Chronic nicotine exposure augments renal oxidative stress and injury through transcriptional activation of p66shc

Abstract

Background: Chronic nicotine (Ch-NIC) exposure exacerbates ischemia/reperfusion (I/R)-induced oxidative stress and acute kidney injury (AKI), and mitochondrial production of reactive oxygen species (ROS) in cultured renal proximal tubule cells (RPTCs). Because Ser36-phosphorylated p66shc modulates mitochondrial ROS production and injury of RPTCs, we hypothesized that Ch-NIC exacerbates AKI by increasing stress-induced phosphorylation of p66shc.

Methods: We first tested whether Ch-NIC augments I/R-AKI-induced expression and phosphorylation of p66shc in vivo. We then examined whether knocking down p66shc, or impairing its Ser36 phosphorylation or binding to cytochrome c, alters the effects of Ch-NIC on oxidative stress (H₂O₂)-induced production of ROS, mitochondrial depolarization and injury in RPTCs in vitro.

Results: We found that Ch-NIC increased the expression of p66shc in the control and ischemic kidneys, but only increased its Ser36 phosphorylation after renal I/R. Knocking down p66shc or impairing phosphorylation of its Ser36 residue, via the S36A mutation (but not the phosphomimetic S36D mutation), blunted Ch-NIC + H2O2-dependent ROS production, mitochondrial depolarization and injury in RPTCs. Additionally, Ch-NIC + H2O2-dependent binding of p66shc to mitochondrial cytochrome c was attenuated by S36A mutation of p66shc, and impairing cytochrome c binding (via W134F mutation) abolished ROS production, mitochondrial depolarization and injury, while ectopic overexpression of p66shc (which mimics Ch-NIC treatment) augmented oxidant injury. We determined that Ch-NIC stimulates the p66shc promoter through p53- and epigenetic modification (promoter hypomethylation).

Conclusions: Ch-NIC worsens oxidative stress-dependent acute renal injury by increasing expression and consequent oxidative stress-dependent Ser36 phosphorylation of p66shc. Thus, targeting this pathway may have therapeutic relevance in preventing/ameliorating tobacco-related kidney injury.

Keywords: ROS; cytochrome c; mitochondria; nicotine; p66shc.

FIGURE 1:

Effects of Ch-NIC on expression and Ser36 phosphorylation of p66shc in control mice and mice subjected to I/R-AKI. Kidney lysates were prepared and blotted with an anti-p66shc (A) and after stripping by an anti-pSer36-p66shc (B) antibody and finally with an anti-actin antibody after serial stripping. Representative examples are shown in the top panels, while the densitometric analysis of the results, normalized to actin, is shown in the bottom graphs. N = 6, *P < 0.05 compared with the vehicle control or as indicated.

FIGURE 2:

Ch-NIC increases the transcription of p66shc via p53- and epigenetic (DNA hypomethylation) mechanisms. (A) To assess transcription of p66shc in response to Ch-NIC and/or H₂O₂, TKPTS cells were transfected with a p66shc promoter luciferase-plasmid together with a Renilla luciferase plasmid. Firefly (p66shc-Luc) and Renilla luciferase activities were determined 24 h after treatment. The values were calculated as firefly/renilla ratios. *P < 0.05 compared with untreated. (B) To determine whether the p66shc promoter is upregulated by p53 and hypomethylation, after transfection with the p66shc-Luc/Renilla plasmids TKPTS cells were treated with either 50 μM PIF or 100 nM 5AZA followed by NIC or H₂O₂ for 24 h. Results were calculated as firefly/Renilla ratios and expressed as percentage of values obtained without PIF or 5AZA treatment. *P < 0.05 compared with the values without PIF or 5AZA.

FIGURE 3:

The role of p66shc in the adverse effects of Ch-NIC in vitro. (A) p66shc knockdown (p66shc k.d. TKPTS) cells were exposed to Ch-NIC for 24 h and then treated with H₂O₂. Immediately after H₂O₂ exposure, ROS production and mitochondrial depolarization (JC-1) were determined, although LDH release was measured 24 h later. The results were compared with control TKPTS cells that were treated similarly and expressed as percentage of responses from control cells. *P < 0.05 compared with untransfected cells. (B) To test whether rescuing p66shc levels—which mimics the effects of Ch-NIC—would restore H₂O₂-induced injury, TKPTS cells were transfected with increasing amounts of a w.t. p66shc plasmid and treated with H₂O₂. Twenty-four hours later, LDH release was determined to assess cellular injury. The results are expressed as percentage of released LDH shown in untreated cells. *P < 0.05 compared with untransfected cells.

FIGURE 4:

The Ser36 phosphorylation of p66shc is necessary for adverse effects of Ch-NIC and binding of p66shc to cytochrome C. (A) TKPTS cells were transfected with the Ser36 phosphorylation-deficient (S36A) or the Ser36 phosphomimetic (S36D) mutant plasmid and exposed to Ch-NIC and H₂O₂ as before and ROS production, mitochondrial depolarization as well as LDH release was determined and compared with vector-transfected (w.t.) cells. Results were compared with w.t. TKPTS cells that were treated similarly and expressed as percentage of responses of control cells. *P < 0.05 compared with w.t. cells. (B) To determine whether the Ser36 phosphorylation of p66shc is needed for p66shc to bind to cytochrome c, TKPTS cells were treated with Ch-NIC and H₂O₂ in the presence or absence of the S36A mutant. Cell lysates were immunoprecipitated with an anti-cytochrome c antibody and immunoblotted with an anti-p66shc antibody. The same blot was re-hybridized with an anti-cytochrome c antibody after stripping. A representative example is shown in (B), while the densitometric analyses of the p66shc/cytochrome c ratios are shown in (C). *P < 0.05 compared with the untreated control or as indicated.

FIGURE 5:

Binding of p66shc to cytochrome c is necessary for Ch-NIC-induced exacerbation of oxidant injury. Cells transfected with a p66shc mutant (W134F), which is impaired in cytochrome c-binding. ROS production, mitochondrial depolarization and cell injury were determined after treatment with Ch-NIC + H₂O₂ as described before. The results are expressed as percentage of untransfected (control) cells that were treated similarly. *P < 0.005.

FIGURE 6:

Proposed effects of Ch-NIC exposure on renal I/R injury. I/R-AKI upregulates the p66shc promoter through p53 and DNA hypomethylation then Ser36-phosphorylates it leading to increased translocation of p66shc to the mitochondria and ultimately increased cytochrome c binding. The result is augmentation of mitochondrial ROS production and mitochondrial depolarization as well as consequent injury. Ch-NIC exacerbates this process by augmenting the expression—but not Ser36 phosphorylation—of p66shc through p53- and DNA hypomethylation-dependent mechanisms.