Increased glomerular prostaglandin E2 (PGE2) production is associated with the progression of diseases such as membranous nephropathy, nephrotic syndrome, and anti-Thy1 nephritis. We investigated the signaling pathways that regulate the synthesis and actions of PGE2 in glomerular podocytes. To study its actions, we assessed the ability of PGE2 to regulate the production of its own precursor, arachidonic acid (AA), in a mouse podocyte cell line. PGE2 dose-dependently reduced phorbol ester (PMA)-mediated AA release. Inhibition of PMA-stimulated AA release by PGE2 was found to be cAMP/PKA-dependent, because PGE2 significantly increased levels of this second messenger, whereas the inhibitory actions of PGE2 were reversed by PKA inhibition and reproduced by the cAMP-elevating agents forskolin and IBMX. PGE2 synthesis in this podocyte cell line increased fourfold at 60 min in response to PMA, coinciding with upregulation of cyclooxygenase (COX)-2 but not COX-1 levels. However, PGE2 synthesis was significantly reduced by COX-1-selective inhibition, yet to a lesser extent by COX-2-selective inhibition. Our findings suggest that PMA-stimulated PGE2 synthesis in mouse podocytes requires both basal COX-1 activity and induced COX-2 expression, and that PGE2 reduces PMA-stimulated AA release in a cAMP/PKA-dependent manner. Such an autocrine regulatory loop might have important consequences for podocyte and glomerular function in the context of renal diseases involving PGE2 synthesis.
- adenosine 3′,5′-cyclic monophosphate
- E-prostanoid receptors
prostaglandin e2 (PGE2) is synthesized by the metabolism of phospholipase A2 (PLA2)-derived arachidonic acid (AA) to prostaglandin G/H2 via cyclooxygenase (COX) isoforms (COX-1 or COX-2), followed by the activity of prostaglandin E synthase (17). Thus, as in many other cell types, phospholipid-derived AA liberated in glomerular podocytes could serve as a substrate for PGE2 synthesis depending on the available complement of COX isoforms. Interestingly, increased podocyte expression of COX-2 has been documented in both a diabetic rat model (20) as well as in a rat subtotal renal ablation model (41). This increased COX-2 expression might therefore translate into elevated PGE2 levels acting on neighboring cells or the podocytes themselves. In such a scenario, COX-2-derived PGE2 might therefore function as an autacoid in podocytes, interacting with one or more of its cell surface receptors.
PGE2 can interact with at least four G protein-coupled E-prostanoid (EP) receptor subtypes that have been cloned and characterized from a variety of species, including human, rabbit, rat, and mouse (1, 2, 5, 12, 16, 28, 32, 42, 44), and are designated EP1, EP2, EP3, and EP4. Several studies have confirmed the presence of both EP1 and EP4 receptor subtypes in glomerular podocytes. The EP4 receptor has been shown by in situ hybridization to be expressed at high levels in the glomerulus of both the mouse and rabbit (5, 33), whereas expression of EP1 and EP2 receptor subtypes could not be detected in either human or rabbit glomeruli (5).
Immunolocalization of the EP3 receptor has also been reported in human glomeruli (25). In contrast, Pavenstadt and coworkers (3) demonstrated the exclusive expression and signaling characteristics for both EP1 and EP4 receptors in a conditionally immortalized murine podocyte cell line. These studies demonstrated that PGE2elicited cAMP production via EP4 receptor populations while transiently increasing intracellular Ca2+ levels, likely via EP1 receptors in mouse podocytes.
Prostaglandin-initiated alterations of podocyte function may contribute to the progression of a variety of glomerular diseases. Evidence for this is derived from studies using nonsteroidal anti-inflammatory drugs (NSAIDs), which block the production of prostaglandins. Interestingly, proteinuria can be reduced by pharmacological treatments with NSAIDs both in experimental models of glomerular injury and in clinical practice (13, 23, 35, 37-39). The mechanism of action of NSAIDs in this context is currently unknown. However, in experimental models of glomerular injury and in the nephrotic syndrome in humans, the ability of NSAIDs to reduce proteinuria has been correlated with reductions in filtration rate, renal blood flow, and urinary PGE2 (9). Regulation of PG production in podocytes might therefore be an important factor in the function of the glomerular filtration barrier under both normal and pathophysiological conditions.
