1 Medical and Anesthesia Services, Massachusetts General Hospital, Charlestown 02129; Departments of 2 Medicine, 3 Anesthesia, and 4 Pathology, Harvard Medical School, Boston 02114; and 5 Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology, Cambridge, Massachusetts 02129
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ABSTRACT |
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Eicosanoids regulate various
cellular functions that are important in physiological and
pathophysiological processes. Arachidonic acid is released from
membranes by phospholipase A2 (PLA2) activity. Activated macrophages derived from mice lacking the 85-kDa group IV
cytosolic PLA2 (cPLA2) have a markedly reduced
release of prostaglandin E2 and leukotrienes B4
and C4. Under basal conditions and after furosemide,
urinary prostaglandin E2 excretion is reduced in
cPLA2-knockout (cPLA2/
) mice.
Serum creatinine, Na+, K+, and Ca2+
concentrations, glomerular filtration rate, and fractional excretion of
Na+ and K+ are not different in
cPLA2
/
and cPLA2+/+
mice. Maximal urinary concentration is lower in 48-h water-deprived cPLA2
/
mice compared with
cPLA2+/+ animals (1,934 ± 324 vs.
3,541 ± 251 mmol/kgH2O). Plasma osmolality is higher
(337 ± 5 vs. 319 ± 3 mmol/kgH2O) in
cPLA2
/
mice that lose a greater percentage
of their body weight (20 ± 2 vs. 13 ± 1%) compared with
cPLA2+/+ mice after water deprivation.
Vasopressin does not correct the concentrating defect. There is
progressive reduction in urinary osmolality with age in
cPLA2
/
mice. Membrane-associated
aquaporin-1 (AQP1) expression, identified by immunocytochemical
techniques, is reduced markedly in proximal tubules of older
cPLA2
/
animals but is normal in thin
descending limbs. However, Western blot analysis of kidney cortical
samples revealed an equivalent AQP1 signal intensity in
cPLA2+/+ and cPLA2
/
animals. Young cPLA2
/
mice have normal
proximal tubule AQP1 staining. Collecting duct AQP2, -3, and -4 were
normally expressed in the cPLA2
/
mice. Thus
mice lacking cPLA2 develop an age-related defect in renal
concentration that may be related to abnormal trafficking and/or
folding of AQP1 in the proximal tubule, implicating cPLA2 in these processes.
mouse; osmolality; kidney concentrating mechanism; prostaglandin; aquaporin; eicosanoid
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INTRODUCTION |
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PHOSPHOLIPASES A2 (PLA2s) are a family of enzymes that participate in lipid digestion, microbial degradation, phospholipid membrane remodeling, and signal transduction (5, 25). The group IV 85-kDa cytosolic form of phospholipase A2, cPLA2 (alternatively, group IVA PLA2), has a preference for arachidonic acid at the sn-2 position of phospholipids (41), translocates to lipid substrates at submicromolar calcium concentrations, and is phosphorylated by mitogen-activated protein kinases (12, 18, 26). Arachidonic acid released by PLA2 activity is subsequently metabolized to the eicosanoids: prostaglandins, thromboxanes, leukotrienes, and cytochrome P-450-derived compounds that are involved in diverse extracellular, physiological, and pathophysiological processes and intracellular signaling pathways (16).
In the kidney, cPLA2 activity participates, through
eicosanoid synthesis, in regulation of vascular tone, glomerular
filtration rate, solute, and water transport and inflammation
(4). Other forms of PLA2 are also present in
the kidney (32), and the functional importance of
cPLA2 relative to other forms of the enzyme is uncertain. We created cPLA2 knockout
(cPLA2/
) mice to evaluate the physiological
role of this enzyme (6). cPLA2
/
mice develop normally with life
spans greater than 1.5 yr. Female cPLA2
/
mice produce small litters, most commonly resulting in dead pups due to
abnormalities in parturition. Peritoneal macrophages from cPLA2
/
animals do not release detectable
amounts of prostaglandin E2 and leukotrienes B4
and C4 in response to lipopolysaccharide treatment or
phorbol myristate acetate and the calcium ionophore, A23187. Thus many
biochemical and physiological functions of cPLA2 cannot be
substituted by other forms of PLA2 .
