Physiol Rev 87: 1083–1112, 2007;
doi:10.1152/physrev.00053.2006.
Mouse Models and the Urinary Concentrating Mechanism
in the New Millennium
ROBERT A. FENTON AND MARK A. KNEPPER
Water and Salt Research Center, Institute of Anatomy, University of Aarhus, Aarhus, Denmark; and
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute,
National Institutes of Health, Bethesda, Maryland
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I. Introduction
A. Transport processes in Henle’s loops that concentrate solutes in the medullary interstitium
B. Osmotic equilibration of collecting duct fluid by vasopressin-regulated water transport
C. Urea recycling
II. Mouse Models
A. Transport proteins involved in urinary concentration and dilution
B. Sodium transporters and channels
C. Potassium channels
D. Chloride channels
E. Aquaporins
F. Collecting duct aquaporins
G. Urea transporters
H. Receptors and signaling molecules
I. Prostaglandins
J. Renin-angiotensin-aldosterone system
K. Osmoprotective genes
L. Miscellaneous
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Fenton RA, Knepper MA. Mouse Models and the Urinary Concentrating Mechanism in the New Millennium.
Physiol Rev 87: 1083–1112, 2007; doi:10.1152/physrev.00053.2006.—Our understanding of urinary concentrating and
diluting mechanisms at the end of the 20th century was based largely on data from renal micropuncture studies,
isolated perfused tubule studies, tissue analysis studies and anatomical studies, combined with mathematical
modeling. Despite extensive data, several key questions remained to be answered. With the advent of the 21st
century, a new approach, transgenic and knockout mouse technology, is providing critical new information about
urinary concentrating processes. The central goal of this review is to summarize findings in transgenic and knockout
mice pertinent to our understanding of the urinary concentrating mechanism, focusing chiefly on mice in which
expression of specific renal transporters or receptors has been deleted. These include the major renal water
channels (aquaporins), urea transporters, ion transporters and channels (NHE3, NKCC2, NCC, ENaC, ROMK,
ClC-K1), G protein-coupled receptors (type 2 vasopressin receptor, prostaglandin receptors, endothelin receptors,
angiotensin II receptors), and signaling molecules. These studies shed new light on several key questions concerning
the urinary concentrating mechanism including: 1) elucidation of the role of water absorption from the descending
limb of Henle in countercurrent multiplication, 2) an evaluation of the feasibility of the passive model of KokkoRector and Stephenson, 3) explication of the role of inner medullary collecting duct urea transport in water
conservation, 4) an evaluation of the role of tubuloglomerular feedback in maintenance of appropriate distal delivery
rates for effective regulation of urinary water excretion, and 5) elucidation of the importance of water reabsorption
in the connecting tubule versus the collecting duct for maintenance of water balance.
I. INTRODUCTION
The mammalian kidney tightly controls systemic
water balance by varying water excretion over a broad
range, chiefly in response to changes in the circulating
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concentration of the antidiuretic hormone vasopressin
(70). These broad adjustments in water excretion can
be achieved without major effects on solute excretion
because of the kidney’s ability to dilute urinary solutes
below plasma osmolality and to concentrate solutes in
0031-9333/07 $18.00 Copyright © 2007 the American Physiological Society
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FIG. 1. Steady-state renal response to varying rates of vasopressin
infusion in conscious rats (70). A water load (4% of body weight) was
maintained throughout the experiments to suppress endogenous vasopressin secretion. Although the urine flow rate was markedly reduced at
higher vasopressin infusion rates, the osmolar clearance changed little.
[Data adapted from Atherton et al. (6).]
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A. Transport Processes in Henle’s Loops That
Concentrate Solutes in the
Medullary Interstitium
It is generally accepted that NaCl is concentrated in
the renal medullary interstitium through the process of
countercurrent multiplication, originally proposed by
Wirz, Kuhn, and colleagues more than 50 years ago (139,
282). This process requires special properties in both the
ascending limb and descending limb of Henle (124). The
ascending limb in the outer medulla (MTAL) is like
the CTAL (described above); it actively transports NaCl,
but water remains in the lumen owing to a lack of aquaporin expression and thus low water permeability (186).
This dilutional effect generates a small transepithelial
osmolality difference (the “single effect” or Einzeleffekt)
that is “multiplied” by the counterflow between the two
limbs of Henle’s loops, resulting in an axially aligned
osmotic gradient that is much larger than the single effect
(see Ref. 70 for details). The properties of the descending
limb of Henle are critical because, for countercurrent
multiplication to work effectively, the luminal fluid must
be close to osmotic equilibrium with the surrounding
interstitium at every point along the descending limb.
How this osmotic equilibration occurs is one point of
controversy (see Fig. 2). Theoretically, the equilibration
could occur by rapid water efflux from the descending
limb, by rapid solute entry into the descending limb, or by
a combination of both processes. Studies of isolated perfused thin descending limbs from the outer medulla revealed that they have high water permeabilities (43, 130),
which are thought to be due to the presence of high levels
of aquaporin-1 in the apical and basolateral plasma membranes of thin descending limb cells (188). However, micropuncture studies addressing the mechanism of osmotic equilibration in the descending limb concluded that
a substantial element of osmotic equilibration is due to
solute entry (106). This controversy has been addressed
using aquaporin-1 (AQP-1) knockout mice (see below).
The countercurrent multiplier mechanism described
in the previous paragraph only functions in the renal outer
medulla and medullary rays of the cortex. In contrast, the
inner medulla lacks a thick ascending limb of Henle and
the ascending limb of Henle’s loop in the inner medulla is
of “thin-limb” morphology. Repeated studies of the inner
medullary thin limb have not been able to uncover evidence of net active NaCl transport (97, 124, 132). However, a substantial axial osmolality gradient has been
demonstrated in the inner medulla of a variety of mammalian species. A major point of controversy has been
regarding the question of how the inner medullary NaCl
gradient is generated. Three hypotheses have been presented to address this question: 1) the “passive” hypothesis of Kokko and Rector (131) and Stephenson (246), in
which energy for concentration of solutes (chiefly NaCl)
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the urine to much greater than plasma osmolality
(Fig. 1). The diluting mechanism is well understood as
being chiefly the function of the cortical thick ascending limb (CTAL) of Henle’s loop, sometimes called the
diluting segment. The CTAL actively transports NaCl
and NH4Cl from the lumen to the peritubular interstitial
space, while (owing to its low water permeability) leaving water behind. In contrast, the urinary concentrating
process is much more complex. The central goal of this
review is to summarize the new knowledge that has
accrued about the urinary concentrating process as a
result of the development and application of gene deletion techniques, both conventional gene knockouts
and cell specific gene knockouts. However, before addressing these mouse models, we provide a brief, basic
summary of our current knowledge about the urinary
concentrating mechanism prior to the advent of knockout mouse technology, with emphasis on extant controversies.
A pedagogically useful, but oversimplified, starting
point is the idea that the concentrating process can be
divided into two separate elements: 1) transport
processes residing in Henle’s loops that concentrate
solutes in the medullary interstitium and 2) vasopressin-regulated water transport in the collecting ducts,
which allows the tubular lumen contents to equilibrate
with the hypertonic medullary interstitium. Although
providing a useful starting point, the reader should be
mindful that in reality these two processes are not truly
separable, particularly with regard to urea, the major
solute responsible for waste nitrogen elimination in
mammals (see below).
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C. Urea Recycling
FIG. 2. Two modes of countercurrent multiplication. Figure depicts
two loops of Henle showing NaCl recycling mode (left) and water
short-circuiting mode (right). NaCl recycling mode concentrates NaCl in
medulla by increasing the mean residence time of Na⫹ and Cl⫺ in
medulla. Water short-circuiting mode (right) concentrates the medulla
by reducing the mean residence time for water molecules in medulla.
Water short-circuiting mode concentrates all solutes present in input to
descending limb of loop.
in the inner medullary interstitium is derived from passive
urea efflux from the inner medullary collecting duct
(IMCD), which concentrates NaCl in the descending limb
lumen via water efflux from the thin descending limb
(225); 2) the “lactate” hypothesis of Thomas and co-workers (89, 261), in which lactate generation from glucose by
glycolysis in inner medullary cells results in net generation of osmotically active particles (one glucose yields
two lactates); and 3) the “mechanico-osmotic induction
hypothesis” (124, 211), in which energy from the peristaltic contractions of the renal pelvic wall is stored by
compression of hyaluronan in the inner medullary interstitium and that this energy is used to concentrate solutes
in the descending limbs and collecting ducts by water
withdrawal when the hyaluronan springs back to its extended state after passage of the peristaltic wave. Recent
results in mice with genetic deletion of the IMCD urea
transporters, UT-A1 and UT-A3, have addressed the first
hypothesis (see below).
B. Osmotic Equilibration of Collecting Duct Fluid
by Vasopressin-Regulated Water Transport
It is generally accepted that vasopressin regulates
water permeability in the renal collecting duct system by
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Urea accumulation in the renal inner medulla depends on passive exit of urea from the IMCDs to the inner
medullary interstitium. The retention of urea in the inner
medullary interstitium depends on recycling processes,
which return urea that would otherwise be lost to the
general circulation to the inner medulla. One recycling
pathway is classical countercurrent exchange, in which
urea absorbed from the ascending vasa recta in the inner
medulla enters the descending vasa recta (22). Additional
recycling pathways have been subsequently proposed
(Fig. 3) (123): 1) direct transfer of urea to the long loops
of Henle in the inner medulla, 2) uptake of urea in the
ascending vasa recta with transfer to the descending
limbs of short loops of Henle in the vascular bundles of
the renal outer medulla, and 3) transfer of urea from the
thick ascending limbs of the outer stripe of the outer
medulla and cortical medullary rays to the neighboring
proximal straight tubules. Some aspects of these recycling pathways have been addressed recently in mice in
which genes encoding the urea transporters UT-A2 and
UT-B have been deleted (see below).
II. MOUSE MODELS
A. Transport Proteins Involved in Urinary
Concentration and Dilution
Our understanding of renal physiology has accelerated in recent years as a result of the advent of the era of
“molecular physiology,” characterized by the development and application of tools to study proteins that can
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stimulating the insertion of aquaporin-2 (AQP-2) water
channels into the apical plasma membrane and by regulating AQP-2 gene expression (186). Furthermore, there
has been evidence in the older physiological literature
that the kidney may be capable of urine concentration in
the absence of vasopressin’s actions in the renal collecting duct (21), which, if true, raises hopes for more effective treatment of patients with X-linked nephrogenic diabetes insipidus (NDI). The development of mice in which
the renal vasopressin V2 receptor (V2R) has been deleted
have begun to address this possibility (see below). Another point of controversy derived from the “micropuncture era” (105) has been the relative importance of water
transport in the connecting tubule and various subsegments of the renal collecting duct in the urinary concentrating mechanism, with regard to renal water conservation. Mouse models in which the V2R or AQP-2 is selectively deleted from the collecting ducts and not the
connecting tubule are addressing this issue (see below).
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mediate renal function. These techniques have led to the
cloning of multiple cDNAs and genes that are thought to
be involved in urinary concentration and dilution. Figure 4
summarizes the renal tubule sites with abundant expression of aquaporins, urea transporters, and ion transporters/channels that are important to the urinary concentrating process. Several of these transporters are molecular
targets for vasopressin action and are also expressed in
low abundance in other renal tubule segments (Fig. 5).
Thus now that the proteins thought to be responsible
for the urinary concentrating mechanism have been identified (Fig. 4) and we are in the “gene-knockout era,”
several important questions remain to be answered in
renal physiology. This review summarizes the features/
phenotype of reported mouse models that are relevant to
the urinary concentrating mechanism with emphasis on
the controversies described above. These models can be
divided into two groups: 1) those in which water balance
appears to be selectively compromised as a result of
disruption of expression of proteins known or suspected
to be involved in the urinary concentrating mechanism,
and 2) those that result in water balance abnormalities
that are due in part to a destructive effect of the gene
deletion on renal structure or that are associated with
broader systemic effects.
B. Sodium Transporters and Channels
1. NHE3
The Na⫹-H⫹ exchanger (NHE3) is the major Na absorptive pathway in the proximal tubule. In addition to
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2. NKCC2
The renal Na⫹-K⫹-2Cl⫺ cotransporter (NKCC2; also
known as “BSC1”) is expressed in the TAL, where it is
localized to the apical membrane of epithelial cells (110,
187). NKCC2 is also expressed in the macula densa (187).
In rat, long-term increases in the circulating vasopressin
concentration result in an increase in NKCC2 protein
abundance in the TAL (118), which is associated with an
increase in the maximal urinary concentrating capacity
(119). In addition, NaCl absorption in the MTAL can be
increased by acute vasopressin administration (86). This
is thought to occur, in part, due to increased apical membrane expression of NKCC2 in association with phosphorylation of the NH2-terminal tail (76, 198).
