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Published ahead of print on October 17, 2007
J Am Soc Nephrol 18: 2937-2944, 2007
© 2007 American Society of Nephrology
doi: 10.1681/ASN.2007010056
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BASIC RESEARCH

Aquaporin-1 Is not Expressed in Descending Thin Limbs of Short-Loop Nephrons

Xiao-Yue Zhai*,{dagger}, Robert A. Fenton{ddagger}, Arne Andreasen§, Jesper Skovhus Thomsen|| and Erik I. Christensen*

Departments of * Cell Biology; § Neurobiology; and || Connective Tissue Biology; {ddagger} The Water and Salt Research Center, Institute of Anatomy, University of Aarhus, Aarhus C, Denmark; and {dagger} Department of Histology and Embryology, China Medical University, Shen Yang, China

Correspondence: Dr. Erik Ilsø Christensen, Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Århus C, Denmark. Phone: +45-89-42-30-57; Fax: +45-86-19-86-64; E-mail: eic{at}ana.au.dk

Received for publication January 16, 2007. Accepted for publication June 19, 2007.


   Abstract
Top
 Abstract
Introduction
 RESULTS
 DISCUSSION
 CONCISE METHODS
 DISCLOSURES
 REFERENCES
 
In mammalian kidneys, aquaporin-1 is responsible for water reabsorption along the proximal tubule and is also thought to be involved in the concentration of urine that occurs in the medulla. It has been suggested, however, that aquaporin-1 is not expressed in the last part of the descending thin limbs of short loop nephrons in rats and mice, and its expression in this region in humans has not been studied. We examined the expression of aquaporin-1 and the urea transporter UT-A2 in serial sections of mouse nephrons in the inner stripe of the outer medulla using immunohistochemistry. In contrast to previous observations, we demonstrate a complete absence of aquaporin-1 along the entire length of descending thin limbs of 90% of short loop nephrons. Conversely, as expected, we identified aquaporin-1 in proximal tubules, descending thin limbs of long loop nephrons, and medullary descending vasa recta. We also observed this abrupt transition from aquaporin-1–positive proximal tubules to aquaporin-1–negative descending thin limbs of short loop nephrons in sections of human and rat kidneys. UT-A2 was restricted to the last 28% to 44% of the descending thin limbs of all short loop nephrons. Because the majority of nephrons are of the short loop variety, our findings suggest that the mechanisms of water transport in the descending thin limbs of short loop nephrons should be reevaluated. Likewise, the roles of aquaporin-1 and UT-A2 in the countercurrent multiplier and water conversation may need to be readdressed.


   Introduction
Top
 Abstract
 Introduction
RESULTS
 DISCUSSION
 CONCISE METHODS
 DISCLOSURES
 REFERENCES
 
Aquaporin-1 (AQP-1), first discovered by Agre and colleagues,1,2 is the highly expressed water channel of the kidney proximal tubules,3 where it is thought to be responsible for constitutive water reabsorption in this nephron segment. AQP-1 is also expressed in the descending thin limb (DTL) of Henle's loop, and, although this localization is consistently generalized in numerous reviews (see, e.g., Nejsum4 and Verkman5) with a few exceptions,6 it has been suggested that the last part of the DTL of short-loop nephrons (SLN) in rats and mice does not express AQP-17,8; however, such a distinction was not made in a study on human kidney.9 The exact localization of AQP-1 in the proximal tubule and DTL is thought to be essential for the urinary concentrating mechanism and, in the case of the latter, specifically the process of countercurrent multiplication. This is underlined by the observation that the water permeability in proximal tubules from AQP-1 knockout mice was reduced five-fold compared with normal mice.10 Similarly, an 8.5-fold reduction in water transport was observed in DTL of AQP-1 knockout mice.11 Together, these reduced water permeabilities contribute to the major urinary concentrating defect observed in these mice.

Histotopographic electron microscopic analyses12,13 and computer-assisted tracing of renal nephrons14 have determined that the DTL of SLN and long-loop nephrons (LLN) consist of four types of epithelial cells. Type 1 epithelium is characterized by a flat, simple epithelium that supposedly covers the entire thin limbs of SLN, whereas the other three epithelial types are distributed successively in the DTL and ascending thin limbs of LLN. Studies of microdissected rat renal tubules15 estimated that the number of AQP-1 molecules per tubule length in short-loop DTL type 1 epithelium was similar to that of segment 1 and segment 2 proximal tubules but seven-fold lower than that of type 2 epithelium of DTL of LLN.

