PHYSIOLOGICAL REVIEWS
Vol. 80, No. 1, January 2000
Printed in U.S.A.
Mammalian Distal Tubule: Physiology, Pathophysiology,
and Molecular Anatomy
ROBERT F. REILLY AND DAVID H. ELLISON
Department of Medicine, University of Colorado School of Medicine and Department of Veterans Affairs
Medical Center, Denver, Colorado
Introduction
Morphological Segmentation
Embryological Development
Physiology of Ion Transport
A. Electrophysiology
B. Na1 and Cl2 transport
C. K1 transport
D. Ca21 transport
V. The Distal Tubule in Human Disease
A. Gitelman’s syndrome
B. Distal disorders of urinary acidification
C. Adapation to diuretic drugs
D. Nephrolithiasis
E. Treatment of nephrolithiasis with distal tubular diuretics
VI. Summary
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II.
III.
IV.
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Reilly, Robert F., and David H. Ellison. Mammalian Distal Tubule: Physiology, Pathophysiology, and Molecular
Anatomy. Physiol. Rev. 80: 277–313, 2000.—The distal tubule of the mammalian kidney, defined as the region
between the macula densa and the collecting duct, is morphologically and functionally heterogeneous. This
heterogeneity has stymied attempts to define functional properties of individual cell types and has led to controversy
concerning mechanisms and regulation of ion transport. Recently, molecular techniques have been used to identify
and localize ion transport pathways along the distal tubule and to identify human diseases that result from abnormal
distal tubule function. Results of these studies have clarified the roles of individual distal cell types. They suggest that
the basic molecular architecture of the distal nephron is surprisingly similar in mammalian species investigated to
date. The results have also reemphasized the role played by the distal tubule in regulating urinary potassium
excretion. They have clarified how both peptide and steroid hormones, including aldosterone and estrogen, regulate
ion transport by distal convoluted tubule cells. Furthermore, they highlight the central role that the distal tubule
plays in systemic calcium homeostasis. Disorders of distal nephron function, such as Gitelman’s syndrome,
nephrolithiasis, and adaptation to diuretic drug administration, emphasize the importance of this relatively short
nephron segment to human physiology. This review integrates molecular and functional results to provide a
contemporary picture of distal tubule function in mammals.
I. INTRODUCTION
The distal tubule of the mammalian kidney, defined
as the nephron segment interposed between the macula
densa region and the first confluence with another
nephron to form the cortical collecting tubule, comprises
several morphologically and functionally heterogeneous
subsegments (see Fig. 1A). It reabsorbs 5–10% of the
filtered sodium and chloride under normal conditions and
participates importantly in net K1 secretion. It plays a
0031-9333/00 $15.00 Copyright © 2000 the American Physiological Society
central role in systemic calcium homeostasis, plays an
important role in systemic magnesium homeostasis, and
participates in net acid secretion. Inherited disorders of
distal cell function lead to systemic abnormalities of extracellular fluid volume and potassium, calcium, and magnesium balance, confirming the importance of the distal
tubule to human physiology and human disease (225). Yet,
until recently, properties of individual distal cell types had
not been identified conclusively. This led both from the
cytological heterogeneity of the segment and from appar277
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ROBERT F. REILLY AND DAVID H. ELLISON
Volume 80
ent differences between properties of distal cells in different species. During the past several years, molecular
tools have permitted investigators to assign functional
properties to specific nephron segments and cell types,
clarifying the molecular basis of physiological function.
Coupled with the identification of human diseases, both
genetic and acquired, that arise from dysfunction of distal
tubule cells, these developments have renewed interest in
the structure and function of the mammalian distal tubule.
II. MORPHOLOGICAL SEGMENTATION
The variable nomenclature applied to the distal portion of the mammalian nephron and segmental and func-
tional differences between mammalian species have led
to confusion about anatomical and functional properties
of the distal tubule. This variability is summarized in
Figure 2, which is adapted from a scheme presented by
the Renal Commission of the International Union of Physiological Sciences (155). Although this scheme was intended to encourage a uniform nomenclature for nephron
segments, the diversity of terms and definitions has persisted. As might be expected, based on nosological inconsistencies, attribution of function to particular segments
may depend on the definition that is employed. One goal
of this review is to provide precise definitions of cell types
and segments with which to correlate functional properties.
Although the definition of “distal tubule” (Fig. 2, see
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FIG. 1. Structure of mammalian nephron. A: organization of nephron including distal tubule. Two types of nephrons
are shown. A superficial nephron, at left, contains proximal and distal tubules that ascend to kidney surface and possess
short loops of Henle. Loops and collecting ducts of superficial nephrons are located within medullary rays (MR). In
superficial nephrons, distal tubule comprises distal convoluted tubule (DCT, shown in white), a short connecting tubule
(CNT, shown as hatched), and a portion of collecting duct epithelium (shown in white) that begins proximal to junction
to form collecting duct. A juxtamedullary nephron (such as that shown at right) has long loops of Henle that descend
into renal medulla. Distal tubules comprise only a DCT and CNT segment. In juxtamedullary nephrons, CNT join to form
branched arcades that ascend through cortical labyrinth (CL) before emptying into collecting duct (CCT). [Modified from
Kriz and Kaissling (155).] B: schematic representation of a cross section through renal cortex. CL is shown as speckled.
Medullary rays are shown in white. Note that DCT and CNT lie within CL. DCT lie close to glomeruli. CNT (arcade
segments) are closely associated with interlobular arteries and veins. [Modified from Kriz and Kaissling (155).]
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MAMMALIAN DISTAL TUBULE
279
asterisk) given in section I is precise, this term has also
been used by anatomists (see Fig. 2, main divisions) to
denote the segment comprising the thick ascending limb
(or distal straight tubule) and distal convoluted tubule
(see below). According to this anatomical definition, the
connecting tubule and cortical and medullary collecting
ducts form the collecting system. Early anatomists and
physiologists, however, also described a “distal convolution” (see Fig. 2, microanatomical terms), a segment that
is distinct from both the proximal convolution and the
straight tubules (112). This distal convolution corresponds to the distal tubule of the micropuncture literature
and comprises primarily the distal convoluted tubule and
connecting tubule (253, 296). Some authors have referred
to the entire region between the macula densa and the
confluence as the “distal convoluted tubule” (190, 191);
this region has then been divided into bright, granular,
and light portions, according to its appearance during
dissection in vitro. In this review, the term distal convoluted tubule is restricted to the segment comprising distal
convoluted tubule cells.
Careful analysis of cell types along the renal distal
tubule and of the segment’s physiological properties, its
hormonal responsiveness, and its response to physiological perturbation indicates that the renal distal tubule
comprises four anatomically discrete subsegments (59,
129, 155). These include a short region of thick ascending
limb (TAL), the distal convoluted tubule (DCT), the connecting tubule (CNT), and the initial portion of the cortical collecting tubule (CCT). These terms (preferred terms
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FIG. 2. Segmentation of mammalian nephron. Scheme modified from that proposed by Renal Commission of
International Union of Physiological Sciences (155). Note that definitions used in this review correspond to those listed
as “preferred terms” except that the term distal tubule (see asterisk) is used to denote nephron segment between region
of macula densa and confluence with another tubule to form collecting duct.
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ROBERT F. REILLY AND DAVID H. ELLISON
posed between the nucleus and apical membrane but do
fill the perinuclear region. The apical membrane is characterized by numerous short microvilli. Corresponding to
the high density of mitochondria and to the extensive
basolateral membrane amplification, the Na1-K1-ATPase
activity is the highest in the DCT of any nephron segment
(141, 229).
The CNT lies just distal to the DCT, arising abruptly
in rabbits and gradually in most other species (133, 155).
The existence of a connecting portion or Verbindungsstück was first suggested by Schweigger-Seidel (59). In all
species, it comprises predominantly two cell types, CNT
cells and intercalated cells. The microanatomical organization of CNT differs between superficial and deeper
nephrons (see Fig. 1A). In superficial nephrons, the CNT
is an unbranched segment that flows into the initial portion of the CCT (the initial collecting tubule) before draining into a collecting duct. In the rat, the CNT of superficial
nephrons was shown to be 0.49 6 0.03 mm in length (67).
In contrast, as shown in Figure 1A, the CNT of midcortical
and juxtamedullary nephrons frequently form branched
structures (termed “arcades”) that ascend through the
cortical labyrinth. These arcades usually run close to an
interlobular artery (Fig. 1B; Ref. 148). Only after one or
more CNT have joined do principal cells appear, indicating transition to the CCT. It has been suggested that some
arcades probably exist in all mammalian species, but the
percentage of nephrons emptying into the collecting duct
via arcades, versus emptying directly, varies. In rat, rabbit, and pig, the majority of deep nephrons drain via
arcades (67, 148, 155). In humans, most nephrons drain
individually (155). In all species, the entire renal distal
tubule lies within the cortical labyrinth (Fig. 1B).
The appearance of CNT cells (see Fig. 3) is the same
whether in superficial distal tubules or in arcades. Connecting tubule cells have been characterized as appearing
intermediate between DCT and principal cells. In species
such as the rat, where transitions from DCT to CNT and
CCT are gradual, distinguishing DCT, CNT, and principal
cells may be difficult, especially near the junctions between segments. However, it is now clear that CNT cells
are distinct morphologically, functionally, and at the molecular level (129, 133, 155, 177, 268). Connecting tubule
cells demonstrate basolateral cell membrane amplification, as do DCT cells. However, in CNT cells, more of the
amplification results from infolding of the basal membrane rather than amplification of lateral cell processes
(as in DCT cells). Thus, in the CNT, there is less cell-cell
contact than in the DCT. Mitochondria appear between
the basal infoldings, but their number is significantly reduced compared with the DCT. The nucleus of CNT cells
is apically oriented, but unlike in DCT cells, mitochondria
may be observed between the nucleus and the apical
membrane. The apical membrane of CNT cells exhibits
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in Fig. 2) will be used to denote these subsegments, unless
otherwise noted. The term renal distal tubule will be used
to indicate the entire region between the macula densa
and the confluence with another tubule.
Figure 1A shows the microanatomical organization
of superficial and juxtaglomerular distal nephrons. In rats
and rabbits, the post macula densa thick ascending limb
of superficial nephrons ascends toward the kidney surface. The length of this post macula densa segment varies
greatly. It is 0 –500 mm in rabbits and 150 6 20 mm in rats
(67). This segment never reaches the kidney surface in
normal rats, rabbits, or humans (155). In certain unusual
strains, such as the Munich Wistar rat, both glomeruli and
post macula densa thick ascending limb segments are
observed at the kidney surface. This feature has been
utilized experimentally to assess glomerular function. In
most other species, however, there is an abrupt transition
from TAL to DCT before the tubule reaches the kidney
surface. After ascending toward the kidney surface, DCT
commonly make “hairpin” turns, after which they return
close to the glomerulus (see Fig. 1A). Although direct
contact of the second loop of the distal tubule with a more
proximal portion of distal tubule or the macula densa is
uncommon, this second portion of the distal tubule frequently does make contact with the afferent arteriole
(68). The physiological significance of this contact is not
clear. The distal convoluted tubule has been shown to be
1.15 6 0.05 mm long in the adult rat, with the entire renal
distal tubule measuring 2.26 6 0.11 mm (67). In all species, the DCT comprises distal convoluted tubule cells; in
most species, intercalated cells begin to appear along the
length of this tubule segment. In rat, mouse, and human,
intercalated cells appear in the latter portion of the DCT
and continue through the connecting and collecting tubules (155); in rabbit, intercalated cells are found only in
the connecting and collecting tubules (155).
Distal convoluted tubule cells (see Fig. 3) are taller
than TAL cells and demonstrate extensive amplification
of the basolateral cell membrane (amplification is defined
as the ratio of basolateral membrane area to apical membrane area). The amplification, twofold in rat and rabbit
(155), results from extensive formation of narrow lamellalike lateral processes. These processes exhibit intermingling with neighboring DCT cells, providing extensive
areas of cell-cell interaction. Encased within these basolateral processes are mitochondria which impart a “palisading” appearance to the cells (129, 133). Distal convoluted tubule cells contain the largest number of
mitochondria per unit length of any cell along the
nephron. In the basal portion of the cell, the lateral cell
processes split into basal ridges that contain bundles of
filaments, believed to anchor the cells to the basement
membrane. Cell nuclei are uniformly located very close to
the apical surface, where they often appear to be flattened
by the luminal membrane. Mitochondria are not inter-
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FIG. 3. Ion transport pathways and cellular morphology of rat distal tubule. Schematic cell models are shown at left.
Transepithelial voltage (VTE) in most proximal portion of DCT is near zero. Magnitude of voltage becomes larger in more
distal segments. Ion transport pathways that have been identified by functional and molecular techniques are shown with
solid black arrows. Ion transport pathways for which functional evidence is present but which lack molecular
confirmation are shown with gray arrows. Each cloned pathway that has been localized to this segment is identified by
a letter. DCT-1 and DCT-2 identify segments of DCT in which Na1/Ca21 is (DCT-2) or is not (DCT-1) expressed. For
details see text. At right are shown DCT and CNT cells. DCT cells are taller with more densely packed mitochondria.
[Morphological drawings from Kriz and Kaissling (155).]
fewer apical projections than does the apical membrane
of DCT cells.
The transition to the initial collecting tubule is abrupt
in the rabbit but appears gradual in the rat, mouse, and
human (58, 59, 129, 155). In the rat, the initial collecting
tubule may be as long as 0.71 6 0.12 mm in superficial
nephrons, but it is shorter (0.22 mm) in deeper nephrons
(67). The epithelium of the initial CNT is indistinguishable
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III. EMBRYOLOGICAL DEVELOPMENT
The human kidney arises from two embryonic tissues, the metanephric blastema and the ureteric bud.
During the process of renal development, the ureteric bud
elongates and branches. At the tips of these branches the
metanephric mesenchyme condenses to form renal vesicles. The renal vesicle elongates to become a commashaped body, then subsequently an S-shaped body before
it fuses with a branch of the ureteric bud to form a
complete nephron (262). After the S-shaped body fuses
with the ampulla of the ureteric bud, this portion of the
nephron undergoes marked elongation, and this structure
is called the connecting piece. The connecting piece runs
from near the glomerulus distal to the macula densa and
enters the collecting duct at a right angle. The site that
corresponds to the junction of the S-shaped body and the
ureteric bud within the nephron is controversial, as is the
embryonic tissue of origin of the connecting piece.
Initial studies that were based on microdissection
concluded that the connecting piece originated from the
metanephric blastema (202). They stated that the CNT
was derived from the S-shaped body and therefore the
metanephric blastema and that the site of union of the
metanephric blastema and the ureteric bud was the junction of the CNT and the collecting duct.
Howie et al. (121) examined this issue using a series
of markers against blood group antigens and cytokeratins
in human kidney. They employed monoclonal antibody
FC 10.2 directed against the type 1 precursor chain of
ABO antigens, a lectin UEA-1 that recognizes the H antigen of the O blood group, monoclonal antibody PKK2
directed against cytokeratins 7, 16, 17, and 19, and monoclonal antibody AE1 generated against cytokeratins 14,
15, 16, and 19. All of these antibodies and lectins stained
both the ureteric bud and the connecting piece, indicating
that the connecting piece had arisen from the ureteric
bud. The authors conclude that the junction of the ureteric bud and the metanephric blastema may be at the
boundary of the TAL and the DCT.
Schmitt et al. (230) recently examined this same
question in the newborn rat using a variety of antibodies
directed against proteins involved either directly or indirectly in ion transport. These workers suggested a third
alternative; the CNT arises as a product of the mutual
induction of the metanephric blastema and the ureteric
bud that creates a unique hybrid epithelium (230).
IV. PHYSIOLOGY OF ION TRANSPORT
A. Electrophysiology
Electrical properties of the DCT, CNT, and initial
portion of the CCD were examined first in vivo using
micropuncture techniques in rats. Subsequently, rabbit,
mouse, and rat kidney tubules have been microperfused
in vitro. Distal convoluted tubules dissected from rabbits
and grown in cell culture have also been studied. There
have been differences between results obtained in different mammalian species and using different experimental
techniques. Although these differences have been emphasized, when data are analyzed in light of more recent
physiological and molecular results, the differences are
outweighed by similarities.