In the present study, we investigated whether differentiated mouse podocytes grown in cell culture can generate PGE2, the major renal prostanoid. Furthermore, we defined a role for PGE2 in regulating the availability of cytosolic PLA2 (cPLA2)-derived AA, the precursor to PGs. Our results provide novel evidence suggesting that PGE2acts as an autacoid to regulate arachidonate release from podocytes.
Cell culture materials, including RPM1 1640, fetal bovine serum (FBS), mouse recombinant γ-interferon, and penicillin/streptomycin, were purchased from Life Technologies (Burlington, ON). [5,6,8,9,11,12,14,15-3H]-arachidonic acid ([3H]AA), 218 Ci/mmol, the PGE2 enzyme immunoassay (EIA) kit, and biodegradable counting scintillant (BCS) were obtained from Amersham Pharmacia Biotech (Baie d'Urfé, QC, Canada). Indomethacin, fatty acid-free bovine serum albumin (BSA), forskolin (FSK), 3-isobutyl-1-methylxanthine (IBMX), type I collagen, sodium dodecyl sulfate (SDS), trichloroacetic acid, protease inhibitor cocktail (containing pepstatin A, bestatin, leupeptin, and aprotinin), diethyl ether, aspirin, and Tris base were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). The rabbit-anti-murine COX-1 polyclonal antibody, the rabbit-anti-murine COX-2 polyclonal antibody, PGE2, bromoenol lactone (HELSS), methyl arachidonyl fluorophosphonate (MAFP), oleyloxyethyl phosphorylcholine (OEL), SC-560, and SC-58125 were purchased from Cayman Chemical (Ann Arbor, MI). The Rp diastereomer of adenosine 3′,5′-cyclic monophosphothioate (Rp-cAMPS) was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). The cAMP assay kit was obtained from Research Diagnostics (Los Angeles, CA).
Culture of conditionally immortalized mouse podocytes was carried out as described in detail previously (26). Briefly, cells were grown on type I collagen-coated plastic tissue culture dishes in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Podocytes were routinely propagated at 33°C in RPMI culture medium supplemented with 10 U/ml mouse recombinant γ-interferon to promote expression of the temperature-sensitive large T antigen. Differentiation was induced by maintaining cultures at 37°C in medium without γ-interferon for at least 7 days. mRNA expression of both EP1 and EP4 receptor subtypes, but not EP2 or EP3 subtypes, was confirmed by RT-PCR as previously reported (3).
Measurement of AA release.
The protocol employed is a modified version of one that we have previously used (19). Cells were plated onto 12-well plastic culture dishes without γ-interferon and allowed to differentiate at 37°C for 7 days. Cultures were serum-starved overnight in RPMI 1640 plus 0.1% FBS, and the membrane phospholipid pools were labeled with 0.3 μCi [3H]AA. After 24 h, cells were washed three times with Hanks' balanced salt solution (HBSS) plus 0.05% fatty acid-free BSA and preincubated for 30 min with this solution containing 20 μM indomethacin to block endogenous prostaglandin synthesis. Subsequently, cells were stimulated for the indicated time periods with the appropriate agent. When the effect of a specific inhibitor was examined, the inhibitor was included during both the preincubation and stimulatory periods. Media were collected and centrifuged at 12,000 g to pellet any cellular debris. An aliquot of the supernatant was measured for [3H]AA content by scintillation counting with 10 ml of BCS. Results were normalized for total label incorporated into the cells by dividing the disintegrations per minute (dpm) of [3H]AA released by the total dpm of [3H]AA incorporated into the cells (obtained by solubilizing the cells in 5% SDS). In some cases results were expressed as percent inhibition of PMA-stimulated AA release.
Cells were grown for 7 days on 12-well cluster dishes at 37°C. Cells were serum-starved overnight with RPMI 1640 containing 0.1% FBS. After serum starvation, cells were preincubated for 30 min with HBSS plus 0.05% BSA containing 20 μM indomethacin and were then exposed for 5 min to HBSS plus 0.05% BSA containing 0.5 mM IBMX to inhibit phosphodiesterase activity. Incubations were next carried out for 10 min in HBSS plus 0.05% BSA containing 0.5 mM IBMX and other agents as specified. Reactions were terminated with the addition of ice-cold 10% (vol/vol) trichloroacetic acid, and after 30 min at 4°C, each sample was washed four times with four volumes of water-saturated diethyl ether and brought to pH 7.4 with Tris base. The cAMP content was determined with a radioimmunoassay kit (DPC; Research Diagnostics) making use of a specific cAMP-binding protein.
