Localization of IP in rabbit kidney and functional role of the PGI2/IP system in cortical collecting duct

Rania Nasrallah1, Rolf M. Nusing2, and Richard L. Hébert1

1 Department of Cellular and Molecular Medicine, Kidney Research Centre, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5; and 2 Department of Experimental Pediatrics and Clinical Pharmacology, Faculty of Medicine, Philipps University of Marburg, 35033 Marburg, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To clarify the role of the PGI2/PGI2 receptor (IP) system in rabbit cortical collecting duct (RCCD), we characterized the expression of IP receptors in the rabbit kidney. We show by Northern and Western blotting that IP mRNA and protein was detectable in all three regions of the kidney. To determine how PGI2 signals, we compared the effects of different PGI2 analogs [iloprost (ILP), carba-prostacyclin (c-PGI2), and cicaprost (CCP)] in the isolated perfused RCCD. PGI2 analogs did not increase water flow (Lp). Although PGI2 analogs did not reduce an established Lp response to 8-chlorophenylthio-cAMP, they equipotently inhibited AVP-stimulated Lp by 45%. The inhibitory effect of ILP and c-PGI2 on AVP-stimulated Lp is partially reversed by the protein kinase C inhibitor staurosporine and abolished by pertussis toxin; no effect was obtained with CCP. In fura 2-loaded RCCD, CCP did not alter cytosolic Ca2+ concentration ([Ca2+]i), but, in the presence of CCP, individual infusion of ILP and PGE2 increased [Ca2+]i, suggesting that CCP did not cause desensitization to either ILP or PGE2. We concluded that ILP and c-PGI2 activate PKC and the liberation of [Ca2+]i but not CCP. This suggested an important role for phosphatidylinositol hydrolysis in mediating ILP and c-PGI2 effects but not CCP in RCCD.

cortical collecting duct; intracellular calcium; prostaglandin I2; transepithelial voltage; vasopressin; water transport


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PROSTAGLANDINS (PG) play an important role in renal physiology and nephropathy. They regulate hemodynamics and tubular transport processes and renin secretion. PGs are metabolites of arachidonic acid, produced by the action of cyclooxygenases (38) and PG synthases (40). Although species differences do exist, the two major PGs produced in the rabbit kidney are prostaglandin E2 (PGE2) and prostacyclin (PGI2), especially in the glomerular region and collecting ducts (3). Abundant work has been accomplished in the study of collecting duct PGE2 responses because of the characterization of the PGE2 receptors [the EP receptor subtypes (EP1, EP2, EP3, and EP4); see Refs. 8 and 30]. For example, the EP1 receptor couples to Gq and activates protein kinase C (PKC), thus inhibiting collecting duct Na+ absorption (5). The EP2 receptor subtype stimulates adenylate cyclase via coupling to Gs. Recent studies have shown that EP2 knockout mice develop salt-sensitive hypertension, suggesting a renal effect (24), but renal expression of this subtype is still debatable. EP3 receptors inhibit adenylate cyclase via a pertussis toxin (PT)-sensitive Gi-coupled mechanism (19, 28). The EP3 subtype is abundant in rabbit cortical collecting duct (CCD; see Refs. 4 and 5), where it antagonizes arginine vasopressin (AVP)-stimulated water absorption (17, 19, 22). Finally, EP4 receptors stimulate adenylate cyclase via Gs. Most evidence suggests that PGE2 activates an EP4 receptor subtype in CCD, which stimulates water reabsorption and amiloride-sensitive current in the absence of vasopressin (5, 12, 16) rather than the EP2 receptor.

Comparable to PGE2, PGI2 is an important vasoactive agent, most recognized for its potent vasodilatory effects and its function as a platelet inhibitor. Most of its actions are mediated by the cell-surface PGI2 receptor (IP), which was cloned in 1994 from mice (29), humans (2), and rats (36). However, with the advent of many pharmacological analogs of PGI2 (41), countless other functions have been associated with this prostanoid, linking it to the regulation of cell fate and gene transcription (15, 23). For example, Gupta et al. (13) reported that PGI2 is involved in tumorigenesis in colorectal cancer as a result of activation of peroxisome proliferator-activated receptors (PPAR). Moreover, a recent study by Hatae et al. (15) in human embryonic kidney cells (HEK-293) suggests a novel PGI2/PPARdelta pathway regulating apoptosis. Although fewer studies are available with respect to the regulation of cellular events by PGI2 in the kidney, biochemical studies have demonstrated various responses to PGI2 in the rodent collecting ducts. For instance, although in the cultured rat inner medullary collecting duct cells (IMCD), PGI2 stimulated cAMP but no inhibition of AVP-dependent cAMP stimulation was obtained (39), an inhibitory effect was observed in freshly isolated IMCD using the selective PGI2 analog cicaprost (CCP; see Ref. 31). This study also showed that calcium stimulation by iloprost (ILP; a less-selective IP agonist) in the microdissected rat IMCD was mediated through the EP1 receptor.

