Alteration in cellular viability, pro-inflammatory cytokines and nitric oxide production in nephrotoxicity generation by Amphotericin B: involvement of PKA pathway signaling F. D. França a , A. F. Ferreira a , R. C. Lara a , J. V. Rossoni Jr c , D. C. Costa c , K. C. M. Moraes d , C. A. Tagliati b and M. M. Chaves a * ABSTRACT: Amphotericin B is one of the most effective antifungal agents; however, its use is often limited owing to adverse effects, especially nephrotoxicity. The purpose of this study was to evaluate the effect of inhibiting the PKA signaling path- way in nephrotoxicity using Amphotericin B from the assessment of cell viability, pro-inflammatory cytokines and nitric oxide (NO) production in LLC-PK1 and MDCK cell lines. Amphotericin B proved to be cytotoxic for both cell lines, as assessed by the mitochondrial enzyme activity (MTT) assay; caused DNA fragmentation, determined by flow cytometry using the propidium iodide (PI) dye; and activated the PKA pathway (western blot assay). In MDCK cells, the inhibition of the PKA signaling path- way (using the H89 inhibitor) caused a significant reduction in DNA fragmentation. In both cells lines the production of interleukin-6 (IL)-6 proved to be a dependent PKA pathway, whereas tumor necrosis factor-alpha (TNF-α) was not influenced by the inhibition of the PKA pathway. The NO production was increased when cells were pre-incubated with H89 followed by Amphotericin B, and this production produced a dependent PKA pathway in LLC-PK1 and MDCK cells lines. Therefore, con- sidering the present study’s results as a whole, it can be concluded that the inhibition of the PKA signaling pathway can aid in reducing the degree of nephrotoxicity caused by Amphotericin B. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: nephrotoxicity; amphotericin B; LLC-PK1; MDCK; PKA; IL-6; TNF-α; NO Introduction Amphotericin B has been the gold standard for treating invasive fungal diseases for many years. This drug combines with cell membrane sterols of host cells, forming pores that leak electro- lytes similar to its antifungal action. This drug’s mechanism of ac- tion can lead to systemic toxicity, including renal (Harmsen et al., 2011), which is the main effect observed in clinical practice. Renal cell lines have been employed as alternative methods for the study of therapeutic products that cause nephrotoxicity (Jung et al., 2009; Lincopan et al., 2005; Pfaller and Gstraunthaler, 1998; Price et al., 2004) and the use of in vitro techniques has en- hanced the comprehension of molecular mechanisms of neph- rotoxicity (Wilmes et al., 2011).The LLC-PK1 (porcine proximal tubular cells) and MDCK cells (canine distal cells) are considered acceptable models to study drug nephrotoxicity (El Mouedden et al., 2000; Ramseyer and Garvin, 2013; Servais et al., 2006; Shin et al., 2010; Yano et al., 2009; Yuan et al., 2011). The protein kinase A (PKA) signaling pathway is involved in the regulation of the cell functions in nearly all types of mamma- lian tissues, including the regulation of cell cycles, apoptosis, proliferation and differentiation (Bichet, 2006). PKA kinase is a serine/threonine in its inactive form that consists of a tetramer comprised of two regulatory subunits (R) and two catalytic subunits (C). Each R subunit contains two binding sites for the 3,5 cyclic adenosine monophosphate (cAMP), a second cellular messenger. Upon binding of the cAMP to the regulatory site, the dissociation of regulatory and catalytic subunits occurs, and two catalytic subunits are released, allowing them to cata- lyze the phosphorylation of proteins in regulatory residues (Gerits et al., 2008). Another aspect of this signaling pathway in- volves the regulation of cytokine gene expression with the cAMP/PKA (Grandjean-Laquerriere et al., 2003). In this context, the development of nephrotoxicity was attributed to the propensity of Amphotericin B to induce pro-inflammatory cy- tokines (Chai et al., 2013) and among the proinflammatory cytokines that were associated with the pathophysiology of nephrotoxicity, *Correspondence to: M.M. Chaves. Departamento de Bioquimica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, 31270-901, Belo Horizonte, MG, Brasil. Email: chavesmm@icb.ufmg.br a Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, 30161-970, Belo Horizonte, MG, Brasil b Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, 31270-901, Belo Horizonte, MG, Brasil c Departamento de Ciências Biológicas, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Moro do Cruzeiro, 35400-000, Ouro Preto, MG, Brasil d Universidade Estadual Paulista ’Júlio de Mesquita Filho‘Instituto de Biociências, Departamento de Biologia, Av 24-A 1515, 13506-900, Rio Claro, SP, Brasil J. Appl. Toxicol. 2014; 34: 1285–1292 Copyright © 2013 John Wiley & Sons, Ltd. Research Article Received: 3 July 2013, Revised: 26 July 2013, Accepted: 4 August 2013 Published online in Wiley Online Library: 18 September 2013 (wileyonlinelibrary.com) DOI 10.1002/jat.2927 1285