Toxicology 404–405 (2018) 10–24 Contents lists available at ScienceDirect Toxicology journal homepage: www.elsevier.com/locate/toxicol Transporter-dependent cytotoxicity of antiviral drugs in primary cultures of T human proximal tubular cells ⁎ Lawrence H. Lasha, , Caroline A. Leeb, Clynn Wilkerb,1, Vishal Shahb,2 a Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201 USA b Ardea Biosciences, San Diego, CA 92121 USA ARTICLE INFO ABSTRACT Keywords: The role of plasma membrane transporters in the nephrotoxicity of two antiretroviral drugs, cidofovir and te- Antiviral drugs nofovir, was studied in primary cultures of human proximal tubular (hPT) cells. Cells were grown on Transwell Nephrotoxicity filter inserts to maintain epithelial polarity and access to either the apical or basolateral plasma membrane. The Human proximal tubular cells function of relevant membrane transporters, organic anion transporter 1 and 3 (OAT1/3), P-glycoprotein Biomarkers (multidrug resistance protein-1; P-gp or MDR1), and organic cation transporter 2 (OCT2), was validated by Membrane transport measurements of apical-to-basolateral and basolateral-to-apical fluxes of furosemide, digoxin, and metformin, respectively. Acute cytotoxicity of cidofovir (0, 10, 50, 150, or 300 μM) in the absence or presence of 500 μM probenecid, tenofovir disoproxil fumarate (0, 20, 90, 180, or 360 μM) in the absence or presence of 500 μM probenecid, or cisplatin (0, 20, 90, 180, or 360 μM) as a positive control in the absence or presence of 500 μM cimetidine, was assessed after 4-h incubations by determinations of release of lactate dehydrogenase (LDH), γ- glutamyltransferase (GGT), N-acetyl-β-D-glucosaminidase (NAG), or Kidney Injury Molecule-1 (KIM-1). Cell death generally agreed with each of the four biomarkers, was generally greater when cidofovir or tenofovir was added to the upper compartment, and was markedly diminished in the presence of the appropriate transport inhibitor. Additionally, the extent of cytotoxicity caused by the two antiviral drugs was similar to that caused by cisplatin. The results demonstrate the importance of plasma membrane transport of antiviral drugs to elicit cytotoxicity in the hPT cell. 1. Introduction laboratory animals (Lacy et al., 1998) and humans (Lalezari et al., 1995) showed protection with probenecid. In fact, co-administration Antiretroviral (ARV) drugs have become key therapeutics against with probenecid and intravenous saline hydration became the normal such infectious agents as human immunodeficiency virus (HIV), hepa- treatment protocol (Lea and Bryson, 1996). An advantage over pre- titis B, cytomegalovirus, and other herpes viruses. As with many che- viously developed ARVs was that cidofovir does not require viral acti- motherapeutics, however, their clinical use is often limited by non- vation (Lalezari, 1997), but is activated by phosphorylation by cellular target organ toxicities. Two of the most important ARVs are cidofovir enzymes to inhibit viral DNA polymerase (Lea and Bryson, 1996). Ci- and tenofovir. dofovir also has efficacy against a broad range of viruses, including Cidofovir was approved by the U.S. Food and Drug Administration refractory and premalignant human papillomavirus (Coremans and in 1996 as an antiviral nucleoside analogue with potent activity against Snoeck, 2009). Despite reports of nephrotoxicity, a carefully designed cytomegalovirus and other herpes viruses. However, it was found to therapeutic regimen can allow cidofovir to be used without ne- exhibit dose-limiting nephrotoxicity (News, 1997). Studies in both phrotoxicity being a limiting problem (Araya et al., 2008). Abbreviations: ARV, antiretroviral; CHO, Chinese Hamster Ovary; CID, collision-induced dissociation; DMEM/F12, Dulbecco’s Modified Eagle’s Medium: Ham’s F12 Medium; DMF, dimethylformamide; DMSO, dimethylsulfoxide; GGT, γ-glutamyltransferase; HIV, human immunodeficiency; hPT, human proximal tubular; IS, internal standard; JAB, apical-to-baso- lateral flux; JBA, basolateral-to-apical flux; KIM-1, Kidney Injury Molecule-1; LC–MS/MS, liquid chromatography-mass spectrometry/mass-spectrometry; LDH, lactate dehydrogenase; MATE1, multidrug and toxin extrusion protein 1; MRM, multiple reaction monitoring; MRP, multidrug resistance-associated protein; NAG, N-acetyl-β-D-glucosaminidase; OAT, organic anion transporter; OATP, organic anion-transporting polypeptide; OCT, organic cation transporter; PBS, phosphate-buffered saline; P-gp, P-glycoprotein (also called MDR1, multidrug resistance protein 1); PT, proximal tubular; TAF, tenofovir alafenamide; TDF, tenofovir disoproxil fumarate; TISP, turbo ionspray ⁎ Corresponding author. E-mail address: [email protected] (L.H. Lash). 