NKCC2 knockout mice have been developed by standard gene targeting techniques (252). However, due to
perinatal fluid wasting and dehydration, the animals are
not viable and die prior to weaning. Treatment of these
mice with indomethacin and the administration of fluid
allowed some mice to survive until adulthood, although
the extreme polyuria, hydronephrosis, and growth retardation could not be abrogated. Studies of NKCC2 heterozygous animals showed essentially no difference from
wild-type mice (251).
Why does deletion of NKCC2 result in such a severe
phenotype, whilst deletion of NHE3, a transporter respon-
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FIG. 3. Pathways of urea recycling in renal medulla. Solid blue lines
represent a short-looped nephron (left) and a long-looped nephron
(right). Transfer of urea between nephron segments is indicated by
dashed red arrows labeled a, b, and c corresponding to recycling pathways described in the text. tAL, thin ascending limb; CD, collecting duct;
DCT, distal convoluted tubule; DL, descending limb; PST, proximal
straight tubule; TAL, thick ascending limb; vr, vasa recta. [Adapted from
Knepper and Roch-Ramel (123).]
the proximal tubule, NHE3 has also been immunolocalized to the outer medullary thin descending limb of Henle
and the TAL (219, 220). In the MTAL, NHE3 activity is
increased by hypotonicity via a phosphatidylinositol 3-kinase (PI-3-K)-dependent pathway, which is inhibited by
vasopressin working though cAMP (81, 82). Thus vasopressin has the net effect to inhibit NHE3 activity in the
MTAL.
NHE3 knockout mice have been developed and are
viable (233) (in contrast to NKCC2 knockout mice, see
below). Their renal phenotype is predominantly associated with the fact that NHE3 is the major Na entry pathway in the proximal tubule (Fig. 4). NHE3 null mice have
a marked reduction in proximal tubule fluid absorption
and a compensatory decrease in glomerular filtration rate
(GFR). This decrease in GFR is the result of activation of
the tubuloglomerular feedback (TGF) mechanism (153).
Metabolic cage studies revealed that with free access
to water, NHE3 null mice manifest a moderate increase in
water intake associated with lower urinary osmolalities
(average of 1,737 mosmol/kgH2O), although maximal urinary osmolality was not evaluated (4). In addition, NHE3
knockout mice have a marked decrease in renal NKCC2
expression, despite elevated plasma vasopressin levels
(36). In conclusion, NHE3 null mice have a mild urinary
concentrating defect that may be associated with a reduction in NKCC2 expression.
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FIG. 4. Major aquaporins, urea transporters, and ion transporters/channels that are important to the urinary concentrating and diluting process.
Figure depicts a schematic overview of a mammalian kidney tubule, showing the solute and water transport pathways in the proximal tubule (PT),
thin descending limb of Henle’s loop (tDL), thick ascending limb (TAL), distal convoluted tubule (DCT), cortical collecting duct (CCD), and inner
medullary collecting duct (IMCD). Tubule lumen side is always on the left side of the cell, whereas the interstitium is on the right side. Arrows
represent direction of movement.
sible for the majority of Na reabsorption in the kidney,
results in a viable mouse capable of maintaining extracellular fluid volume? The answer appears to be in the special role that NKCC2 plays in the macula densa in the
mediation of TGF. An intact TGF system in NHE3 knockout mice allows them to maintain a relatively normal
distal fluid delivery through a reduction in GFR. In contrast, NKCC2 mice cannot compensate in this manner
since the transporter is necessary for the TGF response to
occur. Thus, in NKCC2 knockout mice, the distal nephron
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will be exposed to a NaCl load that drastically exceeds its
absorptive capacity, leading to massive salt wasting and
osmotic diuresis.
3. NCC and ENaC
The thiazide-sensitive Na⫹-Cl⫺ cotransporter (NCC)
and the amiloride-sensitive Na⫹ channel (ENaC) are important targets for the action of aldosterone in the regulation of sodium excretion. Due to their interdependent
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ROBERT A. FENTON AND MARK A. KNEPPER
role, we discuss them together in this section. NCC has
been immunolocalized to the distal convoluted tubule
(DCT) (58, 206), whereas ENaC is predominantly expressed in the connecting tubule, initial collecting tubule,
and the cortical collecting duct (CCD) (85, 151). An increase in circulating vasopressin levels results in increases in abundance of both NCC and the ␤- and ␥-subunits of ENaC (53, 184). In addition, vasopressin acutely
increases Na absorption in the rat CCD by increasing
apical Na entry via ENaC (214, 227, 266), which is proposed to be due to vasopressin-induced trafficking of
ENaC-containing vesicles from intracellular stores to the
apical plasma membrane (240).
NCC knockout mice have a mild phenotype, with a
small decrease in blood pressure (234). On a normal diet,
they appear to have a normal urinary concentrating ability, but upon dietary potassium restriction, the NCC null
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C. Potassium Channels
1. ROMK
The ATP-sensitive, inward rectifier ROMK potassium
channel (Kir1.1) is expressed in the TAL, DCT, connecting
tubule, and collecting duct system, predominantly in the
apical plasma membrane (147, 166, 287). In the TAL,
ROMK plays a critical role in the process of active NaCl
transport and thus the urinary concentrating and diluting
mechanism. The abundance of ROMK in the TAL is increased with chronic vasopressin administration (52). In
the connecting tubule and collecting duct, ROMK is responsible for the process of potassium secretion, thus
regulating urinary potassium excretion and systemic potassium balance. The later process is strongly regulated
by vasopressin (266). This secretory process may be an
indirect consequence of vasopressin’s action to increase
Na entry via ENaC, which results in apical plasma mem-
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FIG. 5. Grid showing sites of expression of water channels, urea
transporters, and ion transporters important to the urinary concentrating process and their regulation by vasopressin.
mice develop hypokalemia and a consequent polyuria that
is associated with an apparent central defect in the regulation of vasopressin secretion (172). It is only after prolonged hypokalemia that the mice develop evidence of
NDI, resulting from a suppression of AQP-2 expression in
the collecting ducts.
Knockout of any of the three ENaC subunits results
in a severe phenotype, with the mice suffering from neonatal death (15, 95, 165). In the ␣-ENaC knockout mice
(95), early death appears to be due to failure to adequately
clear fluid from the pulmonary alveoli after birth, whereas
knockout of the ␤- and ␥-subunits of ENaC results in mice
that die from hyperkalemia and sodium chloride wasting
(15, 165). Interestingly, when ␣-ENaC expression was
deleted selectively from the collecting ducts, leaving intact ENaC expression in the renal connecting tubule and
nonrenal tissues, the mice were viable and exhibited only
a very mild phenotype with little or no inability to maintain fluid homeostasis in the face of salt or water restriction (217). In these mice, after water restriction, urine
osmolality was not different from wild-type controls; thus
it appears that Na absorption from the renal collecting
duct via ENaC does not appear to be necessary for urinary
concentration.
Taken together, NCC deleted only from the DCT or
ENaC deleted only from the collecting duct results in a
very mild phenotype, presumably because one can compensate for the other with regard to sodium balance. In
this respect, double knockout animals could be informative. At present, it remains unclear whether the severity of
the phenotype seen when any ENaC subunit is deleted
globally is due to the importance of ENaC in nonrenal
tissues or is related to the role of ENaC in the connecting
tubule, which is conserved in the collecting duct specific
␣-ENaC knockout mice (217).
KNOCKOUT MOUSE STUDIES OF URINARY CONCENTRATING MECHANISM
that even after a 24-h water deprivation, the knockout
mice were unable to concentrate their urine. This observed polyuria was insensitive to [deamino-Cys1, D-Arg8]vasopressin (dDAVP) administration, indicating NDI.
Clearance studies have shown that the fractional excretion of sodium, chloride, and urea are not different in
knockout mice. Taken together, these data indicate that
the polyuria observed in ClC-K1 null mice is water diuresis and not osmotic diuresis. Solute analysis of the inner
medulla of Clcnk1⫺/⫺ mice determined that the concentrations of urea, Na⫹, and Cl⫺ were approximately half
those of controls, resulting in a significantly reduced osmolality of the papilla (2). Unlike wild-type mice, the
accumulation of these solutes was not increased by water
deprivation.
In conclusion, Clcnk1⫺/⫺ mice showed that rapid
chloride exit from the tAL in the inner medulla is essential
for generating a hypertonic inner medullary interstitium.
As is pointed out by Knepper et al. (124), rapid solute exit
from the tAL is theoretically critical to any process that
concentrates solutes in the inner medulla because of a
need for avoidance of dissipation of solutes from the
inner medulla. Similarly, rapid Na⫹ and urea exit from the
descending limb is a necessity for any inner medullary
concentrating process (124), and the mechanisms involved in the exit of these entities will be an important
area of focus for future research.
E. Aquaporins
D. Chloride Channels
1. ClC-K1
ClC-K1 is a kidney-specific chloride channel that is
localized to the apical and basolateral plasma membranes
of the thin ascending limb of Henle’s loop (tAL) (271).
ClC-K1 expression in both the apical and basolateral
plasma membranes, and examination of its transport
properties, could explain why the tAL possesses an extremely high transepithelial chloride permeability compared with the TAL.
In 1999, Matsumura et al. (163) generated ClC-K1 null
mice (Clcnk1⫺/⫺) and have made use of this model to
examine the role of ClC-K1 in the urinary concentrating
mechanism. Microperfusion studies determined that there
was drastically reduced transepithelial chloride transport
in the tAL of knockout mice. Importantly however,
Clcnk1⫺/⫺ mice had no significant differences in the
plasma concentrations of Na⫹, K⫹, Cl⫺, and HCO⫺
3 and
pH values compared with controls, indicating that a lossof-function mutation of ClC-K1 does not result in hypokalemic alkalosis (unlike CLCNKB mutations, type III
Bartter syndrome). Physiological studies revealed that
Clcnk1⫺/⫺ mice had significantly greater urine volume
and lower urine osmolality compared with controls and
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In the kidney, to date, eight aquaporins have been
localized to various segments of the renal tubule. In this
review we focus only on the mouse models where gene
deletion results in either a urinary concentrating defect or
a reduction in transepithelial water transport (Fig. 6).
1. AQP-1
AQP-1 is localized to the apical and basolateral
plasma membrane of epithelial cells in the proximal tubule (189), where the majority of fluid filtered by the
glomerulus is reabsorbed by an active near-isosmolar
transport mechanism. AQP-1 is also expressed in the thin
descending limb of Henle’s loop (tDL) and the epithelium
of the descending vasa recta (DVR) (186, 188), nephron
segments thought to be involved in countercurrent multiplication and exchange. The constitutively high water
permeability of nephron segments expressing AQP-1 is
consistent with a lack of regulation of AQP-1 by vasopressin (259).
Verkman and colleagues (158) generated a knockout
mouse model of AQP-1 by targeted gene deletion. Compared with wild-type littermates, AQP-1 knockout mice
have a reduced urinary osmolality that is not increased in
response to water deprivation. Indeed, the urinary con-
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brane depolarization and an increase in the electrochemical driving force for K⫹ movement through ROMK (226).
Alternatively, vasopressin may regulate the open probability of ROMK, in a process mediated by the cystic fibrosis transmembrane conductance regulator (CFTR), a
cAMP responsive protein (136, 154).
ROMK knockout mice, developed by Lorenz et al.
(152), manifest early death associated with hydronephrosis and severe dehydration, consistent with the known
role of ROMK in active NaCl absorption in the TAL.
Approximately 5% of these mice survive the perinatal
period, but suffer from metabolic acidosis, hypernatremia, reduced blood pressure, polydipsia, polyuria, and an
impaired urinary concentrating ability. Furthermore,
whole kidney GFR is reduced, apparently as a result of
hydronephrosis, and the fractional excretion of electrolytes is elevated. Micropuncture analysis revealed that the
single-nephron GFR was relatively normal, absorption of
NaCl in the TAL was reduced and the TGF mechanism
was severely impaired. From these animals, a line of mice
has been derived that has a greater survival rate and no
hydronephrosis, although adults have higher water excretion rates (155). Interestingly, these mice do not exhibit
hyperkalemia, indicating that the connecting tubule
and/or collecting duct principal cells must be capable of
secreting K via some other pathway, presumably flowdependent, Ca2⫹-activated K channels referred to as
“maxi-K” channels (283).
