Department of Physiology and Pharmacology, University of Southern Denmark-Odense, DK-5000 Odense C, Denmark
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We investigated the localization of cAMP-coupled prostaglandin E2 EP2 and EP4 receptor expression in the rat kidney. EP2 mRNA was restricted to the outer and inner medulla in rat kidney, as determined by RNase protection assay. RT-PCR analysis of microdissected resistance vessels and nephron segments showed EP2 expression in descending thin limb of Henle's loop (DTL) and in vasa recta of the outer medulla. The EP4 receptor was expressed in distal convoluted tubule (DCT) and cortical collecting duct (CCD) in preglomerular vessels, and in outer medullary vasa recta. Butaprost, an EP2 receptor-selective agonist, dose dependently raised cAMP levels in microdissected DTL and outer medullary vasa recta specimens but had no effect in EP2-negative outer medullary collecting duct segments. Dietary salt intake did not alter EP2 expression in the kidney medulla. These results suggest that PGE2 may act in the resistance vessels and in the DTL and DCT-CCD segments as a paracrine, cAMP-dependent regulator of vascular resistance and tubular transport, respectively.
adenosine 3',5'-cyclic monophosphate; sodium chloride; arteriole
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PROSTANOIDS HAVE IMPORTANT regulatory functions in the kidney. Renal tubular epithelium and interstitial cells synthesize and release predominantly PGE2 in a regulated fashion. PGE2 affects urine concentration ability, promotes salt and water excretion by the renal medulla, maintains renal blood flow and glomerular filtration rate (GFR), particularly under neurohumoral activation, and stimulates renin release (for a recent review, see Ref. 3). PGE2 initiates and maintains these physiological effects through interaction with specific receptors on target cell surface membranes. At least four different PGE2 receptor isoforms exist, designated EP1-4, with various intracellular coupling mechanisms (2, 6, 12, 18, 19, 23, 28, 35). In the kidney, all four known EP receptors are expressed at variable levels (4, 11, 17, 27). The natriuretic and diuretic effects of PGE2 are probably accomplished through interaction with phosphoinositol-coupled EP1 and Gi-coupled EP3 receptors situated on the ascending part of the loop of Henle and the collecting duct system (3, 4, 18, 27). By activation of separate EP1 and EP3 receptors in the collecting ducts, PGE2 inhibits arginine vasopressin (AVP)-stimulated water and sodium transport (10). PGE2 also has the ability to raise cAMP production, at least in distal convoluted tubules (DCT) and cortical collecting duct (CCD) segments (7, 22, 26, 31), leading to an increase in water permeability in CCD (22). These findings suggest the presence of cAMP-coupled EP2 and/or EP4 receptors for PGE2 at specific nephron sites in the kidney cortex. Moreover, both EP2 and EP4 receptors are expressed in the medulla (4, 11, 17), but the exact localization and function are not known. Renal EP2 receptors could play a role in renal salt and water handling, because mice deficient in functional EP2 receptor genes develop salt-sensitive hypertension (13, 30). The mechanism behind the development of hypertension in this model is not clear, but the lack of EP2 receptors in the kidney is a relevant possibility. In contrast to EP1 and EP3 receptors, little is known about the expression and functional role of EP2 and EP4 receptors along the nephron. It therefore appeared relevant to us to determine whether these receptors are expressed along the nephron and, if so, to determine whether changes in expression are involved in renal adaptation to dietary salt intake.
The action of PGE2 on renal blood flow is believed to be direct, through cAMP-coupled vascular receptors, because PGE2 elicits vasodilation in isolated afferent arterioles and in descending vasa recta (8, 20). Recent data have confirmed the preglomerular expression of vasodilator EP receptors (17, 21, 29). However, it is unknown whether EP receptors are present in postglomerular resistance vessels, in particular vasa recta, which are highly sensitive to PGE2 (15, 20, 25). A second purpose of the present study was to investigate renal vascular cAMP-coupled EP receptor expression. To address these issues, we applied RT-PCR analysis to localize EP2 and EP4 receptors in microdissected nephron and vessel samples. Radioimmunoassay measurement of cAMP levels was applied after receptor-specific agonist stimulation.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vivo protocols.
All procedures conformed with the Danish national guidelines for the
care and handling of animals and with the published guidelines from the
National Institutes of Health. Male Sprague-Dawley rats (150-220
g) had free access to standard pathogen-free rat chow (Na: 2 g/kg, Cl:
5 g/kg, Altromin-1310, Lage, Germany) and tap water. In one series,
rats were kept on a high-salt diet (4% Na wt/wt, n = 6), and another group of rats received a NaCl-deficient diet (Altromin,
0.02% Na wt/wt; n = 6) for 10 days. The low-salt diet
(Altromin-1036) contained 150 mg Na+/kg and 3.4 mg
Cl/kg. In the low-salt series, the rats were initially
given an intraperitoneal injection of furosemide (2 mg/kg). The animals were killed by decapitation, trunk blood was sampled in EDTA-coated vials, and organs were rapidly removed, weighed, frozen in liquid nitrogen, and stored at
80°C. Kidneys were separated into major regions by dissection under a stereomicroscope and then snap-frozen in
liquid nitrogen. The medullary rays were contained in the cortical tissue.