The urea transporter UT-A2 is expressed in rats and mice in the inner stripe of the outer medulla (ISOM), where it is localized to the lower portions of the DTL of SLN8,16 and, under prolonged antidiuretic conditions, in the base of the inner medulla, where it is localized to the DTL of LLN.8 UT-A2 is proposed to be involved in urea recycling, a process whereby urea that is reabsorbed from the inner medullary collecting duct (IMCD) is secreted into the descending loop of Henle, causing it to be returned to the collecting duct lumen with the flow of tubule fluid, thereby maintaining a high medullary interstitium urea concentration and allowing the formation of a concentrated urine.17 In this study, we extensively examined the nephron location of both AQP-1 and UT-A2, based to a large extent on traced renal tubules from serial sections of mouse kidneys. Our results show that there is a complete absence of AQP-1 labeling in the type 1 epithelium of short-loop DTL of mouse kidneys, a finding that is also revealed in rat and human kidneys. These findings may have major implications on the understanding of the urinary concentrating mechanism, specifically the role of AQP-1 in countercurrent multiplication mechanisms in the ISOM.


   RESULTS
Top
 Abstract
 Introduction
 RESULTS
DISCUSSION
 CONCISE METHODS
 DISCLOSURES
 REFERENCES
 
AQP-1
No labeling for AQP-1 was detected in the thin limbs of the traced mouse SLN (n = 147 in three kidneys) with type 1 epithelium. In contrast, abundant labeling was observed in the vascular bundles in descending vasa recta and in the inter bundle regions in thin limbs of the traced LLN (n = 50 in three kidneys; Figure 1). This observation was further underpinned by a series of light and electron micrographs demonstrating the transitions from AQP-1–labeled proximal tubules into either nonlabeled, type 1 epithelium or into labeled type 2 epithelium of the LLN (Figures 2 and 3. Exceptions for these observations are demonstrated in Figure 4, where a labeled proximal tubule continues into a labeled relatively thick, type 2–like epithelium that, after a distance (90 to 400 µm), is replaced with a nonlabeled thin type 1 epithelium. These nephrons were identified by tracing as the longest SLN with their bends located close to the border between the outer and inner medulla, and they constitute approximately 10% of all traced SLN.14


Figure 1
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  		Figure 1. Three selected levels from the outer (A), middle (B), and inner part (C) of the ISOM of a mouse kidney showing Epon sections labeled for AQP-1. The DTL of LLN and SLN were identified by tracing and are indicated with either an L or an s. The areas composed of the thin-walled structures constitute the so-called vascular bundles, whereas the areas with the thick-walled tubules placed in between are interbundle regions. The labeled, thin-walled structures in the vascular bundles are the descending vasa recta. None of the traced SLN in the vascular bundles shows labeling for AQP-1, whereas the traced DTL of LLN in the interbundle region are labeled. In the inner part of the ISOM (C), the vascular bundles are smaller than at the earlier levels (A and B). Magnification, x80.

 

Figure 2
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  		Figure 2. Electron micrographs of Epon sections demonstrating ultrastructurally two tubular epithelial transitions of mouse nephrons. (A) Transition from a proximal tubule to type 1 epithelium of the DTL of an SLN. The left inset shows the same transition at light microscope level from the labeled proximal tubule to the nonlabeled DTL of the SLN. The right inset shows ultrastructurally the transition at a higher magnification. (B) Transition from a proximal tubule to type 2 epithelium of the DTL of an LLN. The inset shows the transition at light microscope level of the labeled proximal tubule continuing into the DTL of the LLN. Bars = 5 µm in A and 2 µm in B. Magnifications: x600 in left inset in A; x5600 in right inset in A; x1000 in inset in B.

