Cadmium (Cd2+) induces oxidative stress that ultimately defines cell fate and pathology. Mitochondria are the main energy-producing organelles in mammalian cells, but they also have a central role in formation of reactive oxygen species, cell injury, and death signaling. As the kidney is the major target in Cd2+ toxicity, the roles of oxidative signature and mitochondrial function and biogenesis in Cd2+-related stress outcomes were investigated in vitro in cultured rat kidney proximal tubule cells (PTCs) (WKPT-0293 Cl.2) for acute Cd2+ toxicity (1–30 µM, 24 h) and in vivo in Fischer 344 rats for sub-chronic Cd2+ toxicity (1 mg/kg CdCl2 subcutaneously, 13 days). Whereas 30 µM Cd2+ caused ~50 % decrease in cell viability, apoptosis peaked at 10 µM Cd2+ in PTCs. A steep, dose-dependent decline in reduced glutathione (GSH) content occurred after acute exposure and an increase of the oxidized glutathione (GSSG)/GSH ratio. Quantitative PCR analyses evidenced increased antioxidative enzymes (Sod1, Gclc, Gclm), proapoptotic Bax, metallothioneins 1A/2A, and decreased antiapoptotic proteins (Bcl-xL, Bcl-w). The positive regulator of mitochondrial biogenesis Pparγ and mitochondrial DNA was increased, and cellular ATP was unaffected with Cd2+ (1–10 µM). In vivo, active caspase-3, and hence apoptosis, was detected by FLIVO injection in the kidney cortex of Cd2+-treated rats together with an increase in Bax mRNA. However, antiapoptotic genes (Bcl-2, Bcl-xL, Bcl-w) were also upregulated. Both GSSG and GSH increased with chronic Cd2+ exposure with no change in GSSG/GSH ratio and augmented expression of antioxidative enzymes (Gpx4, Prdx2). Mitochondrial DNA, mitofusin 2, and Pparα were increased indicating enhanced mitochondrial biogenesis and fusion. Hence, these results demonstrate a clear involvement of higher mitochondria copy numbers or mass and mitochondrial function in acute defense against oxidative stress induced by Cd2+ in renal PTCs as well as in adaptive processes associated with chronic renal Cd2+ toxicity.
Oxidative stress Metallothionein Antioxidative enzymes Mitochondrial DNA content Apoptosis
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The authors would like to thank Rosette Beenaerts, Biomedical Research Institute, Hasselt University for her technical assistance and Dr. Michael D. Garrick at the Department of Biochemistry, SUNY, Buffalo, NY 14214, USA for the Fischer 344 rats. This work was supported by Hasselt University [BOF (Bijzonder onderzoeksfonds) projects; BOF08G01] through a PhD grant for Ambily Ravindran Nair and a grant from the Deutsche Forschungsgemeinschaft (DFG TH345/11-1) to Frank Thévenod. Additional funding came from tUL-impulsfinanciering (project toxicology), and Methusalem project (08M03VGRJ).
Conflict of interest
The authors declare that they have no conflict of interest.
Supplemental Table1. Primer details of genes investigated. Column 2: The name of the gene and its official symbol; Column 3: The source of primers- (a) author name and year represents published literature, (b) NCBI/ PrimerBLAST represents primers designed at our lab using the PrimerBLAST software available from www.ncbi.nlm.nih.gov and (c) RTPrimerDB, a database that provides primer sequences and is available from www.rtprimerdb.org; Column 4: The amplicon length of the PCR product; Column 5: The primer efficiency of all genes with two percentages, originating from either pooled control samples or pooled treated samples tested for their efficiency. (PDF 66 kb)
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