Further insight into the elaborate contribution of PGI2 in renal physiology came about from recent work that localized the expression of IP receptors in the kidney, with certain discrepancies between species. For instance, human studies by Komhoff et al. (25) using both in situ hybridization (ISH) and immunohistochemistry with an anti-IP polyclonal antibody showed that IP receptors, both mRNA and protein, are detectable throughout the nephron within the vasculature. Also, the expression was abundant in the glomerular region of the cortex, with very little in the medulla. Only Tamm-Horsfall negative tubules of the outer medulla (nonmedullary thick ascending limb) contained detectable IP (21). This is in contrast to findings in rat kidney (31). The mRNA for IP was readily detected in all three regions of the kidney (glomerular mesangial cells; Nasrallah R and Hébert RL, unpublished observations) and in the vasculature and tubular segments (proximal tubule, medullary thick ascending limb, IMCD). In the mouse, IP expression is mainly restricted to the renal vasculature and glomeruli (33). It is becoming quite evident that species differences do exist with respect to localization and function of the PGI2/IP system along the nephron, thereby expanding the scope of its involvement in distinct renal processes.

To date, there is no molecular evidence for the expression of IP receptors in the rabbit kidney nor any studies clarifying the cellular effects of PGI2 in the rabbit distal nephron. Therefore, the present study focuses on elucidating the functional role of the PGI2/IP system in the rabbit CCD (RCCD). We first characterized the expression of the IP receptor in the rabbit kidney by Northern blotting and Western blotting and then examined the cellular responses to PGI2 analogs [ILP, carba (c)-PGI2, and CCP] in isolated perfused RCCD and their effect on water transport, transepithelial voltage, and cell calcium.


    MATERIALS AND METHODS
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Tissue Preparation

Both kidneys were removed from male New Zealand White rabbits (weighing 2 kg) and were immediately placed in a beaker on ice containing PBS. A small superficial incision was then made in each kidney, and the renal capsule was removed. Under a dissecting microscope, on ice, the kidney was divided into five transverse slices, each 5 mm thick. With high-intensity light, each region of the kidney was then separated with dissecting scissors based on color differences between the various regions (brownish cortex, pinkish outer medulla, and whitish inner medulla). The tissue from each region was then homogenized and used for RNA or protein isolations.

Molecular Studies

RNA isolation and Northern blotting. Total RNA was isolated from the various tissues using the Trizol method, as described by the manufacturer (GIBCO-BRL). Briefly, tissue fragments from each region of the kidney were obtained as described above, placed in 1 ml Trizol reagent, and homogenized for 20 s. After a phase separation with chloroform, the RNA was precipitated using isopropyl alcohol, and the pellet was washed in 70% ethanol. The RNA pellet was then resuspended in diethyl pyrocarbonate-treated H2O and quantified by spectrophotometry at a 260- to 280-nm absorbance ratio. Total RNA (20 µg) from each sample was denatured, loaded on a formaldehyde gel, and electrophoresed in 3-(N-morpholino)propanesulfonic acid buffer for 3 h. The RNA was then transferred to a nitrocellulose membrane using 20× saline-sodium citrate (SSC) and baked for 2 h at 80°C under vacuum. The membranes were prehybridized at 60°C for 3 h in 20 ml hybridization buffer containing 50% formamide, 5× SSC, 3 mg Salmon sperm DNA, 5× Denhardt's solution, and 1% SDS. Using the PrimeIt-II random primer labeling kit (Stratagene), 50 ng of a 920-bp human IP cDNA probe (Cayman Chemicals) were labeled with 3,000 µCi [32P]dCTP, and a hybridization was carried out at 60°C. After a few moderate stringency washes in 2× SSC/0.1% SDS, the signal was visualized by autoradiography. The samples were normalized by detection of beta -actin in each sample. Briefly, the membranes were stripped in boiling 0.5% SDS/H2O for 15 min at room temperature and hybridized with a 1.8-kb 32P-labeled human beta -actin cDNA (Clontech).

Protein isolation and Western blotting. Kidneys from male New Zealand White rabbits were removed, and the cortex was separated from the medullary regions. The spleen was used as a positive control. Protein was isolated by tissue homogenization in a Tris · HCl lysis buffer. Each sample (50 µg of each) was resolved by SDS-PAGE on a polyacrylamide gel and transferred to a Hybond ECL nitrocellulose membrane. The membrane was blocked overnight with a 5% milk/Tris-buffered saline and Tween 20 solution, and the human polyclonal alpha -IP antibody was added at a dilution of 1:4,000 for 3 h at room temperature. Then the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG, and the signal was visualized using ECL. This antibody was derived against a 14-amino acid peptide located at the NH2-terminus of the human IP receptor protein (25). Initial characterization of the antibody by Western blot analysis using human platelets revealed a 52-kDa band by SDS-PAGE, corresponding to the human IP receptor.