 

Figure 3
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  		Figure 3. AQP-1 immunocytochemistry of ultrathin cryosections from a mouse kidney. (A) Immunocytochemistry for AQP-1 of two tubular transitions. (B) One of the transitions from A (top box). Gold labeling for AQP-1 in the proximal tubule at the brush border (BB) and at the basolateral membrane stops at the transition (arrow) into the nonlabeled type 1 epithelium. (C) The other transition from A (bottom box). The gold labeling for AQP-1 in the proximal tubule BB continues (arrow) into the type 2 epithelium at both the apical and basolateral membranes. Bars = 5 µm in A and 1 µm in B and C.

 

Figure 4
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  		Figure 4. Electron micrograph showing the type 2–like epithelium that forms the initial part of the DTL of one of the longest traced mouse SLN (type 3 bend). The section was obtained 55 µm from the transition of the proximal tubule into the DTL. The insets show AQP-1 labeling of the same tubule at the transition (top left), approximately 110 µm from the transition (top middle), and 560 µm from the transition (top right). Bar - 2 µm. Magnifications: x100 in top left; x200 in top middle and right.

 
The observation that the type 1 epithelium of DTL of SLN does not express AQP-1 was further substantiated by investigations of serial paraffin sections from the ISOM of rat and human kidneys. In many images, AQP-1 labeling stopped abruptly at the transition from proximal straight tubules to DTL covered with a very thin epithelium (probably type 1 cells; Figure 5). In contrast, transitions from proximal tubules to DTL with a taller epithelium (probably type 2 cells) showed continuous labeling for AQP-1 similar to what was observed in mice. In addition, double-labeling experiments for AQP-1 and CD34 (expressed in vascular endothelial cells) revealed many unlabeled, thin-walled structures in the interbundle regions of human kidneys (Figure 6), which most likely represent DTL of SLN. In rats, there are more continuous labeled transitions from proximal tubules to DTL covered with thick epithelium (probably type 2) than in mice. Whether these labeled type 2–like epithelia constitute either DTL of LLN or beginnings of DTL of the longest SLN cannot be established without performing a tracing of the nephrons. Taken together, these findings strongly suggest that, similar to the mouse, the DTL of SLN in rat and human kidney are also mainly devoid of AQP-1.


Figure 5
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  		Figure 5. Sections labeled for AQP-1 from rat (A; Epon) and human kidney (B; paraffin). Many cross and elongated tubular profiles exhibit two kinds of transitions: Some from labeled proximal tubules into nonlabeled, thin-walled DTL (probably type 1 epithelium, arrows), whereas others are continuously labeled from the proximal tubules into the relatively thick-walled DTL (probably type 2 epithelium, arrowheads). Magnifications: x150 in A; x300 in B.

 

Figure 6
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  		Figure 6. Paraffin section double labeled for AQP-1 (brown) and CD4 (pink, vascular endothelial marker) in a human kidney. L, labeled, relatively thick DTL of the LLN; S, nonlabeled DTL of the SLN; avr, ascending vasa recta. The arrows mark the double-labeled descending vasa recta. Magnification, x150.

 
To investigate whether hydration status had an effect on AQP-1 localization, we performed immunolocalization studies on kidneys from mice that were either water loaded or water restricted. Under our experimental conditions, the urine osmolality of the water-loaded group was 412 ± 82 mOsmol/kg H2O (n = 3) compared with 3213 ± 276 mOsmol/kg H2O (n = 3) in water-restricted mice. Under these conditions, there was still no AQP-1 labeling of DTL of SLN, whereas labeling of other parts of the nephrons and the descending vasa recta was unchanged (data not shown).

UT-A2
The terminal segment of the DTL of all mouse SLN expresses UT-A2. These labeled segments are located in the inner part of the ISOM (Figure 7), confirming previous studies.8 Uninterrupted labeling continued to the transition between the thin limb and the thick ascending limb (Figure 8). Zhai et al.14 classified mouse SLN into three different types according to their bends. For each of the three types of SLN, the length of the UT-A2–labeled segment was determined in one kidney to be 205 ± 55 µm (n = 13) for nephrons with type 1 bends, 238 ± 91 µm (n = 9) for nephrons with type 2 bends, and 353 ± 119 µm (n = 19) for nephrons with type 3 bends. SLN with either type 1 or type 2 bends originate from glomeruli located an average of 200 and 300 µm from the renal surface, respectively, and with bends located at the middle level of the ISOM. SLN with type 3 bends originate from nephrons with glomeruli located an average of 500 µm from the renal surface and with bends located at various levels of the inner half of the ISOM. Type 1 bends are covered with DTL epithelium continuing a short distance into the ascending limb, type 2 bends have an epithelial transition from DTL into thick ascending limb at the bend, and type 3 bends have a prebend thick ascending limb segment of varying length.14 For all three types of nephrons, UT-A2 labeling begins approximately 470 µm (232 to 623 µm) from the proximal straight tubule. The fraction of UT-A2–labeled thin-limb epithelium constituted 28, 35, and 44% of the total DTL length for the three types of SLN.


