<p>Translating nephrotoxicity risks from rat to human is hampered by species differences in kidney physiology. An in vitro rat kidney platform focused on proximal tubules, the primary site of drug-induced injury, could enable mechanistic, cross-species assessments. Here, we characterized rat primary proximal tubule epithelial cells (rPPTEC) and evaluated their use for nephrotoxicity studies in vitro. rPPTEC were characterized by gene and protein expression, and drug transporter functionality. Next, rPPTEC were exposed to 7 validation compounds (4 nephrotoxicants, 3 non-nephrotoxicants), after which cell viability was assessed with ATP and LDH assays, and high-content imaging. Finally, the toxicity response to compounds known to be more toxic in rat than in human (rat-enhanced nephrotoxicants) was compared in rPPTEC and human renal proximal tubule epithelial cells (HRPTEC). rPPTEC expressed proximal tubule markers (Aqp1, Ggt1), and functionally active drug transporters (P-gp, Oct2, Lrp2/Cubn), but lacked Oat1. The nephrotoxicants cisplatin, puromycin and cyclosporin A, but not tenofovir, induced dose-dependent decreases in ATP, increases in LDH, and alterations in imaging-based parameters. None of the non-nephrotoxicants were associated with major alterations in cell viability nor high-content imaging parameters. rPPTEC were more sensitive to the rat-enhanced nephrotoxicants zoledronate and ibandronic acid, but also to cisplatin and cyclosporin A. Notably, 45% of rPPTEC stained positive for Ki-67, compared to 0.7% of HRPTEC. rPPTEC exhibited a proximal tubular phenotype and correctly identified 6 out of 7 validation compounds. Parallel exposures in rPPTEC and HRPTEC revealed greater sensitivity of rPPTEC towards nephrotoxicants, potentially explained by the greater proliferative capacity of rPPTEC.</p>