Extraction of RNA. Total RNA was extracted from tissue samples, basically according to the acid-guanidinium-phenol-chloroform extraction protocol of Chomczynski and Sacchi (5). RNA pellets were dissolved in diethylpyrocarbonate-treated water, and the yield of RNA was quantified by measuring optical density at 260 nM.
RT-PCR. RT-PCR was performed as described previously (11). In brief, cDNA was supplied with 1 µl of each primer (10 pmol/µl), 2 µl of desoxyribonucleotides (2.5 mmol/l), 2 µl of PCR-buffer (×10), 1 µl of Taq polymerase (1 U), and water to a final volume of 20 µl. The samples were denatured at 95°C for 3 min. PCR amplification of EP2 and EP4 cDNA required the addition of 1 µl of MgCl2 (25 mmol/l) to the standard mixture.
For EP2, primers were 5'-TTC GGA GCA AAA GAA GCC-'3 (sense) and 5'-GAG CGC ATT AGT CTC AGG-'3 (antisense), covering bases 725-1025, 301 bp (2). For EP4, primers were 5'-GGA AGA CTG TGC TCA GTA-'3 (sense) and 5'-GAA GCA AAT TCT TGC CTC-'3 (antisense), covering bases 1007-1246 of rat EP4 cDNA, 240 bp (23; formerly known as the "EP2"-receptor). Restriction sites for BamHI and EcoRI were added to the EP oligomers, which increased the size of the PCR products by 15 bp. The PCR products for EP2 and EP4 receptors have previously been cloned and validated by sequencing (11). For aquaporin-1 (AQP1), 5'-CCA GCG AAA TCA AGA AGA AGG CT-3' (sense) and 5'-CTA TTT GGG CTT CAT CTC CAC C-3' (antisense) covered the translated region, 806 bp (GenBank accession no. L07268). For the parathyroid hormone (PTH) receptor, primers were 5'-GGC TGC ACT GCA CGC GCA A-3' (sense) and 5'-TTG CGC TTG AAG TCC AAC GC-3' (antisense), 817 bp (36). For urea transporter UT-A2, they were 5'-AGC CTA TGC ACT GCC TGT TGG-3' (sense) and 5'-TCC GTG TGA CTG TTC TCC-3' (antisense), 575 bp (24). ForSouthern blotting.
The cloned DNA fragments were excised from the plasmids, and 50 ng were
denatured for 2 min. The following components were added [final
concentrations (in mM)]: 4 dATP, dGTP, and dTTP, respectively; 50 Tris · HCl, pH 8.0; 5 MgCl2, pH 8.0; 1 dithiothreitol; and 200 HEPES, pH 6.6, as well as 0.5 pmol/µl of each
specific primer, 0.4 µg/µl BSA, 1 µCi/µl
[-32P]dCTP (Amersham Pharmacia Biotech, Birkerød,
Denmark), and 0.05 U/µl Klenow to a final volume of 50 µl.
Synthesis was continued for 2 h at room temperature. After
denaturation at 95°C for 2 min, the probe was used. The agarose gels
were equilibrated for 20 min in 1 liter of water followed by 2 × 10 min in NaOH (0.4 mol/l). DNA was transferred by capillary blotting
to Zeta Probe GT membranes (Bio-Rad, Copenhagen, Denmark) for at least
2 h, the membrane was washed in 2× standard sodium citrate (SSC),
air-dried, and baked at 80°C for 30 min. Prehybridization was
performed for 2 h at 42°C in 6× SSC, 5× Denhardt's, 0.25%
SDS, and 100 µg/ml denatured herring sperm DNA, probe was added, and
hybridization was allowed overnight at 42°C. The membrane was rinsed
in 2× SSC and washed for 30 min in 0.1× SSC+0.1% SDS at 65°C.
Autoradiography was performed for 2-4 h.
RNase protection analysis.
Messenger RNA levels for the EP2 receptor and for rat -actin were
assayed by RNase protection assay basically as described (11). In brief, after linearization with
HindIII, the plasmids yielded radiolabeled antisense RNA
transcripts by incubation with SP6 polymerase (Promega) and
[
-32P]GTP (Amersham Pharmacia Biotech) according to
the Promega riboprobe in vitro transcription protocol. RNA probes
(5 × 105 counts/min) were hybridized with samples of
total RNA at 60°C overnight in a final volume of 50 µl. Sequential
digestions were performed with a mixture of RNase A/T1 (Roche,
Hvidovre, Denmark) and proteinase K (Roche). The hybrids were separated
on 8% polyacrylamide gels. Autoradiography was performed at
80°C
for 1-3 days. Radioactivity in the protected probes was
quantitated by excision from the gel and
-counting.