Figure 7
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  		Figure 7. Two selected levels from the middle (A) and inner (B) part of the ISOM of a mouse kidney showing Epon sections labeled for UT-A2. The two levels correspond to the two levels (consecutive sections) shown in Figure 1, B and C. The traced DTL of SLN marked with S in the lumen all are labeled for UT-A2 in the vascular bundles, whereas the traced DTL of the LLN marked with L in the lumen in the interbundle regions are not labeled for UT-A2. Magnification, x80.

 

Figure 8
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  		Figure 8. Micrograph of an Epon section showing the transition (arrows) from UT-A2–labeled DTL into the nonlabeled, thick ascending epithelium of an SLN of the mouse kidney. Magnification, x200.

 
Labeling of serial paraffin sections from the inner part of the ISOM of rat and human kidneys revealed individually exclusive labeling for AQP-1 in DTL of LLN and descending vasa recta and for UT-A2 in DTL of SLN (data not shown) as demonstrated previously in rat and mouse kidneys.8 Thus, in rat and human kidney, short-loop DTL type 1 epithelia do not express AQP-1 but only UT-A2 in the terminal segment of the DTL, which is in agreement with our findings in mice.


   DISCUSSION
Top
 Abstract
 Introduction
 RESULTS
 DISCUSSION
CONCISE METHODS
 DISCLOSURES
 REFERENCES
 
The major finding of this study is that DTL of SLN in mouse, rat, and human kidney to a major extent are devoid of AQP-1 under physiologic, hydrated, or dehydrated conditions. This discovery is potentially of great importance for our understanding of the urinary concentrating mechanism, particularly because previous studies have considered that DTL of both SLN and LLN are highly permeable to water as a result of AQP-1 expression.

Our extensive analysis of AQP-1 expression from 147 DTL of SLN in three different mouse kidneys through their entire length demonstrates conclusively that in this species, DTL type 1 epithelium of SLN does not express AQP-1. This observation was further substantiated by demonstrating direct transitions of AQP-1–labeled pars recta of proximal tubules into unlabeled DTL. The nephrons demonstrating labeling of the first approximately 90 to 400 µm of the DTL that then transition into unlabeled DTL cells are very long SLN that have their bends close to the border between the outer and inner medulla. These very long SLN constitute a minor population of approximately 10% of all SLN.14 These nephrons have a short initial segment covered with type 2–like epithelial DTL cells very similar to the type 2 epithelium covering the DTL of LLN. Our findings in rat and human kidneys demonstrate that the lack of AQP-1 in DTL of SLN is not limited to mice but is probably a general feature of other species; however, in rats, the transitions from pars recta proximal tubule into type 1 epithelium seem to be less frequent than in mice.

Although the majority of our immunolabeling was performed on Epon sections, the AQP-1 labeling observed in these sections was not different from labeling of paraffin sections. Furthermore, the otherwise strong labeling of other nephron segments and of the descending vasa recta precludes the possibility that the lack of labeling of the DTL of SLN could be due to the preparation and treatment of the tissue.

Our findings are in contrast to previous studies that reported AQP-1 expression in SLN.7–9 Also, in a study by Maeda et al.15 on microdissected rat renal tubules quantifying AQP-1 abundance by ELISA, the number of AQP-1 molecules in DTL of SLN was estimated to be equally high per tubule length as segment 1 and 2 proximal tubules but a factor 7 less than that of type 2 epithelium of DTL of LLN. The most plausible reasons for these discrepancies may be difficulties in distinguishing short-loop DTL from descending vasa recta and long-loop DTL morphologically and during microdissection.

