intercluster region includes the
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space that separates CDs of adjacent primary clusters. AQP1-positive segments of DTLs and
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UT-B-expressing DVR lie almost entirely in the intercluster region. ATLs and AVR are
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distributed almost uniformly throughout both the intracluster and intercluster regions (27,
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28). The axial positioning of the AQP1-negative segment of the DTL, relative to the other
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tubular structures, has not been investigated in detail; however, since nearly all prebends
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abut CDs (25), the AQP1-negative DTLs must be repositioned from intercluster to
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intracluster region at some point along their length. In a similar fashion, some proportion
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of ATLs reposition from intracluster to intercluster region in their ascent (25).
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ATLs and prebend segments lie in close association with CDs and AVR to form
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compartments called interstitial nodal spaces (INSs) (27). INSs lie primarily in the
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intracluster region and appear well suited to facilitate mixing of solute reabsorbed from
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ATLs or prebend segments (primarily NaCl) with solute reabsorbed from CDs (primarily
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urea) within a confined space (29). Reabsorption of urea and NaCl would raise the
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osmolality within the INS, thereby driving water reabsorption from CDs. The AVR are well
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positioned to serve as a conduit that carries reabsorbed water out of the inner medulla.
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Our colleagues and we have hypothesized that thin limbs of Henle and blood vessels
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partition NaCl, urea, and water into and out of the INSs in a manner dependent upon their
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transepithelial or transendothelial permeability characteristics, as well as their overall
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architecture (13, 14, 29). Therefore, the type of tubule segments that interact with the INS
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would further promote or constrain inner medullary countercurrent systems. In this study,
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we investigated quantitatively the relationship of the inner medullary thin limbs to the
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INSs, thereby providing insight into their potential contribution to solute mixing and the
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inner medullary urine concentrating mechanism. Our aim was to determine the axial levels
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at which AQP1-negative DTLs and ATLs approach and move away from INSs, so as to more
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clearly understand the structures that form the INSs, and consequent fluid and solute
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partitioning.
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Methods
Animals. Young male Munich-Wistar rats (average weight: 90g) were purchased
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from Harlan Sprague Dawley (Indianapolis, IN). The animals were anesthetized with CO2.
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All experiments were conducted in accordance with The Guide for the Care and Use of
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Laboratory Animals (Washington, DC: National Academy Press, 1996) and approved by the
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Institutional Animal Care and Use Committee.
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Tissue Preparation. Four kidneys from four male Munich-Wistar rats were prepared
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for immunocytochemistry by retrograde perfusion through the aorta with PBS (pH 7.4) for
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5 min, followed by periodate-lysine-paraformaldehyde (.01 M, .075 M, 2%) (22) in PBS (pH
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7.4) for 5 min before removal from the animal. The whole medulla was dissected free, then
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immersed in a fixative for 3 h at 4°C, washed in PBS, dehydrated through an ethanol series,
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and embedded in Spurr’s epoxy resin (Ted Pella). The OM-IM boundary was identified in all
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kidneys on the basis of structural criteria (ie, presence or absence of thick ascending limbs)
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(12). In one kidney, serial 1-μm thick transverse sections were cut beginning near the OM-
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IM boundary and continuing 3000 μm in a papillary direction. Every fifth section was
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placed onto a glass microscope slide for immunohistochemistry (4 sections/slide). In three
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additional kidneys, at 1000 μm below the OM-IM boundary, 2 serial 1-μm thick transverse
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sections were cut and were labeled for immunohistochemistry.
