Descending Thin Limb Research Articles

Expression of SLC4A11 protein in mouse and rat medulla: a candidate transporter involved in outer medullary ammonia recycling.

SLC4A11 is a multifunctional membrane transporter involved with H+ transport, NH 3 and alkaline pH stimulated H+ transport, and water transport. The role of SLC4A11 in the kidney is not well understood. A prior study has shown that in murine kidney, SLC4A11/LacZ staining is primarily in the long‐looped descending thin limb (DTL) as determined by colocalization with aquaporin 1 (AQP1), a protein that is expressed in some, but not all, descending thin limb segments. Using a previously characterized polyclonal antibody, we demonstrate the selective expression of SLC4A11 in the upper DTLs (which are AQP1‐positive) in the outer medulla and inner medulla with little or no expression in the lower DTLs (which are AQP‐1‐null). SLC4A11 also colocalized with AQP1 and the urea transporter UT‐B in the mouse descending vasa recta, but was absent in mouse and rat ascending vasa recta. Mouse, but not rat, outer medullary collecting duct cells also labeled for SLC4A11. Our results are compatible with the hypothesis that in the inner stripe of the outer medulla, SLC4A11 plays a role in the countercurrent transport of ammonia absorbed from the outer medullary thick ascending limb and secreted into the long‐looped DTLs. SLC4A11 can potentially modulate the rate of ammonia transport in the mouse outer medullary collecting duct. Our data suggest functionally unique SLC4A11 pathways in mouse and rat and complement previous studies of DTL Na+, urea and water permeability indicating that the upper and lower DTLs of long‐looped nephrons are functionally distinct.

Deficiency of Carbonic Anhydrase II Results in a Urinary Concentrating Defect.

Carbonic anhydrase II (CAII) is expressed along the nephron where it interacts with a number of transport proteins augmenting their activity. Aquaporin-1 (AQP1) interacts with CAII to increase water flux through the water channel. Both CAII and aquaporin-1 are expressed in the thin descending limb (TDL); however, the physiological role of a CAII-AQP1 interaction in this nephron segment is not known. To determine if CAII was required for urinary concentration, we studied water handling in CAII-deficient mice. CAII-deficient mice demonstrate polyuria and polydipsia as well as an alkaline urine and bicarbonaturia, consistent with a type III renal tubular acidosis. Natriuresis and hypercalciuria cause polyuria, however, CAII-deficient mice did not have increased urinary sodium nor calcium excretion. Further examination revealed dilute urine in the CAII-deficient mice. Urinary concentration remained reduced in CAII-deficient mice relative to wild-type animals even after water deprivation. The renal expression and localization by light microscopy of NKCC2 and aquaporin-2 was not altered. However, CAII-deficient mice had increased renal AQP1 expression. CAII associates with and increases water flux through aquaporin-1. Water flux through aquaporin-1 in the TDL of the loop of Henle is essential to the concentration of urine, as this is required to generate a concentrated medullary interstitium. We therefore measured cortical and medullary interstitial concentration in wild-type and CAII-deficient mice. Mice lacking CAII had equivalent cortical interstitial osmolarity to wild-type mice: however, they had reduced medullary interstitial osmolarity. We propose therefore that reduced water flux through aquaporin-1 in the TDL in the absence of CAII prevents the generation of a maximally concentrated medullary interstitium. This, in turn, limits urinary concentration in CAII deficient mice.

Descending thin limb of the intermediate loop expresses both aquaporin 1 and urea transporter A2 in the mouse kidney.

A new intermediate type of Henle's loop has been reported that it extends into the inner medulla and turns within the first millimeter beyond the outer medulla. This study aimed to identify the descending thin limb (DTL) of the intermediate loop in the adult C57Bl/6 mouse kidney using aquaporin 1 (AQP1) and urea transporter A2 (UT-A2) antibodies. In the upper part of the inner stripe of the outer medulla (ISOM), AQP1 was expressed strongly in the DTL with type II epithelium of the long loop, but not in type I epithelium of the short loop. The DTL of the intermediate loop exhibited weak AQP1 immunoreactivity. UT-A2 immunoreactivity was not observed in the upper part of any DTL type. AQP1 expression was similar in the upper and middle parts of the ISOM. UT-A2 expression was variable, being expressed strongly in the DTL with type I epithelium of the short loop, but not in type II epithelium of the long loop. In the innermost part of the ISOM, AQP1 was expressed only in type III epithelium of the long loop. UT-A2-positive and UT-A2-negative cells were intermingled in type I epithelium of the intermediate loop, but were not observed in type III epithelium of the long loop. UT-A2-positive DTLs of the intermediate loop extended into the UT-A2/AQP1-negative type I epithelium in the initial part of the inner medulla. These results demonstrate that the DTL of the intermediate loop is composed of type I epithelium and expresses both AQP1 and UT-A2. The functional role of the DTL of the intermediate loop may be distinct from the short or long loops.