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centrating defect is so severe in these mice that after 36 h
of water deprivation, the average body weight decreased
by 35% and serum osmolality increased to greater that 500
mosmol/kgH2O. The urinary concentrating defect observed in these mice is likely due to a combination of
different mechanisms. Schnermann et al. (229) used a
combination of isolated tubule microperfusion and freeflow micropuncture to define the role of AQP-1 in proximal tubule water transport and fluid reabsorption. These
studies determined that the transepithelial osmotic water
permeability (Pf) of isolated microperfused proximal tubule S2 segments was fivefold less in AQP-1 knockout
mice, indicating that the major pathway for osmotically
driven transepithelial water transport is through AQP-1.
In addition, the proximal fluid reabsorption rate in AQP-1
knockout mice was approximately half that observed in
control animals. Additional studies in AQP-1 knockout
mice (273) demonstrated that active transport in the absence of a high water permeability increased the transepithelial osmotic gradient in the proximal tubule to
⬃40 mosmol/kgH2O in contrast to the usual gradient of
⬃5 mosmol/kgH2O. However, micropuncture of distal
nephron segments revealed that the single-nephron glomerular filtration rate (snGFR) is reduced in AQP-1
mice, thus reducing distal fluid delivery to approximately the same level as in wild-type mice (229).
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F. Collecting Duct Aquaporins
The production of concentrated urine requires high
collecting duct water permeability, allowing for the osmotically driven movement of water from the lumen to
the interstitium. Transepithelial water transport across
the collecting duct epithelium is generally believed to
occur by a transcellular route with serial passage across
the apical and basolateral plasma membranes. The water
channels responsible for this transport appear to be
AQP-2 in the apical plasma membrane and a combination
of aquaporin-3 (AQP-3) and aquaporin-4 (AQP-4) in the
basolateral plasma membrane (186). Knockout models
have been developed to investigate the roles of these
aquaporins in the urinary concentrating mechanism.
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FIG. 6. Schematic overview of a mammalian kidney tubule, showing
the location of the major aquaporins involved in the urinary concentrating mechanism. See text for description.
If AQP-1 knockout mice have relatively normal distal
fluid delivery (despite defective proximal tubule water
reabsorption), why do they have much greater urinary
flow? It is likely that the answer to this lies in the role of
AQP-1 in countercurrent multiplication. As described earlier, AQP-1 is abundantly expressed in the tDL. The osmotic water permeability of tDLs from AQP-1 knockout
mice is ⬃10-fold reduced compared with control animals
(44), thus confirming the hypothesis that AQP-1 is the
major water channel in tDL and pointing to the possibility
that the countercurrent multiplier mechanism is impaired
as a result of deletion of AQP-1 and diminished water
absorption in the tDL. In addition, in isolated microperfused outer medullary DVRs from AQP-1 knockout mice,
osmotic water permeability was reduced by greater than
50-fold compared with controls (201). The impairment of
water transport in the vasa recta presumably results in a
defect in countercurrent exchange, with concomitant depletion of medullary solutes. Taken together, these results
indicate that the diminished urinary concentrating ability
observed in AQP-1 knockout mice is likely due to a reduced ability to generate and maintain a hypertonic medullary interstitium. This conclusion is supported by the
finding in AQP-1 null mice, based on tissue slice analysis,
that there is a profound decrease in inner medullary solute accumulation (M. A. Knepper and T. Pisitkun, unpublished data). Also consistent with this conclusion is the
finding that there is an almost complete lack of an increase in urine osmolality in AQP-1 null mice after the
administration of vasopressin (158).
In humans, AQP-1 encodes the Colton blood group
antigen (239). Consistent with the finding in knockout
mice, it was observed that Colton-null individuals are
unable to concentrate their urine as effectively as
“normal” subjects when challenged by water deprivation (120).
KNOCKOUT MOUSE STUDIES OF URINARY CONCENTRATING MECHANISM
1. AQP-2
FIG. 7. An overview of multiple pathways within a kidney collecting
duct cell that are involved in the urinary concentrating process. Vasopressin (AVP) binds to the type 2 vasopressin receptor (V2R) in the
basolateral plasma membrane. The GTP-binding protein Gs activates the
synthesis of cAMP via an adenylate cyclase (AC). Binding of cAMP to
the regulatory subunit of protein kinase A (PKA) then causes activation
of the catalytic subunit of PKA, resulting in release of calcium from
intracellular stores. This Ca2⫹ can further regulate other proteins, including calmodulin, protein kinase C (PKC-␣), cytosolic phospholipase
A2 (cPLA2), and nitric oxide synthase (NOS). Genetic deletion of individual components of these pathways in mice can result in a defective
urinary concentrating mechanism. See text for description.
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V2R-mediated signaling leads to the exocytic insertion of
AQP-2-bearing intracellular vesicles into the apical
plasma membrane. These vesicles appear to be recycling
endosomes rather than secretory vesicles (14). In addition, long-term exposure to vasopressin leads to an increase in AQP-2 synthesis via increased gene transcription, increasing the total abundance of AQP-2 in the cells.
These mechanisms ultimately allow an increase in water
reabsorption in the collecting duct.
AQP-2 plays an essential role in the urinary concentrating mechanism. This is underlined in clinical disorders
that result in defects in body water balance (232). AQP-2
is dysregulated in acquired NDI resulting from, for example, lithium treatment or hypercalemia-induced NDI (68,
141, 161). Furthermore, several mutations in the AQP-2
gene can cause the rare disorder of hereditary NDI (autosomal recessive or dominant NDI) (reviewed in Ref. 69).
These clinical disorders have been discussed in detail
elsewhere (223).
Despite the strong evidence implicating an essential
role of AQP-2 in the urinary concentrating mechanism, a
number of suitable mouse models to examine its function
have only recently been developed/discovered. In 2001, a
mouse knock-in model of AQP-2-dependent NDI was generated by inserting a T126M mutation into the mouse
AQP-2 gene by a Cre-loxP strategy (292). This mutation
has been shown to result in a failure of delivery of mature
AQP-2 protein to the apical plasma membrane. Although
the mutant mice appeared normal at birth, they failed to
thrive and generally died within 1 wk. Analysis of the
urine and serum revealed serum hyperosmolality and
low urine osmolality, trademark characteristics of a defective urinary concentrating mechanism. Forward genetic screening of ethylnitrosourea-mutagenized mice isolated another mouse model of NDI with a F204V mutation
(150). These mice survive beyond the neonatal period and
have a much milder form of NDI. Examination of these
mice could result in the identification of molecular chaperones that can correct similar forms of human NDI.
Two other mouse models have been developed that
allow the role of AQP-2 in the adult mouse to be examined. One model, developed by Rojek et al. (216), makes
use of the Cre-loxP system of gene disruption to create a
collecting duct specific deletion of AQP-2, leaving relatively normal levels of expression in the connecting tubule. Another model, developed by Yang et al. (294), with
inducible AQP-2 protein deletion in the kidney, was accomplished by tamoxifen-inducible Cre-recombinase expression in homozygous mice in which loxP sites were
introduced in introns of the mouse AQP-2 gene. The major
phenotype in both of these mice is severe polyuria, with
average basal daily urine volumes approximately equivalent to body weight (216, 294). The high urine output
observed resulted in very low urine osmolality, which was
not increased after water restriction. However, despite
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AQP-2 is abundantly expressed in all renal tubule
segments beyond the DCT, including the connecting tubule (connecting tubule cells), the cortical and outer medullary collecting duct (principal cells), and the IMCD
(IMCD cells) (185, 186). In these cell types, AQP-2 is
found in the apical plasma membrane, subapical vesicles,
and (especially in IMCD cells) in the basolateral plasma
membrane. AQP-2 can be regulated by vasopressin (shortterm and long-term) both by changes in transporter abundance (long-term regulation) and trafficking (short-term
regulation) (reviewed in Ref. 186). The mechanisms behind arginine vasopressin (AVP)-mediated trafficking of
AQP-2 have been discussed in detail elsewhere (reviewed
in Refs. 37, 180), but for the purposes of this review are
summarized as follows (see Fig. 7). Vasopressin binds to
the V2R in the basolateral plasma membrane, resulting in
activation of adenylate cyclase through V2R-coupled
GTP-binding protein Gs. This increases intracellular
cAMP levels and leads to activation of protein kinase A
(PKA), intracellular Ca2⫹ oscillations (297), activation of
myosin light-chain kinase (42) as well as cAMP-dependent, PKA-independent activation of the Rap-GEF Epac
(298). Through multiple (largely unknown) mechanisms,
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2. AQP-3
Xenopus oocyte expression studies have shown that
AQP-3 is not only permeable to water but functions efficiently as a glycerol transporter, thus making it a member
of the so-called aquaglyceroporins (56, 103, 293). In the
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kidney, AQP-3 is localized to the basolateral plasma membranes of the connecting tubule cells and collecting duct
principal cells in cortex and outer medulla (48, 54, 104).
AQP-3 is thought to mediate basolateral exit of water that
enters via AQP-2. Interestingly, AQP-3 is not abundant in
the cytoplasm, and there is no evidence for the short-term
regulation of AQP-3 by vasopressin-induced trafficking.
However, a marked increase in the abundance of AQP-3
mRNA and protein is observed during long-term vasopressin stimulation, such as seen during water deprivation
or vasopressin infusion in Brattleboro rats (54, 104,
176, 259).
AQP-3 knockout mice have been generated by targeted gene deletion and found to have a greater than
threefold reduction in osmotic water permeability of the
basolateral membrane of the CCD compared with wildtype control mice (156). AQP-3 null mice are markedly
polyuric (10-fold greater daily urine volume than controls), with an average urine osmolality of ⬍300 mosmol/
kgH2O. However, unlike AQP-1 or AQP-2 null mice, AQP-3
knockout mice are able to partially raise their urine osmolality after either water deprivation or the administration of dDAVP. Serum electrolyte concentrations from
AQP-3 null mice are not significantly different from controls, although plasma osmolality is mildly elevated. In
contrast to AQP-1 null mice, with a defective countercurrent exchange mechanism, the countercurrent exchange
in AQP-3 knockout mice is virtually intact.
What is the basis for the defective urinary concentrating mechanism in these mice? It is likely that when AQP-3 is
deleted, the reduced osmotic water permeability of the basolateral membrane results in a decrease in transepithelial
water permeability. Since the majority of vasopressin-dependent fluid reabsorption in the antidiuretic kidney is in the
connecting tubule and collecting duct, reduced water permeability is predicted to result in a hyposmolar urine. However, AQP-3 null mice are able to partially raise their urine
osmolality after water deprivation or vasopressin stimulation despite a relatively water-impermeable collecting duct
(156). This response is likely due to the fact that AQP-4 and
not AQP-3 is the predominant basolateral water channel in
the medullary parts of the collecting duct system, allowing a
normal response to vasopressin in the most distal portions
of the collecting duct system.
Another factor leading to increased urinary flow
rates in adult AQP-3 knockout mice is the striking distortion of medullary architecture that progressively destroys
the inner medulla and replaces it with a dilated renal
pelvis (hydronephrosis) (156). The mechanism of hydronephrosis in this model is unknown.
3. AQP-4
AQP-4 is localized to the basolateral plasma membrane of outer medullary collecting duct (OMCD) princi-
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the polyuria, with free access to water, plasma concentrations of electrolytes, urea, and creatinine are not different in knockout mice compared with controls, and
neither was the estimated GFR. Thus, despite having normal renal function (presumably normal active Na⫹ transport), there is a major defect in the urinary concentrating
mechanism in these mice. This defect confirms that
AQP-2 is responsible for the majority of transcellular
water reabsorption at the rate-limiting luminal membrane
of the collecting duct.
A mouse model has also been “discovered” that implicates an essential role of AQP-2 phosphorylation on
transporter function (164). This mouse model has a single
base change in codon 256 of AQP-2, resulting in a serine
to leucine amino acid substitution and loss of AQP-2
phosphorylation at amino acid 256. This mutation results
in an absence of AQP-2 accumulation in the apical plasma
membrane. Phenotypically, the mutant mice have no response to vasopressin and produce large quantities of
hypotonic urine, characteristic of NDI. This mouse model
provides direct genetic evidence that phosphorylation of
AQP-2 at S256 is essential for its apical membrane accumulation and maximal water reabsorption in the collecting duct.
