Human Cortical Distal Nephron: Distribution of Electrolyte...

Human Cortical Distal Nephron: Distribution of Electrolyteand Water Transport Pathways

HELENA LAGGER BINER,* MARIE-PIERRE ARPIN-BOTT,† JOHANNES LOFFING,*XIAOYAN WANG,‡ MARK KNEPPER,‡ STEVE C. HEBERT,§ andBRIGITTE KAISSLING**Anatomical Department, University of Zurich, Zurich, Switzerland; †UMR CNRS 7519, UniversityLouis Pasteur, Strasbourg, France; ‡National Heart, Lung, and Blood Institute, Bethesda, Maryland;and §Department of Cellular and Molecular Physiology, Yale University School of Medicine,New Haven, Connecticut.

Abstract. The exact distributions of the different salt transportsystems along the human cortical distal nephron are unknown.Immunohistochemistry was performed on serial cryostat sec-tions of healthy parts of tumor nephrectomized human kidneysto study the distributions in the distal convolution of thethiazide-sensitive Na-Cl cotransporter (NCC), the � subunit ofthe amiloride-sensitive epithelial Na channel (ENaC), the va-sopressin-sensitive water channel aquaporin 2 (AQP2), andaquaporin 3 (AQP3), the H� ATPase, the Na-Ca exchanger(NCX), plasma membrane calcium-ATPase, and calbindin-D28k (CaBP). The entire human distal convolution and thecortical collecting duct (CCD) display calbindin-D28k, al-though in variable amounts. Approximately 30% of the distalconvolution profiles reveal NCC, characterizing the distal con-voluted tubule. NCC overlaps with ENaC in a short portion at

the end of the distal convoluted tubule. ENaC is displayed allalong the connecting tubule (70% of the distal convolution)and the CCD. The major part of the connecting tubule and theCCD coexpress aquaporin 2 with ENaC. Intercalated cells,undetected in the first 20% of the distal convolution, wereinterspersed among the segment-specific cells of the remainder ofthe distal convolution, and of the CCD. The basolateral calciumextruding proteins, Na-Ca exchanger (NCX), and the plasmamembrane Ca2�-ATPase were found all along the distal convo-lution, and, in contrast to other species, along the CCD, althoughin varying amounts. The knowledge regarding the precise distri-bution patterns of transport proteins in the human distal nephronand the knowledge regarding the differences from that in labora-tory animals may be helpful for diagnostic purposes and may alsohelp refine the therapeutic management of electrolyte disorders.

The cortical distal nephron is the site for fine regulation of saltand water excretion by peptide and mineralocorticoid hor-mones and the site for specific actions of diuretics (1). Inhumans alterations of BP (2–4), as well as some disorders ofsodium, potassium, calcium, magnesium, and volume ho-meostasis (3), have been associated with mutations of genesencoding for salt and water transport proteins in the distalnephron (5–8). These observations emphasize the growingimportance of this nephron portion to human physiology and tohuman disease (9).

According to microanatomical criteria, the distal nephroncomprises a straight part (TAL) and the distal convolution.Structural and functional criteria further subdivide the latterinto the distal convoluted tubule (DCT) and the connectingtubule (CNT) (10). Functionally, the cortical collecting duct

(CCD) is included in the distal nephron. Apical salt entry intothe cells of the distal nephron is mediated by three pathways:in the thick ascending limb of Henle’s loop (TAL) by thebumetanide/furosemide-sensitive Na-K-2Cl cotransporter(NKCC2), in the distal convolution by the thiazide-sensitiveNa-Cl cotransporter (NCC), and by the amiloride-sensitiveepithelial Na channel (ENaC) (1). Vasopressin-regulated watertransport via aquaporin 2 (AQP2) is usually believed to be afunction of collecting ducts (8,11).

Species differences have been reported with respect to thedistribution pattern of NCC, ENaC, and AQP2 within the distalconvolution. In rabbits, the sharp structural transitions from theDCT to the CNT and from the CNT to the CCD preciselycoincide with the substitution of NCC by ENaC and with theonset of AQP2, respectively (12). Within the distal convolutionof rats and mice, structural changes are gradual (13). Overlap-ping of NCC and ENaC at one transition (14,15) and of ENaCand AQP2 (16) at the other have been observed, and segmen-tation becomes a matter of definition. The seemingly relativesmall variations in the distribution pattern among species mightentail distinct differences in the fine control of salt excretion,making extrapolation of data from one species to anotherquestionable.

