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发表于 2009-4-21 13:35 |只看该作者 |倒序浏览 |打印

作者：JohannesLoffing and BrigitteKaissling作者单位：Institute of Anatomy, University of Zurich, CH-8057 Zurich,Switzerland
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      【摘要】
   The final adjustment of renalsodium and calcium excretion is achieved by the distal nephron, inwhich transepithelial ion transport is under control of varioushormones, tubular fluid composition, and flow rate. Acquired orinherited diseases leading to deranged renal sodium and calcium balancehave been linked to dysfunction of the distal nephron. Diuretic drugselicit their effects on sodium balance by specifically inhibitingsodium transport proteins in the apical plasma membrane of distalnephron segments. The identification of the major apical sodiumtransport proteins allows study of their precise distribution patternalong the distal nephron and helps address their cellular and molecularregulation under various physiological and pathophysiological settings.This review focuses on the topological arrangement of sodium andcalcium transport proteins along the cortical distal nephron and onsome aspects of their functional regulation. The availability of data on the distribution of transporters in various species points to thestrengths, as well as to the limitations, of animal models for theextrapolation to humans.   m  g" K2 O. d% R
      【关键词】 thiazidesensitive sodium chloride cotransporter amiloridesensitive epithelial sodium channel epithelial calciumchannel rat mouse* ?$ |1 }! U. e9 E, I. H  p
   INTRODUCTION
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IN THE LAST DECADE, THE MAJOR salt and water transport proteins in the renal distal nephron have beenidentified, and their precise intrarenal localizations were explored byimmunomethods or by in situ hybridization. The resulting data confirmedthat the inventory of the transport proteins in the distal nephron identified so far is the same in all investigated mammalian species (rabbit, rat, mouse, and human). However, the transporter topology along the distal convolution shows subtle species differences, theunawareness of which might have been the cause for occasional discrepancies in the interpretation of experimental data. Furthermore, under altered functional conditions the abundance and extension alongthe distal nephron and intracellular localization of transporter proteins change. The studies also unveiled the strikingly consistent link between the given inventory of apical transport proteins along thedistal nephron and the fine structure of the respective cells. Thispoints to the potential of structural approaches in investigatingfunctional mechanisms in distal nephron electrolyte transport in vivo.

The detailed physiology of the transporters and their involvement inhuman pathophysiology has been discussed in depth in excellent recentreviews ( 33, 44, 53, 95, 107, 110, 114, 138 ). Here, wewill review the distributions in the distal nephron of the majorsodium, calcium, and water transport proteins and regulation of theirrelated functions, in correlation with the structural organization ofthe distal nephron, with the main emphasis on the distal convolution.
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CORTICAL DISTAL NEPHRON/ r7 n* S% Z* J  M9 \. D. F

Microanatomic Organization of the Cortical Distal Nephron; W, a) p9 p) }" x
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The anatomic definition of the distal nephron takes into accountexclusively those tubular portions that originate from the metanephricblastema: the distal straight part [thick ascending limb of Henle'sloop (TAL)], located in the medullary rays, and the convolutedportion, the "distal convolution," located in the corticallabyrinth. The functionally defined "distal nephron" also includes,besides the TAL and the distal convolution, the cortical collectingduct (CCD), which embryologically originates from the most peripheralbranchings of the ureteral bud.

Microanatomically, the distal convolution of superficial nephrons is asimple tube, opening into the most peripheral extensions of a CCD( 70 ). In contrast, distal tubules of deeper nephron generations merge to so-called "arcades" ( 103 ). Theextent of nephron fusion and the ratio between nephrons drainingindividually or through an arcade into a CCD vary among and withinspecies ( 35, 103 ). The arcades ascend within the corticallabyrinth, proximate to the cortical radial vessels, before they openinto a CCD within the medullary rays ( 35, 57 ). The numberof nephrons drained by each CCD averages 11 in the human kidney( 98 ), 6 in rabbits ( 58 ) and rats( 69 ), and 5 in mice ( 71 ).1 K6 C8 k3 M4 U+ @3 a/ Q0 J

The present conventionally used subdivision of the distal convolutioninto the so-called "distal convoluted tubule" (DCT) and the"connecting tubule" (CNT; including arcades) is based on more orless quite obvious structural differences along the distal convolution.They were initially observed by Schweigger-Seidel in 1865 ( 118 ) and disclosed in microdissected preparations of kidneys from rabbits, humans, mice, sheep, cats, pigs, cattle, anddolphins in astounding detail by Peter and Inouye in 1909 ( 103 ). Some 70 years later, Peter's light microscopicobservations were confirmed by detailed electron microscopic studies inrabbits ( 58 ) and extended to rats ( 29 ).

In all species, the TAL epithelium changes at various distancesdownstream of the macula densa abruptly to the DCT, which is theinitial segment of the distal convolution. In rabbits, the entirelength of the DCT epithelium is composed by one cell type, the DCTcells. An exceedingly high density of mitochondria, encased in narrowpalisade-like-arranged, interdigitated lateral cell processes,characterizes them. The DCT cells are abruptly replaced by the"CNT" cells, which by light and electron microscopy appear"lighter" and display among other irregularly arranged basolateralplasma membrane infoldings and fewer mitochondria than do DCT cells( 58 ). Intercalated cells do not show up before thetransition to the CNT and continue all along the CNT and CCD ( 58 ). In deep and intermediate nephrons, the change fromthe DCT to the CNT epithelium regularly occurs a few cells before fusion of two tubules; hence the arcades are entirely made up of CNTepithelium ( 58 ). The transition from the CNT to the CCD isgiven by the abrupt substitution of CNT by CCD cells (principal cells).Basolateral membrane infoldings in CCD cells are restricted to the mostbasal cell portion, and the few mitochondria are normally situated inthe cytoplasm above the infoldings ( 58 ).+ x- u# `7 M3 X- Z  Q/ ~& q( d

