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Am J Physiol Renal Physiol 293: F1571-F1576, 2007. First published August 15, 2007; doi:10.1152/ajprenal.00132.2007
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Hydrogen peroxide stimulates chloride secretion in primary inner medullary collecting duct cells via mPGES-1-derived PGE2

Sunhapas Soodvilai, Zhanjun Jia, and Tianxin Yang

Department of Internal Medicine, University of Utah, and Veterans Affairs Medical Center, Salt Lake City, Utah

Submitted 20 March 2007 ; accepted in final form 30 July 2007


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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We investigated the role and mechanism of H2O2 in regulation of NaCl transport in primary inner medullary collecting duct (IMCD) cells. IMCD cells were isolated from wild-type mice and grown onto semipermeable membranes, and short-circuit current (Isc) was determined by Ussing chamber. Exposure of IMCD cells to H2O2 at a range of 100–300 µM caused a rapid increase in Isc in a time- and dose-dependent manner. This increase was almost abolished by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel inhibitors diphenylamine-2-carboxylic acid (DPC) and CFTRinhibitor-172. In contrast, the magnitude of stimulation was unaffected by the epithelial Na+ channel (ENaC) inhibitor amiloride. The H2O2-induced Cl– secretion was significantly inhibited by indomethacin, as well as by microsomal PGE synthase-1 (mPGES-1) deficiency. Like H2O2, PGE2 treatment induced a twofold increase in Isc that was reduced by the protein kinase A (PKA) inhibitors H-89 and KT5720. These data suggest that H2O2 stimulates CFTR Cl– channel-mediated Cl– secretion through cyclooxygenase- and mPGES-1-dependent release of PGE2 and subsequent activation of PKA.

hydrogen peroxide; microsomal prostaglandin E synthase-1; cystic fibrosis transmembrane conductance regulator

COLLECTING DUCT plays an important role in modifying urine concentration by regulation of water and electrolyte reabsorption. The major electrolytes controlled by this segment are Na+ and Cl– (1, 29). The general mechanism of Na+ reabsorption in collecting duct involves coordinated Na+ transport from lumen across apical membrane via amiloride-sensitive Na+ channel (ENaC), and Na+ exits from the cell to interstitial fluid across the basolateral membrane via Na+-K+-ATPase (22). Unlike Na+, Cl– could be both absorbed and secreted in the collecting duct where Cl– transport occurs through distinct mechanisms (25). First, Cl– could be actively reabsorbed by an electrogenic Cl–/HCO3– exchanger expressed on the apical membrane and then exits the cell via a Cl– channel. Second, Cl– could diffuse passively down an electrochemical gradient via a paracellular pathway. Third, Cl– could be secreted via a cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel expressed on the apical membrane (31).

The collecting duct is responsible for absorption of approximately 2–3% of the filtered fluid and is critically important for final adjustment of urinary Na+, K+, Cl–, H+, water, and urea excretion. This is consistent with the fact that the fluid reabsorption rate in the collecting duct is subjected to regulation by a number of hormones and autocrine/paracrine factors, including aldosterone, insulin, arginine vasopressin, PGE2, nitric oxide, and ANG II (1, 2, 4, 6, 10–12, 15, 17, 18, 26). Emerging evidence suggests that reactive oxygen species (ROS) might represent a novel class of regulators of tubular fluid reabsorption. It has been reported that ROS regulate NaCl reabsorption in isolated, perfused thick ascending limbs through protein kinase C (19–21, 27). Additionally, peroxynitrite (OONO–), a reaction product of superoxide and nitric oxide, inhibits Na+-K+-ATPase activity and paracellular permeability in proximal tubule cells (14). By contrast, the role of ROS in the collecting duct has been studied in much less detail. The observation that manipulation of redox state in the renal medulla influences urinary sodium excretion without an effect on glomerular filtration rate (16, 36) seems to suggest a potential role of ROS in regulation of collecting duct function. Therefore, the present study was undertaken to determine the effect of H2O2 on NaCl transport in inner medullary collecting duct (IMCD) cells.


   MATERIALS AND METHODS
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RESULTS
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Materials. DMEM (ATCC), Snapwell (Costa), collagen type I (Worthington Biochemical), hyaluronidase, amiloride, diphenylamine-2-carboxylic acid (DPC), and epidermal growth factor (EGF) were purchased from Sigma; PGE2, H-89, CFTRinhibitor-172, and KT-5720 were obtained from Calbiochem; and indomethacin was from Cayman Chemicals. All other chemicals were purchased from standard sources and were generally of the highest purity available.