Functional Studies

General microperfusion methods. In vitro microperfusion of isolated CCDs was performed as previously described (17-20, 22). Briefly, rabbits weighing 1.5-2.5 kg were killed by using an intramuscular injection of ketamine (44 mg/kg) and xylazine (11 mg/kg) for anesthesia followed by decapitation. The left kidney was removed quickly, and 1- to 2-mm coronal slices were placed in chilled dissection dishes for freehand dissection. Tubules were transfered to a thermostatically controlled chamber of 1 cm3 volume and cannulated using concentric micropipettes. Bath solution was exchanged continuously at 0.5 ml/min by an infusion pump (Sage; Orion Research, Cambridge, MA) and was maintained at 37°C. The composition of standard bath medium, dissection medium, and isotonic perfusate was as follows (in mM): 105 NaCl, 25 NaHCO3, 10 sodium acetate, 2.3 NaHPO4, 5 KCl, 1.8 CaCl2, 1.0 MgSO4, 8.3 glucose, and 5 alanine (osmolality 300 mosmol/kgH2O). The composition of hypotonic perfusate was identical to that of isotonic perfusate except for the low osmolality (150 mosmol/kgH2O) resulting from the lower NaCl concentration (30 mM) and the addition of [3H]inulin. Both isotonic and hypotonic perfusate also contained 0.2 mg/ml Food, Drug, and Cosmetic dye No. 3 (Aniline and Chemical, Chicago, IL) to detect cell damage and perfusate leak (17-20, 22).

MEASUREMENT OF HYDRAULIC CONDUCTIVITY. The perfusate, which contained [3H]inulin (75 µCi/ml) as a volume marker, was collected in a constriction pipette of known volume (between 90 and 130 nl) and counted for [3H]inulin (New England Nuclear, Boston, MA). The perfusion rate was maintained between 12 and 20 nl/min by adjusting the hydrostatic pressure. At this perfusion rate, osmotic equilibration between bath and lumen did not occur. During the first 45 min of incubation, all tubules were perfused with an isotonic solution similar to the bath. Subsequently, the perfusate was changed to hypotonic perfusate. In control studies, 30 min of further equilibration were allowed and then three collections were made for calculation of basal hydraulic conductivity (Lp). Tubules with a negative basal Lp were discarded. Next, 10 µU/ml AVP were added to the bath, and after a 15-min equilibration period three to four timed collections were made to determine Lp. A stable Lp was usually observed 20-50 min after the addition of AVP. Subsequently, either ILP + AVP, c-PGI2 + AVP, or CCP + AVP was added. After a 15-min equilibration, six more timed collections were made. In each period, the three collections with the greatest calculated Lp were averaged to calculate mean Lp for this period.

MEASUREMENT OF TRANSEPITHELIAL VOLTAGE IN ISOLATED PERFUSED TUBULES. Transepithelial voltage (VT) was measured via a Ringer agarose bridge connected to the perfusion pipette and a calomel electrode. A similar bridge connected the bath to another calomel electrode and completed the circuit. VT (in mV) was measured with an electrometer (model 602; Keithley Instruments, Cleveland, OH) and continuously recorded on a strip-chart recorder (Primeline model R-02; Soltec, Sun Valley, CA).

EXPERIMENTAL PROTOCOLS FOR IN VITRO STUDIES. Each water flux experiment was conducted as follows. After a 45-min equilibration period in which CCDs were perfused with an isotonic solution similar to the bath, the perfusate was changed to a hypotonic solution (150 mosmol/kgH2O). For the experimental group, after an additional 30-min incubation, three collections were made for calculation of basal Lp. Next, the PGI2 analogs were added in the continuous presence to the bath, and, after a 45-min equilibration period, three collections were made to determine the effects of PGI2 analogs on the control Lp. In the experimental studies, 10 µU/ml AVP were added in the presence of ILP, c-PGI2, or CCP, and, after 10 min of incubation, six timed collections were made. The peak Lp was defined as the mean of the three largest contiguous values. Peak Lp was usually observed 15-25 min after the addition of AVP.

Effects of PGI2 analog pretreatment followed by addition of AVP on Lp in RCCD. effect of pgi2 analog preexposure on avp-stimulated lp IN RCCD. After three basal collections were made, 10-7 M ILP, c-PGI2, or CCP was added to the bath for 15 min. Three collections were made and then 10 µU/ml AVP + 10-7 M ILP, 10-7 M c-PGI2, or 10-7 M CCP was added in the continued presence of AVP. After a 10-min equilibration, six more timed collections were made. The three highest contiguous values were averaged in the experimental period.

EFFECT OF PGI2 ANALOGS IN RCCD PREEXPOSED TO AVP ON AVP-STIMULATED Lp. After three basal collections were made, the tubules were pretreated with 10 µU/ml AVP for 15 min. Three collections were made, and, in the continued presence of AVP + 10-7 M ILP, 10-7 M c-PGI2, or 10-7 M CCP, after a 10-min equilibration, six more timed collections were made. The three highest contiguous values were averaged in the experimental period.

EFFECT OF PGI2 ANALOGS ON 8-(4-CHLOROPHENYLTHIO)cAMP-STIMULATED Lp IN RCCD PREEXPOSED TO 8-(4-CHLOROPHENYLTHIO)-cAMP. After three basal collections were made, the tubules were pretreated with 10-5 M 8-(4-chlorophenylthio)cAMP (8-CPT-cAMP) for 15 min. Three collections were made, and in the continued presence of 8-CPT-cAMP, 10-7 M ILP and 10-7 M c-PGI2 were added. After a 10-min equilibration, six more timed collections were made. The three highest contiguous values were averaged in the experimental period.

