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J Am Soc Nephrol 13:836-847, 2002
© 2002 American Society of Nephrology

Human Cortical Distal Nephron: Distribution of Electrolyte and Water Transport Pathways

Helena Lagger Biner*, Marie-Pierre Arpin-Bott{dagger}, Johannes Loffing*, Xiaoyan Wang{ddagger}, Mark Knepper{ddagger}, Steve C. Hebert§ and Brigitte Kaissling*

*Anatomical Department, University of Zurich, Zurich, Switzerland; {dagger}UMR CNRS 7519, University Louis Pasteur, Strasbourg, France; {ddagger}National Heart, Lung, and Blood Institute, Bethesda, Maryland; and §Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut.

Correspondence to: Dr. Brigitte Kaissling, University of Zurich, Anatomical Institute, Division of Vegetative Anatomy, Winterthurerstrasse 190, 8057 Zurich, Switzerland. Phone: 41-1-635-5320 Fax: +41-1-635-5702; E-mail: bkaissl{at}anatom.unizh.ch


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. The exact distributions of the different salt transport systems along the human cortical distal nephron are unknown. Immunohistochemistry was performed on serial cryostat sections of healthy parts of tumor nephrectomized human kidneys to study the distributions in the distal convolution of the thiazide-sensitive Na-Cl cotransporter (NCC), the ß subunit of the amiloride-sensitive epithelial Na channel (ENaC), the vasopressin-sensitive water channel aquaporin 2 (AQP2), and aquaporin 3 (AQP3), the H+ ATPase, the Na-Ca exchanger (NCX), plasma membrane calcium-ATPase, and calbindin-D28k (CaBP). The entire human distal convolution and the cortical collecting duct (CCD) display calbindin-D28k, although in variable amounts. Approximately 30% of the distal convolution profiles reveal NCC, characterizing the distal convoluted tubule. NCC overlaps with ENaC in a short portion at the end of the distal convoluted tubule. ENaC is displayed all along the connecting tubule (70% of the distal convolution) and the CCD. The major part of the connecting tubule and the CCD coexpress aquaporin 2 with ENaC. Intercalated cells, undetected in the first 20% of the distal convolution, were interspersed among the segment-specific cells of the remainder of the distal convolution, and of the CCD. The basolateral calcium extruding proteins, Na-Ca exchanger (NCX), and the plasma membrane Ca2+-ATPase were found all along the distal convolution, and, in contrast to other species, along the CCD, although in varying amounts. The knowledge regarding the precise distribution patterns of transport proteins in the human distal nephron and the knowledge regarding the differences from that in laboratory animals may be helpful for diagnostic purposes and may also help refine the therapeutic management of electrolyte disorders.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cortical distal nephron is the site for fine regulation of salt and water excretion by peptide and mineralocorticoid hormones and the site for specific actions of diuretics (1). In humans alterations of BP (2–4), as well as some disorders of sodium, potassium, calcium, magnesium, and volume homeostasis (3), have been associated with mutations of genes encoding for salt and water transport proteins in the distal nephron (5–8). These observations emphasize the growing importance of this nephron portion to human physiology and to human disease (9).

According to microanatomical criteria, the distal nephron comprises a straight part (TAL) and the distal convolution. Structural and functional criteria further subdivide the latter into the distal convoluted tubule (DCT) and the connecting tubule (CNT) ((10). Functionally, the cortical collecting duct (CCD) is included in the distal nephron. Apical salt entry into the cells of the distal nephron is mediated by three pathways: in the thick ascending limb of Henle’s loop (TAL) by the bumetanide/furosemide-sensitive Na-K-2Cl cotransporter (NKCC2), in the distal convolution by the thiazide-sensitive Na-Cl cotransporter (NCC), and by the amiloride-sensitive epithelial Na channel (ENaC) 1). Vasopressin-regulated water transport via aquaporin 2 (AQP2) is usually believed to be a function of collecting ducts (8,11).

Species differences have been reported with respect to the distribution pattern of NCC, ENaC, and AQP2 within the distal convolution. In rabbits, the sharp structural transitions from the DCT to the CNT and from the CNT to the CCD precisely coincide with the substitution of NCC by ENaC and with the onset of AQP2, respectively (12). Within the distal convolution of rats and mice, structural changes are gradual (13). Overlapping of NCC and ENaC at one transition (14,15) and of ENaC and AQP2 (16) at the other have been observed, and segmentation becomes a matter of definition. The seemingly relative small variations in the distribution pattern among species might entail distinct differences in the fine control of salt excretion, making extrapolation of data from one species to another questionable.