In the rat, experiments performed in vivo showed
that the transepithelial voltage throughout most of the
superficial renal distal tubule is oriented with the lumen negative with respect to the blood (48, 140, 181,
182, 211, 248). Wright (296) first showed that the mag-
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from that observed in true cortical collecting ducts
(CCD). It comprises principal and intercalated cells. Principal cells are organized in a manner similar to that of
CNT cells, but they are lower. Basal infoldings are generally restricted to the basal third of the cells, and the
infoldings do not contain mitochondria. The apical membrane has few microvilli but does contain a prominent
central cilium. Below the apical membrane, a dense meshwork is formed by microtubules and microfilaments; included in this network are aggrephores, which contain
aquaporin for insertion into the apical membrane under
the control of antidiuretic hormone (287).
Intercalated cells appear in the CNT and collecting
duct of all species. In the rabbit, they are not found in the
DCT, but in most other species, including rat, mouse, and
human, they appear in the latter third of the DCT. In the
rat, Dorup (67) showed that intercalated cells appear ;1
mm beyond the macula densa, ;500 mm before DCT cells
disappear (67). In humans, intercalated cells appear along
the latter half of the DCT (268). Along the CNT, intercalated cells comprise from 25–30% (in rat) to 40 – 45% (in
rabbit) of cells. In the CCT, intercalated cells comprise
;40% of cells (155). Unlike CNT and principal cells, which
appear polygonal when viewed by scanning electron microscopy, intercalated cells have a round appearance.
Their apical membranes are densely adorned with microprojections. Their lateral surfaces adhere to adjacent connecting and principal cells by desmosomes. The nucleus
of these cells is at the basal pole (133, 155). Two types of
intercalated cells are recognized morphologically. Type A
cells manifest extensive apical microvilli and numerous
small mitochondria within the subapical region. Type B
cells have fewer apical microvilli, and mitochondria tend
to accumulate in the basal portion of the cell. Although
the ratio of type A to type B cells may vary depending on
the physiological condition of the animal, type A cells are
more numerous in the CNT and type B cells in the CCD
(146, 147, 155).
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MAMMALIAN DISTAL TUBULE
nitude of the transepithelial voltage, measured in vivo,
increases from the most proximal accessible segment
to the most distal segment. This result was subsequently confirmed by others (see Fig. 4) (2, 9, 10, 36, 55,
179, 296). One concern about these experiments is that
they utilized small-tipped Ling-Gerard-type microelectrodes. In contrast, Barratt and colleagues (2, 9, 10)
reported that the transepithelial voltage, measured in
the earliest portions of the superficial distal tubule, is
oriented with the lumen positive compared with the
blood. Those experiments utilized large-tipped electrodes (2, 9, 10). Although differences in electrode size
or filling solution (3, 117) or differences in rat strain (2,
9, 10) may have contributed to differences in results
from different laboratories, Figure 4 suggests a reasonably consistent picture. Whether measured using smalltipped or larger electrodes, the transepithelial voltage
along the superficial rat distal tubule is oriented with
the lumen negative, with respect to the blood, throughout most of its length. The transepithelial voltage of the
most proximal segments is near to zero, but the voltage
becomes negative as fluid courses along the DCT into
the CNT and remains negative into the initial portion of
the CCD.
With the use of new information about structural and
molecular properties of rat superficial distal tubules, it is
now possible to correlate electrophysiological with molecular results showing expression of specific transport
proteins. The transepithelial voltage of the TAL is oriented with the lumen positive with respect to the interstitium. It typically ranges from 5 to 15 mV (107). Thick
ascending limb cells extend 100 –300 mm beyond the macula densa in both rabbit and rat (133). All investigators
have reported that voltage of the CNT is oriented with the
lumen negative with respect to the interstitium. Thus the
transepithelial voltage must change from a lumen positive
to lumen negative orientation between the region of the
macula densa and the CNT. Schnermann et al. (231)
showed that the first 300 mm of rat DCT secretes NaCl in
situ. They suggested that NaCl secretion creates a lumenpositive diffusion potential, owing to the higher permeability of these segments to Na1 than to Cl2. Thus the
most proximal portion of DCT epithelium may be oriented
with the lumen positive with respect to the interestium.
Interestingly, when Wright (296) plotted transepithelial
voltage versus percent length along the superficial distal
tubule, a sigmoidal relation was obtained (see Fig. 4). The
voltage was very low along the first 40% of the tubule
length, rapidly increased in the region between 40 and
60%, and remained constant along the terminal 30% of
tubule length. The region between 40 – 60% of distal length
was shown by Dorup (67) to be the site at which intercalated cells appear in the rat and to comprise primarily
DCT and CNT cells. This region is also the site at which all
three subunits of the epithelial Na1 channel first appear
(283). Thus the transepithelial voltage becomes negative
in the region in which molecular pathways that mediate
electrogenic Na1 transport appear at the apical membrane.
Figure 4A also shows results of transepithelial voltage measurements of rat CCD, perfused in vitro. It is
beyond the scope of this review to discuss properties of
the CCD in detail, but it should be noted that the magnitude of the transepithelial voltage is smaller in collecting
ducts perfused in vitro than in “late” distal tubules, perfused in vivo. This may reflect true differences in properties between collecting ducts and CNT or initial collecting
tubules. A component of the difference is likely to reflect
the environment in which the tubules are studied. Factors
may be present in vivo, such as aldosterone and arginine
vasopressin, that stimulate ion transport and that are
removed during in vitro microperfusion. This is consistent
with the observation that collecting ducts from rats
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FIG. 4. Top: transepithelial voltage (VT, in mV) along rat distal
tubule. Data obtained during in vivo micropunture are indicated by solid
symbols and plotted as a percentage of total distal tubule length (X, Ref.
296; ●, Ref. 179; ■, Ref. 10; , Ref. 117; , Ref. 55). Data obtained by
microperfusing cortical collecting ducts in vitro are presented as open
or gray symbols within 2 ovals. Data collected without mineralocorticoid hormone treatment are indicated with open circles (2MA; {, Ref.
270; E, Ref. 212; h, Ref. 228). Data collected in presence of mineralocorticoid hormone treatment are indicated by shaded symbols (1MA; gray
circle, Ref. 270; ■, Ref. 212). Location of DCT, CNT, and CCT is inferred
from percentage length along distal tubule (67). Bottom: transepithelial
resistance (RT) along rat renal distal tubule. Data obtained during in vivo
micropuncture are indicated by solid symbols (references as in top
panel). Data obtained by microperfusion in vitro are indicated by open
symbols (references as in top panel).
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FIG. 5. Top: transepithelial voltage (VT, in mV) along rabbit distal
tubule, measured during microperfusion in vitro. Data obtained before
1980 are shown as follows: {, Ref. 114; V, Ref. 124. Data obtained since
1980 are shown as follows: }, Ref. 4; ■, Ref. 240; X, Ref. 303; 3, Ref. 279;
, Ref. 280; box with solid circle inside, Refs. 195, 259, 294; box with X
inside, Ref. 222. Data from cortical collecting ducts are grouped according to presence or absence of mineralocorticoid (MA) treatment. Bottom: transepithelial resistance (RT) of DCT, CNT, and CCT segments
perfused in vitro. Symbols are as in top panel. Dotted line shows mean
values. Solid line shows mean values for fractional apical resistances
(fRa) taken from Refs. 195, 279, 303.
time at its midpoint. When the proximal segments were
perfused in vitro, the transepithelial voltage was 0 mV and
was amiloride resistant. In contrast, when the more distal
segments (still “DCT” segments) were perfused in vitro,
the transepithelial voltage was lumen negative (24 mV)
and could be inhibited by amiloride (283). These results
suggest that the large transepithelial voltages obtained
from early studies resulted, in part, from contamination
by CNT cells. A second cause for variability of membrane
voltages is the tubule perfusion rate. The transepithelial
voltage of distal nephron segments varies inversely with
tubule perfusion rate and with perfusion pressure (114,
124, 235). Although the mechanisms by which perfusion
pressure and flow rate affect voltage are not understood
fully, the same effect is observed in vivo, during perfusion
of distal tubules (111). In vivo, the effect appears to result
predominantly from flow-dependent changes in luminal
ion concentration (108). Regardless of the mechanisms by
which luminal flow rate affects the transepithelial voltage,
it appears that most measurements of transepithelial voltage in earlier studies were obtained using lower perfusion
pressures. If one, therefore, uses data from four more
recent studies performed in two different laboratories,
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treated with mineralocorticoid hormones and perfused in
vitro display voltages nearly as large as those observed
along the distal tubule in vivo (see Fig. 4A).
Figure 4B shows measurements of the transepithelial
resistance of rat distal tubules, measured both in vivo and
in vitro. Of note, the resistance is correlated inversely
with the magnitude of the transepithelial voltage and
therefore with the distance along the tubule. De Bermudez and Windhager (61) showed that the resistance of the
distal tubule is reduced by arginine vasopressin, an effect
that is more pronounced along the last 50% of distal
tubule length than along the first half of distal tubule
length. The decline in transepithelial resistance along the
distal tubule probably reflects the larger component of
electrogenic transport in more distal regions. Ion transport across the apical membrane of DCT cells appears to
occur predominantly via electroneutral pathways,
whereas transport across the apical membrane of CNT
cells and principal cells is mediated in large part by
electrogenic pathways. Both arginine vasopressin and aldosterone increase the luminal conductance for Na1
along the rat collecting duct (116, 218). Although the
transepithelial resistance also reflects paracellular resistance, the profile observed along the distal tubule and the
response of that profile to hormonal stimuli suggest that
the axial differences in resistance may reflect cellular
properties.
Transitions between segments of the rabbit distal
nephron are abrupt, making it possible to isolate specific
nephron segments for study in vitro. Data concerning the
transepithelial voltage of rabbit distal tubules in vivo are
not available, so a direct comparison with rat data is not
possible. Unfortunately, despite discrete segmentation of
the rabbit distal nephron, a great deal of variability has
been observed in studies of rabbit distal segments perfused in vitro. This has led to confusion about electrical
and physiological properties of rat and rabbit distal tubules. Figure 5A shows results of voltage measurements
in rabbit DCT, CNT, and collecting ducts. Two studies
during the 1970s indicated that the voltage of the rabbit
DCT is large and oriented with the lumen negative, with
respect to the bath (114, 124). Subsequently, studies conducted during the 1980s and 1990s have obtained voltages
that are much lower (195, 239, 240, 283, 303). There are
several explanations for the differences in transepithelial
voltage between studies performed before 1980 and those
performed subsequently. In the early experiments, the
perfused DCT segments were as long as 800 mm (114). As
noted by Imai and colleagues (241, 243), DCT segments
are usually ,500 mm in length in rabbits, and it is likely
that the longer DCT segments contained CNT cells as well
as DCT cells. Velázquez et al. (283) recently harvested
DCT from rabbits in the usual manner by cutting at the
transition from TAL to DCT and at the junction with the
CNT. Each DCT segment was then transected a second
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Figure 5B also shows the fractional apical resistance of
cells along the distal tubule (ignoring intercalated cells).
The fractional apical resistance declines from DCT to
CNT and then further in the CCT. This indicates that the
apical membrane of principal cells and CNT cells is more
conductive than that of DCT cells. This conclusion is
consistent with results of molecular studies.
Rabbit DCT cells have also been grown in culture as
primary cultures. Whether grown for 15 or 30 days, cells
exhibited a transepithelial voltage oriented with the apical surface negative with respect to the basolateral surface (77, 189). The magnitude of the voltage increased
from 3 to 22 mV from 15 to 30 days. This voltage was
inhibited by the Na1 channel blocker amiloride. The resistance of the epithelial monolayers was 500 Vzcm2 at
15–20 days and ;1,800 Vzcm2 at 30 days (189).
B. Na1 and Cl2 Transport
1. NaCl transport by the distal tubule
Information about the quantitative contribution of
the distal tubule to renal NaCl transport derives primarily
from micropuncture experiments in rats. When volume
status ranges from low to high, Na1 delivery to the superficial distal tubule ranges from 4 to 20% (122, 144, 180).
Because solute but not water is reabsorbed by TAL cells,
the Na1 concentration in the lumen of the first accessible
segments of distal tubules ranges from 35 to 77 mM (108).
It should be recalled, however, that solute delivery to
distal segments accessible to micropuncture reflects the
actions of the post macula densa TAL and the proximal
portion of the DCT. These sites lie proximal to the “earliest” distal puncture site in typical rats. Using Munich
Wistar rats, which have post macula densa segments accessible at the kidney surface, Schnermann et al. (231)
showed that luminal Na1 and Cl2 concentrations rise as
fluid flows from the macula densa into the DCT, owing to
Na1 and Cl2 secretion (231). Thus solute delivery to the
most proximal portions of the DCT may be overestimated
by measurements taken in superficial distal tubules.
When Na1 delivery to the DCT is varied acutely, Na1
transport varies directly. Khuri et al. (144) and Kunau et
al. (158) increased distal Na1 delivery by infusing saline
or urea-saline into hydropenic animals. Khuri et al. (144)
found that 80% of the delivered Na1 load was reabsorbed
along the distal tubule across a wide range of delivered
loads. Kunau et al. (158) reported a similar relation between delivered load and transport when hydropenic animals were volume expanded slightly. When volume expansion was more extreme, however, these investigators
reported that fractional Na1 reabsorption declined to 40%
(158). Although these data suggest that the distal tubule
responds to extracellular volume expansion by reducing
the fractional NaCl reabsorption, Diezi et al. (63) showed
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remarkably similar results are obtained. These indicate
that the transepithelial voltage of the rabbit DCT is quite
low when perfused in vitro (Fig. 5A), a result similar to
that obtained from rat DCT segments perfused in vivo.
When CNT are perfused in vitro, they are usually
obtained from arcade segments (see Fig. 1A), not from
superficial distal tubules. Thus data on the properties of
rabbit CNT cannot be compared directly with data on
properties of “mid” or “late” segments of rat distal tubules. Nevertheless, both morphological and molecular
information suggest that CNT from arcades are similar to
CNT from superficial distal tubules (7, 50, 76, 199). The
transepithelial voltage of CNT from rabbits perfused in
vitro has ranged between 5 and 27 mV, oriented with the
lumen negative with respect to the bath (see Fig. 5A). As
is the case for other tubule segments, the voltage of CNT
varies inversely with perfusion pressure; the highest voltage (227 mV) reported for this segment was obtained at
a very low perfusion rate (124).
The voltage of the rabbit CCT perfused in vitro is
oriented in the lumen-negative direction and is low (average 210 mV). The voltage is higher when animals or
tubules have been treated with mineralocorticoid hormones (235); it is lower when animals have consumed a
high-sodium low-potassium diet or after adrenalectomy
(194). As is the case for the CNT and DCT, the transepithelial voltage varies inversely with fluid flow rate and
perfusion pressure (235).
Differences between properties of rabbit and rat distal tubules have been emphasized (152), yet several of
these differences may not be as great as previously suspected. Figure 5A might be taken to suggest that the
transepithelial voltage does not change along the distal
tubule of the rabbit. However, if one uses only the results
from studies performed after 1980, and if one plots the
data from collecting ducts of aldosterone-treated animals,
the voltage profile is quite similar to that obtained in rat
distal nephrons. One indication that mineralocorticoid
treatment is necessary for in vitro data to more closely
match in vivo data is obtained by comparing the voltage in
the rat “late distal tubule” with the voltage of collecting
ducts perfused in vitro (Fig. 4A). In this case, the data
from the rat late distal tubule perfused in vivo match data
from tubules perfused in vitro only when mineralocorticoid hormones have been used. Thus the voltage profile
along the rabbit distal tubule may be qualitatively similar
to the profile observed in rat. The proposed similarities in
electrical profiles between rat and rabbit distal tubules
are strongly supported by recent observations on expression of electroneutral and electrogenic transport proteins
by rat and rabbit tubules (7, 199).