Microdissection of rat nephron segments and microvessels.
Nephron segments for RT-PCR analysis and for receptor studies were
obtained by microdissection of rat kidney tissue from male Sprague-Dawley rats (180-220 g) (36). The rats were
anesthetized with mebumal, and both kidneys were perfused through the
aorta with 20 ml cold saline followed by 20 ml of DMEM with 0.1% BSA and collagenase A (1 mg/ml-0.2 U/mg, Roche). Thin coronal slices were
cut, and the slices were incubated in 25 ml DMEM with 0.1% BSA and 1 mg/ml collagenase A for 25 min at 37°C with modest shaking. Next, the
tissue was washed twice with DMEM containing 10% FCS and kept on ice.
Microdissection was done under a stereomicroscope by using sharpened
forceps. The nephron segments were identified by their localization in
kidney regions and by their appearance. We isolated segments of the
proximal convoluted tubule (PCT), proximal straight tubule (PST),
descending thin limb of Henle's loop (DTL) from outer medulla, thin
limbs of Henle's loop (TL) from inner medulla, medullary thick
ascending limb of Henle's loop (mTAL), cortical thick ascending limb
of Henle's loop (cTAL), DCT, CCD, outer medullary collecting duct
(OMCD), and inner medullary collecting duct (IMCD). Single glomeruli
and preglomerular vessels were isolated by grasping the cortical radial
arteries with attached afferent arterioles and carefully removing all
tubular tissue and all glomeruli before RNA extraction. For RNA
isolation, 30-50 "branching points" were pooled. Vasa recta
bundles were dissected from the outer medulla and were identified on
the basis of the typical parallel arrangement of small-diameter
structures with irregular cell spacing (see Fig. 7A). Most
rat vasa recta bundles are of the simple type that do not contain
tubules in the core, but DTL (type 1 cells) can be associated with the
bundle periphery in the complex type of bundles (14). We
used the core for RNA isolation and carefully inspected the samples to
rule out attached tubules. The isolated segments were transferred in
3-5 µl DMEM to 500 µl fresh DMEM in a 24-well cell culture
plate on ice. Identical segments were pooled, and the total length of
tubule was assessed by a calibrated micrometer scale built into the
ocular. For isolation of RNA, at least 10 mm of tubule segments were
pooled, except for the DCT segments, of which 5 mm were pooled, and
then transferred to 400 µl guanidinium thiocyanate (4 mol/l)
solution, and 12 µg yeast tRNA were added as carrier. Samples were
stored at 80°C until RNA extraction by the
acid-guanidinium-phenol-chloroform extraction protocol
(5). RNA from at least 5 mm of microdissected samples was
used for RT.
Incubation studies.
All procedures of anesthesia and surgery were identical to those used
above for RNA isolation except for the addition of the cyclooxygenase
inhibitor indomethacin (10 µmol/l) to all solutions to inhibit
endogenous prostanoid synthesis. For the incubation studies, the
microdissected samples were transferred to 500 µl DMEM containing
0.1% BSA in a cell culture dish on ice. The tubules and vasa recta
samples were selected from this pool, and a total length of 2-3 mm
(2-5 single tubules) and up to 2 cm of vasa recta (1-2
bundles) were used for incubation, respectively. The samples were
stored on ice in a total volume of 20 µl in a 1.5-ml cup until all
segments had been distributed. Each experiment was carried out by using
segments dissected from one animal. Experiments were only performed
when sufficient amounts of tubule segments for a complete dose-response
series were obtained no more than 2 h after the animal was killed.
The segments were preincubated for 5 min at 37°C in a heating block.
Then, 20 µl preheated DMEM mix containing 1 mmol/l of the
phosphodiesterase inhibitor IBMX and appropriate test substances were
added. The reaction was continued for 5 min. The incubation was
terminated by quickly placing the tubes on ice and adding 1 ml of
absolute ethanol containing 20 mmol/l HCl. The samples were extracted
overnight at 20°C.
Plasma renin concentration. Renin was measured by ultramicroassay (16). In short, 10 µl plasma were diluted 20, 40, and 80 times by Tris buffer containing human albumin. Five-microliter samples were incubated for 24 h at 37oC with 20 µl of a mixture of renin substrate, ANG I antibody, and buffer followed by RIA of generated ANG I. Renin concentration is expressed in Goldblatt units (GU) compared with renin standards from the Institute for Medical Research (MRC, Holly Hill, London, UK).
