Human Kidney Tubuloids Model of Repeated Cisplatin-induced Cellular Senescence and Fibrosis for Drug Screening

In the pursuit of 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 remains challenging. Here, we present an innovative approach to generate epithelial-like structures known as “tubuloid” using primary human renal proximal tubular epithelial cells (hRPTECs) cultured from human resected kidneys, as a refined alternative. We evaluated the efficacy of tubuloids using cisplatin treatment. Tubuloids showed highly differentiated structures. Exposure to cisplatin increased ?H2AX, Kidney Injury Molecule-1 (KIM-1) and Cleaved Caspase-3, markers for DNA damage response, epithelial damage and apoptosis respectively. Repeated cisplatin administration resulted in upregulation of the senescence markers. Additionally, increased secretion of inflammatory cytokines, indicating the induction of a senescence-associated secretory phenotype (SASP) were induced Supernatant collected from cisplatin-treated tubuloids induced myofibroblast activation, indicating the onset of renal fibrosis. We successfully established a tubuloid-based model of cisplatin-induced kidney injury using hRPTECs. Tubuloids can replicate cellular senescence, SASP, and fibrosis, which can recapitulate the phenotypes of chronic kidney disease (CKD). Furthermore, tubuloids provide a novel platform for studying the response of renal epithelial cells to toxins and therapeutics and offer innovative strategy for drug screening in a human-based fashion. In this study, we also performed bulk RNA sequencing on the tubuloids generated using established protocol to verify their cellular composition. Overall design: Human renal proximal tubular epithelial cells derived from resected kidneys were seeded onto ultra-low-attachment plates, and Matrigel was added two days later. The cells were then expanded for two weeks in culture medium containing various growth factors to generate tubuloids. RNA was extracted from these tubuloids and subjected to bulk RNA sequencing.