Although data by Obermuller et al. (17) demonstrated thatNCC characterizes the initial portion of the distal convolution

Received October 2, 2001. Accepted December 4, 2001.The first two authors contributed equally to the study.Correspondence to: Dr. Brigitte Kaissling, University of Zurich, AnatomicalInstitute, Division of Vegetative Anatomy, Winterthurerstrasse 190, 8057 Zurich,Switzerland. Phone: 41-1-635-5320 Fax: �41-1-635-5702; E-mail:[email protected]

1046-6673/1304-0836Journal of the American Society of NephrologyCopyright © 2002 by the American Society of Nephrology

J Am Soc Nephrol 13: 836–847, 2002

in the human kidney as well, the exact organization of thehuman distal convolution is incompletely known. Therefore,the aim of the study presented here was to show by immuno-chemistry the distribution patterns along the human distalconvolution of a set of apical salt and water transport proteins(NCC, ENaC, AQP2). Because transcellular sodium and cal-cium transport are closely interrelated (18), we included in ourstudy the localization of proteins involved in transcellularcalcium reabsorption, the basolateral Na�-Ca2� exchanger(NCX), the plasma membrane Ca2�-ATPase (PMCA), and thecytoplasmic protein calbindin-D28k (CaBP). Our results dem-onstrate that as in other mammalian species, the apical trans-port proteins in the human distal nephron are serially arranged,but their detailed distribution patterns differ markedly fromthose in laboratory animals.

Materials and MethodsThe 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, respec-tively, and 1 woman, 50 yr old at time of surgery, were investigated.The preoperative creatinine values were in all cases in the normalrange (7 to 14 mg/L). For at least 6 mo before surgery, the 3 menreceived antihypertensive therapy with thiazide diuretics.

Tissue ProcessingImmediately 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, ad-justed 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, thecortical 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 wasstored at �80°C until further processing.

Production of Anti-Human NCC AntibodyTo produce a suitable rabbit polyclonal antibody to human NCC,

rabbits were immunized to a synthetic peptide (sequence PGEP-RKVRPTLADLHSFLKQEGC-COOH), corresponding to a portionof the amino acid terminal tail of human NCC. The peptide waspurified via HPLC and conjugated to keyhole limpet hemocyanin asdescribed previously (19). The resulting antisera were affinity purifiedon a column on which the same peptide was covalently linked to themedium (agarose beads). These antibodies were screened by immu-noblotting. One of the antibodies (A857) strongly recognized NCC onimmunoblots (see Results).

ImmunoblotImmunoblotting was used to screen existing antibodies for recog-

nition of the orthologous protein targets in human tissue and tocharacterize the new anti-human NCC antibody described above.Homogenates were prepared from cortex, outer medulla, and innermedulla of a human kidney removed because of tumor. The proteinconcentration was measured, and the samples were solubilized inLaemmli sample buffer as described previously (19). Sodium dodecylsulfate–polyacrylamide gel electrophoresis was performed on 7.5 or10% polyacrylamide gels (Ready Gels, Bio-Rad, Hercules, CA), and

the proteins were transferred from the gel electrophoretically to ni-trocellulose membranes. Membranes were probed overnight at 4°Cwith the respective primary antibodies, then exposed to secondaryantibody (Donkey anti-rabbit IgG conjugated with horseradish perox-idase, Pierce no. 31458, diluted to 1:5000) for 1 hr at room temper-ature. Sites of antibody-antigen reaction were visualized by means ofa luminal-based enhanced chemiluminescence substrate (LumiGlo;Kirkegaard and Perry Laboratories, Gaithersburg, MD) before expo-sure to x-ray film (no. 165-1579; Eastman Kodak, Rochester, NY).

Staining StrategyFor immunohistochemistry, we cut at least 10 series of 5-�m-thick

cryostat sections from each kidney (HM 500 OM, Zeiss, Germany).Each section series was made from a different, randomly orientedcortical tissue block and comprised between 18 and 39 consecutivesections. All sections of a series were placed successively onto num-bered slides (three sections per slide) and double immunostained.

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

Immunostaining ProcedureAfter pretreatment for 10 min with 10% normal goat serum in

phosphate-buffered saline, the sections were incubated at 4°C forapproximately 15 h in a humidified chamber with a mixture ofanti-CaBP and one of the other primary antibodies (Table1) diluted inphosphate-buffered saline–1% bovine serum albumin. For detectionof the proton ATPase sections were treated before incubation withsodium dodecyl sulfate 5% during 10 min (20) at 4°C.

