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.