Although data by Obermüller et al. (17) demonstrated that NCC characterizes the initial portion of the distal convolution in the human kidney as well, the exact organization of the human distal convolution is incompletely known. Therefore, the aim of the study presented here was to show by immunochemistry the distribution patterns along the human distal convolution of a set of apical salt and water transport proteins (NCC, ENaC, AQP2). Because transcellular sodium and calcium transport are closely interrelated (18), we included in our study the localization of proteins involved in transcellular calcium reabsorption, the basolateral Na+-Ca2+ exchanger (NCX), the plasma membrane Ca2+-ATPase (PMCA), and the cytoplasmic protein calbindin-D28k (CaBP). Our results demonstrate that as in other mammalian species, the apical transport proteins in the human distal nephron are serially arranged, but their detailed distribution patterns differ markedly from those in laboratory animals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The protocol of this study was approved by the ethics committee of the Medical University of Strasbourg. Healthy parts of 4 tumor-nephrectomized kidneys from 3 men, aged 74, 69, and 59 yr, respectively, and 1 woman, 50 yr old at time of surgery, were investigated. The preoperative creatinine values were in all cases in the normal range (7 to 14 mg/L). For at least 6 mo before surgery, the 3 men received antihypertensive therapy with thiazide diuretics.

Tissue Processing
Immediately after kidney extirpation and removal of the tumor, the healthy tissue was cut into pieces approximately 0.5 to 1 cm thick. The pieces were immersed for 3 h at 4°C in freshly prepared 3% paraformaldehyde, dissolved in 0.1 cacodylate buffer (pH 7.4, adjusted to 300 mosmols with sucrose), containing 0.5 g/L of picric acid. After rinsing for at least 12 h at 4°C in the cacodylate buffer, the cortical tissue was cut into slices approximately 2 to 3 mm thick, which were mounted onto thin cork plates, frozen in liquid propane, and cooled to -196°C with liquid nitrogen. The frozen tissue was stored at -80°C until further processing.

Production of Anti-Human NCC Antibody
To produce a suitable rabbit polyclonal antibody to human NCC, rabbits were immunized to a synthetic peptide (sequence PGEPRKVRPTLADLHSFLKQEGC-COOH), corresponding to a portion of the amino acid terminal tail of human NCC. The peptide was purified via HPLC and conjugated to keyhole limpet hemocyanin as described previously (19). The resulting antisera were affinity purified on a column on which the same peptide was covalently linked to the medium (agarose beads). These antibodies were screened by immunoblotting. One of the antibodies (A857) strongly recognized NCC on immunoblots (see Results).

Immunoblot
Immunoblotting was used to screen existing antibodies for recognition of the orthologous protein targets in human tissue and to characterize the new anti-human NCC antibody described above. Homogenates were prepared from cortex, outer medulla, and inner medulla of a human kidney removed because of tumor. The protein concentration was measured, and the samples were solubilized in Laemmli sample buffer as described previously (19). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed on 7.5 or 10% polyacrylamide gels (Ready Gels, Bio-Rad, Hercules, CA), and the proteins were transferred from the gel electrophoretically to nitrocellulose membranes. Membranes were probed overnight at 4°C with the respective primary antibodies, then exposed to secondary antibody (Donkey anti-rabbit IgG conjugated with horseradish peroxidase, Pierce no. 31458, diluted to 1:5000) for 1 hr at room temperature. Sites of antibody-antigen reaction were visualized by means of a luminal-based enhanced chemiluminescence substrate (LumiGlo; Kirkegaard and Perry Laboratories, Gaithersburg, MD) before exposure to x-ray film (no. 165-1579; Eastman Kodak, Rochester, NY).

Staining Strategy
For immunohistochemistry, we cut at least 10 series of 5-µm-thick cryostat sections from each kidney (HM 500 OM, Zeiss, Germany). Each section series was made from a different, randomly oriented cortical tissue block and comprised between 18 and 39 consecutive sections. All sections of a series were placed successively onto numbered slides (three sections per slide) and double immunostained.

Each section was immunostained for CaBP and counterstained with a primary antibody directed against one of the given apical or basolateral transport proteins in the following order: 1, anti-NKCC2; 2, anti-NCC; 3, anti-ß subunit of ENaC; 4, anti-proton ATPase; 5, anti-AQP2; 6, anti-NCX; 7, anti-NKCC2; and 8, anti-NCC. Thus, at a distance of approximately 30 µm, the given transport proteins were displayed up to 6 times over a tissue depth of a maximum of 200 µm. Additional section series were used for colocation of the apical transport proteins with the basolateral proteins aquaporin 3 (AQP3), PMCA, and Na-K-ATPase.