Animals. mPGES-1 mutant mice were originally generated by Trebino et al. (30). This mouse colony was propagated at the University of Utah and maintained on a mixed DBA/1lacJ*C57BL/6 background. All protocols employing mice were approved by the University of Utah Institutional Animal Care and Use Committee and were conducted in accordance the guidelines and principles of this Committee.

Cell culture. Primary cultures of renal IMCD cells were generated with a modification of a previous study (9). In brief, mice were killed by cervical dislocation, and kidneys were quickly removed under sterile condition. The renal medulla was dissected, minced, and digested for 60 min in 10 ml of medium (DMEM) containing 0.2% collagenase type I, 0.2% hyaluronidase, and 0.025% trypsin-EDTA at 37°C with shaking. After incubation, 20 ml of sterile distilled water was added for 20 min to lyse cells other than collecting duct cells by osmotic shock (100 mosmol/kgH2O). Cells were then centrifuged at 1,000 rpm for 5 min, the supernatant was discarded, and the pellet was resuspended in the modified medium (DMEM, 10% fetal bovine serum, 20 ng/ml EGF, and 100 U/ml penicillin G-streptomycin sulfate). Cells were seeded onto semipermeable membranes (12-mm-diameter Snapwell) for electrophysiological studies. After the cells reached confluence, usually in 7–10 days, the cells were incubated in serum-free media for at least 24 h before experiments. The cell monolayers were confirmed to be confluent by development of high resistance. The transepithelial resistance (Rte) and the transepithelial voltage (Vte) of cultures were examined using EVOM epithelial volt-ohmmeter and a set of two stick STX electrodes (Wold Precision Instruments, Sarasota, FL). Only cell monolayers that develop high resistance >2,000 ohm/cm2 were used for electrophysiological studies.

Electrophysiological transepithelial measurements. Snapwell semipermeable membranes containing confluent cell monolayers were mounted in an Ussing chamber (Physiologic Instruments, San Diego, CA), and both surfaces of the cell monolayer were bathed in DMEM maintained at 37°C and equilibrated in 5% CO2-95% O2 to keep pH at 7.4. Vte was clamped at zero using a VCC600 voltage-clamp apparatus (Physiologic Instruments), and then the short-circuit current (Isc) was recorded using Ag-AgCl electrode in agar brides. Positive Isc reflects the active transport of cation (Na+) from apical side to basolateral side of media or transport of anion (Cl–) from basolateral to apical side of media.

Enzyme immunoassay. Culture medium samples were centrifuged for 5 min at 10,000 rpm and diluted 1:5 with enzyme immunoassay buffer. Concentrations of PGE2 were determined by enzyme immunoassay (Cayman Chemicals).

Data analysis. Data are summarized as means ± SE. Statistical analysis was performed using one-way ANOVA or Student's t-test as appropriate.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Basal electrical parameters of primary IMCD cells. The confluent monolayer of IMCD cells grown on semipermeable membranes were mounted in Ussing chamber for a 60-min equilibration period. The basal Isc, Vte, and Rte were 2.37 ± 0.11 µA/cm2, 7.16 ± 0.58 mV, and 2,563 ± 146 ohm/cm2, respectively (n = 50). The Isc developed in this cell type appeared to be mainly due to amiloride-sensitive epithelial sodium channel (ENaC) since the final addition of 100 µM amiloride to the apical media decreased Isc by 72.0 ± 2.0%. To verify responsiveness of the isolated IMCD cells to a defined stimulus, we determined the effect of aldosterone on Isc in these cells. Exposure to 1 µM aldosterone for 24 h caused an increase of Isc from 2.53 ± 0.22 to 4.67 ± 0.22 µA/cm2 (n = 10), and this stimulation was inhibited by apical administration of amiloride, indicating involvement of ENaC.