EFFECT OF PGE2 AND SULPROSTONE ON 8-CPT-cAMP-STIMULATED Lp IN RCCD PREEXPOSED TO 8-CPT-cAMP. After three basal collections were made, the tubules were pretreated with 10-5 M 8-CPT-cAMP for 15 min. Three collections were made, and in the continued presence of 8-CPT-cAMP, 10-7 M PGE2 or 10-7 M sulprostone (SLP) was added. After a 10-min equilibration, six more timed collections were made. The three highest contiguous values were averaged in the experimental period.

EFFECT OF PGI2 ANALOGS ON AVP-STIMULATED Lp IN RCCD PREEXPOSED TO PT. Before three basal collections were made, RCCD were pretreated with 500 ng/ml PT alone for 60 min. After basal collections, the CCD was exposed to 10 µU/ml AVP, and Lp was determined. Finally, ILP + AVP, c-PGI2 + AVP, and CCP + AVP were added to the bath, and peak Lp was determined. Six collections were made, and the three highest contiguous values were averaged in the experimental period.

EFFECT OF PGI2 ANALOGS ON AVP-STIMULATED Lp IN RCCD PREEXPOSED TO THE PKC INHIBITOR STAUROSPORINE. Before three basal collections were made, the tubules were pretreated with 10-7 M staurosporine (SSP) alone for 15 min before basal collections. After basal collections, the CCD was exposed to 10 µU/ml AVP, and Lp was determined. Finally, ILP + AVP, c-PGI2 + AVP, and CCP + AVP were added to the bath, and peak Lp was determined. Six collections were made, and the three highest contiguous values were averaged in the experimental period.

For measurement of intracellular calcium concentration ([Ca2+]i) in isolated perfused CCDs, the tubules were perfused in vitro as previously described (17-20, 22). Briefly, this method is similar to that described above with the following differences: 1) perfusate was Ca2+ and Mg2+ free; 2) the bath perfusion chamber was a special low-volume (0.150 ml) chamber to allow for rapid fluid exchange; and 3) the bath solution was preheated in a water-jacketed line, and flow rate was between 0.5 and 2.5 ml/min. Tubules were bathed in 2.5 µM fura 2-AM (Molecular Probes, Eugene, OR; see Refs. 11 and 37) for 45 min at 30°C (flow rate 0.5 ml/min). After tubules were loaded, the bath temperature was increased to 37°C and the flow rate was increased to 2.5 ml/min; CCDs were allowed to equilibrate for 20-30 min. Intracellular fura 2 fluorescence intensity was measured using continuous rapidly alternating excitation (20 ms/reading) from dual monochrometers set at 340 and 380 nm (Deltascan; Photon Technology International, New Brunswick, NJ). The monochrometer output was coupled to the inverted microscope using a 400-nm dichroic mirror and a ×100 lens (Nikon fluor oil immersion). Fluorescent emission of light >435 nm was measured by photon counting. Before loading with fura 2, CCD autofluorescence and background light were measured (<10% of fluorescent emission in fura 2-loaded tubules), and this value was continuously subtracted from all measurements. The corrected emission intensity ratio, using 340- and 380-nm excitation (340/380 ratio, R), was monitored continuously (11, 37).

Experimental protocols. After fura 2 loading and equilibration, a baseline reading of 100-200 s was taken in standard bath medium. The tubules were then exposed to the different prostanoids for 100-150 s. At the end of each experiment, an in situ calibration of [Ca2+]i was performed. The bath medium was changed to a Ca2+- and Mg2+-free isotonic solution containing 2 mM EGTA and 10 µM 4-bromo-A-23187. After a stable 340- to 380-nm ratio (minimum ratio, Rmin) was achieved, the bath was changed back to normal bath medium (1.8 mM Ca2+) and 10 µM 4-bromo-A-23187, and the ratio was again allowed to stabilize (maximum ratio, Rmax).

EFFECTS OF 10-7 m ccp, ilp, and pge2 on liberation of [ca2+]i IN RCCD. A baseline reading of 100-150 s was taken in standard bath medium. The tubules were then exposed to 10-7 M CCP for 100-150 s and then, in the continuous presence of CCP, the tubules were exposed to 10-7 M ILP followed by 10-7 M PGE2 for the same period of time.

Calculations

Volume reabsorption and Lp. Net volume reabsorption (Jv) was calculated as Jv = (Vi - Vo)/L, where Vi is the perfusion rate (nl/min), Vo is the collection rate (nl/min), and L is the tubule length. Vo was measured directly, and Vi was calculated as: Vi = Vo (cpmo/cpmi), where cpmo and cpmi are perfusate and collected fluid 3H counts per minute per nanoliter, respectively. Lp [10-7 cm/(atm · s)] was calculated according to Dubois et al. (9)
L<SUB>p</SUB><IT>=</IT>(1<IT>/RTS</IT>)<IT>×</IT>(1<IT>/</IT>O<SUB>b</SUB>)<SUP>2</SUP> × {O<SUB>b</SUB> × (<IT>V</IT><SUB>i</SUB><IT>−V</IT><SUB>o</SUB>)

 + O<SUB>i</SUB> × <IT>V</IT><SUB>i</SUB> × ln [(O<SUB>b</SUB> − O<SUB>i</SUB>) × <IT>V</IT><SUB>i</SUB>/(O<SUB>b</SUB> × <IT>V</IT><SUB>o</SUB> − O<SUB>i</SUB> × <IT>V</IT><SUB>i</SUB>)]}
where R is the gas constant, T is · K, S is the CCD lumen surface area (assumed luminal diameter of 20 µm), and Ob and Oi represent the osmolality of the bath and perfusate, respectively.

