Angiotensin II induces calcium-mediated autophagy in podocytes through enhancing reactive oxygen species levels.

As well known, abnormalities of Angiotensin II (Ang II) is closely related with glomerular damage. This study was to investigate whether Ang II could affect autophagy in podocytes via oxidative stress, and whether autophagy had a positive role in protecting podocytes impaired by Ang II. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that Ang II induced podocyte death. The measurements of malondialdehyde (MDA) and H2 O2 levels, and flow cytometry assay revealed that Ang II considerably increased reactive oxygen species (ROS) generation in podocytes. Meaningfully, treatment with ROS scavenger N-(mercaptopropionyl)-glycine (N-MPG) could inhibit podocyte death and attenuate accumulation of ROS induced by Ang II. The patch-clamp experiments indicated that Ang II increased the current of transient receptor potential canonical 6 (TRPC6). Moreover, measurement of Fluo-3 image showed that Ang II increased intracellular Ca2+ level, as N-MPG and La3+ impeded Ang II induced Ca2+ influx. Acridine orange staining indicated that Ang II induced accumulation of acidic vacuoles. Beclin-1 and LC3 are essential for autophagosome formation. Furthermore, as one of the selective substrates for autophagy, P62 plays a key role in the formation of cytoplasmic proteinaceous inclusion. Western blot assay presented that Ang II obviously elevated LC3-II/LC3-I ratio and expression of beclin-1, and reduced expression of P62. Meanwhile, N-MPG expectedly down-regulated autophagy in Ang II-treated podocytes. Rapamycin can enhance the level of autophagy by inhibiting mTOR, and 3-methyladenine (3-MA) can inhibit autophagosome formation through blocking class III phosphatidylinositol 3-kinase. MTT assay exhibited that rapamycin significantly enhanced the cell viability, while 3-MA considerably reduced it in Ang II-treated podocytes. Consequently, this study demonstrated that Ang II could increase TRPC6 induced Ca2+ influx and enhance autophagy through increasing ROS levels in podocytes, and autophagy could protect Ang II-treated podocytes. Improving TRPC6 channels and autophagy may become a new targeted therapy to relieve glomerular damage induced by Ang II.