Coenzyme Q10 protects renal proximal tubule cells against nicotine-induced apoptosis through induction of p66shc-dependent antioxidant responses

Abstract

Chronic nicotine exposure (via smoking, E-cigarettes) increases oxidative stress in the kidney that sensitizes it to additional injury in experimental models and in the renal patient. The pro-apoptotic p66 protein-via serine36 phosphorylation that facilitates its mitochondrial translocation and therein cytochrome c binding-generates oxidative stress that leads to injury of renal proximal tubule cells during chronic nicotine exposure. Coenzyme Q10-a clinically safe antioxidant-has been used against nicotine/smoke extract-associated oxidative stress in various non-renal cells. This study explored the anti-oxidant/anti-apoptotic effect of Coenzyme Q10 on nicotine-induced oxidative stress and its impact on p66shc in cultured rat renal proximal tubule cells (NRK52E). We studied the anti-oxidant effect of 10 µM Coenzyme Q10 using various mutants of the p66shc gene and also determined the induction of selected anti-oxidant entities (antioxidant response element, promoter of the manganese superoxide dismutase gene) in reporter luciferase assay during oxidative stress induced by 200 µM nicotine. Our studies revealed that Coenzyme Q10 strongly inhibits nicotine-mediated production of reactive oxygen species and consequent apoptosis that requires serine36 phosphorylation but not mitochondrial translocation/cytochrome c binding of p66. While both nicotine and Coenzyme Q10 stimulates the p66shc promoter, only nicotine exposure results in mitochondrial translocation of p66. In contrast, the Coenzyme Q10-stimulated and non-mitochondrial p66 activates the anti-oxidant manganese superoxide dismutase promoter via the antioxidant response elements and hence, rescues cells from nicotine-induced oxidative stress and consequent apoptosis.

Nicotine Exposure Augments Renal Toxicity of 5-azacytidine Through p66shc: Prevention by Resveratrol

“…It is that the Ser36 phsophorylated p66shc does not translocate to the mitochondria -as during its prooxidant function -but rather activates the ARE and hence, a set of antioxidant genes that harbors the ARE in their promoter. This phenomenon has been described in erythroleukemia cells upon hemin treatment (25) and by us in renal proximal tubule cells upon coenzyme Q10 treatment (17). Some studies demonstrated that RES protects keratinocytes (26) and prostate cells (27) from injury via activating (Ser36 phosphorylating) p66shc.…”

“…To prevent Serine36 phosphorylation or mitochondrial cytochrome c binding of p66shc, cells were transfected with a Serine-36 phosphorylation-deficient mutant p66shc (S36A) or a cytochrome c-binding-deficient mutant p66shc (W134F) as described elsewhere (16). Nrf2 expression was knocked down by a Nrf2 siRNA (Santa Cruz, Ja Jolla, CA) using Lipofectamine 3000 (Life Technologies, Grand Island, NY, USA) as previously described (17). HO-1 expression was knocked down by an HO-1 siRNA (Santa Cruz, La Jolla, CA) using Lipofectamine 3000 (Life Technologies, Grand Island, NY, USA).…”

Chronic Nicotine Exposure Reduces Antioxidant Function of Simvastatin in Renal Proximal Tubule Cells

“…Cells were transfected with either of the following reporter luciferase plasmids: HO1 promoter (20); CRE, ELK1 (Agilent Technologies, Santa Clara, CA) or MnSOD promoter (21). Cells were also co-transfected with a renilla luciferase plasmid (Promega, Madison, WI, USA) using Lipofectamine 3000 as suggested by the manufacturer (Thermo Fisher Scientific) and described elsewhere (19). After treatment, as described above, firefly and renilla luciferase activities were determined by a Dual Luciferase assay kit (Promega) and calculated as firefly/renilla ratios and expressed as a percentage that of the control.…”

“…Plasmid transfection. Some cells were also transfected with a shorthairpin p66shc (shp66shc) (19) to knockdown p66shc expression or with a plasmid containing constitutively activated CREB (VP16CREB) (22) in order to overexpress CREB.…”

Differential roles of 3-Hydroxyflavone and 7-Hydroxyflavone against nicotine-induced oxidative stress in rat renal proximal tubule cells

“…Nrf2 expression was knocked down by a Nrf2 siRNA (Santa Cruz, Ja Jolla, CA) using Lipofectamine 3000 (Life Technologies, Grand Island, NY) as we reported earlier [23]. To knockdown MnSOD expression, NRK52E cells were transfected with a short-hairpin (sh)MnSOD plasmid (Addgene, Cambridge, MA, USA) using Lipofectamine 3000 as we described elsewhere [24].…”