To evaluate the importance of cPLA2 in the kidney, we
measured a number of renal functional parameters in
cPLA2/
mice compared with litter mate
cPLA2+/+ and cPLA2+/
mice. We found that there is a significant age-dependent
urinary-concentrating defect without a defect in diluting capability.
This defect is associated with a significant age-dependent reduction in
proximal tubule membrane-associated aquaporin-1 immunocytochemical
staining of the plasma membrane with no obvious effects on thin
descending limb aquaporin-1 or collecting duct aquaporin-2, -3, or -4.
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METHODS |
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Animals. Group IV cytosolic cPLA2-deficient mice were generated by targeted disruption of the exon encoding amino acids 187-231 in the cPLA2 gene as previously described (6). Embryonic stem (ES) cells, carrying the targeted mutation, were derived from a 129/Sv mouse strain and introduced into blastocysts from a C57/B6 mouse, yielding lines with a mixed 129/Sv and C57/B6 background. Genotyping was performed by Southern blot analysis, using DNA from tail samples, and was confirmed by Western blotting of extracts from multiple organs (42). In all experiments littermate controls were used to eliminate any concerns that differences in genetic background might contribute to the effects seen.
Female mice were used for all the studies due to the high content of PGE2 in seminal fluid that can contaminate urine (33). Mice were housed in a sterile environment with 12:12-h light-dark cycles and had free access to sterile food and water. Manipulation and experiments were done in a barrier facility with full gowning and sterile gloves.Serum and urine parameters. Creatinine, sodium, potassium, and calcium measurements were assessed in plasma and 24-h urine samples. Animals were placed in individual metabolic cages with access to water. Urine was collected under mineral oil in preweighed vials. With the mice under anesthesia, blood was withdrawn from the retroorbital plexus by using heparinized capillary tubes (Drummond Scientific), and plasma was separated by centrifugation.
Glomerular filtration rate (GFR) was estimated by creatinine clearance. Plasma and urinary creatinine were measured using a Beckman creatinine analyzer 2 (Beckman Instruments, Fullerton, CA). Sodium and potassium were determined with a flame photometer (SS3326 Radiometer, Copenhagen, Denmark). Ionic calcium was measured from whole blood with an ion-sensitive calcium/pH analyzer (model 634, Ciba-Corning, Wilmington, DE) at pH 7.4. Urinary protein concentration was measured with the Bradford method (7). Fractional excretion (FE) of sodium and potassium, and osmolar and free water clearances were calculated by using standard formulas.Water deprivation studies. Fresh urine was obtained by gentle bladder massage from animals with free access to food and water, as well as from fasted animals (30). Samples were collected into clean tubes, kept at 4°C, and assayed within 24 h of collection. Osmolality was measured with a vapor pressure osmometer (model 5500 Wescor, Logan, UT). To minimize stress, blood was withdrawn from the saphenous vein of the unanesthetized animal by using an almost painless method (21). Animals were weighed with a digital scale (Scout, Ohaus, Florham Park, NJ). In some experiments 0.5-1 U of vasopressin (American Regent Laboratories, Shirley, NJ), at a concentration of 20 U/ml, was delivered subcutaneously (sc) in the back of the animals or intramuscularly (im) in the left thigh using a 30-gauge needle.
PGE2 measurements. Urine samples were collected under mineral oil over 4 h either under basal conditions or after an intraperitoneal injection of 2 mg/kg of furosemide (Astra USA, Westborough, MA). PGE2 concentration was determined by using a bicyclo-PGE2 enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). Briefly, samples were derivatized to transform urine PGE2 into the more stable compound, bicyclo-PGE2. After dilution to the optimal concentration, samples were mixed with bicyclo-PGE2, conjugated to a tracer molecule (acetylcholinesterase), and incubated with rabbit anti-bicyclo-PGE2 antiserum for competitive binding. Samples were placed in wells coated with a mouse monoclonal anti-rabbit antibody that attaches anti-PGE2 antibody and the tracer to the well. Color change was measured spectrophotometrically.
