Development of in-vitro human and rat proximal tubule cell models as a platform for drug transporter and drug-drug interaction studies

Abstract

The kidney plays a key role in the systemic clearance of new molecular entities (NMEs). Approximately 32% of drugs exhibit significant renal elimination. It is estimated that nephrotoxicity accounts for 8 % of pre-clinical and 9% of clincal safety failures in drug development. Current pre-clinical models used to screen NMEs are poor predictors of human nephrotoxicity. The focus of my project is thus to develop predictive in-vitro rat and human primary proximal tubule cell models as a platform drug transporter and drug-drug interaction (DDI) studies. Primary human and rat proximal tubule cells (PTCs) were isolated from renal cortex using a combination of enzymatic digestion and density centrifugation. The isolation procedure was optimised to maximise cell yield and viability. Human and rat PTCs cultured on Transwell® inserts formed confluent monolayers with low paracellular permeability. Quantitative PCR showed mRNA expression of key renal transporters, OAT1, OAT3, OATP4C1, OCT2, BCRP, MATE1, MATE2-K, MDR1, MRP2, URAT1, NaPi-IIa, NaPi-IIc and PiT2, in human PTC monolayers. Orthologs of these transporters were also detected in rat PTC monolayers. The utility of human and rat PTC monolayers as predictive in-vitro models of proximal tubular drug handling were demonstrated using radiolabeled [3H]-tenofovir (TFV). Human and rat PTC monolayers exhibited a cell-to-media ratio greater than 1, which indicated uptake and accumulation of TFV across the basolateral membrane. We also observed a predominant absorptive pathway of TFV. The transporters mediating the transport of TFV were identified using a cocktail of transporter inhibitors. The basolateral uptake of TFV was mediated by OATP4C1 and OAT1. TFV had low affinities for the apical efflux transporters MRP2, MRP4, MDR1 and BCRP. The novel identification of OATP4C1 as a TFV transporter has led Gilead to develop assays for investigating OATP4C1-mediated DDIs, and the FDA to recognise OATP4C1 as a key renal transporter. The handling of radiolabelled inorganic [32P]-phosphate (Pi) by human and rat PTC monolayers was also investigated. Pi flux measurement revealed a net absorptive pathway of Pi across human and rat PTC monolayers. Uptake of Pi across the apical membrane was sodium dependent, saturable, and inhibited by parathyroid hormone, fibroblast growth-factor 23 and -klotho. Apical uptake of Pi was also inhibited by TFV in a saturable manner. This suggests that the mechanisim of TFVinduced hypophosphatemia is not via TFV-induced nephrotoxicity, but TFV inhibition of Pi reabsorption. The outcomes of this work have initiated a patient clinical trial. This finding could have a large translational impact, as over 14.9 million HIV-patients are pescribed TFV and TFV-related hypophosphatemia affects 30 % of patients. The data highlight the importance of developing holistic cell based models of the proximal tubule. The outcomes of this work demonstrate the power of translational science to have an impact on how the pharmaceutical industry operates.