Calculation of [Ca2+]i. [Ca2+]i was calculated as [Ca2+]i = Kd(R - Rmin)/(Rmax - R)(380min/380max), assuming that the dissociation constant (Kd) value for the fura 2-Ca2+ complex is 224 nM at 37°C (11, 37). Because this apparent Kd might display shifts in the intracellular environment, the data are represented not only as [Ca2+]i but also as a percent increase in [Ca2+]i above basal levels, which is independent of Kd (11, 37).

Reagents

AVP, EGTA, PGE2, and 8-CPT-cAMP were purchased from Sigma (St. Louis, MO). [3H]inulin was purchased from New England Nuclear. Fura 2-AM and 4-bromo-A-23187 were purchased from Molecular Probes. c-PGI2 and ILP were purchased from Cayman Chemical (Ann Arbor, MI). CCP was a generous gift from Berlex Pharmaceuticals (Lachine, Quebec, Canada). SLP was a generous gift from Berlex Laboratories (Cedar Knolls, NJ).

Statistics

Student's t-test for unpaired data was used when only two unrelated treatment groups were compared. To determine the statistical significance of differences between more than two groups, ANOVA and the Student-Newman-Keul's multiple-comparison test were used. Differences of P < 0.05 were considered statistically significant. Data are presented as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Localization of IP Receptor mRNA in Rabbit Kidney

This study localized the expression of IP receptors within different regions of the rabbit kidney. As shown in Fig. 1, IP mRNA was detected in all three regions of the rabbit kidney (cortex, outer medulla, and inner medulla) using a human cDNA probe from the coding region of the human sequence. A single band of 2.5 kb was observed by autoradiography, comparable to the predicted size of 2.4 kb in humans (2, 28).


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Fig. 1.   Detection of PGI2 receptor (IP) mRNA by Northern blotting in different rabbit kidney regions. A: autoradiograph showing an ~2.5-kb signal for the IP receptor mRNA in total RNA preparations from three kidney region samples loaded from 3 different rabbits. B: corresponding signals for human beta -actin mRNA detection.

Localization of IP Protein in Rabbit Kidney

Having demonstrated the expression of IP receptor mRNA throughout the rabbit kidney, we then examined the expression of IP receptor protein in different regions of the kidney. In Fig. 2, IP protein was highly detectable throughout the rabbit kidney (cortex and medulla) at levels comparable to the spleen. A single band of 52 kDa was obtained in each sample.


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Fig. 2.   Detection of IP protein in rabbit kidney regions by Western blotting. A: representative autoradiograph showing IP protein expression in lysates of cortex and medullary regions (pooled outer and inner) of the rabbit kidney. The spleen was used as a positive control. B: protein was normalized by detection of beta -actin in each sample (n = 3).

Effects of ILP, c-PGI2, and CCP on Hydrosmotic Water Flow

The calculation of Lp is described in MATERIALS AND METHODS [the units are 10-7 cm/(atm · s)], and for the sake of brevity, will not be written after each value.

Effects of PGI2 Analog Pretreatment on AVP-Induced Lp

PGI2 analogs failed to alter water permeability (data not shown). However, we had previously shown that 10-7 M PGE2 increased Lp (17). This demonstrates a major difference between the actions of these prostanoids in RCCD. On the other hand, 23 pM AVP stimulates Lp to 290 ± 14. In tubules pretreated with different PGI2 analogs, the AVP response was inhibited by almost 50% [139 ± 10 with CCP, 150 ± 11 with ILP, and 152 ± 12 with c-PGI2 (n = 9, P < 0.005, Fig. 3)]. Thus, despite the lack of stimulatory effect on basal water flow in RCCD, PGI2 analogs significantly inhibit AVP-stimulated Lp.


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Fig. 3.   PGI2 analogs attenuate arginine vasopressin (AVP)-stimulated hydraulic conductivity (Lp). The effect of AVP (black bar) on Lp is plotted. Three PGI2 analogs [cicaprost (CCP; gray bar), iloprost (ILP; dark gray bar), and carba-prostacyclin (c)-PGI2 (light gray bar)] were administered before addition of AVP to examine their effect on the AVP response. Data are presented as means ± SE. *P < 0.005; no. of experiments (n) are indicated on x-axis.

Effect of PGI2 Analog Posttreatment on Established AVP-Induced Lp

Water flow in RCCDs was prestimulated by the addition of 23 pM AVP, which increased Lp from basal levels of 9 ± 2 to 290 ± 14 (n = 13). Subsequent addition of 10-7 M ILP, c-PGI2, or CCP inhibited AVP-stimulated water permeability to a similar extent, which represents an inhibition of 45% compared with the AVP time control (n = 6; Fig. 4).