In rats and mice, the situation is different. On the basis of serial1-µm sections, Crayen and Thoenes ( 29 ) reconstructed thedistal convolution of a superficial rat nephron, from its beginningshortly downstream of the macula densa to the first confluence withanother tubule. They distinguished by light microscopy andultrastructure a total of four cell types; types 1-3 correspond toDCT, CNT, and CCD (principal) cells and type 4 to intercalated cells.Type 1 (DCT) cells exclusively comprised the first part of the tubularportion. In the direction of the flow, these cells became intermingledwith intercalated cells. Then, type 2 cells progressively replaced type1 cells, and type 3 cells progressively replaced type 2 cells.Furthermore, cell height, basolateral cell membranes, and mitochondrialdensity of cell types 1-3 gradually decreased in flow direction.Because of the gradual structural changes, Crayen and Thoenes concludedthat in rats segmentation of the distal convolution can be made onlyarbitrarily. A decade later, the lack of sharp segment borders in therat distal convolution was confirmed by Dorup ( 35 ), andexcellent ultrastructural descriptions of each of the four cell typeswere given by Madsen and Tisher ( 83 ). Dorup( 35 ) also reported, in addition to the gradual segmenttransitions, ultrastructural distinctions between the DCT cells in theearly and late part of the DCT. These were corroborated by theobservation of the abundant presence of caveolin in late DCT cells( 17 ), but not in early DCT cells, consistent with thestructural abundance of caveolae on the basolateral plasma membranes ofcells in this tubular region ( 57 ).4 [" `$ S) X8 |1 ^7 ^
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In mice (Fig. 1 ), the distal convolutionseems to be similarly organized as it is in rats; however, systematicultrastructural studies are not available. Ultrastuctural descriptionsof the transitional regions in the human distal convolution are lacking as well.* S0 @8 E/ s* @9 S

Fig. 1. Mouse cortical distal nephron portions. a, d,and g : 1-µm Epon sections. b, c, e, f, h, and i : Cryostatsections. a - c : Arrows, transition from thethick ascending limb (T) to the early distal convoluted tubule (D1); P,proximal tubule. b and c : Doubleimmunofluorescence for bumetanide-sensitive sodium-2 chloride potassiumcotransporter (NKCC2) and thiazide-sensitive sodium-chloridecotransporter (NCC). d - f : Approximatetransition from the late distal convoluted tubule (DCT; D2) to theconnecting tubule (CN). e and f : Double labelingfor H ATPase, prominent in intercalated cells, andaquaporin-2 (AQP2), confined to connecting tubule cells. g - i : Transition from CN to the corticalcollecting duct (CD) in the medullary rays. h and i : Consecutive cryostat sections stained for epithelialsodium channel (ENaC) and AQP2. Bars = ~ 50 µm.

Distribution of Transport Proteins Along the Distal Nephron

The following apical transport systems have been shown byimmunomethods and/or in situ hybridization to be confined in the kidneyexclusively to the distal nephron: the bumetanide-sensitive sodium-2chloride potassium cotransporter (NKCC2) ( 62, 82, 97 ), thethiazide-sensitive sodium-chloride cotransporter (NCC) ( 3, 96, 105 ), the amiloride-sensitive epithelial sodium channel (ENaC) ( 37 ), the recently discovered epithelialcalcium channel (ECaC1; CaT2; TRPV5) ( 54, 101 ), and thevasopressin-sensitive water channel [aquaporin-2 (AQP2)] ( 66, 94 ). The basolateral sodium/calcium exchanger (NCX) ( 96, 108 ) and the plasma membrane Ca-ATPase (PMCA) ( 13, 14 ), as well as the cytoplasmic calcium-binding proteincalbindin D 28k ( 10, 13, 112 ) are, in contrastto other nephron portions, particularly abundant in some parts of thedistal nephron.# T* {4 T  q. n) Z, i

Mapping the apical transport systems along the distal nephron ofrabbits ( 77 ), rats ( 26, 55, 116 ), mice( 19, 65, 78 ), and humans ( 11 ) revealed theirserial arrangement. NKCC2 is confined to the TAL, including the maculadensa ( 62, 82, 97 ), and distinguishes this segment fromall others and in all species. Salt subtraction from the tubular fluidvia NKCC2 in the (water impermeable) TAL is the precondition forurinary concentration. Solute reabsorption by the subsequent distalconvolution is the premise for fine-tuning of renal electrolyteexcretion. It proceeds by the concerted action of the apicaltransporters NCC, ENaC, ECaC1, and AQP2, as well as others notmentioned in this context. Of these transporters, NCC is withoutexception the most upstream transporter in the distal convolution andreplaces the NKCC2 exactly at the structural transition from the TAL tothe DCT ( 3, 96, 97, 105 ) (Fig. 1, a - c )., `$ l: ^/ ?* S4 A7 }

What differs markedly among the species is the site of start-off alongthe distal convolution of ENaC, ECaC1, and AQP2 expression. In therabbit kidney (Fig. 2, top; Fig. 4, a and b ),immunostaining for ENaC starts where in situ hybridization for NCC mRNAstops ( 77 ). NCC immunostaining, made with antibodiesdirected against a synthetic peptide, is replaced abruptly by ECaC1immunostaining (Fig. 4, a and b; LoffingJ and Loffing D, unpublished observations). Immunostaining witha monoclonal antibody against a metolazone-binding protein continuesbeyond the stop of the in situ signal for some distance into the CNT( 3 ), where it colocalizes with ECaC1 ( 52 ).The abrupt start of coexpression with ENaC of the vasopressin-sensitive water channel AQP2 (Fig. 3, a and b ) sharplymarks the beginning of the CCD ( 77 ), which structurally isdiscernible by abrupt replacement of CNT cells by CCD cells (principalcells). The basolateral NCX is immunohistochemically detectableexclusively in the CNT ( 3, 108 ), and calbindinD 28k is intermediate in the DCT, rises sharply with thebeginning of the CNT, and continues somewhat more weakly along the CCD( 77 ).5 v2 Q7 q; p% I1 n% a( S
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Fig. 2. Transitional regions from the DCT to the connectingtubule in a rabbit, mouse, and human. All sections are cryostatsections. Top : rabbit consecutive sections (modified fromRef. 77 ). Left, in situ hybridization for NCCmRNA, confined to the DCT (D); right, immunostaining forENaC in CN; arrowheads, definite stop of NCC mRNA and the abruptbeginning of apical ENaC staining. Middle : mouse sections(modified from Ref. 78 ). Shown are double immunostainingfor NCC and ENaC, exclusive presence of NCC immunostaining in D1, andcoexpression of NCC and ENaC by cells in D2 (arrows). Bottom : human consecutive sections (modified from Ref. 11 ). Shown are immunostaining for NCC and ENaC, exclusiveNCC immunostaining in early profiles of distal convoluted tubule (D1),and weak NCC staining colocalized with ENaC in late profiles (D2).Bars = ~50 µm.8 B5 y, e4 \5 R
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Fig. 3. Farthest upstream sites of AQP2 in the distal nephronof a rabbit, mouse, and human. All sections are cryostat sectionsshowing immunofluorescence. a and b : Rabbitsections (modified from Ref. 77 ). Shown are doublelabeling for calbindin D 28k (CB) and AQP2, resepctively,and abrupt start of AQP2 immunostaining, coincident with sharp decreaseof CB immunostaining at the sharp transition (arrowheads) from CN toCD. D, DCT. c and d : Mouse (unpublishedobservations). Shown are double labeling for NCC and AQP2,respectively, intermingling of NCC- and AQP2-positive cells(arrowheads) at the end of DCT (D2), and beginning of the CN, where NCCdisappears. e and f : Human consecutive cryostatsections (modified from Ref. 11 ) labeled for ENaC andAQP2, respectively. Shown is the lack of AQP2 immunostaining in mostupstream ENaC-labeled CN profiles (CN*). Bars = ~ 50 µm.  d+ i" `9 w6 A9 i. s: E