Statistics.
When several sets of data were compared at the same time (e.g., data
from the salt-intake groups), a one-way ANOVA was used. If the ANOVA
was significant at the 5% level, differences among data sets were
established by using 95% confidence intervals. When two sets of data
were compared, an unpaired Student's t-test was used.
P 0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Linearity of the EP2 mRNA assay was tested with total RNA from rat
spleen in the 3- to 24-µg range. EP2 receptor expression is
reportedly high in spleen (2, 19). Hybridization of the labeled EP2 antisense RNA probe with total RNA from rat spleen resulted
in a significant hybridization product, with a size of the hybrid that
corresponded to the expected value. Notably, the probe was completely
digested in the absence of template RNA, and there was no unspecific
hybridization (Fig. 1A, tRNA).
The assay was linear in a range from 12 to 24 µg total RNA (Fig.
1A). Significant EP2 expression was detected with the probe
in total RNA samples from the inner and outer medulla from rat kidney, whereas expression was not obvious in cortex, with the use of up to 48 µg total RNA for the assay (n = 4, Fig.
1B). -Actin was used as an RNA loading control, and it
was strongly and equally expressed in all kidney regions (Fig.
1B).
|
Next, EP2 and EP4 receptor expression along the rat nephron was
determined by RT-PCR analysis of microdissected rat nephron segments
and glomeruli (Fig. 2). Both EP2 and EP4
receptors were expressed in glomeruli; on the basis of serial dilution,
EP4 would seem to be much more highly expressed than EP2 (Fig.
2A). In a first screening analysis of nephron segments, EP2
transcripts were found essentially in the descending medullary part of
the loop of Henle: in DTL and in TL dissected from the inner medulla (Fig. 2B, top). The significant amplification
product seen in the mTAL segment was falsely positive, as shown later
(Fig. 3B). The EP4 receptor
was predominantly expressed in DCT and CCD (Fig. 2B,
middle). Weaker signals for EP4 were seen in OMCD. -actin was equally amplified in all segments investigated (Fig. 2B,
bottom). To confirm the finding on EP2 mRNA localization,
separate samples of DTL and mTAL segments obtained in four different
animals were analyzed (Fig. 3). EP2 expression was detected in all DTL
segments where RT was included, and no amplification products were seen in the absence of RT (Fig. 3A, DTL4-RT) or cDNA. In
contrast, the finding of EP2 expression in mTAL could not be reproduced in repeated experiments (Fig. 3B), whereas actin was present
in all mTAL samples (Fig. 3B). This discrepancy was most
likely caused by contamination of the first mTAL sample with DTL
segments.
|
|
The correct identification of the EP2- and EP4-positive nephron
segments was validated by analysis of marker transcript localization (Fig. 4). AQP1 expression was detected in
DTL and TL but not in mTAL (Fig. 4A). The urea transporter
UT-A2 was expressed in TL as reported (34) and,
unexpectedly, also in mTAL specimens. UT-A2 was not detectable in DTL
samples. This identifies our samples as DTL segments from long loops of
Henle because UT-A2 is expressed exclusively in short loops of Henle in
the outer medulla (34). Actin was expressed equally in all
the tested segments (Fig. 4A). The EP4 receptor was found
consistently in DCT and CCD, weakly in OMCD, and not found in IMCD
(Fig. 4B). The PTH receptor was expressed in DCT, whereas
CCD was PTH receptor negative (Fig. 4B), as reported
(36). The collecting duct system strongly expressed AQP2
as expected (not shown). Actin was expressed in all the tested segments
(Fig. 4B). Thus the EP4 receptor is expressed in the cortical distal nephron and CCD.
|
To determine whether cAMP-coupled EP2 and EP4 receptors are expressed
in the renal resistance vasculature, we dissected out preglomerular
vessels from rat kidney cortex and vasa recta bundles from the outer
medulla. RNA was analyzed by RT-PCR and subsequent Southern blotting
(Fig. 5). EP4 receptor was strongly
expressed in the preglomerular resistance vessels and in outer
medullary vasa recta bundles (3 of 3 and 3 of 3 separate preparations,
respectively), whereas EP2 receptors were most consistently detected in
outer medullary vasa recta bundles (3 of 3 preparations, respectively). In preglomerular vascular samples, one of three PCR reactions was
clearly positive for EP2. Rat -actin was equally amplified from the
three preparations of preglomerular vessels and the vasa recta bundle
preparations (Fig. 5).
|
To investigate whether the mRNA localization data were relevant at the
level of functional EP2 receptors, we examined the effect of the
specific EP2 receptor agonist butaprost on cAMP levels in
microdissected nephron segments and outer medullary vasa recta bundles.