Immunostaining Procedure
After pretreatment for 10 min with 10% normal goat serum in phosphate-buffered saline, the sections were incubated at 4°C for approximately 15 h in a humidified chamber with a mixture of anti-CaBP and one of the other primary antibodies (Table1) diluted in phosphate-buffered saline–1% bovine serum albumin. For detection of the proton ATPase sections were treated before incubation with sodium dodecyl sulfate 5% during 10 min (20) at 4°C.


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  		Table 1. Primary antibodies used in immunostaining
 
For detection of the binding sites of the primary antibodies, the sections were incubated in a humidified chamber at 20°C during 45 min with a mixture of the 2 respective secondary antibodies, goat–anti-rabbit IgG, and goat–anti-mouse IgG, conjugated with CY3 and FITC, respectively (Jackson Immuno Research Laboratories, West Grove, PA). Per 1 ml of the working dilution of the secondary antibodies, 2 µg of 4,6-diamidino-2-phenylindole dihydrochloride (Boehringer, Mannheim, Germany) was added for nuclear staining. After repeated rinsing with phosphate-buffered saline, the sections were postfixed in paraformaldehyde 3% for 10 min at 20°C. Finally, coverslips were mounted with Dako-Glycergel (Dakopatts, Glostrup, Denmark) to which 2.5% 1,4-diazabicyclo (2,2,2) octane (Dabco; Sigma, St. Louis, MO) was added as a fading retardant.

Nonspecific binding of primary and secondary antibodies was checked by making incubations with the nonimmune sera and by omitting the primary antibody, respectively. Applications of the diverse rabbit antisera and mouse monoclonal antibodies directed against differential, defined antigens were additional internal controls. The specificity of the primary antibodies for the respective human antigens has been shown previously. For anti-human ß-ENaC and anti-human NCC, the respective controls are given above.

Analyses of Sections
Sections were studied by epifluorescence with a Polyvar II microscope (Reichert Jung, Vienna, Austria). Images were acquired with a charge-coupled device camera (Visicam 1280, Visitron Systems, Puching, Germany) and processed by Image-Pro (Media Cybernetics, Silver Spring, MD) and Photoshop (Adobe, San Jose, CA) software.

Assessment of Fractional Distributions of Transport Proteins along the Distal Convolution
In humans, CaBP is detectable in the distal convolution and in the CCD (21). Thus, in a given section, CaBP labeling reveals 100% of the profiles of the distal convolution. In the first section of a series, we randomly numbered all CaBP-immunostained tubular profiles located within the cortical labyrinth (approximately 10 per section) and tracked the corresponding profiles over the uninterrupted series of 18 to a maximum of 39 sections (tissue depth of approximately 100 to 200 µm). CaBP-positive profiles of CCD were excluded from the calculations because of their histotopographic situation in the medullary rays. The sequential counterstaining of the sections with antibodies against differential transport proteins allowed us to attribute to each CaBP-positive tubular profile one or more of the given transport proteins; we were also able to establish their sequence along the distal convolution. The collected numbers of profiles with a given transport protein were expressed as fraction of collected numbers of the CaBP-positive profiles (approximately 70 to 120 per kidney). Because the tubular diameters along the distal convolution were approximately similar, the fractional values for profiles with the given transport protein should correspond to the respective fractional tubular length of the distal convolution.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Antibody Characterization
Previously described antibodies directed to sequences corresponding to NCC and the three ENaC subunits (19) were screened by using immunoblots of human tissue to determine which ones recognize the orthologous human proteins. Among these, only the antibody to rat–ß-ENaC recognized the human protein (Figure 1). The newly raised polyclonal antibody directed against a synthetic peptide corresponding to a portion of the amino terminal tail for human NCC was tested by immunoblot and preadsorption controls. In the immunoblot (Figure 1), the antibody recognized a single band of appropriate molecular weight, which was restricted to the cortex. The blot corresponded to that previously described (19). The preadsorption was performed with an IgG concentration of 0.15 µg/ml with or without 1 mg of the immunizing peptide per 30 ml of diluted antibody. The antibody was incubated with the peptide overnight at 4°C. The staining patterns of the tissues of both anti-NCC antibodies used in this study were identical.



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  		Figure 1. Immunoblots of homogenates from renal cortex, outer medulla, and inner medulla of human kidney probed with anti–Na-Cl cotransporter (NCC) and anti–ß-epithelial Na channel (ENaC) antibodies. Each lane was loaded with 10 µg of total protein for NCC blot and 20 µg of protein for ß-ENaC blot.

