Renal Tubular Organoids, "Tubuloids," Derived from Dialysis-Dependent Patients' Resected Kidneys Exhibit Cellular Senescence and Express CKD-Specific Genes

Background: Renal tubular organoids (tubuloids) developed by our technology have the potential to serve as an alternative, more human-like kidney model of human pathophysiology. Tubuloids are made from patient kidney-derived primary cells, not stem cells, and mimic kidney tubules. Here, cells from unfunctional kidneys of dialysis patients could be 3D engineered. Furthermore, single-cell RNA sequencing (scRNA-seq) was employed to comprehensively analyze these tubuloids, identify CKD-associated phenotypes, and discover novel disease-specific markers. Methods: 5 patients receiving hemodialysis and 5 patients with normal kidney function were joined to this study with written informed consents. Using kidneys obtained from patients by surgery, primary cultures of renal tubular epithelial cells were selectively grown. Growth factors and Matrigel were then added. After several days of culture, tubuloids were obtained. In primary cultured cells in 2D condition and tubuloids, Western blotting was used to determine the phenotypes. ScRNA-seq was performed on tubuloids. Results: At the beginning of 3D culture, the growth speed of normal tubuloids is faster than that of renal cells from unfunctional kidneys. Eventually, however, the size difference between normal renal tubuloids and tubuloids from dialysis patients is not very discernible. Western blot analysis revealed that ESKD-derived tubuloids exhibited significantly higher expression of p16, a marker of senescence, compared to non-CKD tubuloids. ScRNA-seq analysis identified VCAM1 expression, a marker of CKD, in ESKD tubuloids. Two novel genes (Gene X and Gene Y) were identified that were specifically upregulated in ESKD tubuloids. Conclusion: We successfully established renal tubular organoids ("tubuloids") from dialysis-dependent ESKD kidneys. These tubuloids exhibit CKD-specific characteristics, including markers of cellular senescence and tubular injury, and have the potential to serve as a physiologically relevant model for studying CKD pathogenesis. Moreover, the discovery of novel CKD-specific genes highlights the potential of tubuloids for advancing our understanding of CKD and identifying new therapeutic targets. Funding: Private Foundation Support, Government Support – Non-U.S.