First, EP2 mRNA-positive DTL segments were compared with EP2
mRNA-negative OMCD segments (Fig. 6).
Butaprost significantly increased cAMP formation in DTL (Fig.
6A). A maximal 10-fold stimulation was observed at a
concentration of 105 mol/l (control 20.6 ± 8.1 vs.
198 ± 17.1 fmol/mm × 5 min, n = 7). In
identical dose-response experiments with OMCD segments, butaprost did
not significantly change cAMP production (n = 6; Fig.
6B), and cAMP was at the detection limit of the assay. In contrast, the adenylyl cyclase activator forskolin (10 µmol/l) significantly increased cAMP production in both segments (control 20.6 ± 8.1 vs. 83.4 ± 11.1 in DTL and 6.4 ± 2.2 vs.
135.9 ± 45.4 fmol/mm × 5 min in OMCD, n = 6; Fig. 6). These data suggest that both DTL and OMCD segments are
endowed with adenylyl cyclase, but functional EP2 receptors are not
present in OMCD.
|
Subsequently, it was examined whether butaprost affects cAMP levels in
outer medullary vasa recta bundles (Fig.
7C). Outer medullary vasa
recta bundles and single outer medullary vasa recta (Fig.
7A) expressed the EP2 receptor, and PCR amplification was only noted in the presence of RT (Fig. 7B). Because of the
limited amount of tissue that could be dissected in a reasonable time, only a single concentration of butaprost was tested. In individual experiments (n = 3 rats; 2-4 separate
samples/condition), butaprost (104 mol/l) invariably
increased cAMP production but to different absolute cAMP levels (Fig.
7C). The geometric mean of the control cAMP level was 1.4 vs. 37.8 after incubation with butaprost. Forskolin stimulated basal
cAMP levels from 1.4 to 60.2 (geometric mean; data not shown).
|
In the next series of experiments, we tested the hypothesis that
dietary salt intake regulates expression of the EP2 receptor (Fig.
8). After 10 days on high- or low-salt
intake, the two groups of rats had plasma renin concentrations that
were significantly different (low salt: 2.92 ± 0.5 mGU/ml vs.
high salt: 0.14 mGU ± 0.04 mGU/ml; n = 6).
Although there was a tendency for a high-salt diet to lower EP2 mRNA
abundance in both outer and inner medulla (Fig. 8), this was not
statistically significant at the 5% level (P = 0.06).
Thus dietary salt content had no effect on the renal medullary EP2 mRNA
level.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We report here on the localization of the cAMP-coupled PGE2 EP2 and EP4 receptors in rat kidney. EP2 receptor mRNA transcripts were found predominantly in the outer and inner medulla of rat kidney in accordance with our previous report (11). RT-PCR analysis and cAMP measurements showed that the DTL is a major site of EP2 expression along the rat nephron. In contrast, EP4 receptor expression was detected in DCT and CCD. In addition to the tubular epithelium, the EP4 receptor was localized in both cortical preglomerular resistance vessels and outer medullary vasa recta bundles, whereas the EP2 receptor was found most reproducibly in outer medullary vasa recta. Glomeruli expressed both EP receptor isoforms.
The various effects of PGE2 on kidney function are mediated by at least four distinct receptors with different intracellular couplings (2, 6, 12, 18, 19, 23, 28, 35). Recent data suggest that EP2 receptors, but probably also EP4 receptors, are involved in PGE2-mediated blood pressure regulation, potentially by an effect on renal salt handling (13, 30) or vascular reactivity (1, 39). Previous attempts to localize the EP2 receptor by Northern blotting or in situ hybridization of kidney tissue from different species have not yielded conclusive evidence (2, 4, 12). More sensitive RT-PCR analysis and immunolocalization showed EP2 expression in both rat and human kidney (11, 17, 19). In human kidney, the EP2 recptor was not detected in the tubular system but only in the smooth muscle of small-caliber vessels (17). This finding, which is different from the present observations, could reflect species differences between rat and human kidneys. Alternatively, it might reflect different sensitivities of the methods employed to detect EP2. The present set of data shows agreement between the EP2 localization obtained by RNAse protection analysis and by RT-PCR analysis, because vasa recta bundles and single vasa recta and DTL are restricted to the kidney medulla. Moreover, butaprost, a highly specific EP2 receptor agonist with low potency (6), markedly and selectively stimulated cAMP formation in microdissected DTL segments. In agreement, previous reports have shown that PGE2 potently increases cAMP formation in freshly isolated DTL segments from rat kidney (31-33). Thus it is justified to conclude that functional EP2 receptors are expressed by the DTL in rat kidney. Most of the DTL specimens that we dissected from the outer medulla continued into the inner medulla. The absence of the urea transporter UT-A2 in the specimens confirms that they belong to long loops of Henle. In the outer medulla, UT-A2 is expressed only in short loops (34). An important question regards the functional role the EP2 receptor plays in the DTL. Medullary PGE2 synthesis is increased in many physiological settings, including salt loading (37). To our knowledge, prostaglandin-dependent, cAMP-mediated effects on transepithelial transport of NaCl, water, or urea in the DTL have not been reported thus far. In the TAL, PGE2 inhibits reabsorption of NaCl through interaction with the EP3 receptor (3), and data suggest that the EP3 receptor itself is regulated by salt intake (11). Although the present data were not statistically significant, there was a tendency for EP2 expression to be decreased by a high-salt intake, and vice versa. A goal of future studies should be an examination of the impact of PGE2 on hydraulic conductivity and on epithelial transport of urea in isolated DTL segments.