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Immunohistochemistry. Kidneys were prepared for immunohistochemistry as
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described previously. Two sets of serial, transverse sections were prepared for each kidney
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– one set labeled for nephrons and CDs, and one set labeled for vasa recta and CDs. In the
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first set, nephrons and CDs were labeled by indirect immunohistochemistry using affinity-
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purified polyclonal or monoclonal mouse antibodies against the COOH-terminal of the
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human water channel AQP1 (diluted 1:100, raised in mouse, provided by Abcam #9566, or
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in chicken, provided by Dan Stamer, Duke University); the rat kidney-specific chloride
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channel ClC-K (diluted 1:200, raised in rabbit, Alomone #ACL-004); the human water
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channel AQP2 (diluted 1:100, raised in goat, Santa Cruz #SC-9882); and either a bovine
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monoclonal antibody raised against purified αβ-crystallin (diluted 1:50, raised in mouse,
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Stressgen #SPA-222) or fluorescein-labeled wheat germ agglutinin (Vector Laboratories
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#FL-1021). AQP1 is generally expressed in cells of DTLs, and AQP2 is generally expressed
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in the principal cells of CDs. Therefore, AQP1 and AQP2 antibodies can serve as markers for
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DTLs and CDs, respectively. The ClC-K antibody used in these studies binds to epitopes of
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both ClC-K1 and ClC-K2, however, there is no evidence that ClC-K2 is expressed in the inner
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medullary thin limbs. Therefore, in the inner medulla this antibody serves to identify ClC-
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K1. Because ClC-K1 is expressed only in ATLs in the inner medulla (29, 32), this antibody
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serves as a marker for these tubules. αB-crystallin is expressed in DTLs, ATLs, and CDs, and
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wheat germ agglutinin labels all tubules and vessels. Therefore, the latter two probes serve
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as common markers for tubular and vascular structures.
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In the second set of sections, DVR and AVR were labeled by indirect
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immunohistochemistry using affinity-purified polyclonal antibodies against rat urea
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transporter UT-B (diluted 1:200, raised in rabbit, provided by Jeff Sands and Janet Klein,
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Emory University); rat PV-1, a plasmalemmal vesicle protein (diluted 1:500, raised in
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chicken, provided by Radu Stan, Dartmouth College). PV-1 is a component of the fenestral
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diaphragm and its physiological function is presently poorly understood. In the rat inner
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medulla, the AVR are fenestrated and believed to have diaphragms, therefore PV-1
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antibodies serve as a marker for AVR. Some DVR express UT-B and AQP1; these AQP1-
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positive DVR can be distinguished from DTLs by their relatively thin endothelia and by
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their relatively small diameter compared to DTLs. CDs were labeled for AQP2 as described
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above. Image overlays showing both blood vessels and nephrons together were obtained
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from 2 sections offset from each other by no more than 5 μm.
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Before antibody application, Spurr resin was etched by applying to each slide a
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solution consisting of 5 g NaOH, 5 ml 100% ethanol, and 5 ml propylene oxide for 3 min
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(21) followed by washing with 100% ethanol and distilled water. Sections were then
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treated with 0.2% Triton-X100 in PBS (PBS/0.2%Triton) for 2 min and 1% SDS in PBS for 5
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min. They were then washed with PBS/0.2% Triton 3 x 5 min. Next they were treated with
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a blocking solution consisting of 5% BSA, 1% normal donkey serum, and 0.2% Triton
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diluted in PBS. Primary antibodies diluted in the blocking solution were then applied
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simultaneously overnight at 4°C followed by three 5 min washes with PBS/Triton.
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Secondary antibodies produced in donkey and conjugated to FITC, TRITC, CY5, or
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DAPI (Jackson ImmunoResearch, diluted 1:200 or 1:100 in PBS/Triton) were applied
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simultaneously for 2 hr at room temperature ending with three 5-min washes with
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PBS/Triton. Sections were mounted with Dako fluorescent mounting medium (Carpinteria,
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CA) and were viewed with epifluorescence microscopy using 10x or 20x objectives.
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Image Analysis. Serial images were obtained by capturing AQP1, AQP2,
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ClC-K1, αB-crystallin, FITC-wheat germ agglutinin, UT-B, or PV-1 indirect
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immunofluorescence from each tissue section. Quantitative analyses were carried out on
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the images using Photoshop (Adobe) and the Image Processing Toolkit (Reindeer Graphics)
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and Amira visualization software (Visage Imaging).
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Quantitative analyses consisted of measuring the lateral distance between each loop
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of Henle and its nearest CD. For four kidneys, the distance between each AQP1-positive
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DTL and each AQP1-negative DTL and its nearest CD neighbor was measured for a single
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section from 1000 microns below the OM-IM boundary. For a single secondary CD cluster
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from one kidney, the distance between each segment of the thin limb of Henle and its
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nearest CD neighbor was measured for every section (600 sections for a total of 3000
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microns) beginning at the OM-IM boundary and continuing in a papillary direction.