1 Current address: Heron Therapeutics, 12707 High Bluff Drive, Suite 200, San Diego, CA 92130, USA. 2 Current address: Organovo Holdings, Inc., 6275 Nancy Ridge Drive, Suite 110, San Diego, CA 92121, USA. https://doi.org/10.1016/j.tox.2018.05.002 Received 19 December 2017; Received in revised form 10 April 2018; Accepted 2 May 2018 Available online 05 May 2018 0300-483X/ © 2018 Elsevier B.V. All rights reserved. L.H. Lash et al. Toxicology 404–405 (2018) 10–24 Effective transport into renal proximal tubular (PT) cells is an im- 2. Materials and methods portant component for many nephrotoxic agents. Cidofovir is transported into renal PT cells by the human organic anion transporter 1 (hOAT1; 2.1. Experimental design SLC22A6), as demonstrated in Chinese Hamster Ovary (CHO) cells stably transfected with hOAT1 cDNA (Ho et al., 2000). Intracellular accumu- For all studies, cells were grown to approximately 90% confluence ® lation of cidofovir in transfectants was > 100-fold higher than in par- on collagen-coated Transwell inserts (6-well, 0.4-μm pore size) with ental CHO cells, and this accumulation was associated with enhanced serum-free, hormonally-defined Dulbecco’s Modified Eagle’s Medium: cytotoxicity, which was reduced by addition of the OAT1 inhibitor pro- Ham’s F12 Medium (DMEM/F12; 1/1; 3.0 ml in upper compartment, benecid. This is consistent with the clinical data described above, 1.5 ml in lower compartment), as done previously for transport studies showing that co-administration of cidofovir with probenecid diminished in hPT cells (Lash et al., 2006). As hPT cells grow with their brush nephrotoxicity. Diminished capacity for excretion is another important border facing upwards and their basal membrane on the growth sur- factor: Cundy et al. (1996) found a prolonged elimination phase for ci- face, the upper compartment is functionally analogous to the tubular dofovir in African Green Monkeys, consistent with a long intracellular lumen whereas the lower compartment is functionally analogous to the half-life for the phosphorylated form of the drug. interstitial fluid and nearby perfusing capillaries. Accordingly, for Although most of the mechanistic studies on cidofovir have been transport studies, addition of drug to the lower compartment followed performed in transfected cell lines, Ortiz et al. (2005) showed that ci- by measurement of drug in the upper compartment allows one to dofovir caused a time- (peak at 7 days) and concentration- (10–40 μg/ measure basolateral-to-apical flux (JBA) whereas addition of drug to the ml) dependent incidence of apoptosis in primary cultures of human upper compartment followed by measurement of drug in the lower proximal tubular (hPT) cells. Such a pattern was concluded to be con- compartment allows one to measure apical-to-basolateral flux (JAB). sistent with that of clinical toxicity. Apoptosis was prevented by pro- Similarly, for cytotoxicity studies, addition of drug to the lower com- benecid, again demonstrating the critical role of membrane transport in partment along with appropriate transport inhibitors allows one to as- cytotoxicity. sess the importance of basolateral plasma membrane transporters in Tenofovir, originally given as the disoproxil fumarate preparation determining cytotoxicity whereas addition of drug to the upper com- (TDF), was the first nucleotidic inhibitor of HIV reverse transcription partment along with appropriate transport inhibitors allows one to as- and became available in 2001. It has been extensively used worldwide sess the importance of apical plasma membrane transporters in de- and is the most prescribed ARV drug. Early clinical trials suggested that termining cytotoxicity. TDF was safer than other ARVs. Even as early as 2001, however, cases We took a conservative approach by evaluating toxicity at relatively of TDF-induced nephrotoxicity were reported and such occurrences high concentrations and a short incubation time to detect toxicity. As were considered a potentially serious dose-limiting side effect (Foisy transporters are involved in the uptake of these drugs, their con- et al., 2013; Gupta et al., 2012; Hall, 2013; Mitra et al., 2014; centrations would mimic their accumulation and if toxicity could be Monteagudo-Cho et al., 2012; Mouton et al., 2016; Tourret et al., 2013). detected in a short-term incubation, then this decreases the risk of As noted in a recent review (Murphy and Valentovic, 2017), changes in missing a true toxicity effect. Moreover, cytotoxicity of other ne- dosing of tenofovoir are needed in the presence of renal impairment. phrotoxic chemotherapeutic agents has previously been determined at Risk factors for development of tenofovir-induced nephrotoxicity in- 1, 2, 4, and 24 h of incubation (Gallardo-Godoy et al., 2016; Huang