The congruency of structural segmentation and distribution pattern ofapical transporters along the rabbit distal nephron are explicit. Thedistribution pattern of apical transporters along the rat distalconvolution mirrors the above-described lack of sharp structuraltransitions in this species. DCT cells in this late portion differ insome structural aspects from those in the early portion (see above) andcoexpress, in addition to NCC, the apical channels ENaC( 116 ) and ECaC1 ( 55 ). Furthermore, theydisplay a very high abundance of cytoplasmic calbindin D 28k ( 76, 105 ) and basolateral NCX ( 96 ). In maleSprague-Dawley rats, the fractional length of the late NCC-displayingtubular portion, defined by its high abundance of basolateral NCX, has been estimated to amount to ~20% of the total NCC-positive portion ( 26 ).' d6 j1 i, e$ a3 K  F5 X- h

A further significant difference between rats and rabbits concerns thedistribution of AQP2 in the distal nephron. In rats, immunostaining forAQP2 has been detected to start with the break-off of NCC-staining( 77 ). Occasionally, AQP2-positive cells may beintermingled even with the last few NCC-positive cells. This observation agrees with the former structural data on intermingling ofcell types along the distal convolution ( 29, 35 ).Quantitative estimation by Western blot analysis of AQP2 inpreparations of isolated rat CNT and CCD revealed ~60% AQP2abundance in the CNT from that in the CCD ( 66 ), consistentwith weaker AQP2 immunostaining along the CNT than in the CCD of rats( 26, 66, 77 ). The CCD cells in the successive segmentcoexpress ENaC and AQP2, but not ECaC1. In the cortex of maleSprague-Dawley rats, the fractional volumes of DCT, CNT, and CCDcorrespond to ~1:1:1 ( 26 ).
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In mice, the distribution of NCC, ENaC, ECaC1, and AQP2 (Fig. 1, c and f; Fig 2, middle; Fig 3, c and d; Fig. 4, c and d ) resembles that in rats. Inthe late part of the DCT, the apical transporters NCC, ENaC, and ECaC1are coexpressed ( 78 ) and, additionally, the mostdownstream NCC-positive cells might even coexpress AQP2 (Loffing J,unpublished observations). A significant abundance ofcytoplasmic calbindin D 28k, basolateral NCX ( 19, 78 ), and PMCA ( 78 ) has also been demonstrated byimmunomethods in the early portion of the DCT. Nevertheless, a markedjump in abundance of these latter proteins was consistently observed at the sites where, in addition to NCC, coexpression of ENaC and ECaC1began (Fig. 5 ) ( 78 ). Thefunctional link between apical ECaC1 and NCX and PMCA is furtherevidenced by the parallel decreases in apical ECaC1 and basolateral NCXand PMCA immunostaining along the CNT and their simultaneous cessationat the histotopographically recognized CCD( 78 ). Single cells, coexpressing ECaC1, NCX, PMCA,and calbindin D 28k, are occasionally interspersed inthe CCD epithelium ( 78 ). In female C57/BL6 mice, thefractional volumes of early DCT, late DCT, and CNT correspond to~2:1:2 ( 75 ).
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Fig. 4. NCC and ECaC1 in the distal nephron of rabbit and mouse(unpublished observations). All sections are cryostat sections showingdouble immunostaining with a rabbit-anti-NCC antibody (characterized inreference Ref. 11 ) and a guinea pig anti-ECaC1 antibody(characterized in Ref. 54 ). a and b :Rabbit sections. Arrowheads, abrupt replacement of apical NCC by apicalECaC1 labeling. Basement membrane staining is due to binding of rabbitimmunoglobulins to the secondary anti-rabbit antibody. c and d: Mousesections. Shown are strong apical coexpression of NCC and ECaC1 in lateD2 profiles and cytoplasmic labeling for ECaC1 in NCC-negative CNprofiles. Bars = ~ 50 µm.% P, E2 w3 X1 x4 J0 W. d! ~/ _
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Fig. 5. ECaC1 and basolateral sodium-calcium exchanger (NCX)double immunolabeling in mouse sections (modified from Ref. 78 ). All sections are cryostat sections. Shown are theabrupt onset of apical ECaC1 staining in D2, coincident with abruptrise in NCX immunostaining (arrowheads) and gradual translocation ofECaC1 staining from apical toward cytoplasmic sites in the CN,paralleled by a decrease in basolateral NCX staining. Bars = ~50µm.
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In humans, the tubular portion with overlap of NCC and ENaC is rathershort. NCC characterizes ~30-35% of profiles of the distalconvolution and ENaC ~70-75% ( 11 ). AQP2 does notdirectly supersede the NCC, but the initial ~15% of the CNT [atleast the portions before the fusion with arcades display ENaC alone(Fig. 3, e and f )] ( 11 ). Thedistribution of ECaC1 along the human nephron has not been assessed byimmunostaining so far. Similarly to mice, NCX, PMCA, and calbindinD 28k are traceable to varying extents even in the initialportion of the DCT, and, in pronounced difference from all otherspecies, continue in significant abundance along the corticalcollecting duct ( 11 ).
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Definition of the Distal Nephron Based on Distribution ofTransport Proteins) o! E7 V/ k6 @4 H: c: s; t/ U

The clear-cut structural and functional organization of the rabbitdistal nephron constitutes a simple model, which aids in anunderstanding of the more complex distal nephron organization in rats,mice, and humans (Fig. 6 ). In all fourmammalian species analyzed so far, of the proteins discussed in thisarticle, NKCC2 is the most upstream apical salt transporter andunequivocally defines the TAL. The succeeding salt transporter is theNCC, the onset and end of which in the distal convolution ofall species are clearly discernible, and which defines the DCT. Thenext apical ion transporters in the series are ENaC and ECaC1. Inrabbits, they abruptly replace NCC in the apical membrane. In rats,mice, and humans, ENaC and ECaC1 seem to be "pushed" more or lessupstream along the distal convolution into the NCC-displaying lateportion of the DCT, giving rise to a portion with apical coexpression of NCC, ENaC, and ECaC1. Accordingly, in these latter species the DCTcan be further subdivided into an early and a late portion. Besides thecoexpression with NCC of the apical transporters ENaC ( 11, 79, 116 ) and ECaC1 ( 55, 78 ), the second portion differsfrom the first, e.g., by the presence of intercalated cells ( 35, 83 ), by very prominent cytoplasmic calbindinD 28k ( 76, 105 ), by basolateral NCX( 96 ) and PMCA ( 78 ), as well as by discretestructural differences ( 35 ). The conspicuous onset ofbasolateral NCX immunostaining in the NCC-positive segment of the rat( 96 ) prompted the groups of Bachmann ( 3 ) andEllison ( 40 ) to propose the subdivision of the DCT intoDCT1 (NCX negative) and DCT2 (NCX positive). According to thiscriterion, neither in mice ( 19 ) nor in humans could suchsubdivision of the DCT be made. The onset of traceability of ENaC andECaC1, as far as has been investigated to date, coincides approximatelywith the marked start (rat) ( 96 ) or rise (mouse)( 78 ) in NCX, PMCA, and calbindin D 28k immunostaining. The coexpression of NCC and ENaC has been used as wellto distinguish the late (~DCT2) from the early (only NCC expressing)DCT (~DCT1) ( 78 ). That the late part of the DCT variesin length among species, probably also among species strains, possiblyalong with age, sex, and other factors, has been emphasized in aneditorial comment by Wade ( 134 ).