For detection of the binding sites of the primary antibodies, thesections were incubated in a humidified chamber at 20°C during 45min with a mixture of the 2 respective secondary antibodies, goat–anti-rabbit IgG, and goat–anti-mouse IgG, conjugated with CY3 andFITC, respectively (Jackson Immuno Research Laboratories, WestGrove, PA). Per 1 ml of the working dilution of the secondaryantibodies, 2 �g of 4,6-diamidino-2-phenylindole dihydrochloride(Boehringer, Mannheim, Germany) was added for nuclear staining.After repeated rinsing with phosphate-buffered saline, the sectionswere 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 waschecked by making incubations with the nonimmune sera and byomitting the primary antibody, respectively. Applications of the di-verse rabbit antisera and mouse monoclonal antibodies directedagainst differential, defined antigens were additional internal controls.The specificity of the primary antibodies for the respective humanantigens has been shown previously. For anti-human �-ENaC andanti-human NCC, the respective controls are given above.

Analyses of SectionsSections were studied by epifluorescence with a Polyvar II microscope

(Reichert Jung, Vienna, Austria). Images were acquired with a charge-

J Am Soc Nephrol 13: 836–847, 2002 Human Distal Nephron 837

coupled device camera (Visicam 1280, Visitron Systems, Puching, Ger-many) and processed by Image-Pro (Media Cybernetics, Silver Spring,MD) and Photoshop (Adobe, San Jose, CA) software.

Assessment of Fractional Distributions of TransportProteins along the Distal Convolution

In humans, CaBP is detectable in the distal convolution and in theCCD (21). Thus, in a given section, CaBP labeling reveals 100% ofthe profiles of the distal convolution. In the first section of a series, werandomly numbered all CaBP-immunostained tubular profiles locatedwithin the cortical labyrinth (approximately 10 per section) andtracked the corresponding profiles over the uninterrupted series of 18to a maximum of 39 sections (tissue depth of approximately 100 to200 �m). CaBP-positive profiles of CCD were excluded from thecalculations because of their histotopographic situation in the medul-lary rays. The sequential counterstaining of the sections with antibod-ies against differential transport proteins allowed us to attribute toeach CaBP-positive tubular profile one or more of the given transportproteins; we were also able to establish their sequence along the distalconvolution. The collected numbers of profiles with a given transportprotein were expressed as fraction of collected numbers of the CaBP-positive profiles (approximately 70 to 120 per kidney). Because thetubular diameters along the distal convolution were approximatelysimilar, the fractional values for profiles with the given transportprotein should correspond to the respective fractional tubular length ofthe distal convolution.

ResultsAntibody Characterization

Previously described antibodies directed to sequences cor-responding to NCC and the three ENaC subunits (19) werescreened by using immunoblots of human tissue to determinewhich ones recognize the orthologous human proteins. Amongthese, only the antibody to rat–�-ENaC recognized the humanprotein (Figure 1). The newly raised polyclonal antibody di-rected against a synthetic peptide corresponding to a portion of

the amino terminal tail for human NCC was tested by immu-noblot and preadsorption controls. In the immunoblot (Figure1), the antibody recognized a single band of appropriate mo-lecular weight, which was restricted to the cortex. The blotcorresponded to that previously described (19). The preadsorp-tion was performed with an IgG concentration of 0.15 �g/mlwith or without 1 mg of the immunizing peptide per 30 ml ofdiluted antibody. The antibody was incubated with the peptideovernight at 4°C. The staining patterns of the tissues of bothanti-NCC antibodies used in this study were identical.

Table 1. Primary antibodies used in immunostaining

Antigen Host Dilution Source

ApicalNKCC2, bumetanide-sensitive Na�-K�-2Cl cotransporter Rabbit 1:500 M.A. KnepperNCC, thiazide-sensitive Na�-Cl� cotransporter (A857) Rabbit 1:40,000 M.A. KnepperNCC, thiazide-sensitive Na�-Cl� cotransporter Rabbit 1:500 S.C. HebertENaC, � subunit of amiloride-sensitive epithelial Na channel (L558) Rabbit 1:500 M.A. KnepperAQP2, vasopressin-sensitive water channel, Aquaporin 2 Rabbit 1:500 M.A. Knepper

Apical and basolateralH�-ATPase (B2) proton-ATPase Rabbit 1:500 M.A. Knepper

BasolateralNCX, Na�-Ca2� exchanger Rabbit 1:1000 Swant, BellinzonaAQP3, Aquaporin 3 Rabbit 1:500 M.A. KnepperNa�-K�-ATPase Rabbit 1:500 Upstate Biotechnology,