Figure 5B shows the transepithelial resistance of rabbit distal segments, perfused in vitro. Although the transepithelial resistance is highest in the CCT, this value
reflects both transcellular and paracellular resistances.
285
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ROBERT F. REILLY AND DAVID H. ELLISON
2. Mechanisms of Na1 and Cl2 transport
Pathways for Na1 and Cl2 transport across the
mammalian distal tubule have been investigated using
functional, optical, electrophysiological, immunological,
radioligand, and molecular techniques. These pathways
are shown in Figure 3. Sodium-potassium-ATPase is expressed at the basolateral membrane of DCT cells, CNT
cells, and principal cells (139). It provides the primary
driving force for Na1 transport across all three segments
by keeping the cellular Na1 concentration low and the
cellular K1 concentration high. The Na1-K1-ATPase also
contributes to making the inside of the cell electronegative with respect to the outside, both because it is electrogenic (moves 3 Na1 out and 2 K1 in) and because it
generates large ion concentration gradients. Distal convoluted tubule cells express the highest Na1-K1-ATPase
activity of any nephron segment (69, 80, 96, 141, 166), an
observation that mirrors the extensive amplification of
their basolateral cell membrane and the abundance of
mitochondria (134, 166). Connecting tubule cells also express high levels of Na1-K1-ATPase activity. The Na1-K1ATPase activity of principal cells is much lower but is
subject to considerable regulatory variation (166).
A major pathway by which Na1 enters DCT cells
across the apical membrane is the thiazide-sensitive NCC
(see Fig. 3) (7, 23, 199, 206, 301). Thiazide diuretics were
shown more than 30 years ago to inhibit Na1 transport
along the superficial distal tubule (159), but the nature of
the transport pathway(s) inhibited by these diuretics remained obscure. Velázquez et al. (278) showed that Na1
transport by distal tubules is dependent on the luminal
Cl2 concentration and that Cl2 transport is dependent on
the luminal Na1 concentration (278). These data suggested close coupling between Na1 and Cl2 transport by
A) DCT.
distal tubules. Stokes (260) reported the existence of a
thiazide-sensitive electroneutral Na1-Cl2 cotransport
pathway in the bladder of the winter flounder. Ellison et
al. (78) reported that a thiazide-sensitive electroneutral
NCC mediates the majority of Na1 and Cl2 transport by
DCT. Gamba et al. (95) used an expression cloning strategy to isolate a NCC from the bladder of the winter
flounder. Northern blot analysis, using the probe from the
flounder bladder, indicated that a related transcript is
present in rat and mouse renal cortex. The thiazide-sensitive NCC was subsequently cloned from rat (94), human
(246), mouse (160), and rabbit (281).
The mammalian renal thiazide-sensitive NCC has a
core size of 110 kDa, but because it is glycosylated runs
between 125 and 180 kDa on Western blots (23, 206). The
protein is believed to comprise 12 membrane-spanning
domains, with amino and carboxy termini within the cytoplasm. The protein is glycosylated on two asparagine
residues on the fourth extracellular loop (198); deletion of
both glycosylation sites (but not either one alone) creates
a protein that is not functional. The sites on the cloned
protein at which thiazide diuretics, Na1, and Cl2 bind
have not been identified. Tran et al. (271) showed that Cl2
competitively inhibits [3H]metolazone binding to kidney
membranes, suggesting that the diuretic binds to the anion site on the transporter. The NCC is homologous to the
bumetanide-sensitive Na1-K1-2Cl2 cotransporters (both
secretory and absorptive isoforms, Ref. 125), and to the
more recently described K1-Cl2 cotransporter (105).
These transporters are part of a gene family, the cation
chloride cotransporters (118); the genes for this family
are identified as SLC12, and the thiazide-sensitive NCC is
SLC12A3.
Recently, Chang and Fujita (40) proposed a kinetic
model of the thiazide-sensitive NCC. According to this
model, mechanisms underlying the binding of ions and
diuretics can be approximated by a state diagram. The
transport protein is postulated to possess two ion binding
sites: one for anions and DCT diuretics and one for cations. Rate constants can be devised that permit the behavior of the model system to correspond to the behavior
of the protein under a variety of physiological conditions.
Functional data from rat microperfusion studies in
vivo (52, 78) and isotopic studies of mouse DCT cells
grown in vitro (100) indicate that the thiazide-sensitive
NCC is expressed predominantly by DCT cells. In contrast, some results of experiments using microperfusion
of rabbit distal segments in vitro have suggested expression by other cell types. In rabbit nephron segments,
dissected and perfused in vitro, thiazide-sensitive Na1Cl2 cotransport was detected in CNT but not in DCT (238,
240, 303) by some, but not all (280), investigators. Some
(267) but not other (220) investigators detected thiazidesensitive Na1-Cl2 transport in rat CCD perfused in vitro.
In those experiments, pretreatment of animals with min-
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that acute volume expansion does not alter Na1 transport
along the distal tubule when Na1 and Cl2 delivery rates
are controlled by microperfusion. The bulk of the increased NaCl transport along the distal tubule that occurs
when luminal delivery increases results from enhanced
transcellular transport via the thiazide-sensitive Na1-Cl2
cotransporter (NCC). In microperfused rat distal tubules,
raising the luminal NaCl concentration twofold increased
transepithelial Na1 transport by a factor of 3; this increase could be blocked entirely by luminal chlorothiazide (79). The dependence of transepithelial NaCl transport on luminal NaCl concentration probably results from
a dependence of the thiazide-sensitive NCC on extracellular Na1 and Cl2 concentrations (95).
Very recently, a numerical model of the renal distal
tubule has been presented (41). The model incorporated
40 initial parameters and could successfully predict the
behavior of the tubule during exposure to thiazide diuretics and amiloride.
Volume 80
January 2000
MAMMALIAN DISTAL TUBULE
by the DCT, but the method for identifying this segment
was not stated explicitly. Subsequent experiments utilizing in situ PCR showed expression of the a-subunit of
ENaC along the rat TAL and DCT (47); the same subunit
was detected by PCR of immortalized mouse DCT cells
(47). Other data, however, suggest that ENaC is not expressed uniformly by DCT cells. First, experiments in
developing (230) and mature rats (76), using antibodies
generated by Duc et al. (71), did not detect ENaC expression along the proximal portion of the DCT (the DCT-1).
In the rabbit, nephron-segment PCR detected only the
a-subunit of ENaC in DCT cells; b and g were not detected (283). The physiological significance of a-ENaC
expression alone by DCT cells remains unclear. The
a-subunit of ENaC is sufficient to generate a small but
finite Na1 conductance, but conductance is increased
severalfold by coexpression of the b- and g-subunits.
Noncoordinated expression of ENaC subunits has also
been detected in the lung (87), and it is possible that
expression of b and g is regulated physiologically. Evidence against an important component of apical Na1
entry via Na1 channels in DCT cells comes from results of
chronic thiazide diuretic infusion. Loffing et al. (172)
showed that chronic infusion of thiazide diuretics causes
apoptosis and degeneration of cells in the proximal portion of the DCT (the DCT-1). In contrast, cells of the more
distal portion of the DCT (the DCT-2) retained more
normal morphology during chronic thiazide treatment.
The authors interpreted these data as suggesting that Na1
entry into DCT-1 cells (early distal tubule) occurs almost
entirely via the thiazide-sensitive NCC. In contrast, multiple pathways permit Na1 to enter DCT-2 cells (middistal tubule). Sodium entry pathways other than the
thiazide-sensitive NCC and ENaC have been detected in
immortalized mouse DCT cells. Mouse DCT cells express
Na1/H1 exchange activity, Na1/Ca21 exchange activity,
and Na1-Pi transport (99, 102). The majority of Na1 uptake by these cells, however, is mediated by the thiazidesensitive NCC and amiloride-sensitive Na1 channels.
Sodium appears to exit DCT cells across the basolateral membrane via the ubiquitous Na1-K1-ATPase, discussed above (see Fig. 3). Until recently, mechanisms of
Cl2 exit remained obscure. Chloride is raised above electrochemical equilibrium within the DCT cell by the NCC.
Recent data suggest that one important pathway for Cl2
to exit the cell across the basolateral cell membrane is a
Cl2 channel (or channels) of the CLC family. Vandewalle
et al. (285a) detected both CLC-K1 and CLC-K2 in microdissected Sprague-Dawley rat DCT segments. Immunocytochemical analysis, using an antibody that recognizes
both CLC-K1 and CLC-K2, demonstrated abundant labeling of the DCT basolateral membrane (285a). In contrast,
Uchida et al. (272) did not detect expression of the CLC-K
by DCT segments using a different anbibody (272). Very
recently, Yoshikawa et al. (302) used in situ hybridization,
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eralocorticoid hormones was necessary to induce the
thiazide-sensitive Na1 and Cl2 transport.
The cloning of the mammalian NCC has permitted
sites of NCC expression to be determined at the molecular level. Whether assessed by in situ hybridization (7,
199), nephron-segment PCR (281, 301), or immunocytochemistry (23, 145, 206), the thiazide-sensitive NCC is
expressed predominantly, if not exclusively, by DCT cells.
This pattern of expression has been observed in rat,
mouse, rabbit, and human. Thus, from a molecular standpoint, the NCC is expressed by DCT cells in all mammalian species examined to date. In rabbit, NCC expression
is limited to DCT cells and ends abruptly at the transition
to CNT. In human, rat, and mouse, expression of NCC
extends from the proximal end of the DCT into a transitional segment. This transitional segment, referred to as
the DCT-2 (199), shares properties of the distal convoluted and CNT and will be described more fully below (23,
199).
Cloning of the thiazide-sensitive NCC has provided
definitive proof that a single protein couples Na1 and Cl2
transport. Data obtained by in vivo microperfusion of rat
distal tubules indicated that the half-maximal luminal
concentrations for transport were 9 mM for Na1 and 12
mM for Cl2 (278). These values contrast with Michaelis
constant values of 25 for Na1 and 14 for Cl2, obtained by
Eadie-Hofstee plots of data from Xenopus oocytes injected with the flounder transport protein (95). It is not
clear whether the difference in mean affinity constant for
Na1 reflects genuine physiological differences or differences in experimental technique.
There is evidence for other apical Na1 and Cl2 entry
pathways in DCT cells. Wang et al. (289) showed that the
addition of formate or oxalate to the lumen of distal
tubule segments increases Na1 and volume absorption.
They showed that this effect occurs in the “early” part of
the distal tubule, presumably in the DCT. The effect could
be inhibited by DIDS and by ethylisopropylamiloride, but
not by thiazide diuretics. They interpreted these data as
indicating that rat DCT cells express a Na1/H1 exchanger,
a Cl2/formate exchanger, a Cl2/oxalate exchanger, and a
carbonate/oxalate exchanger. Recently, molecular confirmation that a Na1/H1 exchanger is expressed at the
apical membrane of rat DCT cells was presented. NHE-2
was shown to be expressed at the apical membrane of rat
DCT cells (103), where it is coexpressed with the NCC
(39). Although NHE-3 has been detected at the apical
surface of TAL cells, it does not appear to be expressed by
DCT (6).
The possibility that DCT cells express amiloride-sensitive Na1 channels has been a subject of controversy.
Shortly after the epithelial Na1 channel (ENaC) was
cloned, Duc et al. (71) studied its expression in rat kidney
using in situ hybridization and immunocytochemistry.
Those studies detected expression of a-, b-, and g-ENaC
287
288
ROBERT F. REILLY AND DAVID H. ELLISON
hybridization (7) or nephron segment PCR (281). These
results suggest that the component of thiazide-sensitive
Cl2 transport detected when rabbit CNT are microperfused in vitro does not reflect high-level expression of the
NCC. They indicate that the major site of NCC expression
in rabbit, as in rat, is the DCT and not the CNT. The
functional effects of thiazides in rabbit CNT may reflect
low-level NCC expression or expression of thiazide-sensitive transporters that are structurally distinct from the
NCC. One electroneutral Na1 transporter shown recently
to be expressed by CNT cells is NHE-2 (39). Its functional
significance in this segment is, at this time, unclear. The
Na1/H1 exchanger NHE-1 has been detected at the basolateral membrane of rabbit CNT cells (17).
Whereas data suggesting expression of electroneutral NCC by rabbit CNT cells have been contradictory,
evidence for expression of Na1 channels at the apical
membrane of CNT has been more consistent. Rabbit CNT
segments demonstrate a lumen-negative transepithelial
voltage and electrogenic Na1 transport when perfused in
vitro (4, 124, 239 –241). Although it has not been possible
to isolate and perfuse CNT from rats, micropuncture data,
reviewed above, suggest that the magnitude of the transepithelial voltage increases along the superficial CNT in
rat. Furthermore, both rabbit (283) and rat CNT segments
have been shown to express ENaC at the apical membrane (71, 76, 230).
A role of CNT cells in mediating transepithelial chloride transport has not been well established. As discussed
above, data from rabbit CNT perfused in vitro suggest that
a component of transepithelial Na1 and Cl2 transport by
this segment is sensitive to thiazide diuretics (240), yet
expression of NCC by CNT cells (or intercalated cells) has
been minimal in both rabbit or rat (7, 199, 283). The
chloride channel CLC-K2 is expressed at the basolateral
cell membrane of CNT (285a) (302). Connecting tubule
cells, whether in the superficial distal tubule or in arcade
segments, also express the vasopressin-regulated water
channel and the vasopressin receptor (50, 148). Thus the
junction between the DCT and CNT represents the transition between the diluting segment and the vasopressinsensitive portion of the nephron.
3. Regulation of Na1 and Cl2 transport
As discussed in section IVB1, the superficial distal
tubule reabsorbs ;5–10% of the filtered NaCl load under
many conditions. This means that NaCl transport by the
superficial distal tubule is load dependent. In fact, luminal
NaCl delivery appears to be the predominant regulator of
NaCl cotransport acutely. Sodium reabsorption by distal
tubules does not change during acute volume expansion if
ion delivery is maintained constant (63); in contrast, extracellular fluid volume expansion depresses proximal
solute and volume reabsorption independent of changes
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combined with immunocytochemistry with known markers, to identify sites of CLC-K expression. Whereas
CLC-K1 was expressed in the medulla, CLC-K2 was expressed throughout the TAL, DCT, and CNT (302).
Another pathway by which Cl2 may exit from DCT
cells is a K1-Cl2 cotransporter. Functional evidence for
K1-Cl2 cotransport across DCT cells is discussed in section IVC2, but there is also molecular evidence for renal
expression of K1-Cl2 cotransporters (105, 120, 168). One
K1-Cl2 cotransporter isoform, KCC1, was recently detected in DCT segments of rat kidney by in situ hybridization (168).
Other Na1 entry pathways have been detected in
DCT cells in some species. The “housekeeping” isoform of
the Na1/H1 exchanger, NHE-1, has been localized by
immunocytochemistry to the basolateral surface of DCT
cells in rabbit (17). Immortalized mouse DCT cells grown
in vitro express a Na1/Ca21 exchanger (102, 292; see
below), which has also been detected in rat DCT-2 segments in situ (199). Rabbit DCT cells do not express the
Na1/Ca21 exchanger (7), but mouse (173) and probably
human (199) DCT cells resemble those in rat.
Distal convoluted tubule cells do not express aquaporin-2, the vasopressin-sensitive water channel (50). The
DCT therefore functions as the most distal portion of the
diluting segment.
1
2
B) CNT. Mechanisms of Na and Cl transport by CNT
cells have been studied using in vitro microperfusion,
optical techniques, electrophysiological techniques, and
molecular techniques. The major Na1 and Cl2 transport
pathways are shown in Figure 3. Early studies showed
that rabbit CNT isolated and perfused in vitro transport
Na1 and K1 and exhibit a transepithelial voltage oriented
in the lumen-negative direction (4, 124). Rates of Na1
transport were reported to be ;20 pmolzcm21zs21, which
is three or four times as great as rates in isolated CCD but
similar to rates in isolated DCT segments perfused in vitro
(4). Like DCT segments, CNT express the Na1-K1-ATPase
at their basolateral surface. Although Na1-K1-ATPase expression levels are lower than in the DCT, they are higher
than in the CCT (141).