The latest mouse model to study the role of AQP-2
has helped us to understand the molecular mechanisms
behind AQP-2-dependent autosomal-dominant nephrogenic diabetes (AD-NDI) insipidus (AD-NDI). Using a
“knockin” strategy, and based on previous studies identifying three frameshift mutations in the COOH terminus of
AQP-2 that result in AD-NDI (140), Sohara et al. (242)
have created a mouse model that expresses 76 amino
acids of the COOH terminus of a human AD-NDI mutant
AQP-2 (763–772del), fused to the “wild-type” 254 NH2terminal amino acids of mouse AQP-2. Mice that are
heterozygous for the mutation exhibited a severely impaired urinary concentrating ability, but after dehydration
were able to moderately increase their urine osmolality, a
milder phenotype indicative of AD-NDI compared with
autosomal-recessive NDI. Furthermore, the mutant AQP-2
was missorted to the basolateral plasma membrane and
formed heteroligomers with wild-type AQP2, resulting in
a dominant-negative effect on the normal apical sorting of
wild-type AQP-2. Additional studies in this mouse model
determined that the phosphodiesterase 4 inhibitor rolipram
was partially able to restore concentrating ability, indicating that phosphodiesterase inhibitors may be useful drugs
for the treatment of AD-NDI.
KNOCKOUT MOUSE STUDIES OF URINARY CONCENTRATING MECHANISM
4. AQP-7
AQP-7 is a member of the aquaglyceroporins and
effectively transports both glycerol and water (102). In
the kidney, AQP-7 is abundantly expressed on the apical
plasma membrane of the proximal straight tubules (101,
181). AQP-7 knockout mice have reduced water permeability in the proximal tubule brush-border membrane
(241). However, AQP-7 null mice do not exhibit a urinary
concentrating defect or water balance abnormality. Because AQP-1 has been shown to be the major water
transport pathway in the proximal tubule, it is not surprising that there is no abnormality in water excretion in
AQP-7 knockout mice. However, AQP-1/AQP-7 doubleknockout mice have been generated and showed a significantly greater urine output compared with AQP-1 null
mice, leading the authors to speculate that the amount of
Physiol Rev • VOL
water reabsorbed through AQP-7 in the proximal straight
tubules is physiologically substantial, although direct
measurements of proximal tubule water transport were
not reported (241). The greater water excretion observed
in the AQP-1/AQP-7 double-knockout mice compared
with AQP-1 solo-knockout mice was accompanied by a
proportional decrease in urine osmolality, suggesting that
the total excretion of osmolar substances was not altered
in these mice.
G. Urea Transporters
In mammals, ⬃90% of waste nitrogen is normally
excreted by the kidney as urea, the balance being attributable to ammonium and uric acid. The majority of this
urea is generated in the liver via the urea-ornithine cycle.
In humans and animals, under most circumstances, dietary protein intake greatly exceeds that necessary for the
support of anabolic processes; thus excess quantities of
urea are generated that need to be excreted. This urea
constitutes a large osmotic load to the kidney. Most solutes excreted in such large amounts, for example, mannitol (7), would obligate large amounts of water excretion
by causing an osmotic diuresis. However, under normal
circumstances, urea does not induce an osmotic diuresis.
Studies in the 1930s by Gamble et al. (71) demonstrated
“An economy of water in renal function referable to urea,”
which provided early evidence for a unique role of urea in
the urinary concentrating mechanism.
The process of urea accumulation in the medulla has
been thoroughly studied, and it is generally accepted that
urea accumulation is dependent on facilitated urea transport across the epithelium of the IMCD (125). This urea
transport process and regulation have been reviewed extensively (65, 221, 222) and thus are not covered here.
Central to urea transport within the kidney are UT-A and
UT-B facilitative urea transporters. Recently, three mouse
models have been created with selective deletion of different urea transporter isoforms. These mouse models
have shed new light on the role of urea in the urinary
concentrating mechanism.
1. UT-A1 and UT-A3
UT-A1 and UT-A3 are expressed exclusively in IMCD
cells. Immunochemical methods have localized UT-A1 to
the cytoplasm and apical region of the IMCD (66, 190),
whereas UT-A3 is localized both intracellularly and in the
basolateral membrane (248, 260) (Fig. 8). In 2004, we
developed a mouse model that allowed us to specifically
assess the role of inner medullary urea transport in kidney
function (63) by deleting both UT-A1 and UT-A3 by standard gene targeting techniques (UT-A1/3⫺/⫺ mice).
A) ROLE OF IMCD UREA TRANSPORTERS IN THE URINARY CONCENTRATING MECHANISM. Isolated perfused tubule studies
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pal cells and IMCD cells (258). AQP-4 is most abundant in
the inner medulla, with a gradual decrease in expression
towards the cortex (258). In mice, AQP-4 has also been
reported to be expressed in the basolateral plasma membrane of proximal tubule S3 segments (274). Renal medullary AQP-4 mRNA abundance was found to be increased
in response to water restriction (176) or vasopressin infusion (34), whereas there was no change in protein expression in response to either manipulation (259).
AQP-4 null mice have been generated by standard
gene deletion methods (157). Isolated perfused tubule
studies determined that there is a fourfold decrease in
IMCD osmotic water permeability in AQP-4 null mice
relative to wild-type mice, indicating that AQP-4 is responsible for most of the basolateral membrane water movement in this segment (45). Despite this reduced water
permeability in the IMCD, in hydrated mice, there was no
difference in urine osmolality compared with controls
and no difference in serum electrolyte concentrations.
However, there was a significant reduction in maximal
urine osmolality in AQP-4 null mice after a 36-h water
deprivation, and this reduced urine osmolality could not
be further increased by vasopressin administration, indicating a mild urinary concentrating defect (157). Why
does deletion of AQP-4 have such a profound effect on
water permeability of the IMCD, with only a modest decrease in urinary concentrating ability? The answer to this
is based on the normal distribution of water transport
along the collecting duct. Micropuncture studies performed under antidiuretic conditions demonstrated that
the amount of water reabsorbed osmotically in the late
distal tubule (connecting tubule plus initial collecting tubule) is much greater than that absorbed in the medullary
nephron segments (145). Thus, since AQP-4 in mouse is
expressed predominantly in the medullary collecting
ducts, deletion of AQP-4 results in only a mild defect in
kidney water absorption.
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ROBERT A. FENTON AND MARK A. KNEPPER
demonstrated a complete absence of phloretin-sensitive
and vasopressin-regulated urea transport in IMCD segments from UT-A1/3⫺/⫺ mice (63), thus providing confirmation of the role of these transport proteins in IMCD
urea transport. The prime conceptual framework outlining the proposed contribution of collecting duct urea
transport to the urinary concentrating mechanism is derived largely from a model of urea handling proposed in
the 1950s by Berliner et al. (22). They hypothesized that
luminal urea in the IMCD is osmotically ineffective because of a high IMCD urea permeability that, abetted by
countercurrent exchange processes, allows urea to accumulate to high concentrations in the inner medullary interstitium. Thus, although urea accumulates to 1 M or
more in the lumen, it accumulates to a similar concentration in the inner medullary interstitium, thereby preventing an osmotic diuresis. Indeed, UT-A1/3⫺/⫺ mice on either a normal protein (20% protein by weight) or highprotein (40%) diet had a significantly greater fluid intake
and urine flow than wild-type animals, resulting in a decreased urine osmolality (63, 64). However, UT-A1/3⫺/⫺
mice on a low-protein diet did not show a substantial
degree of polyuria. In this latter condition, hepatic urea
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FIG. 8. Localization of UT-A urea transporters in the mouse renal
tubule. A schematic representation of the mouse nephron is shown.
UT-A1 is localized to the terminal portion of the IMCD and is both
intracellular and in the apical domain. UT-A2 is localized to the thin
descending limbs of Henle’s loop in both the outer medulla and inner
medulla. UT-A3 is localized to the terminal portion of the IMCD and is
both intracellular and in the basolateral domains.
production is low and urea delivery to the IMCD is predicted to be low, thus rendering collecting duct urea
transport immaterial with regard to water balance. Studies investigating the maximal urinary concentrating capacity of UT-A1/3⫺/⫺ mice showed that after an 18-h
water restriction, mice on a 20 or 40% protein intake are
unable to reduce their urine flow to levels below those
observed under basal conditions, resulting in volume depletion and loss of body weight (64). In contrast, UT-A1/3⫺/⫺
mice on a 4% protein diet were able to maintain fluid
balance. Thus the concentrating defect in UT-A1/3⫺/⫺
mice is caused by a urea-dependent osmotic diuresis;
greater urea delivery to the IMCD results in greater levels
of water excretion. Overall, these results demonstrate
that the primary role of IMCD urea transporters in the
urinary concentrating mechanism is in their ability to
prevent a urea-induced osmotic diuresis.
B) ROLE OF IMCD UREA TRANSPORTERS IN SODIUM CHLORIDE
ACCUMULATION IN THE INNER MEDULLA. In 1959, Kuhn and
Ramel (139) proposed the classical countercurrent multiplier model, the basis for the urinary concentrating mechanism (see sect. I). The gradient that drives the countercurrent multiplier in the outer medulla, thus concentrating the urine, is dependent on the active reabsorption
of NaCl in the water-impermeable TAL. However, the
mechanism that concentrates NaCl in the inner medulla
interstitium and thus water reabsorption from the collecting ducts remains controversial, as the tAL is apparently
incapable of measurable rates of active NaCl transport
(97, 132). Various mechanisms have been offered to explain NaCl accumulation in the inner medulla (121, 124,
228, 261), with the most influential model independently
proposed by Stephenson (246) and by Kokko and Rector
in 1972. In this mechanism, known as the “passive countercurrent multiplier mechanism,” the rapid reabsorption
of urea from the IMCD generates a high urea concentration in the inner medullary interstitium, resulting in ureainduced water absorption in the descending limb of Henle
generating a transepithelial gradient for the passive reabsorption of NaCl from the tAL. In addition, if tAL urea
permeability is extremely low (almost zero), then NaCl
that has been reabsorbed from the tAL will not be replaced by urea and the ascending limb fluid will be dilute
relative to other nephron segments. This dilution process,
analogous to that observed in the outer medulla, is proposed to constitute a “single effect” that can be multiplied
by the counterflow between the ascending and descending limbs of Henle’s loops.
If the passive model of NaCl accumulation in the
inner medulla functions as proposed, abolition of rapid
passive urea absorption in the IMCD as seen in UT-A1/3⫺/⫺
mice would be expected to eliminate NaCl accumulation
in the inner medulla. However, two independent experiments in UT-A1/3⫺/⫺ mice failed to corroborate the view
that inner medullary NaCl accumulation depends on fa-
KNOCKOUT MOUSE STUDIES OF URINARY CONCENTRATING MECHANISM
2. UT-A2
Under normal conditions, UT-A2 mRNA and protein
are expressed in the inner stripe of the outer medulla (Fig.
8), where it is localized to the lower portions of the tDL of
short loops of Henle (66, 237, 278). Under prolonged
vasopressin action, UT-A2 mRNA and protein can also be
detected in the inner medulla, being localized to the tDL
of long loops of Henle. Multiple studies have shown that
UT-A2 mRNA and protein can be regulated in response to
changes in circulating vasopressin concentration (34, 66,
278), and recent studies have determined that UT-A2mediated urea transport can be acutely regulated by
cAMP (207). The presence of vasopressin receptors has
not been reported in the descending limb of Henle’s loop,
raising the possibility that the effects of vasopressin are
indirect, e.g., mediated by autacoids released by vasopressin-responsive cells.
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What is the predicted role of UT-A2 in the urinary
concentrating mechanism and why is the thin limb localization of UT-A2 important for the kidneys concentrating
ability? The large amount of urea that is reabsorbed from
the IMCD (via UT-A1 and UT-A3) is trapped in the inner
medullary interstitium by countercurrent exchange processes. Any urea that “escapes” the inner medulla via the
ascending vasa recta (AVR) can exit via the fenestrated
endothelium of the AVR. However, the close proximity of
the AVR and short-loop descending limbs in the vascular
bundles of the inner stripe theoretically can permit transfer of this urea from the AVR into the descending limbs
via UT-A2 (Fig. 3). This mechanism is predicted to be
important for the concentrating mechanism by ensuring
efficient recycling of urea, thus making urea available to
distal sites of short-looped nephrons. Evidence for urea
recycling was obtained from the micropuncture studies of
Lassiter et al. (145, 146), showing that there is a large
amount of net urea secretion into the short loops of
Henle, resulting in the delivery of large amounts of urea to
the superficial distal tubule that often exceeds the filtered
load. UT-A2 in the inner medullary portion of the tDL may
also recycle urea absorbed from the ascending thin limb
of Henle [which is highly permeable to urea (43) despite
the absence of any known urea transporter] and from the
AVR. The enhanced countercurrent exchange between
ascending and descending structures would help maintain
a high urea concentration in the inner medullary interstitium and thus the driving force for the osmotic extraction
of water.