Fig. 6. Distribution patterns of apical and basolateral transport proteinsalong distal nephron in rabbit, rat, mouse, and human (modified fromRef. 11 ). G, glomerulus; TAL, thick ascending limb; CNT,connecting tubule; CCD, cortical collecting duct; MR, medullary ray.Colored bars, schematic representation of transport protein locations;arrows, continuation of given transporter along the CCD; light shading,consistent weak immmunostaining (for axial gradients in proteinabundance and changes in intracellular locations, see text). Apicaltransport proteins: light green, NKCC2; dark green, NCC; red, ENaC;orange, ECaC1 (not yet traced in humans); blue, AQP2. Basolateraltransport proteins: orange, coexpression of plasma membrane Ca-ATPase(PMCA) and NCX. Site of onset of immunostaining varies among and withinspecies.! c" ?/ }5 c( v4 }% y* r

The end of NCC immunostaining can be used in all species to mark thebeginning of the CNT. In rabbits, it comes along with the onset of ENaCand ECaC1 and basolateral NCX immunostaining and in rats and mice withonset of additional AQP2 coexpression (see Fig. 6 ). In all species, thepresence of AQP2 and the histotopographical location of the tubule inthe medullary ray (or in superficial nephrons the proximity to therenal capsule) defines a CCD. The overall length of the distalconvolution (DCT and CNT) can be conveniently estimated by doubleimmunostaining for NCC and NCX ( 26, 96 ) or NCC andcalbindin D 28k ( 11 ).! g* l, A5 T+ H0 B
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Relevance of Specific Transporter Topology

The specific sequence of electrolyte transporters along the distalnephron probably guarantees sodium recovery under a large range ofphysiopathological situations. It seems to imply the possibility ofpartially compensating for inadequate salt reabsorption by adaptingsalt reabsorption via other and differentially regulated salt transportsystems in downstream tubular portions. For instance, impairment ofNaCl reabsorption in the TAL, by whatever means, drastically increasesthe NaCl load in the downstream segments of the TAL, which respond withhigher salt reabsorption rates, and over longer periods, with increasedtransport capacity and associated epithelial hypertrophy( 70 ). Such compensatory mechanisms along the nephron arethought to occur also under impaired sodium uptake due to mutations ofgenes for some transport proteins (e.g., NKCC2 in Barrter syndrome, NCCin Gitelman syndrome) ( 49, 107 ).& T7 @. u# v: f8 x

Although at first glance the species differences in the distributionpattern of transporters appear to be rather trivial, their functionalrelevance might be substantial. The definite (rabbit) or gradual (rat,mouse, human) changes in the distribution of specific transportpathways along the distal nephron are also reflected in respectivedistribution patterns of sensitivities to various peptide hormones( 87 ). For instance, the sites of vasopressin sensitivityalong the distal nephron, assessed in preparations of isolated distaltubules from rabbit, rat, mouse, and human kidneys ( 87 ),match precisely in all cases the respective sites of AQP2 occurrence.The upstream shifting of vasopressin-sensitive water retrieval into theENaC-displaying CNT may be functionally relevant for at least tworeasons. It might favor ENaC-mediated sodium entry into the cells, bycreating a favorable gradient for sodium entry by water subtractionfrom the (salt diluted) tubular fluid in the CNT. The differentialhormonal control of AQP2 and ENaC activity opens subtle and complexpossibilities for regulatory interactions. Furthermore, the AQP2upstream shifting of vasopressin-regulated water subtraction in theCNTs might go along with a considerable gain in the overall waterreabsorption ( 66 )., {' Y8 n0 N2 y

Thus the final result of solute excretion depends not only on thepresence and abundance of given transport proteins in the kidney butalso on their species-specific topological arrangement.

CONTROL OF TRANSPORT ACROSS APICAL PROTEINS

The differences in the specific distribution patterns of transportproteins are probably evolutionary responses to species-specific livingconditions. Evidently, within this given frame there is room forindividual adaptation to the actual needs. Immunomethods and/ormorphology can disclose the nephron sites in which, under specificfunctional conditions, in vivo adaptive changes took place.

Regulation of NCC-Mediated Salt Reabsorption
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NCC mediates electroneutral NaCl uptake into the DCT cells.Because renal NCC is confined to the DCT, data on NCC derived fromwhole-organ homogenates truly reflect corresponding changes in NCCabundance in this segment. As long as specific anti-NCC antibodies werenot available, abundance of NCC was often assessed by its specificbinding to metolazone, a thiazide-like diuretic ( 5 ).Changes in metolazone binding protein, NCC abundance, or NCC mRNA havebeen observed under many different pathophysiological or clinicalsituations (for a review, see Refs. 47, 67,and 107 ). What might be the common denominator under thedifferential conditions, controlling specifically apical NCC abundanceand NCC-mediated cellular salt uptake?

Tubular sodium load and flow rates. Some data from the time when neither[ 3 H]metolazone-binding studies nor NCC antibodies wereavailable suggested that tubular salt load and/or flow rate in the DCTdirectly controls sodium reabsorption in the DCT. Rabbits under chronichigh-sodium, reduced-potassium diets (with respective chronic very lowendogenous plasma levels of aldosterone) revealed marked hypertrophy ofthe DCT epithelium, indicating chronically upregulated transportcapacity in the DCT epithelium ( 59 ). Micropuncture studiesin rats under furosemide-induced impairment of NaCl reabsorption (withsimultaneous replacement of salt and fluid loss) confirmed that themarked increases in surface basolateral plasma membrane and inmitochondrial volume in the distal convolution cells mirrorcorrespondingly increased sodium reabsorption rates ( 40, 60, 122 ). In situ hybridization ( 96 ) as well as RNAseprotection assays ( 140 ) disclosed upregulation of mRNA forNCC under the given conditions. Others were unable to detect anyincrease for NCC mRNA ( 1, 88 ). Indications for NCCmRNA-independent regulation of NCC protein abundance, e.g., bymechanisms that control protein translation and/or stability, have beenobtained in some studies ( 1, 137 ), but so farintracellular processing and trafficking of NCC in vivo have not beenaddressed. Studies in rats under the above experimental conditionsdemonstrated the involvement of IGF-1 and IGF binding protein 3 in thehypertrophy of the DCT epithelium ( 68 ).