Hydration status affects osteopontin expression in the rat kidney.

Osteopontin (OPN) is a secretory protein that plays an important role in urinary stone formation. Hydration status is associated with the development of urolithiasis. This study was conducted to examine the effects of dehydration and hydration on OPN expression in the rat kidney. Animals were divided into three groups, control, dehydrated, and hydrated. Kidney tissues were processed for light and electron microscope immunocytochemistry, in situ hybridization, and immunoblot analysis. Dehydration induced a significant increase in OPN protein expression, whereas increased fluid intake induced a decrease in protein expression. Under control conditions, OPN protein and mRNA expression were only detected in the descending thin limb (DTL). Dehydration induced increased expression in the DTL and the development of detectable expression in the thick ascending limb (TAL). In contrast, OPN expression levels declined to less than the controls in the DTL after hydration, while no expression of either protein or mRNA was detectable in the TAL. Immunoelectron microscopy demonstrated that hydration status altered tubular ultrastructure and intracellular OPN expression in the Golgi apparatus and secretory cytoplasmic vesicles. These data confirm that changes in oral fluid intake can regulate renal tubular epithelial cell OPN expression.

Alternative channels for urea in the inner medulla of the rat kidney.

The ascending thin limbs (ATLs) and lower descending thin limbs (DTLs) of Henle's loop in the inner medulla of the rat are highly permeable to urea, and yet no urea transporters have been identified in these sections. We hypothesized that novel, yet-unidentified transporters in these tubule segments could explain the high urea permeability. cDNAs encoding for Na(+)-glucose transporter 1a (SGLT1a), Na(+)-glucose transporter 1 (NaGLT1), urea transporter (UT)-A2c, and UT-A2d were isolated and cloned from the Munich-Wistar rat inner medulla. SGLT1a is a novel NH2-terminal truncated variant of SGLT1. NaGLT1 is a Na(+)-dependent glucose transporter primarily located in the proximal tubules and not previously described in the thin limbs. UT-A2c and UT-A2d are novel variants of UT-A2. UT-A2c is truncated at the COOH terminus, and UT-A2d has one exon skipped. When rats underwent water restriction for 72 h, mRNA levels of SGLT1a increased in ATLs, NaGLT1 levels increased in both ATLs and DTLs, and UT-A2c increased in ATLs. [(14)C]urea uptake assays performed on Xenopus oocytes heterologously expressing these proteins revealed that despite having structural differences from their full-length versions, SGLT1a, UT-A2c, and UT-A2d enhanced urea uptake. NaGLT1 also facilitated urea uptake. Uptakes were Na(+) independent and inhibitable by phloretin and/or phloridzin. Our data indicate that there are several alternative channels for urea in the rat inner medulla that could potentially contribute to the high urea permeabilities in thin limb segments.

Architecture of the human renal inner medulla and functional implications.

The architecture of the inner stripe of the outer medulla of the human kidney has long been known to exhibit distinctive configurations; however, inner medullary architecture remains poorly defined. Using immunohistochemistry with segment-specific antibodies for membrane fluid and solute transporters and other proteins, we identified a number of distinctive functional features of human inner medulla. In the outer inner medulla, aquaporin-1 (AQP1)-positive long-loop descending thin limbs (DTLs) lie alongside descending and ascending vasa recta (DVR, AVR) within vascular bundles. These vascular bundles are continuations of outer medullary vascular bundles. Bundles containing DTLs and vasa recta lie at the margins of coalescing collecting duct (CD) clusters, thereby forming two regions, the vascular bundle region and the CD cluster region. Although AQP1 and urea transporter UT-B are abundantly expressed in long-loop DTLs and DVR, respectively, their expression declines with depth below the outer medulla. Transcellular water and urea fluxes likely decline in these segments at progressively deeper levels. Smooth muscle myosin heavy chain protein is also expressed in DVR of the inner stripe and the upper inner medulla, but is sparsely expressed at deeper inner medullary levels. In rodent inner medulla, fenestrated capillaries abut CDs along their entire length, paralleling ascending thin limbs (ATLs), forming distinct compartments (interstitial nodal spaces; INSs); however, in humans this architecture rarely occurs. Thus INSs are relatively infrequent in the human inner medulla, unlike in the rodent where they are abundant. UT-B is expressed within the papillary epithelium of the lower inner medulla, indicating a transcellular pathway for urea across this epithelium.

