Renal cells exposed to cadmium in vitro and in vivo: normalizing gene expression data

Cadmium (Cd) is a toxic metal with a long half-life in biological systems. This half-life is partly as a result of metallothioneins (MTs), metal-binding proteins with a high affinity for Cd. The high retention properties of the kidneys reside in proximal tubular cells that possess transport mechanisms for Cd-MT uptake, ultimately leading to more Cd accumulation. Researchers have studied MT-metal interactions using various techniques including quantitative real-time PCR (qPCR), an efficient tool for quantifying gene expression. Often a poor choice of reference genes, which is represented by their instability and condition dependency, leads to inefficient normalization of gene expression data and misinterpretations. This study demonstrates the importance of an efficient normalization strategy in toxicological research. A selection of stable reference genes was proposed in order to acquire reliable and reproducible gene quantification under metal stress using MT expression as an example. Moreover, in vitro and in vivo setups were compared to identify the influence of toxicological compounds in function of the experimental design. This study shows that glyceraldehyde-3-phosphate dehydrogenase (Gapdh), tyrosine monooxygenase/tryptophan5-monooxygenase activation-protein, zeta polypeptide (Ywhaz) and beta-actin (Actb) are the most stable reference genes in a kidney proximal tubular cell line exposed to moderate and high Cd concentrations, applied as CdCl2. A slightly different sequence in reference gene stability was found in renal cells isolated from rats in vivo exposed to Cd. It was further shown that three reference genes are required for efficient normalization in this experimental setup. This study demonstrates the importance of an efficient normalization strategy in toxicological research. Copyright (c) 2014 John Wiley & Sons, Ltd. The choice of reference genes for gene quantification is an important pre-requisite for carrying out new studies. A good normalization leads to biological relevant data interpretation as the detected variation in gene expression originates from biological rather than technical differences. The present study therefore serves as a baseline for renal cadmium (Cd) toxicity studies both in vitro and in vivo and the selected pool of reference genes that proved to be stable can be included in new experimental setups.