Steroid hormones. mineralocorticoids and glucocorticoids. In rabbits, drastically increased endogenous aldosterone levelsassociated with chronic low-sodium, high-potassium intake did notincrease the transport machinery in the DCT but did markedly in the CNT( 59 ). Adaptive structural and functional hypertrophy inthe DCT epithelium after furosemide-induced inhibition of salt reabsorption in the TAL occurred in adrenalectomized rats with clampedplasma levels of mineralocorticoid and glucocorticoid hormones tosimilar extents as in intact rats ( 60, 122 ). These collected observations suggested that changes in salt delivery and flowrate by themselves might be sufficient to initiate modulation of salttransport rates in the DCT. Nevertheless, several studies advocated theimplication of aldosterone in the control of apical NCC abundanceand/or NCC-mediated salt reabsorption. In rat kidneys, low dietarysodium intake, which promotes rises of endogenous aldosterone plasmalevels, as well as exogeneous aldosterone application increased theabundance of NCC protein, as assessed by Western blotting andimmunohistochemistry ( 64 ). Increased binding of [ 3 H]metolazone to kidney homogenates was observed afterexogenously applied aldosterone ( 22 ) but not with dietarysodium restriction ( 21 ). Velazquez et al.( 128 ) observed that the low electroneutral sodiumtransport activity, measured in vivo in the early distal convolution ofadrenalectomized rats, can be restored to normal and further increasedby replacement of aldosterone and/or high doses of glucocorticoids( 128 ). The increase in NCC-mediated sodium transport andNCC protein after furosemide treatment or dietary sodium restrictionwas significantly lower when aldosterone receptors were blocked byspironolactone ( 1, 93 ). This experimental setting providedindirect evidence for the role of aldosterone in the observed changes;however, the possible contribution of systemic or upstream renalparameters to the given observations has not been experimentally excluded.( H0 R; S8 j+ ]: Y/ |+ b" y- V
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MINERALOCORTICOID RECEPTORS AND11 - HYDROXYSTEROID DEHYDROGENSASE TYPE 2. Canonically, a direct role of mineralocorticoid hormones in the controlof apical NCC abundance and of NCC-mediated NaCl transport requires thepresence in the DCT of mineralocorticoid receptors (MR) and of theenzyme 11 -hydroxysteroid dehydrogensase type 2 (11 -HSD2), whichconfers mineralocorticoid specificity to the MR by the rapidmetabolization of glucocorticoids. MR and 11 -HSD2 have been revealedby various methods in the distal nephron of rats, rabbits, and humans(for a review, see Refs. 42 and 107 ) and11 -HSD2 also in mice ( 19 ). Whereas MR seem to bepresent all along the rat DCT and CNT ( 15 ), 11 -HSD2 isnot detectable in the early (ENaC-lacking) portion (DCT1) but is welldetectable in the second DCT portion (DCT2), in the CNT, and CD( 15, 116 ). These observations indicate that the effects ofaldosterone on NCC-mediated sodium transport might differ along theaxis of the DCT, and they corroborate the suggestion that the DCT mayexhibit some promiscuity with respect to the selectivity formineralocorticoids and glucocorticoids ( 107 ). Reilly andEllison ( 107 ) proposed that the latter may stimulateNCC-mediated sodium transport primarily in the early DCT (DCT1),whereas aldosterone may stimulate sodium transport predominantly in thelate DCT (displaying NCC and ENaC ), where MR and 11 -HSD2 are bothhighly expressed. Putatively, control of NCC abundance by aldosteronevia MR-independent noncanonical pathways is conceivable.

SEX HORMONES. Sex hormones seem to affect directly or indirectly NCC-mediated salttransport. RT-PCR ( 74 ) and autoradiographic ( 30, 124 ) experiments pointed to the expression of estrogen receptors in rat kidneys. Chen et al. ( 23 ) and Verlander et al.( 130 ) demonstrated that estrogens given to ovarectomizedrats increase [ 3 H]metolazone binding sites and NCCabundance, respectively, in the renal cortex. These observations wereused to explain why female rats respond with a more pronounced diuresisto thiazides than do male rats ( 23 ). Whether increasedNCC-mediated sodium transport contributes to the sodium retention seenunder estrogen therapy (e.g., the contraceptive pill) is not known." q  Q; M+ E) w8 I1 ^8 k) A: E
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Peptide hormones. The presence of receptors for peptide hormones such as calcitonin, PTH,and isoproterenol had been shown indirectly by the increases in cAMPactivity after application of these hormones to morphologicallydefined, microdissected distal tubule segments ( 87 ).Blakely et al. ( 12 ) reported increased[ 3 H]metolazone binding in kidney homogenates ofcalcitonin-treated rats./ k0 U/ _0 v+ n4 ]" g

Angiotensin II is involved in the control of arterial blood pressureand whole body sodium homeostasis. Independently from its effect onadrenal aldosterone secretion, angiotensin II stimulates sodiumreabsorption in the distal tubule ( 135 ). It has beendiscussed that the hypertrophy in the DCT under increased salt load ofthe DCT (after furosemide treatment, combined with high salt and water load) might be ascribed, in part, to an effect of angiotensin II( 6 ). The role of angiotensin II in NCC regulation can be deduced from recent experiments in mice with targeted disruption of theangiotensin II type 1 (AT 1 ) receptor ( 18 ).Unlike wild-type mice, these AT 1 knockout mice lackincreases in NCC protein abundance in response to a low dietary sodiumintake. Thus in the rodent DCT part of the adaptation to low dietarysodium intake is possibly mediated by increased angiotensin II levels.) ~; m9 Y6 G8 F+ @1 C& D4 R

Inhibition of NCC-mediated transport. Thiazide diuretics are frequently used in the treatment ofhypertension. Thiazides inhibit the uptake of NaCl into the DCT cell bybinding to NCC. Prolonged reduction of sodium entry into the cellsresults in chronically lower transport rates, structurally reflected bylowering of the epithelium with a reduction of the active-salttransporting machinery ( 70 ). In the DCT of mice, treatmentwith thiazides is indeed associated with lowering of DCT epithelium(Valderrabano V and Loffing J, unpublished observations), andNCC knockout mice have a marked hypotrophy of the DCT epithelium ( 117 ). However, treatment of rats with metolazone for 3 days or with hydrochlorothiazide induced massive apoptosisexclusively in the early DCT ( 76 ). In rats treatedcontinuously for 2 and 4 wk with metolazone, the epithelium of theearly DCT was markedly simplified, displaying fewer basolateralmembranes and mitochondria, and de- and regenerating cells. Theseobservations suggest chronically lower transport rates and corroborateformer data by Morsing et al. ( 89 ), who measured thatpost-chronic in vivo blockade of NaCl transport by thiazides reducedtransport capacity of rat distal tubules, despite a significantlyincreased number of thiazide binding sites. The authors pointed outthat increases in the number of thiazide receptors are not necessarilysynonymous with increases in transport activity. The different effectsof thiazides on the DCT in rats (apoptosis) and mice(hypotrophy) once again emphasize species differences, even betweenclosely related species.