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The volume shrinkage factor for ethanol dehydration of rat medullary tissue has
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been reported to be approximately 20% (1) and the linear shrinkage factor has been
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reported to be approximately 20 to 25% (6). Thus, the linear distances that we report
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would underestimate the distances measured for fresh tissue by a maximum of
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approximately 20 – 25%.
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Statistical analyses. Data combined from three or more samples are reported as
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mean and standard error (SE), (N = number of replicates). The statistical significance of
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differences between means for each category of two data sets was determined with two-
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sample t-test (p<0.05) and significance of differences between means for multiple data sets
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was determined with ANOVA and Duncan’s post-hoc test (p<0.05).
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Results
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medulla. To quantify the distribution of DTLs and ATLs in the intercluster and intracluster
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regions, and their proximity to INSs, the distance from the basal membrane of each DTL or
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ATL to the basal membrane of the nearest CD was measured in transverse tissue sections.
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Distances were measured for 200 AQP1-positive DTLs, 200 AQP1-negative DTLs, and 200
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ATL/prebends from the inner medullas of 4 kidneys, from 4 different animals, in an area of
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about 1 mm2, at 1000 microns below the OM-IM boundary. A depiction of nearest CD
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distance for AQP1-positive DTLs is shown in Fig. 1 (inset). ClC-K1-positive segments of the
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terminal DTL (prebend segment) were included with ATLs as these are indistinguishable
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from each other. Distances were allocated into bins to more clearly show the lateral
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distribution (Fig. 2). About 95% of AQP1-positive DTLs lie at a distance greater than 6 μm
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from their nearest CDs (mean of 15.3 ± 0.9 μm for 200 segments ± SE), whereas about 75%
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of AQP1-negative DTLs lie less than 6 μm from their nearest CD (4.5 ± 0.3 μm). About 75%
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of ATL/prebends lie less that 6 μm from their nearest CD (4.4 ± 0.3 μm). The mean distance
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of the AQP1-positive DTLs from the nearest CD (15.3 μm) is significantly greater than the
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distances of AQP1-negative or ATL/prebend (4.5 μm and 4.4 μm) (P<0.05; ANOVA),
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whereas the latter two distances are not significantly different from each other. We have
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previously shown that prebend and equivalent length postbend segments of the ATL abut
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CDs along their entire lengths (25).
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Compartmentation of thin limbs of loops of Henle in the outer zone of the inner
Interestingly, the number density of ATLs, and to some extent the number density of
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AQP1-negative DTLs, declines with increasing distance from the CD cluster at a near
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exponential rate (compare histograms in Fig. 2). This relationship can be explained as
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follows. Loops of Henle form “bends” at all levels along the corticopapillary axis, and the
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total loop population of the rat medulla declines in number at progressively deeper depths.
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This decline in loop number approximates that of an exponential “loop decay rate” (9, 17,
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30). As noted, we have previously shown that all loop bends abut CDs within CD clusters
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(25). As we show below, the AQP1-negative descending and ascending segment of each
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loop lie progressively closer to CDs at progressively lower axial levels. Therefore, at any
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axial level an exponential relationship, at least roughly comparable to the loop decay rate,
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might also be predicted to occur between the number density of loop segments and their
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distance from the CD cluster for the total population of loops.
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In contrast, the number density of AQP1-positive DTLs is distributed almost
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normally around a mean of about 12 µm (Fig. 2). Therefore, each AQP1-positive DTL lies
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within the intercluster region, its distance from the CD cluster being proportional to its
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loop length.