Effect of H2O2 on Isc in IMCD cell monolayers. We first examined the dose response of H2O2 on Isc. Following the equilibrium period, H2O2 was added to the basolateral side of the IMCD cell monolayers. As shown in Fig. 1A, at 1 min, H2O2 dose-dependently increased Isc that was detectable at 100 µM and maximal at 300 µM (from 2.72 ± 0.22 to 2.95 ± 0.21 and to 3.60 ± 0.21 µA/cm2). The stimulatory effect of H2O2 on Isc was reversible since after washout of H2O2 the Isc returned to the basal level. H2O2 at 300 µM was chosen as a concentration for subsequent experiments. Next, we determined the time course of H2O2-induced Isc. After a 60-min equilibration period, IMCD cell monolayers were incubated with 300 µM H2O2 for various periods of time, and the Isc was recorded. As shown in Fig. 1B, H2O2 at 300 µM induced a rapid and transient increase in Isc, peaking at 1 min. Apical administration of H2O2 had a similar stimulatory effect on the Isc (data not shown). A positive Isc may reflect the active transport of Na+ transport from apical to basolateral side of the monolayer or Cl– transport from basolateral to apical side of the monolayer. Therefore, we next determined whether the increase of Isc in response to H2O2 was mediated by Na+ or Cl– transport. First, the effect of H2O2 was determined when the ENaC was inhibited by amiloride. After the equilibration period, amiloride (final concentration at 100 µM) was added to the apical side of monolayers for 5 min to inhibit ENaC, followed by addition of 300 µM H2O2 to the basolateral side. As shown in Fig. 2, H2O2 increased the Isc in the presence of amiloride, indicating amiloride-insensitive currents. Next, we examined whether Cl– transport contributed to the H2O2-stimulated Isc using the nonspecific (CFTR) Cl– channel blocker DPC and the specific CFTR inhibitor, CFTRinhibitor-172. Addition of 1 mM DPC or 20 µM CFTRinhibitor-172 on top of amiloride almost abolished the H2O2-stimulated Isc. These results indicated that CFTR plays a major role in H2O2-stimulated amiloride-insensitive Isc. To validate this result, we pretreated the IMCD cell monolayers with 1 mM DPC for 5 min, followed by application of 300 µM H2O2 at the basolateral side. In the presence of DPC, the H2O2-induced Isc was completely eliminated (Fig. 3).


Figure 1
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  		Fig. 1. Effect of H2O2 on short-circuit current (Isc) in primary inner medullary collecting duct (IMCD) cell monolayers. A: dose-response. B: time course. In experiment in A, the monolayers were equilibrated for 60 min, and then the incubated with various concentrations of H2O2 at 1 min; then the Isc was recorded. In the experiment in B, after equilibration period, the monolayers were incubated with 300 µM H2O2 at different time intervals. Each time point represents mean ± SE of Isc for 9 monolayers (A) and 7 monolayers (B). *Significantly different from control as determined by pair t-test (P < 0.01).

 

Figure 2
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  		Fig. 2. Effect of cystic fibrosis transmembrane conductance regulator (CFTR) inhibitors on H2O2-stimulated Isc in the presence of 100 µM amiloride in primary IMCD cell monolayers. The stimulation effect of 300 µM H2O2 was diminished in the presence of 1 mM diphenylamine-2-carboxylic acid (DPC) or 20 µM CFTRinhibitor-172 (C-172). Results are expressed as means ± SE for 10 monolayers. *P < 0.01 compared with control. **P <0.05 compared with H2O2-treated group.

 

Figure 3
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  		Fig. 3. Effect of H2O2 in the presence of DPC in Isc in primary IMCD cell monolayers. The monolayers were equilibrated for 60 min, and then 1 mM DPC was added to the apical side of media for 5 min. H2O2 (300 µM) was applied to the basolateral of membrane. After 25-min incubation, 100 µM amiloride was added to apical side of membrane. Results are means ± SE of Isc for 9 monolayers. *P < 0.01 compared with control.

 
Role of PGE2 in H2O2-stimulated Isc. H2O2 has been reported to stimulate PGE2 release from proximal tubule and collecting duct cells (7, 35), raising a possibility that PGE2 may mediate the H2O2-stimulated Cl– transport. To test this hypothesis, the monolayers were preincubated with 10 µM indomethacin, a nonselective cyclooxygenase (COX) inhibitor, for 30 min and then with 100 µM amiloride for 5 min, followed by a final addition of 300 µM H2O2 to the basolateral side. Indomethacin had no effect on basal Isc but significantly attenuated the H2O2-stimulated Isc (Fig. 4A), indicating the requirement of COX activity. PGE2 is the major metabolite of arachidonic acid produced in the kidney by the activity of COX, which exists in two isoforms (COX-1 and COX-2). To examine involvement of a specific COX isoform, we determined the effect of SC-560, a COX-1 inhibitor, and Cay10404, a COX-2 inhibitor, on the H2O2-induced Cl– current. For COX inhibitors and H2O2 treatments, the Isc were recorded after the monolayers were incubated with 0.5 µM SC-560 or 1 µM Cay10404 for 30 min or 300 µM H2O2 for 1 min. For the COX inhibitors plus H2O2, the monolayer were preincubated with COX inhibitors for 30 min, followed by adding H2O2 for 1 min. As shown in Fig. 4B, exposure to SC-560 completely abolished the effect of H2O2. In contrast, exposure to Cay10404 only slightly affected H2O2-induced Cl– current. To evaluate the contribution from PGE2, the effect of H2O2 was determined in IMCD cell monolayers isolated from mPGES-1 –/– mice. The H2O2-stimulated Isc was significantly attenuated in mPGES-1 –/– IMCD cells compared with the +/+ cells (Fig. 5A). To support the contention that H2O2 induced PGE2 release mediated by mPGES-1, the effect of H2O2 on PGE2 release was determined in mPGES-1 –/– IMCD cells compared with the +/+ cells. As shown in Fig. 5B, H2O2 stimulated PGE2 release in the +/+ IMCD cells. This stimulation was diminished in mPGES-1 –/– IMCD cells. This result indicated that H2O2-induced PGE2 release mediated by mPGES-1.