Water-pumping turbulent flow in thin segments of the loop of Henle concentrates urine

In the mammalian kidney, urine is concentrated by passing through the thin descending limb (TDL) of Henle's loop, correlated with an osmotic gradient in the interstitial fluid that is increased from the outer medulla (OM) to the tip of the in- ner medulla (IM). The water-pumping turbulence hypothe- sis states that both the urinary concentrat- ing and the osmotic gradient of the renal medulla result from turbulent urine flow in the TDL. In turbulent flow, molecules move crosswise as well as along the tubule so H 2 O molecules are driven out through aquaporins; this concentrates urine. Since the TDL has fewer aquaporins in the IM than in the OM, the IM interstitium re- ceives less water; this can establish an osmotic gradient. Turbulence in thin seg- ments results from a greater Reynolds' number (RN), the measure of tendency for turbulence, calculated based on velocity, density, and viscosity of urinary flow and tubule diameter. RN (and the tendency for turbulence) of urinary flow in the TDL is approximately 2 times the RN in a thick segment. It makes the flow a water-pump and results in urinary concentration and the osmotic gradient of the renal medulla. INTRODUCTION The mammalian kidney continuously changes water excretion and urinary concentration depending on different conditions in order to maintain a nearly constant blood plasma osmolality. For most mammals that do not have free access to water, the ability to change wa- ter excretion is essential for survival 1 . In order to change urinary concentration, nephrons have several mechanisms, the best known of which is urinary concen- tration through the generation of a pro- gressive osmotic gradient extending from the cortico-medullary boundary to the inner medullary tip 2 and a hyperos-

Three-dimensional reconstruction of the rat nephron

This study gives a three-dimensional (3D) structural analysis of rat nephrons and their connections to collecting ducts. Approximately 4,500 2.5-μm-thick serial sections from the renal surface to the papillary tip were obtained from each of 3 kidneys of Wistar rats. Digital images were recorded and aligned into three image stacks and traced from image to image. Short-loop nephrons (SLNs), long-loop nephrons (LLNs), and collecting ducts (CDs) were reconstructed in 3D. We identified a well-defined boundary between the outer stripe and the inner stripe of the outer medulla corresponding to the transition of descending thick limbs to descending thin limbs and between the inner stripe and the inner medulla, i.e., the transition of ascending thin limbs into ascending thick limbs of LLNs. In all nephrons, a mosaic pattern of proximal tubule (PT) cells and descending thin limb (DTL) cells was observed at the transition between the PT and the DTL. The course of the LLNs revealed tortuous proximal "straight" tubules and winding of the DTLs within the outer half of the inner stripe. The localization of loop bends of SLNs in the inner stripe of the outer medulla and the bends of LLNs in the inner medulla reflected the localization of their glomeruli; i.e., the deeper the glomerulus, the deeper the bend. Each CD drained approximately three to six nephrons with a different pattern than previously established in mice. This information will provide a basis for evaluation of structural changes within nephrons as a result of physiological or pharmaceutical intervention.

Transepithelial water and urea permeabilities of isolated perfused Munich-Wistar rat inner medullary thin limbs of Henle's loop

