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. 2016 Dec 22;6:39513. doi: 10.1038/srep39513

Figure 1. Ang II increases the glomerular permeability and enhances the interaction of β-arrestin to nephrin.

Figure 1

(a) Ang II increases the glomerular permeability significantly in FVB mice (n = 5 per data point, p < 0.004 tested by Kruskal-Wallis test). The glomerular permeability was measured by FITC-Ficoll 70 appearance in the urine before (white columns, 0 min), 60 minutes after the begin of Ang II stimulation (black columns, 60 min) and after additional 60 minutes of Ang II stimulation or 60 minutes of Ang II discontinuation washout (grey columns, 120 min or +60 min). The measured FITC-Ficoll 70 concentrations in the urine were referenced to the urine creatinine concentration. Pretreatment with candesartan blocks the Ang II mediated increased glomerular permeability (control n = 9, 0 min vs 60 min p = 0.35, 60 min vs 120 min p = 0.87; Ang II n = 9, 0 min vs 60 min p = 0.0036, 60 min vs 120 min p = 0.18, 0 min vs 120 min p = 0.16; Ang II + washout n = 5, 0 min vs 60 min p = 0.038, 60 min vs 120 min p = 0.25, 0 min vs 120 min p = 0.58; Ang II + ARB n = 3, 0 min vs 60 min p = 0.99). (b) Systolic blood pressure was controlled by tail cuff measurement. No significant systolic blood pressure differences between the Ang II treated mice and the controls were found. (c) Mouse glomeruli were isolated from kidneys and stimulated with Ang II. Thereafter β-arrestin was immunoprecipitated from glomerular lysates and nephrin was shown to coimmunoprecipitate with β-arrestin. Ang II enhances the interaction of β-arrestin to nephrin significantly (n = 4, p = 0.03). Comparable protein amounts for β-arrestin, nephrin and actin were ensured by Western blotting. (hc – IgG heavy chain). (d) Densitometry of the by β-arrestin2 coimmunoprecitated nephrin. (e) Kidneys from mice treated with Ang II were stained for nephrin and β-arrestin2. The merged images from Ang II treated animals showed a pronounced colocalization of nephrin with β-arrestin2 after Ang II stimulation in comparison to the control. The scale bar represents 20 μm. (f) Kidneys from mice treated with Ang II were stained for EEA-1. Under Ang II stimulation an enhanced EEA-1 expression is noted. The scale bar represents 20 μm.