PLA2 activity measurement.
1-Stearoyl-2-[14C]arachidonyl phosphatidylcholine
([14C]PC) and phosphatidylethanolamine
([14C]PE) were obtained from Amersham. Kidneys were
excised and washed in iced PBS, snap frozen, and stored at 80°C
until the time of homogenization. Dounce homogenization was performed
using 20-30 strokes in 1 ml of lysis buffer containing (in mM) 120 NaCl, 1 EDTA, 10% glycerol, and 50 Tris · HCl at pH 9.0. The
crude lysate was briefly sonicated (Heat Systems-Ultrasonics), and an
aliquot of each specimen was centrifuged at 100,000 g for
1 h at 4°C. The supernatant was recovered as the S-100 cytosolic
protein fraction. Activity against vesicles containing
[14C]PC was assayed at 37°C for 30 min in 100-µl
reactions with (in mM) 5 CaCl2, 1 EDTA, 100 NaCl, and 75 Tris · HCl at pH 9.0. Activity against vesicles containing
[14C]PE was assayed at 37°C for 120 min in 150-µl
reactions with (in mM) 3.3 CaCl2, 66 Tris · HCl at
pH 9.0.(3). Dithiothreitol (DTT) was used in
some experiments at 2 mM. Reactions were quenched in Dole's reagent,
and the free arachidonic acid was extracted by using a modified Bligh
and Dyer technique (40). Radioactivity was counted in a
liquid scintillation counter.
Immunostaining.
Mice were anesthetized with 65 mg/kg of pentobarbital sodium (Abbott
Laboratories, North Chicago, IL). A catheter was placed into the heart,
and animals were perfused and fixed with paraformaldehyde (4%)-lysine-periodate containing 5% sucrose in 0.1-M sodium phosphate buffer, pH 7.4. Kidneys were removed and further fixed by immersion overnight. Once fixed, kidneys were transferred to 30% sucrose/PBS at
4°C overnight and then embedded in Tissue-Tek OCT compound (Miles,
Elkart, IN) and frozen in liquid nitrogen. Three-micron frozen sections
were cut with a cryomicrotome (2800 Frigocut E, Leica, Deerfield, IL).
Sections were mounted on Fisher Superfrost Plus slides and stored at
20°C until immunostaining. After 10 min of rehydration in PBS,
sections were treated with 1% BSA in PBS for 20 min at room
temperature. In an attempt to enhance antibody staining, other kidney
sections were pretreated with 1% SDS for 4 min before BSA blocking
(9). Sections were incubated with the primary antibody at
a 1:100 dilution for 2 h at room temperature in a moist chamber.
After three consecutive washes of 5 min each with PBS, slices were
incubated with secondary goat anti-mouse IgG-FITC at a 1:60 dilution
for 60 min. Slides were mounted in Vectashield (Vector Laboratories
Burlingame, CA) and sealed with a coverslip. Tissue sections were
examined and photographed with a Nikon FXA photo microscope.
Immunoblotting.
Under anesthesia, mice were perfused with PBS administered via
intracardiac puncture, and kidneys were removed. With the aid of a
stereomicroscope, 1-mm-thick slices from cortex, outer medulla, and
inner medulla were prepared. Samples were transferred into lysis buffer
(in mM) Triton 1%, 150 NaCl, 50 Tris · Cl, pH 8, and 1 EGTA
with protease inhibitor (Complete, Boehringer, Mannheim, Germany),
homogenized with several passes through a sequence of 22, 25, and 27-G
needles, and stored at 20°C until processing. Lysates were spun for
5 min at 5,000 rpm. Thirty micrograms of protein were loaded on an SDS
polyacrylamide 10% gel and transferred to a polyvinylidene difluoride
membrane (Millipore, Bedford, MA) for 1 h at 100 V. After
blocking for 1 h with 5% nonfat milk and 0.05% Tween-20 in PBS,
membranes were incubated for 1 h with primary antibody (1:1,000
dilution), washed five times with 0.05% Tween-20 in PBS over 35 min,
and incubated with goat anti-rabbit horseradish peroxidase-conjugated
secondary antibody at 1:10,000 dilution (Sigma, St. Louis, MO) in
blocking solution for 1 h. After five washes with PBS, transfers
were developed by using the ECL-Plus Western blotting detection system
(Amersham International, Buckinghamshire, UK).