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Fig. 4.   Mechanism of attenuation of AVP-stimulated Lp by PGI2 analogs. Three PGI2 analogs [ILP (A), c-PGI2 (B), and CCP (C)] were used to inhibit AVP-stimulated water flow. Open bars, percentage inhibition by each analog; gray bars, effect of pertussis toxin (PT) pretreatment on this inhibition; black bars, staurosporine (SSP) pretreatment. **P < 0.01 and *P < 0.05; n are indicated on the x-axes.

PT Reverses Inhibitory Effects of ILP, c-PGI2, and CCP on AVP-Stimulated Water Flow

CCDs were pretreated with 500 ng/ml PT (1 h, 37°C). PT irreversibly inactivates the inhibitory guanine nucleotide-binding regulatory protein (Gi; see Refs. 18 and 19), and this protocol has previously been shown to block the inhibitory effect of PGE2 on AVP-induced Lp in the RCCD (18, 19). Compared with untreated tubules, PT pretreatment for 1 h did not increase AVP-stimulated Lp. However, PT pretreatment blocked the inhibitory action of the PGI2 analogs by 70 ± 15% compared with the control value (n = 5, P < 0.01; Fig. 4). Thus PT significantly reversed the inhibitory effect of PGI2 analogs on AVP-induced Lp in RCCD, indicating that their effect is strongly mediated by a Gi-coupled pathway.

SSP Partially Reverses Inhibitory Effects of ILP and c-PGI2, but not of CCP, on AVP-Stimulated Water Flow

CCDs were pretreated with 10-7 M SSP, a potent PKC inhibitor (17, 19). SSP was added to the bath 15 min before the initiation of Lp measurements and was present throughout the subsequent experiment. Compared with untreated tubules, SSP pretreatment did not significantly decrease AVP-stimulated Lp. On the other hand, SSP pretreatment partially but significantly reversed the inhibitory action of 10-7 M ILP and c-PGI2 by 35 ± 8% compared with SSP + AVP time controls (n = 8 and 6, P < 0.05; Fig. 4). Of interest, CCP did not activate PKC, since the inhibitory effect of CCP on AVP-induced Lp was not partially abolished by SSP (Fig. 4).

Effect of PGI2 Analog Posttreatment on Established 8-CPT-cAMP-Stimulated Lp

We next examined the effect of the cell-permeable cAMP analog 8-CPT-cAMP on water permeability. 8-CPT-cAMP (10-5 M) increased Lp to 315 ± 17. The subsequent addition of 10-7 M c-PGI2 or ILP did not inhibit 8-CPT-cAMP-stimulated Lp (Fig. 5). These results show that PGI2 analogs did not inhibit AVP-induced Lp at a post-cAMP level in RCCD.


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Fig. 5.   Effect of various prostanoids on established 8-(4-chlorophenylthio)cAMP (8-CPT-cAMP)-stimulated Lp. Different prostaglandin analogs were added to rabbit CCD, after Lp was stimulated by 8-CPT-cAMP. Data are presented as means ± SE. A: effect of sulprostone (SLP; black bars) and PGE2 (gray bars) on 8-CPT-cAMP-stimulated Lp (open bars; n = 6, *P < 0.05). B: effect of c-PGI2 (black bars) and 10-7 M ILP (gray bars) on 8-CPT-cAMP-induced Lp (open bars) (n = 6, P = not significant).

Effect of PGE2 and SLP Posttreatment on Established 8-CPT-cAMP-Stimulated Lp

We next examined the effect of the cell-permeable cAMP analog 8-CPT-cAMP on water permeability. 8-CPT-cAMP (10-5 M) increased Lp to 305 ± 17, subsequent addition of 10-7 M PGE2 decreased 8-CPTcAMP-stimulated Lp to 185 ± 20 (n = 6, P < 0.05, Fig. 5), and, in the second group, 10-7 M SLP inhibited 8-CPT-cAMP-stimulated Lp from a control value of 295 ± 22 to 170 ± 21 (n = 6, P < 0.05; Fig. 5). These results demonstrated that PGE2 and SLP decreased AVP-induced Lp at a post-cAMP level in RCCD.

Effect of PGI2 Analog Posttreatment after Infusion of AVP on VT

We examined the effect of PGI2 analogs on the VT of rabbit tubules. Our laboratory has previously shown that the depolarization of the tubules by PGE2 was the result of the decrement of sodium transport from the lumen to the bath (12, 18). Also, our group and others have demonstrated a hyperpolarization in response to AVP (data not shown). As can be seen in Fig. 6A, in the presence of AVP, 10-7 M ILP caused a depolarization of the tubules measured from -14.5 ± 3.5 to -5 ± 2.5 mV, but no effect was obtained after the infusion of 10-7 M CCP (Fig. 6B). These results are in perfect agreement with our present findings, since ILP increases the liberation of [Ca2+]i and not CCP, as described below; this may explain the absence of depolarization of the tubules by CCP.