Anatomically, the ratio of SLN to LLN is 82:18 in mice,14 70:30 in rats, and 85:15 in humans (for a review, see Bankir and de Rouffignac18). Our data suggest that approximately 82% of all DTL in mice do not possess AQP-1. This figure may be somewhat lower for rats in part because of the difference in the ratio between SLN and LLN. Also, it is not known whether the rat has the very long type 3 SLN similar to those found in mice.14 Whereas the AQP-1–deficient DTL are located inside the vascular bundles in mice and at the periphery in rats, the DTL of LLN are located in the inter bundle regions of the ISOM in both species.14,19,20 In human kidney, both types of DTL are located in the inter bundle regions.21

From a physiologic perspective, the lack of AQP-1 in short DTL is intriguing. As originally described by Kuhn and Ramel,22 the urinary concentrating mechanism depends on the process of countercurrent multiplication, whereby the loops of Henle serve as a countercurrent multiplier system that generates and maintains a high medullary osmotic gradient and allows the osmotic extraction of water from the collecting duct system (see Knepper and Gamba23 for review). For the countercurrent multiplication to work effectively, osmotic equilibration in the descending limbs must occur and the tubule fluid of the DTL must become progressively more concentrated as it flows toward the papilla, a feature confirmed by early micropuncture studies.24 The mechanism by which osmotic equilibration occurs could be by solute entry, water exit, or both (discussed by Levine et al.25). Indeed, numerous studies in several species using the isolated perfused tubule technique have shown that the water permeability of DTL of LLN is extremely high as a result of the high abundance of AQP-111,26,27; however, to our knowledge, only one study measuring the water permeability of DTL of SLN has been performed,28 presumably because of the thin/fragile wall of the type 1 epithelium making intact tubules virtually impossible to isolate manually. This single study, performed in hamsters, indicated that the DTL of SLN are indeed permeable to water, but their osmotic water permeability is much lower than that of the DTL of LLN. Importantly, in these experiments, the authors stipulated that they could not discount the possibility that one or more of their perfused "type 1 epithelia" are indeed "type 2 epithelia"; therefore, the actual osmotic water permeability of the DTL of SLN may be even lower than actually reported. These results lead us to speculate that the lower water permeability observed in DTL of SLN is due to a lack of AQP-1. In addition, our results suggest, in part, that both the mechanism of solute entry and water removal may occur in the DTL of SLN and that osmotic equilibration can occur as a result of solute entry coupled to a small amount of water efflux independent of AQP; however, further studies examining the water permeability of SLN are required to clarify this issue, although such investigations are likely to be extremely technically challenging.

If AQP-1 is not expressed in DTL of SLN but the loops are permeable to water,28 then what accounts for this observed water permeability? Studies by Chou et al.11 clearly showed that even in the absence of AQP-1, DTL of LLN have a relatively high water permeability, and this may also be the case for DTL of SLN. One possible explanation for the observed water permeability28 is that another AQP water channel localizes to this nephron segment. Six other AQP have been localized to the mammalian nephron, but it is unlikely that any of these are suitable candidates because they all have been extensively studied; however, the possibility exists that another, as-yet-unidentified AQP localizes to the DTL of SLN, and further studies are required to investigate this. Another possibility for such a high water permeability is a paracellular pathway, but because the tight junctions of type 1 epithelium are characterized by a deep junctional complex12 containing several ramified junctional strands, characteristic of "tight " tight junctions, this pathway seems unlikely to be responsible. The final potential explanation is water movement through the lipid bilayer; however, because type 1 epithelia do not contain abundant convoluted apical and basolateral membranes, like the type 2 epithelium, the contribution of this pathway to membrane permeability remains speculative. In contrast, if the DTL of SLN are not permeable to water (because only one study has ever reported such findings), then further studies are required to investigate the nature of solute concentration along the descending limb.