 
Immunohistochemical Analyses
The renal tissue used in this study was taken from the healthy parts of 4 tumor nephrectomized human kidneys from patients aged between 50 and 75 yr. The histology of the tumor revealed in all 4 cases a clear-cell renal carcinoma, stages 1 to 3, grades 2 to 4. In the case of the 50-yr-old woman, the tumor was predominantly of the rhabdosarcomatoïde variety. The overall aspect of the investigated renal tissue showed no overt pathologic changes in either the glomeruli or the interstitium. Most tubules were open. A few of them (mostly proximal tubules) contained cellular debris. Remnants of the tumor were absent.

Calbindin-D28k
The distribution in the human kidney of CaBP has been formerly described (21). Our data confirm the absence of immunohistochemically detectable CaBP in the TAL and its presence in the entire distal convolution and CCD. CaBP immunostaining was mostly rather weak in the initial portion of the distal convolution and rather strong in the major portion of the distal convolution. Yet more or less strongly stained portions alternated. CaBP immunostaining slightly decreased along the CCD. Intercalated cells were CaBP negative.

Distribution of Apical Transport Proteins in the Distal Convolution
The onset of cytoplasmic CaBP immunostaining (Figure 2a) coincided with the abrupt substitution of apical NKCC2 staining (Figure 2b), the transport protein characterizing the TAL (including the macula densa and the short postmacula segment of the TAL) by distinct apical NCC labeling of the tubular cells (Figure 2c). This point unequivocally marks the beginning of the distal convolution. The NCC positive profiles were often strikingly clustered (Figure 3). NCC labeling of the apical membrane was prominent over the major portion of the NCC-positive segment and slightly weakened just before its disappearance. Intriguingly, in three of the four investigated kidneys, the NCC-positive profiles revealed more or less abundant accumulations of autofluorescent phagolysosomes in their cytoplasm (Figures 2, d and e, and 4b), .



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  		Figure 2. Photomicrographs of consecutive Cryostat sections illustrating the transition from Na-K-2Cl cotransporter (NKCC2) to Na-Cl cotransporter (NCC). Sections are immunostained for calbindin-D28k (a), for NKCC2 (b, insert, d), and for NCC (c, e). The clear-cut substitution of NKCC2 (b, d) by NCC (c, e) coincides with the onset of weak calbindin staining (a) and marks the beginning (arrows) of the distal convoluted tubule (DCT). The insert in (b) shows a macula densa in the end portion of the thick ascending limb of Henle’s loop (TAL). (d, e) Autofluorescent lysosomes (arrowheads) are apparent exclusively in the NCC-positive profiles; the asterisk in the inset in (b) indicates TAL cells immediately at the transition from the TAL to the DCT. Bars, 50 µm.

 


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  		Figure 3. Photomicrograph of cryostat section illustrating the Na-Cl cotransporter (NCC) in the cortical labyrinth. The cryostat section is immunostained for NCC. The NCC-positive profiles are obviously clustered. Bar, 50 µm.

 


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  		Figure 4. Photomicrographs of consecutive cryostat sections illustrating the transition from the Na-Cl cotransporter (NCC) to epithelial Na channel (ENaC). Sections are immunostained for NCC (a) and for ENaC (b). The immunostained profiles are numbered in flow direction. The approximate transition from the distal convoluted tubule to the connecting tubule is indicated by arrows. Note the autofluorescent lysosomes in the NCC-displaying profiles 1 and 2. Insets: overlap of NCC and ENaC. Bar, 50 µm.

 
From the collected number of CaBP-labeled profiles in the cortical labyrinth (100%), approximately 30% displayed NCC. In a few profiles (less than 5% of the distal convolution), we observed coexpression for NCC and ENaC. In these profiles, immunostaining for NCC and ENaC was rather weak (Figures 4 and 5). At sites of coexpression of NCC and ENaC, the latter was occasionally found to be located in the apical cell membrane, and it was weak in the cytoplasm (Figure 4). The remainder—approximately 70% of the distal convolution—displayed heavy cytoplasmic immunostaining for ß-ENaC. The ENaC-positive profiles in the cortical labyrinth were preferentially located in the vicinity of the cortical radial vessels (Figure 6a).



