Potential involvement of P2Y2 receptor in diuresis of postobstructive uropathy in rats

Abstract

AVP resistance of the medullary collecting duct (mCD) in postobstructive uropathy (POU) has been attributed to increased production of PGE2. P2Y2 receptor activation causes production of PGE2 by the mCD. We hypothesize that increased P2Y2 receptor expression and/or activity may contribute to the diuresis of POU. Sprague-Dawley rats were subjected to bilateral ureteral obstruction for 24 h followed by release (BUO/R, n = 17) or sham operation (SHM/O, n = 15) and euthanized after 1 wk or 12 days. BUO/R rats developed significant polydipsia, polyuria, urinary concentration defect, and increased urinary PGE2 and decreased aquaporin-2 protein abundance in the inner medulla compared with SHM/O rats. After BUO/R, the relative mRNA expression of P2Y2 and P2Y6 receptors was increased by 2.7- and 4.9-fold, respectively, without significant changes in mRNA expression of P2Y1 or P2Y4 receptor. This was associated with a significant 3.5-fold higher protein abundance of the P2Y2 receptor in BUO/R than SHM/O rats. When freshly isolated mCD fractions were challenged with different types of nucleotides (ATPgammaS, ADP, UTP, or UDP), BUO/R and SHM/O rats responded to only ATPgammaS and UTP and released PGE2, consistent with involvement of the P2Y2, but not P2Y6, receptor. ATPgammaS- or UTP-stimulated increases in PGE2 were much higher in BUO/R (3.20- and 2.28-fold, respectively, vs. vehicle controls) than SHM/O (1.68- and 1.30-fold, respectively, vs. vehicle controls) rats. In addition, there were significant 2.4- and 2.1-fold increases in relative mRNA expression of prostanoid EP1 and EP3 receptors, respectively, in the inner medulla of BUO/R vs. SHM/O rats. Taken together, these data suggest that increased production of PGE2 by the mCD in POU may be due to increased expression and activity of the P2Y2 receptor. Increased mRNA expression of EP1 and EP3 receptors in POU may also help accentuate PGE2-induced signaling in the mCD.

Fig. 1.

Characterization of postobstructive uropathy in rats. Sprague-Dawley rats were subjected to bilateral ureteral obstruction for 24 h followed by release (BUO/R) or sham operation (sham). Water intake (A), urine output (B), and urine osmolality (C) were monitored before surgical procedures (day 0) and 7 or 8 days and 12 days after release of obstruction. Since there were no significant differences in these parameters 7 or 8 days after release, data were combined and are presented as day 7/8. Values are means ± SE of number of rats shown in parentheses. *P < 0.001; **P < 0.05 vs. corresponding day 0 value (by ANOVA).

Fig. 2.

Changes in protein abundances of aquaporin-2 (AQP-2) and P2Y₂ receptor in rat inner medulla during postobstructive uropathy. BUO/R and sham rats (n = 4/group) were euthanized on day 12. Whole inner medullary tissue samples were immunoblotted using specific antibodies to AQP-2 or P2Y₂ receptor. A: immunoblots of AQP-2 and P2Y₂ receptor in sham and BUO/R rats. Equal amounts of proteins from 1 rat per lane were loaded onto gels. Equality of protein loading was checked by staining loading gels with Coomassie blue or probing blots for protein abundance of β-actin. AQP-2 protein blots show 29-kDa native band and a smear of glycosylated band extending between 35 and 50 kDa. In P2Y₂ receptor protein blots, 45- and 105-kDa bands are specific, as documented previously (21, 24, 49). B: densitometry of bands in immunoblots. Immunoblots were digitized, and pixel densities of the bands were determined. Combined density values (means ± SE) of 29- and 35- to 50-kDa protein bands of AQP-2 and 47- and 105-kDa protein bands of P2Y₂ receptor in sham and BUO/R groups were normalized to density of respective β-actin bands. Results are plotted as percentage of mean values in the sham group. *P < 0.01; **P < 0.05 vs. sham.

Fig. 3.

Increased excretion of urinary PGE₂ metabolites during postobstructive uropathy. BUO/R and sham rats were euthanized on day 8 or 12. Aliquots of 24-h urine samples were collected and processed for assay of PGE₂ metabolite. Nanograms of urinary PGE₂ metabolite per 24 h are presented as percentage of values in sham rats on the corresponding days: 17.1, 17.2, and 9.9 ng urinary PGE₂/24 h on days 0, 8, and 12, respectively. Values are means ± SE of number of rats in parentheses. *P < 0.01 vs. sham on the corresponding day.

Fig. 4.

Changes in relative expression of cyclooxygenases 1 and 2 (COX-1 and COX-2) in rat inner medulla during postobstructive uropathy. BUO/R and sham rats (n = 4/group) were euthanized on day 12. RNA was extracted and reverse transcribed, and cDNA samples were amplified with gene-specific primer pairs for COX-1, COX-2, or β-actin using real-time PCR. Expression of COX enzymes was normalized to that of β-actin and plotted as percentage of mean values in sham group. Values are means ± SE. *P < 0.04; (unpaired t-test); **P < 0.03 (Mann-Whitney test) vs. corresponding sham.

Fig. 5.

Changes in relative expression of prostanoid E (EP) receptor subtypes 1 and 3 (EP₁ and EP₃) in rat inner medulla during postobstructive uropathy. BUO/R (n = 4) and sham (n = 3) rats were euthanized on day 8. RNA was extracted and reverse transcribed, and cDNA samples were amplified with gene-specific primer pairs for EP₁ and EP₃ receptors or β-actin using real-time PCR. Expression of EP receptor subtypes was normalized to that tk;4of β-actin to obtain relative expression values. Values are means ± SE. *P < 0.03; **P < 0.05 vs. corresponding sham (by unpaired t-test).

Fig. 6.

Changes in relative expression of P2Y receptor subtypes in rat inner medulla during postobstructive uropathy. BUO/R (n = 4) and sham (n = 3) rats were euthanized on day 8. RNA was extracted and reverse transcribed, and cDNA samples were amplified with specific primer pairs for P2Y₁, P2Y₂, P2Y₄, or P2Y₆ receptor or β-actin using real-time PCR. Expression of P2Y receptor subtypes was normalized to that of β-actin to obtain relative expression values. Values are means ± SE. *P < 0.03 vs. corresponding sham (by Mann-Whitney test).

Fig. 7.

Changes in the pattern of nucleotide-stimulated PGE₂ release by inner medullary collecting duct preparations during postobstructive uropathy. BUO/R and sham rats (n = 5/group) were euthanized on day 8. Inner medullas from each group were pooled separately and processed. Fractions enriched in collecting ducts were freshly prepared from pooled inner medullas. Aliquots of fractions were warmed to 37°C and then challenged with each nucleotide at 50 μM for 20 min. PGE₂ released into the medium was assayed and normalized to protein contents of the corresponding incubations. Values are means ± SE of triplicate incubations. A: pattern of nucleotide-stimulated PGE₂ release in sham rats plotted as percentage of mean values in vehicle incubations. *P < 0.01 vs. vehicle. B: pattern of nucleotide-stimulated PGE₂ release in BUO/R rats plotted as percentage of mean values in vehicle incubations. *P < 0.001 vs. vehicle.