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.

慢性代谢性酸中毒（Chronic metabolic acidosis）在肾脏疾病患者中十分常见，且与肾衰竭进展密切相关。随着肾功能下降，肾单位（nephron）质量丢失，近端肾小管会出现适应性肥大与高代谢状态。我们此前在猪LLC-PK1细胞的慢性酸中毒体外模型中证实，氨生成增加（即氧化高代谢）与肾小管细胞功能障碍及氧化应激标志物升高存在关联。本研究旨在探究慢性酸中毒对HK-2细胞中氧化应激诱导的基因与蛋白质表达变化的影响。实验中将HK-2细胞以4×10⁴个/孔的密度接种于6孔板，于添加了5.5 mM葡萄糖、2 mM L-谷氨酰胺、5 μg/ml胰岛素、5 μg/ml转铁蛋白、5 ng/ml亚硒酸钠、0.4 μg/ml氢化可的松、5 ng/ml表皮生长因子（epidermal growth factor）、100 U/ml青霉素、100 μg/ml链霉素及10%胎牛血清（FCS）的DMEM/Ham’s F12培养基中培养72小时。随后将培养基更换为用12.5 mM双（羟甲基）氨基甲烷（bis-Tris）缓冲至pH 7.4、7.0或6.7的生长培养基，继续培养48小时。再将培养基替换为经12.5 mM双（羟甲基）氨基甲烷缓冲至对应pH的无血清培养基（SFM），培养24小时后收集细胞并提取RNA。我们分别通过3次独立实验，采用Tri-reagent（英国Sigma公司）按照制造商推荐的操作流程从HK-2细胞中提取总RNA；将各重复样本的RNA混合后，使用RiboGreen RNA定量试剂（RiboGreen RNA Quantitation Reagent，英国Molecular Probes、Invitrogen公司）测定RNA含量，并通过变性琼脂糖-甲醛凝胶电泳验证总RNA的完整性。