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  		Figure 6. Photomicrographs of consecutive cryostat sections illustrating the epithelial Na channel (ENaC)- and aquaporin 2 (AQP2)-coexpressing profiles in the cortical labyrinth. Sections are immunostained for ENaC (a), AQP2 (b, d), and Na-Ca exchanger (NCX) (c). The ENaC- and AQP2-positive profiles are preferentially located around the cortical radial artery (A). The asterisk distinguishes between ENaC-positive profiles, which lack immunostaining for AQP2. The arrow in (b) points to the abrupt onset of AQP2. (c, d) Confluence of two tubules in the cortical labyrinth; the arrow in (c) indicates the direction of the tubular fluid flow. AQP2 appears shortly downstream the confluence (arrows in d); the NCX labeling varies in intensity along its occurrence (c). Bars, 50 µm.

 


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  		Figure 5. Photomicrographs of consecutive cryostat sections illustrating the distribution of calcium-ATPase and calbindin-D28k (CaBP) Na-Cl cotransporter (NCC), epithelial Na channel (ENaC), and Na-Ca exchanger (NCX) in the cortical labyrinth. Sections are immunostained for CaBP (a), NCC (b), ENaC, (c) and NCX (d). The profiles are numbered in the direction of flow. The weak calbindin staining in profiles 1 and 2 coincides with weak NCX staining. NCC labels the apical membrane of profiles 1 to 4. In profiles 3 and 4, weak NCC labeling overlaps with intermediate (3) to strong (4) ENaC, CaBP, and NCX labeling. Bar, 50 µm.

 
Most of the ENaC-labeled profiles also labeled for AQP2; apical AQP2 immunostaining started abruptly, usually at the site of the first confluence of tubules (Figure 6). AQP2 was never seen coexpressed with NCC. In all cases, the AQP2-positive portion of the distal convolution was preceded by a tubular portion (approximately 20% of the distal convolution), displaying ENaC alone. Of the CaBP-positive profiles in the cortical labyrinth, approximately 55% displayed immunostaining for AQP2.

Basolateral Proteins
NCX labeled the basolateral membranes of cells almost all along the distal convolution. At the beginning of the distal convolution, staining was barely detectable; then it progressively increased in the direction of the flow and consistently showed a steep increase a short distance before ENaC became detectable (Figure 5, c and d). Further downstream, strongly and weakly stained portions alternated (Figure 6c). NCX was found in the basolateral plasma membranes of at least 95% of CaBP-positive profiles in the labyrinth.

The distribution of the plasma membrane calcium ATPase, PMCA (22,23) located in the basolateral membranes, coincided with that of NCX—that is, it was exceedingly weak at the beginning of the distal convolution and increased in flow direction. AQP3 was seen in the basolateral membranes of all profiles that expressed the vasopressin-regulated water channel AQP2 in their apical aspect.

Intercalated Cells
Intercalated cells were recognized by their strong labeling for proton ATPase. They were negative for all other investigated proteins. At the beginning of the distal convolution, intercalated cells were never observed (Figure 7a). They were first met in low frequency in the second one-third of the NCC-stained tubular portion (Figure 7b), clearly before onset of detectable ENaC (Figure 7c), and increased in frequency approximately in parallel with the increasing basolateral NCX labeling. They were found in approximately 80% of CaBP-positive profiles in the labyrinth.



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  		Figure 7. Photomicrographs of consecutive cryostat sections illustrating the intercalated cells in the distal convolution. Sections are immunostained for Na-Cl cotransporter (NCC) (a), for H + ATPase (b), and for epithelial Na channel (ENaC) (c). The profiles are numbered in the direction of flow. Intercalated cells appear in NCC-labeled segment 2 and increase in abundance further downstream in the weakly NCC-labeled profile 3, in which the most upstream ENaC labeling is seen. Bar, 50 µm.

 
Medullary Rays
All profiles of the CCD were positive for ENaC, AQP2, and CaBP. In their basolateral membranes, they displayed strong AQP3 immunolabeling and intermediate NCX and PMCA immunolabeling (Figure 8). They all displayed intercalated cells. Figure 9 schematically summarizes the distributions along the human cortical distal nephron of the apical and basolateral transport proteins that are analyzed in this study.



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  		Figure 8. Photomicrographs of consecutive cryostat sections illustrating the cortical collecting duct (CCD). Sections are immunostained for epithelial Na channel (ENaC) (a), aquaporin 2 (AQP2) (b), calcium-ATPase and calbindin-D28k (CaBP) (c), plasma membrane Ca2+-ATPase (PMCA) (d), Na-Ca exchanger (NCX) (e), and aquaporin 3 (AQP3) (f). CCD with opening of an arcade; arrows in (a) and (b) indicate direction of urinary flow. Note colocalization of ENaC, AQP2, and AQP3 and of CaBP, PMCA, and NCX within the same cells. Bar, 50 µm.