Net Cl2 transport by rabbit CNT perfused in vitro
was reduced 31% by 1024 M trichlomethiazide, suggesting
that the thiazide-sensitive NCC is expressed by this segment (240). Ouabain-induced swelling of CNT cells was
prevented by the combination of trichlomethiazide and
amiloride but not by either agent alone (237). These data
were interpreted as a further indication that rabbit CNT
cells express thiazide-sensitive NCC activity. Surprisingly,
levels of NCC expression by rabbit CNT segments were
either very low and variable (when detected by nephron
segment PCR; H. Velázquez, personal communication) or
absent (when detected by in situ hybridization). This is in
contrast to NCC. This protein demonstrated robust expression in the rabbit DCT, whether detected by in situ
Volume 80
MAMMALIAN DISTAL TUBULE
January 2000
1. Regulation of NaCl transport by the distal
convoluted tubule
TABLE
NaCl
Transport
[3H]metolazone
Binding
NCC Message
NCC Protein
Hormones
1 (275)
1 (43, 275)
Glucocorticoids
Estrogen
Adrenomedullin
Angiotensin II
Amylin
Calcitonin
1 (275)
1 (286)
1 (43, 275)
1 (43)
1 (21)
1 (74)
1 (21)
1 (20)
Furosemide
1 (79, 255)
1 (45, 199)
1 (199)
(7) (192)
HCTZ
2 (193)
1 (45, 193)
2 (172)
1 (290)
7 (Ellison,
unpublished
data)
1 (145)
1 (286)
Diuretics
1 (Ellison,
unpublished
data)
7 (172)
in agreement with very early studies that were later ignored. An example is the report of Hierholzer et al. (119)
who studied the effects of adrenal steroids on Na1 and K1
transport by superficial distal tubules of rats. Their data
indicated that adrenalectomy increases the tubule fluidto-plasma Na1 ratio in distal but not proximal tubules
(see Fig. 6A). Although these changes were later ascribed
to effects on connecting and collecting duct cells, inspection of the data reveals that the mineralocorticoid effects
are as large along the first 40% of tubule length (which
would comprise exclusively DCT cells, Ref. 67) as they
are along the last 40%. The effects of adrenalectomy could
be reversed completely by infusing aldosterone, indicating that they resulted from mineralocorticoid action.
These changes in luminal NaCl concentration occurred
without any change in transepithelial voltage. When
Metabolic and dietary factors
Metabolic
alkalosis
Metabolic
acidosis
Low NaCl intake
1 (83)
2 (83)
1 (79)
7 (45)
Other factors
Ischemia
Renal nerves
a-Adrenergic
receptors
1 (15)
1 (99)
2 (12)
2 (291)
2 (73)
1, Increased; 2, decreased; 7, unchanged. Unless otherwise
noted, these factors appear to increase thiazide-sensitive component of
Na1 and Cl2 transport by distal tubules. Other hormones that have been
examined include glucagon (and somatostatin) and parathyroid hormone. Neither of these has altered the thiazide-sensitive component of
Na1 or Cl2 transport (56, 187). NCC, Na1-Cl2 cotransport; HCTZ, hydrochlorothiazide
in luminal delivery. Because it was difficult to demonstrate acute regulation of NaCl reabsorption by distal
tubules, few studies were conducted to examine the
chronic regulation of ion transport by the superficial distal tubule. More recent data, however, indicate that ion
transport by the distal tubule is regulated by physiological
control systems. These physiological regulators include
steroid and peptide hormones, metabolic and dietary factors, and pathological events, such as ischemia. Factors
purported to regulate NCC function are shown in Table 1.
A) DIETARY NACL INTAKE AND ADRENAL STEROID HORMONES.
Mineralocorticoid hormones contribute importantly to
Na1 homeostasis under both normal and pathological
conditions. Although glucocorticoid hormone receptors
are present throughout the nephron (269), mineralocorticoid receptors are expressed primarily by distal nephron
segments (269). Coupled with clearly evident effects of
mineralocorticoids on principal cells, these results have
been used to suggest that mineralocorticoids act predominantly along the collecting duct and not in the DCT (70,
219, 249). This view, however, is now being modified in
light of more recent results. These more recent results are
1
FIG. 6. K secretion and cell types along distal tubule. Top: luminal
K1 delivery (expressed as fractional K1 delivery) along superficial distal
tubules of rats, as determined by micropuncture. [From Wright (296).]
Bottom: data from Dorup (67) showing locations of different cell types
along superficial distal tubule of rats. TAL, thick ascending limb cell;
DCT, distal convoluted tubule cell; CNT, connecting tubule cell; PC,
principal cell; IC, intercalated cell. Note that most K1 secretion occurs
between 30 and 70% of tubule length, a region that comprises primarily
DCT and CNT cells.
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Aldosterone
289
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ROBERT F. REILLY AND DAVID H. ELLISON
of high (aldosterone infusion) or low (NaCl restriction)
extracellular fluid volume. Taken together, these results
indicate that both the activity and the abundance of the
NCC are regulated by adrenal steroid hormones. It appears that high levels of either glucocorticoid hormones
or mineralocorticoid hormones increase thiazide-sensitive transport activity.
Aldosterone specificity is maintained in target tissues
because the metabolic enzyme 11b-hydroxysteroid dehydrogenase is expressed. Endogenous glucocorticoid and
mineralocorticoid hormones bind to the mineralocorticoid receptor with equal affinity. Glucocorticoid hormones typically circulate at concentrations that are 100to 1,000-fold higher than mineralocorticoid horomone
concentrations. 11b-Hydroxysteroid dehydrogenase metabolizes glucocorticoid hormones in mineralocorticoid
target cells, leaving mineralocorticoids intact. Recently,
Bostanjoglo et al. (23) showed that 11b-hydroxysteroid
dehydrogenase colocalizes with the NCC in rat distal
tubules. In rabbit, Velázquez et al. (281) showed that
11b-hydroxysteroid dehydrogenase message colocalizes
with message for the NCC in DCT segments (281). In the
rat, although 11b-hydroxysteroid dehydrogenase was expressed by DCT cells, expression was not uniform (23). It
was present at low to undetectable levels in the DCT-1
(the most proximal portion of the the DCT) and at higher
levels along the DCT-2 (the more distal portion of the
DCT). Even along the DCT-2, however, expression of
11b-hydroxysteroid dehydrogenase was lower than expression along the CNT.
Dietary NaCl intake is a powerful determinant of
circulating aldosterone levels. Although the effects of aldosterone on NCC abundance and activity suggest that
dietary NaCl intake may regulate NCC abundance and
activity, these effects have not been as consistent. In rats,
dietary NaCl restriction has been reported to increase
thiazide-sensitive Na1-Cl2 transport by threefold (79) and
to increase NCC protein expression twofold (145). Dietary NaCl restriction, however, was not shown to increase the number of [3H]metolazone binding sites in
kidney cortex (82) or to increase NCC message expression (192). In the rabbit, dietary NaCl loading, which
suppresses aldosterone secretion, increases the fractional
volume of DCT cells and increases basolateral cell membrane amplification (134) and Na1-K1-ATPase activity
(166). These morphological effects of high dietary NaCl
intake are accompanied by increases in the transport
capacity of DCT cells, perfused in vitro (243).
These data can be used to construct the following
hypothesis concerning regulation of ion transport by the
distal tubule of the rat kidney. Adrenal steroid hormones
increase NCC activity at least in part by increasing NCC
protein expression. This action of mineralocorticoid hormones may be predominantly along the DCT-2, a segment
that expresses significant levels of 11b-hydroxysteroid
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viewed in light of more recent information (reviewed
below), these results are consistent with an effect of
adrenalectomy and minerlocorticoid hormones on activity of the NCC in DCT cells.
Studies using isolated dissected rabbit kidney tubules detected minimal binding of [3H]aldosterone to DCT
segments (70, 84 – 86). When binding was examined in situ
by autoradiography, however, binding along the DCT (and
TAL) was detected, albeit at higher aldosterone concentrations (1.5–20 nM) (85). Similar results were obtained in
rats, in which binding to DCT segments was detectable at
2 but not 0.2 nM [3H]aldosterone (85). More recently, after
the cloning of steroid receptors, it has become possible to
localize mineralocorticoid receptors at the molecular
level. Antibodies generated against the mineralocorticoid
receptor were found to localize to the DCT, the CNT, and
the CCT in rat (23, 156), human (247), and rabbit (174). In
the only experiments to use nephron-segment PCR to
detect mineralocorticoid receptors, neither DCT nor CNT
segments were tested (269). Thus, in all species in which
molecular evidence has been obtained, mineralocorticoid
receptors were shown to be expressed by DCT cells (and
CNT cells) as well as principal cells.
More recent functional and molecular data confirm
the importance of mineralocorticoid hormones in regulating NaCl transport by DCT (see Table 1). Stanton et al.
(250) reported that low physiological concentrations of
mineralocorticoid hormones increased Na1 transport by
the superficial distal tubule, but this was attributed to an
effect on the late distal tubule (250). Ellison et al. (79)
reported that dietary NaCl restriction increased thiazidesensitive Na1-Cl2 cotransport capacity in rats. Chen et al.
(43) and Velázquez et al. (275) reported that mineralocorticoid and glucocorticoid hormones both increase the
number of [3H]metolazone binding sites when administered to adrenalectomized animals (see Fig. 6D). The
number of [3H]metolazone binding sites was taken as an
indication of the number of thiazide-sensitive NCC. The
combination of glucocorticoid and mineralocorticoid hormones increased the number of receptors more than did
either hormone alone. Velázquez et al. (275) showed that
mineralocorticoid hormones strongly simulate thiazidesensitive NCC activity (see Fig. 6C). In those experiments,
thiazide-sensitive Na1-Cl2 cotransport was barely detectable in adrenalectomized rats. High physiological doses of
either aldosterone or dexamethasone increased transport
capacity by up to 20-fold (275). The increase in ion transport did not result only from increases in luminal NaCl
delivery, because they persisted when the luminal NaCl
delivery was controlled by microperfusion. Recently, Kim
et al. (145) showed that aldosterone increased expression
of thiazide-sensitive NCC (see Fig. 6B), as determined by
Western blot and immunocytochemistry of rat kidney
cortex. The effects of aldosterone to increase expression
of the NCC protein were observed whether in the setting
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MAMMALIAN DISTAL TUBULE
servation correlated with an increased sensitivity to thiazide diuretics in female animals. They noted that
ovariectomy reduced the number of [3H]metolazone receptors, suggesting that estrogens increase expression of
activated NCC. Verlander et al. (286) reported that estrogen increases the amount of NCC protein in rat kidney
cortex and increases the complexity of the apical plasma
membrane of the DCT. In preliminary results, Verlander
and colleagues (271) showed that estrogens increase expression of the NCC in immortalized mouse DCT cells,
grown in vitro. These data were interpreted as suggesting
that the effects of estrogen to increase NCC expression
are a direct effect of the hormone on receptors in distal
tubule cells.
C) PEPTIDE HORMONES. Three peptide hormones have
been reported to increase the density of [3H]metolazone
binding sites in rat kidney. Amylin is a peptide secreted by
b-cells of the pancreas in response to nutrient stimuli.
Administration of this peptide increased the density of
[3H]metolazone binding sites 32–58% (21). Administration
of the structurally related peptide adrenomedullin also
increased [3H]metolazone density by ;30% (21). The
physiological significance of these observations remains
unclear.
Angiotensin II increases, and [Sar1,Ile8]ANG II decreases, Na1, fluid, and HCO2
3 transport along the superficial distal tubule (290). A component of this effect was
attributed to cells of the early distal tubule, presumably
DCT cells. It was suggested that the increase in Na1
transport reflected stimulation of Na1/H1 exchange, although data were not presented to support this directly.
Along the late distal tubule, probably primarily the CNT,
angiotensin II stimulation of Na1 transport was believed
to reflect increases in amiloride-sensitive electrogenic
Na1 transport. Some other peptide hormones have been
shown not to affect Na1 transport along the distal tubule.
These include glucagon, somatostatin, and parathyroid
hormone (8, 56, 187).
D) DIURETIC DRUGS. Chronic diuretic treatment has profound effects on the structure and function of the DCT.
Kaissling and colleagues (130 –132) showed that chronic
furosemide infusion in rats causes hypertrophy and hyperplasia of DCT cells. Diuretic-induced hypertrophy was
associated with increases in basolateral cell membrane
amplification (132) and Na1-K1-ATPase activity (226).
The functional activity of the thiazide-sensitive NCC was
increased approximately threefold after 1 wk of furosemide infusion (79). The effects of chronic diuretic infusion on ion transport and cell morphology were shown to
occur even if circulating mineralocorticoid and glucocorticoid levels were maintained constant (135, 255). The
effects were also shown to occur even if arginine vasopressin concentrations were maintained at high and constant levels by hormonal infusion. Based on earlier experiments in rabbits, wherein high dietary NaCl intake led to
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dehydrogenase. As in the collecting duct (134), the effect
of mineralocorticoid hormones may be enhanced when
luminal NaCl delivery is high. Thus the effects of dietary
NaCl restriction (high aldosterone/low distal NaCl delivery) on NCC abundance are blunted, compared with the
effects of exogenous mineralocorticoid administration
(high aldosterone/high distal NaCl delivery). Based on the
absence of effects of dietary NaCl restriction on NCC
mRNA levels (192), these effects of mineralocorticoid
hormones on NCC protein abundance may occur predominantly at the level of protein synthesis or protein stability. Glucocorticoid hormones may act predominantly
along the DCT-1, a segment that expresses very low levels
of 11b-hydroxysteroid dehydrogenase. Nevertheless, the
rat DCT may be more promiscuous than either the CNT or
CCT with respect to steroid hormone specificity because
levels of 11b-hydroxysteroid dehydrogenase expression
are intermediate, lower than in the CNT, and higher than
in more proximal segments. These variations may have
physiological relevance. When aldosterone concentrations are increased moderately, electrogenic Na1 transport along the CNT and CCT may be stimulated via the
action of mineralocorticoids. When volume depletion is
more extreme, circulating mineralocorticoid levels may
be sufficient to stimulate Na1-Cl2 cotransport along the
more distal portions of the DCT. When volume status is
severely reduced, stress-induced glucocorticoid secretion
may stimulate Na1 reabsorption along the entire distal
tubule and in more proximal segments. Based on molecular and morphological similarities, regulation of DCT
cells in humans may be similar to that in rodents.
The CNT is the site at which the enzyme 11b-hydroxysteroid dehydrogenase is expressed at its highest level
(23). Mineralocorticoid receptors are also expressed by
CNT cells (23). These data suggest that aldosterone participates importantly in regulating ion transport by CNT
(130, 131, 134, 166).
It is more difficult to construct a unifying hypothesis
that explains all the results from experiments in rabbit. In
the rabbit, high dietary NaCl intake increases DCT cell
size, probably by increasing distal NaCl delivery and NaCl
transport. Nevertheless, rabbit DCT cells coexpress the
thiazide-sensitive NCC with 11b-hydroxysteroid dehydrogenase. One possibility is that dietary NaCl loading drives
high-level transepithelial Na1 transport along the DCT
even in the absence of aldosterone. This leads to morphological and functional changes. When low dietary NaCl
intake raises aldosterone in the setting of low NaCl delivery, aldosterone may preserve NaCl reabsorption, preventing volume depletion. It appears, however, that transepithelial transport of salt is the primary stimulus to cell
growth.
B) SEX STEROIDS. Chen et al. (44) reported that the
densities of [3H]metolazone receptors were higher in kidneys from female than male rats (see Table 1). This ob-
291
292
ROBERT F. REILLY AND DAVID H. ELLISON
tegrity. They suggested that thiazide diuretics, by blocking the major Na1 entry pathway, disrupt this process
leading to structural changes.