In kidney cortex, the EP4 receptor is strongly expressed in glomeruli of several species (4, 11, 12, 27), but tubular sites of EP4 expression have not been identified before. Our data suggest that EP4 receptors are expressed in the distal nephron and early collecting duct system, in DCT and CCD. Previous reports support these data by showing that, in the absence of AVP, PGE2 stimulates cAMP production in cultured DCT cells (7), CCD cells (22, 26), and microdissected CCD segments (31), an effect not mimicked by EP2-specific butaprost (22). Thus altogether the available data suggest the presence of EP4 receptors in DCT and CCD segments. Some data imply segregation of distinct PGE2-coupling mechanisms to different poles of the epithelial cells in this nephron segment. On the one hand, perfusion of CCD segments with PGE2 induces an increase in water permeability (22). On the other hand, abluminal addition of PGE2 inhibits the AVP-induced increase in water permeability (10). The physiological significance of raising cAMP in DCT is related to an enhanced reabsorption of magnesium (7) and probably also calcium.
It is generally believed that PGE2 protects overall renal perfusion. In particular, renal medullary perfusion is markedly sensitive to cyclooxygenase inhibitors under physiological conditions (15, 25). This effect is most likely directly mediated because PGE2 potently dilates isolated and in situ preconstricted afferent arterioles (8, 21, 29), as well as isolated descending vasa recta in vitro (20). Glomerular expression of EP4 receptors has been extensively documented (4, 11, 12, 27), and recently EP2 expression and butaprost sensitivity were also reported in rat glomeruli (9). Our data confirm these observations and extend them to include the major resistance vessel segments. Thus we found EP4 receptor expression in both preglomerular resistance vessels and the vasa recta of the kidney outer medulla, which is in agreement with recent data from rat and human kidney (17, 21, 29). In contrast to EP4, we most consistently detected EP2 receptors in the vasa recta from the outer medulla that, in addition, were sensitive to butaprost. In previous reports (21, 29), EP2 receptors were not found in preglomerular rat vessels. Our data suggest the coexistence of EP2 and EP4 receptors in outer medullary vasa recta. The reason for the existence to two separate cAMP-coupled receptors in this vascular segment is not resolved by the present experiments. The receptors could be located in different cells, because the descending vasa recta consist of both a continuous layer of endothelial cells that control water, NaCl, and urea transport and a layer of contractile pericytes involved in regulation of medullary vascular resistance. Thus it is possible that EP2 receptors are involved in PGE2-mediated regulation of water and urea permeability of DTL and vasa recta, whereas EP4 receptors are involved in PGE2-mediated regulation of vascular tone.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by grants from the Danish Health Science Research Council (9903058, 9902742), the Novo Nordisk Foundation, the Foundation of 23-9-1909, the Danish Heart Association (98-1-2-8-22583, 99-2-2-36-22743), the Ms. Ruth T. E. König Petersens Research Foundation for Kidney Diseases, the Danish Medical Association Research Fund, and the Overlægerådets Legatudvalgs Fond.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: B. L. Jensen, Dept. of Physiology and Pharmacology, Odense Univ., Winsløwparken 21, 3, DK-5000, Odense C, Denmark (E-mail: bljensen{at}health.sdu.dk).
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 28 June 2000; accepted in final form 24 January 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Audoly, LP,
Tilley SL,
Goulet J,
Key M,
Nguyen M,
Stock JL,
McNeish JD,
Koller BH,
and
Coffman TM.
Identification of specific EP receptors responsible for the hemodynamic effects of PGE2.
Am J Physiol Heart Circ Physiol
277:
H924-H930,
1999
2.
Boie, Y,
Stocco R,
Sawyer N,
Slipetz DM,
Ungrin MD,
Neuschafer-Rube F,
Puschel GP,
Metters KM,
and
Abramovitz M.
Molecular cloning and characterization of the four rat prostaglandin E2 receptor subtypes.
Eur J Pharmacol
340:
227-241,
1997[ISI][Medline].
3.
Breyer, MD,
and
Breyer RM.