Figure 4
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  		Fig. 4. Effect of H2O2 and indomethacin (Indo) (A) and selective cyclooxygenase (COX)-1 and COX-2 inhibitors (B) on Isc in the presence of 100 µM amiloride in primary IMCD cell monolayers. A: effect of H2O2 and Indo on Isc in the presence of 100 µM amiloride. Results are expressed as means ± SE for 12 monolayers. Conditions of incubation are given under each bar. For Indo and H2O2 treatments, Isc was recorded after the monolayers were incubated with 10 µM Indo for 30 min or 300 µM H2O2 for 1 min. For Indo plus H2O2, the monolayer were preincubated with 10 µM Indo for 30 min, followed by adding H2O2 for 1 min. B: effect of 0.5 µM SC-560 and 1 µM Cay10404 on H2O2-induced Isc. Conditions of incubation are given under each bar (see details in text). *P < 0.01 compared with control. **P < 0.05 compared with H2O2-treated group.

 

Figure 5
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  		Fig. 5. Effect of H2O2 on Isc in the presence 100 µM amiloride in primary microsomal PGE synthase-1 (mPGES-1) –/– IMCD cells (A), and the effect of H2O2 on PGE2 release (B). A: effect of H2O2 on Isc in the presence of 100 µM amiloride in primary mPGES-1 –/– IMCD cells. The IMCD cell monolayers were isolated from mPGES-1 –/– mice [knockout (KO)] and the mPGES-1 +/+ mice [wild type (WT)]. After equilibration period, 100 µM amiloride was added to the apical side, and then 300 µM H2O2 was added to the basolateral side of media. Results are expressed as means ± SE for 9 monolayers for WT and 10 monolayers for KO. *P < 0.01 compared with control. #P < 0.01 compared with H2O2-treated group of WT. B: effect of H2O2 on PGE2 release in mPGES-1 –/– and mPGES-1 +/+ IMCD cells. The cells were incubated with 300 µM H2O2 for 15 min, and then the concentration of PGE2 in the medium was determined by ELISA. Results are expressed as means ± SE (n = 3). *P < 0.05 compared with control.

 
We examined the effect of PGE2 on Cl– transport in IMCD cell monolayers. As shown in Fig. 6, 1 µM PGE2 induced a significant increase in Isc in the presence of amiloride. Given the fact that cAMP is a major signaling pathway of PGE2 (28, 32), we examined the effect of PKA inhibition on PGE2-stimulated Cl– current. Before administration of H2O2, the cells were preincubated with vehicle, 5 µM H-89, or 5 µM KT5720 for 30 min, followed by 100 µM amiloride for 5 min. As shown in Fig. 6, H-89 and KT5720 alone produced no effect on Cl– current. PGE2 stimulated Cl– current by twofold, and this effect was reduced by the PKA inhibitors H-89 and KT5720, indicating PGE2-induced Cl– current mediated at least by PKA activity.


Figure 6
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  		Fig. 6. Effect of PGE2 and protein kinase A (PKA) inhibitors on Isc in the presence of 100 µM amiloride in primary IMCD cell monolayers. Conditions of incubations are given under each bar (see detail in text). For the PKA inhibitors plus PGE2, the cells were preincubated with inhibitors (5 µM H-89 or 5 µM KT5720) for 30 min, and then the 1 µM PGE2 was applied at basolateral side. Results are presented as means ± SE for 6 monolayers. *P < 0.01 compared with control. **P < 0.05 compared with PGE2-treated group.

