Cyclooxygenase-2-dependent prostacyclin formation and blood pressure homeostasis: targeted exchange of cyclooxygenase isoforms in mice

Abstract

Rationale: Cyclooxygenase (COX)-derived prostanoids (PGs) are involved in blood pressure homeostasis. Both traditional nonsteroidal antiinflammatory drugs (NSAIDs) that inhibit COX-1 and COX-2 and NSAIDs designed to be selective for inhibition of COX-2 cause sodium retention and elevate blood pressure.

Objective: To elucidate the role of COX-2 in blood pressure homeostasis using COX-1>COX-2 mice, in which the COX-1 expression is controlled by COX-2 regulatory elements.

Methods and results: COX-1>COX-2 mice developed systolic hypertension relative to wild types (WTs) on a high-salt diet (HSD); this was attenuated by a PGI(2) receptor agonist. HSD increased expression of COX-2 in WT mice and of COX-1 in COX-1>COX-2 mice in the inner renal medulla. The HSD augmented in all strains urinary prostanoid metabolite excretion, with the exception of the major PGI(2) metabolite that was suppressed on regular chow and unaltered by the HSD in both mutants. Furthermore, inner renal medullary expression of the receptor for PGI(2), but not for other prostanoids, was depressed by HSD in WT and even more so in both mutant strains. Increasing osmolarity augmented expression of COX-2 in WT renal medullary interstitial cells and again the increase in formation of PGI(2) observed in WTs was suppressed in cells derived from both mutants. Intramedullary infusion of the PGI(2) receptor agonist increased urine volume and sodium excretion in mice.

Conclusions: These studies suggest that dysregulated expression of the COX-2 dependent, PGI(2) biosynthesis/response pathway in the renal inner renal medulla undermines the homeostatic response to a HSD. Inhibition of this pathway may contribute directly to the hypertensive response to NSAIDs.

Figure 1. Effects of normal or HSD on blood pressure and renin expression in COX-1>COX-2, COX-2 KO and WT mice

A. Systolic blood pressure (BP) was measured in conscious COX-1>COX-2, WT and COX-2 KO mice by tail cuff (n = 9–12 for each group). Mice (6–7 weeks old) were fed either a normal chow diet (0.7% NaCl) or a HSD (8% NaCl) for 2 weeks. *, p<0.05 vs WT controls; #, p<0.05 vs normal diet. B. Quantitative real time RT-PCR was performed to analyze renin mRNA expression in the kidneys from COX-1>COX-2, WT and COX-2 KO mice. *, p<0.05 vs WT controls; #, p<0.05 vs normal diet, n=4–6.

Figure 2. Effect of high salt intake on PG biosynthesis in COX-1>COX-2, COX-2 KO and WT mice

Twenty four hour urine samples from COX-1>COX-2, COX-2 KO and WT male mice (8 weeks old) were collected, the urinary PGE₂ metabolite-PGE-M (A), PGI₂ metabolite PGI-M (B) PGD₂ metabolite-PGD-M (C), Tx₂ metabolite-Tx-M (D) were measured. Data are presented as mean ± SEMs, *, p<0.05 vs WT controls (n=7–20).

Figure 3. Effect of high salt intake on COX-1 and COX-2 expression in renal tissue obtained from COX-1>COX-2, COX-2 KO and WT mice

COX-1 and COX-2 mRNA in renal cortex (A, B) and inner medulla (C, D) were determined by real time RT-PCR; HS, HSD. *, p<0.05 vs WT controls; #, p<0.05 vs normal diet, n=4–6.

Figure 4. Effect of high salt intake on the capacity of renal medulla to generate PGs

Mice were fed a HSD or normal chow diet for 2 weeks, the renal medulla was dissected and the PG profile (A, PGE₂; B, PGD₂; C, 6-keto-PGF_1α; D, PGF_2α) was analyzed by mass spectrometry. *, p<0.05 vs WT controls, n=5–8; #, p<0.05 vs normal diet group, n=5–8.

Figure 5. HSD decreases the medullary IP receptor expression in COX-1>COX-2, COX-2 and WT mice

A. IP mRNA levels in medulla from COX-1>COX-2, COX-2 KO and WT mice were quantitated by real time RT-PCR; HS, HSD treatment. #, p<0.05 vs normal diet; *, p<0.05 vs WT controls, n=4–6. B. Representative Western blot of medullary IP receptor in COX-1>COX-2, COX-2 KO and WT mice. N, Normal salt diet; H, HSD. C. Relative level of IP protein to β-actin expression. #, p<0.05 vs normal diet, n=4.

Figure 6. COX protein expression and PG production by cultured RMICs from COX-1>COX-2 and WT mice

RMICs grown to sub-confluence were subjected to gradual changes in media osmolality from 330 to 630 mOsm/kg H₂O. After 24 h, the cells were incubated with arachidonic acid (20 μM) for 15 min. The cell lysates were prepared for Western blot of COX-1 and COX-2 (A) and the supernatants underwent PG analysis (B) by mass spectrometry. *, p<0.05 vs WT; #, p<0.01 vs 330 mOsm/kg H₂O group; n=6, repeated 3 times.

Figure 7. Activation of the IP by cicaprost evokes natriuresis in anesthetized mice

The right kidney was removed 1 wk before the experiment. The mice were anesthetized and catheterized as described in Methods. After 1 hr equilibration, cicaprost (10 ng/h) was infused into the renal medulla over 120 min. Urine was collected every 30 min before and during cicaprost infusion. Urine volume (UV, A) and urinary sodium excretion (U_NaV, B) were determined. Values presented as means ± SEM. *, p<0.05 vs basal level, n=6.