Cells were grown for 7 days on type I collagen-coated 96-well cluster dishes at 37°C and serum-starved overnight with RPMI 1640 containing 0.1% FBS. After serum starvation, cells were preincubated for 30 min with or without 20 μM indomethacin, 0.1 mM aspirin (to irreversibly inactivate COX activity), and 0.01–1,000 nM SC-58125 or 0.01–1,000 nM SC-560, followed by addition of 100 nM PMA for 60 min at 37°C in RPMI-1640 plus 0.1% FBS. Cell supernatants were assayed for PGE2 by EIA using a specific anti-PGE2 antibody according to the manufacturer's protocol (Amersham Pharmacia Biotech).
Western blotting of COX isoforms in mouse podocytes.
Cells were grown for 7 days on type I collagen-coated 100-mm dishes at 37°C. Cells were then serum-starved overnight with RPMI 1640 containing 0.1% FBS and then stimulated for 1 h with 100 nM PMA. Cells were then rinsed twice with ice-cold PBS and scraped off the plates, followed by centrifugation at 12,000 g. Cell pellets were resuspended and sonicated briefly in lysis buffer consisting of PBS and protease inhibitor cocktail. Samples containing 50 μg of total cellular protein [as determined by Bio-Rad protein assay method (Mississauga, Ontario) with BSA as a standard] were diluted with 2× Laemmli buffer, electrophoresed on 8% resolving gels, and transferred to nitrocellulose membranes using a Bio-Rad Mini Transblot system. Western analysis was carried out by using either a rabbit polyclonal anti COX-2 antibody (1:250 dilution; Cayman Chemical) or a rabbit polyclonal anti COX-1 antibody (1:250 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in conjunction with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution). Blots were developed with a Supersignal West Pico chemiluminescent substrate (Pierce, Rockford, IL) and Kodak X-Omat Blue XB-1 film (Mandel Scientific, Guelph, ON, Canada).
Real-time RT-PCR of COX isoforms in mouse podocytes.
Cells were grown for 7 days on type I collagen-coated 100-mm dishes at 37°C. Podocytes were then serum-starved overnight with RPMI 1640 containing 0.1% FBS and stimulated for 1 h with 100 nM PMA. RNA was extracted by using an RNeasy kit according to the manufacturer's instructions (Qiagen, Valencia, CA). COX-1 and COX-2 mRNA levels were determined by real-time RT-PCR using TaqMan One-Step RT-PCR master mix reagents (Applied Biosystems, Branchburg, NJ) and an ABI Prism 7000 sequence detection system. Reactions were carried out by using 50 ng of total podocyte RNA under the following conditions: 48°C for 30 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Primers and TaqMan probe for murine COX-1 were as follows: forward primer, 5′-CCA GAA CCA GGG TGT CTG TGT-3′; reverse primer, 5′-GTA GCC CGT GCG AGT ACA ATC-3′; probe, 6FAM-CGC TTT GGC CTC GAC AAC TAC CAG TG-TAMRA. Primers and TaqMan probe for murine COX-2 were as follows: forward primer, 5′-GGG TGT CCC TTC ACT TCT TTC A-3′; reverse primer, 5′-TGG GAG GCA CTT GCA TTG A-3′; probe, 6FAM-TGT GCA AGA TCC ACA GCC TAC CAA AAC A-TAMRA. Primers and probes were verified for specificity by BLASTing against the National Center for Biotechnology Information database. Reactions yielded single amplicon products of predicted size. Values were normalized to GAPDH mRNA levels in each sample as determined by a TaqMan Rodent GAPDH control reagent kit (Applied Biosystems).
Data are expressed as means of duplicate determinations from individual experiments and are presented as means ± SE wheren ≥ 4 or as means ± SD where n = 3 experiments. Statistical significance was accepted atP < 0.05 as determined by ANOVA followed by a Newman-Keuls multiple comparison test or was alternatively determined by a paired t-test where appropriate. IC50 and EC50 values were calculated by nonlinear regression analysis by assigning a line of best fit to each data set using GraphPad Prism software.
PMA stimulates AA release from mouse podocytes.
