Sub-chronic exposure to fluoride impacts the response to a subsequent nephrotoxic treatment with gentamicin Mariana Cárdenas-González a , Tania Jacobo Estrada a , Rafael Rodríguez-Muñoz b , Jonatan Barrera-Chimal c , Norma A. Bobadilla c , Olivier C. Barbier a and Luz M. Del Razo a * ABSTRACT: Fluoride is an important groundwater contaminant, and more than 200 million people are exposed to high fluoride levels in drinking water, the major source of fluoride exposure. Exposure above 2 ppm of fluoride is associated with renal impair- ment in humans. In rats, moderate levels of fluoride induce kidney injury at early stages in which the glomerular filtration rate (GFR) is not altered. In the present study, we investigated if sub-nephrotoxic stimulus induced by fluoride might impact the re- sponse to a subsequent nephrotoxic treatment with gentamicin. Male Wistar rats (~21 days) were exposed to 0, 15 or 50 ppm of fluoride through drinking water during 40 days. Afer that, rats were co-exposed to gentamicin (40 mg kg –1 day –1 , 7 days). Gentamicin induced a marked decrease in the GFR and an increase in urinary levels as well as the protein and mRNA expression of biomarkers of early kidney injury, such as Kim-1. Interestingly, gentamicin nephrotoxicity was less pronounced in groups previously exposed to fluoride than in the group only treated with gentamicin. Fluoride induced Hsp72, a cytoprotective molecule, which might have improved the response against gentamicin. Moreover, fluoride decreased the expression of megalin, a molecule necessary for inter- nalization of gentamicin into the proximal tubule, potentially reducing gentamicin accumulation. The present results suggest that fluoride reduced gentamicin-induced nephrotoxicity by inducing a compensatory response carried out by Hsp72 and by decreasing gentamicin accumulation. These findings should not be interpreted to suggest that fluoride is a protective agent as megalin deficiency could lead to serious adverse effects on the kidney physiology. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: fluoride; gentamicin; kidney injury; Kim-1; megalin; Hsp72 Introduction Fluoride is naturally present in mineral complexes of many bodies of water (Whitford, 1983). Fluoride ions are released from these fluoride-containing minerals into the groundwater, which is the main source of fluoride exposure (ATSDR, 2003). The World Health Organization has established 1.5 ppm as the maximum limit for fluoride concentration in drinking water, a level considered bene- ficial for its cariostatic effects (WHO, 2006). Nevertheless, it has been estimated that more than 200 million people from 25 coun- tries, including China, India, Mexico and Argentina, are exposed to high fluoride concentrations (>1.5 ppm) through drinking water (WHO, 2006). Chronic fluoride exposure above 2 ppm has been associated with renal impairment (Xiong et al., 2007). Given that renal excretion is the primary pathway for fluoride elimination, the kidney is, therefore, one of the main target organs for fluoride toxicity (Whitford, 1994). In the kidney, fluoride induces oxidative stress and peroxidation of the cell membrane lipids (Guan et al., 2000; Karaoz et al., 2004). Furthermore, experimental animal stud- ies have shown that the proximal tubule, which is primarily local- ized in the renal cortex, is the segment of the nephron that is most susceptible to damage by fluoride exposure (Usuda et al ., 1998; Dote et al., 2000). In a previous study carried out in rats, we have demonstrated that exposure to 15 and 50 ppm of fluoride in- duced a sub-nephrotoxic effect only detected using very sensitive and specific biomarkers of kidney injury. Neither serum creatinine (SCr) nor estimated glomerular filtration rate (eGFR) levels were modified after fluoride exposure. However, urinary levels of kidney injury molecule-1 (Kim-1), clusterin (Clu), osteopotin (OPN), heat shock protein 72 (Hsp72), β-2-microglobulin (B2M) and cystatin-C (CysC) were significantly increased after fluoride exposure. More- over, while fluoride-induced tubular damage a repair process was in progress (Cárdenas-González et al., 2013). The cumulative nehprotoxicity induced by a sub-toxic stimulus has an effect on the response to a subsequent treatment with a potentially damaging dose with the same or a related stressor *Correspondence to: Luz M. Del Razo, Departamento de Toxicología. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN).Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, México, D.F. 07360. E-mail: ldelrazo@cinvestav.mx a Departamento de Toxicología, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México, D. F., Mexico b Departamento de Fisiología, Biofísica y Neurociencias. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México, D. F., Mexico c Unidad de Fisiología Molecular. Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México and Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México, D. F., Mexico J. Appl. Toxicol. 2015 Copyright © 2015 John Wiley & Sons, Ltd. Research Article Received: 23 March 2015, Revised: 1 May 2015, Accepted: 2 May 2015 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jat.3186