Lake Placid, NYPMCA, plasma membrane Ca-ATPase Mouse 1:1 Sigma, St. Louis, MOCytoplasmic and nuclearCaBP, calbindin-D 28k Mouse 1:10000 Swant, BellinzonaCaBP, calbindin-D 28k Rabbit 1:5000 Swant, Bellinzona

Figure 1. Immunoblots of homogenates from renal cortex, outermedulla, and inner medulla of human kidney probed with anti–Na-Clcotransporter (NCC) and anti–�-epithelial Na channel (ENaC) anti-bodies. Each lane was loaded with 10 �g of total protein for NCC blotand 20 �g of protein for �-ENaC blot.

838 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 836–847, 2002

Immunohistochemical AnalysesThe renal tissue used in this study was taken from the

healthy parts of 4 tumor nephrectomized human kidneys frompatients aged between 50 and 75 yr. The histology of the tumorrevealed in all 4 cases a clear-cell renal carcinoma, stages 1 to3, grades 2 to 4. In the case of the 50-yr-old woman, the tumorwas predominantly of the rhabdosarcomatoıde variety. Theoverall aspect of the investigated renal tissue showed no overtpathologic changes in either the glomeruli or the interstitium.Most tubules were open. A few of them (mostly proximaltubules) contained cellular debris. Remnants of the tumor wereabsent.

Calbindin-D28kThe distribution in the human kidney of CaBP has been

formerly described (21). Our data confirm the absence ofimmunohistochemically detectable CaBP in the TAL and itspresence in the entire distal convolution and CCD. CaBPimmunostaining was mostly rather weak in the initial portionof the distal convolution and rather strong in the major portionof the distal convolution. Yet more or less strongly stainedportions alternated. CaBP immunostaining slightly decreasedalong the CCD. Intercalated cells were CaBP negative.

Distribution of Apical Transport Proteins in theDistal Convolution

The onset of cytoplasmic CaBP immunostaining (Figure 2a)coincided with the abrupt substitution of apical NKCC2 stain-ing (Figure 2b), the transport protein characterizing the TAL(including the macula densa and the short postmacula segmentof the TAL) by distinct apical NCC labeling of the tubular cells(Figure 2c). This point unequivocally marks the beginning ofthe distal convolution. The NCC positive profiles were oftenstrikingly clustered (Figure 3). NCC labeling of the apicalmembrane was prominent over the major portion of the NCC-positive segment and slightly weakened just before its disap-pearance. Intriguingly, in three of the four investigated kid-neys, the NCC-positive profiles revealed more or less abundantaccumulations of autofluorescent phagolysosomes in their cy-toplasm (Figures 2, d and e, and 4b), .

From the collected number of CaBP-labeled profiles in thecortical labyrinth (100%), approximately 30% displayed NCC.In a few profiles (less than 5% of the distal convolution), weobserved coexpression for NCC and ENaC. In these profiles,immunostaining for NCC and ENaC was rather weak (Figures4 and 5). At sites of coexpression of NCC and ENaC, the latterwas occasionally found to be located in the apical cell mem-brane, and it was weak in the cytoplasm (Figure 4). Theremainder—approximately 70% of the distal convolution—displayed heavy cytoplasmic immunostaining for �-ENaC.The ENaC-positive profiles in the cortical labyrinth were pref-erentially located in the vicinity of the cortical radial vessels(Figure 6a).

Most of the ENaC-labeled profiles also labeled for AQP2;apical AQP2 immunostaining started abruptly, usually at thesite 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 atubular portion (approximately 20% of the distal convolution),displaying ENaC alone. Of the CaBP-positive profiles in thecortical labyrinth, approximately 55% displayed immunostain-ing for AQP2.

Figure 2. Photomicrographs of consecutive Cryostat sections illus-trating the transition from Na-K-2Cl cotransporter (NKCC2) to Na-Clcotransporter (NCC). Sections are immunostained for calbindin-D28k(a), for NKCC2 (b, insert, d), and for NCC (c, e). The clear-cutsubstitution of NKCC2 (b, d) by NCC (c, e) coincides with the onsetof weak calbindin staining (a) and marks the beginning (arrows) of thedistal convoluted tubule (DCT). The insert in (b) shows a maculadensa in the end portion of the thick ascending limb of Henle’s loop(TAL). (d, e) Autofluorescent lysosomes (arrowheads) are apparentexclusively in the NCC-positive profiles; the asterisk in the inset in (b)indicates TAL cells immediately at the transition from the TAL to theDCT. Bars, 50 �m.


