Macrosep advance device

Glycated albumin (MGA) was prepared as described (50 (link)). Briefly, 500 μM methylglyoxal (Sigma, St. Louis, MO, US) was incubated with 100 uM BSA (Sigma) dissolved in phosphate buffered saline (PBS) for 24 hours, then washed on 10 kDa filters (Macrosep® Advance Device, Pall Life Sciences, MI, USA) to remove excess methyl glyoxal, reconstituted with DMEM/F12 serum free media and passed through a 0.2 μmicron filter. TGF-β (5 nanograms (ng)/ml, Peprotech, NJ, USA) was prepared according to manufacturer to treat cells in a subset of experiments. Cells were co-treated with one or combinations of the following: Ang-(1-7) (100 nM, Bachem, CA, USA), D-Ala7-Ang-(1-7) (10 μM, DAL, Bachem), ERK1/2 kinase inhibitor, PD 98059 (1 μM, PD, Sigma), TGF- β receptor kinase inhibitor; SB525334 (1 μM, SB, Selleckchem, TX, USA), the AT₁ receptor antagonist losartan (1 μM, Merck Pharmaceuticals, NJ, USA), the renin inhibitor aliskerin (1 μM, Tocris) and the ACE inhibitor lisinopril (1 μM, Sigma).