Prostaglandin E receptors and the kidney.
Am J Physiol Renal Physiol
279:
F12-F23,
2000
4.
Breyer, MD,
Davis L,
Jacobsson HR,
and
Breyer R.
Differential localization of prostaglandin E receptor subtypes in human kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F912-F918,
1996
5.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidinium-thiocyanate-phenol-chloroform extraction.
Annal Biochem
162:
156-159,
1987[ISI][Medline].
6.
Coleman, RA,
Smith WL,
and
Narumiya S.
VIII. International Union of Pharmacology Classification of Prostanoid Receptors: properties, distribution, and structure of the receptors and their subtypes.
Pharmacol Rev
46:
205-229,
1994[ISI][Medline].
7.
Dai, LJ,
Bapty B,
Ritchie G,
and
Quamme GA.
PGE2 stimulates Mg2+ uptake in mouse distal convoluted tubule cells.
Am J Physiol Renal Physiol
275:
F833-F839,
1998
8.
Edwards, RM.
Effects of prostaglandins on vasoconstrictor action in isolated renal arterioles.
Am J Physiol Renal Fluid Electrolyte Physiol
248:
F779-F784,
1985
9.
Hartner, A,
Pahl A,
Brune K,
and
Goppelt-Struebe M.
Upregulation of cyclooxygenase-1 and the PGE2 receptor EP2 in rat and human mesangioproliferative glomerulonephritis.
Inflamm Res
49:
345-354,
2000[ISI][Medline].
10.
Hebert, RL,
Jacobson HR,
Fredin D,
and
Breyer MD.
Evidence that separate PGE2 receptors modulate water and sodium transport in rabbit cortical collecting duct.
Am J Physiol Renal Fluid Electrolyte Physiol
265:
F643-F650,
1993
11.
Jensen, BL,
Mann B,
Skøtt O,
and
Kurtz A.
Differential regulation of renal prostaglandin receptor mRNAs by dietary salt intake in the rat.
Kidney Int
56:
528-537,
1999[ISI][Medline].
12.
Katsuyama, M,
Nishigaki N,
Sugimoto Y,
Morimoto K,
Negishi M,
Narumiya S,
and
Ichikawa A.
The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and Northern blot analysis.
FEBS Lett
372:
151-156,
1995[ISI][Medline].
13.
Kennedy, CRJ,
Zhang Y,
Brandon S,
Guan Y,
Coffee K,
Funk CD,
Magnuson MA,
Oates JA,
Breyer MD,
and
Breyer RM.
Salt-sensitive hypertension and reduced fertility in mice lacking the prostaglandin EP2 receptor.
Nature Med
5:
217-220,
1999[ISI][Medline].
14.
Kriz, W,
and
Kaissling B.
Structural organization of the mammalian kidney.
In: The Kidney: Physiology and Pathophysiology, edited by Seldin DW,
and Giebisch G.. New York: Raven, 1985, p. 265-306.
15.
Lemley, KV,
Schmitt SL,
Holliger C,
Dunn MJ,
Robertson CR,
and
Jamison RL.
Prostaglandin synthesis inhibitors and vasa recta erythrocyte velocities in the rat.
Am J Physiol Renal Fluid Electrolyte Physiol
247:
F562-F567,
1984
16.
Lykkegaard, S,
and
Poulsen K.
Ultramicroassay for plasma renin concentration in the rat using the antibody trapping technique.
Anal Biochem
75:
250-259,
1976[ISI][Medline].
17.
Morath, R,
Klein T,
Seyberth HW,
and
Nüsing RM.
Immunolocalization of the four prostaglandin E2 receptor proteins EP1, EP2, EP3 and EP4 in human kidney.
J Am Soc Nephrol
10:
1851-1860,
1999
18.
Narumiya, S,
Sugimoto Y,
and
Ushikubi F.
Prostanoid receptors: structures, properties, and functions.
Physiol Rev
79:
1193-1226,
1999
19.
Nemoto, K,
Pilbeam CC,
Bilak SR,
and
Raisz LG.
Molecular cloning and expression of a rat prostaglandin E2 receptor of the EP2 subtype.
Prostaglandins
54:
713-725,
1997[ISI][Medline].
20.
Pallone, TL.
Vasoconstriction of outer medullary vasa recta by angiotensin II is modulated by prostaglandin E2.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F850-F857,
1994
21.
Purdy, KE,
and
Arendshorst WJ.
EP1 and EP4 receptors mediate prostaglandin E2 actions in the microcirculation of rat kidney.
Am J Physiol Renal Physiol
279:
F755-F764,
2000
22.
Sakairi, Y,
Jacobson HR,
Noland TD,
and
Breyer MD.
Luminal prostaglandin E receptors regulate salt and water transport in rabbit cortical collecting duct.