Basolateral ProteinsNCX labeled the basolateral membranes of cells almost all

along the distal convolution. At the beginning of the distalconvolution, staining was barely detectable; then it progres-sively increased in the direction of the flow and consistentlyshowed a steep increase a short distance before ENaC becamedetectable (Figure 5, c and d). Further downstream, stronglyand weakly stained portions alternated (Figure 6c). NCX wasfound in the basolateral plasma membranes of at least 95% ofCaBP-positive profiles in the labyrinth.

The distribution of the plasma membrane calcium ATPase,PMCA (22,23) located in the basolateral membranes, coin-cided with that of NCX—that is, it was exceedingly weak atthe beginning of the distal convolution and increased in flowdirection. AQP3 was seen in the basolateral membranes of allprofiles that expressed the vasopressin-regulated water channelAQP2 in their apical aspect.

Intercalated CellsIntercalated cells were recognized by their strong labeling

for proton ATPase. They were negative for all other investi-gated proteins. At the beginning of the distal convolution,intercalated cells were never observed (Figure 7a). They werefirst met in low frequency in the second one-third of theNCC-stained tubular portion (Figure 7b), clearly before onsetof detectable ENaC (Figure 7c), and increased in frequencyapproximately in parallel with the increasing basolateral NCXlabeling. They were found in approximately 80% of CaBP-positive profiles in the labyrinth.

Medullary RaysAll profiles of the CCD were positive for ENaC, AQP2, and

CaBP. In their basolateral membranes, they displayed strongAQP3 immunolabeling and intermediate NCX and PMCA

immunolabeling (Figure 8). They all displayed intercalatedcells. Figure 9 schematically summarizes the distributionsalong the human cortical distal nephron of the apical andbasolateral transport proteins that are analyzed in this study.

DiscussionOur study provides a comprehensive analysis on the immu-

nohistochemical localization along the human distal convolu-tion of the major renal Na� and water transporting proteins, theNCC, ENaC, and AQP2, and of basolateral proteins involvedin regulated, transcellular transport of Ca2� ions. In contrast torabbits, tubular portions with coexpression of different salttransporters and of vasopressin-regulated water channels existin human distal convolution, but their relative extents seem to

Figure 3. Photomicrograph of cryostat section illustrating the Na-Clcotransporter (NCC) in the cortical labyrinth. The cryostat section isimmunostained for NCC. The NCC-positive profiles are obviouslyclustered. Bar, 50 �m.

Figure 4. Photomicrographs of consecutive cryostat sections illus-trating the transition from the Na-Cl cotransporter (NCC) to epithelialNa channel (ENaC). Sections are immunostained for NCC (a) and forENaC (b). The immunostained profiles are numbered in flow direc-tion. The approximate transition from the distal convoluted tubule tothe connecting tubule is indicated by arrows. Note the autofluorescentlysosomes in the NCC-displaying profiles 1 and 2. Insets: overlap ofNCC and ENaC. Bar, 50 �m.


Figure 5. Photomicrographs of consecutive cryostat sections illustrat-ing the distribution of calcium-ATPase and calbindin-D28k (CaBP)Na-Cl cotransporter (NCC), epithelial Na channel (ENaC), and Na-Caexchanger (NCX) in the cortical labyrinth. Sections are immuno-stained for CaBP (a), NCC (b), ENaC, (c) and NCX (d). The profilesare numbered in the direction of flow. The weak calbindin staining inprofiles 1 and 2 coincides with weak NCX staining. NCC labels theapical membrane of profiles 1 to 4. In profiles 3 and 4, weak NCClabeling overlaps with intermediate (3) to strong (4) ENaC, CaBP, andNCX labeling. Bar, 50 �m.