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Compartmentation of reconstructed thin limbs of loops of Henle along their entire
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inner medullary lengths. We then analyzed all of the loops of Henle expressing inner
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medullary AQP1, that were associated with one secondary CD cluster from one kidney. This
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analysis included loops that formed their bends at least 1200 μm but no more than 3500
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µm below the OM-IM boundary. Inclusion within the CD cluster was determined by
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Euclidean distance mapping (30) (Fig. 1). Nearest neighbor CD distances were determined
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for each DTL and its corresponding ATL (N=42) in serial transverse sections at 5 μm
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intervals along the entire inner medullary axial length of each loop. One example that
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illustrates results from a single loop of Henle is shown in Fig. 3. The AQP1-positive segment
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of the DTL lies furthest away from the CDs (19.41 μm) at the OM-IM boundary and
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approaches CDs as it descends (Fig. 3A). This loop expresses no detectable AQP1 at levels
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deeper than 955 μm below the outer medulla, and at this point the AQP1-negative segment
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lies closer to the CDs (5.37 μm) than does most of the AQP1-positive segment. The loop
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increasingly contacts CDs towards its terminus and at the prebend segment. The ascending
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segment of this loop (Fig. 3B) makes contact with CDs for most of its length as it ascends,
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but moves away from the CDs as it nears the OM-IM boundary.
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Because the 42 loops of Henle analyzed were of varying lengths, and in order to
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assess the average points along the corticopapillary axis at which DTLs enter and ATLs exit
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the intracluster region, DTL and ATL lengths were fractionated into 8 proportional
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segment lengths for comparison. Each segment is equivalent to 12.5% of each DTL or ATL
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total length. The loop displayed in Fig. 3 is shown in Fig. 4 with its length fractionated and
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the corresponding nearest CD distance for each interval. The patterns of loop proximity to
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CDs for non-fractionated loops are similar to the patterns seen for fractionated loops. The
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nearest CD distances at each of the nine fractional points are shown for all 42 loops of
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Henle studied in Fig. 5. At the OM-IM boundary, the DTLs lie at a mean of 8.7 microns from
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the nearest CD, and at the terminal portion of the AQP1-negative DTL, they lie at a mean of
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0.8 microns away from the nearest CD. ATLs lie at a mean of 0.95 microns away from the
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nearest CD as they begin to ascend and at a mean of 4.2 microns from the nearest CD at the
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OM-IM boundary.
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Another summary displaying the relationships of all loops of the single secondary
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CD cluster is shown in Fig. 6. This figure shows the nearest neighbor CD distances for each
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defined descending segment (AQP1-positive segment beginning at the OM-IM boundary,
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beginning of the AQP1-negative segment, beginning of the prebend segment, and the loop
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bend), and for the equivalent intervals of the corresponding ATLs. At the OM-IM boundary
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the AQP1-positive segments of the DTLs lie at a mean of 8.7 μm away from their nearest
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CD, lying in the intercluster region. At the beginning of their AQP1-negative segments they
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were within a mean of about 5 μm from their nearest CD, and at the beginning of the
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prebend segment and at the loop bend they were at a mean of less than 1 μm from their
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nearest CD. For this particular CD cluster, the ATLs lie, on average, at a closer or equal
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distance to the nearest CD, compared to the DTL segments at each of the four levels shown.
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They were only about 7.5 µm from the nearest CD at the OM-IM boundary.
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Solute secretion in the intercluster region. Parallel arrangements of AVR and DVR
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that are juxtaposed within vascular bundles support countercurrent exchange in the
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intercluster region (29, 33). This process involves urea and NaCl secretion into DVR (19,
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23, 24). Fluid reabsorbed by AQP1-expressing DTLs and DVR potentially dilutes the
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interstitial fluid of the intercluster region, thereby reducing the driving force for solute
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secretion into DVR. However, the AVR are juxtaposed with descending segments in a
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manner that likely minimizes dilution of the intercluster interstitium. Within the
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intercluster region, at least one AVR intervenes between each DTL and any neighboring
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DVR, thereby minimizing abutments between the two structures (Fig. 7). To quantify the
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interactions between these segments, we measured the length of circumferential abutment
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between each DTL and any abutting DVR or AVR, across an area of 0.08 μm2 at a depth of
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about 500 μm below the outer medulla, for the inner medulla from each of 3 kidneys. The
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length of abutment between each AQP1-positive DTL and AVR (along their circumference)
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was 13.1 ± 0.8 µm (Mean ± SE), whereas there was virtually no abutment between each
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AQP1-positive DTL and DVR (0.7 ± 0.5 μm, Mean ± SE; significantly different from DTL-to-
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AVR abutments, p<0.05).