UT-A2 knockout mice have recently been developed,
and some aspects of their renal phenotype have been
described (272). On a normal level of protein intake (20%
protein), the UT-A2 null mice do not have significant
differences in daily urine output compared with control
mice and, even after a 36-h period of water deprivation,
differences in urine output and urine osmolality are not
observed. Furthermore, UT-A2 knockout mice do not
have an impairment of urea or chloride accumulation in
the inner medulla (272). These results are surprising,
considering the role that UT-A2 has been proposed to play
in maintaining a high inner medullary urea concentration.
However, on a low-protein diet (4% protein), UT-A2 null
mice have a mildly reduced maximal urinary concentrating capacity compared with wild-type controls and a significant reduction in urea accumulation in the inner medulla.
The findings from these initial studies in UT-A2
knockout mice are surprising, especially considering the
strongly induced upregulation of UT-A2 under antidiuretic conditions. Clearly from these experiments, under
basal conditions, UT-A2 makes a minimal contribution to
urea accumulation in the inner medullary interstitium and
does not play a major role in the formation of concentrated urine. However, when urea supply to the kidney is
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cilitated urea transport in the IMCD. In an initial experiment, the mean urea, Na⫹, Cl⫺, and K⫹ concentrations
were measured in whole inner medulla tissue isolated
from water-restricted UT-A1/3⫺/⫺ mice and wild-type littermates (63). In UT-A1/3⫺/⫺ mice there was a significantly reduced inner medullary urea concentration. However, there were no reductions in the mean Na⫹, Cl⫺, or
K⫹ concentrations. In a second study, osmolality, urea,
and Na⫹ concentrations were measured in the cortex,
outer medulla, and two levels of the inner medulla from
UT-A1/3⫺/⫺ and wild-type mice (64) fed either a low- (4%)
or high-protein (40%) diet. In wild-type mice, changing the
dietary protein intake from 4 to 40% resulted in a greater
tissue osmolality that was attributable solely to a greater
accumulation of urea in the inner medulla. However, at all
levels of the corticomedullary axis, sodium concentrations were unaffected by changes in dietary protein intake. Furthermore, UT-A1/3⫺/⫺ mice had a substantially
attenuated corticomedullary osmolality gradient and no
urea gradient on either diet. Despite this difference in
urea concentration, the corticomedullary sodium gradients of UT-A1/3⫺/⫺ mice were virtually identical to wildtype mice. Thus marked medullary urea depletion resulting from either dietary protein restriction or deletion of
collecting duct urea transporters does not affect the ability of the kidney to form a corticomedullary sodium
chloride gradient. We conclude from these studies in
UT-A1/3⫺/⫺ mice that NaCl accumulation in the inner
medulla is not reliant on either IMCD urea transport or
the accumulation of urea in the IMCD interstitium. Thus
the passive concentrating model in the form originally
proposed by Stephenson and by Kokko and Rector, where
NaCl reabsorption from Henle’s loop depends on a high
IMCD urea permeability, is apparently not the mechanism
by which NaCl is concentrated in the inner medulla.
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limited, UT-A2 may be important for maintaining a high
concentration of urea in the inner medulla and thus maximal urinary concentrating ability.
enter erythrocytes via non-UT-B pathways as they enter
the medulla, but could not exit fast enough to equilibrate
with the surrounding interstitium as the erythrocytes are
carried back to the general circulation.
3. UT-B
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H. Receptors and Signaling Molecules
1. V2R
AVP is essential in the regulation of body fluid homeostasis. In response to small increases in plasma osmolality or a reduction in the effective circulating blood
volume, AVP is released by the posterior pituitary gland
and promotes water reabsorption in the kidney collecting
duct, enhanced urinary concentration in the TAL, and
vasoconstriction via four subtypes of receptors (93). The
antidiuretic effects of AVP result from a cascade of
events, initialized by the binding of AVP to the V2R in the
collecting duct and TAL (reviewed in Refs. 37, 186). In the
collecting duct, this binding causes activation of G-coupled proteins, a rise in intracellular cAMP, and eventually,
through several mechanisms, the exocytic insertion of
AQP-2 water channels into the apical plasma membrane
(Fig. 7). This dramatically increases water permeability
(typically 8- to 10-fold) and allows the osmotically driven
movement of water from the kidney tubule lumen into the
kidney interstitium, promoting water retention and
thereby lowering plasma osmolality. Physiological studies, receptor binding, and radioactive tracer studies have
shown that V2R is subject to internalization in response to
binding of vasopressin (28, 92, 98 –100). Internalization
may also be involved in the vasopressin escape phenomenon, a physiological adaptation to prevent water intoxication with prolonged vasopressin action (262).
Inactivating V2R mutations in humans cause a rare
kidney disease known as X-linked nephrogenic diabetes
insipidus (XNDI), an AVP-insensitive form of diabetes
insipidus that is inherited in an X-linked manner (223).
XNDI patients produce large volumes of dilute urine, are
polydipsic, and in the case of an inadequate water supply,
can become severely hypernatremic. Failure to recognize
the disease in affected boys soon after birth can result in
abnormalities of the central nervous system, owing to
severe dehydration.
In 2000, Yun et al. (300) created a mouse model of
XNDI by introducing a nonsense mutation (Glu242stop)
known to cause XNDI in humans into the mouse genome.
This particular mutation was chosen as it has been shown
that the encoded mutant receptor is retained intracellularly and completely lacks functional activity (231), thus
mimicking the functional properties of many other disease-causing V2R mutants.
Male V2R mutant mice (V2R⫺/y) typically died within
7 days after birth (300). Urine osmolalities, collected from
the bladders of 3-day-old pups, were significantly lower
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In contrast to the multiple UT-A isoforms described
above, the mouse UT-B gene encodes only a single protein. UT-B is expressed exclusively throughout the kidney
medulla in the basolateral and apical (luminal) regions of
the DVR endothelial cells (209, 268, 288). In humans, UT-B
protein carries the Kidd blood group antigen, and subjects
lacking this antigen [JK (a⫺,b⫺) individuals] have a dramatically reduced urea permeability of red blood cells
and a mild urinary concentrating defect (224). UT-B is
thought to contribute to the urinary concentrating mechanism by allowing any urea that “escapes” the inner medullary interstitium via the venous AVR, to be recycled
back into the DVR, thus not allowing this urea to return to
the general circulation. This complex intrarenal urea recycling between AVR and DVR, in combination with recycling of urea between UT-B and UT-A2 (in thin descending limbs of Henle’s loop), is thought to maintain the high
inner medullary interstitium urea concentration and thus
the driving force for passive water reabsorption.
In 2002, Yang et al. (291) developed a mouse model
with genetic deletion of UT-B that has enabled the role of
UT-B in the urinary concentrating mechanism to be thoroughly investigated. However, since the physiology of the
UT-B knockout mice has been recently discussed very
extensively elsewhere (290), for the purposes of this article only a brief overview is given. Erythrocytes from
UT-B knockout mice have an ⬃45-fold lower urea permeability compared with those from controls. Under basal
conditions (normal protein diet), daily urine output in
UT-B null mice is significantly higher and urine osmolality
significantly lower compared with wild-type mice. However, when UT-B knockout mice are subjected to water
deprivation for 36 h, they are able to concentrate their
urine, although to a lesser extent than controls. Knockout
mice have a significantly lower urine urea concentration
(although in same proportion to other solutes) and significantly higher plasma urea; thus their urine-to-plasma
urea ratio (U/P) is more severely reduced than that of
other solutes. This reduced capacity to concentrate urea
compared with other solutes indicates that the UT-B null
mice have a “urea-selective” urinary concentrating defect
(13). This diminished ability to concentrate urea is highlighted by a lower inner medullary urea concentration
compared with other solutes. Taken together, these data
indicate that the urinary concentrating defect observed in
UT-B null mice is due to impaired urea recycling in the
vasa recta, although at present, it is unclear whether the
loss of urea transport in red blood cells also contributes
to this phenomenon. The latter could occur if urea can
KNOCKOUT MOUSE STUDIES OF URINARY CONCENTRATING MECHANISM
2. Bradykinin B2 receptor
Kinins are produced by proteolytic cleavage of a
protein precursor, kininogen, by the enzyme kallikrein.
They act as “local hormones” by activating the release of
endothelium-derived relaxing factor and prostaglandins
(39). Kinins act through a family of G protein-coupled
receptors, the B1 and B2 receptors, with the B2 receptor
mediating vasodilation, diuresis, and natriuresis (213). In
the kidney, kallikrein is localized to the distal nephron
including the DCT, connecting tubule, and initial collecting tubule (210). It has been proposed that kinins are
formed chiefly in the lumen of the collecting ducts (276).
However, in isolated perfused tubule experiments, bradykinin is only effective in decreasing vasopressin-stimulated NaCl and water transport when added to the peritubular environment and not when added to the lumen
(265, 266).
It has been proposed that renal kinins counteract the
hydrosmotic effect of AVP, and tubule perfusion studies
have shown that kinins added to the peritubular bath can
decrease AVP-stimulated water reabsorption in the cortical collecting duct (235). Furthermore, in Brattleboro
rats, the kallikrein inhibitor aprotinin augments the renal
response to AVP (113, 114).
Bradykinin B2 receptor knockout mice have been
developed and their renal phenotype partially characterized (3, 27). During basal conditions there is no significant
difference in either urine volume or urine osmolality between knockout mice and controls, suggesting that renal
kinins play little role in regulation of water excretion
under normal conditions. However, after a 24-h fluid restriction, urine volume was significantly lower and urine
osmolality significantly higher in knockout mice. Furthermore, subcutaneous administration of dDAVP results in a
significantly greater increase in urine osmolality in knockout mice compared with controls. Taken together, these
results suggest that water restriction or V2-receptor stimulation has a greater urinary concentrating effect in B2
Physiol Rev • VOL
receptor knockout mice than in controls, suggesting that
endogenous kinins acting through B2 receptors oppose
the antidiuretic effect of AVP in vivo (33).
3. Integrin ␣1␤1
Integrins are transmembrane receptors for extracellular matrix components and are thought to play an important role in regulating cytoskeletal organization (208),
as well as in the src-dependent activation of mitogenactivated protein (MAP) kinases (84). One member of the
integrin family, integrin ␣1␤1, acts as a collagen binding
receptor and is highly expressed in the glomerulus and
along the renal tubule (137, 277). Studies of integrin ␣1
null mice have shown that integrin ␣1␤1 plays a regulatory
role in the hypertonicity-mediated accumulation of osmolytes in the kidney inner medulla (168). Integrin ␣1
knockout mice have reduced osmolyte accumulation under control, diuretic, and antidiuretic conditions. These
changes are probably caused by a reduced expression of
TonEBP, a result of impaired signaling pathways associated with reduced p38 activation and increased ERK activation. In addition, under basal conditions, urine osmolality is significantly reduced in integrin ␣1 null mice
compared with controls, although maximal urinary concentrating ability is not affected (168). The mechanism
behind this concentrating defect is not understood. We
speculate that it may be due, at least in part, to a reduced
expression of other proteins involved in the urinary concentrating mechanism (see sect. IIK1).
4. Heterotrimeric G protein subunit, Gs␣
Heterotrimeric G proteins function in signal transduction pathways as molecular switches. Each G protein
is composed of ␣-, ␤-, and ␥-subunits, the products of
separate genes. The ␣-subunit binds guanine nucleotides
and interacts with specific receptors and effectors (243,
244). The ␣-subunit of Gs (Gs␣) is ubiquitously expressed
and is known to couple multiple receptors to the stimulation of adenylyl cyclase and specific ion channels (see
Fig. 7). For example, in the kidney, vasopressin binds to
the V2R in the basolateral plasma membrane, resulting in
the activation of adenylyl cyclase through the V2R-coupled Gs. Adenylyl cyclase activation increases intracellular cAMP levels and ultimately increases water reabsorption in the collecting duct (see above and Ref. 37).
Gs␣ knockout (GSKO) mice have been created by
targeted disruption of the Gnas gene (299). Homozygote
knockout mice do not develop beyond the embryonic
stage, but heterozygotes survive and manifest an interesting phenotype that is dependent on whether the mutant
gene is derived from the mother (m⫺/p⫹) or father
(m⫹/p⫺). This phenotypic variability is due to genomic
imprinting (281). Heterozygotic mice have a significantly
reduced expression of Gs␣ protein abundance in the outer
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than controls. Serum electrolyte analysis revealed that
V2R⫺/y pups have increased Na⫹ and Cl⫺ levels, indicative
of a severe state of hypernatremia. In control mice, an
intraperitoneal injection of dDAVP resulted in a significant increase in urine osmolality, whereas no effect was
observed in V2R⫺/y mice. Analysis of adult female V2R⫹/⫺
mice revealed that the mice have polyuria, polydipsia, and
a reduced urinary concentrating ability, clear symptoms
of XNDI, although milder than in hemizygous males. Furthermore, females have an ⬃50% decrease in total AVP
binding capacity, resulting in an ⬃50% decrease in vasopressin-induced intracellular cAMP levels. Taken together, the results obtained from this loss-of-function mutation in the V2R clearly show that the V2R is necessary
for normal regulation of water excretion.