Figure 6. Photomicrographs of consecutive cryostat sections illustrat-ing the epithelial Na channel (ENaC)- and aquaporin 2 (AQP2)-coexpressing profiles in the cortical labyrinth. Sections are immuno-stained for ENaC (a), AQP2 (b, d), and Na-Ca exchanger (NCX) (c).The ENaC- and AQP2-positive profiles are preferentially locatedaround the cortical radial artery (A). The asterisk distinguishes be-tween ENaC-positive profiles, which lack immunostaining for AQP2.The arrow in (b) points to the abrupt onset of AQP2. (c, d) Confluenceof two tubules in the cortical labyrinth; the arrow in (c) indicates thedirection of the tubular fluid flow. AQP2 appears shortly downstreamthe confluence (arrows in d); the NCX labeling varies in intensityalong its occurrence (c). Bars, 50 �m.


be less than in rats and mice (Figure 10). From all profiles ofthe human distal convolution, approximately 30% displayedNCC, approximately 75% ENaC, and approximately 55%AQP2. One salient difference in the distribution pattern ofapical transporters between humans and rabbits is the upstreamshift of vasopressin-dependent water channels, AQP2, into theCNT. Another relevant difference between humans and labo-ratory animals (rabbits, rats, mice) is the apparently muchlonger tubular portion in humans involved in transcellularCa2� transport (i.e., the entire distal convolution and the CCD)(Figure 10).

Abrupt substitution of apical transporters, or their gradualreplacement and overlapping of different transporters, is man-ifest in corresponding sharply and gradually, respectively, oc-

curring changes in epithelial structure. The structural differ-ences along the distal convolution of many mammalian specieshad been observed and was described for the first time by KarlPeter in 1909 (24), who studied microdissected nephrons. Hebrilliantly depicted the gradual transitions along the humandistal nephron (Figure 11), which match well with the now-described distribution pattern of transport proteins in the hu-man distal convolution.

Figure 7. Photomicrographs of consecutive cryostat sections illustrat-ing the intercalated cells in the distal convolution. Sections are im-munostained for Na-Cl cotransporter (NCC) (a), for H � ATPase (b),and for epithelial Na channel (ENaC) (c). The profiles are numberedin the direction of flow. Intercalated cells appear in NCC-labeledsegment 2 and increase in abundance further downstream in theweakly NCC-labeled profile 3, in which the most upstream ENaClabeling is seen. Bar, 50 �m.

Figure 8. Photomicrographs of consecutive cryostat sections illustrat-ing the cortical collecting duct (CCD). Sections are immunostainedfor epithelial Na channel (ENaC) (a), aquaporin 2 (AQP2) (b), calci-um-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 samecells. Bar, 50 �m.


Putative Influences on the Distribution Pattern ofTransport Proteins

We assume that the described distribution pattern, found tobe the same in all four human kidneys examined in this study,is representative of the human nephron. In addition, the givenindividual conditions—diet, age, and preoperative therapy—might have affected the specific antigen abundance and loca-tions along the segments and within the cells and might havecaused some structural alterations.

The activity of amiloride-sensitive ENaC in the apical mem-brane is rate limiting for ENaC-mediated transepithelial so-dium transport. In the human kidney, we found ENaC almostexclusively at cytoplasmic sites. In laboratory animals, thislocation of ENaC is associated with low plasma levels ofmineralocorticoids or high dietary salt intake, whereas underhigh aldosterone or low salt intake, ENaC is shifted fromcytoplasmic sites to the apical cell membrane (25–27). Clinicaldata about these parameters from the patients from whom thekidneys were taken are not available. Other pathophysiologicconditions (e.g., those associated with hypertension) affectexpression and abundance of salt transporting proteins. Forinstance, the abundance of ENaC and NCC was increased inhypertensive obese Zucker rats compared with normotensiverat strains (28).

Our observations that approximately the second 55% of thedistal convolution, the major part of the CNT, display thevasopressin-dependent water channel AQP2, agree with databy Chabardes et al. (29), who revealed vasopressin 2–receptor–

mediated vasopressin responsiveness (30) in the human lateDCT (CNT). Coleman et al. (31) suggested that the upstreamextension of detectable AQP2 along the CNT might be mod-ulated by the chronic plasma levels of vasopressin. In chroni-cally vasopressin-substituted Brattleboro rats, Coleman et al.(31) recorded AQP2 immunoexpression further upstream thanin untreated Brattleboro rats that genetically lack vasopressin.Protein abundance of AQP2 in the renal cortex of fasting ratswas found to be reduced by approximately 60% (32).

In the inner medulla of senescent rats AQP2 and AQP3expression was found to be downregulated by 80 and 50%,respectively, and AQP2 was redistributed from the apicalmembrane to intracellular compartments (33). Marked upregu-lation of AQP2 abundance was revealed in kidneys and inapical plasma membranes of collecting ducts in rats withdiabetes mellitus experimentally induced by streptozotocin(34). These few examples from experimental animals shouldillustrate that numerous functional and pathophysiologic fac-tors modulate the abundance and the extension of a giventransporter along the segment, with corresponding functionalconsequences. It is conceivable that this applies to humans aswell.

