Urinary Excretion of Tetrodotoxin Modeled in a Porcine Renal Proximal Tubule Epithelial Cell Line, LLC-PK1

Abstract

This study examined the urinary excretion of tetrodotoxin (TTX) modeled in a porcine renal proximal tubule epithelial cell line, LLC-PK1. Time course profiles of TTX excretion and reabsorption across the cell monolayers at 37 °C showed that the amount of TTX transported increased linearly for 60 min. However, at 4 °C, the amount of TTX transported was approximately 20% of the value at 37 °C. These results indicate that TTX transport is both a transcellular and carrier-mediated process. Using a transport inhibition assay in which cell monolayers were incubated with 50 µM TTX and 5 mM of a transport inhibitor at 37 °C for 30 min, urinary excretion was significantly reduced by probenecid, tetraethylammonium (TEA), l-carnitine, and cimetidine, slightly reduced by p-aminohippuric acid (PAH), and unaffected by 1-methyl-4-phenylpyridinium (MPP+), oxaliplatin, and cefalexin. Renal reabsorption was significantly reduced by PAH, but was unaffected by probenecid, TEA and l-carnitine. These findings indicate that TTX is primarily excreted by organic cation transporters (OCTs) and organic cation/carnitine transporters (OCTNs), partially transported by organic anion transporters (OATs) and multidrug resistance-associated proteins (MRPs), and negligibly transported by multidrug and toxic compound extrusion transporters (MATEs).

Use of Physiologically Based Kinetic Modeling-Facilitated Reverse Dosimetry to PredictIn VivoAcute Toxicity of Tetrodotoxin in Rodents

“…Furthermore, the PBK modeling data of this study revealed that up to 86% of TTX clearance in the kidney could be ascribed to active transport by the proximal tubule cells. This active transport of TTX was previously also demonstrated in the renal proximal tubule cell line LLC-PK1 ( Matsumoto et al , 2017 ). In this in vitro study, TTX was shown to be primarily transported by the organic cation transporters and the organic cation/carnitine transporters.…”

“…The glomerular filtration (GF) was added to the model according to the equation: GF = GFR × (CVK × fub in vivo ), where GFR is the GF rate, which is 5.2 ml/min/kg bw for rat and 14 ml/min/kg bw for mouse ( Walton et al , 2004 ), CVK is the concentration of TTX in the kidney compartment and fub in vivo is the fraction of TTX unbound in the in vivo situation. As mentioned by Matsumoto et al (2017) , TTX seems to be a substrate for some active transporters in the proximal tubule cells in the kidney. Since it is unknown which transporter has the highest contribution it was decided to work with an estimated apparent overall V max and K m ; 1 V max and 1 K m for all transporters involved.…”

“…After running model predictions including only GF as the excretion pathway, data for the apparent overall V max and K m were estimated based on manual input of V max and K m searching for the optimal transporter efficiency (TE = V max / K m in µl/min/mg protein) by fitting to the available in vivo kinetic data. Matsumoto et al (2017) reported Papp values for the bidirectional transport of TTX over an LLC-PK1 kidney cell layer. Future use of such Papp values to define the in vivo kinetic parameters for urinary excretion of TTX in a PBK model requires definition of the scaling factor(s) needed to convert these in vitro Papp values to the kinetic constants for active transport of TTX in the kidney in vivo .…”

“…Thus, this study aimed to evaluate the potential of using in vitro toxicity data obtained with primary rat neonatal cortical cells on a multielectrode array (MEA) assay or an effect study in mouse neuro-2a cells combined with PBK model-based reverse dosimetry to predict the in vivo acute neurotoxicity of TTX in rodents. As TTX is hardly metabolized, highly hydrophilic, and has been identified as a substrate for organic cation transporters in the kidneys ( Matsumoto et al , 2017 ), active renal transport can be expected to contribute substantially to the in vivo TTX kinetics and has to be accounted for in the PBK models to be developed to facilitate the QIVIVE.…”

Including active excretion in physiologically based kinetic models for new approach methodologies

“…Upon oral exposure, TTX absorption is rapid but incomplete (around 7%), there is hardly metabolism of TTX in the liver and with its highly hydrophilic characteristic TTX is predominantly excreted via the urine (Hong et al 2017;Hong et al 2018). Due to the charges on the molecule it is foreseen that excretion of TTX, next to glomerular filtration, will proceed via active transporters in the renal proximal tubule epithelial cells, most likely the OCT2 transporter with a lesser contribution of OCTN1/2, MATE and OATs (Matsumoto et al 2017). Using PBK modelling the active transport of TTX will be further unravelled.…”