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For a single medulla, we traced six loops that formed their bends at a mean depth of
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2600 µm below the outer medulla (Fig. 8, longer loops), and four loops that formed their
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bends at a mean depth of 675 µm below the outer medulla (Fig 9, shorter loops), and
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measured the same abutments at regular intervals. For these ten loops, the DTL abutment
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with AVR was very high, whereas the DTL abutment with DVR was very low, consistent
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with the sectional analysis of 3 kidneys at 500 µm below the outer medulla (see above).
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Estimation of effective permeabilities for Na and urea. On the basis of the
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morphometric data presented in this study, we estimated effective permeabilities (Peff) for
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Na and urea between the intracluster and intercluster regions, and between two structures
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that lie in the same or different regions. Effective permeability between two structures was
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calculated as 1/Peff = 1/P1 + 1/P2 + 1/P3, where subscripted P values represent
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permeabilities of the two structures and the interstitial compartment. Tubular transmural
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permeability values (Table 1) were taken from Layton et al (16) (14). For Peff calculations,
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diffusivities in water are reduced by a factor of 5 due to interstitial viscosity (15). Path
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lengths between 2 points in the transverse dimension are shown in Table 2. The path
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length between the two regions was taken as the mean maximum distance between an
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AQP1-positive DTL and its nearest CD (15.3 μm), as reported above.
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Passive solute fluxes between two compartments depend largely on solute
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concentrations in the various compartments, and these depend at least in part on luminal
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fluid and solute flow rates; nonetheless, the Peff estimates (Table 3) provide potentially
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useful indices of the variability and relative degree of solute flows that could occur between
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compartments. The Peff estimates suggest that there is a slight preferential flow of urea
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from the CD into the “nearbend” intracluster ATLs relative to flow into more distal
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intercluster ATLs (16% difference). As a result of the large difference in transepithelial
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urea permeabilities between AQP1-positive and AQP1-negative DTL, the Peff for urea
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between CD and intercluster AQP1-negative DTL is markedly greater than the Peff between
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CD and AQP1-positive DTL (180% difference). This permeability difference will increase
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slightly as the AQP1-negative DTL continues to descend and gradually approach the CDs.
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Overall, the highest Peff for urea exists between AVR and AQP1-negative DTLs, regardless of
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whether or not either structure lies in the intercluster or intracluster region. Na
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reabsorbed from the intracluster ATL flows preferentially into the intracluster AVR,
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relative to the intercluster AVR (35% difference). Na permeabilities between other
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compartments are nearly equal, indicating little or no preferential flows arise on the basis
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of permeability characteristics.
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Discussion
The inner medullary loop of Henle follows clearly defined pathways in its descent
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and ascent along the corticopapillary axis. Inner medullary AQP1-positive DTLs lie
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predominantly in the intercluster region, where they have minimal interactions with CDs
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and interstitial nodal spaces. This architecture is depicted in Fig. 1 and described
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quantitatively in Fig. 2 (shown for a level 1000 μm below the outer medulla). As shown in
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Fig. 5, nearest CD distance measurements for reconstructed DTLs indicate that, regardless
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of loop length, and throughout their descent, the AQP1-negative DTL segments become
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progressively more closely associated with the intracluster region, and, therefore, also with
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the interstitial nodal spaces. This relationship becomes statistically significant near the
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midpoint of the total DTL axial length (AQP1-positive plus AQP1-negative segments). The
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number density of CDs remains relatively unchanged with depth through the first 3 mm of
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the rat inner medulla, whereas the number density of loops of Henle declines by ~20-30%
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(9). Therefore, the lateral movement of AQP1-negative DTLs towards the CDs is not due
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simply to the well known narrowing of the papilla. These results support previous
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qualitative estimates that the DTLs of loops with shallow bends lie closer to CDs than do
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the DTLs of loops with deep bends (28).
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Fluid reabsorption from, and solute secretion into the AQP1 and UT-B-positive DVR
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in the outer inner medulla are considered to concentrate luminal solutes and reduce the
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fluid load delivered to the deep inner medulla. Although the positioning of AQP1-positive
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DTLs outside the intracluster region minimizes the potential impact of their reabsorbed
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fluid on solute mixing within the INSs, it could be detrimental to fluid and solute flows into
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or out of DVR within the intercluster region. Here again, as with the intracluster region, the
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urine concentrating mechanism is aided by AVR architecture. As can be seen in Fig. 7, at
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least one fenestrated, highly water permeable AVR generally is positioned between each
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AQP1-positive DTL and its nearby DVR within the intercluster region. These AVR can be
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distinguished from interconnecting capillaries by the fact that they typically do not abut
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CDs (33). Fluid reabsorbed from DTLs may tend to be selectively routed into AVR.