Antibodies. An antibody against whole human red cell aquaporin-1 was raised in rabbits as previously described (38). A polyclonal antibody against the last 16 amino acids of the COOH-terminal region of aquaporin-2 conjugated with keyhole limpet hemocyanin was raised in rabbits and affinity purified as described (15, 37). Aquaporin-3 and -4 antibodies were raised in rabbits against a 15-amino acid COOH-terminal peptide, coupled to keyhole limpet hemocyanin. A goat anti-rabbit horseradish peroxidase-conjugated secondary antibody was used for Western blotting. A goat anti-rabbit IgG FITC conjugated (Kirkegard and Perry, Gaithersburg, MD) was used for immunostaining. Bodipy 581/591 phalloidin conjugated at a 1:40 dilution was used to stain F-actin filaments at the brush border membrane (Molecular Probes. Eugene, OR).
Statistical analysis. Data are expressed as means ± SE. Comparisons between groups were performed using analysis of variance and unpaired Student's t-test, assuming unequal variances. A P value <0.05 was considered significant.
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RESULTS |
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Serum and urine physiological parameters with free access to food
and water.
In animals allowed free access to food and water, there were no
differences among cPLA2+/+, +/,
and
/
mice of ages 216 ± 11 (n = 28), 224 ± 12 (n = 25), and 247 ± 7 (n = 24) days, respectively, in the following measured
variables: plasma sodium, potassium and creatinine
concentration, creatinine clearance, and FE of sodium and
potassium (Table 1).
|
Serum and urine osmolality after water deprivation. Eicosanoids have been implicated in the regulation of urinary concentration due to their effects on medullary blood flow, thick ascending limb transport function, and antidiuretic hormone (ADH) antagonism. We evaluated whether there was an altered ability to concentrate the urine after water deprivation for 48 h. Baseline blood samples were collected 48 h before water withdrawal, to avoid circulatory changes and anesthesia at the time of initiation of dehydration. After 48 h of deprivation, water was returned to the cage and 3 h later food was added. Blood was sampled after 48 h of water restriction and after 3 and 24 h of restoration of water. Urine was sampled just before the withdrawal of food and water, and then again after 48 h of water deprivation and 3 and 24 h after restoration of water.
cPLA2+/+ mice increased their urinary osmolality from 2,446 ± 114 to 3,541 ± 251 mmol/kgH2O during water restriction. In contrast, cPLA2
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Effects of vasopressin in antidiuretic and water-diuretic mice.
When animals (mean age: 428 ± 22 days) were given 1.5 ml of
distilled water ip there was a dramatic reduction in urinary
concentration to ~100 mmol/kgH2O in both
cPLA2/
(112 ± 5 mmol/kg,
n = 7) and +/+ mice (126 ± 17 mmol/kg, n = 8). Two hours after water administration, when urinary osmolalities were comparably low, one
cPLA2+/+ (120 mmol/kgH2O) and one
cPLA2
/
mouse (91 mmol/kgH2O)
were injected im with 1 U of vasopressin. Urinary osmolality was
increased 1 h later in both cPLA2+/+
(1,277 mmol/kgH2O) and cPLA2
/
(1,188 mmol/kgH2O) mice, indicating that animals of both
genotypes respond acutely to vasopressin.
|
Urinary PGE2 production.
Urinary PGE2 concentration and excretion rate were measured
in cPLA2+/+ and /
mice under
basal conditions and following treatment with furosemide. Furosemide
was given to enhance PGE2 production. PGE2
concentration was lower in urine from
cPLA2
/
(252 ± 8 days) mice compared
with cPLA2+/+ (239 ± 8 days) littermates
(Fig. 5A). Total
PGE2 excretion in 4 h was also reduced in
cPLA2
/
mice. In response to furosemide,
there was no increase in urinary PGE2 excretion in
cPLA2
/
animals (Fig. 5B). The
decreased urinary PGE2 excretion in
cPLA2
/
mice compared with
cPLA2+/+ mice was seen in both young (222 ± 5 days) and old (400 ± 38 days) animals (data not shown).
|
Kidney PLA2 activities.