Am J Physiol Renal Fluid Electrolyte Physiol
269:
F257-F265,
1995
23.
Sando, T,
Usui T,
Tanaka L,
Mori K,
Sasaki Y,
Fukuda Y,
Namba T,
Sugimoto Y,
Ichikawa A,
Narumiya S,
and
Nakao K.
Molecular cloning and expression of rat prostaglandin E receptor EP2 subtype.
Biochem Biophys Res Commun
200:
1329-1333,
1994[ISI][Medline].
24.
Shayakul, C,
Knepper MA,
Smith CP,
DiGiovanni SR,
and
Hediger MA.
Segmental localization of urea transporter mRNAs in rat kidney.
Am J Physiol Renal Physiol
272:
F654-F660,
1997
25.
Solez, K,
Fox JA,
Miller M,
and
Heptinstall RH.
Effects of indomethacin on renal inner medullary plasma flow.
Prostaglandins
7:
91-97,
1974[Medline].
26.
Sonnenburg, WK,
and
Smith WL.
Regulation cyclic AMP metabolism in rabbit cortical collecting tubule cells by prostaglandins.
J Biol Chem
263:
6155-6160,
1988
27.
Sugimoto, Y,
Namba T,
Shigemoto R,
Negishi M,
Ichikawa A,
and
Narumiya S.
Distinct cellular localization of mRNAs for three subtypes of prostaglandin E receptor in kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F823-F828,
1994
28.
Takeuchi, K,
Abe T,
Takahashi N,
and
Abe K.
Molecular cloning and intrarenal localization of rat prostaglandin E2 receptor EP3 subtype.
Biochem Biophys Res Commun
194:
885-891,
1993[ISI][Medline].
29.
Tang, L,
Loutzenhiser K,
and
Loutzenhiser R.
Biphasic actions of prostaglandin E2 on the renal afferent arteriole.
Circ Res
86:
663-670,
2000
30.
Tilley, SL,
Audoly LP,
Hicks EH,
Kim HS,
Flannery PJ,
Coffmann TM,
and
Koller BH.
Reproductive failure and reduced blood pressure in mice lacking the EP2 prostaglandin E2 receptor.
J Clin Invest
103:
1539-1545,
1999
31.
Torikai, S,
and
Kurokawa K.
Distribution of prostaglandin E2-sensitive adenylate cyclase along the rat nephron.
Prostaglandins
21:
427-438,
1981[Medline].
32.
Torikai, S,
and
Kurokawa K.
Effect of PGE2 on the cell cyclic AMP content in the thin descending limb of Henle of the rat.
Miner Electrolyte Metab
10:
21-25,
1984[ISI][Medline].
33.
Umemura, S,
Smyth DM,
and
Pettinger WA.
Regulation of renal cellular cAMP levels by prostaglandins and 2-adrenoceptors: microdissection studies.
Kidney Int
29:
703-707,
1986[ISI][Medline].
34.
Wade, JB,
Lee AJ,
Liu J,
Ecelbarger CA,
Mitchell C,
Bradford AD,
Terris J,
Kim GH,
and
Knepper MA.
UT-A2: a 55-kDa urea transporter in thin descending limb whose abundance is regulated by vasopressin.
Am J Physiol Renal Physiol
278:
F52-F62,
2000
35.
Watabe, A,
Sugimoto Y,
Honda A,
Irie A,
Namba T,
Negishi M,
Ito S,
Narumiya S,
and
Ichikawa A.
Cloning and expression of cDNA for a mouse EP1 subtype of prostaglandin E receptor.
J Biol Chem
268:
20175-20178,
1993
36.
Yang, T,
Hassan S,
Huang YG,
Smart AM,
Briggs JP,
and
Schnermann JB.
Expression of PTHrP, PTH/PTHrP receptor, and Ca2+-sensing receptor mRNAs along the rat nephron.
Am J Physiol Renal Physiol
272:
F751-F758,
1997
37.
Yang, T,
Singh I,
Pham H,
Sun D,
Smart A,
Schnermann JB,
and
Briggs JP.
Regulation of cyclooxygenase expression in the kidney by dietary salt intake.
Am J Physiol Renal Physiol
274:
F481-F489,
1998
38.
Yu, AS,
Hebert SC,
Brenner BM,
and
Lytton J.
Molecular characterization and nephron distribution of a family of transcripts encoding the pore-forming subunit of Ca2+ channels in the kidney.
Proc Natl Acad Sci USA
89:
10494-10498,
1992[Abstract].
39.
Zhang, Y,
Guan Y,
Schneider A,
Brandon S,
Breyer RM,
and
Breyer MD.
Characterization of murine vasopressor and vasodepressor prostaglandin E2 receptors.
Hypertension
35:
1129-1134,
2000