Activation of protein kinase C via tumor-promoting phorbol esters (PMA) is known to promote the release of AA in a variety of cell types. To investigate AA release in mouse podocytes, we incubated cells with PMA and measured [3H]AA release into the medium. As shown in Fig. 1, Aand B, PMA caused a substantial dose-dependent (EC50 = 6.5 nM) release of AA that was statistically significant as early as 10 min (PMA, 6.01 ± 1.01% of total [3H]AA incorporated; control, 2.79 ± 0.21% of total [3H]AA incorporated; P < 0.01) and plateaued around 60 min (PMA, 31.67 ± 2.02% of total [3H]AA incorporated; control, 12.74 ± 0.70% of total [3H]AA incorporated; P < 0.001). The total [3H]AA incorporated into the cells was not different between treatment groups. PMA-stimulated AA release was significantly reduced by the cPLA2 inhibitor MAFP (5 μM) (Fig. 1 C; PMA alone, 7.10 ± 0.40% of total [3H]AA incorporated; PMA + MAFP, 4.00 ± 0.31% of total [3H]AA incorporated; P < 0.01) but not by the Ca2+-independent PLA2 inhibitor HELSS (15 μM) (6.30 ± 0.49% of total [3H]AA incorporated; not significant vs. PMA alone) or the secretory PLA2 inhibitor OEL (15 μM) (5.60 ± 0.26% of total [3H]AA incorporated; not significant vs. PMA alone).
We next evaluated whether PGE2 could elicit AA release. Cells were incubated with 1 μM PGE2 in the presence of 20 μM indomethacin to block endogenous PG production. Figure1 D shows that the addition of 1 μM PGE2 had no significant effect on AA release compared with control (control, 1.20 ± 0.20% of total [3H]AA incorporated; PGE2, 1.29 ± 0.28% of total [3H]AA incorporated). The EP1/EP3-selective agonist sulprostone (1 μM) likewise had no significant effect on AA release (1.22 ± 0.22% of total [3H]AA incorporated). Furthermore, bradykinin (BK; 1 μM) (1.43 ± 0.28% of total [3H]AA incorporated), endothelin-1 (1 μM), angiotensin II (1 μM), TNF-α (1 μg/ml), ATP (1 μM), IL-1β (1 μg/ml), and transforming growth factor-β (1 μg/ml) did not stimulate AA release (not shown).
PGE2 inhibits PMA-stimulated AA release from mouse podocytes.
We next investigated a role for PGE2 in regulating PMA-stimulated AA release from mouse podocytes. As shown in Fig.2, coincubation of podocyte cultures with PGE2 and PMA led to a concentration-dependent and significant reduction in AA release, reaching 32.7 ± 4.9% inhibition with 1 μM PGE2 (P < 0.05 vs. PMA alone) with an IC50 of 87 nM. The EP1/EP3-selective agonist sulprostone failed to reproduce the effect of PGE2 (2.5 ± 0.5% inhibition, not significant vs. PMA alone), suggesting that the remaining EP subtype present in these cells (i.e., the EP4 receptor) mediates the inhibitory actions of PGE2 on PMA-induced AA release.
Inhibition of PKA restores PMA-stimulated AA release.
To determine whether cAMP might be involved in PGE2-mediated inhibition of AA release, we first assessed the ability of PGE2 to elicit cAMP production in these cells. As shown in Fig. 3, PGE2 significantly and dose-dependently increased cAMP levels from 0.7 ± 0.3 pmol/well to 5.4 ± 0.8 pmol/well (1 μM PGE2, P < 0.001 vs. control) with an EC50 of 52 nM. This PGE2-stimulated cAMP increase was not reproduced by the EP2-selective agonist butaprost (0.8 ± 0.4 pmol/well; not significant). Incubation of the cells with the cAMP-elevating agents FSK and IBMX increased cAMP levels from 0.7 ± 0.3 pmol/well to 20.2 ± 1.3 pmol/well (P < 0.001 vs. control). As shown in Fig.4, this combination of cAMP-elevating agents significantly reduced PMA-stimulated AA release (PMA, 8.4 ± 0.8% of total label incorporated vs. FSK + IBMX, 5.2 ± 0.3% of total label incorporated, P < 0.001).
To evaluate the possible involvement of cAMP-dependent protein kinase (PKA) in mediating the inhibitory actions of PGE2, we employed the PKA-selective inhibitor Rp-cAMPS (22). Preincubation of the cells with 100 μMRp-cAMPS 1 h before addition of PGE2/PMA reduced PGE2-mediated inhibition of PMA-induced AA release (Fig. 5; PMA, 8.4 ± 0.5% of total label incorporated vs. PMA + PGE2, 6.4 ± 0.4% of total label incorporated, P < 0.01; PMA + PGE2 + Rp-cAMPS, 7.6 ± 0.7% of total label incorporated, P < 0.05 vs. PMA + PGE2), thereby supporting a role for PKA in mediating the inhibitory actions of PGE2.