E) ISCHEMIA. Beaumont et al. (12) noted that warm
ischemia reduced the number of [3H]metolazone binding
sites in rat kidney cortex. As little as 10 min of warm
ischemia was sufficient to reduce the number of receptors
significantly, and 30 min reduced them close to background. Wang et al. (291) studied the effects of ischemia
in situ on expression of message for NCC. They found
reductions in NCC mRNA expression 24 h after an ischemic insult, but not before. Edelstein et al. (73) showed
that ischemia and reperfusion led to reductions in NCC
protein expression that began within 15 min and lasted
more than 24 h. These findings suggest that decreases in
NCC expression may contribute to the high fractional
Na1 excretion that characterizes ischemic renal injury.
F) ACID-BASE ABNORMALITIES. Recently, Fanestil et al. (83)
reported that chronic administration of NaHCO3 increased and chronic NH4Cl decreased the density of receptors for [3H]metolazone.
C. K1 Transport
1. K1 transport by the distal tubule
Filtered K1 is reabsorbed along the proximal tubule
and loop of Henle; ;10% of filtered K1 reaches the DCT.
Early micropuncture work established the superficial distal tubule as an important site of K1 secretion. Many
studies indicated that K1 secretion along the superficial
distal tubule accounted for most or all of urinary K1
excretion under a variety of conditions. In fact, Jamison et
al. (126) summarized the existing literature as indicating
that the “principal site of the regulation of K1 excretion is
the distal tubule.” He noted that the collecting duct probably plays an important role as well because K1 delivery
to the end of the superficial distal tubule could not fully
account for K1 excretion under some conditions (126).
Wright (297) reviewed data concerning sites of K1 secretion and emphasized the importance of the late distal
tubule. He noted that this segment probably comprises
two structurally distinct segments, the CNT and the initial
portion of the CCD. He raised the possibility that ion
transport mechanisms in the two segments might be different.
More recently, regulation of K1 excretion has been
viewed as determined primarily by principal cells of the
cortical (and initial) collecting tubule (104, 299). This
view has resulted from morphological and functional data
demonstrating conclusively that principal cells secrete
K1. Also most of the factors that regulate K1 excretion
have been shown to regulate K1 secretion by principal
cells. This review does not address the important role of
principal cells and of the CCD in renal K1 homeostasisis,
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hypertrophy of DCT cells, and based on the ability of
furosemide to induce structural changes even when steroid hormone levels were controlled, Stanton and Kaissling (256) postulated that the effects resulted from increases in distal NaCl delivery and NaCl transport. These
observations, however, do not exclude a role of steroid
hormones in contributing to the changes observed during
chronic diuretic administration. Chen et al. (43) and later
Obermüller et al. (199) showed that chronic furosemide
infusion increased the density of [3H]metolazone receptors by up to 100%, suggesting the chronic furosemide
infusion increases expression of the NCC. Obermüller et
al. (199) detected an increase in NCC mRNA by in situ
hybridization after chronic diuretic infusion. Moreno et al.
(192), however, did not detect a significant increase in
NCC message after once-daily furosemide administration.
Whether the difference in results reflected more intense
stimulation in the setting of constant diuretic infusion is
unknown. Of note, Moreno et al. (192) reported that NCC
message levels increased after dehydration. The authors
suggested that the results of Obermüller et al. (199) may
have reflected effects of dehydration. Venkatachalam and
co-workers (150) showed that chronic loop diuretic infusion increases expression of both insulin-like growth factor (IGF) and insulin-like growth factor binding protein
(IGFBP) in distal tubules. These investigators postulated
that the morphological changes that occur during chronic
diuretic infusion result, at least in part, from activation of
growth factors. Beck et al. (14) studied the effects of
inhibiting angiotensin converting enzyme on diureticinduced hypertrophy of the distal nephron. Administration of an angiotensin-converting enzyme inhibitor did
not block the development of hypertrophy, but instead
converted the predominant effect from one in which tubule cells thickened to one in which tubule length increased (14).
If increased transepithelial ion flux drives the structural and functional effects of chronic loop diuretic infusion, then administration of diuretics that block such
transport would be expected to have opposite effects.
Morsing et al. (193) administered thiazide diuretics chronically to rats and measured the functional activity of the
DCT. After 10 days of thiazide infusion, the ability of the
distal tubule to reabsorb Na1 and Cl2 was reduced significantly. Surprisingly, this reduction was associated
with an increase in the number of [3H]metolazone binding
sites. A similar result was obtained by Chen et al. (45),
who found that chronic thiazide infusion increased the
number of [3H]metolazone binding sites (45). Loffing et al.
(172) showed that chronic thiazide infusion in rats led to
apoptosis of DCT cells and destruction of the epithelium.
This was associated with decreases in the amount of NCC
protein, as determined by Western blot. They postulated
that DCT cells are dependent on Na1 entry via the apical
membrane to maintain their structural and functional in-
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MAMMALIAN DISTAL TUBULE
293
nor will it provide a detailed description of factors that
regulate K1 secretion by principal cells. Mechanisms and
control of K1 excretion by the kidney and by the collecting duct have been reviewed extensively (104, 299). Instead, this review emphasizes the important role that DCT
and CNT cells play in K1 homeostasis and discusses
insights that derive from a synthesis of recent molecular
results with earlier physiological results.
Micropuncture studies indicated that the luminal
concentration of K1 along the first 20 –30% of the superficial distal tubule is low (297). This portion of the
tubule is lined by DCT cells (67). In one study, when
early distal tubules were perfused separately from late
distal tubules, rates of K1 secretion by early tubules
were found to be low and did not reach statistical
significance (251). These data suggested that the DCT
does not contribute importantly to K1 secretion. More
recently, however, this view has been revised.
Velázquez et al. (277) microperfused segments of the
DCT that lay within the initial 40% of tubule length.
These segments, which would be expected to comprised only DCT cells (67), did secrete K1 at low but
significant rates under control conditions. When the
luminal Cl2 concentration was reduced, rates of K1
secretion rose substantially (see below).
A plot of the luminal K1 concentration along the
length of the distal tubule juxtaposed with the axial
distribution of cell types (see Fig. 7) shows that a large
percentage of K1 secretion occurs between 20 and 80%
of tubule length. This portion of the distal tubule comprises primarily DCT, CNT, and intercalated cells. As
discussed in section IVC2, pathways that can participate
in K1 secretion have been identified in both DCT-2 cells
and in CNT cells. In view of the fact that deep nephrons
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1
FIG. 7. Effects of adrenal steroid hormones on Na concentrations along distal tubule. A: data are shown as TF/P
Na1 concentrations from adrenalecoctomized (right hatch) and control (left hatch) animals. Note that luminal Na1
concentrations are higher along entire distal tubule of adrenalectomized animals. Data from adrenalectomized animals
replaced with aldosterone are given as solid triangles. [From Hierholzer (119).] B: protein expression of thiazide-sensitive
Na1-Cl2 cotransproter in control animals and animals treated with aldosterone (Aldo). [From Kim et al. (145).] C:
chlorothiazide (CTZ)-sensitive Na1 transport in distal tubules of adrenalectomized animals treated with replacement or
high physiological levels of dexamethasone and aldosterone [From Velázquez et al. (275).] D: number of [3H]metolazone
binding sites in kidney cortex from animals treated as in C.
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ROBERT F. REILLY AND DAVID H. ELLISON
Volume 80
join each other via arcades lined with CNT cells, it is
likely that CNT cells play a central role in renal K1
homeostasis.
2. Mechanisms of K1 transport
Two mechanisms of K1 secretion across
the apical membrane of DCT cells have been proposed
(Fig. 3). Based on the effects of low luminal Cl2 concentrations to stimulate K1 secretion, Velázquez et al. (277)
proposed that a K1-Cl2 cotransport pathway is expressed
at the luminal membrane of DCT cells. A second apical K1
secretory pathway is a K1 channel. Antibodies against
ROMK show expression at the apical membrane of DCT-1,
DCT-2, and CNT cells (see Fig. 3) (186, 300). It seems
likely that the low rates of K1 secretion by the DCT-1 (the
early distal tubule) that are observed in vivo under typical
conditions reflect the absence of electrogenic Na1 reabsorption and the low transepithelial voltage in this segment (as discussed in sect. IVC1) rather than the absence
of K1 secretory pathways.
Along the DCT-2, K1 secretion is more rapid and
contributes importantly to net K1 excretion under typical
conditions. This results because Na1 channels are expressed by DCT-2 cells at the apical membrane (47, 102,
230). These channels depolarize the membrane, driving
K1 from cell to lumen. This group of cells also expresses
the thiazide-sensitive NCC at the apical membrane as well
as mineralocorticoid receptors and the enzyme 11b-hydroxysteroid dehydrogenase (23, 230).
B) CNT CELLS. Connecting tubule cells are distinguished
at the molecular level by the absence of the thiazidesensitive NCC at the apical membrane and the presence of
the Na1/Ca21 exchanger at the basolateral membrane
(see Fig. 3; Ref. 199). Connecting tubule cells are designed
optimally to secrete K1, with both Na1 channels and K1
channels expressed at their apical membrane (71, 230,
300). Furthermore, their abundance of mitochondria and
more amplified basolateral membrane, compared with
principal cells, would be expected to confer a large ion
transport capacity. Interestingly, recent immunocytochemical experiments have suggested that CNT cells express the highest levels of the enzyme 11b-hydroxysteroid
dehydrogenase (23).
C) PRINCIPAL CELLS. Clearly, principal cells of the initial
and CCD contribute importantly to systemic K1 homeostasis. Their role has been reviewed recently (104)
and is not emphasized here.
A) DCT CELLS.
Many factors regulate K1 secretion along the distal
tubule. These have been reviewed recently (104, 299).
Factors that act from the peritubular side to increase K1
secretion along the distal tubule include elevations of K1
(253), aldosterone (88), vasopressin (89), and angiotensin
II (290). A low H1 concentration also stimulates K1 secretion (252). Basolateral factors that inhibit K1 secretion
along the distal tubule include metabolic acidosis (252)
and hypokalemia (253).
Factors that influence K1 secretion along the distal
tubule from the luminal side include the luminal concentrations of Na1, K1, and Cl2 and the luminal fluid flow
rate (299). Luminal inhibitors of K1 secretion include
organic cations, such as amiloride, triamterene, trimethoprim, and pentamadine, as well as inorganic cations, such as Ca21 (see Fig. 8). Because these factors have
received less attention, but appear to be potent and clinically significant, they are discussed in more detail in
section IVC3.
Increases in distal flow rate and luminal Na1 concentration both stimulate K1 secretion by distal tubules. It is
likely that reducing the luminal Na1 concentration below
a critical level inhibits K1 secretion by its effects on
cellular Na1 concentration and activity of the Na1-K1ATPase pump (108). This is supported by the parallel
reductions in K1 secretion and transepithelial voltage that
occur when luminal Na1 concentration is reduced below
40 mM (108). As noted by Good et al. (108), however, the
luminal Na1 concentration in fluid entering the distal
tubule usually ranges from 38 to 77 mM, even under
conditions of low dietary NaCl intake. Thus the luminal
Na1 concentration would not limit K1 secretion along the
distal tubule unless severe extracellular fluid volume contraction reduced the luminal Na1 concentration below 40
mM. It should be emphasized that the luminal Na1 concentration can and does frequently decline below 40 mM
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3. Regulation of K1 transport
1
FIG. 8. Regulation of K
transport by DCT and CNT cells. K1
secretion is inhibited by Ca21, trimethoprim, and pentamadine because
these cations exert blockade of apical Na1 channels. A K1-Cl2 cotransport pathway in apical membrane appears to participate importantly in
net K1 secretion only when luminal Cl2 concentration is low. Under
these conditions, Cl2 that enters cell driven by activity of Na1-Cl2
cotransporter recycles across apical membrane, driving continued K1
secretion. Adding a DCT diuretic (DCTD) will then inhibit net potassium
secretion (see text for details). 2, Inhibitory effect; 1, stimulatory
effect.
January 2000
MAMMALIAN DISTAL TUBULE
Cl2-free solution (285). They showed that this effect occurred primarily along the early distal tubule and suggested that it reflected transport via an apical K1-Cl2
cotransport pathway. Figure 8 shows a model apical
membrane of a DCT cell (a DCT-2 cell). According to this
hypothesis, the K1-Cl2 cotransport pathway is activated
only when the luminal Cl2 concentration is low. In this
case, Cl2 recycles back into the cell via the thiazidesensitive NCC. Under conditions of low luminal Cl2 concentrations, DCT diuretics should inhibit K1 secretion by
reducing the availability of Cl2 in the cell. This hypothesis
was supported by data showing that chlorothiazide inhibited K1 secretion during perfusion with a Cl2-free solution. Chlorothiazide was also shown to reduce K1 excretion during infusion of sodium sulfate (which reduces
urinary Cl2 concentrations) (276).
Organic cations may also affect K1 secretion by the
distal tubule. Amiloride blocks epithelial Na1 channels
and thereby reduces K1 secretion along the distal tubule
and collecting duct. Triamterene appears to act in much
the same manner (37). More recently, it has been recognized that other organic cations, used primarily as antimicrobial substances, can also block epithelial Na1 channels and cause K1 retention. Both trimethoprim (46, 227,
282) and pentamadine (149) have been shown to block
Na1 channels (see Fig. 8), inhibit K1 secretion, and cause
hyperkalemia in patients. It is postulated that these drugs,
like amiloride, enter the pore region of Na1 channels
(203) leading to channel blockade. It seems likely that
other organic cations may have similar “amiloride-like”
effects on the distal tubule.
D. Ca21 Transport
1. Ca21 transport by the distal tubule
Total body Ca21 homeostasis is maintained by the
interplay of three processes: intestinal absorption, bone
resorption and formation, and urinary excretion. Net intestinal absorption of Ca21 amounts to ;200 mg of the
normal dietary intake of 1,000 mg. In the steady state, this
net absorption is matched by urinary excretion. As a
result, 10,600 mg of the ;10,800 mg (98%) of Ca21 filtered
daily must be reabsorbed by the kidney (57). Thereby,
renal Ca21 reabsorption plays an important role in Ca21
homeostasis.
It is generally accepted that the bulk of the filtered
load of Ca21 (60 –70%) is reabsorbed in the proximal
tubule largely by passive means through the paracellular
pathway and only ;10 –15% of the filtered load reaches
the distal tubule. The mammalian distal nephron, despite
reabsorbing only ;10% of the filtered load of Ca21, is the
primary target site for all of the major Ca21 regulatory
hormones (57). In addition, most or all of the systemic
factors that affect renal Ca21 excretion also affect Ca21
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along the collecting duct. If the same relation between
Na1 concentration and K1 secretion holds in that segment, then luminal Na1 may be limiting to K1 excretion,
even under conditions of mild extracellular fluid contraction.
Fluid flow rate also affects K1 secretion along the
distal tubule (110, 143). In a series of microperfusion
experiments, when the luminal flow rate was increased
from 6 to 26 nl/min, K1 secretion nearly doubled (110). In
contrast to the case of luminal Na1 concentration, discussed above, this range of flow rates is well within the
range observed under free flow conditions of hydropenia
and mild volume expansion. Thus the flow rate of fluid
entering the distal tubule is likely to have a large effect on
K1 secretion on a day to day basis. An important mechanism mediating the flow-induced increase in K1 secretion results from changes in luminal K1 concentration.
When the flow rate is low, luminal K1 concentrations
increase dramatically along the length of the distal tubule,
owing to continued secretion. In contrast, when the flow
rate is high, luminal K1 concentrations remain lower. The
lower luminal K1 concentration permits continued K1
secretion along the length of the distal tubule (298).
