Renal concentrating defect in mice lacking group IV cytosolic phospholipase A2

Patricio Downey1,2, Adam Sapirstein1,3, Eileen O'Leary1,5, Tian-Xiao Sun1,2, Dennis Brown1,4, and Joseph V. Bonventre1,5

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

For quantitation, cryostat sections were immunostained with antibodies against aquaporin-2, -3, and -4 by using a protocol identical to that described for aquaporin-1. Antibodies were affinity purified by using a commercially available kit (Pierce, Rockford, IL). After immunostaining, sections were examined on a Nikon FXA photomicroscope, equipped with an Optronix 3-bit color CCD camera and IP Lab Spectrum image analysis software. Sections of three control and three cPLA2 knockout mice were examined. Four digital images of the renal medulla were captured from each stained section, and five perpendicularly sectioned cells from each image were quantified. All sections for a given aquaporin were immunostained at the same time, and all images were captured in a single session by using identical camera parameters of exposure time, gain, aperture, contrast, and brightness. These parameters were set so that the brightest regions of fluorescence were not saturated (i.e., pixel intensity was ~200, where 0 is black and 255 is saturated). Images were stored and analyzed using IP Lab Spectrum image analysis software.

A linear region of interest was drawn from the apex to the base of each analyzed cell, and a curve showing fluorescence intensity against length in microns was obtained for each cell. The peak value for each curve was noted, and the width of each curve was measured at an arbitrary level representing a pixel value of 50% of the maximum value. The width of each peak is taken to represent the scatter of fluorescent pixels about the plasma membrane, and the peak value represents an estimation of the greatest local concentration of each aquaporin in the cell. Any change in the mean peak width for aquaporin-2 would be interpreted to represent insertion into or removal of protein from the apical plasma membrane. Any change in the peak intensity measured for aquaporin-3 and -4 would be taken as an indication that more, or less, protein was present at the cell surface, because aquaporin-3 and -4 have not been detected in intracellular storage vesicles and are believed to be constitutively expressed at the cell surface.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).

                              
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Table 1.   Renal functional parameters in cPLA2+/+, cPLA2+-, and cPLA2-/- mice

Urinary protein excretion was similar, 0.5 ± 0.1, 0.6 ± 0.1, and 0.7 ± 0.2 mg/24 h, in cPLA2+/+, +/-, and -/- animals, respectively. In addition, serum-ionized calcium was 1.25 mmol/l in each of the three groups. The weight of cPLA2-/- mice was equivalent to that of cPLA2+/+ and +/- animals of similar ages (data not shown).

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-/- animals had significantly lower urine osmolalities before (1,419 ± 236 mmol/kgH2O) and after water deprivation (1,934 ± 324 mmol/kgH2O) (Fig. 1A). Plasma osmolality increased in cPLA2+/+, +/-, and -/- mice but was statistically greater in cPLA2-/- animals than in +/+ and +/- animals (Fig. 1B). During 48 h of water deprivation, cPLA2-/- mice lost a greater percentage of their basal body weight, 20 ± 2%, than did cPLA2+/+ littermates, 13 ± 1%, (Fig. 2).


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Fig. 1.   Urinary and plasma osmolality in water-deprived mice. A: urinary osmolality. Urinary osmolality was measured before and after 48 h of water deprivation. Data are from 8 animals, 292 ± 20 days of age. * and #: P < 0.05 compared with wild-type littermates at 0- and 48-h time points, respectively. B: plasma osmolality. Plasma osmolality was measured before and after 48 h of water deprivation, and at 3 and 24 h after animals were given access to water in 4 of the 8 animals used in each group in Fig. 1A. Age: 299 ± 29 days. *P < 0.05 when the cytosolic phospholipase A2-knockout (cPLA2-/-) animals are compared with cPLA2+/- or cPLA2+/+ animals.



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Fig. 2.   Weight changes after water deprivation. Animal weight was measured at time 0 and after 24 and 48 h of water deprivation. The percent change in body weight was calculated in 8 animals from each genotype. Initial weight was similar in the 3 groups. Age: 292 ± 20 days. *P < 0.05.

The concentrating defect in cPLA2-/- mice increased with age. A group of animals followed over time had much lower urine osmolalities at 320 days of life than at 253 days of life (Fig. 3). When 8-wk-old mice were water deprived for 48 h, changes in urinary and plasma osmolality, and weight loss were comparable between cPLA2+/+ and cPLA2-/- (data not shown).


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Fig. 3.   Changes in maximal urinary osmolality with aging. Urine osmolality measured after 48 h of water deprivation in the same animals at 3 different times during 68 days of monitoring. *P < 0.05 comparing cPLA2-/- with cPLA2+/+ mice. (n = 4/group).

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.

