Cell culture, drug, and peptide treatments:
Renal pre-glomerular smooth muscle cells (RPGSMCs) were isolated from Wistar-Kyoto rats as previously described (Zhu and Jackson, 2017) and cultured at 37ºC in SmGm-2 fully supplemented growth medium (Lonza, CC-3181) containing 0.5% FBS and SmGm-2 SingleQuot (Lonza, CC-3182) reagents and passaged using 1X trypsin–EDTA (Gibco, 10779413) dissolved in 1X PBS. During drug treatments, RPGSMCs were washed twice with 1X PBS and cultured in serum and growth factor starved Dulbecco’s Modified Eagle Medium/Ham’s F12 (DMEM/F12, Sigma, D6421) media containing: 100 U/mL penicillin/streptomycin (Gibco, 15140-122), 1.6 mM L-glutamine (Gibco, 25030-081), 200 μM L-ascorbic acid, 5 μg/mL apo-transferrin, and 6.25 ng/mL sodium-selenite. Losartan (Cayman Chemicals, 124750-99-8), PD123319 (Sigma, 136676-91-0), and AS18428456 FoxO inhibitor (Cayman, A15871) were dissolved in dimethyl sulfoxide (DMSO, D8418), while Angiotensin II (Ang II, Sigma, A9525) peptide was dissolved in sterile deionized distilled water for stock solutions prior to treatment. Treatment concentrations for Losartan, PD123319, AS1842856, and Ang II were 100 nM, 100 nM, 1 μM and 1 μM, respectively. Control treatments involved 0.1% DMSO treatment for 48 hours prior to harvesting. For NO stimulation experiments, cells were pretreated with 10 μM sildenafil citrate (Sigma, PZ0003) for 45 minutes to inhibit cGMP-specific phosphodiesterase 5 activity, and then stimulated with the NO-donor, diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, Cayman, 82100), for 15 minutes prior to lysis.