Regulation of ENaC-Mediated Sodium Transport

The second major sodium transporter in the distal convolution isENaC. ENaC is the major player in aldosterone-regulated sodium reabsorption, not only in the kidneys but also in other organs (e.g.,distal colon, salivary glands) ( 131 ). In the kidney, all ENaC-positive tubule portions have been subsumed under the term "aldosterone-sensitive distal nephron" (ASDN) ( 81 ),which comprises the CNT and CD in all species and includes, in somespecies at least, the late part of the DCT (see above). The essentialrole of ENaC in control of salt and volume homeostasis is highlighted by the fact that some hereditary forms of severe salt-sensitive arterial hypertension (Liddle's syndrome) and severe renal salt wasting (pseudohypoaldosteronism type I) are related to gain- andloss-of-function mutations, respectively, of ENaC genes (for a review,see Ref. 110 ).& ]& _' i4 @" X' r' n2 O1 I
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Sodium uptake into the cell via ENaC is electrogenic and can bemeasured by corresponding sodium currents that are inhibited byamiloride. ENaC-related sodium transport favors potassium secretion, which proceeds most probably via the apical renal outer medulla potassium channel ROMK, coexisting with ENaC in the same cells ( 136 ). Putative potassium reabsorption by intercalatedcells, which are regularly interspersed among the ENaC-expressingcells, might modify the rigid link between sodium reabsorption via ENaC and K secretion via ROMK.

ENaC is composed of three subunits (,, ) ( 110 ).All three subunits have been located along the ASDN ( 37 ),although in differential imunnohistochemical abundance andintracellular sites ( 50 ). Interestingly, -ENaC, but not - and -ENaC, has been evidenced by RT-PCR in microdissectedrabbit DCTs; however, amiloride-sensitive sodium currents could not bedetected in this segment ( 129 ). Expression of -ENaCmRNA has been also revealed by in situ RT-PCR in the mouse TAL and DCT( 25 ).5 f. \) C% K' B- K" Q. X

Regulation of renal sodium transport via ENaC could intervene 1 ) directly at the level of single channels in the membrane, by changing the channel gating kinetics; in vitro studies in amphibian cells and heterologous expressions systems suggested this possibility (for a review, see Refs. 38 and 48 ), but toour knowledge, such effects have not been described so far for the ASDNin vivo; or 2 ) at the level of density of functional apicalchannels. This can result either from altered synthesis rates of ENaCsubunits and/or from altered exo- and endocytosis rates of alreadypresent ENaC subunits.

Role of synthesis of ENaC subunits. Although the exact stoichiometry of the subunits in the channel isstill debated, there seems to be consensus that the -subunit plays apivotal role in the assembling of functionally active channels( 120 ). In the kidney, primarily the abundance of -ENaC appears to be regulated by aldosterone. Exogenous aldosterone application increases the abundance of -ENaC at the mRNA and proteinlevel ( 84, 123 ). Dietary sodium restriction, which increases endogenous aldosterone production, has been shown to induce -ENaC in some ( 84, 85, 140 ) but not all studies( 109, 123 ). Based on previous studies in Xenopuslaevis A6 cells, it has been proposed that the induction of -ENaC might be a prerequiste for the apical translocation of ENaC( 86 ). Sufficient availability of -ENaC may allow fullassembly of ENaC channels and their subsequent release from theendoplasmic reticulum and delivery to the cell surface. The inductionof -ENaC (at least in nonadrenalectomized rats) in response toaldosterone, however, is rather small, and it is conceivable that invivo the induction of -ENaC alone does not account for the apicaltargeting of all three ENaC subunits. Interestingly, Nielsen andco-workers ( 93 ) recently reported that MR inhibition byspironolactone blunts the induction of -ENaC but does not preventthe apical redistribution of all three ENaC subunits in response to alow-sodium diet. Short-term adaptation of the kidneys to 24 h ofdietary sodium restriction apparently does not involve significantupregulation of -ENaC ( 85 ). However, amiloride-sensitive renal sodium reabsorption was shown to be significantly increased after 4 h of sodium restriction( 46 ).+ B2 z" F; L: L& B9 ?: B, w

Other hormones besides aldosterone affect the synthesis rate of ENaCsubunits as well, and thereby they might also intervene in the controlby aldosterone of ENaC-mediated sodium reabsorption. For example, inthe rat kidney in vivo chronic increases in vasopressin levels induce - and -ENaC at the mRNA level ( 92 ), in agreement with studies in rat CCD cells in vitro ( 34 ). Similarly,water restriction in normal rats and chronic infusion of dDAVP (astable analog of vasopressin) into Brattleboro rats (a rat strain that lacks endogenous vasopressin) increase - and -ENaC proteinabundance ( 39 ).- i' U# v+ i" o  K$ O5 t
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Angiotensin II rapidly stimulates ENaC activity in isolated distalnephron segments ( 135 ), an effect most likely mediated byAT 1 receptors ( 104 ). Such acute effects ofangiotensin II are unlikely to be mediated by an increased ENaCsynthesis rate, but recent experiments in AT 1 receptorknockout mice suggest that angiotensin II might have a direct impact onENaC abundance as well. Despite elevated plasma aldosterone levels, theknockout mice express less -ENaC than wild-type mice( 18 ).% e3 s% P0 W  d* ~0 k