 
Effect of PKA inhibition on H2O2-stimulated Isc. Following the demonstration of PKA as a mediator of PGE2-induced stimulation of Cl– current, we examined the involvement of PKA in H2O2-stimulated Isc in the presence of amiloride. For the PKA inhibitor plus H2O2, the IMCD cell monolayers were preincubated with 5 µM of H-89 or KT5720 for 30 min, and then with 100 µM amiloride for additional 5 min, followed by administration of a final 300 µM H2O2 to the basolateral side. As shown in Fig. 7, H2O2 significantly increased Cl– current, which was eliminated by exposure to H-89 or KT5720, whereas exposure to H-89 or KT5720 alone had no effect on Cl– current. These results support the contention that the H2O2-induced stimulation of Cl– transport required PKA activity.


Figure 7
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  		Fig. 7. Effect of PKA inhibitors on H2O2-stimulated Isc in the presence of 100 µM amiloride in primary IMCD cell monolayers. Conditions of incubations are similar to that of PGE2 experiment except using H2O2 instead of PGE2 (see detail in text). For the PKA inhibitors plus H2O2, the cells were preincubated with inhibitors (5 µM H-89 or 5 µM KT5720) for 30 min; the 300 µM H2O2 was applied at basolateral side. Results are presented as means ± SE for 6 monolayers. *P < 0.01 compared with control. **P < 0.05 compared with H2O2-treated group.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Although in vivo evidence supports a potential role of ROS in regulation of fluid metabolism in the renal medulla, direct evidence for redox-dependent regulation of NaCl transport in the collecting duct cells is still lacking. The present study investigated the role and mechanism of H2O2 in regulation of NaCl transport in primary IMCD cells using an Ussing chamber. We provide evidence that H2O2 induces a direct stimulatory effect on Isc that was almost abolished by the CFTR Cl– channel inhibitors DPC and CFTRinhibitor-172 but was insensitive to the ENaC inhibitor amiloride. We further provide evidence for involvement of mPGES-1-derived PGE2 in mediating the increased Cl– secretion in response to H2O2.

Although various immortal collecting duct cells are widely used for studying NaCl transport processes, the transformation with constitutively active oncogenes may make the cells significantly different from the native collecting duct cells. Therefore, we utilized the primary cultures of IMCD cells. The primary IMCD cell monolayers developed high resistance and negative luminal voltage. These cells exhibited responsiveness to aldosterone in terms of increases in amiloride-sensitive Isc and also expressed ENaC protein as assessed by immunoblotting (data not shown). These properties characterized the primary IMCD cells as a useful model for studying NaCl transport processes in the collecting duct despite some evidence that IMCD may process relatively low Na+ transport capacity compared with the proximal part of the collecting duct.

The present study examined the effect of H2O2 on Isc, which reflects NaCl transport in cultured collecting duct cells. We found that exposure to H2O2 increased Isc in a dose- and time-dependent manner in primary IMCD cell monolayers. The dose-dependent effect was observed at a range of 100–300 µM H2O2, while a further increase above this range was associated with cytotoxicity (data not shown). The effect of H2O2 was transient, lasting shorter than 15 min possibly because of the activation of antioxidant systems such as catalase, which converts H2O2 to H2O (33). The positive Isc could be contributed by reabsorption of cations (e.g., Na+) or secretion of anions (e.g., Cl–). The stimulation of Isc in response to H2O2 was unlikely to be mediated by ENaC as this phenomenon persisted in the presence of amiloride on the apical side. This leaves a possibility that the increased Isc might be due to amiloride-insensitive Na+ reabsorption or Cl– secretion. The fact that addition of the CFTR inhibitors DPC or CFTRinhibitor-172 on the top of amiloride abolished the H2O2-stimulated Isc suggests involvement of CFTR rather than amiloride-insensitive Na+ transport. These data are consistent with the observation that H2O2 stimulated the CFTR Cl– channel in human airway epithelial cells (5). The lack of an effect of H2O2 on ENaC-mediated Na+ reabsorption was also consistent with a previous study showing that H2O2 had no effect on Na+ reabsorption in isolated perfused thick ascending limb (21).