To better understand the role that water and urea fluxes play in the urine concentrating mechanism, we determined transepithelial osmotic water permeability (Pf) and urea permeability (Purea) in isolated perfused Munich-Wistar rat long-loop descending thin limbs (DTLs) and ascending thin limbs (ATLs). Thin limbs were isolated either from 0.5 to 2.5 mm below the outer medulla (upper inner medulla) or from the terminal 2.5 mm of the inner medulla. Segment types were characterized on the basis of structural features and gene expression levels of the water channel aquaporin 1, which was high in the upper DTL (DTLupper), absent in the lower DTL (DTLlower), and absent in ATLs, and the Cl-(1) channel ClCK1, which was absent in DTLs and high in ATLs. DTLupper Pf was high (3,204.5 ± 450.3 μm/s), whereas DTLlower showed very little or no osmotic Pf (207.8 ± 241.3 μm/s). Munich-Wistar rat ATLs have previously been shown to exhibit no Pf. DTLupper Purea was 40.0 ± 7.3 × 10(-5) cm/s and much higher in DTLlower (203.8 ± 30.3 × 10(-5) cm/s), upper ATL (203.8 ± 35.7 × 10(-5) cm/s), and lower ATL (265.1 ± 49.8 × 10(-5) cm/s). Phloretin (0.25 mM) did not reduce DTLupper Purea, suggesting that Purea is not due to urea transporter UT-A2, which is expressed in short-loop DTLs and short portions of some inner medullary DTLs close to the outer medulla. In summary, Purea is similar in all segments having no osmotic Pf but is significantly lower in DTLupper, a segment having high osmotic Pf. These data are inconsistent with the passive mechanism as originally proposed.

Urea permeabilities in defined segments of rat renal inner medullary thin limbs of Henle's loops

Urea permeabilities and resulting urea fluxes in specific segments of thin limbs of Henle's loops are critical to concentrating urine in rat renal inner medulla (IM) but have not yet been well‐defined. Immunohistochemistry has shown that urea transporter UT‐A2 is expressed in rat descending thin limbs (DTLs) near outer medullary (OM)‐IM border but not more than 0.5 mm below that point. No other urea transporters have been identified in rat IM. To understand the role that urea fluxes may play in the urine concentrating mechanism, we determined urea permeabilities (Purea) from 14C‐urea fluxes in perfused rat DTLs and ascending thin limbs (ATLs) isolated from 1) initial 2.5 mm below OM‐IM border (upper IM) and 2) terminal 2.5 mm of the IM (lower IM). Regardless of flux direction, Purea (x 10−5 cm/s) in upper IM was 49.6 ± 7.7 (mean ± SE) in DTLs and 192.9 ± 23.6 in ATLs. In lower IM, it was 208 ± 31 in DTLs and 225 ± 38 in ATLs. Phloretin (0.25 mM) had no effect on Purea in upper DTLs, suggesting that Purea was not due to the UT‐A2 transporter. Purea is similar (~200 × 10−5 cm/s) in lower DTLs and upper and lower ATLs, all segments that lack water channel AQP1 and have no osmotic water permeability, but is significantly lower (~50 × 10−5 cm/s) in upper DTLs, segments that express AQP1 and have high osmotic water permeabilities. Pathways or transporters accounting for urea permeabilities are unknown.

Gene expression in the thin limbs of Henle of the waterrestricted rat

Sodium and water homeostasis and the formation of concentrated urine depend upon the generation of a steep osmotic gradient within the renal medulla. Although the mechanism of this process remains controversial, the descending thin limbs (DTLs) and ascending thin limbs (ATLs) of the loops of Henle play important roles. Our goal was to identify genes that may be involved in the urine concentrating mechanism and to quantify their transcript levels in the DTLs and ATLs. Using real‐time PCR, we analyzed transcripts in isolated DTLs and ATLs of water‐restricted rats. Claudin 4 (CLDN4), sodium‐dependent glucose transporter 1 (NaGLT1), sodium glucose cotransporter 1 (SGLT1), and sodium myo‐inositol cotransporter 1 (SMIT1) were present in both types of tubules and their levels increased in the ATLs after 72h of water restriction. Chloride channel (Clcnka) mRNA in the ATLs and urea transporter mRNA (UT‐A2) in the DTLs increased. We also identified a novel variant of SGLT1 and four variants of the arginine vasopressin receptor 2 (AVPR2). Our results indicate that in addition to the previously characterized aquaporins and urea transporters, several other genes and their products may be involved in the movement of ions and solutes in the thin limbs of Henle. Further studies will determine how these genes contribute to urine concentration. NIDDK DK083338

Relationship of three‐dimensional architecture of thin limbs of Henle's loops to the renal inner medullary concentrating mechanism

