ET-1 increases reactive oxygen species following hypoxia and high-salt diet in the mouse glomerulus

Abstract

Aim: This study was designed to determine whether ET-1 derived from endothelial cells contributes to oxidative stress in the glomerulus of mice subjected to a high-salt diet and/or hypoxia.

Methods: C57BL6/J control mice or vascular endothelial cell ET-1 knockout (VEET KO) mice were subjected to 3-h exposure to hypoxia (8% O₂) and/or 2 weeks of high-salt diet (4% NaCl) prior to metabolic cage assessment of renal function and isolation of glomeruli for the determination of reactive oxygen species (ROS).

Results: In control mice, hypoxia significantly increased urinary protein excretion during the initial 24 h, but only in animals on a high-salt diet. Hypoxia increased glomerular ET-1 mRNA expression in control, but not in vascular endothelial cell ET-1 knockout (VEET KO) mice. Under normoxic conditions, mice on a high-salt diet had approx. 150% higher glomerular ET-1 mRNA expression compared with a normal-salt diet (P < 0.05). High-salt diet administration significantly increased glomerular ROS production in flox control, but not in glomeruli isolated from VEET KO mice. In C57BL6/J mice, the ETA receptor-selective antagonist, ABT-627, significantly attenuated the increase in glomerular ROS production produced by high-salt diet. In addition, chronic infusion of C57BL6/J mice with a subpressor dose of ET-1 (osmotic pumps) significantly increased the levels of glomerular ROS that were prevented by ETA antagonist treatment.

Conclusion: These data suggest that both hypoxia and a high-salt diet increase glomerular ROS production via endothelial-derived ET-1-ETA receptor activation and provide a potential mechanism for ET-1-induced nephropathy.

Keywords: endothelin; glomerulus; high-salt diet; hypoxia; kidney; reactive oxygen species.

Figure 1

Protein and albumin excretion in C57BL6/J mice maintained on a normal (a,c; 0.4% NaCl) or high (b,d;4% NaCl) salt diet; 24-hr urine collections were obtained before (baseline) and after a 3-hr exposure to hypoxia. Separate groups of mice on each diet were also treated with the ET_A selective receptor antagonist, ABT-627, for the duration of the study. *P<0.05 vs. ABT-627 mice on the same diet at the same time point.

Figure 2

Nephrin excretion in C57BL6/J mice maintained on a normal (a) or high salt (b) diet as measured in 24 hr urine collections before (baseline) and after 3-hr of hypoxia (8% O₂). A separate group of mice were also treated with the ET_A receptor antagonist, ABT-627. *P<0.05 vs. ABT-627 mice on the same diet at the same time point.

Figure 3

Urinary ET-1 excretion in mice maintained on a high salt diet with or without treatment with BT-627.

Figure 4

ET-1 mRNA measured in the glomerulus (a), outer (b), and inner (c) medulla of VEET KO or flox control mice. Separate groups of mice were subjected to 3-hr of hypoxia (8% O₂) immediately prior to harvesting tissue. *** P < 0.001 vs. VEET KO hypoxia. Renal cortical superoxide levels measured by HPLC from C57BL6/J mice exposed to hypoxia (8% O₂) and normoxic controls (d). **P<0.01 vs. normoxia.

Figure 5

PMA-stimulated ROS production in glomeruli from C57BL6/J mice infused subcutaneously with ET-1 (2pmol/kg/min) or saline for one week (osmotic mini-pump). Half of the animals were also treated with the ET_A receptor antagonist, ABT-627 (5mg/kg/day) in drinking water. *P<0.05 vs. saline treated animals not treated with ABT-627.

Figure 6

PMA-stimulated ROS production in glomeruli of mice maintained for 2 wks. on a normal (0.4% NaCl) or high (4% NaCl) diet. Results in panel a are from VEET KO and Flox control mice while results in panel b are from C57BL6/J mice treated with our without the ETA receptor antagonist, ABT-627. *P<0.05 vs. control mice on high salt.

Figure 7

Glomerular ROS production in Flox and VEET KO mice on a high salt diet. A subset of animals were exposed to 3 hours of hypoxia (8% O₂) *P<0.05 vs. HS VEET KO