Establishment of a Human Patient–Derived Proximal Tubule Microphysiological System (MPS) for in vitro Nephrotoxicity Drug Screening

Recently, the FDA and NIH have launched new initiatives to reduce animal testing in drug development, emphasizing the urgent need for alternative technologies, including in vitro culture systems, for non-clinical safety evaluation. As nephrotoxicity remains one of the major concerns in drug development, there is an increasing demand for in vitro systems that closely recapitulate human kidney function. To address this need, we aimed to establish a novel in vitro proximal tubule–mimicking system using primary renal tubular epithelial cells (hRTECs) derived from human nephrectomy specimens and a custom-designed microfluidic device capable of reproducing physiological luminal flow.

hRTECs were isolated from the non-tumor regions of nephrectomy specimens obtained from patients with renal or ureteral cancer. Renal cortex tissues were manually minced, digested with collagenase, and cultured in serum-free medium supplemented with epidermal growth factor. After passaging, cells were seeded onto a microfluidic device from a 3D-printed replica mold at a density of 1.0 × 10⁵ cells/mL, and continuous medium perfusion at 0.7 µL/min was applied to mimic urinary flow. From day 10, repeated exposure to cisplatin (2.0 µg/mL) was used to induce nephrotoxicity, followed by treatment with dapagliflozin (20 µM), sodium-glucose co-transporter-2 (SGLT2) inhibitors known to exert renoprotective effects.

Under continuous perfusion, hRTECs showed markedly enhanced expression of differentiation markers such as acuaporine1 (AQP1) and Na⁺/K⁺-ATPase compared with static cultures, forming a denser and tighter epithelial barrier structure, while non-perfusion condition did not differentiate the cells when compared with normal 2D-cultured condition. Repeated cisplatin administration disrupted the epithelial architecture and significantly upregulated kidney injury molecule-1 (KIM-1), successfully reproducing nephrotoxic injury within the microfluidic system. In contrast, post-treatment with dapagliflozin preserved epithelial differentiation and suppressed KIM-1 upregulation, indicating functional renoprotection within the device.

We successfully developed a human patient–derived proximal tubule in vitro microphysiological system (MPS) that closely mimics urinary flow and promotes advanced epithelial differentiation. This platform not only recapitulates cisplatin-induced nephrotoxicity but also reproduces pharmacological recovery following SGLT2 inhibitor treatment. Our system represents a promising tool for modeling renal pathophysiology, including chronic kidney disease (CKD), and may serve as a valuable alternative to animal testing in preclinical drug evaluation.