The data in Fig. 5 suggest that there is PLA2 activity in
the kidneys of cPLA2/
mice. We partially
characterized the PLA2 activity in total kidney as well as
cytosolic fractions from cPLA2+/+ and
/
littermates (Fig. 6).
As expected, when phosphatidylcholine (PC) is used as a substrate there
is little activity in the cPLA2+/+ total
homogenates or cytosolic fraction. This is consistent with the absence
of cPLA2 from these homogenates (6). By
contrast, when phosphatidylethanolamine (PE) is used as a substrate
there is activity in the cPLA2
/
as well as
+/+ homogenates and cytosolic fractions. The activity
against PE in the
/
animals, which do not have group
IIA PLA2 or cPLA2, indicates that there are
other active forms of PLA2, perhaps group V
PLA2 or a calcium-independent form of PLA2. The
greater PE-directed PLA2 activity in
cPLA2+/+ compared with
/
mouse
kidneys is probably due to cPLA2 in the +/+
animals because PE is also a substrate for cPLA2.
|
Aquaporin immunofluorescence studies.
To determine the potential role of aquaporin water channels in the
concentrating defect found in the cPLA2/
animals, frozen kidney sections were immunostained with antibodies against aquaporin-1, -2, -3, and -4. In
cPLA2+/+ mice intense aquaporin-1 staining was
found at the apical and basolateral membranes of the proximal tubules
in the cortex and outer medulla and thin descending limbs in the outer
and inner medulla (Fig.
7A). In
cPLA2
/
mice the membrane expression of
aquaporin-1 was strongly reduced in the proximal tubules. In contrast,
aquaporin-1 staining appeared normal in thin descending limbs of
cPLA2
/
animals, comparable to the staining
pattern in cPLA2+/+ mice (Fig. 7B).
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DISCUSSION |
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Eicosanoids play many key physiological roles in the kidney. They affect renal blood flow regulation, GFR, renin secretion, and sodium and water excretion (4). PLA2 catalyzes the first step in eicosanoid synthesis, the release of arachidonic acid from membrane phospholipids (39, 44).
The group IV PLA2 was originally characterized by our group in mesangial cells and purified from rat kidney (18, 19). It is also found in tubular epithelium (20). cPLA2 is translocated to membranes by calcium and activated by phosphorylation. It is under hormonal, growth factor, and cytokine influence (19, 24, 43).
To understand the physiological relevance of cPLA2, we
generated cPLA2-null mice and partially characterized these
mice (6). Macrophages from these mice have markedly
reduced PGE2, LTB4, and LTC4
production in response to lipopolysaccharide or phorbol ester and
A23187. In this report we have characterized the renal function of
cPLA2/
mice. We found a significant
urinary-concentrating defect that is associated with an abnormality in
the aquaporin-1 water channel in proximal tubules.
The reduction in urinary PGE2 concentration and excretion
rate in cPLA2/
mice under basal and
furosemide-stimulated conditions argues that cPLA2 makes an
important contribution to renal eicosanoid production. We used urinary
excretion to estimate renal PGE2 synthesis. Only 12% of
plasma PGE2 is filterable, and less than 2% is excreted unchanged in urine, so the systemic contribution to urinary excretion is very low. Urinary prostaglandins are derived mainly from medullary synthesis (14, 27).
Furosemide produces a significant increase in urinary
PGE2 compared with the basal urinary
excretion in cPLA2+/+ animals. In contrast,
there is no increase in urinary PGE2 in cPLA2/
animals. Thus the
furosemide-inducible increase in renal PGE2 synthesis is
dependent on cPLA2. Our data indicate that a primary, but
not exclusive, source of arachidonic acid used for renal production of
PGE2 is cPLA2 .