To evaluate whether the concentrating defect was related to a central decrease in ADH production, mice were supplemented with subcutaneous vasopressin 0.5-U each 12 h during the 48-h period of water deprivation. Vasopressin did not correct the concentrating defect in cPLA2-/- animals as reflected by the lower urinary osmolality and higher plasma osmolality in the cPLA2-/- mice given vasopressin compared with cPLA2+/+ littermates treated in an identical fashion (Figs. 4, A and B).


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Fig. 4.   Urinary and plasma osmolality in water-deprived mice supplemented with vasopressin. Four cPLA2+/+, +/-, and -/- mice were deprived of water for 48 h and simultaneously injected with vasopressin subcutaneously (sc) each 12 h. Urinary (A) and plasma (B) osmolalities were measured before and after water restriction. At the end of the period of water restriction animals were allowed free access to water (H2O). Three hours later, mice were allowed free access to food. Age: 321 ± 36 days. *P < 0.05 comparing cPLA2-/- with either cPLA2+/- or cPLA2+/+ mice.

To evaluate whether the concentration defect was associated with other functional defects of the kidney that might be related to a reduction in filtration, GFR was measured in young and old animals. GFR was similar at 57 days of life in cPLA2+/+ and cPLA2-/- animals, 7.7 ± 1.03 vs. 9.1 ± 1.14 µl · min-1 · g-1 (n = 4), respectively, measured over 24 h. At 465 days of life GFR was lower than at 57 days in each group but remained similar between cPLA2+/+ and -/- mice (3.6 ± 1.1 and 3.1 ± 0.5 µl · min-1 · g-1, respectively, n = 4).

No differences in fractional sodium excretion were found between cPLA2+/+ and -/- mice under basal conditions, 0.32% ± 0.01 in cPLA2+/+ vs. 0.43% ± 0.1 cPLA2-/-, or after water deprivation, 0.33% ± 0.04 vs. 0.32% ± 0.1, in cPLA2+/+ animals aged 454 ± 16 days and cPLA2-/- animals aged 466 ± 10 days, respectively.

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).


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Fig. 5.   Urinary PGE2 concentration (A) and excretion (B) before and after furosemide administration. Studies were performed in 4 cPLA2+/+ and -/- mice over a period of 4 h. Basal collections were made and then animals were administered furosemide. There were no differences in PGE2 concentration or excretion between cPLA2+/+ and +/- mice in pooled urine collections (data not shown). Animals were injected intraperitoneally with 2 mg/kg of furosemide. Excretion was calculated as the product of concentration times volume. Age: 246 ± 7 days. *P < 0.05 comparing cPLA2+/+ with cPLA2-/- mice. # P < 0.05 comparing PGE2 excretion before and after furosemide administration in cPLA2+/+ mice.

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.


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Fig. 6.   PLA2 activity of cPLA2+/+ and -/- kidney homogenates and S100 fractions. The PLA2 activities of total and cytosolic protein fractions derived from the kidneys of cPLA2+/+ and -/- mice are displayed as pmol · mg protein-1 · min-1. Incubations were performed at 38°C for 30 min with [14C]phosphatidylcholine (PC) or 120 min with [14C]phosphatidylethanolamine (PE) as substrate. A final concentration of 2 mM dithiothreitol (DTT) was added in some experiments to inhibit PLA2 specific activity as indicated (n = 4 for cPLA2+/+, n = 2 for cPLA2-/-).

DTT had a significant, albeit small, effect to reduce PLA2 activity in tissue homogenates but had no effect on PLA2 activities in cytosolic fractions from either cPLA2+/+ or cPLA2-/- mice. This indicates that there is PLA2 activity dependent on disulfide bonds for its integrity, and it is consistent with the presence of a small molecular mass (12-14 kDa) sPLA2 in the particulate fraction of kidney homogenates. The residual DTT-insensitive PLA2 activity in the cytosolic and particulate fractions of cPLA2-/- mouse kidney may represent a calcium-independent PLA2.

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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Fig. 7.   Localization of aquaporin-1 (AQP1) by indirect immunofluorescence in the outer medulla of mouse kidney. In all images, the top of the micrograph shows proximal tubule S3 segments in the outer stripe (OS; *), whereas the bottom of each micrograph shows the inner stripe (IS) with positive-stained thin descending limbs (TDLs) of Henle (arrows). In some images, regions of transition from the S3 segment to the TDLs are visible. A: outer medulla from a cPLA2+/+ mouse kidney (356 days old). In the OS, both apical and basolateral plasma membranes of proximal tubule S3 segments are heavily stained. In the IS (bottom), S3 segments are absent, but TDLs of Henle are brightly stained. B: outer medulla from a cPLA2-/- mouse (376 days old). In the OS, proximal tubule S3 segments show only a very weak staining for AQP1, whereas TDLs of Henle in the IS are heavily stained. C: outer medulla from the same cPLA2-/- mouse kidney shown in B, except that the kidney section was treated with SDS (an antigen retrieval technique) before immunostaining. Although staining intensity is not restored to control levels (compare with A), the AQP1 labeling is considerably increased compared with the nonrelated cPLA2-/- kidney section shown in B. D: outer medulla from a younger cPLA2-/- mouse (64 days old), showing apparently normal levels of AQP1 staining in S3 proximal tubule segment. AQP1 staining patterns in S3 segment and TDLs are comparable to those seen in cPLA2+/+ mice. No concentrating defect was apparent in functional studies of cPLA2 -/- mice at this younger age. Bar = 20 µm.