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ROBERT A. FENTON AND MARK A. KNEPPER
5. Protein kinase C
Protein kinase C (PKC) is a member of a family of
closely related kinases that phosphorylate serine or threonine residues in proteins (183). It acts on numerous
intracellular proteins and influences multiple cellular
functions such as protein expression, ion transport, hormone release, and regulation of receptor proteins (183).
The PKC family consists of 10 different isozymes that are
classified according to their primary structure: conventional (␣, ␥, ␤I, ␤II), novel (␦, ⑀, ␩/L, ␪), and atypical
(␨, ␫/␭). Several of these isoforms are expressed in the
kidney (50, 59, 109, 111, 204, 212), where they have been
proposed to play a role in vascular and glomerular function, renin gene transcription, and tubular transport (5, 17,
23, 46, 88, 296).
PKC-␣ is predominantly expressed in the kidney,
where it is localized to the glomerulus, CCD intercalated
cells, and the medullary collecting duct (212). In the
Physiol Rev • VOL
IMCD, PKC-␣ is not directly activated by vasopressin as
judged by translocation assays (46), but is regulated indirectly by intracellular calcium (Fig. 7). Insights into the
role of PKC-␣ in kidney function have been uncovered by
the use of PKC-␣ knockout mice (PKC␣-KO) (148, 296).
Under basal conditions (free access to water), PKC␣-KO
mice have a greater urinary flow rate and lower urinary
osmolality, associated with a greater urinary vasopressinto-creatinine ratio. In addition, the significantly lower urinary osmolality in PKC␣-KO mice persists after a 36-h
water deprivation. Taken together, these results indicate
that the lower urinary concentrating ability in PKC␣-KO
mice is independent of water intake. Despite the obvious
phenotype, the precise role of PKC-␣ on urinary concentrating ability remains undetermined. However, it seems
likely that the effect is in the collecting duct or thick
ascending limb, the segments most centrally involved in
the urinary concentrating process. Studies directly assessing abundance, localization, and phosphorylation state of
the major transporters involved in the concentrating
mechanism in the knockout mice are needed to determine
the mechanism of the effect of deletion of PKC-␣ on water
excretion.
6. Serine/threonine phosphatase, calcineurin A␣
The calcium- and calmodulin-dependent serine/threonine protein phosphatase calcineurin has been implicated in numerous cellular processes and Ca2⫹-dependent signal transduction pathways (218). Calcineurin is a
heterodimeric protein composed of the catalytic subunit
calcineurin A and a Ca2⫹-binding regulatory subunit, calcineurin B. The A subunit can exist as three closely related forms: ␣, ␤, and ␥. In the kidney, the proximal
tubules and collecting ducts express predominantly the
␣- and ␤-isoforms, with the distal tubules containing the
highest calcineurin activity corresponding to the ␣-isoform (269). Calcineurin A␣ (CN-A␣) colocalizes with
AQP-2 in the collecting duct and has been proposed to be
involved in the vasopressin-stimulated AQP-2 trafficking
mechanism (80), further emphasized by recent studies in
CN-A␣ knockout mice.
Compared with wild-type controls, CN-A␣ knockout
mice (79) have a higher serum osmolality, a reduced urine
osmolality, and an impaired response to concentrate their
urine after vasopressin administration. Furthermore,
abundance of a phosphorylated form of AQP-2 (S256),
known to be required for release of AQP-2 from the
endoplasmic reticulum (ER) (186), is decreased compared with controls, resulting in minimal accumulation of
AQP-2 in the apical plasma membrane. Taken together,
these results show that calcineurin is required for normal
phosphorylation of AQP2, and loss of calcineurin activity
disrupts AQP-2 trafficking. The mechanism behind these
seemingly paradoxical results is presently unclear, but
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medulla, resulting in reduced glucagon-stimulated cAMP
production (55). In addition, NKCC2 protein abundance is
significantly less in the outer medulla of GSKO mice, as is
the abundance of type VI adenyl cyclase. In response to a
single intramuscular injection of dDAVP, GSKO mice have
a significantly lower urine osmolality compared with controls. However, after thirsting for 48 h, there was no
significant difference in outer medullary NKCC2 abundance or urine osmolality compared with wild-type mice,
suggesting that the impairment of outer medullary NKCC2
expression in GSKO mice could be overcome by a chronic
increase in circulating vasopressin (55). Taken together,
these results provide additional evidence for the importance of NKCC2 abundance in the TAL of the outer medulla of rodents as a determinant of urine concentrating
capacity, due to its involvement in countercurrent multiplication by the loop of Henle.
Recently, another mouse model has been developed
that further implicates an essential role of Gs␣ in the
vasopressin signaling cascade and ultimately the urinary
concentrating mechanism. Mice that express Cre recombinase in the kidney medulla (Cre recombinase driven by
the renin promoter) were crossed with mice in which
exon 1 of the Gnas gene was “floxed,” resulting in mice
with deletion of Gs␣ in the medullary collecting duct (41).
These mice had reduced basal concentrating ability that
did not respond to vasopressin, indicative of NDI.
Interestingly, human patients with heterozygous inactivating mutations of the Gs␣ gene (pseudohypoparathyroidism type Ia) do not have a decreased urinary
concentrating capacity after overnight fluid restriction or
in response to exogenous vasopressin (173). This suggests that the impairment of countercurrent multiplication in humans may be less severe than in mice with
analogous mutations.
KNOCKOUT MOUSE STUDIES OF URINARY CONCENTRATING MECHANISM
calcineurin may be involved in dephosphorylating AQP-2
during transit from the ER to the Golgi as previously
proposed.
7. Nitric oxide synthase
Physiol Rev • VOL
can promote the membrane insertion of AQP-2 into the
apical membrane (reviewed in Ref. 37); however, in isolated perfused CCD tubules, NO has been demonstrated
to inhibit vasopressin-stimulated osmotic water permeability (72). Furthermore, in isolated perfused IMCD tubules, cGMP reduces vasopressin-stimulated water permeability (191), and in the TAL, NO has a strong effect to
increase the expression of NKCC2 (270). In summary, we
speculate that the decreased concentrating ability in the
n/i/eNOS⫺/⫺ mice could be due to the loss of the stimulatory effect of NO on NKCC2 expression and the consequent reduction in countercurrent multiplication in the
outer medulla.
8. Endothelin-1
Within the kidney, the collecting duct is the major
site of endothelin-1 (ET-1) synthesis (127), where it is
thought to be an autocrine inhibitor of AVP-stimulated
water reabsorption (similar to bradykinin). Several studies, performed in vitro, have shown that exogenous ET-1,
through activation of the ETB receptor, reduces AVPstimulated water permeability in the CCD (264) and can
inhibit AVP-stimulated cAMP accumulation (57, 129) and
osmotic water permeability in the IMCD (177, 193). Due to
the effects of ET-1 on the renal vasculature (alteration of
renal plasma flow and GFR), it has proven to be difficult
to assess the role of ET-1 in regulating CD water permeability in vivo. However, low doses of systematically administered ET-1, which do not significantly alter renal
hemodynamics, have been shown to increase water excretion (77, 230).
Recently, studies in mice with CD-specific knockout
of ET-1 (CD ET-1 KO) have provided direct evidence for
a role of ET-1 in regulating renal water excretion (74).
Under basal conditions, CD ET-1 KO mice have a normal
urine volume, urine osmolality, and plasma Na⫹ concentration and osmolality. However, plasma AVP concentration is reduced by 35% in CD ET-1 KO mice. Knockout
mice also have an impaired ability to excrete an acute but
not a chronic water load. Administration of dDAVP results in greater increases in urine osmolality, V2R and
AQP-2 mRNA, and AQP-2 phosphorylation in CD ET-1 KO
mice compared with controls. Furthermore, IMCD suspensions isolated from CD ET-1 KO mice have enhanced
AVP and forskolin-stimulated cAMP accumulation. Taken
together, these results show that the CD ET-1 KO has an
increased renal sensitivity to the urinary concentrating
effects of AVP, indicating that ET-1 functions as an autocrine regulator of AVP action in the CD. Why absence of
ET-1 results in these defects in the urinary concentrating
mechanism is unknown; however, several possible explanations have been suggested. One explanation is that a
reduction in ET-1 results in reduced activation of the ETB
receptor and a lack of an ETB receptor-mediated down-
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Nitric oxide (NO) is a short-lived gaseous free radical
that passes freely through plasma membranes and produces multiple intracellular actions, either directly by
nitrosylation of proteins or by means of a soluble guanylate cyclase cGMP-mediated pathway (29, 78, 96, 169)
(Fig. 7). NO is formed from L-arginine by three distinct
members of the NO synthase (NOS) family: neuronal
(nNOS; also known as NOS1), inducible (iNOS; also
known as NOS2), and endothelial NOS (eNOS; also
known as NOS3). All three NOS isoforms are expressed in
the kidney; nNOS and eNOS are localized mainly in renal
tubules, collecting ducts, and glomeruli [nNOS is abundantly expressed in macula densa cells, and its expression
is stimulated in conditions with high levels of renin (174,
200)], whereas iNOS is found predominantly in renal tubules and collecting ducts (133–135).
Mouse models with deletion of single or double NOS
isoforms have been extensively studied, but because of
the substantial compensatory interactions among the
NOS isoforms, the role of NO in the kidney has been
difficult to assess (reviewed in Refs. 94, 162, 175). However, elegant studies by Morishita et al. (171) using triple
NOS-deleted animals have proven to be extremely informative in our understanding of the role of NO in water
homeostasis. These so-called “n/i/eNOS⫺/⫺” mice have
markedly reduced survival and fertility rates. In addition,
they are polydipsic and excrete a large volume of hypotonic urine. Although plasma vasopressin levels are not
different from controls, n/i/eNOS⫺/⫺ mice have a reduced
antidiuretic response to exogenous vasopressin and reduced vasopressin-induced cAMP production in the collecting ducts, leading to decreased amounts of AQP-2 in
the plasma membrane of collecting duct cells. Over a
period of time, the n/i/eNOS⫺/⫺ mice develop structural
abnormalities in glomeruli and renal tubules. Taken together, the phenotype of the n/i/eNOS⫺/⫺ mice closely
resembles NDI. At present, the cause of the defective
urinary concentrating mechanism in the n/i/eNOS⫺/⫺
mice is unknown. Importantly, however, the occurrence
of the functional abnormalities preceded that of structural abnormalities. Previous studies have shown that
(consistent with the knockout mice results) NO stimulates cAMP production by means of cGMP-dependent
activation of adenylate cyclase in isolated rat kidney,
presumably in the proximal tubule (90). In addition, it is
also conceivable that NO increases cAMP accumulation
by interfering with cAMP degradation through cGMPregulated cAMP phosphodiesterase activity (18). In the
collecting duct, there is morphological evidence that NO
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ROBERT A. FENTON AND MARK A. KNEPPER
regulation of cAMP production, thus increasing water
permeability. Another explanation is that a lack of ET-1
results in decreased levels of collecting duct NO production, which in itself has been shown to decrease AVPstimulated water permeability in isolated cortical CD (see
above and Ref. 72). However, recent data suggest that
neither ET-1-mediated NO production or NO donors inhibit AVP-stimulated cAMP accumulation, suggesting that
NO does not mediate ET-1 inhibition of IMCD cAMP
production (249).
9. Endothelin A receptor
I. Prostaglandins
1. Group IV cytosolic phospholipase A2
Cytosolic phospholipase A2 (cPLA2) is a member of
the phospholipase A2 (PLA2) family, a heterogeneous
group of enzymes that are involved in lipid digestion,
phospholipid membrane remodeling, and signal transduction (25). cPLA2 catalyzes the first step in eicosanoid
Physiol Rev • VOL
2. PGE2 E-prostanoid receptors, EP1 and EP3
PGE2 is a locally acting autacoid produced from the
enzymatic metabolism of arachidonic acid (see above and
Ref. 25). In the kidney, PGE2 is the predominant cyclooxygenase metabolite and the major prostanoid product
excreted in the urine. PGE2 has several known effects in
the kidney, including inhibiting the actions of vasopressin. For example, in the CCD, PGE2 inhibits cAMP production and reduces vasopressin-stimulated water reabsorption (32). The effects of PGE2 are mediated by four
classes of PGE2 E-prostanoid (EP) receptors (EP1
through EP4) (30); cell surface receptors that belong to a
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The mechanism by which ET-1 exerts its effects on
the urinary concentrating mechanism (described above)
is not fully understood, but it is thought to result from an
initial interaction with either of the two ET receptors
expressed in the kidney collecting duct, ETA and ETB (47,
128, 197, 257). In vitro, exogenous ET-1 inhibition of AVP
is mediated through the ETB receptor (57, 129, 264). However, whether the ETA receptor plays a role in regulation
of water excretion in vivo is a subject of controversy. This
controversy, in part, is augmented by a relative lack of
ETA receptors compared with ETB receptors in collecting
duct cells (⬃1:4, ETA;ETB in CD) (57). A mouse model
with selective deletion of the ETA receptor in the CD (CD
ETA KO mice) has been utilized to address this controversy (75).
