ABSTRACT
A close link between stress protein up-regulation and oxidative damage may provide a novel therapeutic tool to counteract nephrotoxicity induced by toxic metals in the human population, mainly in children, of industrialized countries. Here we analysed the time course of the expression of several heat shock proteins, glucose-regulated proteins and metallothioneins in a rat proximal tubular cell line (NRK-52E) exposed to subcytotoxic doses of inorganic mercury and lead. Concomitantly, we used morphological and biochemical methods to evaluate metal-induced cytotoxicity and oxidative damage. In particular, as biochemical indicators of oxidative stress we detected reactive oxygen species (ROS) and nitrogen species (RNS), total glutathione (GSH) and glutathione-S-transferase (GST) activity. Our results clearly demonstrated that mercury increases ROS and RNS levels and the expressions of Hsp25 and inducible Hsp72. These findings are corroborated by evident mitochondrial damage, apoptosis or necrosis. By contrast, lead is unable to up-regulate Hsp72 but enhances Grp78 and activates nuclear Hsp25 translocation. Furthermore, lead causes endoplasmic reticulum (ER) stress, vacuolation and nucleolar segregation. Lastly, both metals stimulate the over-expression of MTs, but with a different time course. In conclusion, in NRK-52E cell line the stress response is an early and metal-induced event that correlates well with the direct oxidative damage induced by mercury. Indeed, different chaperones are involved in the specific nephrotoxic mechanism of these environmental pollutants and work together for cell survival.
Subject(s)
Heat-Shock Proteins/metabolism , Kidney Tubules, Proximal/drug effects , Lead/toxicity , Mercuric Chloride/toxicity , Oxidative Stress/drug effects , Animals , Apoptosis/drug effects , Biomarkers/metabolism , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Chaperone BiP , Glutathione/metabolism , Glutathione Transferase/metabolism , HSP27 Heat-Shock Proteins/metabolism , HSP72 Heat-Shock Proteins/metabolism , Kidney Tubules, Proximal/metabolism , Kidney Tubules, Proximal/pathology , Metallothionein/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Molecular Chaperones/metabolism , Necrosis , Nitric Oxide/metabolism , Rats , Reactive Oxygen Species/metabolism , Time FactorsABSTRACT
In this study, we investigated the influence of inorganic lead (Pb(II)), an environmental pollutant having nephrotoxic action, on the focal adhesion (FA) organization of a rat kidney epithelial cell line (NRK-52E). In particular, we evaluated the effects of the metal on the recruitment of paxillin, focal adhesion kinase, vinculin and cytoskeleton proteins at the FAs complexes. We provided evidences that, in proliferating NRK-52E cell cultures, low concentrations of Pb(II) affect the cell adhesive ability and stimulate the disassembly of FAs, thus inhibiting the integrin-activated signalling. These effects appeared to be strictly associated to the Pb-induced arrest of cell cycle at G0/G1 phase also proved in this cell line.
Subject(s)
Cytoskeletal Proteins/metabolism , Environmental Pollutants/toxicity , Focal Adhesions/drug effects , Kidney/drug effects , Lead/toxicity , Actins/drug effects , Animals , Cell Line , Cell Proliferation/drug effects , Cytoskeletal Proteins/analysis , Epithelial Cells/chemistry , Epithelial Cells/drug effects , Kidney/cytology , Microtubules/drug effects , RatsABSTRACT
Tubular epithelium represents the primary target of mercuric ions (Hg(2+)) nephrotoxicity. Although widely investigated, the mechanisms of Hg(2+) cell uptake, accumulation and excretion all along the nephron remain largely unknown. In the present study, native distal tubular-derived Madin-Darby canine kidney (MDCK) cells exposed to subcytotoxic (micromolar) HgCl(2) concentrations were used for investigating specific mechanisms involved in the tubular response to toxic metals. Inductively coupled plasma-mass spectrometry (ICP-MS) was firstly used for assessing HgCl(2) solubility and then for quantifying Hg(2+) cell uptake. Exposed to HgCl(2), MDCK cells showed a rapid, but transient, Hg(2+) accumulation. The metallic cation was found to affect cell density and morphology, being these effects related to the dose and the time of exposure. In parallel, an Hg(2+)-induced up-regulation of endogenous MRP1 and MRP2 export pumps, a significant HgCl(2)-dependent induction of protective cellular thiols and an increase in the glutathione conjugates metabolism were also observed. The functional suppression of MRPs activity, obtained by MK-571 treatment, increased the Hg(2+) cell content and the sensitivity of MDCK cells to HgCl(2). Our results demonstrate that, in MDCK cells, inorganic Hg(2+) promotes the activation of specific detoxifying pathways that may, at least partly, depend on the activity of MRP transporters.