Studies of mathematical models have given great insight into the role of a short portion of the DTL of SLN that is water impermeable but urea permeable. One such recent study, by Layton et al.,29 clearly showed that a water-impermeable segment of the DTL of SLN is actually beneficial to maximal urinary concentrating ability. In their studies, the replacement of a short water-impermeable and highly urea-permeable segment (so-called SDL2 segment) with a highly water-permeable and moderately urea-permeable segment (so-called SDL1 segment) reduced collecting duct osmolality, outer medullary free water absorption, and efficiency. The authors’ explanation for their findings was that the presence of the water-impermeable segment increases collecting duct fluid osmolality near the outer medulla–inner medulla boundary by eliminating the load that would otherwise arise from fluid absorbed from the terminal portion of the SDL. With respect to our findings, it would be interesting to model mathematically the same processes in a DTL of an SLN that has water permeability equivalent to that observed in DTL of LLN of AQP-1 knockout mice to examine the overall effect on concentrating ability.

In addition to our findings with respect to AQP-1, our studies determined that the facilitative urea transporter UT-A2 is expressed in all three types of SLN. On the basis of our tracing studies, the expression begins approximately 470 µm from the transition of the proximal straight tubule into the DTL and continues to the transition between the thin limb and the thick ascending limb. For water extraction from the vasopressin-sensitive collecting duct, a corticomedullary osmolality gradient must exist within the kidney, with a maximum osmolality at the tip of the inner medulla. In the inner medulla, this gradient is composed mainly of urea,30 which is passively reabsorbed from the IMCD via the urea transporters UT-A1 and UT-A3. Although the process of countercurrent exchange minimizes dissipation of urea from the inner medullary interstitium, it cannot completely eliminate urea loss because the volume flow rate of blood in the ascending vasa recta exceeds that in the descending vasa recta. Thus, in addition to countercurrent exchange, urea recycling is believed to provide an important means of maintaining a high level of urea in the renal inner medulla.17 Urea recycling occurs when the urea that is reabsorbed from the IMCD is re-secreted into the loop of Henle, causing it to be returned to the collecting duct lumen with the flow of tubule fluid. A major element of urea secretion into Henle's loop is believed to occur via transfer from the vasa recta (containing another facilitative urea transporter, UT-B) to the DTL of SLN in the vascular bundles of the outer medulla, where these two structures are closely associated.14,20 Our localization of UT-A2 suggests that intrarenal urea recycling must occur in a very specific location, although recent findings from knockout mice studies suggest that urea secretion into the DTL of Henle's loop does not seem to be as important in medullary urea accumulation as previously believed.31

This study has demonstrated that the majority of DTL of SLN in mouse, rat, and human kidneys do not express AQP-1, contrary to the general opinion. These findings are of great importance for the understanding of the urine concentrating mechanisms. We have further obtained data giving the exact lengths of the expression of UT-A2 in the same part of the nephron, data of importance for the mathematical modeling of transport mechanisms in the kidney.


   CONCISE METHODS
Top
 Abstract
 Introduction
 RESULTS
 DISCUSSION
 CONCISE METHODS
DISCLOSURES
 REFERENCES
 
Preparation of Renal Tissue
The tissue preparation of the mouse kidneys has previously been described in detail.14 Briefly, kidneys from male C57/BL/6J mice, 25 g body weight, were fixed by perfusion through the abdominal aorta with 1% glutaraldehyde in 0.06 M sodium cacodylate buffer and 4% hydroxyethyl starch. Tissue blocks cut perpendicular to the longitudinal axis of the kidney were postfixed for 1 h in 1% OsO4 and embedded in Epon 812. From each kidney, a total of 2500, 2.5-µm-thick consecutive sections were obtained from the surface to the papillary tip and stained with toluidine blue. Renal tissue was also obtained from rats or mice fixed by perfusion as described or with 2% paraformaldehyde and embedded in either Epon or paraffin or frozen in liquid nitrogen for cryosectioning, respectively, by standard methods. In addition, renal sections were obtained from paraffin-embedded normal human kidneys (autopsy materials). All animal experiments were carried out in accordance with provisions for the animal care license provided by the Danish National Animal Experiments Inspectorate. Image recordings, alignment, and digital tracing were performed as described previously in detail.14

Effect of Hydration Status on AQP-1 Expression
Six mice were housed individually in normal cages and received free access to drinking water and pelleted diet for several days before experimental manipulation. Subsequently, the mice received a fixed daily ration of gelled diet containing either 2 ml of deionized water (water restricted) or 8 ml of deionized water (water loaded) as described previously.32 The mice did not have access to supplemental drinking water during this period. After 48 h, spot urine was collected, urine osmolalities were determined using a vapor pressure osmometer, and kidneys were processed for Epon embedding as detailed previously.