An intriguing observation in three of the four kidneys is thestriking accumulation of autophagolysosomes exclusively inthe DCT (Figures 2 and 4). In early electron microscopicstudies on renal tissue samples from healthy young men (35),lysosomes were not described in DCT cells, yet lipofuscin or

Figure 9. Schematic representation of distributions of transport proteins and intercalated cells along the human cortical distal nephron. Coloredbars, the extension of a given transporter along the cortical distal nephron; shading of bars, gradual increases and decreases of immunostainingalong the tubules; thick vertical lines, sharp onset or end of transport protein immunostaining. MR, medullary ray; TAL, thick ascending limbof 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.


“degeneration” pigment was occasionally observed. The renaltissue we studied was taken from much older people, andputatively, the masses of phagolysosomes seen in the DCTmight be related to the age of the patients. However, thedefinite distribution of phagolysosomes in the DCT insteadsuggests that their occurrence might be correlated with agentsthat specifically affect the cells of this segment. Two of thepatients had been chronically treated with thiazides and anotherwith unspecified antihypertensive therapy, which likely in-cluded diuretics. For the fourth patient in whom accumulationsof phagolysosomes in the DCT were not apparent, no diureticor antihypertensive therapy was recorded.

Thiazides specifically inhibit Na-Cl entry via NCC, dis-played in the kidney exclusively by the DCT. The findings inthe human kidneys strikingly resembled those found in ratstreated for 3 d and longer with thiazide diuretics. In theotherwise unaffected rat kidneys, we observed apoptosis andmassive accumulations of large autophagocytotic lysosomesexclusively in DCT cells (36). Meta-analyses (37,38) haverevealed a significant correlation between thiazide treatmentand the (altogether low) risk for renal cell carcinoma, inparticular in women. Verlander et al. (39) demonstrated that inrats, NCC expression is regulated by estrogens.

Functional Implications of Serial Arrangement andCoexpression of Transport Proteins

The activity of the transport pathways in the apical mem-brane, which is rate limiting for transcellular transports, iscontrolled by luminal salt delivery, flow rates, or both and byplasma levels of various hormones among other means (40).Coexpression by the same cells of two differentially controlledtransport systems might enhance by mutual interaction oftransports 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, aneffect on transport rates at downstream sites. Therefore, thesequential arrangement of apical transport proteins along thedistal convolution is important for modulating the overall ef-fect of regulatory agents such as hormones and diuretics. Suchmechanisms may be relevant in therapeutic considerations(e.g., in treatment with diuretics). For instance, inhibition ofsalt reabsorption by the TAL (e.g., furosemide treatment) nec-essarily results in a higher solute load delivered to the imme-diately downstream segment, the DCT, as long as salt and fluidlosses are replaced. Transport rates by the NCC-displayingDCT epithelium seem to respond primarily to luminal sodiumdelivery (41,42). The chronically higher solute load in the DCTof rats entails upregulation of NCC expression (17,43) andeventually hypertrophy of the DCT epithelium (44), whichendows the DCT with a capacity for higher salt transport rates(41,45). These might partially compensate the impaired saltreabsorption by the TAL. Such a mechanism might contributeto the decreasing efficiency of loop diuretics in humans receiv-ing prolonged diuretic treatment (46). The usually increasedaldosterone plasma levels that occur during diuretic treatmentmight be another factor involved in the observed adaptation ofthe DCT (47); data suggest that NCC might be a previouslyunrecognized target for regulation by aldosterone (19,48).

The established target for mineralocorticoid-regulated saltreabsorption in the nephron is the sodium transport channelprevailing in the CNT and CCD, downstream of the DCT: theamiloride-sensitive ENaC (49). Data from laboratory animalssuggest that the aldosterone-initiated insertion of ENaC fromintracellular pools into the apical membrane might be influ-enced by luminal factors (26)—among others, by the tubularsolute load or the flow rate (50). Thus, luminal factors puta-tively constitute a means for modulating aldosterone-controlledsalt reabsorption. Immediately downstream from the first join-ing together of CNT in the cortical labyrinth, the human CNTcells coexpress both ENaC and AQP2. ENaC-mediated cellularsodium entry might generate a positive osmotic gradient forwater entry into the cells (51), and vasopressin-stimulatedwater entry into the cells might sustain the gradient for sodiumentry into the cells (52,53).

