MO008NEPHROTOXIC MECHANISMS OF GADOLINIUM: IMPLICATIONS FOR THE USE OF GADOLINIUM-BASED CONSTRAST AGENTS
Authors: Reis Sousa Nícia , Santos-Silva Alice , Coimbra Susana , Valente Maria João 
Published: May 2021

Abstract

Background and Aims
Gadolinium-based contrast agents are widely used for magnetic resonance imaging, and although they may be considered well tolerated at recommended dosing levels, recent evidences support the deposition of free gadolinium in the tissues, and its slow release into circulation, resulting in long-term toxicity, which is aggravated in renal patients. The kidney, as the major excretion organ of these agents, and particularly the proximal tubule, as a common location of xenobiotics’ bioaccumulation, may be key targets of gadolinium’s deleterious effects. This study aimed at unveiling the nephrotoxic potential and the underlying mechanisms of toxicity of gadolinium, using an in vitro model of normal human proximal tubular cells (HK-2 cell line).

Method
HK-2 cells were exposed for 24 h to a wide concentration range of gadolinium, in the form of trichloride hexahydrate, and cell viability was assessed to estimate the half maximal effective concentration (EC50) and the subtoxic concentration eliciting 1 % of cell death (EC01). These ECs were further used to determine the effects of gadolinium on the cell’s oxidative status, mitochondrial function, cell death mechanisms and lipid deposition, through variable colorimetric and fluorometric assays. Expression of the pro-inflammatory gene IL6 was also determined through quantitative PCR.

Results
Gadolinium induced cell death in a concentration-dependent manner, with estimated EC01 and EC50 of 3 and 340 µM, respectively. When compared to control cells, the subtoxic concentration showed no significant effects in terms of reactive oxygen and nitrogen species (ROS and RNS) production, total glutathione levels (tGSH) or total antioxidant status (TAS). On the other hand, the EC50 showed significant disruption of the oxidative status of the cell, with over 60 % depletion of tGSH cell contents (p &lt; 0.01) and a significant decrease of TAS (p &lt; 0.05). However, this effect was not accompanied by an increase, but rather by a significant decline in ROS and RNS production of about 50 % below control (p &lt; 0.0001). At the EC50, but not at EC01, it was also observed the disturbance of the mitochondrial and energetic homeostasis, as showed by the increment of intracellular free calcium levels, hyperpolarization of the mitochondrial membrane, and decay of the ATP levels (p &lt; 0.0001). Cell death induced by gadolinium was characterized by typical morphological changes of late apoptosis and necrosis, with a significant increase in propidium iodide uptake and lactate dehydrogenase leakage (p ≤ 0.0001) at EC50. The presence of neutral lipids-containing vesicles was observed in the cytoplasm of cells exposed to gadolinium using the fluorescent dye BODIPY, already noticeable at the subtoxic concentration. Cells exposed to gadolinium also showed increased expression of IL6, though this effect was only significant at the EC01 (p &lt; 0.05).

Conclusion
Gadolinium showed marked cytotoxic potential at micromolar levels in HK-2 cells. This cytotoxicity was characterized by increased oxidative stress independent of ROS and RNS production and mitochondrial dysfunction followed by cell death via late apoptosis and necrosis. At a subtoxic concentration, gadolinium was also able to elicit the accumulation of lipidic vesicles within the cells’ cytoplasm, and to trigger a pro-inflammatory response. Although it is still unclear which amount of gadolinium is in fact released from the complexes commonly used as contrast agents, this study shows that gadolinium ion has direct nephrotoxic potential, with noteworthy manifestations at subtoxic concentrations.

Acknowledgments
This work was supported by Applied Molecular Biosciences Unit (UCIBIO), financed by national funds from FCT/MCTES (UIDB/04378/2020), by North Portugal Regional Coordination and Development Commission (CCDR-N)/NORTE2020/Portugal 2020 (Norte-01-0145-FEDER-000024).