Endothelial mineralocorticoid receptor ablation does not alter blood pressure, kidney function or renal vessel contractility

Figures

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  Fig 1. Evaluation of Nr3c2 excision in endothelial mineralocorticoid receptor knockout mice (EC-MR KO) and in littermate controls (WT). Expression of mRNA of Nr3c2 from isolated aortic endothelial cells in EC-MR KO and WT mice (n = 6 per group). (B) Expression of mRNA from the abdominal aorta, mesenteric artery and renal artery. Amplification of mRNA transcribed from the Nr3c2 gene using a primer set spanning exons 2–4 would yield only one fragment of 253 bp in mice carrying the floxed Nr3c2 alleles, whereas mice expressing both the floxed Nr3c2 gene as well as the Cre recombinase driven by Tie2 promoter would yield another fragment of 113 bp. (C-E) Immunohistochemical analysis of kidney from intercrosses of mice expressing Tie2-Cre and the LacZ/eGFP reporter gene (Z/EG). eGFP expression is detected in cells positive for Cre recombinase. (C) Cre-dependent excision allowing eGFP expression can be detected in arterioles within kidney (denoted A). (D) eGFP positive cells were also present in the renal microvasculature, in the vasa recta bundels in the outer medulla (indicated by arrows) and (E) afferent arterioles (AA) located adjacent to the macula densa (MD), in glomerular (G)…

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  Fig 2. Evaluation of mean arterial pressure (MAP) and heart rate (HR) between wildtype WT and EC-MR KO during baseline and AngII infusion. Baseline (A) MAP and (B) HR were measured (D1-N3) in EC-MR KO and WT mice (n = 14–18 per group). (C) MAP and (D) HR were measured for 7 days (D3-D10) after start of AngII infusion (60 ng/kg/min) (n = 7–9 per group). (E) MAP and (F) HR were also measured for 4 days (D4-D7) after infusion of angiotensin II (AngII 100 ng/kg/min) (n = 7–9 per group). All data are mean ± SEM.

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  Fig 3. Determination of contractility and endothelial function in the renal artery in WT and EC-MR KO mice. Effect on contraction to the thromboxane analog U46619 of (A) WT and EC-MR KO at baseline (n = 8–12 per group), (B) WT and EC-MR KO after 2 weeks of angiotensin II (AngII) infusion (n = 7–8 per group) and (C) WT and EC-MR KO after 4 weeks of AngII infusion (n = 11–7 per group). Effect on acetylcholine (Ach)-induced endothelial-dependent relaxation of renal arteries from WT and EC-MR KO at baseline (n = 7–11 per group), (E) WT and EC-MR KO after 2 weeks of AngII infusion (n = 7–8 per group), and (F) WT and EC-MR KO after 4 weeks of AngII infusion (n = 11–7 per group). Effect on endothelial-independent relaxation by the NO donor, sodium nitroprusside (SNP) in (G) WT and EC-MR KO at baseline (n = 7–11 per group), (H) WT and EC-MR KO after 2 weeks of AngII infusion (n = 7–8 per group), and (I) WT and EC-MR KO after 4 weeks of AngII infusion (n = 11–7 per group). All data are mean ± SEM.

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  Fig 4. Vascular response of afferent arterioles and renal plasma flow (RPF) of WT and EC-MR KO mice. (A) Depolarized-induced vasoconstriction to 100 mM K+ solution of WT and EC-MR KO (n = 6–5 per group). (B) Secondary dilatory responses of afferent arterioles of WT and EC-MR KO (n = 6–5 per group). (C) Effective RPF calculated from para-aminohippurate clearance before and after 60 ng/kg/min infusion of angiotensin II (AngII) of WT and EC-MR KO (n = 5–7 per group).

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  Fig 5. Evaluation of renal function and Na+ handling. Renal function was evaluated in WT and EC-MR KO after 4 weeks of angiotensin II infusion by osmotic minipumps (1000 ng/kg/min) in metabolic cages. (A) Water intake, (B) Food intake, (C) urine volume, (D) serum Na+ concentration, (E) Urinary Na+ excretion, (F) estimated glomerular filtration rate determined by creatinine clearance, (G) fractional excretion of Na+. (H) Urinary Na+/ K+ ratio (n = 8 per group). All data are mean ± SEM.

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  Fig 6. Evaluation of renal damage. Renal damage was evaluated following 4 weeks of angiotensin II (AngII) infusion by osmotic minipumps (1000 ng/kg/min) in metabolic cages. (A) Albumin/creatinine ratio between WT and EC-MR KO littermates (n = 8 per group). (B-C) Histopathological changes evaluated by PAS staining in WT and EC-MR KO mice after 4 weeks of AngII infusion. (B) Evaluation of histopathological changes between WT and EC-MR KO were determined using PAS stained kidney crossections. Overall, no marked changes in structure could be seen between genotypes.