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

Abstract

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.

Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions

“…An additional problem is encountered when normalizing gene expression in experimentally manipulated tissues; in the context of this study, animals that had been exposed to a variety of chemicals, often at toxicological dose levels. Experimental manipulations can adversely affect gene expression levels in various tissues, including genes normally regarded as ‘stable housekeeping genes’ ( Das, Banerjee & Shapiro, 2013 ; DuBois et al, 2013 ; Martínez-Beamonte et al, 2011 ; Nair et al, 2014 ; Pohjanvirta et al, 2006 ; Santin et al, 2013 ; Silver et al, 2008 ; Taki, Abdel-Rahman & Zhang, 2014 ). Here, we have also shown that the expression of endogenous reference genes such as Tbp , B2m and Gapdh are affected in tissues having been exposed to chemicals during development.…”

Peer Review #2 of "Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions (v0.1)"

“…An additional problem is encountered when normalizing gene expression in experimentally manipulated tissues; in the context of this study, animals that had been exposed to a variety of chemicals, often at toxicological dose levels. Experimental manipulations can adversely affect gene expression levels in various tissues, including genes normally regarded as 'stable housekeeping genes' (Das et al 2013;DuBois et al 2013;Martínez-Beamonte et al 2011;Nair et al 2014;Pohjanvirta et al 2006;Santin et al 2013;Silver et al 2008;Taki et al 2014). Here, we have also shown that the expression of endogenous reference genes such as Tbp, B2m and Gapdh are affected in tissues having been exposed to chemicals during development.…”

Chemical Contaminants in Poultry Meat and Products

“…The tubular damage is seen as a result of exposure to Cd at low dose for a long period or at high dose for a short period. "s a result, glomerular iltration speed is decreased and inally renal impairment is seen [31]. International "gency on Cancer Research I"RC classiies Cd as human carcinogenic Group 1 [3 ]; while European Union Food Safety "uthority EFS" also underlined this fact stating, and exposure of Cd would lead increased risk for lung, endometrium, urinary bladder, and breast cancer [33].…”