We attempted to determine the intracellular mediators of H2O2-induced activation of CFTR in cultured IMCD cells with emphasis on prostaglandins. ROS have been reported to stimulate PGE2 release from both collecting duct and proximal tubule cells (7, 35). We found that H2O2-stimulated Isc was significantly attenuated by the nonselective COX inhibitor indomethacin, indicating a requirement of COX activity for the activation of CFTR. The involvement of specific COX isoforms was tested using the COX-1 inhibitor SC-560 and the COX-2 inhibitor Cay10404. Exposure of IMCD cells to SC-560 completely abolished the effect of H2O2, whereas Cay-10404 had no obvious effect. These results indicated that COX-1 but not COX-2 may play a role in H2O2-induced Cl– secretion in collecting duct cells. This result is consistent with the observation that COX-1 predominates in the collecting duct, while COX-2 is restricted to renal medullary interstitial cells (3, 8).

The COX-1-derived product involved in H2O2-induced Cl– secretion appeared to be PGE2. This can be inferred from the observation that collecting duct cells lacking mPGES-1, a well-characterized PGE synthase, exhibited a significant attenuation of H2O2-stimulated Isc in the presence of amiloride. In support of this finding, exposure to H2O2 induced a rapid release in wild-type IMCD cells but not in mPGES-1-deficient IMCD cells. Given the residual response in mPGES-1-deficient cells relative to the complete blockade with COX-1-selective or nonselective COX inhibitors, it seems reasonable to speculate that other PGs than the E series may play a minor role in H2O2-induced Cl– secretion.

Commensurate with the role of endogenous PGE2 derived from mPGES-1 is the observation that exposure to exogenous PGE2 significantly elevated Isc in the presence of amiloride, mimicking the effect of H2O2. These findings are consistent with the previous report that PGE2 stimulated Cl– secretion in the collecting duct cells (23, 32). Another possibility exists that mPGES-1-mediated Cl– secretion may be relevant to the pathogenesis of cyst formation and enlargement in polycystic kidney disease (PKD). This possibility seems to warrant future investigation using mPGES-1 inhibitors or mPGES-1 –/– mice.

mPGES-1 is a novel PGE synthase with an established role in pain and inflammatory responses via coupling to COX-2 (27). Emerging evidence suggests that mPGES-1 may play a physiological role in facilitating renal salt excretion (7). Within the kidney, mPGES-1 predominates in the distal nephron, a site for COX-1 but not COX-2 expression. These colocalization data seem to suggest coupling of mPGES-1 with COX-1 but not COX-2 in the collecting duct (3, 24, 34), clearly different from that in inflammatory cells. In support of this notion, the present study provides the first functional evidence for coupling of mPGES-1 with COX-1 but not COX-2 in renal collecting duct cells in the setting of ROS-induced Cl– secretion.

The mechanism of PGE2 action appeared to involve activation of PKA as PGE2-stimulated Isc was significantly reduced by the PKA inhibitors H-89 and KT5720. However, the incomplete inhibition of the effect of PGE2 by PKA inhibitors indicated involvement of a PKA-independent pathway. The exact mechanism of PKA-dependent activation of CFTR is unknown. PKA may directly or indirectly phosphorylate CFTR, leading to trafficking of the channel to the cell membrane or increased open probability of the channel. It is unlikely, however, that the stimulatory effect of H2O2 involves increased synthesis of the channel given the rapid response to H2O2. The mechanism for PGE2-dependent activation of the cAMP/PKA pathway, particularly the type of EP receptors involved, still remained uninvestigated in the present study. The biological action of PGE2 is mediated by G protein-coupled E-prostanoid receptors designated EP1, EP2, EP3 and EP4, among which EP2 and EP4 act primarily via cAMP. Since EP4 but not EP2 is expressed in the collecting duct, it seems reasonable to speculate that EP4 may be responsible for PGE2-dependent activation of CFTR.

CFTR is abundantly expressed along the nephron with unclear physiological function. However, there is a rich literature documenting pathological roles of renal CFTR, particularly in PKD. It has been reported that CFTR mediates Cl– secretion from the cyst epithelia, which appears to be a critical event for the fluid accumulation and enlargement of the cysts (13). The elucidation of the H2O2/PGE2/CFTR pathway in regulation of Cl– secretion from renal collecting duct cells may help understand the pathophysiology of PKD and may also offer a unique therapeutic opportunity for this devastating disease.


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 ABSTRACT
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This work was supported by National Institutes of Health Grants HL-079453, DK-066592 and DK-069490, and by Merit Review from the Department of Veterans Affairs (to T. Yang).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Yang, Univ. of Utah and Veterans Affairs Medical Center, 30 N. 1900 E., Rm. 4R312, Salt Lake City, UT 84132 (e-mail: tianxin.yang{at}hsc.utah.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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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