Endothelin-1 contributes to the progression of renal injury in sickle cell disease via reactive oxygen species

Abstract

Background and purpose: Endothelin-1 (ET-1) is increased in patients with sickle cell disease and may contribute to the development of sickle cell nephropathy. The current study was designed to determine whether ET-1 acting via the ETA receptor contributes to renal injury in a mouse model of sickle cell disease.

Experimental approach: Adult, humanized HbSS (homozygous for sickle Hb) mice had increased ET-1 mRNA expression in both the cortex and the glomeruli compared with mice heterozygous for sickle and Hb A (HbAS controls). In the renal cortex, ETA receptor mRNA expression was also elevated in HbSS (sickle) mice although ETB receptor mRNA expression was unchanged. Ligand binding assays confirmed that sickle mice had increased ETA receptors in the renal vascular tissue when compared with control mice.

Key results: In response to PKC stimulation, reactive oxygen species production by isolated glomeruli from HbSS sickle mice was increased compared with that from HbSA controls, an effect that was prevented by 1 week in vivo treatment with the selective ETA antagonist, ABT-627. Protein and nephrin excretion were both elevated in sickle mice, effects that were also significantly attenuated by ABT-627. Finally, ETA receptor antagonism caused a significant reduction in mRNA expression of NADPH oxidase subunits, which may contribute to nephropathy in sickle cell disease.

Conclusions and implications: These data support a novel role for ET-1 in the progression of sickle nephropathy, specifically via the ETA receptor, and suggest a potential role for ETA receptor antagonism in a treatment strategy.

Figure 1

ET‐1 mRNA expression in heterozygous control and SCD mice in isolated glomeruli (A) and renal cortex (B). ET_A (C) and ET_B (D) receptor mRNA expression was measured in the renal cortex. Data are mean ± SEM. *P < 0.05; n = 5, 6 or 7.

Figure 2

Specific ET‐1 (A) and ET‐3 (B) binding in membrane preparations from dissected renal vasculature. B_max for the ET_A receptor was calculated as the difference between B_max for ET‐1 and ET‐3 binding (C), while ET_B receptor binding was equated to maximum ET‐3‐specific binding (D). Data are mean ± SEM. *P < 0.05; n = 9, 10 or 11.

Figure 3

Representative luminescence reading in glomeruli isolated from control, SCD and SCD mice treated with the ET_A receptor antagonist, ABT‐627. (A) Total ROS luminescence was calculated from the area under the luminescence curve and normalized to mg protein (B). Panels C and D depict the average protein and nephrin excretion, respectively, for the three groups of mice. Data are mean ± SEM. *P < 0.05 versus control; ^† P < 0.05 versus untreated sickle mice; n = 6 or 8.

Figure 4

Expression of NADPH oxidase subunits, NOX4 and p47^phox, in renal cortex and glomeruli of SCD mice treated with or without the ET_A receptor antagonist, ABT‐627. Data are mean ± SEM. *P < 0.05; n = 6 or 7.