Phenotypic Variability of Urine-Derived Stem Cells and Their Effect on Renal Inflammation and Fibrosis in Renal Epithelial Cells and UUO Mice

Background: Chronic kidney disease (CKD) is characterized by persistent inflammation and fibrosis, leading to progressive renal dysfunction. Urine-derived stem cells (USCs) have shown potential in modulating these processes, but their phenotypic heterogeneity may influence therapeutic effects. Objective: This study aimed to evaluate the effects of distinct USC phenotypes on renal inflammation and fibrosis in kidney cells and a CKD mouse model. Hypothesis: We hypothesized that distinct USC phenotypes would differentially affect inflammation and fibrosis in kidney cells and a CKD mouse model. Methods: USCs from three donors were classified into normal, tissue-like, and swarmy phenotypes based on growth pattern and morphology. For further classification, surface marker expression (CD73, CD90, CD105, and CD146) was analyzed. Due to the low abundance of the tissue-like phenotype, CM from normal, swarmy, and mixed USC populations were used for functional experiments. HKC-8 cells were treated with CM and stimulated with tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) to induce inflammation or transforming growth factor-beta (TGF-β) to induce fibrosis. The effect of CM on inflammation and fibrosis was evaluated by qPCR and western blotting analysis. A 7-day unilateral ureteral obstruction (UUO) mouse model was used to induce kidney fibrosis. The left ureter was ligated, and USCs were administered subcapsularly. Kidneys were harvested on day 7, and inflammation and fibrosis were evaluated by qPCR, western blotting, and immunohistochemistry. Results: Surface marker analysis revealed no differences between the normal and swarmy phenotypes, while the tissue-like phenotype exhibited lower expression of all markers. CM from the normal phenotype demonstrated the strongest anti-fibrotic and anti-inflammatory effects, significantly reducing fibronectin ( FN ), collagen type I alpha 1 ( COL1A1 ), and alpha-smooth muscle actin ( ACTA2 ), as well as inflammatory markers monocyte chemoattractant protein-1 ( MCP1 ) and interleukin-6 ( IL-6 ) expression. CM from swarmy and mixed phenotypes had a less pronounced anti-fibrotic effect compared to the normal phenotype. In UUO mice, treatment with USCs of the normal phenotype reduced fibrosis-related markers. Conclusion: USC phenotype influences their effects on renal inflammation and fibrosis. The normal phenotype showed the strongest anti-fibrotic and anti-inflammatory effects in vitro, and reduced fibrosis in UUO mice. Ethics statement: Urine samples were collected with approval from the South Denmark Region Committee on Biomedical Research Ethics and the Danish Data Protection Agency. Animal experiments were conducted in compliance with the Danish Animal Welfare Act and approved by the Danish Animal Experiments Inspectorate. Funded by the Novo Nordisk Foundation. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.