Stress-related gene expression in HK-2 cells exposed to pH 7.4, 7.0 or 6.7

Chronic metabolic acidosis occurs commonly in patients with diseased kidneys and is linked with progression to renal failure. As renal function declines there is loss of nephron mass and adaptive proximal tubular hypertrophy and hypermetabolism. We have previously demonstrated in an in vitro model of chronic acidosis in porcine LLC-PK1 cells increased ammonia generation (oxidative hypermetabolism) linked with tubular cellular dysfunction and increased markers of oxidative stress. The aim of this study was to determine the effect of chronic acidosis in HK-2 cells on changes in gene and protein expression stimulated by oxidative stress. HK-2 cells were were seeded onto 6 well plates at a density of 4x104 cells/well and cultured for 72 h in growth medium (DMEM/Ham s F12 medium supplemented with 5.5 mM glucose, 2 mM L-glutamine, 5 ug/ml insulin, 5 ug/ml transferrin, 5 ng/ml sodium selenite, 0.4 ug/ml hydrocortisone, 5 ng/ml epidermal growth factor, 100 U/ml penicillin, 100 ug/ml streptomycin, and 10% FCS). The medium was then replaced by growth media buffered to pH 7.4, 7.0 or 6.7 with 12.5 mM bis-Tris and cells were cultured for further 48 h. Media were changed to serum free media (SFM) buffered by 12.5 mM bis-Tris to pH 7.4, 7.0 or 6.7 and cells were harvested and RNA isolated after 24 h. Total RNA was isolated from HK-2 cells on 3 different occasions using Tri-reagent (Sigma, UK) as recommended by the manufacturer's protocol. The RNA from the respective replicate samples were pooled together and the RNA content was measured using RiboGreen RNA Quantitation Reagent (Molecular Probes, Invitrogen, UK). The integrity of the total RNA was verified on a denaturing agarose-formaldehyde gel.