PROJECT SUMMARY The current pathway for drug discovery is associated with costs of $2.55 billion and between 10-15 years of development for a single drug to reach the market. The challenges in predicting drug toxicities and efficacies are attributed to inherent species differences in drug-metabolizing enzyme activities and cell-type-specific sensitivities to toxicants. Organs-on-a-chip are an emerging technology in disease modeling and screening therapeutics to address discrepancies between animal models and human clinical trials. They utilize tissue engineering, fluid mechanics, and biomaterials to replicate in vivo architectures and functions of complex organs and tissues. The renal proximal tubule (PT) in vivo is exposed to fluid flow and mechanical stress (pressure, stretch, shear) and these stimuli play an important role in maintaining cellular phenotype and homeostasis. Currently, available prototypes fall short of replicating the in vivo environment because they often fail to mimic the physiological forces. Therefore, these models have had limited success in predicting drug-induced nephrotoxicity. In this proposal, we will bioengineer and evaluate a dynamic platform of the PT and study the effects of drugs and tubular dysfunction to establish its potential for translational research. Human renal proximal tubule cells (hRPTECs) will be cultured within gelatin methacryloyl (GelMA) hydrogels under physiological shear and pressure. These devices will also incorporate the diversity in the patient population by using hRPTECs from multiple donors to determine the impact of age, sex, and racial differences on nephrotoxicity effects. Drugs will be classified based on their nephrotoxic risk (high, intermediate, and low) and the platform will incorporate automated readouts to reflect cellular function and viability. Together, this will help investigate more accurate pharmacological and pathological responses and to determine the utility of in vitro perfusion models. Secondly, a more complex and novel bioengineered platform will be developed. This design contains a 3D PT tubule and 3D vascular vessels surrounded by pericyte vascular networks. The platform will then be subjected to physiological shear stress and pressure to demonstrate the flow loop can accurately mimic cellular organization, establishment of tight junctions, maintenance of barrier function, and selective transport as seen in vivo. This device composes of a co-culture of hRPTECs, human umbilical vein endothelial cells (hUVECs), and human dermal fibroblasts (hDF) within a GelMA hydrogel to model an environment where both reabsorption and secretion functions are replicated. Lastly, this proposal investigates the translational potential of PT tissue chips through demonstration of a PT diabetic nephropathy model and engineering multi-well PTs to facilitate high- throughput studies. The organ-on-a-chip developed in this study will provide an enabling technology that has broad applications i...