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Functional studies have shown net fluid reabsorption from DVR occurs in the axial descent
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(34), yet overall fluid outflows from the inner medulla via AVR exceed fluid inflows via
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DVR, in support of mass balance, thus this architecture would have an effect consistent
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with functional studies.
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Our previous studies have indicated that ATLs are not randomly distributed
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laterally across the inner medulla (28, 30) and our present results indicate that they tend
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to be more densely distributed in the intracluster region (shown in Fig. 2 at a level 1000
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μm below the outer medulla). Distance measurements of reconstructed ATLs associated
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with a single secondary CD cluster indicate, that, as they ascend, the ATLs remain within
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the intracluster region for most of their length. However, the most distal ascending
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segments of some loops remain in the intracluster region whereas others move into the
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intercluster region. Apparently, a greater fractional ascending segment of those loops with
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the deepest bends moves into the intercluster region, whereas a greater fractional
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ascending segment of shorter loops tends to remain within the intracluster region.
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A recent mathematical model has suggested that interstitial urea concentrations of
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intracluster INSs could significantly exceed urea concentrations of the intercluster region
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(16). This model provides a hypothetical framework for evaluating loop architecture with
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reference to urea and water fluxes. The AQP1-negative DTLs of rat and chinchilla are
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relatively water impermeable (3, 4); however, previous studies in hamster and rat have
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shown that tubular fluid osmolality continues to increase as the DTL descends (5, 7, 18,
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20). Near the bend, tubular fluid and interstitial fluid osmolality are nearly equivalent.
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Thus, solutes must be secreted into the AQP1-negative segment of DTLs as they approach
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the nodal spaces. Urea entering the DTL lumen could be derived from several medullary
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urea-recycling pathways, pathways that include both DVR and CDs (10), and perhaps ATLs
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(29). Significant, but less urea secretion occurs into the AQP1-positive DTL segment, where
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transepithelial urea permeabilities are lower than those of the AQP1-negative DTL
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(Dantzler, Evans, and Pannabecker, unpublished) (2). We hypothesize that three-
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dimensional architecture plays a role in this process, since at any transverse level a given
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structure in the intercluster region is distant from a CD, whereas a structure in the
367
intracluster region is near a CD. When the DTL lies in close proximity to a CD (as occurs for
368
the intracluster region), urea secretion into the DTL will be favored, as this region likely
369
provides a more favorable peritubular-to-lumen concentration gradient.
370
The ATL of rat and chinchilla has very high transepithelial urea permeability,
371
therefore, the magnitude of NaCl reabsorption in excess of urea secretion (11, 31) could
372
potentially be limited if the ATLs were to border interstitial nodal spaces for too long a
373
distance (16). This could markedly diminish the urine concentrating mechanism. In their
374
ascent, ATLs of some loops leave the intracluster region and move laterally towards the
375
intercluster region. A low ATL transepithelial urea permeability would minimize urea
376
reabsorption, leading to excessive urea outflow from the medulla that would be
377
detrimental to the urine concentrating mechanism. In contrast, a high ATL transepithelial
378
urea permeability would be beneficial to the urine concentrating mechanism, since it will
18
379
allow urea reabsorption. Countercurrent urea flows between ATLs and DTLs or DVR would
380
minimize urea loss from the inner medulla. Reabsorption would be maximized and loss of
381
urea would be minimized if a substantial portion of the ATL resides in a region of low urea
382
concentration, i.e. in the intercluster region – a region distant from CDs (Fig. 10). As ATLs
383
are almost completely impermeable to water, urea reabsorption would dilute luminal fluid,
384
thereby contributing to net water outflow from the inner medulla.
385
In summary, we hypothesize that loops enter and exit clusters so as to maximize
386
overall interactions of DTLs, prebends, and ATLs with vessels and CDs associated with
387
interstitial nodal spaces, thereby optimizing fluid and urea reabsorption from CDs (Fig. 10).