CD ETA KO mice have no differences in urinary
sodium excretion, plasma aldosterone, plasma renin activity, or systemic blood pressure (75). Under basal conditions, CD ETA KO mice have a normal water intake,
urine volume, and urine osmolality, but their plasma AVP
concentration is increased greater than twofold compared with controls. In contrast to CD ET-1 KO mice (74),
CD ETA KO mice have a greater ability to excrete an
acute, but not a chronic, water load, and IMCD suspensions from CD ETA KO mice have a reduced AVP- and
forskolin-stimulated cAMP accumulation. Taken together,
these data suggest that activation of the CD ETA receptor
opposes the diuretic effect of ET-1, most likely through
increased sensitivity to AVP-induced cAMP accumulation.
Furthermore, these results suggest that ET-1 exerts its
diuretic actions through either activation of the CD ETB
receptor or through paracrine effects.
synthesis (Fig. 7), the release of arachidonic acid from
membrane phospholipids (149). Eicosanoids (e.g., prostaglandins) are thought to play key roles in the kidney,
including regulation of sodium and water excretion, renin
secretion, and the regulation of renal blood flow and GFR
(73, 83, 178). cPLA2 is activated by increases in intracellular calcium, which results in its translocation to intracellular membranes including the nuclear membrane.
To evaluate the physiological role of cPLA2 in the
kidney, physiological studies have been performed on
cPLA2 knockout mice (cPLA2⫺/⫺) (26, 51). cPLA2⫺/⫺ mice
have reduced urinary prostaglandin E2 (PGE2) excretion
under both basal conditions and after furosemide treatment, suggesting that a primary source of the arachidonic
acid used for renal production of PGE2 is cPLA2. Despite
evidence that prostaglandins produced in the renal cortex
can modulate GFR and renal blood flow (73), cPLA2⫺/⫺
mice have a normal GFR, and their fractional excretion of
Na⫹ and K⫹ is not different from controls. Urine osmolality in cPLA2⫺/⫺ mice is significantly lower than controls,
both under basal conditions and after 48 h of water deprivation. Water deprivation also results in a significantly
greater increase in plasma osmolality and a greater percentage loss in body weight in cPLA2⫺/⫺ mice. Vasopressin does not correct this urinary concentrating defect.
cPLA2⫺/⫺ mice have normal expression of the collecting
duct aquaporins AQP-2, AQP-3, and AQP-4, but immunofluorescence staining revealed a marked reduction in
staining of apical membrane AQP-1 in proximal tubules.
This reduced immunostaining may be due to abnormal
trafficking of AQP-1 or abnormal protein folding or protein-protein interactions. However, AQP-1 expression appeared to be normal in the tDL. Thus the concentrating
defect is most likely via a different mechanism than seen
in AQP-1 knockout mice in which the concentrating defect is believed to be due to reduced AQP-1 expression/
function in the descending limb of Henle (158). AQP-1
expression in vasa recta was not investigated in the
cPLA2⫺/⫺ mice, and we speculate that the concentrating
mechanism could be due to reduced AQP-1 expression in
the DVR.
KNOCKOUT MOUSE STUDIES OF URINARY CONCENTRATING MECHANISM
ance (107, 192). In overview, renin is secreted by the
kidney and cleaves angiotensinogen to angiotensin I,
which is converted by the angiotensin converting enzyme
(ACE) to angiotensin II. Angiotensin II binds to AT1 and
AT2 receptors in kidney to produce its physiological effects. The RAAS system is involved in regulating renal
tubular functions, e.g., modulating sodium/water reabsorption, regulating the renal microcirculation, and regulating aldosterone production by the adrenal glands (275).
1. Renin 1C (Ren1c)
In several mouse strains often used for genetic manipulation studies (e.g., SV129), two renin genes lie in
tandem on the same chromosome (Ren2 and Ren1d),
complicating the interpretation of experimental data (238,
289). However, a mouse model has recently been developed with genetic deletion of the renin gene in the
C57BL/6 strain of mice that, like humans, express only
one renin gene (Ren1c). This model has allowed the function of renin in the urinary concentrating mechanism to
be examined (253).
Ren1c knockout mice (Ren1c⫺/⫺) have no renin
mRNA expression in the kidney, and plasma renin is
undetectable (253). ANG I and ANG II are also undetectable in plasma, whereas daily urinary aldosterone excretion is ⬍10% of the levels observed in wild-type mice. As
predicted, Ren1c⫺/⫺ mice have a lower blood pressure
compared with controls. Structural analysis of kidneys
from Ren1c⫺/⫺ mice revealed thickening of renal arterial
walls, fibrosis, and severe hydronephrosis with marked
atrophy of the renal medulla. Furthermore, only 20% of
homozygote pups live to adulthood, with the remainder
dying of dehydration within a few days of birth. Under
basal conditions, Ren1c⫺/⫺ mice have a greater daily water intake, fivefold greater urine volume, and a lower urine
osmolality compared with controls. After 12 h of water
deprivation, knockout mice lose ⬎20% of their body
weight and are still unable to concentrate their urine. The
vasopressin analog dDAVP has no effect on urine osmolality in Ren1c⫺/⫺ mice, whereas it increases osmolality
severalfold in wild-type mice. Minipump infusion of ANG
II to Ren1c⫺/⫺ mice restores blood pressure to wild-type
levels, but does not restore the kidney’s urinary concentrating ability.
Taken together, the NDI observed in the Ren1c⫺/⫺
mice is likely due chiefly to the medullary atrophy and
hydronephrosis that they develop. The cause of these
structural abnormalities is unknown, but it seems possible that the renin-angiotensin system plays an important
role in renal development.
J. Renin-Angiotensin-Aldosterone System
2. Angiotensinogen
The renin-angiotensin-aldosterone system (RAAS)
plays a critical role in maintaining fluid-electrolyte bal-
Angiotensinogen (Atg) null mice have been created
independently in two different laboratories, and studies of
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large family of G protein-coupled receptors. Each EP
receptor not only selectively binds PGE2, but also preferentially couples to different signal transduction pathways,
including activation of phosphatidylinositol hydrolysis
(EP1 receptor), stimulation of cAMP generation, via Gq
(EP2 and EP4 receptors), and inhibition of cAMP generation, via Gi (EP3 receptors).
In the kidney, the EP1 receptor is localized to the
connecting tubule and throughout the collecting duct (16,
170), where it can modulate phosphatidylinositol and intracellular calcium concentrations via the G-coupled protein Gq. The role of EP1 receptors in urinary concentrating
processes has been examined by the use of EP1 knockout
mice (EP1⫺/⫺) (116). Despite reduced urinary osmolality
and reduced medullary interstitial osmolality after water
deprivation, EP1⫺/⫺ mice responded equivalently to wildtype control mice in concentrating their urine following
dDAVP administration. These data, coupled with reduced
urinary AVP levels and equivalent AQP-2 trafficking compared with controls, suggest that PGE2 modulates urine
concentration via the EP1 receptor, not in the kidney
collecting duct, but by promoting AVP synthesis in the
hypothalamus.
The EP3 receptor is highly expressed in the medullary and cortical thick ascending limbs (254 –256), as well
as the cortical and medullary collecting duct (31, 250),
where it couples to Gi proteins and inhibits adenylyl
cyclase activity (Fig. 7). The role of EP3 receptors in renal
function has been examined by the use of EP3 knockout
mice (EP3⫺/⫺) (67). EP3 knockout mice have a normal
GFR and renal blood flow, suggesting that EP3 receptors
do not have a primary role in regulating hemodynamics in
the whole kidney. Under basal conditions, with free access to water, EP3⫺/⫺ mice have a similar water intake,
urine volume, and urine osmolality compared with controls. However, after administration of the cyclooxygenase inhibitor indomethacin, wild-type mice significantly
increased their urine osmolality, whereas EP3⫺/⫺ mice did
not, suggesting that under basal conditions, PGE2 modulates the urine osmolality through the EP3 receptor.
Despite evidence to suggest that EP3 receptors mediate the effects of PGE2 to inhibit the actions of vasopressin in the kidney, both water restriction and dDAVP
administration had a similar effect on urinary concentrating function in knockout mice and controls. Taken together, these results suggest that the EP3 receptor is not
essential for the normal regulation of urinary concentrating mechanisms in response to various physiological stimuli and that EP3-mediated effects of PGE2 on urinary
concentration are not directly dependent on vasopressin.
1101
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ROBERT A. FENTON AND MARK A. KNEPPER
3. ACE
Inactive ANG I is converted by the ACE to ANG II
(275). ACE exists as a single polypeptide with two homologous catalytic domains that are active against ANG I. In
addition, ACE is a membrane-bound ectoenzyme (49), i.e.,
both catalytic domains are found outside of cells, whereas
the remainder of the protein is bound to the cell membrane. Thus, although significant ACE activity is found in
plasma, the majority of the enzyme is bound to tissues
such as the vascular endothelium (20). ACE is also found
in urine bound to exosomes (205).
Two “forms” of ACE null mice have been generated.
The so-called ACE.1 strain of mice has a complete absence of both circulating and tissue ACE (60, 138).
Whereas ACE.2 mice express a form of ACE that lacks the
COOH-terminal half of the molecule, and although catalytically active, the ACE is entirely secreted from cells,
resulting in mice with significant plasma ACE activity but
no tissue-bound enzyme (61). Both strains of ACE null
mice have low systolic blood pressures and an inability to
concentrate the urine, both before and after water deprivation. Similar to Ren1c and Agt null mice, ACE.1 mice
have major abnormalities in renal structure, including
marked medullary and papillary atrophy and greater hydronephrotic lesions (60). In contrast, ACE.2 mice have a
relatively preserved renal medulla and papilla (61).
The ACE.2 mouse strain has provided insights into
the role of the RAAS system in the urinary concentration
mechanism, without the complication of interpreting results in mice with renal abnormalities (e.g., ACE.1, Ren1c,
Physiol Rev • VOL
and Agt null mice). Indeed, the greater urine output and
reduced urine osmolality in the ACE.2 strain of mice
suggests that the RAAS plays a direct role in urinary
concentration. It is likely that multiple factors are responsible. Reduced NaCl reabsorption and natriuresis presumably impair concentrating ability by causing an osmotic
diuresis. Furthermore, the associated reduction of ANG II
levels may alter medullary blood flow, which may affect
osmolar gradients (62). Finally, ANG II has been shown to
be involved in the regulation of collecting duct urea transport (112), which could be disrupted in the ACE.2 strain
of mice.
4. Type 1 angiotensin receptors
Pharmacological studies suggest that the effects of
ANG II on water homeostasis are mediated by the type 1
(AT1) angiotensin receptors (263, 275). In mice there are
two AT1 receptors termed AT1A and AT1B. Mice with
combined AT1A-AT1B receptor deficiency have a marked
atrophy of the renal medulla, similar to that seen in
ACE.1, Ren1c, and Agt null mice (see above). This is
accompanied by a profound inability to concentrate the
urine (196, 267). The renal function of mice lacking the
AT1A receptor alone has been more thoroughly investigated (195). These mice have a defect in urinary concentration manifested by an inability to increase urinary osmolality to levels seen in controls after thirsting. Basal
(free access to water) plasma vasopressin levels of AT1A
receptor knockout mice are similar to controls and increase normally after water deprivation, suggesting that
the defect in urine concentration is intrinsic to the kidney.
However, after dDAVP administration, AT1A receptor
knockout mice rapidly and significantly increase their
urine osmolality, although the maximal urine osmolality
observed is substantially lower than controls. Thus the
absence of AT1A receptors does not eliminate the actions
of vasopressin to augment water permeability in the distal
nephron; instead, the defect appears to be related to the
generation of a maximal osmolar gradient.