Another factor that alters K1 excretion in humans
and animals is diuretic drugs. Loop and DCT diuretics
(thiazides and others) increase urinary K1 excretion and
can cause hypokalemia. A factor that contributes to diuretic-induced K1 wasting is contraction of the extracellular fluid volume and hyperaldosteronism (293). Another
factor is the increase in distal flow rate. If distal Na1
delivery is markedly reduced, increases in distal Na1
delivery may also contribute. Loop diuretics inhibit K1
reabsorption along the TAL and increase K1 delivery to
the DCT (122, 284). Thus a component of the K1 wasting
that results from loop diuretic administration reflects direct actions of the drugs, yet DCT diuretics such as the
thiazides do not alter K1 secretion directly (284), at least
under normal conditions. However, DCT diuretics are
potent kaliuretic drugs. One factor contributing to this
effect is that DCT diuretics increase Ca21 absorption by
the DCT. This reduces the luminal concentration of Ca21
in the late distal tubule. Calcium acts in the distal tubule
to inhibit K1 secretion indirectly, by blocking Na1 channels (see Fig. 8, Ref. 201). This observation may explain
part of the difference in kaliuretic potency of loop and
DCT diuretics. In contrast to the effects of DCT diuretics,
loop diuretics increase distal Ca21 delivery, which would
be expected to inhibit K1 secretion.
Under some conditions, however, thiazide diuretics
may inhibit K1 secretion by the distal tubule. When the
luminal concentration of Cl2 in the distal tubule is low,
the relation between luminal Na1 concentration and K1
secretion changes. Velázquez et al. (285) showed that
raising luminal Na1 concentration from 40 to 150 mM
stimulates K1 secretion by distal tubules perfused with a
295
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ROBERT F. REILLY AND DAVID H. ELLISON
2. Mechanisms of Ca21 transport
The nephron segment localization of the Ca21 transport-related proteins has been investigated using functional, immunological, and molecular methods (see Fig.
9). Discrepancies exist between studies, some of which
may be due to differences in the species examined (7,
199) or the methods used (28, 30, 65, 66, 92, 242, 265, 273).
The effects of PTH on distal tubular Ca21 transport
are well known. Parathyroid hormone binds to its receptor in the basolateral membrane and increases transepithelial Ca21 flux. Two recent studies employed molecular
methods (isolated nephron segment RT-PCR and in situ
hybridization) to localize the PTH receptor in rat kidney.
Expression was noted in the glomerulus, proximal tubules, cortical thick ascending limb (cTAL), and the DCT,
but not in the CCD (216). (165). The CNT was not addressed as a distinct segment in these studies.
Early radioligand binding studies suggested that 1,25dihydroxyvitamin D3 receptors were expressed only by
cells of the distal tubule (142). Kumar et al. (157) recently
raised antibodies to the cloned 1,25-dihydroxyvitamin D3
receptor and confirmed that the highest levels of expression in human kidney were along the DCT, and CCD, with
lower but significant expression in the proximal tubule
(157).
FIG. 9. Calcium transport pathways in DCT and CNT cells. Calcium
entry across apical membrane is driven by steep electrical and chemical
gradients. Calcium entry pathway appears to be dihydropyridine sensitive and activated by hyperpolarization. Calcium is extruded into interstitial fluid by Na1/Ca21 exchanger (213, 214) and Ca21-ATPase (22).
Calcium transport pathways in these cells that have been cloned are
identified by solid arrows and letters. Calcium transport pathways that
have been identified functionally, but have not been cloned, are shown
with gray arrows.
Calbindins D28K and D9K are intracellular Ca21
binding proteins that are thought to participate in shuttling Ca21 from the apical to the basolateral membrane
(115). In the rabbit and rat, calbindin D28K is expressed
predominantly in the DCT and CNT (266). One study has
also shown significant levels of expression in the CCD of
rat (215). Calbindin D9K in rat is expressed in the cTAL,
DCT, CNT, and CCD (18, 232). Interestingly, while calbindin D28K is expressed throughout the cytoplasm, calbindin D9K expression is concentrated in the basolateral
aspect of the cell cytoplasm.
The recently discovered Ca21 sensing receptor is
another gene that is involved in regulated Ca21 transport
(217). Reverse transcription-PCR studies in rat kidney
show that this receptor is expressed in the glomerulus,
proximal convoluted tubule, proximal straight tubule,
cTAL, DCT, and CCD (216). Immunofluorescence studies
reveal that the Ca21-sensing receptor may be trafficked
either to the apical or the basolateral membrane depending on the tubular segment. In proximal tubule and medullary collecting duct, it is expressed in the apical membrane, whereas in the loop of Henle, distal tubule, and
CCD, expression was detected in the basolateral membrane (216). In the rat inner medullary collecting duct, the
receptor colocalizes with aquaporin-2, indicating that it
plays a role in vasopressin-mediated water permeability
(221). In the thick limb of Henle, high external Ca21
concentrations at the basolateral membrane activate the
receptor. This results in inhibition of the apical K1 channel through the action of a cytochrome P-450 metabolite
of arachidonic acid. Without apical K1 recycling, Na1-K1-
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reabsorption by the distal nephron. Micropuncture studies have shown that Ca21 reabsorption in the distal
nephron must be active given that the luminal concentration of Ca21 is below that of blood and the transepithelial
voltage is lumen negative (164). The passive permeability
of the distal nephron for Ca21 is very low. As the amount
of Ca21 delivered to the distal tubule increases there is a
proportionate increase in reabsorption (55, 113). The primary nephron site of this adaptive response is the early
distal tubule in the rat.
As discussed in section II, there are appreciable species differences in the molecular structure of the distal
nephron. In addition, a variety of functional differences
have been observed. In the rabbit, calcitonin (1 nM) significantly increased Ca21 flux from 1.58 6 0.29 to 4.45
6 1.01 pmol/min but had no effect in either the CNT or the
CCD. Whereas parathyroid hormone (PTH; 1 nM) increased Ca21 flux in the CNT from 2.28 6 0.35 to 9.44
6 1.13 pmol/min but had no effect in either the DCT or the
collecting duct (242). In the rabbit, the CNT is the predominant site of both PTH-stimulated adenylyl cyclase
and Ca21 reabsorption. In contrast, in humans and rats,
PTH stimulates adenylyl cyclase in the DCT. In isolated
perfused rabbit CNT, PTH increased intracellular Ca21 by
a mechanism that was dependent on extracellular Ca21
(27). This suggested that PTH activates an apical Ca21
entry process that is subsequently followed by extrusion
of Ca21 across the basolateral membrane.
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MAMMALIAN DISTAL TUBULE
3. Regulation of Ca21 transport
Transepithelial Ca21 reabsorption in the distal
nephron involves the coordinated interplay of at least
three transport pathways and two intracellular Ca21 binding proteins (see Fig. 9). The transport rate is regulated by
both peptide (PTH, calcitonin) and steroid hormones
(1,25-dihydroxyvitamin D3) as well as by the G proteincoupled Ca21-sensing receptor as well.
The combination of a 1,000-fold inward concentration gradient and the cell-negative membrane potential
provides a large electrochemical driving force favoring
the entry of Ca21 across both the apical and basolateral
membrane. Calcium entry across the apical membrane is
believed to be mediated via Ca21 channels driven by this
gradient (32). Despite the high flux of Ca21 in the distal
nephron, only one laboratory has successfully patched
Ca21 channels from the apical membrane of microdissected tubules (264). These authors examined singlechannel currents in microdissected CNT from rabbit. The
patch-clamp data demonstrated a highly selective, 25-pS,
cAMP-sensitive channel. Open probability peaked at 70
mV and declined with either hyperpolarization or depolarization. Spontaneous open probability is negligible, is
stimulated to ;2% by agonists, and is reversibly inhibited
by dihydropyridine antagonists. Calcium channels have
also been patched from primary cultures of rabbit DCT
(208) and an immortalized mouse distal tubular cell line
(185). In rabbit primary cultures, an 8-pS channel with no
voltage dependence that was blocked by lanthanum, verapamil, and nifedipine was identified. In the mouse, a
2.1-pS channel whose open probability was increased by
hyperpolarizing voltages and inhibited by nifedipine but
not verapamil was detected.
On a molecular level, Yu et al. (304) employed isolated nephron segment RT-PCR to isolate a variety of
Ca21 channel a1-subunits from rat kidney (304). The predominant isoform detected in the distal nephron was a1A
or CaCh4. The predominant b-subunit detected was b4
(304). The significance of this finding remains unclear
given the a1A-isoform is insensitive to dihydropyridines.
Antisense oligonucleotides directed against a and b Ca21
channel subunits were employed to identify those isoforms that mediated Ca21 entry into the mouse cell line
discussed above. An antisense oligonucleotide complementary to the a1C-isoform sequence inhibited the rise of
intracellular Ca21 induced by chlorothiazide but not PTH,
whereas an antisense oligonucleotide complimentary to
the b3-sequence inhibited the rise of intracellular Ca21
induced by chlorothiazide and PTH. These results suggest
that the a1C- and b3-isoforms play a role in Ca21 uptake in
this cell line. However, one cannot be certain that these
Ca21 channel subunits mediate an apical Ca21 entry process given that these cells are not polarized and that PTH
may also activate Ca21 channels in the basolateral membrane of the distal nephron (27).
Recently, Hoenderop et al. (119a) have expression
cloned an apical membrane Ca21 channel from a primary
culture of rabbit kidney connecting tubule and collecting
duct. This novel epithelial Ca21 channel (ECaC) is a distant relative of the recently cloned capsaicin receptor and
the transient receptor potential-related ion channel. Expression was detected predominantly in the proximal
small intestine, distal nephron, and placenta. In kidney,
the channel is expressed in the apical membrane of the
distal nephron and colocalizes with calbindin D28. Lanthanum, cadmium, and manganese inhibit channel activity, whereas barium, magnesium, and strontium do not.
L-type Ca21 channel antagonists do not block the channel.
These properties mimic those of transepithelial transport
in the primary cultures. Interestingly, lowering the extracellular pH also inhibits the channel. Metabolic acidosis is
a well known inhibitor of distal nephron transepithelial
calcium transport. These authors subsequently showed
that channel activation is hyperpolarization dependent,
that conductance is regulated by Ca21, and that the channel desensitizes during repetitive stimulation (1196).
Calcium exit across the basolateral membrane must
be active and is carried out by at least two transporters,
the Na1/Ca21 exchanger and the PMCA. Two studies
suggest that the Na1/Ca21 exchanger, and not the Ca21ATPase, is responsible for the majority of basolateral
Ca21 exit (19, 242). Shimizu et al. (242) used in vitro
microperfusion in rabbit to show that the Na1/Ca21 exchanger plays a major role in the net flux of Ca21 from
distal tubular segments in the basal state and in response
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2Cl2 cotransport activity is decreased and the medullary
countercurrent gradient is reduced, as is paracellular
Ca21 transport. This provides a mechanism for urinary
Ca21-mediated regulation of water permeability, which
may aid in preventing the formation of Ca21 stores.
The plasma membrane Ca21-ATPases (PMCA) are a
family of proteins first described by Schatzmann in 1966
(223). They are P-class ATPases that depend on the formation of an aspartyl phosphate intermediate and are
inhibited by vanadate. Borke et al. (22) raised antibodies
against the purified human erythrocyte PMCA and detected expression along the basolateral membrane of human DCT (22). Stauffer et al. (257) using Western analysis
of whole kidney detected expression of PMCA-1 and -4 in
human, but not PMCA-2 and -3 (257). In the rat, PMCA-1
was detected but not PMCA-2 and -3. Expression of
PMCA-4 was not examined. Magocsi et al. (178) employed
nephron segment RT-PCR to examine the expression of
PMCA-1, -2 and -3 in rat kidney (178). PMCA-2 was predominantly expressed in DCT and cTAL, whereas PMCA-1
was predominantly expressed in glomeruli. Expression of
PMCA-3 has been detected principally in the thin descending limb of Henle (38).
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ROBERT F. REILLY AND DAVID H. ELLISON
Brunette and colleagues (26) recently examined the
effects of calbindins on membrane Ca21 transport in distal nephron membrane vesicles. They showed that calbindin D9K increased ATP-dependent Ca21 transport by
basolateral cell membranes, an effect that is similar to
one reported previously in duodenal cells. In contrast,
calbindin D28K stimulated Ca21 uptake across apical
membrane vesicles. These studies suggest that expression
of the Ca21 binding proteins by distal tubule cells may
stimulate transepithelial transport both by increasing
Ca21 diffusion and through direct interactions with transport proteins.
Studies in cultured cell lines derived from the distal
nephron have also contributed significantly to the understanding of how PTH and 1,25-dihydroxyvitamin D3 regulate transepithelial Ca21 transport. Two groups, Bindels
et al. (19) and Gesek and Friedman (101), have used
immunodissected cell lines in rabbit and mouse, respectively, to investigate PTH-stimulated Ca21 transport.
These studies verified that PTH increased transepithelial
Ca21 transport in rabbit (19) and suggest that both protein
kinase A and protein kinase C participate in this process
(91).
The rabbit primary culture used by Bindels et al. (19)
is immunodissected with a monoclonal antibody to an
unknown “distal tubule antigen” and contains a mix of
CNT and CCD cells. The murine cell line used by Pizzonia
et al. (205) was immunodissected with a monoclonal antibody directed against the Tamm-Horsfall protein, a glycoprotein expressed in the cTAL and DCT. This cell line
was subcloned and classified as originating from the DCT
based on the expression of the thiazide-sensitive NaCl
cotransporter (101).
Clearance studies in rats show that 1,25-dihydroxyvitamin D3 increases renal Ca21 reabsorption across a wide
range of serum Ca21 concentration (53). The molecular
mechanisms responsible for these effects remain unknown. The effect of 1,25-dihydroxyvitamin D3 on transepithelial Ca21 transport appears to vary depending on
the species examined. It increases transepithelial Ca21
transport directly in the rabbit (19), whereas in the mouse
it has no direct effect but potentiates PTH-induced Ca21
uptake (93).
V. THE DISTAL TUBULE IN HUMAN DISEASE
A. Gitelman’s Syndrome
Gitelman’s syndrome (Online Mendelian Inheritance
in Man no. 263800) is an autosomal recessive disorder of
hypokalemia, metabolic alkalosis, hypocalciuria, and hypomagnesemia. The triad of hyperaldosteronism, hypokalemia, and hyperplasia of the juxtaglomerular apparatus
was described by Bartter and colleagues in 1960 (209) and
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to stimulation by PTH. When Na1 was removed from the
bathing fluid or 0.1 mM ouabain was added, net flux was
dramatically reduced both in the baseline and PTH-stimulated states, suggesting that the basolateral exit of Ca21
was mediated, at least in part, by a Na1-dependent secondary active transport process. Further proof that Na1/
Ca21 exchange plays a major role in basolateral Ca21 exit
in distal nephron was also provided by Bindels et al. (19)
in primary cultures of cells derived from both the CNT
and the CCD of rabbit. Transcellular Ca21 transport was
shown to depend on basolateral Na1 and was inhibited by
ouabain (0.1 mM) to the same extent as basolateral Na1
removal. The authors concluded that ;70% of active Ca21
transport was driven by basolateral Na1/Ca21 exchange
and 30% by another mechanism, presumably a Ca21-ATPase.
On the basis of the sequence of the previously reported canine cardiac Na1/Ca21 exchanger (196), a combination of RT-PCR and cDNA library screening was used
to isolate two overlapping cDNA that spanned the entire
coding region for a rabbit renal Na1/Ca21 exchanger
(213). A comparison of the deduced amino acid sequences
of the exchangers revealed 95% amino acid identity. The
majority of the amino acid differences occurred in two
short regions. The first region, the initial 30 amino acids,
was shown to represent a signal sequence (72); only 17 of
these amino acids were conserved. The second region
spanned amino acids 602– 633; only 10 of these amino
acids were conserved. This was followed by a segment of
28 amino acids (649 – 676) that is not present in the rabbit
renal exchanger. Subsequently, Schulze and co-workers
(153, 154) showed that these changes were the result of
alternative splicing. The exchanger is most highly expressed in distal nephron (29, 214, 304).