 


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  		Figure 9. Schematic representation of distributions of transport proteins and intercalated cells along the human cortical distal nephron. Colored bars, the extension of a given transporter along the cortical distal nephron; shading of bars, gradual increases and decreases of immunostaining along the tubules; thick vertical lines, sharp onset or end of transport protein immunostaining. MR, medullary ray; TAL, thick ascending limb of Henle’s loop; DCT, distal convoluted tubule; CNT, connecting tubule; CCD, cortical collecting duct; G, glomerulus; NKCC2, bumetanide-sensitive Na-K-2Cl cotransporter; NCC, thiazide-sensitive Na-Cl cotransporter; ENaC, amiloride-sensitive epithelial Na channel; AQP2, vasopressin-dependent water channel aquaporin 2; ICc, intercalated cells; NCX, Na-Ca exchanger; PMCA, plasma membrane Ca2+-ATPase; CaBP, calcium binding protein calbindin-D28k.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study provides a comprehensive analysis on the immunohistochemical localization along the human distal convolution of the major renal Na+ and water transporting proteins, the NCC, ENaC, and AQP2, and of basolateral proteins involved in regulated, transcellular transport of Ca2+ ions. In contrast to rabbits, tubular portions with coexpression of different salt transporters and of vasopressin-regulated water channels exist in human distal convolution, but their relative extents seem to be less than in rats and mice (Figure 10). From all profiles of the human distal convolution, approximately 30% displayed NCC, approximately 75% ENaC, and approximately 55% AQP2. One salient difference in the distribution pattern of apical transporters between humans and rabbits is the upstream shift of vasopressin-dependent water channels, AQP2, into the CNT. Another relevant difference between humans and laboratory animals (rabbits, rats, mice) is the apparently much longer tubular portion in humans involved in transcellular Ca2+ transport (i.e., the entire distal convolution and the CCD) (Figure 10).



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  		Figure 10. Schematic comparison between rabbit, mouse/rat, and humans of organization of the distal convolution. The bars represent the distribution pattern of apical (1 to 3) and basolateral (4) transporters, and intercalated cells (green, dotted) along the distal convolution and along cortical collecting ducts (CCD). G, glomerulus; DCT, distal convoluted tubule; CNT, connecting tubule; 1, thiazide-sensitive Na-Cl cotransporter (NCC); 2, epithelial Na channel (ENaC); 3, vasopressin-dependent water channel aquaporin 2 (AQP2); 4, calcium transporting proteins (Na.Ca exchanger, plasma membrane calcium ATPase, calcium binding protein calbindin-D28k). The basic distribution pattern of transporters in the distal convolution is displayed by rabbits. In rats and mice, ENaC is shifted upstream into the DCT, and AQP2 is shifted upstream all along the CNT. In humans, NCC and ENaC overlap very little; in the most upstream portion of the CNT, AQP2 is not detectable. Calcium-transporting proteins in humans extend along the entire distal convolution and, in contrast to laboratory animals, along the CCD.

 
Abrupt substitution of apical transporters, or their gradual replacement and overlapping of different transporters, is manifest in corresponding sharply and gradually, respectively, occurring changes in epithelial structure. The structural differences along the distal convolution of many mammalian species had been observed and was described for the first time by Karl Peter in 1909 (24), who studied microdissected nephrons. He brilliantly depicted the gradual transitions along the human distal nephron (Figure 11), which match well with the now-described distribution pattern of transport proteins in the human distal convolution.



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  		Figure 11. Original drawings of an isolated human nephron by Karl Peter (1909) (22). (A) View from the glomerulus. (B) View from the cortical collecting duct (CCD). The coloration is that of the original; we added the lettering and arrows. A, cortical radial artery; G, glomerulus; uncolored, proximal tubule; yellow, TAL; light to dark brown, distal convolution; violet, collecting duct; 1, thick ascending limb of Henle’s loop (TAL); 2, approximate transition to the distal convoluted tubule; 3, approximate transition to the connecting tubule (CNT); 4, approximate transition to the CCD; asterisk, opening of the illustrated nephron into the CCD within the medullary ray. Note the correct histotopographical location of the TAL, passing between the glomerular arterioles, and of the CNT, ascending in the cortex in close vicinity to the cortical radial artery.

