Recapitulation of Cellular Senescence, Inflammation, and Fibrosis in Human Kidney-Derived Tubuloids by Repeated Cisplatin Treatment

Background: In the pursuit of more accurate pathophysiological models for assessing renal drug response, the development of kidney organoids derived from human pluripotent stem cells represents a significant step forward. However, recapitulating aging/senescence associated pathophysiology within these models remains challenging. Here, we present an innovative approach to generate more homogeneous epithelial-like structures known as “tubuloid” using primary human renal proximal tubular epithelial cells (hRPTECs) cultured from human resected kidneys as a refined alternative and try to evaluate their ability to show aging/senescence associated pathophysiology. Methods: Primary hRPTECs were established from the non-tumor kidney tissue removed from patients with malignancies with written informed consents. Cells were cultured on ultra-low attachment plates in media containing basement membrane gel and multiple growth factors. To assess the disease-modeling potential of tubuloids, we evaluated the efficacy of tubuloids using cisplatin treatment at three different concentrations: 0.2, 2.0, and 20.0 µg/mL. Results: Upon exposure to cisplatin, γH2AX expression increased in a dose-dependent manner, indicating DNA damage. Cisplatin treatment also resulted in the expression of Kidney Injury Molecule-1 (KIM-1) and Cleaved Caspase-3, which are indicators of kidney injury and apoptotic signaling, respectively. Repeated cisplatin administration resulted in upregulation of the cellular senescence marker p16, alongside increased secretion of inflammatory cytokines IL-1β and IL-6, indicating the induction of a senescence-associated secretory phenotype (SASP). Furthermore, supernatant collected from repeated cisplatin treated tubuloids induced myofibroblast activation, indicating the onset of renal fibrosis. Conclusion: We successfully established a tubuloid-based model that goes beyond the immediate effects of cisplatin nephrotoxicity to simulate the transition from AKI to CKD, or even model CKD itself, using hRPTECs. Tubuloids can recapitulate cellular senescence, SASP, and fibrosis, making them a promising pathophysiological model for chronic kidney disease (CKD) providing insights into the disease’s fibrotic mechanisms. Funding: Private Foundation Support, Government Support – Non-U.S.