Cadmium-induced nephrotoxicity: from defense strategy to acclimation

Cadmium (Cd) indirectly induces reactive oxygen species (ROS) by (1) a displacement of redox-active metals, (2) depletion of redox scavengers, (3) inhibition of anti-oxidant enzymes and (4) inhibition of the electron transport chain. This ultimately results in mitochondrial damage leading to loss of function or cell death in multiple organs. A disturbance of the redox balance by Cd at the cellular level has been studied repeatedly in different experimental set-ups including differentiated cells at the whole animal level (including humans), tissue level, primary cell cultures and/or cell lines as well as non-differentiated cells. However the outcome of these results as to where the oxidative balance gets compromised at the cellular level and leads to pathologies at the level of the organ depends on a multitude of experimental and environmental conditions. It is important to keep in mind that the comparison of results from different experiments should be done with caution as many studies use high concentrations of Cd that might cause an effect in cell lines, but are irrelevant in terms of environmentally realistic exposures in animals. Several parameters that are inherently connected to the experimental set-up such as the route of Cd administration, the speciation of Cd applied and different cell types to be studied all determine the final outcome of Cd toxicity. In my study, research on kidney cell lines for example, results in direct exposure of Cd on these cells, while an oral or sub-cutaneous administration of Cd to rats in vivo results in Cd absorption by blood and other organs before it reaches the kidney resulting in changes of initial Cd speciation and concentration applied. Nevertheless, in terms of translational toxicology, it is highly interesting to compare the effects of Cd exposure levels in vitro to those in vivo, when Cd concentrations are determined for both experimental set-ups in a comparable manner. Cadmium has a half-life of 15-30 years in the kidney, and considerable damage is mostly confined to the apical domain of the proximal tubular cells (PTCs). These cells possess large amounts of mitochondria, an important source of ROS, but also a highly sensitive organelle to increased ROS levels. As such, mitochondrial alterations are involved in both damaging as well as adaptation responses. This makes the WKPT-0293 cell line derived from the S1 segment of PTCs of the rat a good model to study Cd toxicity, as they also possess transporters and receptors for Cd. The mechanism of Cd-induced ROS production in association with mitochondrial alterations leading to adaptation responses or cell death is not fully elucidated, allthough it is known that Cd can directly damage mitochondria. In general, the aim of this study was to investigate and compare the molecular and cellular responses in renal cells in vitro and in vivo exposed to different doses of Cd in an acute respectively subchronic way. The study specifically focused on the role of oxidative stress and mitochondria, and how they modulate different defense strategies and help cells to acclimate to Cd stress. Gene expression analyses of antioxidant genes and mitochondrial genes formed the core technology of this study and therefore it was very important that the results obtained from qPCR are accurate and reliable. For this purpose, an additional analysis to select appropriate reference genes was performed in both cell lines and kidney tissues of in vitro and in vivo experiments respectively. The choice of reference genes for gene quantification is an important pre-requisite for carrying out new studies. From a set of eight commonly used reference genes, it was found that glyceraldehyde-3-phosphate dehydrogenase (Gapdh), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (Ywhaz) and beta-actin (Actb) were the most stable reference genes in the in vitro experimental set-up, while Gapdh, Ywhaz and peptidylprolyl isomerase A /cyclophilin A (Ppia) were the stable set in vivo. It was shown that the stability and hence selection of reference genes varied between both experimental set-ups even when both the cells/tissues originated from rat. Subsequently all the qPCR results of transcripts analyzed in vitro or in vivo were normalized to their respective set of stable reference genes. The in vitro experiment made use of Cd free medium and of 1, 10 and 30 μM of Cd concentrations that caused low, moderate or high amounts of damage to PTCs. In this case, cells without or with visible alterations were obtained to study the antioxidant signature and role of mitochondria during Cd exposure and/or subsequent adaptation. It was shown that at lower Cd concentrations (1 μM CdCl2), mitochondrial DNA (mtDNA) content and anti-apoptotic gene expression increased with a decrease in reduced glutathione (GSH) content. At higher concentrations (10 and 30 μM CdCl2), there was an increase in hydrogen peroxide (H2O2) and glutathione disulfide (GSSG) content, metallothionein (MT) transcript levels, pro-apoptotic gene induction together with loss of mitochondrial DNA content and function as well as depletion of GSH. From these observations, we noticed that alterations in the balance between the pro- and anti-oxidant status of the cell can lead to cell signalling for survival or damage to cells, called the oxidative challenge. With increasing ROS and thereby cell stress, the mitochondrial system can collapse, leading to further damage of mitochondria and ROS production culminating in apoptosis and/or necrosis. Both mitochondrial abundance and a shift from GSH to GSSG as a response to Cd induced oxidative stress are probably involved in the final outcomes towards long-term exposure and were explored in more detail in the in vivo study. In the in vivo study, when rats were treated for 13 days with sub-chronic Cd exposure (1 mg CdCl2/kg bw), the GSSG/GSH ratio remained the same indicating the maintenance of the cellular redox state. Superoxide dismutase 2 and GSH immunohistochemical stainings together with gene expression analyses of antioxidants revealed their up-regulated activities, which points towards the cell’s attempt to defend, and adapt to the Cd-induced oxidative challenge. It is clear that under Cd stress, a new redox equilibrium is installed in the PTCs. The increased expression of genes responsible for regulation and biogenesis of mitochondria indicates a positive regulation for mitochondrial biogenesis. This was also reflected in terms of increased mtDNA content in Cd-treated animals as compared to control animals, which might be the result of an increasing energy demand of the cell necessary for adaptive and or survival mechanisms under Cd stress. Whereas increased caspase activity in Cd-treated animals points towards enhanced apoptosis under Cd stress, the simultaneous up-regulation of both pro- and antiapoptotic genes might represent the varying activities of damaged and undamaged cells present in the heterogeneous kidney tissue. Overall, the in vivo experimental results suggested a clear and obvious involvement of mitochondria in defending against the Cd-induced oxidative stress during long-term subchronic exposure. In conclusion, a comparison of the oxidative stress signature between acute in vitro versus sub-chronic in vivo Cd exposure shows a clear shift in terms of defense and acclimation. It was shown that GSH is a first line of defense in Cd toxicity both in vitro and in vivo due to its chelating and antioxidant properties. There is also a parallel activation of mitochondrial biogenesis in order to meet the increasing energy demand of the cells under stress. Other acclimation strategies i.e. chelation by MTs and antioxidant defense mechanisms come into play during exposure to higher Cd concentrations or prolonged exposure. Once the Cd stress is beyond the withholding limit, these mechanisms fail and the cell enters a point of no return leading to irreversible damage and eventually Cdinduced pathologies.

Publication year:2013

Accessibility:Open