Clusters of collecting ducts (CDs) form the lateral organizing structural motif for the arrangement of thin limbs of Henle's loops and of vasa recta in the renal inner medulla (IM). Within CD clusters (intracluster region), CDs, ascending vasa recta (AVR), and ascending thin limbs (ATLs), form transverse arrays of compartments called interstitial nodal spaces (INSs). The purpose of this study was to investigate quantitatively the relationship of inner medullary thin limbs of Henle's loops to the intracluster region and INSs. With serial section of the IM, Amira imaging software was used to determine the points AQP1‐null descending thin limbs (DTLs) and ATLs enter and exit INSs by tracking DTLs and ATLs as they descended/ascended through the IM. Their distance to the nearest CD was measured on each section. DTLs and ATLs contacting CDs were assumed to lie within the intracluster region, where they may contribute to the generation of an IM osmotic gradient by fluid and solute mixing within an INS. 40 AQP1‐null DTLs were on average 8.6 μm away from their nearest CDs at the base of the IM, and were on average 1.3 μm from CDs at the terminal portion of their length. 21 ATLs were on average 1.79 μm away from CDs as they began to ascend and were 8.02 μm on average from CDs at the IM base. The points at which DTLs and ATLs enter and exit CD clusters is likely related to NaCl reabsorption that occurs at the prebend and ATL.NIH DK 083338; NSF IOS‐0952885.

Expression of ciliary neurotrophic factor and its receptor in experimental obstructive nephropathy

Ciliary neurotrophic factor (CNTF) is well known as a growth/survival factor of neuronal tissue. We investigated the expression of CNTF and its specific receptor alpha (CNTFRα) in a unilateral ureteral obstruction (UUO) model. Complete UUO was produced by left ureteral ligation in Sprague-Dawley rats. The animals were sacrificed on days 1, 3, 5, 7, 14, 21, and 28 after UUO. The kidneys were fixed, and processed for both immunohistochemistry and in situ hybridization. CNTF immunoreactivity in sham-operated kidneys was observed only in the descending thin limb (DTL) of the loop of Henle. In UUO kidneys, CNTF expression was induced in the S3 segment (S3s) of the proximal tubule from day 1, and progressively expanded into the entire S3s and a part of the convoluted proximal tubules, distal tubules (DT), and glomerular parietal epithelium up to day 7. Upregulated CNTF expression was maintained to day 28. From day 14, the inner medullary collecting duct showed weak CNTF immunoreactivity. The CNTFRα mRNA hybridization signal in sham-operated kidneys was weakly detected in the DTL, DT, medullary thick ascending limb, and in a few S3s cells. After UUO, CNTFRα mRNA expression increased progressively in both the renal cortex and the medulla up to day 7 and increased expression was maintained until day 28. The results suggest that the S3s may be the principal induction site for CNTF in response to renal injury, and that CNTF may play a role in chronic renal injury.

Urine concentrating mechanism in the inner medulla of the mammalian kidney: role of three‐dimensional architecture

The urine concentrating mechanism in the mammalian renal inner medulla (IM) is not understood, although it is generally considered to involve countercurrent flows in tubules and blood vessels. A possible role for the three-dimensional relationships of these tubules and vessels in the concentrating process is suggested by recent reconstructions from serial sections labelled with antibodies to tubular and vascular proteins and mathematical models based on these studies. The reconstructions revealed that the lower 60% of each descending thin limb (DTL) of Henle's loops lacks water channels (aquaporin-1) and osmotic water permeability and ascending thin limbs (ATLs) begin with a prebend segment of constant length. In the outer zone of the IM (i) clusters of coalescing collecting ducts (CDs) form organizing motif for loops of Henle and vasa recta; (ii) DTLs and descending vasa recta (DVR) are arrayed outside CD clusters, whereas ATLs and ascending vasa recta (AVR) are uniformly distributed inside and outside clusters; (iii) within CD clusters, interstitial nodal spaces are formed by a CD on one side, AVR on two sides, and an ATL on the fourth side. These spaces may function as mixing chambers for urea from CDs and NaCl from ATLs. In the inner zone of the IM, cluster organization disappears and half of Henle's loops have broad lateral bends wrapped around terminal CDs. Mathematical models based on these findings and involving solute mixing in the interstitial spaces can produce urine slightly more concentrated than that of a moderately antidiuretic rat but no higher.