388
A lateral separation of descending and ascending segments of each loop of Henle is
389
depicted in Fig. 10 to emphasize loop passages through both intercluster and intracluster
390
regions. This is true for some loops; however, descending and ascending segments of other
391
loops may abut each other within the same regions as they pass along the corticopapillary
392
axis (26). The morphometric data presented in this study enable us to estimate the Peff
393
associated with loop of Henle, CD, and AVR structure-to-structure Na and urea flows of the
394
“outer inner medulla”, thereby providing potential insights into preferential solute flows in
395
the transverse dimension. The impact of the Peff will be more thoroughly assessed with a
396
comprehensive mathematical simulation of medullary fluid and solute flows.
397
19
398
399
Acknowledgements
This study was supported by National Institutes of Health Grant DK-083338.
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22
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494
Table 1. Parameter values.*
DTL+
DTLATL
CD
DVR
AVR
Na
urea
PNa
Purea
Diffusivity**
10-5 cm/sec
10-5 cm/sec
10-5 cm2/sec
0
0
80
1
76
750
13
180
190
35
360
690
0.3
0.276
*Values from references (13, 16).
**For P
eff calculations, diffusivity values are reduced by 5-fold (from
the correct values of 1.5 for Na and 1.38 for urea) due to interstitial
viscosity (15).
Abbreviations: see Table 3.
495
496
23
497
498
Table 2. Path length between structures.*
Structure
1
CD
CD
CD
CD
CD**
CD
CD
ATLintra
ATLintra
DTL+
DVR
DTL-inter
DTL-intra
Structure
2
DTL+inter
DTL-inter
DTL-intra
AVRinter
AVRintra
ATLinter
ATLintra
AVRinter
AVRintra
AVRintra
AVRintra
AVRintra
AVRintra
microns
15.3
15.3
4.5
15.3
0.5
15.3
4.4
15.3
0.5
15.3
15.3
15.3
0.5
*Mean path length at 1 mm below the outer
499
medulla.
** From reference (27).
Abbreviations: see Table 3.
24
500
501
Table 3. Effective permeabilities (Peff) between regions and structures.
Structure 1
DTL+
DTL+
DTL-inter
DTL-inter
DTL-intra
CD
CD
CD
CD
CD
CD
CD
ATLintra
ATLintra
CD
Rintra
Structure 2
AVRinter
AVRintra
AVRinter
AVRintra
AVRintra
AVRinter
AVRintra
ATLinter
ATLintra
DTL+inter
DTL-inter
DTL-intra
AVRinter
AVRintra
DVRinter
Rinter
Effective
Permeability (10-5
cm/sec)
PNa
Purea
12.76
11.92
142.76
79.69
139.16
0.99
28.12
1.00
33.11
0.98
25.39
0.99
29.41
9.01
25.21
27.97
52.82
71.43
0.98
27.11
196.08
180.39
Abreviations: DTL+, AQP1-positive DTL;
DTL-, AQP1-negative DTL; intra,
intracluster; inter, intercluster; R, region.
502
25
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
Figure Captions
Figure 1. Immunolocalization of AQP2, AQP1, and ClC-K in transverse section from rat
inner medulla, located about 400 μm below the outer medulla. Five primary clusters
(outlined by white lines) make up a single secondary CD cluster. Intercluster boundaries
(white lines) are determined by Euclidean distance map technique; intracluster boundaries
(red lines) are polygons drawn by eye. The combined area of the intercluster regions of the
five primary clusters is 0.0235 mm2 and the combined area of the intracluster regions is
0.0169 mm2 (8). Black box outlines area shown in inset. Inset: Arrow indicates nearestneighbor distance between AQP1-positive DTL and CD. Scale bars, 100 μm; inset, 25 µm.
Figure modified from (30).
Figure 2. Average number of AQP1-positive DTLs, AQP1-negative DTLs, and ATLs grouped
according to their nearest-neighbor CD distance (mean ± SE, n=4 kidneys, from 4 animals).
The numbers of segments were grouped into bins that each span 3 μm. Nearest neighbor
CD distances for 200 of each segment, from each kidney, were measured on a transverse
tissue section located about 1000 μm below the OM-IM boundary. The number of ATLs per
bin declines exponentially with respect to nearest CD distance (y=73.3e-1E-03x; R2=0.94). See
Results for additional details.
