Thromboxane prostanoid receptors enhance contractions, endothelin-1, and oxidative stress in microvessels from mice with chronic kidney disease

Abstract

Cardiovascular disease is frequent in chronic kidney disease and has been related to angiotensin II, endothelin-1 (ET-1), thromboxane A2, and reactive oxygen species (ROS). Because activation of thromboxane prostanoid receptors (TP-Rs) can generate ROS, which can generate ET-1, we tested the hypothesis that chronic kidney disease induces cyclooxygenase-2 whose products activate TP-Rs to enhance ET-1 and ROS generation and contractions. Mesenteric resistance arterioles were isolated from C57/BL6 or TP-R+/+ and TP-R-/- mice 3 months after SHAM-operation (SHAM) or surgical reduced renal mass (RRM, n=6/group). Microvascular contractions were studied on a wire myograph. Cellular (ethidium: dihydroethidium) and mitochondrial (mitoSOX) ROS were measured by fluorescence microscopy. Mice with RRM had increased excretion of markers of oxidative stress, thromboxane, and microalbumin; increased plasma ET-1; and increased microvascular expression of p22(phox), cyclooxygenase-2, TP-Rs, preproendothelin and endothelin-A receptors, and increased arteriolar remodeling. They had increased contractions to U-46,619 (118 ± 3 versus 87 ± 6, P<0.05) and ET-1 (108 ± 5 versus 89 ± 4, P<0.05), which were dependent on cellular and mitochondrial ROS, cyclooxygenase-2, and TP-Rs. RRM doubled the ET-1-induced cellular and mitochondrial ROS generation (P<0.05). TP-R-/- mice with RRM lacked these abnormal structural and functional microvascular responses and lacked the increased systemic and the increased microvascular oxidative stress and circulating ET-1. In conclusion, RRM leads to microvascular remodeling and enhanced ET-1-induced cellular and mitochondrial ROS and contractions that are mediated by cyclooxygenase-2 products activating TP-Rs. Thus, TP-Rs can be upstream from enhanced ROS, ET-1, microvascular remodeling, and contractility and may thereby coordinate vascular dysfunction in chronic kidney disease.

Keywords: cyclooxygenase; mitochondria; oxidative stress; thromboxane; vascular remodeling.

Figure 1

Concentration-response relationships for phenylephrine (Panel A), U-46,619 (Panel B), endothelin-1 (Panel C), and cellular and mitochondrial reactive oxygen species generation with 10⁻⁷ mol·L⁻¹ endothelin-1 (Panel D) in mesenteric resistance arterioles from SHAM (open circles and broken lines or open boxes) and reduced renal mass mice (closed circles and continuous lines or shaded boxes). Comparing groups: *, P<0.05; **, P<0.01, ***, P<0.005. E:Dht; ethidium: dihydroethidium fluorescence ratio.

Figure 2

Contractile concentration responses to endothelin-1 in mesenteric resistance arterioles from mice with reduced renal mass after bath addition of: Panel A. Reactive oxygen species inhibitors: vehicle, tempol (10⁻⁴ mol·l⁻¹) or mitoTEMPO (10⁻⁴ mol·l⁻¹) or Panel B, cyclooxygenase (COX) inhibitors: SC-560 (10⁻⁶ mol·l⁻¹; COX-1 inhibitor), paracoxib (10⁻⁵ mol·l⁻¹; COX-2 inhibitor) or SC-560 plus paracoxib; or Panel C, thromboxane inhibitors: OKY-046NA (10⁻⁵ mol·l⁻¹; thromboxane A₂ synthase inhibitor), or SQ-29,548 (10⁻⁶ mol·l⁻¹; thromboxane-prostanoid inhibitor). Compared with vehicle: *, P<0.05; **, P<0.01; ***, P<0.005

Figure 3

Effect of thromboxane-prostanoid receptors and reduced renal mass on contractile responses in mesenteric resistance arterioles. Data from SHAM (open symbols) or RRM (shaded symbols) in TP-R +/+ (circles) or TP-R −/− (square) mice. Mean± SEM values (n=6 per group). Compared with TP-R +/+: *P<0.05; ***, P<0.005.

Figure 4

Effect of thromboxane-prostanoid receptors and reduced renal mass on cellular and mitochondrial reactive oxygen species generation with 10⁻⁷ mol·l⁻¹ endothelin-1 in mesenteric resistance arterioles. Mean± SEM values (n=6 per group) for cellular ROS generation (Panel A) and mitochondrial ROS generation (Panel B) from TP-R +/+ or TP-R −/− mice from SHAM (open boxes) or RRM (shaded boxes) groups. Compared to TP-R +/+: *P<0.05.

Figure 5

A flow diagram to demonstrate the potential central role for thromboxane-prostanoid receptors in mediating the increased endothelin-1, reactive oxygen species and microvascular remodeling that may contribute to progression of chronic kidney disease.