Functional studies have shown that the acute administration of physiological doses of PTH significantly increased Na1/Ca21 exchange activity in renal basolateral
membrane vesicles from rats (127), rabbits (25), and dogs
(236). In all of these studies, PTH increased the maximum
velocity of the transporter, without influencing the
Michaelis constant. This effect was mimicked by dibutyryl
cAMP in the rabbit (25), but not in the dog (236). The
signal transduction pathways that mediate PTH effects on
Na1/Ca21 exchange remain to be elucidated.
The transcellular route between the luminal entry of
Ca21 into the distal nephron and its basolateral extrusion
is unknown. One possibility is that Ca21 diffuses through
the cytosol. However, the concentration gradient between
the apical and basolateral membranes may be too small to
support significant transcellular unidirectional flux. Calbindin D28K may aid in this process. It may also act as a
cellular buffer during changes in transepithelial Ca21
transport. The expression of calbindin D28K is regulated
by 1,25-dihydroxyvitamin D3, and each molecule binds
four Ca21.
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MAMMALIAN DISTAL TUBULE
at any age but that hypokalemia, hypocalciuria, and hypomagnesemia are uniform features of the disease. Furthermore, they suggest that patients with Gitelman’s syndrome have lower mean arterial pressures than normal
controls.
Based on the autosomal recessive inheritance and
the phenotypic features of the syndrome, Simon et al.
(246) postulated that Gitelman’s syndrome results from
defective Na1 and Cl2 transport by the mutant gene
product. In partial confirmation of this hypothesis, Schultheis et al. (233) detected several phenotypic features of
Gitelman’s syndrome in an NCC-knockout mouse. Null
mice exhibited lower rates of urinary Ca21 excretion and
lower serum magnesium concentrations than did wildtype mice. In addition, the mean arterial pressure of the
null mice was lower than the pressure of wild-type mice
during dietary Na1 depletion. The size and number of
DCT cells was reduced in null mice, and the number of
mitochondria in DCT cells appeared to be reduced. Surprisingly, however, the arterial pH and PCO2, and the
serum bicarbonate and K1 concentrations did not differ
between null mice and controls. Furthermore, NCC
knockout did not affect levels of renin mRNA or serum
aldosterone. The authors suggest that the mild phenotype
observed in the knockout animals is similar to the phenotype in humans. The data of Simon et al. (245), discussed above, however, suggest that there are important
differences in phenotype between the knockout mouse
and human patients who suffer from Gitelman’s syndrome. Most importantly, hypokalemia is consistently observed in humans, and the mean arterial pressure is usually reduced, even when patients consume a normal-salt
diet (245).
Mutations in many regions of the NCC gene have
been reported to cause the Gitelman’s phenotype. Kunchaparty et al. (160) compared the properties of wild-type
and mutant NCC clones, expressed in Xenopus ooyctes.
In contrast to the wild-type clone, which imparted thiazide-sensitive 22Na uptake, a clone containing a Gitelman’s mutation that deletes the carboxy-terminal 54
amino acids (R948X) did not increase Na1 uptake above
levels achieved in water-injected oocytes. Thus this Gitelman’s mutation completely abrogates the ability of the
protein to transport salt. The mechanism by which Gitelman’s mutations impair transport was investigated using
the oocyte expression system. The NCC is normally glycosylated at two asparagine residues, located on the
fourth extracellular domain. The NCC R948X was found
to be translated normally by the oocytes, but it was not
glycosylated. Furthermore, the mutant protein was not
delivered to the plasma membrane. These data suggest
that at least one Gitelman’s mutation leads to misfolding
of the protein, activating the “quality control” system of
the endoplasmic reticulum. The quality control system is
able to detect misfolded proteins, targeting them for deg-
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later given the name Bartter’s syndrome. Gitelman later
described an inherited disorder characterized by hypokalemia, hypomagnesemia, and normal blood pressure
(106). For many years, Gitelman’s syndrome and Bartter’s
syndrome were considered to be one in the same. Bettinelli’s careful analysis of phenotypic features, however,
indicated clearly that Gitelman’s syndrome is distinct
from classical Bartter’s syndrome (16). These workers
differentiated the disorders on the basis of urinary Ca21
excretion (Gitelman’s patients had urine Ca21-to-creatinine ratios ,0.20) and serum magnesium concentration
(Gitelman’s patients had plasma magnesium concentrations ,0.75 mM). Bettinelli (16) also reported that patients with Bartter’s syndrome were more often products
of pregnancies complicated by polyhydramnios or premature delivery. Many Bartter’s patients also had short stature, polyuria, polydipsia, and a tendency to dehydration
during infancy and childhood. In contrast, patients with
Gitelman’s syndrome had tetanic episodes.
Because the phenotype of Gitelman’s syndrome resembles the consequences of thiazide diuretic therapy,
the thiazide-sensitive NCC was examined as a candidate
gene. Simon et al. (246) showed complete linkage of
Gitelman’s syndrome to the locus encoding the human
thiazide-sensitive NCC. Seventeen different molecular
variants were identified that were predicted to alter the
structure of the encoded protein (246). Other groups have
subsequently reported mutations in the same gene that
were linked to this disorder (138, 167, 183, 207, 263). In a
preliminary report, Simon et al. (245) collected a total of
82 distinct mutations that were associated with Gitelman’s syndrome. Nearly all of these mutations are nonconservative and affect amino acids that have been conserved throughout evolution. To date, mutations in the
thiazide-sensitive NCC have been detected in most cases
of Gitelman’s syndrome that have been screened; mutations in other genes have not been shown to cause the
Gitelman’s phenotype. Thus the syndrome appears to be
caused by mutations in a single gene.
Identification of the gene defect responsible for Gitelman’s syndrome has permitted the phenotype to be defined more clearly. In general, Gitelman’s syndrome is a
mild disease, but recent case reports indicate that adverse
outcomes, such as hypokalemic rhabdomyloysis, can occur (197). Simon et al. (245) showed that affected patients
were diagnosed at a mean age of 22.5 yr (range 0.75– 65
yr). The mean serum K1 concentration was 2.6 mM (range
1.0 –3.5 mM), the serum bicarbonate was 30.7 mM (range
22.0 –37.4 mM), the serum Mg1 was 1.2 mM (range 0.3–1.7
mM), and the urine Ca21-to-creatinine ratio was 0.088
(range 0.0001– 0.36). In addition, 83% of patients had diastolic blood pressures below the 50th percentile for age
and sex. Fifty-one percent of patients had diastolic blood
pressures below the 25th percentile (P , 0.001). These
findings emphasize that Gitelman’s syndrome can present
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ROBERT F. REILLY AND DAVID H. ELLISON
Volume 80
radation, often via an ubiquitin-dependent interaction
with the proteasome. In preliminary experiments, at least
10 other Gitelman’s mutations were shown to disrupt
function of the protein by causing misprocessing (Ellison,
unpublished data). Thus most cases of Gitelman’s syndrome appear to result because misfolding of the protein
activates the quality control system of the endoplasmic
reticulum. This leads to a failure of delivery to the plasma
membrane.
Identification of a single gene defect that is responsible for most or all cases of Gitelman’s syndrome
implies that the phenotypic features of the disease all
result from dysfunction of this protein. The observation
that the gene for the thiazide-sensitive NCC is expressed predominantly by renal tissue and not elsewhere (184; Ellison, unpublished data) implies that all
of the phenotypic features of Gitelman’s syndrome result from abnormalities of renal function. Hypokalemia
and alkalosis probably develop because extracellular
fluid volume contraction results from loss of Na1 and
Cl2. It is further exacerbated because Na1 delivery into
the collecting duct is increased (owing to the absence
of Na1-Cl2 cotransport by DCT cells, see Fig. 10).
Patients with Gitelman’s syndrome generally have high
levels of plasma renin activity (16) that enhance aldosterone secretion and favor K1 and H1 secretion along
the distal tubule and collecting duct. Mild contraction
of the extracellular fluid volume also reduces the blood
pressure, as observed by Simon et al. (245). An early
observation was that patients with Bartter’s and Gitelman’s syndromes are resistant to the effects of angiotensin II. This resistance probably results from chronically elevated levels of plasma renin and angiotensin II
leading to a desensitization phenomenon. It should be
noted that patients with Gitelman’s syndrome exhibit
levels of plasma renin activity that are not as high as
the levels observed in patients with classical Bartter’s
syndrome. This is likely to reflect the fact that Bartter’s
syndrome results from dysfunction of TAL cells (via
disruption of either the Na1-K1-2Cl2 cotransporter, the
apical K1 channel, or the basolateral Cl2 channel).
Macula densa cells are specialized TAL cells that suppress renin secretion when transcellular NaCl transport
occurs (31). Thus plasma renin activity is stimulated in
Bartter’s syndrome both because extracellular fluid volume is contracted and because the macula densa signal
is blocked.
The pathogenesis of the hypocalciuria and hypomagnesemia remains less fully understood. As discussed
above, blocking Na1 entry across DCT cells leads to
increases in Ca21 reabsorption, probably by increasing
Ca21 entry, via a voltage-activated luminal Ca21 channel
(see Fig. 10) and secondarily by stimulating basolateral
Na1/Ca21 exchange. These effects are believed to occur
because the intracellular Cl2 concentration declines, owing to the absence of an apical Cl2 entry pathway. The
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FIG. 10. Postulated mechanism of
electrolyte defects in Gitelman’s syndrome. DCT-1 (top) and DCT-2 (bottom)
cells in normal (left) and Gitelman’s
(right) individuals are shown. Under normal conditions, cellular Cl2 leaves cell
via basolateral channels and depolarizes
cell. In DCT-2 cells, Na1 limits Ca21 exit
by inhibiting Na1/Ca21 exchanger. As
discussed in text, many Gitelman’s mutations impair protein processing, generating Na1-Cl2 cotransporters that do not
reach apical membrane. Cellular Na1 and
Cl2 concentrations decline, then, because major NaCl entry pathway is nonfunctional. This hyperpolarizes cell, activating apical Ca21 entry pathways and
stimulating basolateral Na1/Ca21 exchanger (which is electrogenic). Na1/
Ca21 exchanger is also stimulated by
lower cell Na1 activity. Extracellular
fluid volume depletion stimulates aldosterone, which stimulates DCT-2 cells to
absorb Na1 and secrete K1. This effect is
magnified because electroneutral pathways are absent in these aldosterone-sensitive cells. Magnesium wasting may involve paracellular movement, driven by
transepithelial voltage.
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MAMMALIAN DISTAL TUBULE
FIG. 11. Mechanisms of acid secretion by a-intercalated cells. H1 are secreted across luminal membrane by vacuolar H1-ATPase and by H1/K1 exchanger
(295). Both of these transporters utilize ATP. HCO2
3
leaves cell via activity of a Cl2/HCO2
3 exchanger (234).
2
2
Cl can recycle via a Cl channel (200).
B. Distal Disorders of Urinary Acidification
The secretion of H1 by tubular epithelial cells serves
at least two purposes: 1) the reabsorption of filtered
bicarbonate and 2) the excretion of the dietary proton
load. The distal tubule is largely responsible for the latter,
via the acid-excreting a-intercalating cell shown in Figure
11. Aldosterone stimulates Na1 reabsorption in the distal
nephron by increasing apical Na1 entry via the amiloridesensitive epithelial Na1 channel and Na1 exit via the
basolateral Na1-K1-ATPase. This promotes H1 secretion
by establishing a more favorable electrochemical gradient. In addition, aldosterone stimulates H1 secretion directly (261).
Defects in any of the a-intercalated cell transport
proteins shown in Figure 11, the epithelial Na1 channel,
or the mineralocorticoid receptor could result in a metabolic acidosis. To date, distal acidification defects as a
result of either acquired or inherited defects in the H11
ATPase, the Cl2/HCO2
3 exchanger, the epithelial Na
channel, and the mineralocorticoid receptor have been
described. Autosomal dominant familial distal renal tubular acidosis is caused by mutations in the renal Cl2/HCO2
3
exchanger (33) (137). Mutations in the amiloride-sensitive
epithelial Na1 channel cause autosomal recessive
pseudohypoaldosteronism type 1 (PHA-1) (42), whereas
mutations in the mineralocorticoid receptor result in autosomal dominant PHA-1 (98).
Several groups have reported that immunohistochemical staining of kidney in patients with distal renal
tubular acidosis and Sjogren’s syndrome failed to detect
staining of H1-ATPase in intercalated cells (11, 49). Intercalated cells were present in these patients, and antibody
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lower intracellular Cl2 hyperpolarizes the cell, enhancing
Ca21 entry via the voltage-activated channel. The hyperpolarization also stimulates the basolateral Na1/Ca21 exchanger, because this transporter is electrogenic. Finally,
lower intracellular Na1 concentrations stimulate the Na1/
Ca21 exchanger by increasing the Na1 gradient across the
basolateral membrane. All of these factors probably participate in the increased Ca21 transport.
Gitelman’s syndrome is associated with Mg21 wasting. This suggests that the cellular events that enhance
Ca21 absorption inhibit Mg21 absorption. Surprisingly, if
immortalized DCT cells are hyperpolarized, as postulated
to occur in Gitelman’s patients, Mg21 uptake increases
rather than decreases (60, 210). Recently, a paracellular
magnesium transport protein, named paracellin, has been
cloned (245a). As an alternative hypothesis, therefore,
Figure 10 postulates that Mg21 wasting in Gitelman’s
syndrome occurs via alterations in paracellular Mg21
movement. According to this model, Gitelman’s syndrome
converts some DCT cells from predominantly electroneutral cells to cells that reabsorb Na1 in an electrogenic
manner. As discussed in section IVB3A, these cells are also
responsive to the actions of aldosterone. This combination of the dominance of electrogenic ion transport pathways, the stimulation by aldosterone, and the increased
luminal Na1 concentrations all favor electrogenic Na1
reabsorption. This greatly increases the magnitude of the
transepithelial voltage. The high transepithelial voltage
would favor Mg21 secretion, via the paracellular pathway.
Until mechanisms of Mg21 transport by kidney cells are
better understood, however, the pathogenesis of Mg21
wasting in patients with Gitelman’s syndrome will remain
speculative.
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ROBERT F. REILLY AND DAVID H. ELLISON
Volume 80
to the H1-ATPase was not detected in serum. In addition,
in two patients, the Cl2/HCO2
3 exchanger was also not
detected in the basolateral membrane by immunofluorescence. T cell-mediated damage to intercalated cells has
been postulated.
C. Adapation to Diuretic Drugs
Diuretic drugs are effective therapy for edematous
disorders and hypertension. However, several adaptations to diuretic treatment may affect the ability of these
drugs to lead to natriuresis. When NaCl reabsorption
along the TAL is inhibited by loop diuretics, the NaCl
concentration in fluid that enters the distal tubule is
greatly increased. In one study, the Na1 concentration in
fluid entering the distal tubule of rats rose from 42 to 140
mM during acute loop diuretic infusion (122). The increased luminal NaCl concentration drives increased Na1
absorption along the distal tubule (from 148 to 361 pmol/
min) (122) because NaCl transport by the distal tubule is
load dependent (as discussed above). This increase in
solute reabsorption by segments that lie distal to the site
of diuretic action is the first form of diuretic adaptation
and limits the intrinsic potency of the diuretic drug. The
net effect of acute diuretic administration on urinary Na1
and Cl2 excretion, therefore, reflects the sum of effects in
the diuretic-sensitive segment (inhibition of NaCl reabsorption) and in diuretic-insensitive segments (secondary
stimulation of NaCl reabsorption).