Figure 3. Nearest neighbor CD distances for descending and ascending segments of a single
loop of Henle, as they descend and ascend the corticopapillary axis. (A) The AQP1-positive
DTL lies distant from the CDs during the initial descent from 0 to about 700 μm and
thereafter approaches the CDs, the AQP1-negative DTL begins contacting CDs towards its
terminus (below about 1700 μm), and the prebend lies close to or in contact with CDs for
most of its descent. (B) The ATL makes contact with CDs for most of its ascent between
1000 and 2500 μm, but moves away from CDs as it nears the OM-IM boundary.
Figure 4. Nearest neighbor CD distances for descending and ascending segments of a single
loop of Henle (same loop shown in Fig. 3) at fractionated lengths along the corticopapillary
axis. (A) The DTL lies farthest away from the CDs at the OM-IM boundary and it nearly
contacts CDs at its terminus (the prebend). (B) The ATL lies near CDs for most of its length
upon ascent but moves away from CDs as it nears the OM-IM boundary.
Figure 5. Nearest neighbor CD distances for descending and ascending segments of loops
of Henle from a single secondary CD cluster determined at fractionated axial lengths (mean
± se; N=42 loops). (A) DTLs are farthest away from CDs at the OM-IM boundary, where they
lie in the intercluster region, and gradually approach CDs at deeper levels. (B) ATLs ascend
in relatively close contact with CDs for about 38% of their length immediately after the
bend (the fraction lying between 0.625 to 1 on the abcissa) before moving away from CDs.
Points labeled with common numerals are not significantly different from each other.
26
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
Figure 6. Nearest neighbor CD distances for descending segments of loops of Henle from a
single secondary CD cluster (same loops shown in Fig. 5; mean ± se; N=42 loops). The
nearest neighbor CD distances were determined for the AQP1-positive segment at the OMIM boundary, at the beginning of the AQP1-negative segment, at the beginning of the
prebend segment, and at the loop bend. The nearest CD distances for ATLs at equivalent
levels were also determined. Points labeled with common numerals are not significantly
different from each other.
Figure 7. Immunolocalization of AQP2, AQP1, UT-B, and PV-1 in transverse section from
rat inner medulla, located about 250 μm below the outer medulla. A single vascular bundle
(enclosed by white circle) and associated DTLs lie in the intercluster region. AVR nearly
always lie between AQP1-positive DTLs and UT-B-positive DVR. Scale bar 50 μm.
Figure 8. Lengths of inner medullary loop of Henle circumferential abutment with DVR or
AVR. Segments included DTLs, ATLs, and prebend segments from loops forming bends at
about 2000 μm below the outer medulla. Circumferential abutment lengths were measured
at 500 µm intervals below the outer medulla. Absence of a column signifies no abutment.
Values are mean ± SE, N=6 loops from a single inner medulla. DTL abutments with DVR are
essentially zero, and significantly less than all DTL abutments with AVR. ATL abutments
with DVR are significantly higher than DTL abutments with DVR at 500 and 1000 µm below
the outer medulla, but not at 1500 and 2000 μm below the outer medulla.
Figure 9. Lengths of inner medullary loop of Henle circumferential abutment with DVR or
AVR. Segments included DTLs, ATLs, and prebend segments from loops forming bends at
about 500 μm below the outer medulla. Circumferential abutment lengths were measured
at 500 µm intervals below the outer medulla. Absence of a column signifies no abutment.
Values are mean ± SE, N=4 loops from a single inner medulla. DTL abutments with DVR are
essentially zero, and significantly less than all DTL abutments with AVR. ATL abutments
with DVR are not significantly different from DTL abutments with DVR at all levels.
Figure 10. Schematic organization of loops of Henle and CDs in the upper inner medulla of
rat kidney. DTLs lie in the intercluster region for most of their length and enter the
intracluster region at a short proximal distance above the prebend segment. ATLs lie in the
intracluster region for the innermost 50% of their length, with equal numbers of ATLs lying
in the intracluster and intercluster regions for the outermost 50% of their length.
27