Antibodies
A commercial rabbit anti–AQP-1 (AB3065; Chemicon Int., Temecula, CA) was used at a dilution of 1:400. Rabbit anti–UT-A28 was used at dilutions of 1:200 to 1:800. Mouse anti–AQP-1 (ab6994) was obtained from Abcam (Cambridge, UK), and mouse anti-human CD34 was purchased from Immunotech (Marseilles, France) and used at dilutions of 1:100 and 1:400, respectively.

Immunohistochemistry
A total of 20, 2.5-µm-thick Epon sections (50 µm between the sections) from the start of the ISOM until the inner medulla were re-embedded in Epon and sectioned into two 0.5-µm-thick sections for each of the 2.5-µm-thick sections. Thus, the sections span the entire length of the DTL of SLN. The re-embedded sections originated from one of the three kidneys that have been three-dimensionally reconstructed and traced by Zhai et al.14 The sections were pretreated as described previously,33 and one glass was incubated with anti–AQP-1 and the other with anti–UT-A2, followed by incubation with peroxidase-conjugated secondary anti-rabbit antibody (DAKO A/S, Glostrup, Denmark). Sections from similar selected zones from the two other mice traced by Zhai et al. were treated identically. Consecutive paraffin sections from rat and human kidneys were incubated similarly with the same antibodies. Epon and paraffin sections from rat and paraffin sections from human kidneys were examined at the border of the inner and outer stripes of the outer medulla to visualize the transitions from the proximal tubule to the DTL. The sections were examined using a Leica (Herlev, Denmark) DMR microscope equipped with a Leica DFC320 camera. Images were acquired by the Leica TFC TWAIN 6.1.0 driver and processed using Adobe Photoshop 8.0 (Adobe, San José, CA). The areas examined from the traced kidneys were selected so as to include as many of the traced tubules as possible.

Length Measurements
The lengths of the DTL of mouse SLN labeled for UT-A2 were calculated as the sum of the Euclidian distance starting at the initial labeling until the transition into the thick ascending limb as described previously in detail.14

Electron Microscopy
On the basis of the tracing and immunolabeling, sections representing the transitions between different cell types were selected for ultrastructural analysis. These sections were re-embedded in Epon, sectioned in 50-nm-thick sections, and observed in a Philips CM 100 electron microscope. Ultrathin cryosections, 80 nm thick, incubated with rabbit anti–AQP-1 and subsequently with goat anti-rabbit 10-nm gold particles (British BioCell Int., Cardiff, UK), were likewise examined in the electron microscope.


   DISCLOSURES
Top
 Abstract
 Introduction
 RESULTS
 DISCUSSION
 CONCISE METHODS
 DISCLOSURES
REFERENCES
 
None.


   Acknowledgments
 
The work was supported in part by the Danish Medical Research Council, the University of Aarhus, the Novo-Nordisk Foundation, the Biomembrane Research Center, National Natural Science Foundation of China (contract 30640011), and the European Commission (EU Framework Program 6, EureGene, contract 05085). R.A.F. is supported by a Marie Curie Intra-European Fellowship, the Carlsberg Foundation (Carlsbergfonden), and the Danish National Research Foundation (Danmarks Grundforskningsfond).

The skilful technical assistance of Pia Nielsen, Hanne Sidelmann, and Inger Kristoffersen is gratefully acknowledged. We are thankful to Dr. Mark A. Knepper (National Heart, Lung, and Blood Institute, Bethesda, MD) for helpful discussions.


   Footnotes
 
Published online ahead of print. Publication date available at www.jasn.org.

See related editorial, "Aquaporin 1, Urea Transporters, and Renal Vascular Bundles," on pages 2798–2800.


   REFERENCES
Top
 Abstract
 Introduction
 RESULTS
 DISCUSSION
 CONCISE METHODS
 DISCLOSURES
 REFERENCES
 

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