Abstract
We have previously shown that carboplatin induces inflammation and apoptosis in renal tubular cells (RTCs) through the activation of the nuclear factor of activated T cells-3 (NFAT3) protein by reactive oxygen species (ROS), and that the ROS-mediated activation of NFAT3 is prevented by N-acetyl cysteine and heme oxygenase-1 treatment. In the current study, we investigated the underlying molecular mechanisms of the protective effect of L-carnitine on carboplatin-mediated renal injury. Balb/c mice and RTCs were used as model systems. Carboplatin-induced apoptosis in RTCs was examined using terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling. We evaluated the effects of the overexpression of the peroxisome-proliferator-activated receptor alpha (PPARα) protein, the knockdown of PPARα gene, and the blockade of AMPK activation and PPARα to investigate the underlying mechanisms of the protective effect of L-carnitine on carboplatin-mediated renal injury. Carboplatin reduced the nuclear translocation, phosphorylation, and peroxisome proliferator responsive element transactivational activity of PPARα. These carboplatin-mediated effects were prevented by L-carnitine through a mechanism dependent on AMPK phosphorylation and subsequent PPARα activation. The activation of PPARα induced cyclooxygenase 2 (COX-2) and prostacyclin (PGI2) synthase expression that formed a positive feedback loop to further activate PPARα. The coimmunoprecipitation of the nuclear factor (NF) κB proteins increased following the induction of PPARα by L-carnitine, which reduced NFκB transactivational activity and cytokine expression. The in vivo study showed that the inactivation of AMPK suppressed the protective effect of L-carnitine in carboplatin-treated mice, indicating that AMPK phosphorylation is required for PPARα activation in the L-carnitine-mediated protection of RTC apoptosis caused by carboplatin. The results of our study provide molecular evidence that L-carnitine prevents carboplatin-mediated apoptosis through AMPK-mediated PPARα activation.
Highlights
The quaternary ammonium compound, L-carnitine (L-trimethyl-3-hydroxy-ammoniabutanoate), is synthesized in cells from lysine and methionine precursors [1], and is required for the transport of fatty acids from the cytosol into the mitochondria during lipid catabolism
Carboplatin increased the levels of proteins involved in apoptotic and inflammatory signaling in renal tubular cells (RTCs), including hypo-pNFAT3, Bcl-xS, caspase 3, and p65/p50, and the levels of these proteins were significantly reduced in L-carnitine-treated cells
We observed that the protective effect of L-carnitine coincided with increased levels of peroxisome-proliferator-activated receptor alpha (PPARa) and cyclooxygenase 2 (COX-2), whereas the levels of PPARa and COX-2 were reduced in cells treated using carboplatin alone (Figures 1)
Summary
The quaternary ammonium compound, L-carnitine (L-trimethyl-3-hydroxy-ammoniabutanoate), is synthesized in cells from lysine and methionine precursors [1], and is required for the transport of fatty acids from the cytosol into the mitochondria during lipid catabolism. The predominant doselimiting toxicities of carboplatin are bone marrow suppression and ototoxicity caused by free-radical oxidative injury [9] Using both gain- and loss-of-function strategies, we previously showed that the activation of the transcription factor, nuclear factor of activated T cells-3 (NFAT3), induces RTC apoptosis, and that NFAT3-mediated apoptosis in RTCs is blocked by HO-1 gene therapy and N-acetyl cysteine (NAC) treatment [10]. The antioxidant activities of L-carnitine warrant further investigation to determine whether it might provide protection against carboplatin-mediated renal injury
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