Three‐dimensional architecture of the renal inner medulla of the desert rodent Dipodomys merriami: potential impact on the urinary concentrating mechanism

We hypothesize that the inner medulla (IM) of Dipodomys merriami, a desert rodent that concentrates its urine to over 6000 mOsm/Kg water, provides extreme examples of architectural features most necessary for production of highly concentrated urine. Three‐dimensional architecture of vasculature and nephron segments in the IM was assessed with digital reconstructions from physical tissue sections. Descending and ascending vasa recta (DVR, AVR), descending thin limbs (DTLs), ascending thin limbs (ATLs), and collecting ducts (CDs) were identified by indirect immunofluorescence using antibodies that label segment‐specific proteins associated with solute and water transport and lectins (UT‐B, wheat germ lectin, AQP1, ClC‐K1, and AQP2, respectively). CD clusters form the central organizing motif in the IM. Transverse sections along the corticopapillary axis show nonuniform distribution of AQP‐1‐positive DTLs and uniform distribution of ATLs. Countercurrent exchange occurs only at the perimeter of CD clusters as DVR lie distant from CDs. Electron microscopy shows fenestrated AVR adhering to CDs. This architectural arrangement raises the possibility that fluid and solute reabsorbed from CDs diffuses preferentially into AVR. An additional factor increasing and defining axial compartmentation, and thereby restricting diffusive exchange, would be interstitial cell architecture. DK16294

Osmotic water permeabilities in specific segments of rat inner medullary thin limbs of Henle's loops

Three‐dimensional reconstructions of thin limbs of Henle's loops in rat renal inner medulla (IM) involving antibody to constitutive water channel aquaporin 1 (AQP1) have revealed that descending thin limbs (DTLs) of loops extending deeper than 1.0 mm express AQP1 for first 40% of their length, but fail to express it for remaining 60%. Ascending thin limbs (ATLs) and prebend segments do not express AQP1. Therefore, we examined water permeabilities in isolated perfused segments of DTLs from specific regions of IM and in ATLs. We perfused DTL segments from two IM regions: 1) final 2.0‐2.5 mm, where all DTLs lack AQP1, and 2) initial 2.5 mm. In initial 2.5 mm, we chose DTLs extending beyond first 1 mm because all of these should contain AQP1. DTLs from AQP1‐null region showed no osmotic water permeability (Pf = ‐292 ± 166 μm/s; mean ± SE; n=4). ATLs also showed no osmotic water permeability (Pf = 69 ± 69 μm/s; mean ± SE; n = 10). In contrast, DTL segments from initial 2.5 mm had mean osmotic water permeability of 1670 ± 169 μm/s (mean ± SE; n = 6). However, values for individual tubule segments varied from about 1200 to 2100 μm/s. Since there is no way to determine if segments perfused expressed AQP1 for their entire length, it seems likely that segments with lower values had regions that did not express AQP1. These data indicate that presence or absence of AQP1 assessed by immunocytochemistry accurately reflects differences in water permeability. DK16294

Role of three-dimensional architecture in the urine concentrating mechanism of the rat renal inner medulla

Recent studies of three-dimensional architecture of rat renal inner medulla (IM) and expression of membrane proteins associated with fluid and solute transport in nephrons and vasculature have revealed structural and transport properties that likely impact the IM urine concentrating mechanism. These studies have shown that 1) IM descending thin limbs (DTLs) have at least two or three functionally distinct subsegments; 2) most ascending thin limbs (ATLs) and about half the ascending vasa recta (AVR) are arranged among clusters of collecting ducts (CDs), which form the organizing motif through the first 3-3.5 mm of the IM, whereas other ATLs and AVR, along with aquaporin-1-positive DTLs and urea transporter B-positive descending vasa recta (DVR), are external to the CD clusters; 3) ATLs, AVR, CDs, and interstitial cells delimit interstitial microdomains within the CD clusters; and 4) many of the longest loops of Henle form bends that include subsegments that run transversely along CDs that lie in the terminal 500 microm of the papilla tip. Based on a more comprehensive understanding of three-dimensional IM architecture, we distinguish two distinct countercurrent systems in the first 3-3.5 mm of the IM (an intra-CD cluster system and an inter-CD cluster system) and a third countercurrent system in the final 1.5-2 mm. Spatial arrangements of loop of Henle subsegments and multiple countercurrent systems throughout four distinct axial IM zones, as well as our initial mathematical model, are consistent with a solute-separation, solute-mixing mechanism for concentrating urine in the IM.