Regulation of ENaC activity by redistribution of ENaC subunits. In rodent kidneys, dietary sodium restriction causes animmunohistochemically traceable redistribution of ENaC subunits from intracellular compartments toward the apical cell surface ( 79, 84 ). These observations correlate well with previous patch-clamp studies in isolated rat collecting ducts by Pacha and co-workers ( 99 ), who recorded increases in the number of open sodiumchannels in the apical plasma membrane after 1 wk of dietary sodiumrestriction ( 99 ). The increases in active channels were inparallel to increases in endogenous plasma aldosterone levels( 99 ) and were observable even within 15 h aftersodium restriction ( 45 ). Immunohistochemistry revealedthat in adrenalectomized rats within less than 4 h after onesingle aldosterone injection, the apical ENaC density increased ( 81 ). The rapid upregulation of apical ENaC activity andabundance indicates that such changes might be relevant not only forthe long-term adaptation of renal sodium excretion but also in the adaptation to circadian variations of dietary sodium intake( 45 ).
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The molecular mechanisms underlying the rapid accumulation of ENaC inthe apical cell surface are not well understood. Data suggestinvolvement of the serum and glucocorticoid-regulated kinase SGK1,which is an aldosterone-induced protein ( 20, 90 ). SGK1induction by aldosterone is clearly dose dependent ( 119 ) and occurs even under small variations in endogenous plasma aldosterone levels provoked by alterations in dietary sodium intake( 56 ). Coexpression of ENaC with SGK1 in X. laevis oocytes manifestly stimulates ENaC activity ( 20, 90 ) and cell surface abundance ( 32 ). The latter isthought to be related to SGK1-dependent phosphorylation andinactivation of the ubiquitinligase Nedd4-2 that downregulatesENaC ( 31, 121 ). The importance and regulation of SGK1 andNedd4-2 under various experimental conditions are summarized inseveral recent reviews ( 61, 73, 80, 100, 120 ).

The induction of SGK1 by aldosterone, detectable by immunomethodsexclusively in the ENaC-positive DCT2, CNT, and CD cells ( 81 ), precedes the apical targeting ( 81 ) andfunctional activation of ENaC ( 9 ). An in vivo role of SGK1in ENaC regulation can be deduced from recent findings made in SGK1knockout mice ( 141 ). However, the salt-loosing phenotypeof the SGK1 knockout mice is mild compared with that of MR knockoutmice ( 7 ) or that of the -, -, or -ENaC knockoutmice ( 110 ). These observations in SGK1 knockout micesuggest that SGK1 might be an important, but not the sole, playernecessary for apical ENaC activity.6 i- j; d( o9 M& b9 |" E
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Axial differences in ENaC density. Under standard conditions of sodium intake, all three subunits aretraceable in the apical membranes only in the initial parts of the ASDN( 77, 79 ). Farther downstream in particular, - and -subunits seem to vanish from the apical membrane and become increasingly prominent in intracellular compartments ( 50, 77, 79, 84 ).
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Former studies on net sodium reabsorption in microperfused rat tubules( 27, 106, 125 ) and in isolated rabbit tubules( 2 ) demonstrated that in upstream portions of the ASDN(late DCT and CNT; formerly designated together as "late distaltubule"), sodium transport was consistently present and several timeshigher than in CCDs located downstream. The axial decrease in thebasolateral Na-K-ATPase ( 63 ) and the morphologicalcorrelate of the sodium extrusion apparatus ( 59 ), i.e.,the basolateral membrane infoldings and the mitochondrial volumes, alsomirrors progressive decreases in the sodium transport rates along theASDN. The functional and morphological data match the progressiveimmunohistochemical decrease along the ASDN of the apical expression ofENaC. Under chronic high-salt intake, associated with low aldosteronelevels, apical ENaC is barely detectable, even at the very beginning ofthe ASDN ( 79 ). Under moderate standard salt intake byanimals in European laboratories, apical -, -, and -ENaC arefound to extend at best along the DCT2 and the early CNT ( 77, 79 ). Under salt restriction, inducing rises in endogeneousaldosterone plasma levels, apical ENaC is detectable also in the CCDand outer medullary CD ( 79 )./ D2 o' |& r% J6 Y0 m. `. q
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What causes the axial differences in apical ENaC and, consequently, insodium transport along the ASDN? Theoretically, an axially decreasinggradient of apical ENaC localization might be connected withcorrespondingly distributed MR density along the ASDN. However, formerdata by Farman et al. ( 43 ) as well as Doucet and Katz( 36 ) showed similar densities or even rather increasing MRdensities along the ASDN in rats. Equal sensitivity for aldosteronealong the collecting system is also suggested by thealdosterone-dependent induction of SGK1 and -ENaC, which occurreduniformly all along the collecting system ( 81 ). This implies that other factors superimpose the systemic changes in aldosterone levels and contribute to the differential regulation ofapical ENaC abundance along the distal nephron. Whetherdifferential sensitivities along the ASDN for other hormones, known toaffect ENaC function (e.g., insulin, vasopressin, angiotensin II),exist and whether these hormones influence ENaC surface expression are not known. Extracellular proteases [e.g., kallikrein,channel-activating protease-1 (CAP1)] ( 24, 126, 133 )secreted into the tubular fluid might contribute to axiallydifferential regulation of ENaC-mediated salt transport. Kallikrein issynthezized in the most upstream portions of the ASDN( 132 ). Prostasin, the human homologue of X. laevis CAP1, has been recently shown to be increased in urine inhumans under conditions of increased plasma aldosterone levels ( 91 ). However, the extracellular proteases seem to affectchannel activity ( 24, 126, 133 ) rather than ENaCcell-surface expression.

What necessarily changes in tubular flow direction is the tubular fluidcomposition. Osmolarity changes and intra- and extracellular ionconcentrations (e.g., for sodium, calcium) have been discussed to playa role in ENaC regulation ( 48 ). Also, effects of tubular flow rate on ENaC-mediated sodium transport in isolated rabbit collecting tubules have been reported ( 113 ).% Y! Q9 P# x$ u6 n* `9 g
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Taken together, the marked gradient in apical ENaC density, assessed byin vivo experiments, agrees with functional data on sodium transport indifferent ASDN portions. The causes for this gradient along the ASDNare still unclear. The findings suggest that local factors along thetubule, presumably via the tubular fluid composition, importantlymodify the effects of systemic factors on ENaC-mediated transport.