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Fig. 6.   Effect of PGI2 analog posttreatment on AVP-mediated transepithelial voltage. A representative tracing showing the effect of 10-7 M ILP (A) and 10-7 M CCP (B) on depolarization of the tubules in the presence of AVP. The magnitude of transepithelial voltage is measured in mV. In A, the scale of 10 mV is indicated; n = 6.

Effects of PGI2 Analogs on Liberation of [Ca2+]i in Isolated RCCD

Because ILP and c-PGI2 both activate PKC, we assessed the effects of PGI2 analogs on the liberation of [Ca2+]i. Addition of 10-7 M CCP to fura 2-loaded CCDs did not increase [Ca2+]i (Fig. 7). In contrast, in the continuous presence of CCP, successive addition of 10-7 M ILP increased [Ca2+]i from a basal value of 71 ± 31 to 285 ± 36 nM. Likewise, 10-7 M PGE2 stimulated [Ca2+]i from a basal value of 90 ± 37 to 305 ± 41 nM (n = 3; Fig. 7). Therefore, in RCCD, ILP stimulates [Ca2+]i but not CCP.


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Fig. 7.   Effects of PGI2 analogs on the liberation of intracellular calcium concentration ([Ca2+]i) in isolated rabbit CCD. Representative tracing of fura 2-loaded rabbit CCD shows the effect of 10-7 M CCP on the release of [Ca2+]i and of 10-7 M ILP and 10-7 M PGE2 in the continuous presence of CCP. The nM increment in [Ca2+]i above baseline values is plotted (vertical axis) vs. time in s (horizontal axis); n = 3.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study characterizes the role of the PGI2/IP system in the rabbit kidney. Although cDNA for the rabbit IP receptor has not yet been cloned, the sequences for the human, rat, and mouse counterparts are available (Geneblast). The human IP receptor is encoded by a message of 2.4 kb (2, 28), and the gene containing three exons is located on chromosome 19q13.3 (10). To determine whether IP mRNA is present in the rabbit kidney, we used a probe within the COOH terminus of the human cDNA, a region that is quite homologous to the rodent sequences as follows: 74.3 and 75.9% in mice and rats, respectively. Using this probe, we have shown that the IP receptor is in fact present in all three regions of the rabbit kidney, with a greater abundance in the outer medullary region. These results are consistent with previous findings in rats by RT-PCR and ISH showing the highest expression in outer medullary tubules (31) and in human kidneys, where IP expression was present throughout the nephron in vasculature, glomeruli, and distal tubules (25). In contrast, in the mouse, ISH revealed intense IP signals within the renal vasculature but no tubular staining (33).

To further characterize the localization of IP receptors in the rabbit kidney, we used a human polyclonal anti-IP antibody to identify protein expression in the kidney cortex and medulla. These findings are consistent with previous reports in the rat (31) showing a ubiquitous distribution of the IP receptor throughout the nephron, thereby establishing a possible role for IP in diverse renal processes, such as regulating the glomerular filtration rate, glomerular hemodynamics, renin release, and salt and water transport in the collecting duct. However, like previous inconsistencies reported for the localization of the EP4 receptor subtype in human collecting ducts (27), there remain certain uncertainties with respect to the expression of IP receptors in different species, especially in the medullary thick ascending limb (25, 31). This brings up an important question regarding the function of this system in different segments of the nephron. More work is needed to clarify this issue and better establish a role for PGI2/IP in the kidney and its contribution to the maintenance of homeostasis or pathogenesis of renal diseases.

Having characterized the expression of IP receptors in the rabbit nephron, we pursued our work by examining the physiological role of the PGI2/IP system in cellular signaling and transport properties of the RCCD. Our findings indicate that ILP and c-PGI2 have different effects than CCP on water transport, VT, and cell calcium in the isolated perfused RCCD. Perhaps the most impressive differences in the functional effects of these three PGI2 analogs were: 1) alone, they did not increase water flow; 2) they did not decrease AVP-stimulated Lp; 3) ILP and c-PGI2 depolarized the VT of the tubules, whereas CCP did not; 4) ILP and c-PGI2 activated PKC, whereas CCP did not; and 5) finally, ILP increased the liberation of intracellular calcium, whereas CCP did not. These results are consistent with previous studies done in our laboratory demonstrating that even 10-5 M SLP failed to increase PKC/cAMP generation in RCCDs (19). In contrast, PGE2 potently stimulates cAMP generation at comparable concentrations (19). Because water permeability in the CCD is thought to be mediated by cAMP activation of protein kinase A, the different effects of the PGI2 analogs and PGE2 on cAMP metabolism probably account for the different effects on basal water permeability.