Both strains of mice used to create the
cPLA2/
line, C57BL/6J and SV/129, have a
natural mutation resulting in the absence of group IIa secretory
PLA2 (23). Thus arachidonic acid used to
generate the residual amount of renal PGE2 in the
cPLA2
/
mice does not derive from group IIa
or group IV cPLA2. We identified DTT-inhibitable and
DTT-noninhibited activity in the cPLA2
/
mice. Potential identities of these forms include group V
PLA2 and calcium-independent cPLA2, which have
been identified in rabbit proximal tubules (34, 35). The
calcium-independent form has preferential selectivity for phospholipids
with arachidonic acid at the sn-2 position. Its role in
eicosanoid metabolism under normal conditions has not been clarified.
Because prostaglandins produced in the renal cortex can modulate GFR
and renal blood flow, we evaluated whether
cPLA2/
mice had significant changes in GFR
when compared with littermates (16). There were no
differences between age-matched cPLA2+/+ and
/
mice in plasma creatinine or creatinine clearance in
either young (57 ± 4 days of age) or older (14 months of age)
mice. Plasma creatinine levels after 48 h of water restriction did
not change compared with basal levels (data not shown). At 14 mo of
age, creatinine clearance was lower compared with younger mice, but values remained similar between cPLA2+/+ and
/
mice whether they were in the older or younger group.
Thus age-related differences in GFR cannot explain the difference in
concentrating ability between genotypes.
Prostaglandins have direct actions on tubular sodium, chloride, and
water transport in a number of nephron segments. PGE2 can
directly inhibit sodium transport in microperfused thick ascending limbs and collecting ducts (13, 45). In the inner
medullary collecting duct, PGE2 can inhibit
Na+-K+-ATPase activity (22). It
has been reported that cPLA2 can reduce sodium transport in
the thick ascending limb of Henle's loop by decreasing
Na+/2Cl/K+ cotransporter function
through inhibition of apical K+ channels (2).
We found similar urinary excretion levels of sodium and potassium, and
the FE of both ions was not different in +/+ and
/
animals.
PGE2 can influence water transport, indirectly by
increasing medullary blood flow and inhibiting sodium transport, and
directly through antagonism of ADH action in the cortical collecting
tubule (17, 31). Administration of vasopressin had no
significant effect on urinary or plasma osmolality changes after water
deprivation, consistent with the fact that the concentrating defect was
not due to impaired central release of ADH. The reduction in urinary concentration is probably related to the reduction in eicosanoid production in cPLA2/
mice. Thus, although
acute cyclooxygenase blockade with reduced PGE2 production
enhances antidiuresis (1), a chronic change in eicosanoid
production has a net diuretic effect.
Despite their concentrating defect, cPLA2/
mice had no impairment of diluting capacity, indicating that their
ability to turn off the production of vasopressin, dilute the urine in
the distal nephron, and maintain impermeability of the collecting duct
to water was unimpaired, consistent with the absence of histological pathology in the medulla.
We propose that the concentrating defect seen in the
cPLA2/
mouse is due to abnormalities
observed in proximal tubule aquaporin-1. Aquaporin-1 is expressed in
proximal tubules and thin descending limbs in the apical as well as in
the basolateral membrane. Water moves into the cell and exits via the
aquaporin-1 water channel. Reduction in aquaporin-1 functional
expression will reduce the effective surface for reabsorbing filtered
water in the cortex, resulting in greater water delivery to the
medullary thin descending limb where the presence of normal amounts of
aquaporin-1 will result in increased water removal. This enhanced water
flow will provide an added burden for water removal from the medulla,
taxing the countercurrent isolation function of the vasa recta and
effectively limiting the amount of medullary-concentrating gradient
that can be maintained.
