Adverse Effects of Nicotine and Immunosuppression on Proximal Tubular Epithelial Cell Viability, Tissue Repair and Oxidative Stress Gene Expression

Abstract

Renal dysfunction in non-renal transplantation is a major arbiter of poor late allograft outcomes. Tobacco recidivism is an important modifiable risk marker for cardiac allograft loss, but its effects on renal dysfunction remain poorly studied. In a 96-well plate, 10(-5) proximal tubular epithelial (PTE) cells (HK-2, American Type Culture Collection) were cultured overnight and treated with sirolimus (SRL; 100 nmol/liter), nicotine (N; 10(-7) mol/liter) and mycophenolate mofetil (MMF; 10 micromol/liter), alone or in combination for 24 hours. Cell viability was quantified by treatment with tetrazolium salt WST-1 and calculated as the difference in percent inhibition with respect to the optical densitometry (OD) of treated and untreated cells. Gene and protein expression was analyzed using real-time polymerase chain reaction and Western blot techniques. OD decreased with SRL (-52.7 +/- 2.85%), N (-47.3 +/- 3.84%) and MMF (-53.3 +/- 2.4%) in isolation. Further reduction in OD occurred when N was combined with SRL (-63 +/- 2.3%, p < 0.04), MMF (-64.3 +/- 1.45%, p < 0.02) or the combination of SRL and MMF (-78.2%, p < 0.007). Compared with control, treatment of PTE cells with N increased mRNA expression of transforming growth factor-beta (TGF-beta; 10-fold), connective tissue growth factor (CTGF; 25-fold), osteopontin (OPN; 10-fold) and NADPH oxidase components (p22(phox), NOX-1 and Rac-1 at 18-, 16- and 12-fold, respectively). The pre-treatment of cells with inhibitor of superoxide generator diphenylene iodonium (DPI) reversed these effects. Nicotine adversely amplified the effects of SRL and MMF on tissue repair and oxidative stress markers, subsequently modulating PTE viability. However, caution is advised in extrapolating these in vitro findings to the human model.