Although PGI2 analogs (ILP, c-PGI2, and CCP) had no effect on basal water permeability, they equipotently inhibited AVP-prestimulated water flow. However, there are significant differences between the mechanisms used by these, involving both a PT-sensitive (Gi) mechanism and an SSP-sensitive mechanism (PKC activation), except for CCP, which did not activate PKC. In the present study, ILP was shown to inhibit AVP-induced Lp, which is in apparent contradiction to the report that c-PGI2 did not inhibit the increase in water permeability elicited by AVP (20). The factor that may explain this discrepancy is that, in the previous study, the effects of c-PGI2 were investigated in the rat IMCD (39). There are significant differences in transport systems and hormonal modulation in the IMCD and the CCD as well as species differences (20, 39). Although the types of IP and EP receptors and the functional consequences may differ in the IMCD and the CCD, the cells comprising these distinct nephron segments seem to have in common the expression of separate receptors for these prostanoids (20, 39). Moreover, the inhibitory action of PGI2 analogs on AVP-induced Lp is abolished by PT, consistent with the activation of a receptor coupling to Gi, e.g., EP3 (19). Interestingly, we previously observed that the inhibitory effects of 10-7 M SLP are completely reversed by PT but unaffected by SSP (20). Whether or not the inhibitory effect of PGI2 analogs is mediated by the PGE2 receptor EP3, the IP receptor coupling to Gi instead of Gs or an as of yet uncloned putative "IP3" receptor remains to be determined.

Because our laboratory had previously reported that PGI2 analogs potentially activated different putative receptor subtypes, which we called "IP1" and IP3 in RCCD (22), in a fashion independent of EP receptor subtypes, the current study further examines how PGI2 analogs signal and their mechanisms of action in RCCD. To date, there is no molecular evidence for any subtypes or splice variants of the IP receptor. In a previous study in different rat kidney preparations, we were unable to detect any IP receptors homologous to the cloned rat IP cDNA (31). Also, we demonstrated that in the rat IMCD, ILP did increase intracellular calcium levels, whereas CCP did not. However, this was inhibited by pretreatment with AH-6809, a selective EP1 antagonist. Thus ILP and PGE2 both increased calcium levels via the same receptor.

The effects of ILP and PGE2 on changes in [Ca2+]i were qualitatively similar to the effects seen on AVP Lp. ILP was found to stimulate an increase in [Ca2+]i at a concentration of 10-7 M. This is consistent with what has been shown regarding agonist activity of ILP and c-PGI2 in other cells (8, 34). Previously, we have shown that PT had no effect on the increase in [Ca2+]i and the subsequent inhibition of Na+ reabsorption (18). Furthermore, 10-7 M SLP only slightly increases [Ca2+]i in contrast to the dramatic effects of 10-7 M PGE2 on [Ca2+]i. Since no effect on [Ca2+]i was observed in response to CCP, the more selective IP agonist, it appears that ILP and PGE2 are acting on similar receptors, consistent with our findings in the rat IMCD (31).

A broad body of literature suggests that the induction of water permeability by AVP or 8-CPT-cAMP occurs via the exocytic insertion of water-permeable channels at the apical membrane of the collecting duct cell (1, 6, 14). Removal of AVP or 8-CPT-cAMP causes cell cAMP levels to fall, resulting in the retrieval of this water-permeable membrane. In light of this model, we would like to offer the following interpretation of our data. In Fig. 5A, after water flow is established, PGI2 can only inhibit the maintenance of a high water permeability by decreasing cell cAMP levels and does not independently stimulate membrane retrieval. Hence, PGI2 is ineffective in reducing water permeability in CCDs once fully established by exogenous 8-CPT-cAMP, since under these circumstances it cannot alter cell cAMP levels. However, PGI2 can decrease AVP-established water flow; therefore, it is capable of decreasing cell cAMP levels via its inhibitory action on adenylyl cyclase. In Fig. 5B, after water flow is established, PGE2 can only inhibit the maintenance of a high water flow by decreasing cell cAMP levels and does not independently stimulate membrane retrieval. Nevertheless, PGE2 and its analog SLP appear to interfere with the hydrosmotic response of AVP by at least two mechanisms (first, by modulating cAMP generation, and second, by interfering with a cAMP-stimulated process, leading to the onset of water flow at a pre-cAMP level). Of importance, we have previously shown that both of these effects are reversed by SSP and may be in part mediated by activation of PKC (17). Although this interpretation is compatible with the current data, there may be other reasons to explain the present results.

In summary, using three of the structural analogs of PGI2, we have provided evidence for two functionally distinct PGI2 responses that stimulate both Ca2+/PKC and Gi-coupled pathways in the RCCD. Nevertheless, it remains unclear whether these two effects are mediated by a single or multiple receptors, because of the fact that the same IP receptor may be coupling to different G proteins or PGI2 actions may be mediated by the EP receptors. In contrast, there is a clear-cut distinction between the capacity of ILP/c-PGI2 and CCP to activate PKC/Ca2+ and depolarize the VT. Additional studies will be required to better characterize the distinct receptor systems involved and to determine whether IP splice variants/subtypes or other non-IP receptors can be identified that selectively modulate water permeability, cell calcium, and VT in the rabbit CCD.


    ACKNOWLEDGEMENTS

This research was supported by the Kidney Foundation of Canada and by the Medical Research Council of Canada (MT-14103).


    FOOTNOTES

Address for reprint requests and other correspondence: R. L. Hébert, Dept. of Cellular and Molecular Medicine, Faculty of Medicine, Rm. 1337, 451 Smyth Rd., Univ. of Ottawa, Ottawa, ON, Canada K1H 8M5 (E-mail: rlhebert{at}uottawa.ca).

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.

May 7, 2002;10.1152/ajprenal.00020.2002

Received 15 January 2002; accepted in final form 4 May 2002.


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