The immunocytochemical staining pattern for aquaporin-1 in the proximal
tubule indicates that, in cPLA2/
mice, the
aquaporin protein is in a form that is not well recognized by the
anti-aquaporin-1 antibody. This could be due to protein misfolding or
to the masking of recognized epitope(s) by other proteins that might
associate with aquaporin during its intracellular trafficking and
processing. Another possibility is that aquaporin-1 is located
diffusely in intracellular compartments (e.g., the rough endoplasmic
reticulum) due to a trafficking defect and is less visible than when it
is concentrated at the cell membrane. These possibilities are not
mutually exclusive and together might account for the findings. In any
case, the reduction in aquaporin-1 staining intensity in proximal
tubules of cPLA2
/
mice is also age related,
because aquaporin-1 was normally expressed and distributed in kidneys
of younger mice in which no concentration defect was apparent when they
were water deprived. The fact that treatment of tissue sections with
SDS, a denaturing agent, partially restores immunocytochemical
reactivity for aquaporin-1 in the proximal tubule provides
evidence that at least part of the defect in aquaporin-1 is
related to protein misfolding or masking of the recognized epitope. No
abnormalities in the expression and localization of other renal
aquaporins (2, 3, and 4) were detectable between
cPLA2+/+ and cPLA2
/
mice, indicating that the aquaporin defect was restricted to proximal
tubule aquaporin-1. Furthermore our data attest to the importance of
proximal tubule water absorption for maximal urinary concentration. It
is not clear why the aquaporin-1 defect is seen only in older animals.
There is precedent for age-dependent changes in aquaporin -2 and -3 expression in rats, but in these studies age had no effect on
aquaporin-1 expression (36). The defect in membrane
aquaporin-1 that we see in the cPLA2
/
mice
is specific to that form of aquaporin. Trafficking of other proteins to
cell membranes has been shown to be decreased by accumulated oxidative
stress, a feature of aging (29). The absence of
cPLA2 may confer increased sensitivity of aquaporin-1
trafficking to such factors.
Although cPLA2/
mice develop a
concentrating defect when water deprived similar to that of mice
lacking aquaporin-1 (AQP1
/
) (28), some
interesting differences between these two knockout models are found.
cPLA2
/
mice develop a less severe
urinary-concentrating defect, achieving higher urinary osmolalities,
lower plasma osmolalities, and less weight loss when water deprived for
48 h than AQP1
/
mice. The more severe defect in
AQP1
/
mice is probably related to the absence of
aquaporin-1, not only in proximal tubules but also in the thin
descending limb (10) and vasa recta.
In conclusion, mice lacking the group IV cytosolic form of
cPLA2 synthesize less PGE2 in the kidney and
develop a urine-concentrating defect as they age. Immunofluorescence
staining reveals a marked reduction in apical membrane aquaporin-1
staining in proximal tubules of cPLA2/
mice. Abnormal trafficking, protein folding, or protein-protein interactions that result in masking of epitopes of aquaporin-1 in
proximal tubules may result in diminished proximal tubule water reabsorption, which in turn impairs maximal urinary concentration in
cPLA2
/
mice. Changes in trafficking or
folding of aquaporin-1 may be related to alterations in lipid
composition of intracellular or plasma membrane compartments resulting
from the absence of cPLA2 (11). These data
confirm the importance of proximal water reabsorption to the urinary
concentration mechanism and implicate cPLA2 in the
trafficking and/or folding of aquaporin-1.
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ACKNOWLEDGEMENTS |
---|
We are grateful for the technical assistance of Robert Tyszkowski and Margaret McLaughin.
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FOOTNOTES |
---|
P. Downey was a recipient of a fellowship training program award from the International Society of Nephrology. Drs. Bonventre and Brown were supported by National Institutes of Health MERIT Award DK-39773 (J. V. Bonventre), DK-38452 (J. V. Bonventre and D. Brown), and NS-10828 (J. V. Bonventre). A. Sapirstein was supported by National Institutes of Health Grant DK-02493.
Present address for P. Downey: Departamento de Nefrologia, Pontificia Universidad Catolica de Chile, Diagonal Paraguay 361, Torre 10, Santiago, Chile.
Address for reprint requests and other correspondence: J. V. Bonventre, Suite 4002, Massachusetts General Hospital East, 149 13th St., Charlestown, MA 02129 (E-mail: joseph_bonventre{at}hms.harvard.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 July 2000; accepted in final form 5 December 2000.
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