Podocytes generate PGE2 via both COX-1 and COX-2.
To determine whether PMA-stimulated AA release could promote COX-induced synthesis of PGE2 in this podocyte cell line, we performed an EIA on the cell supernatants after acute incubation with phorbol ester. As shown in Fig. 6, incubation of podocyte cultures with 100 nM PMA for 60 min increased PGE2 synthesis fourfold (control, 39.0 ± 3.2 pmol/well; PMA, 159.7 ± 11.6 pmol/well, P < 0.01). The COX-2 inhibitor SC-58125 blocked PGE2 production at concentrations >0.1 μM. However, despite the relatively high dosage required for inhibition, this compound is very selective with IC50 values >10 μM for COX-1 and 50 nM for COX-2 (29). Because the COX-2 gene is known to be inducible by PMA, we irreversibly inactivated endogenous COX by preincubating the cultures with 0.1 mM aspirin for 30 min to determine whether PMA stimulation was causing increased COX-2 expression, which might thereby contribute to the observed PGE2 synthesis (18,27). As shown in Fig. 6, aspirin preincubation markedly reduced PMA-stimulated PGE2 production (aspirin, 33.1 ± 1.5 pmol/well; aspirin + PMA, 69.5 ± 2.8 pmol/well,P < 0.01 vs. both aspirin alone and PMA alone). In contrast, the COX-1-selective inhibitor SC-560 blocked PGE2production with a much higher potency, yielding an IC50 of 7.5 nM. The reported IC50 values for SC-560 are 9 nM for COX-1 and 6.3 μM for COX-2 (30).
To further confirm the identity of the COX isoform(s) that contribute to PMA-stimulated PGE2 synthesis, we performed Western blotting and real-time RT-PCR. As shown in Fig.7 A, low yet detectable expression of COX-2 in podocyte cell lysates was observed by Western blotting with a polyclonal anti-murine COX-2 antibody. COX-2 expression was markedly increased after 1 h of stimulation with PMA. A rabbit anti-murine COX-1 antibody detected expression of this isoform in cell lysates by Western blot analysis that did not change after 1 h of incubation with PMA. Finally, real-time RT-PCR was carried out by using TaqMan COX-1 and COX-2 probes with total RNA derived from podocytes incubated with or without 100 nM PMA for 1 h. When normalized to amplified GAPDH as an internal control, COX-2 mRNA levels were significantly increased (COX-2 + PMA, 63.2 ± 2.4 arbitrary units; COX-2 control, 1.0 ± 0.1; P < 0.001), whereas COX-1 mRNA levels remained constant after 1 h of PMA incubation (Fig. 7 B).
Prostaglandins are important lipid mediators exerting influence over a variety of renal functions, including the regulation of vascular tone and fluid homeostasis. Prostaglandins have been implicated in the development of proteinuria in the nephrotic syndrome. It is thought that changes in the ultrafiltration coefficient (K f) via modification of podocyte foot processes result in a compromised barrier to protein (10). The intracellular signaling cascades that regulate both the synthesis and actions of prostaglandins in podocytes, including the most abundant renal prostaglandin, PGE2, have not been fully elucidated.
In the present study, phorbol ester significantly and acutely increased cPLA2-catalyzed liberation of AA, the precursor to PGE2, more than fourfold from a differentiated mouse podocyte cell line. In contrast, we failed to observe any significant increases in AA release in response to either PGE2 or the EP1/3 receptor agonist sulprostone. These observations may be explained by insufficient expression of EP1 receptors in this cell line, despite the detection of EP1 mRNA by RT-PCR in the present study (see experimental procedures), thereby confirming the results of Bek et al. (3). These same studies reported that EP1 receptor-mediated Ca2+ elevations were sporadic and not observable for many of the cells tested. The absence of PGE2-mediated AA release is also consistent with the findings of others who were unable to detect podocyte EP1 receptor mRNA by in situ hybridization in rabbit and human tissue (5, 21). Our results indicating a lack of PGE2-mediated AA release could be a reflection of the cell line employed or, simply, the EP1 receptor may not play a significant role in mediating the actions of PGE2 in podocytes.