 
Putative Influences on the Distribution Pattern of Transport Proteins
We assume that the described distribution pattern, found to be the same in all four human kidneys examined in this study, is representative of the human nephron. In addition, the given individual conditions—diet, age, and preoperative therapy—might have affected the specific antigen abundance and locations along the segments and within the cells and might have caused some structural alterations.

The activity of amiloride-sensitive ENaC in the apical membrane is rate limiting for ENaC-mediated transepithelial sodium transport. In the human kidney, we found ENaC almost exclusively at cytoplasmic sites. In laboratory animals, this location of ENaC is associated with low plasma levels of mineralocorticoids or high dietary salt intake, whereas under high aldosterone or low salt intake, ENaC is shifted from cytoplasmic sites to the apical cell membrane (25–27). Clinical data about these parameters from the patients from whom the kidneys were taken are not available. Other pathophysiologic conditions (e.g., those associated with hypertension) affect expression and abundance of salt transporting proteins. For instance, the abundance of ENaC and NCC was increased in hypertensive obese Zucker rats compared with normotensive rat strains (28).

Our observations that approximately the second 55% of the distal convolution, the major part of the CNT, display the vasopressin-dependent water channel AQP2, agree with data by Chabardès et al. (29), who revealed vasopressin 2–receptor–mediated vasopressin responsiveness (30) in the human late DCT (CNT). Coleman et al. (31) suggested that the upstream extension of detectable AQP2 along the CNT might be modulated by the chronic plasma levels of vasopressin. In chronically vasopressin-substituted Brattleboro rats, Coleman et al. (31) recorded AQP2 immunoexpression further upstream than in untreated Brattleboro rats that genetically lack vasopressin. Protein abundance of AQP2 in the renal cortex of fasting rats was found to be reduced by approximately 60% (32).

In the inner medulla of senescent rats AQP2 and AQP3 expression was found to be downregulated by 80 and 50%, respectively, and AQP2 was redistributed from the apical membrane to intracellular compartments (33). Marked upregulation of AQP2 abundance was revealed in kidneys and in apical plasma membranes of collecting ducts in rats with diabetes mellitus experimentally induced by streptozotocin (34). These few examples from experimental animals should illustrate that numerous functional and pathophysiologic factors modulate the abundance and the extension of a given transporter along the segment, with corresponding functional consequences. It is conceivable that this applies to humans as well.

An intriguing observation in three of the four kidneys is the striking accumulation of autophagolysosomes exclusively in the DCT (Figures 2 and 4). In early electron microscopic studies on renal tissue samples from healthy young men (35), lysosomes were not described in DCT cells, yet lipofuscin or "degeneration" pigment was occasionally observed. The renal tissue we studied was taken from much older people, and putatively, the masses of phagolysosomes seen in the DCT might be related to the age of the patients. However, the definite distribution of phagolysosomes in the DCT instead suggests that their occurrence might be correlated with agents that specifically affect the cells of this segment. Two of the patients had been chronically treated with thiazides and another with unspecified antihypertensive therapy, which likely included diuretics. For the fourth patient in whom accumulations of phagolysosomes in the DCT were not apparent, no diuretic or antihypertensive therapy was recorded.

Thiazides specifically inhibit Na-Cl entry via NCC, displayed in the kidney exclusively by the DCT. The findings in the human kidneys strikingly resembled those found in rats treated for 3 d and longer with thiazide diuretics. In the otherwise unaffected rat kidneys, we observed apoptosis and massive accumulations of large autophagocytotic lysosomes exclusively in DCT cells (36). Meta-analyses (37,38) have revealed a significant correlation between thiazide treatment and the (altogether low) risk for renal cell carcinoma, in particular in women. Verlander et al. (39) demonstrated that in rats, NCC expression is regulated by estrogens.

Functional Implications of Serial Arrangement and Coexpression of Transport Proteins
The activity of the transport pathways in the apical membrane, which is rate limiting for transcellular transports, is controlled by luminal salt delivery, flow rates, or both and by plasma levels of various hormones among other means (40). Coexpression by the same cells of two differentially controlled transport systems might enhance by mutual interaction of transports the possibilities for fine regulation of salt excretion.

