A dual blocker of endothelin A/B receptors mitigates hypertension but not renal dysfunction in a rat model of chronic kidney disease and sleep apnea

Abstract

Obstructive sleep apnea is characterized by recurrent episodes of pharyngeal collapse during sleep, resulting in intermittent hypoxia (IH), and is associated with a high incidence of hypertension and accelerated renal failure. In rodents, endothelin (ET)-1 contributes to IH-induced hypertension, and ET-1 levels inversely correlate with glomerular filtration rate in patients with end-stage chronic kidney disease (CKD). Therefore, we hypothesized that a dual ET receptor antagonist, macitentan (Actelion Pharmaceuticals), will attenuate and reverse hypertension and renal dysfunction in a rat model of combined IH and CKD. Male Sprague-Dawley rats received one of three diets (control, 0.2% adenine, and 0.2% adenine + 30 mg·kg^-1·day^-1 macitentan) for 2 wk followed by 2 wk of recovery diet. Rats were then exposed for 4 wk to air or IH (20 short exposures/h to 5% O₂-5% CO₂ 7 h/day during sleep). Macitentan prevented the increases in mean arterial blood pressure caused by CKD, IH, and the combination of CKD + IH. However, macitentan did not improve kidney function, fibrosis, and inflammation. After CKD was established, rats were exposed to air or IH for 2 wk, and macitentan feeding continued for 2 more wk. Macitentan reversed the hypertension in IH, CKD, and CKD + IH groups without improving renal function. Our data suggest that macitentan could be an effective antihypertensive in patients with CKD and irreversible kidney damage as a way to protect the heart, brain, and eyes from elevated arterial pressure, but it does not reverse toxin-induced tubule atrophy.

Keywords: chronic kidney disease; endothelin; intermittent hypoxia; macitentan; obstructive sleep apnea.

Fig. 1.

Macitentan (MACI) prevents increases in mean arterial pressure (MAP) in a rat model of chronic kidney disease (CKD) and sleep apnea. Over 7 wk of study, weekly 2-day averages of MAP (A) and heart rate (HR; B) and weekly body weight (C) and hematocrit (Hct%; D) are shown. Data are expressed as means ± SE. Results were analyzed by two-way repeated-measures ANOVA followed by Tukey’s multiple-comparison test. A: *P < 0.0001, CKD/sham and CKD/intermittent hypoxia (IH) vs. control/sham groups; +P < 0.0001, CKD + MACI/sham vs. CKD/sham or CKD + MACI/IH vs. CKD/IH groups. #Times 4–7 vs. time 3 (P = 0.02 control/IH; P < 0.01 MACI/IH; P = 0.03 CKD/IH; P < 0.02 CKD + MACI/IH). B: *P < 0.05 all CKD groups vs. the control/sham group. C: *P < 0.0005, all CKD groups vs. the control/sham group. D: *P < 0.005, all CKD groups vs. the control/sham group. *Times 5–6 P < 0.02 CKD/sham and CKD + MACI/sham vs. control/sham groups. n = 7 in all groups except for n = 6 in the control/IH group. B, baseline; A, after 2-wk adenine diet; R, after 2 wk of recovery.

Fig. 2.

Macitentan (MACI) does not prevent adenine-induced kidney dysfunction. Over 7 wk of study, weekly blood urea nitrogen (BUN) (A) and blood creatinine (B) are shown. At time 7, urinary volume (C), free water clearance (C_H2O) (D), creatinine clearance (C_Cre; E), and urinary protein (F) are shown. Data are expressed as means ± SE. A and B show results analyzed by two-way repeated-measures ANOVA. C–F show results analyzed by one-way ANOVA followed by a Tukey’s multiple-comparison test. *P < 0.05 vs. the control/sham group. n = 7 in all groups except n = 6 in the control/intermittent hypoxia (IH) group. B, baseline; A, after 2-wk adenine-diet; R, after 2 wk of recovery; CKD, chronic kidney disease.

Fig. 3.

Chronic kidney disease (CKD) increases pre-pro-endothelin (ET)-1 mRNA levels in the kidney cortex without affecting plasma ET-1 levels. At time 7, pre-pro-ET-1 mRNA levels in the kidney cortex (A), medulla (B), lungs (C), and ET-1 plasma levels (D) are shown. Data are expressed as means ± SE. Results were analyzed by one-way ANOVA followed by a Tukey’s multiple-comparison test. *P < 0.05 vs. the control/SHAM group. IH, intermittent hypoxia; MACI, macitentan.

Fig. 4.

Macitentan (MACI) does not prevent adenine-induced kidney interstitial fibrosis. Representative images of AZAN-stained kidneys to identify fibrosis (blue) in each of the seven groups are shown. Images on the left are ×4, in the middle are ×20 with no mask, and on the right are ×20 and masked using the HALO system to indicate areas of fibrosis. A summary graph with HALO-calculated percentages of fibrosis for entire sagittal cross sections from 7−8 kidneys from each group is shown. Data are expressed as means ± SE. Results were analyzed by one-way ANOVA followed by a Tukey’s multiple-comparison test. *P < 0.05 vs. the control/sham group. CKD, chronic kidney disease; IH, intermittent hypoxia.

Fig. 5.

Macitentan (MACI) does not prevent chronic kidney disease (CKD)-induced increases in renal CD3⁺ T cell infiltration. Left: total cell count (blue) and CD3⁺ signal (yellow) as detected by HALO software. Right: zoomed-in region depicted by the square in the middle. The summary graph shows HALO-calculated percentages of CD3⁺ cells. Data are expressed as means ± SE. Results were analyzed by one-way ANOVA followed by a Tukey’s multiple-comparison test. *P < 0.05 vs. the control/sham group.

Fig. 6.

Macitentan (MACI) does not prevent adenine-induced increases in renal macrophage (CD68⁺). Left: total cell count (blue) and CD68⁺ signal (yellow) as detected by HALO software. Right: zoomed-in region depicted by the square in the middle. The summary graph shows HALO-calculated percentages of CD68⁺ cells. Data are expressed as means ± SE. Results were analyzed by one-way ANOVA followed by a Tukey’s multiple-comparison test. *P < 0.02 vs. the control/sham group. CKD, chronic kidney disease.

Fig. 7.

Macitentan (MACI) does not prevent adenine-induced kidney inflammation. IL-6 and nitric oxide synthase isoform 2 (NOS2) mRNA levels were measured in the kidney cortex and medulla. Data are expressed as means ± SE. Results were analyzed by one-way ANOVA followed by a Tukey’s multiple-comparison test. *P < 0.01 vs. the control/sham group. CKD, chronic kidney disease.

Fig. 8.

Macitentan (MACI) reverses hypertension but not renal dysfunction in rats from chronic kidney disease (CKD) and CKD/intermittent hypoxia (IH) groups. A: mean arterial pressure (MAP). B: heart rate (HR). C: blood urea nitrogen (BUN). D: blood creatinine. E: creatinine clearance (C_Cre). F: urinary protein. n = 7 except for MACI/IH and CKD/IH with n = 6. Data are expressed as means ± SE. Results were analyzed by one-way ANOVA followed by a Tukey’s multiple-comparison test. MAP decreased in all rats that received MACI (CKD + MACI/sham, MACI/IH, and CKD + MACI/IH groups, times 1–2 vs. time 0, P < 0.05). +P < 0.05, CKD groups without MACI vs. CKD groups with MACI. *P < 0.05, CKD groups vs. the control/sham group.