Interaction of Transcellular Ca2� and Na� TransportTranscellular Ca2� transport requires, in addition to apical

entry pathways, Ca2�-extrusion machinery, NCX, and PMCA,in the basolateral membranes and cytoplasmic calcium-bindingproteins to uphold the driving force for apical Ca2� entry (54).The presence of these proteins in established sodium-transport-

Figure 10. Schematic comparison between rabbit, mouse/rat, andhumans of organization of the distal convolution. The bars representthe distribution pattern of apical (1 to 3) and basolateral (4) transport-ers, and intercalated cells (green, dotted) along the distal convolutionand along cortical collecting ducts (CCD). G, glomerulus; DCT, distalconvoluted tubule; CNT, connecting tubule; 1, thiazide-sensitiveNa-Cl cotransporter (NCC); 2, epithelial Na channel (ENaC); 3,vasopressin-dependent water channel aquaporin 2 (AQP2); 4, calciumtransporting proteins (Na.Ca exchanger, plasma membrane calciumATPase, calcium binding protein calbindin-D28k). The basic distri-bution pattern of transporters in the distal convolution is displayed byrabbits. In rats and mice, ENaC is shifted upstream into the DCT, andAQP2 is shifted upstream all along the CNT. In humans, NCC andENaC overlap very little; in the most upstream portion of the CNT,AQP2 is not detectable. Calcium-transporting proteins in humansextend along the entire distal convolution and, in contrast to labora-tory animals, along the CCD.


ing epithelia provides evidence of the additional function ofthese epithelia in transcellular Ca2� transport.

In the human distal nephron, we used immunohistochemis-try to reveal these proteins all along the DCT and CNT,although in varying quantity, and in addition in the CCD. Inrabbits, the Ca2�-extruding machinery was consistently detect-able in the CNT (54,55); in rats (17,23) and mice (10,14), thiswas in addition in the last portion of the DCT, but not in theCCD. In these species, the sites with the highest manifestation

of the calcium-extruding machinery displayed, in addition tosodium entry pathways (NCC, ENaC) (13), the recently dis-covered renal epithelial calcium channel, ECaC1 (56,57). Inthe 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 forCa2� (1,18,57). For instance, inhibition of sodium transport,via NCC by thiazides or via ENaC by amiloride (58,59), is

Figure 11. Original drawings of an isolated human nephron by Karl Peter (1909) (22). (A) View from the glomerulus. (B) View from thecortical 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’sloop (TAL); 2, approximate transition to the distal convoluted tubule; 3, approximate transition to the connecting tubule (CNT); 4, approximatetransition to the CCD; asterisk, opening of the illustrated nephron into the CCD within the medullary ray. Note the correcthistotopographical location of the TAL, passing between the glomerular arterioles, and of the CNT, ascending in the cortex in closevicinity to the cortical radial artery.


associated with increased transcellular calcium reabsorption,inciting hypocalciuria (1,18). Hypocalciuria is also the leadingsymptom in humans with genetic loss-of-function mutations inthe NCC gene, known as Gitelman syndrome (2). The mani-festation in the human distal nephron of the Ca2�-extrudingmachinery over a much longer tubular portion than in labora-tory animals might disclose a human-specific regulation ofrenal calcium excretion. Alternatively, it might be related toprolonged diuretic therapy of the patients, causing Ca2� entryat sites of inhibited Na� entry. Future studies on experimentalanimals under prolonged thiazide and amiloride treatmentshould elucidate this question.

The organization of the transport pathways of the humandistal convolution shows a sequential distribution of NCC,ENaC, and AQP2 similar to that of laboratory animals. How-ever, the detailed arrangement of the apical transport pathwaysalong the distal convolution with respect to ENaC and AQP2shows distinct differences to other species. Furthermore, inhumans, the entire distal convolution (and, unlike in laboratoryanimals, the CCD as well) seems to be involved in transcellularcalcium reabsorption. The knowledge of the precise distribu-tion patterns of transport proteins in the human distal nephronmay be a help in diagnosis as we seek to better understanddisorders of salt homeostasis and to refine their therapeuticmanagement.

AcknowledgmentsWe thank Professors Jaquemin and Saussine, Service d’Urologie,

Hopital Universitaire, Strasbourg, France, for placing at our disposalthe renal tissue of tumor nephrectomized kidneys and enabling us toundertake this study. The investigations were supported by the SwissNational Foundation (grant 31-47742-96 to BK).

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