Antigen epitope access and/or abnormal protein folding may explain aquaporin-1 staining differences at the proximal tubule between cPLA2-/- and cPLA2+/+ mice. Slices were pretreated with SDS to maximize antigen exposure as previously described (9). Though staining with anti-aquaporin-1 antibodies was increased in SDS-treated cPLA2-/- proximal tubule membranes, a clear reduction in staining pattern remained apparent in proximal tubules of cPLA2-/- compared with cPLA2+/+ mice (Fig. 7C). An identical staining pattern was obtained after affinity purification of the aquaporin-1 antiserum by using a column prepared with a peptide consisting of the COOH-terminal 15 amino acids of aquaporin-1.

Because the concentration defect is age related it was important to evaluate whether the abnormal aquaporin-1 staining was age related. Kidneys of 8-wk-old animals were studied. Aquaporin-1 staining was normal in proximal tubules and thin descending limbs of cPLA2-/- animals, comparable to their cPLA2+/+ littermates (Fig. 7D). To clarify whether differences in immunocytochemical staining seen in older animals is due to a reduction in total amount of protein or to an abnormal aquaporin-1 processing, localization, or presentation to the antibody, we immunoblotted extracts from kidney cortex, outer medulla, and inner medulla from both cPLA2-/- and cPLA2+/+ mice. We found no differences in the aquaporin-1 signal intensity in any corresponding regions from cPLA2+/+ and -/- mice (Fig. 8). These findings suggest that either abnormal cellular distribution of aquaporin-1 or folding leading to epitope masking is the cause of the immunofluorescence findings.


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Fig. 8.   AQP1 immunoblotting in cortex (C), OS, and IS. Extracts from dissected C, OS, and IS of the outer medulla were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and blotted with anti-AQP1 antibodies. Extracts were made from 2 cPLA2+/+ (1 and 2) and 2 cPLA2-/- mice (3 and 4). Arrowheads identify AQP1; arrows mark the glycosylated form of AQP1. Age: 457 ± 11 days.

A strong staining of actin at the brush border apical membrane in both cPLA2+/+ and -/- animals was evident when tissue was stained with bodipy-conjugated phalloidin, indicating that the apical brush border was intact in both cPLA2-/- and +/+ mice (not shown).

Aquaporin-2 was localized at the apical pole of renal collecting duct principal cells in both cPLA2-/- and +/+ mice in a pattern previously described (8), with no detectable differences seen between mice of different genotypes (Figs. 9, A and B). Aquaporin-3 and -4 were located at the basolateral pole of collecting duct principal cells predominantly at the inner medulla in a pattern indistinguishable between cPLA2+/+ and -/- mice (Figs. 9, C-F). Quantification of the aquaporin-2, -3, and -4 staining patterns revealed no significant differences in either intensity of staining, or the cellular distribution of staining between cPLA2+/+ and -/- mice (Figs. 10, A and B).


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Fig. 9.   Comparison of aquaporin staining in cryostat sections from cPLA2+/+ mice (A, C, E) and cPLA2-/- mice (B, D, F). Sections from comparable kidney regions were stained with antibodies against AQP2 (A, B), AQP3 (C, D), and AQP4 (E, F). No differences could be detected for any of these aquaporins between control and knockout mice. Intense staining for AQP2 was detected in the apical region of principal cells of all kidney regions. Representative images from the IS of the outer medulla are shown here. For AQP3 (IS) and AQP4 (inner medulla), staining was basolateral.



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Fig. 10.   Quantification of the immunofluorescence staining for AQP2, AQP3, and AQP4 in kidneys from cPLA2+/+ and cPLA2-/- mice. A linear region of interest was drawn from the apex to the base of each cell as described in METHODS. A linear measurement of the pixel intensity along each cell was obtained, and the peak intensity was noted. In addition, the width of each peak was measured in microns at a pixel intensity that was 50% of the maximum value for each curve. A: mean values for peak intensity for AQP2, -3, and -4. B: mean values for the width of the peak. The peak width provides an indication of how tightly the aquaporins are associated with the plasma membrane. Note that the peak width is the greatest for AQP2, which is expressed not only at the cell surface, but also on intracellular vesicles. This results in a broader band of staining than for AQP3 and -4, which are concentrated only on the plasma membrane, with no significant localization on intracellular vesicles. No significant differences in either peak intensity or peak width were detectable between cPLA2+/+ and cPLA2-/- mice for any of the aquaporins depicted.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

We are grateful for the technical assistance of Robert Tyszkowski and Margaret McLaughin.


    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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