Angiotensin II Increases Nephrin Endocytosis via AT1-Receptor ERK 1/2 Activation

Background: Albuminuria is characterized by a disruption of the glomerular filtration barrier, which is composed of the fenestrated endothelium, the glomerular basement membrane, and the slit diaphragm. Nephrin is a major component of the slit diaphragm. Apart from hemodynamic effects, Angiotensin II (Ang II) enhances albuminuria by β-Arrestin2 dependent nephrin endocytosis. Methods: 1. Isolated mouse glomeruli were stimulated with Ang II and co-immunopreciptiation of nephrin with β-Arrestin 2 performed. 2. Differentiated murine podocytes were stimulated with Ang II and biotinylation assay with immunoprecipitated nephrin performed. 3. HEK293T cells were pretreated with AT1-receptor blockers candesartan and irbesartan before treatment with Ang II. 4. An AT1-receptor mutant, defective of EGFR transactivation and β-Arrestin 2 recruitment, was used as well as mutant β-Asrrestin2 K11/K12R. Different ERK 1/2 inhibitors were tested. Immunofluorescence imaging was performed in Cos7 cells. Results: Ang II increases nephrin-β-Arrestin2 binding in murine glomeruli and endocytosis in murine podocytes. Blocking the AT1 receptor with candesartan and irbesartan reduces the Ang II-mediated nephrin-β-Arrestin2 interaction. The inhibition of MAPK ERK 1/2 blocks Ang II-enhanced nephrin-β-Arrestin2 binding. ERK 1/2 signaling, which follows AT1 receptor activation, is mediated by G-protein signaling, EGFR transactivation, and β-Arrestin2 recruitment. A mutant AT1 receptor defective in EGFR transactivation and β-Arrestin2 recruitment reduces the Ang II-mediated increase in nephrin β-Arrestin2 binding. The mutation of β-Arrestin2K11,K12, critical for AT1 receptor binding, completely abrogates the interaction with nephrin, independent of Ang II stimulation. β-Arrestin2K11R,K12R does not influence nephrin cell surface expression. Conclusion: The data presented here deepen our molecular understanding of a blood-pressure-independent molecular mechanism of AT-1 receptor blockers (ARBs) in reducing albuminuria by nephrin endocytosis. MAP kinase signalling events play a relevant role in Ang II mediated nephrin endocytosis.