As the acute effect of a diuretic declines, a period of
NaCl retention usually commences. This phenomenon,
often termed postdiuretic NaCl retention, has been reviewed recently (75). One mechanism by which diuretic
drugs may increase the tendency for NaCl retention directly, without participation of changes in extracellular
fluid volume, involves diuretic-induced activation of ion
transporters within the diuretic-sensitive nephron segment. Within 60 min of thiazide administration, the number of thiazide-sensitive NCC in kidney cortex (measured
as the number of [3H]metolazone binding sites) increases
substantially (45). The techniques used to estimate the
number of transporters in these experiments do not permit one to determine whether the increased number reflects insertion of preexisting transporters from a subapical storage pool or activation of transporters that are
present but inactive in the apical membrane. Recent immunocytochemical studies show that a subapical compartment contains thiazide-sensitive NCC in DCT cells of
rats (206), suggesting that a shuttle system may regulate
functional activity of the thiazide-sensitive NCC in the
distal tubule. An increase in the number of activated ion
transporters at the apical membrane would be expected
to increase the transport capacity so that when diuretic
concentrations decline, increased Na1 and Cl2 transport
would result.
Another mechanism by which diuretic drugs may
enhance the tendency to NaCl retention directly involves
stimulation of transport pathways in nephron segments
that lie distal to the target of diuretic action (segments
that are insensitive to the diuretic drug). For example, the
number of thiazide-sensitive Na1-Cl2 transporters in the
kidney (and presumably in the DCT) increases within 60
min after a loop diuretic has been administered (45).
Because thiazide-sensitive transporters are expressed
only by nephron segments that do not express loop diuretic-sensitive pathways, the increased number of thiazide-sensitive NCC is believed to result from increases in
salt and water delivery to DCT cells.
Another factor that can enhance the reabsorptive
capacity of the distal tubule is adaptive changes in the
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FIG. 12. Effects of continuous loop
diuretic infusion on rat kidney. Loop diuretic infusion increased number of thiazide-sensitive Na1-Cl2 cotransporters
[data from Chen et al. (45)], rate of thiazide-sensitive Na1 transport along distal
tubule [data from Ellison et al. (79)],
abundance of thiazide-sensitive Na1-Cl2
cotransporter (NCC) mRNA (top, furosemide-treated kidney cortex; bottom,
control kidney cortex) [data from Obermüller et al. (199)], and Na1-K1-ATPase
activity along distal convoluted tubule
[data from Scherzer et al. (226)].
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MAMMALIAN DISTAL TUBULE
delivery to the collecting duct is increased in the presence
of high levels of circulating aldosterone, principal cell
hypertrophy develops; when salt delivery is high in the
absence of aldosterone secretion, hypertrophy is absent.
This indicates that aldosterone plays a permissive role in
the development of cellular hypertrophy in this aldosterone-responsive renal epithelium. Although recent experiments suggest that aldosterone does affect ion transport
by cells of the DCT, and aldosterone almost certainly
contributes to adaptations along the CCT, hypertrophy of
DCT cells has been shown to occur during chronic loop
diuretic infusion even when changes in circulating mineralocorticoid, glucocorticoid, and vasopressin levels are
prevented (135).
One intriguing hypothesis is that cellular ion concentrations regulate epithelial cell growth directly (256). Increases in Na1 uptake across the apical plasma membrane precede cell growth in the TAL during treatment
with antidiuretic hormone (24), in principal cells of the
CCT during treatment with mineralocorticoid hormones
(131, 204), and in the DCT during treatment with loop
diuretics (79, 135). Although the cause of the increased
Na1 uptake varies, changes in the intracellular Na1 concentration appear to precede growth in each example
(13). This hypothesis predicts that blockade of apical Na1
entry would lead to atrophy of epithelial cells. Chronic
treatment of rats with DCT diuretics reduces activity of
Na1-K1-ATPase and Na1 transport capacity of DCT segments (97, 193), but these experiments are complicated
by other structural effects of chronic DCT diuretic treatment, discussed below. Regardless of the proximate stimulus for DCT cell growth, recent experiments have shown
that immunoreactivity for IGF-I and for an IGFBP
(IGFBP-I) increases during chronic treatment of rats with
loop diuretics (150). The changes in IGF-I expression
appeared not to result from changes in IGF-I mRNA expression, but rather appeared to reflect posttranscriptional events. IGFBP-I mRNA was increased by threefold
18 h after loop diuretic treatment was initiated. IGF-I has
been shown to participate in regeneration of injured or
ischemic renal tissue and promotes cell proliferation and
differentiation in vitro. Whether these changes in IGF
expression mediate the effects of diuretics on distal
nephron structure remains to be established.
When DCT diuretics are administered chronically,
Na1-K1-ATPase activity in the DCT is reduced (97), and
the capacity of DCT cells to reabsorb Na1 and Cl2 declines (193). However, chronic administration of DCT
diuretics to rats leads to profound changes in cellular
morphology; DCT cells undergo apoptosis and necrosis
with resulting interstitial fibrosis. Chronic treatment also
leads to the disappearance of normal polarization of thiazide-sensitive NCC proteins. Under normal conditions,
immunoreactivity for the thiazide-sensitive NCC is restricted to the apical membrane and to a small subapical
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nephron itself. When solute delivery to the distal tubule is
increased chronically, distal cells undergo both hypertrophy and hyperplasia. Infusion of furosemide into rats
continuously for 7 days increased the percentage of renal
cortical volume occupied by DCT cells by nearly 100% (79,
132, 135). Biochemical and functional correlates of these
structural changes are shown in Figure 12. Chronic loop
diuretic administration increases the Na1-K1-ATPase activity in the distal convoluted and CCT (226, 288) and
increases the number of thiazide-sensitive NCC, measured as the maximal number of binding sites for [3H]metolazone (45, 199). In one study, chronic furosemide
treatment increased expression of mRNA encoding the
thiazide-sensitive NCC, as detected by in situ hybridization (see Fig. 12) (199). In another study, however, mRNA
expression of the thiazide-sensitive NCC as well as the
ouabain-sensitive Na1-K1-ATPase was not affected by
chronic furosemide infusion, when detected by Northern
analysis (188). Distal tubule cells that express high levels
of transport proteins and are hypertrophic have a higher
Na1 and Cl2 transport capacity than normal tubules;
compared with tubules from normal animals, tubules of
animals treated chronically with loop diuretics can absorb Na1 and Cl2 up to three times more rapidly than
control animals. Even salt and water delivery is fixed by
microperfusion (Fig. 12); when distal tubules are presented with high NaCl loads, as occurs during loop diuretic administration in vivo, Na1 and Cl2 absorption
rates approach those commonly observed only in the
proximal tubule (79). Recently, it has been observed that
chronic treatment of rats with loop diuretics also results
in significant hyperplasia of cells along the distal nephron.
Whereas mitoses of renal tubule epithelial cells are infrequent in adult kidneys, distal tubules from animals treated
with furosemide chronically demonstrate prominent mitoses; increased synthesis of DNA in these cells was
confirmed by showing increases in labeling of DCT cells
with bromodeoxyuridine and proliferating cell nuclear
antigen (171).
The diuretic-induced signals that initiate changes in
distal nephron structure and function are poorly understood. Several factors, acting in concert, may contribute
to these changes; these include diuretic-induced increases
in Na1 and Cl2 delivery to distal segments, effects of
extracellular fluid volume depletion on systemic hormone
secretion and renal nerve activity, and local effects of
diuretics on autocrine and paracrine secretion. Increased
production of angiotensin II or increased secretion of
aldosterone resulting from increases in renin activity may
contribute to hypertrophy and hyperplasia. Angiotensin is
a potent mitogen; angiotensin II receptors have not been
localized definitively to DCT cells, but recent functional
studies do suggest that DCT cells express angiotensin II
receptors. Aldosterone also promotes growth of responsive tissues under some circumstances (136); when salt
303
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ROBERT F. REILLY AND DAVID H. ELLISON
Volume 80
pool of vesicles. During chronic treatment with DCT diuretics, the protein is distributed uniformly throughout
the cell. Surprisingly, based on the severe morphological
degenerative changes in tubular morphology, chronic thiazide administration results in an increase in the density
of [3H]metolazone binding sites (functional thiazide-sensitive transporters) in kidney cortex (97) despite a decline
in mRNA expression for the transporter (171); this may
reflect an increased ability of diuretic to bind to degenerating receptors that have been internalized into cells during the diuretic treatment.
Although experimental data concerning structural
and functional responses of the distal nephron to chronic
treatment with diuretic drugs come predominantly from
studies employing experimental animals, Loon et al. (175)
reported that chronic treatment with loop diuretics in
humans enhanced ion transport rates in the distal tubule.
They estimated the transport capacity of the DCT as the
portion of Na1 and Cl2 reabsorption that could be inhibited by thiazide diuretics. When furosemide was administered to volunteers for 1 mo, the enhancement in Na1
excretion that occurred resulted from a dose of thiazide
diuretic that was significantly larger. Although these data
are necessarily indirect, they are entirely consistent with
the data derived from experimental animals given loop
diuretics chronically. The extracellular fluid volume-independent component of NaCl retention that occurs after
loop diuretic administration (5) may also reflect changes
in distal nephron structure and function.
D. Nephrolithiasis
Nephrolithiasis accounts for ;7–10 of every 1,000
hospital admissions in the United States and is a cause of
considerable morbidity (1). It has been estimated that
there is a genetic component involved in up to 45% of
cases, and hypercalciuria is a major risk factor.
Recently, a common molecular etiology for three rare
X-linked hypercalciuric syndromes characterized by
nephrolithiasis (Dent’s disease, X-linked recessive nephrolithiasis, and X-linked recessive hypophosphatemic
rickets) was identified using a positional cloning approach (169). Although these syndromes do differ slightly
in their clinical presentation, they are all characterized by
proximal tubular dysfunction, nephrocalcinosis, nephrolithiasis, and progressive renal failure. The defect is in the
CLC-5 gene, which is a member of the CLC family of
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FIG. 13. Model of CLC-5 channel showing sites of missense mutations (in gray) that have been reported to cause
Dent’s disease nephrolithiasis (224). A large deletion (between lines indicated with D), splice site mutations (at S), and
an insertion (arrow) have also been reported to cause this syndrome.
January 2000
MAMMALIAN DISTAL TUBULE
immunolocalize CLC-5. Luyckx et al. (176) showed that
CLC-5 is expressed in the S3 segment of the proximal
tubule and in the medullary TAL (176). Devuyst et al. (62)
immunolocalized CLC-5 to the proximal tubule, TAL, and
CCD intercalated cells (62). In both studies, CLC-5 was
shown to be associated with endosomes of the receptormediated endocytic pathway. An inward Cl2 flux is required in these vesicles to dissipate the positive charge
that results from the secretion of protons into the interior
of the vacuole by a H1-ATPase. It is likely that defects in
protein trafficking due to dysfunction of CLC-5 are responsible for the phenotype of these disorders.
E. Treatment of Nephrolithiasis With Distal
Tubular Diuretics
The treatment of choice in patients with Ca21-containing stones and hypercalciuria is thiazide diuretics.
Two randomized placebo-controlled trials have shown
that thiazides reduce the risk of Ca21 lithiasis by 54 and
58% (81, 161). It is thought that thiazides reduce the risk
of stone formation by increasing distal tubular reabsorption of Ca21. Interestingly, the majority of patients in
these studies were not hypercalciuric. Therefore, it may
be possible that thiazides not only reduce the risk of
recurrent nephrolithiasis by lowering urinary Ca21 but
may also act via other mechanisms as well.
At least two different mechanisms have been postulated to explain the effect of thiazides to increase distal
tubular Ca21 transport. Brunette et al. (34, 35) have identified two kinetically distinct Ca21 uptake pathways in the
luminal membrane of distal tubules. The first is a highaffinity, low-capacity component. The maximum velocity
of this component is increased by preincubation with
PTH. The second is a low-affinity, high-capacity component whose maximum velocity is enhanced by hydrochlorothiazide (162). Hydrochlorothiazide and PTH produced
an additive stimulation of Ca21 uptake. Calcium channel
blockers alone did not affect Ca21 transport; however,
when added with PTH, they completely abolish the PTHinduced stimulation of Ca21 uptake.
Hydrochlorothiazide has no effect on basolateral
Ca21 transport mediated by either the Na1/Ca21 exchanger or the Ca21-ATPase (163). Only when hydrochlorothiazide was incubated with apical membrane vesicles
of the distal tubule was an increase in Ca21 uptake noted.
Sodium was required for the effect. Sodium decreased
Ca21 uptake in a dose-dependent manner, and hydrochlorothiazide partially reestablished Ca21 uptake to those
observed in Na1-free media. It was concluded that hydrochlorothiazide decreased the inhibitory effect of Na1 on
Ca21 uptake in the luminal membrane of the distal tubule.
Gesek and Friedman (100) also examined the mechanism whereby hydrochlorothiazide increases distal
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voltage-gated Cl2 channels (see Fig. 13). More recently, a
fourth disorder, low-molecular-weight proteinuria associated with hypercalciuric nephrocalcinosis in Japanese
children, was discovered to be caused by a defect in the
same gene (170).
There are at least nine known members of the CLC
gene family, the first of which, CLC-1, was expression
cloned from Torpedo marmorata by Jentsch et al. (128).
Other members of the family include CLC2–7, CLC-Ka,
and CLC-Kb. Defects in the expression of at least two
additional members of the CLC gene family are associated
with human disease. CLC-1 is mutated in both the dominant and recessive forms of congenital myotonia (151),
and mutations in CLC-Kb were reported to cause some
cases of Bartter’s syndrome (244).
CLC-5 has a high selectivity for Cl2, is strongly outwardly rectifying, and is activated at inside-positive membrane voltages exceeding 10 –20 mV. It is expressed predominantly in the kidney in humans (90); in the rat, the
transcript is also expressed in brain, liver, and lung (258).
To date, 19 mutations have been reported in 22 different families (224). The majority of these have been
expressed in Xenopus laevis oocytes by the Jentsch group
and have been found to have either no or marked reduced
Cl2 channel activity (see Fig. 13). Interestingly, there
appears to be little correlation between the phenotype
and the nature of the severity of the Cl2 channel defect.
Even within families, individuals sharing the same mutation can be either mildly or severely affected.
It is clear that this group of disorders is caused by a
lack of expression of CLC-5. How this Cl2 channel defect
results in low-molecular-weight proteinuria, moderate hypercalciuria, an exaggerated calciuretic response to an
oral Ca21 load, defective urinary concentrating ability, an
acidification defect with acid loading, nephrocalcinosis,
and ultimately renal failure remains unclear. The best
arguments that these abnormalities are due to a primary
renal abnormality as opposed to nonspecific renal injury
from an increased filtered load of Ca2- are as follows: 1) in
humans the major site of expression of CLC-5 is the
kidney, and 2) of patients that have undergone renal
transplantation for these disorders, there has been no
evidence of recurrent disease in the allograft.
Obermüller et al. (200) employed in situ hybridization
to show that CLC-5 is expressed by type A intercalated
cells in the CNT and collecting duct (200). This would
indicate that the highest level of expression of the CLC-5
transcript is in the intercalated cell in CNT and CCD.
However, on the basis of the clinical presentation, it
would appear that the CLC-5 gene product is likely expressed more widely than distal nephron. The low-molecular-weight proteinuria is suggestive of proximal tubular
expression that was below the level of detection of the in
situ hybridization technique that we employed. More recently, two laboratories used polyclonal antibodies to
305
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ROBERT F. REILLY AND DAVID H. ELLISON
7.
8.
9.
10.
11.
12.
13.
VI. SUMMARY
14.
The distal tubule is a short nephron segment between
the region of the macula densa and the confluence to form
the collecting duct. Its importance in sodium, chloride,
potassium, calcium, and magnesium balance is emphasized by human diseases that result from ion transport
defects along its length. During the past 10 years, nearly
all of the dominant ion transport proteins that control its
function have been identified and cloned. This has permitted earlier physiological information to be reassessed
based on molecular information. It has also permitted
specific transport functions to be assigned to specific cell
types within this extremely heterogeneous tubule segment.
Address for reprint requests and other correspondence:
D. H. Ellison, Univ. of Colorado Health Sciences Center, Renal
Section/111C, VA Medical Center, 1055 Clermont St., Denver, CO
80220 (E-mail: [email protected] or www.uchsc.edu/sm/
renal/epithelial).
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