Changes of transport rates at a given site necessarily have, via changes of tubular fluid composition, flow rate, or both, an effect on transport rates at downstream sites. Therefore, the sequential arrangement of apical transport proteins along the distal convolution is important for modulating the overall effect of regulatory agents such as hormones and diuretics. Such mechanisms may be relevant in therapeutic considerations (e.g., in treatment with diuretics). For instance, inhibition of salt reabsorption by the TAL (e.g., furosemide treatment) necessarily results in a higher solute load delivered to the immediately downstream segment, the DCT, as long as salt and fluid losses are replaced. Transport rates by the NCC-displaying DCT epithelium seem to respond primarily to luminal sodium delivery (41,42). The chronically higher solute load in the DCT of rats entails upregulation of NCC expression (17,43) and eventually hypertrophy of the DCT epithelium (44), which endows the DCT with a capacity for higher salt transport rates (41,45). These might partially compensate the impaired salt reabsorption by the TAL. Such a mechanism might contribute to the decreasing efficiency of loop diuretics in humans receiving prolonged diuretic treatment (46). The usually increased aldosterone plasma levels that occur during diuretic treatment might be another factor involved in the observed adaptation of the DCT (47); data suggest that NCC might be a previously unrecognized target for regulation by aldosterone (19,48).

The established target for mineralocorticoid-regulated salt reabsorption in the nephron is the sodium transport channel prevailing in the CNT and CCD, downstream of the DCT: the amiloride-sensitive ENaC (49). Data from laboratory animals suggest that the aldosterone-initiated insertion of ENaC from intracellular pools into the apical membrane might be influenced by luminal factors (26)—among others, by the tubular solute load or the flow rate (50). Thus, luminal factors putatively constitute a means for modulating aldosterone-controlled salt reabsorption. Immediately downstream from the first joining together of CNT in the cortical labyrinth, the human CNT cells coexpress both ENaC and AQP2. ENaC-mediated cellular sodium entry might generate a positive osmotic gradient for water entry into the cells (51), and vasopressin-stimulated water entry into the cells might sustain the gradient for sodium entry into the cells (52,53).

Interaction of Transcellular Ca2+ and Na+ Transport
Transcellular Ca2+ transport requires, in addition to apical entry pathways, Ca2+-extrusion machinery, NCX, and PMCA, in the basolateral membranes and cytoplasmic calcium-binding proteins to uphold the driving force for apical Ca2+ entry (54). The presence of these proteins in established sodium-transporting epithelia provides evidence of the additional function of these epithelia in transcellular Ca2+ transport.

In the human distal nephron, we used immunohistochemistry to reveal these proteins all along the DCT and CNT, although in varying quantity, and in addition in the CCD. In rabbits, the Ca2+-extruding machinery was consistently detectable in the CNT (54,55); in rats (17,23) and mice (10,14), this was in addition in the last portion of the DCT, but not in the CCD. In these species, the sites with the highest manifestation of the calcium-extruding machinery displayed, in addition to sodium entry pathways (NCC, ENaC) (13), the recently discovered renal epithelial calcium channel, ECaC1 (56,57). In the human nephron, ECaC1 has not yet been located.

Under conditions of impaired sodium transport, voltage-gated channels in the apical membrane can be activated that, under settings of regular sodium transport, are not operative for Ca2+ (1,18,57). For instance, inhibition of sodium transport, via NCC by thiazides or via ENaC by amiloride (58,59), is associated with increased transcellular calcium reabsorption, inciting hypocalciuria (1,18). Hypocalciuria is also the leading symptom in humans with genetic loss-of-function mutations in the NCC gene, known as Gitelman syndrome (2). The manifestation in the human distal nephron of the Ca2+-extruding machinery over a much longer tubular portion than in laboratory animals might disclose a human-specific regulation of renal calcium excretion. Alternatively, it might be related to prolonged diuretic therapy of the patients, causing Ca2+ entry at sites of inhibited Na+ entry. Future studies on experimental animals under prolonged thiazide and amiloride treatment should elucidate this question.

The organization of the transport pathways of the human distal convolution shows a sequential distribution of NCC, ENaC, and AQP2 similar to that of laboratory animals. However, the detailed arrangement of the apical transport pathways along the distal convolution with respect to ENaC and AQP2 shows distinct differences to other species. Furthermore, in humans, the entire distal convolution (and, unlike in laboratory animals, the CCD as well) seems to be involved in transcellular calcium reabsorption. The knowledge of the precise distribution patterns of transport proteins in the human distal nephron may be a help in diagnosis as we seek to better understand disorders of salt homeostasis and to refine their therapeutic management.


   Acknowledgments
 
We thank Professors Jaquemin and Saussine, Service d’Urologie, Hôpital Universitaire, Strasbourg, France, for placing at our disposal the renal tissue of tumor nephrectomized kidneys and enabling us to undertake this study. The investigations were supported by the Swiss National Foundation (grant 31-47742-96 to BK).


   Footnotes
 
The first two authors contributed equally to the study.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication October 2, 2001. Accepted for publication December 4, 2001.




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