Pannabecker, Thomas L., and William H. Dantzler. Threedimensional architecture of inner medullary vasa recta. Am J Physiol Renal Physiol 290: F1355-F1366, 2006. First published December 27, 2006 doi:10.1152/ajprenal.00481.2005The manner in which vasa recta function and contribute to the concentrating mechanism depends on their three-dimensional relationships to each other and to tubular elements. We have examined the three-dimensional architecture of vasculature relative to tubular structures in the central region of rat kidney inner medulla from the base through the first 3 mm by combining immunohistochemistry and semiautomated image acquisition techniques with graphical modeling software. Segments of descending vasa recta (DVR), ascending vasa recta (AVR), descending thin limb (DTL), ascending thin limb (ATL), and collecting duct (CD) were identified with antibodies against segment-specific proteins associated with solute and water transport (urea channel B, PV-1, aquaporin-1, ClC-K1, aquaporin-2, respectively) by immunofluorescence. Results indicate: 1) DVR, like DTLs, are excluded from CD clusters that we have previously shown to be the organizing element for the inner medulla; 2) AVR, like ATLs, are nearly uniformly distributed transversely across the entire inner medulla outside of and within CD clusters; 3) DVR and AVR outside CD clusters appear to be sufficiently juxtaposed to permit good countercurrent exchange; 4) within CD clusters, about four AVR closely abut each CD, surrounding it in a highly symmetrical fashion; and 5) AVR abutting each CD and ATLs within CD clusters form repeating nodal interstitial spaces 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 inner medullary osmotic gradient. three-dimensional reconstruction; PV-1; urea channel B; aquaporin; ClC-K; ␣B-crystallin; countercurrent multiplier; concentrating mechanism; vasa recta AMONG ALL FUNCTIONAL DOMAINS within the kidney, the concentrating mechanism in the inner medulla (IM) is perhaps the most complicated and, certainly, the least well understood. Recent characterization of proteins associated with epithelial and endothelial membrane transport in defined vascular and tubular segments of the IM has provided new information for models of the concentrating mechanism. However, interactions of the transport of fluid and small solutes that these proteins enable depend profoundly on the three-dimensional (3-D) architecture of the nephrons, collecting ducts (CDs), blood vessels, and interstitial cells and matrix. We have previously described some of the 3-D relationships of the inner medullary thin limbs of Henle's loops and CDs and the location of some of the transport or channel proteins along them (19,20). This information helped us to propose one possible model for the concentrating mechanism in the IM (9). However, these studies did not include information on the inner medullary vascula...

We used a mathematical model of the urine concentrating mechanism of rat inner medulla (IM) to investigate the implications of experimental studies in which immunohistochemical methods were combined with three-dimensional computerized reconstruction of renal tubules. The mathematical model represents a distribution of loops of Henle with loop bends at all levels of the IM, and the vasculature is represented by means of the central core assumption. Based on immunohistochemical evidence, descending limb portions that reach into the papilla are assumed to be only moderately water permeable or to be water impermeable, and only prebend segments and ascending thin limbs are assumed to be NaCl permeable. Model studies indicate that this configuration favors the targeted delivery of NaCl to loop bends, where a favorable gradient, sustained by urea absorption from collecting ducts, promotes NaCl absorption. We identified two model modes that produce a significant axial osmolality gradient. One mode, suggested by preliminary immunohistochemical findings, assumes that aquaporin-1-null portions of loops of Henle that reach into the papilla have very low urea permeability. The other mode, suggested by perfused tubule experiments from the literature, assumes that these same portions of loops of Henle have very high urea permeabilities. Model studies were conducted to determine the sensitivity of these modes to parameter choices. Model results are compared with extant tissue-slice and micropuncture studies.

Functional reconstruction of inner medullary thin limbs of Henle and collecting ducts (CDs) has enabled us to characterize distinctive three-dimensional vertical and lateral relationships between these segments. We previously reported that inner medullary descending thin limbs (DTLs) that form a bend at a distance greater than approximately 1 mm below the inner medullary base express detectable aquaporin (AQP) 1 only along the initial 40% of the segment before the bend, whereas ClC-K1 is expressed continuously along all ascending thin limbs (ATLs), beginning with the prebend segment. We have now reconstructed individual CDs that are grouped together in single clusters at the base of the inner medulla; CDs belonging to each separate cluster coalesce into a single CD in the deep papilla. DTLs are positioned predominantly at the periphery of each individual CD cluster at all levels of the inner medulla and are absent from within the cluster. In contrast, ATLs are distributed near uniformly among the CDs and DTLs at all levels of the inner medulla. A second population of inner medullary DTLs averages approximately 700 microm in length from base to bend and, as previously reported, expresses no detectable AQP1 and expresses ClC-K1 continuously beginning with the prebend segment. ATLs located within the interior of the CD clusters arise predominantly from these short AQP1-null inner medullary DTLs, suggesting there may be functional interdependence between IMCD1 segments and short-length inner medullary thin limbs exhibiting minimal water permeability along their descending segments. AQP1-expressing DTLs and CDs are apparently separated into two structurally distinct lateral compartments. A similar lateral compartmentation between the ATLs and CDs is not apparent. This architectural arrangement indicates that fluid and solutes may be preferentially transported transversely between multiple inner medullary compartments.

Digital three-dimensional (3-D) functional reconstructions of inner medullary nephrons were performed. Antibodies against aquaporins (AQP)-1 and -2 and the chloride channel ClC-K1 identified descending thin limbs (DTLs), collecting ducts (CDs), and ascending thin limbs (ATLs), respectively, through indirect immunofluorescence. Tubules were labeled in transverse sections and assembled into 3-D arrays, permitting individual tubule or combined surface representations to depths of 3.3 mm to be viewed in an interactive digital model. Surface representations of 75 tubules positioned near the central region of the inner medulla were reconstructed. In most DTL segments that form loops below 1 mm from the inner medullary base, AQP1 expression begins at the base, becomes intermittent for variable lengths, and continues nearly midway to the loop. The terminal DTL segment exhibiting undetectable AQP1 represents nearly 60% of the distance from the medullary base to the tip of the loop. AQP1 expression was entirely undetectable in shorter long-looped DTLs. ClC-K1 is expressed continuously along the terminal portion of all DTLs reconstructed here, beginning with a prebend region approximately 164 microm before the bend in all tubules and continuing through the entire ascent of the ATLs to the base of the inner medulla. CDs express AQP2 continuously and extensive branching patterns are illustrated. 3-D functional reconstruction of inner medullary nephrons is capable of showing axial distribution of membrane proteins in tubules of the inner medulla and can contribute to further development and refinement of models that attempt to elucidate the concentrating mechanism.

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