Quantitative analysis of functional reconstructions reveals lateral and axial zonation in the renal inner medulla

Three-dimensional functional reconstructions of descending thin limbs (DTLs) and ascending thin limbs (ATLs) of loops of Henle, descending vasa recta (DVR), ascending vasa recta (AVR), and collecting ducts (CDs) permit quantitative definition of lateral and axial zones of probable functional significance in rat inner medulla (IM). CD clusters form the organizing motif for loops of Henle and vasa recta in the initial 3.0-3.5 mm of the IM. Using Euclidean distance mapping, we defined the lateral boundary of each cluster by pixels lying maximally distant from any CD. DTLs and DVR lie almost precisely on this independently defined boundary, placing them in the intercluster interstitium maximally distant from any CD. ATLs and AVR lie in a nearly uniform pattern throughout intercluster and intracluster regions, which we further differentiated by a polygon around CDs in each cluster. Loops associated with individual CD clusters show a similar axial exponential decrease as all loops together in the IM. Because approximately 3.0-3.5 mm below the IM base CD clusters cease to form the organizing motif, all DTLs lack aquaporin 1 (AQP1), and all vasa recta are fenestrated, we have designated the first 3.0-3.5 mm of the IM the "outer zone" (OZ) and the final 1.5-2.0 mm the "inner zone" (IZ). We further subdivided these into OZ-1, OZ-2, IZ-1, and IZ-2 on the basis of the presence of completely AQP1-null DTLs only in the first 1 mm and on broad transverse loop bends only in the final 0.5 mm. These transverse segments expand surface area for probable NaCl efflux around loop bends from approximately 40% to approximately 140% of CD surface area in the final 100 microm of the papilla.

AVOIDING A ‘SUPERFICIAL’ UNDERSTANDING OF THE EXCRETION OF WATER; importance of ‘thought’ experiments.

Our objective is to define how the kidney handles a water load by asking two questions. First, “What is the actual signal for the release of vasopressin (VP)?” Our data suggest that the signal is related to the arterial and not the venous plasma Na concentration (PNa). In more detail, when humans consumed identical water loads after an overnight fast slowly (in 2‐hr) or rapidly (0.5‐hr), there was an equal venous PNa, but a 4‐mM lower arterial PNa 30‐min after rapid water ingestion; only these latter subjects had a large water diuresis (12±1 ml/min). Second, “How can we estimate of the volume of filtrate delivered the distal nephron to understand what the urine flow rate should be when VP does not act’? We found a large discrepancy in this estimation using 3 different data sets in rats. First, the (TF/P)Inulin rose 2‐fold between the PCT and the DCT. Second, the medullary interstitial osmolality was ~2500‐mOsm/kg H2O in water‐deprived rats. Third, AQP1 was not found in the descending thin limbs (DtL) of superficial nephrons. We conclude that apparent discrepancies in data can be resolved by recognizing heterogeneity of sampling. First, a lower arterial PNa is needed to induce a water diuresis; hence water that was ingested slowly can be retained for future heat dissipation. Second, washout of the medulla can only influence loops of Henle that have water permeability. Although this seems not to apply to superficial nephrons, examining other data such as the (TF/P)Inulin implies that the DtL is permeable to water even if AQP‐1 were truly absent in superficial nephrons. These points indicate that deeper understanding requires integration of all relevant data.

Quantitative analysis of renal inner medullary structure

3‐D architecture of vessels and nephrons of rat renal inner medulla (IM) was analyzed quantitatively. IM outer zone (first 3–3.5 mm) has collecting duct (CD) clusters with borders defined by Euclidean distance map as organizing motif. Descending thin limbs (DTLs) of Henle's loops and descending vasa recta (DVR) are located outside CD clusters on these independently defined borders. DTLs in first 1 mm lack AQP1 and in remaining 2–2.5 mm lack AQP1 for last 60%. Ascending thin limbs (ATLs) and ascending vasa recta (AVR) are inside and outside CD clusters. ∼50% of AVR tightly abut CDs. Nodal spaces inside clusters are bordered laterally by CDs, AVR, and prebends or ATLs, and vertically by interstitial cells. Surface area of prebends plus corresponding length of ATL (probable site of NaCl efflux) decreases vertically with periodic peaks and valleys. Peaks (av. ∼2500 μm2 greater than valleys) may correspond to location of nodal spaces. In IM inner zone (last 1.5–2 mm), CD clusters disappear, all DTLs lack AQP1, all vasa recta are fenestrated, and loops are too few to show periodic prebend oscillations. Number of AVR abutting CDs increases with increasing CD diameter so that abutting surface area is unchanged. Nodal spaces are fewer, but larger. Final 0.5 mm has loops with narrow bends and loops with transverse bends. Transverse bends prevent surface area/medullary volume of prebends plus corresponding ATLs from decreasing. DK16294