The cause of the concentrating defect observed in
mice lacking AT1 receptors remains unclear. However,
similarly to the effects observed in ACE knockout mice
(see above), it is unlikely to be the result of renal tubule
structural abnormalities, as the apparent inability to concentrate the urine is prevalent in both mice lacking both
AT1A-AT1B receptors (major renal hypertrophy) or those
lacking AT1A receptors alone (no major structural defects). However, since AT1 receptors are expressed in the
proximal tubule (179), where they regulate solute and
fluid reabsorption, interruption of the direct epithelial
actions of ANG II could alter the distal delivery of solutes,
thus urinary concentrating ability. AT1 receptors are also
expressed in the renal collecting duct (87), where they are
involved in regulation of ENaC activity (203), ENaC abun-
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both models have shown a similar phenotype. Thus an
overview of the two models is presented (117, 194).
Knockout mice have no detectable angiotensinogen
or angiotensin peptides, and therefore lack a functional
RAAS. Atg⫺/⫺ mice have chronic hypotension, increased
renal renin expression, and an abnormal renal morphology with marked atrophy of the renal inner medulla.
Atg⫺/⫺ mice have a higher basal urine output and lower
urine osmolality than controls, and although 24 h of water
deprivation causes a small decrease in urine volume,
urine osmolality is unchanged. Administration of dDAVP
results in only a small increase in urine osmolality in
Atg⫺/⫺ mice. Furthermore, both urinary excretion and
plasma levels of vasopressin are significantly higher in
mutant mice than in controls. Taken together, the results
indicate that the abnormality in Atg⫺/⫺ mice is not due to
the absence of an ANG II-mediated stimulatory effect
on the synthesis and secretion of vasopressin in the hypothalamus, but a form of NDI. The cause of the NDI is
likely to be multifactoral, similar to those of the Ren1c⫺/⫺
mice (see above). The structural abnormality of the renal
medulla provides further evidence for a possible role of
the renal-angiotensin system on renal development.
KNOCKOUT MOUSE STUDIES OF URINARY CONCENTRATING MECHANISM
dance (24, 35), AQP-2 expression (142), and the abundance of the UT-A1 and UT-A3 urea transporters (279).
5. Aldosterone synthase
Physiol Rev • VOL
transport in the TAL, e.g., adrenalectomized rats have a
30% reduction in Na⫹ reabsorption in the TAL that can be
restored by aldosterone treatment, but not glucocorticoids (245, 284). Therefore, lack of aldosterone would be
expected to blunt the countercurrent multiplication process. Finally, a marked reduction in the abundance of
AQP-3 in the CD has been demonstrated in aldosteronedeficient rats, which may impact on collecting duct water
permeability (143).
K. Osmoprotective Genes
1. OREBP/TonEBP
The kidney inner medullary tissue is extremely hypertonic, facilitating water reabsorption from the collecting ducts. Renal medullary cells protect themselves from
this hypertonic stress by increasing the synthesis or import of several organic osmolytes, including sorbitol (synthesized by the enzyme aldose reductase, see below),
betaine (imported by the betaine/␥-aminobutyric acid
transporter), myo-inositol (imported by the Na⫹-dependent myo-inositol transporter), taurine (imported by the
taurine transporter), and glycerylphosphorylcholine (reviewed in Refs. 38, 182). The tonicity responsive transcriptional regulation of these genes is modulated by the
osmotic response element binding protein (OREBP)/
tonicity responsive element binding protein (TonEBP)
(126, 167). OREBP/TonEBP is highly expressed in the
kidney inner medulla and the inner stripe of the outer
medulla, and hypertonic stimulation results in the rapid
translocation of OREBP/TonEBP to the nucleus, where it
binds with osmotic response elements (OREs) in the promoter region of the osmoprotective genes and stimulates
gene transcription (40) (Fig. 9).
OREBP/TonEBP knockout mice die during embryonic development; however, a transgenic mouse line that
overexpresses a dominant negative form of OREBP/
TonEBP (OREBPdn) in the collecting duct has uncovered
a novel role of the OREBP/TonEBP in the urinary concentrating mechanism (144). OREBPdn transgenic mice
had significantly higher daily urine output and lower urine
osmolality compared with nontransgenic controls. However, when subjected to either water deprivation for 24 h,
or vasopressin administration, OREBPdn transgenic mice
are able to increase urine osmolality, although to a lesser
extent than controls. Although OREBPdn transgenic mice
have major structural damage to the kidney, it is unlikely
that this is the sole cause of the defective urinary concentrating mechanism as measurements of concentrating
ability performed before the onset of hydronephrosis also
show a defect. OREBPdn transgenic mice have drastically
reduced expression levels of UT-A1, UT-A2, and AQP-2 in
the inner medulla. These transporters play essential roles
in the urinary concentrating mechanism (see above), and
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Aldosterone is an important regulator of body fluid
homeostasis and electrolyte balance. The role of aldosterone in the kidney has been discussed extensively elsewhere (see Refs. 108, 215), so for the purposes of this
review only a basic oversight is given. Aldosterone-dependent regulation of Na⫹ reabsorption and K⫹ secretion
takes place in distal portions of the nephron: the DCT,
CNT, and CCD. Aldosterone regulates several major proteins involved in the urinary concentrating mechanism,
including ROMK, ENaC, and the Na⫹-K⫹-ATPase (reviewed extensively in Refs. 8, 122, 215, 280).
To assess the effects of decreased amounts or the
absence of aldosterone on kidney function, Makhanova
et al. (159) disrupted the gene coding for aldosterone
synthase (AS) expressed chiefly in the zona glomerulosa
of the adrenal cortex. AS catalyzes an essential step in the
synthesis of aldosterone, involving the 11␤-hydroxylation
of 11-deoxycortisol (11-DOC) to form corticosterone,
which is further hydroxylated to form 18-hydroxycorticosterone, before oxidation at position C-18 to give aldosterone. AS⫺/⫺ mice have low blood pressure and abnormal
electrolyte homeostasis, including increased plasma concentrations of K⫹, Ca2⫹, and Mg2⫹ and decreased concen⫺
trations of HCO⫺
3 and Cl . Examination of renal function
⫺/⫺
showed that AS
mice have a reduced diluting capacity,
a higher urine output, decreased urine osmolality, and an
impaired maximal urinary concentrating ability compared
with controls (159). In addition, the absence of aldosterone results in several compensatory changes, including
induction of the RAAS and elevated plasma glucocorticoid levels.
The impaired urinary concentrating mechanism observed in the AS⫺/⫺ mice is likely to be the result of a
combination of several different factors; however, it is
unlikely to be the result of hydronephrosis, as the reduced
concentrating capacity was also apparent in heterozygote
animals that do not suffer from structural defects in the
kidney. The most likely cause of the concentrating defect
is that the absence of aldosterone impairs NaCl reabsorption in the CNT and CD (via ENaC), thus reducing the
driving force for the osmotic reabsorption of water from
the cortical portion of the collecting duct system. In the
classic formulation of Stephenson (247), there are three
major determinants of concentrating ability: active transport of NaCl from the TAL, water permeability of the
medullary collecting ducts, and the rate of fluid delivery
to the beginning of the medullary collecting ducts. Decreased activity of ENaC in the CNT and CCD would
affect the third factor. In vitro microperfusion experiments have also shown that aldosterone can affect Na⫹
1103
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ROBERT A. FENTON AND MARK A. KNEPPER
bitransgenic mouse (295), it has been shown that AR
deficiency in the kidney inner medulla alone is responsible for the concentrating defect observed in AR knockout
mice, thus ruling out a more general effect of AR gene
deletion (ubiquitously expressed in several other tissues
including brain) on kidney function. AR null mice have
significant renal cellular and structural abnormalities, including cell shrinkage, disorganized tubular and vascular
structures, and segmental atrophy. These abnormal structural features in AR knockout mice are most likely the
cause of the defective urinary concentrating mechanism.
L. Miscellaneous
it is likely that the polyuria observed in OREBPdn transgenic mice is due, at least in part, to their reduced expression.
2. Aldose reductase
Aldose reductase (AR) is abundantly expressed in
the renal medulla (reviewed in Refs. 38, 182). It is a
member of the “osmoresponsive genes” and can be regulated by the TonEBP/OREBP. AR catalyzes the conversion of glucose to sorbitol, a major organic osmolyte in
the renal medulla. Renal medullary cells accumulate large
amounts of sorbitol, and other organic osmolytes, to compensate for the large interstitial hypertonicity that cells
are subjected to during osmotic stress. Thus AR is
thought to have an osmoprotective function in the kidney.
Furthermore, it is also thought to play a role in kidney
development (236).
AR-deficient mice (1, 91) have a urinary concentrating defect, with polydipsia and polyuria, which is only
partially overcome by vasopressin administration, thus
representing a mild form of NDI. The mechanism behind
these defects is not fully understood. One group has
reported that AR null mice have hypercalciuria, hypercalcemia, and hypermagnesemia and that the hypercalcemia
may be partially responsible for the urinary concentrating
defect observed (1). However, another group failed to
corroborate these findings (91). Recently, by the use of a
Physiol Rev • VOL
1. Foxa1
Foxa1 is a member of the winged helix family of
transcription factors and is expressed in the kidney collecting duct (19). Potential Foxa1 binding sites are evident in several genes expressed in the kidney, including
the V2R and several subunits of the Na⫹-K⫹-ATPase (199,
202). Foxa1 null mice have a urinary concentrating defect,
with reduced urine osmolality and volume depletion (19).
In addition, administration of vasopressin has no effect on
the urinary concentrating ability of Foxa1 knockout mice,
indicating a mild form of NDI. Analysis of the mRNA
expression levels for several major genes involved in the
urinary concentrating mechanism failed to identify a direct downstream target for Foxa1 that could account for
the observed phenotype. Thus Foxa1 null mice are a
unique model of NDI and provide the first example of a
mutation in a transcription factor gene that can lead
to a defect in renal water homeostasis. However, further
studies are required to assess whether the observed phenotype is due to a primary defect in the kidney or a result
of the other metabolic abnormalities observed in these
mice.
2. Urate oxidase
Urate oxidase (Uricase) is an enzyme found in liver
peroxisomes of several mammalian species and catalyzes
the conversion of uric acid to allantoin (286), which is
more soluble and easily excreted by the kidney. Uricasedeficient mice have a urinary concentrating defect, with
an approximately sixfold greater urine volume compared
with controls (115, 285). Furthermore, even after a 12-h
water deprivation, uricase-deficient mice cannot concentrate their urine above 900 mosmol/kgH2O. Except for
moderate azotemia, adult uricase-deficient mice do not
show signs of renal insufficiency, except for a mildly
elevated serum urea concentration. It has been suggested
that the renal concentrating defect in uricase-deficient
mice results from renal damage induced by the excretion
of uric acid in amounts that exceed its solubility in urine.
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FIG. 9. Putative role of osmoprotective genes in the urinary concentrating mechanism. The tonicity responsive element binding protein
(TonEBP) is highly expressed in the kidney inner medulla and the inner
stripe of the outer medulla, and hypertonic stimulation (during antidiuresis) results in the activation of TonEBP. This activation requires
nuclear redistribution, dimerization, and phosphorylation. In the nucleus, TonEBP binds to TonE enhancer elements and stimulates gene
transcription of several osmoprotective genes, including the sodiummyo-inositol cotransporter (SMIT), aldose reductase (AR), and the
sodium-chloride-betaine cotransporter (BGT1), allowing renal cells to
adapt to the high osmolality by accumulating intracellular osmolytes
(reducing intracellular ionic strength). TonEBP also stimulates transcription of the UT-A urea transporter gene, AQP-2, and heat-shock
protein 70 (Hsp70). Genetic deletion of either TonEBP or AR results in
defective urinary concentrating ability. See text for description.
KNOCKOUT MOUSE STUDIES OF URINARY CONCENTRATING MECHANISM
However, it is also possible that the urinary-concentrating
defect observed is due to osmotic diuresis.
3. Tamm-Horsfall protein
ACKNOWLEDGMENTS
We are grateful to Professor Donald Kohan, Dr. Shinichi
Uchida, and Dr. Jurgen Schnermann for critical reading of the
manuscript and Ken Kragsfeldt for help with illustrations.
Address for reprint requests and other correspondence:
R. A. Fenton, Water and Salt Research Center, Institute of
Anatomy, Univ. of Aarhus, Aarhus, Denmark (e-mail: ROFE
@ana.au.dk).
GRANTS
The Water and Salt Research Centre at the University of
Aarhus is established and supported by the Danish National
Research Foundation (Danmarks Grundforskningsfond). Support for R. A. Fenton is provided in part by a Marie Curie
Fellowship, the Carlsberg Foundation (Carlsbergfondet), the
Nordic Council (the Nordic Centre of Excellence Programme in
Molecular Medicine), and the Danish National Research Foundation. Funding to M. A. Knepper was provided by the Intramural Budget of the National Heart, Lung, and Blood Institute
(National Institutes of Health Project ZO1-HL-01285).
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