Sites of Calcium Transport in the Distal Nephron7 m3 K% N2 [  e8 |7 d, w

Former micropuncture experiments and studies in isolated tubulesof rabbits attributed active, transcellular movement of calcium to thedistal segments downstream of the macula densa (reviewed in Refs. 44, 53, and 107 ). Theobservation that, in humans, the tubular portion with a high abundanceof the calcium-extruding proteins PMCA and NCX, andcalbindinD 28k, is much longer than in rabbits, rats, andmice (see Fig. 6 ) suggests that, in humans, relevant transcellularcalcium transport may occur over a much longer tubular portion thanin laboratory animals, i.e., all along the distal convolution and alsoin the cortical collecting duct.
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The specific apical calcium channels in the distal tubules remainedelusive for a long time. Verapamil (dihydropyridine)-sensitive (cardiacL-type) calcium channels have been implicated in transcellular calciummovements ( 4, 72 ). In an immortalized mouse DCT cell line,antisense oligonucleotides directed against the 1c - or the 3 -subunits of verapamil-sensitive calcium channelsinhibited the rise in intracellular calcium concentrations in responseto PTH or chlorothiazide ( 4 ). Targeted disruption of the 3 -subunit in gene-modified mice blunted (in vivo) thehypocalciuric action of thiazides ( 8 ). These data couldsupport the hypothesis that verapamil-sensitive calcium channels indeedmay be implicated in transcellular calcium transport in the distalnephron. However, the predominantly cytoplasmic and basolateraldistribution pattern of 1c -subunits of the cardiacL-type calcium channel is more compatible with a role of these channelsin intracellular and membrane signaling processes rather than intranscellular calcium movement ( 142 ). The epithelialcalcium channel ECaC1 exhibits highly selective calcium permeability,is activated by hyperpolarization, and yet is insensitive to verapamil( 53 ). A homologous channel was identified from rat kidneyby Peng et al. ( 101 ) and called "calcium transporter2" (CaT2) ( 101 ), to distinguish it from the apicalcalcium channel CaT1 in intestine described earlier ( 102 ).

Interestingly, immunohistochemical ECaC1 traceability in the kidney ofrabbits ( 52 ), rats ( 55 ), and mice( 78 ) goes precisely in parallel with immunohistochemicalabundance of PMCA and NCX in distal nephron portions. Particularly wellevident is the axial decrease in ECaC1 localization in the apicalmembrane from the most upstream ECaC1-positive portions towardcytoplasmic domains in portions further downstream in mice( 78 ) and the parallel decreases in basolateral NCX andPMCA (Fig. 5 ). These observations suggest a parallel reduction ofcalcium transport rates. Whether the intracellulary localized ECaC1molecules are recruited to the apical plasma membrane, and in responseto which stimulus, is unknown as yet. Like NCC (see above), ECaC1 alsoappears to be upregulated by estrogens ( 127 ). Consistentwith a role of ECaC1 in regulated transcellular calcium transport isthe response in vivo at the mRNA and protein level to vitaminD 3 ( 55 ) and the reduced ECaC1 expression inkidneys of 25-hydroxyvitamin D 3 -1 -hydroxylase knockoutmice ( 51 ). These observations, taken together, speak infavor of ECaC1 as the key apical calcium channel in renal transcellular calcium transport in vivo ( 53 ). However, the notion thatNCX and other proteins involved in transcellular calcium movement aredetectable, although in comparably lower abundance, in distal siteslacking ECaC1 suggests that these tubular portions and other apicaltransporters may contribute to some calcium homeostasis as well.

Interaction of sodium and calcium transport along the distalconvolution. Costanzo and Windhager ( 27, 28 ) demonstrated in a seriesof microperfusion experiments that the acute application of thiazides or amiloride has a direct stimulatory effect on transcellular calciumreabsorption in the renal distal convolution. The obvious inverserelationship of sodium and calcium transport in the distal convolutionhas been explained by two rationales. 1 ) Ion transport inhibition by thiazide and amiloride hyperpolarizes the cells, andthereby activates calcium channels within the apical plasma membrane( 44 ). The hyperpolarization of the cells is thought to beeither indirectely related to the lowered chloride entry via NCC,causing an enhanced chloride influx across chloride channels, or isdirectly related to impaired sodium entry via ENaC, respectively. 2 ) The reduced apical sodium entry lowers intracellularsodium concentration and thus may increase the driving force forbasolateral Na/Ca exchange ( 44 ). These mechanisms couldexplain the hypocalciuric effect of thiazide diuretics and amiloride,as well as the reduced urinary calcium excretion found in patients withloss-of-function mutations for NCC (Gitelman syndrome) ( 44, 115 ). However, alternative explanations for the hypocalciuriaseen under these conditions are conceivable. For example, reducedurinary calcium excretion may be secondary to extracellular volumecontraction, contributing to enhanced sodium and calcium reabsorptionin the proximal tubules ( 16, 139 ).. s; R2 L5 a+ J0 K8 @6 O, a
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CONCLUDING REMARKS
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The cortical distal nephron is the site of fine regulation ofrenal electrolyte excretion. Along this nephron portion, three different salt transporters are arranged in series: the twocotransporters NKCC2 and NCC, and ENaC. In addition, within the distalconvolution ENaC is consistently coexpressed (as far as is known) withECaC1, and in some species also with the vasopressin-sensitive water channel AQP2. Because the transport activity of each of these transporters is differentially controlled, the serial arrangement mightguarantee, teleologically speaking, optimal electrolyte recovery undera vast range of environmental conditions.4 k* p! L* W* m1 E/ F6 E

Although the inventory and the basic sequence of transporters along thedistal cortical nephron are the same in kidneys of the investigatedmammalian species, there exist, however, subtle differences that mightbe functionally relevant. The distinctions pertain to the extent ofcoexpression of NCC with ENaC and ECaC1, ranging from not existent inthe rabbit to considerable in the mouse, and to the lengths of overlapof ENaC and AQP2. The species-specific distributions of transportproteins along the distal nephron coincide with the respectivestructural organization, either with sharp (rabbit) or with more orless progressive (mouse, rat human) structural segment transitions inthe distal convolution.

These species differences present some problems. One is a semantic oneregarding criteria for subdivision of the distal convolution. Thesegments DCT, CNT, and CCD are clearly demarcated by structure andcongruent distribution of transport proteins only in rabbits. The moreor less gradual structural changes and the partial coexpressions oftransporters in the other species make segmentation a matter ofdefinition. Thus interpretation of functional data from the distalnephron needs precise criteria defining the investigated nephron portion.

A presumably more challenging problem is implicated in the functionalconsequences of the different species-specific topological arrangementsof the transporters. Any maneuver affecting a given transporteractivity in the distal nephron will result in slightly differentsecondary changes along the nephron and in the final urinaryelectrolyte excretion pattern in each species. These differences mightbe amplified by the exceedingly functional plasticity of the distalconvolution, in particular of the DCT, triggered by the manifoldfactors mentioned in this article and probably by many others notdiscussed here.7 u5 i* X0 }3 N# e: J
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Thus despite the large congruence among the species with respect to theoccurrence of transport proteins in the distal convolution, thedistinct differences in the topology of transporters along the distalconvolution limit direct extrapolation of data from one species to theother. Discrepancies between the urinary excretion pattern of humanssuffering from a defined gene defect for a given transport protein andgenetically engineered mouse models with the respective defect mightlie, in part, in the different species-specific organizations of thedistal convolution and of the kidney.

ACKNOWLEDGEMENTS. h- ^4 Q3 D5 H/ _; e$ U. K  F( @. Z, b
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The authors thank Dr. D. Loffing-Cueni for contributing Fig. 4.# C8 W; ~/ J) Q" i
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