The EP3 receptor has yet to be observed specifically in podocytes; however, its expression has been detected in human glomeruli by an immunohistochemical approach (25). The mouse podocyte cell line used in the present study does not exhibit EP3 receptor mRNA (3), thereby making the involvement of this receptor in the PGE2-mediated effects in these cells unlikely. Several lines of evidence point to abundant EP4 receptor subtype expression and signaling in podocytes. For example, in situ hybridization studies of rabbit, human, and mouse glomeruli have revealed ample expression of EP4 mRNA in the podocytes (4, 33). Using immunohistochemical approaches, Nusing and colleagues (25) demonstrated podocyte expression of the EP4 receptor in human kidney sections employing an EP4 receptor polyclonal antibody. The podocyte cell line employed in the current study has been shown to express functional EP4 receptors (3). In the present study, we confirmed the presence of (by RT-PCR, see experimental procedures), and butaprost-insensitive cAMP elevation via, the EP4 receptor. Finally, the effects of PGE2 on cAMP levels cannot be accounted for by the prostacyclin (IP) receptor because of its absence in this cell line (3).
The role of PGE2 in podocyte biology remains speculative. However, its ability to raise intracellular cAMP levels may directly influence the availability of its precursor, AA. Studies in other cell types have shown that G protein-coupled receptor agonists can reduce AA release via elevations in cAMP. For example, in MDCK-D1cells, elevation of cAMP significantly reduced nucleotide-stimulated P2Y receptor-mediated AA release in a PGE2-dependent manner (43). Likewise, we have previously shown that BK-stimulated AA release is inhibited via a cAMP-dependent signaling cascade (19). Our present findings suggest that an analogous mechanism is present in podocytes, because PGE2dose-dependently reduced PMA-stimulated AA release in a cAMP/PKA-dependent manner. Such regulation of AA release from podocytes might limit substrate availability for subsequent COX-mediated prostaglandin synthesis in various glomerular diseases. Of the two cyclooxygenase isoforms, COX-1 is regarded as a constitutively expressed isoform (8, 11). In the kidney, it is found in renal vasculature, mesangial cells, and the collecting duct (31). In contrast, renal COX-2 expression is more restricted, being localized to the cells of the macula densa, cortical thick ascending limb, and medullary interstitium (14). COX-2 expression can be induced in many cell types in response to various stimuli, including phorbol esters (6, 7, 24, 34, 36,40). There is also evidence for in vivo upregulation of COX-2 in the podocytes following anti-Thy1 nephritis (15), in a diabetic rat model (20), and in a rat model of subtotal renal ablation (41). Our results indicate that in mouse podocytes, both COX isoforms contribute to acute PGE2production. Nearly two-thirds of the PGE2 produced within 1 h of PMA stimulation is derived from the activity of basal COX-1 levels. However, within that hour PMA rapidly stimulates COX-2 expression that subsequently contributes to the remaining one-third of the PGE2 production observed. Synthesis of such PGE2, if it were produced in vivo by the podocytes, would likely act locally because circulating prostaglandin levels are efficiently metabolized after a single pass through the lungs. PGE2 generated by podocytes may therefore act in an autocrine or paracrine fashion via cell surface EP4receptors, ultimately limiting its own production. This regulatory mechanism may restrict the extent of physiological effects mediated by the podocytes, including morphological alterations that lead to proteinuria.
In summary, our data support the hypothesis of COX-1- and COX-2-dependent PGE2 synthesis in mouse podocytes and the possibility that this prostanoid, acting through the EP4receptor, reduces AA release in a cAMP/PKA-dependent manner in response to PMA. Such a regulatory loop might directly impact podocyte function under both normal and pathophysiological conditions that require changes in PGE2 synthesis.
We thank Dr. Peter Mundel for the kind gift of the murine podocyte cell line. We offer special thanks to Dr. Kevin D. Burns for critical assessment of this manuscript.
This work was supported by Canadian Institutes of Health Research Grant MOP-44013. C. R. J. Kennedy is the recipient of a Biomedical Scholarship from the Kidney Foundation of Canada.
Address for reprint requests and other correspondence: C. R. J. Kennedy, Ottawa Health Research Institute, Division of Nephrology, Ottawa Hospital and Univ. of Ottawa, 451 Smyth Rd. Rm. 1317, Ottawa, Ontario, Canada K1H 8M5 (E-mail:).
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.
First published September 25, 2002;10.1152/ajpcell.00024.2002
- Copyright © 2003 the American Physiological Society