ABSTRACT: Calcineurin inhibitors (CNIs) are indispensable for transplantation immunosuppression, yet cyclosporine A (CsA) produces renal toxicity. Voclosporin (VOC), a VOC analog, is proposed to be less nephrotoxic, but mechanisms remain unclear. We compared CsA and VOC effects on primary human proximal tubule epithelial cells (PTECs) cultured both in 2D monolayers and in a 3D kidney microphysiological system (MPS). We evaluated cell viability, mitochondrial membrane potential (ΔΨm), injury biomarkers, and inflammatory cytokines using multiplex immunoassays. RNA sequencing was performed on 3D-cultured PTECs to identify differentially expressed genes and pathways. In 2D PTECs, viability and KIM-1 did not distinguish CsA from VOC. In 3D MPS, CsA increased aggregate biomarker deviations, whereas VOC responses remained near vehicle (CsA was ~1.8-fold higher than VOC; two-way ANOVA drug main effect, F = 71.99, p < 0.0001). Confocal tomography showed CsA-associated mitochondrial fragmentation and a ~ 40% reduction in ΔΨm, whereas VOC preserved network architecture and ΔΨm (p < 0.0001). RNA-seq identified 1188 CsA-specific and 185 VOC-specific differentially expressed genes, with 304 shared. Pathway analysis indicated CsA enrichment of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress, p21-associated G2/M checkpoint arrest, and ferroptosis-prone signatures, while VOC mainly induced ER chaperone and ER-associated degradation gene programs without activating canonical UPR sensors and showed limited cell-cycle suppression. A physiologically relevant 3D kidney MPS revealed sublethal tubular stress from CsA that is masked in 2D culture, including mitochondrial depolarization, proteostatic stress, and ferroptosis priming. A matched exposure, VOC preserved mitochondrial and proteostasis and elicited a narrower, adaptive ER quality control response. These data support VOC as a nephron-sparing immunosuppressant and 3D MPS as a mechanism-based platform for evaluating renal safety of drugs and nominating early sub-lethal tubular injury biomarkers.
