Organoids are 3-dimensional (3D) stem cell-derived cultures that offer a variety of technical advantages compared to traditional 2-dimensional (2D) cell cultures. Although murine models have proved useful in biomedical research, rodent models often fail to adequately mimic human physiology and disease progression, resulting in poor preclinical prediction of therapeutic drug efficacy and toxicity. An interesting alternative is to use the canine model in research, due to its numerous similarities to humans (shared environment, intact immune system, and development of civilization diseases). The use of canine organoids in drug testing and disease modeling has been limited by the number of models as well as the depth of characterization. Therefore, we believe these types of models can expedite drug testing and create a platform for personalized medicine.
Here, we report the establishment, maintenance, and molecular characterization of six adult-stem cell-derived canine organoid cell lines including endometrium, pancreas, urinary bladder, kidney, lung, and liver from two genetically related canines (B816 and B818). Characterization of these lines was done using multiple techniques including immunohistochemistry (UPKIII, TTF-1) and bulk RNA-seq. Furthermore, scRNA-seq was utilized on a subset of the organoids to identify organoid specific transcriptomic signatures including lung, pancreas, kidney, and bladder.
In total, six tissues and organoid lines from each donor were characterized, allowing for a unique, multi-organ comparison between these two individuals and identification of specific cell types within the organoids. Bulk RNA-seq revealed tissue-specific transcriptomic profiles, with organoids enriched in proliferation-related genes and tissues enriched in inflammation-related genes. Principal component analysis showed organ-based clustering, while scRNA-seq identified diverse epithelial subtypes.
These organoids begin to establish a platform for reverse translational research, reducing reliance on live animal testing. By leveraging genetically related donors, it highlights tissue-specific variations, facilitating applications in personalized medicine, disease modeling, and pharmacology to bridge veterinary and human research gaps.
Copyright © 2025 Zdyrski, Gabriel, Ospina, Nicholson, Catucci, Melvin, Wickham, Sahoo, Dao, Aguilar Meza, Ralston, Bedos, Bastian, Honold, Piñeyro, Pawlak, Corbett, Douglass, Allenspach and Mochel.

Obesity is a significant public health concern associated with numerous health risks. Infections are a major complication of obesity, but the mechanisms driving increased infection risk remain unclear. Using a diet-induced obesity mouse model in male and female C57BL/6 mice, we show that high-fat diet feeding heightens vulnerability to uropathogenic E. coli (UPEC) urinary tract infection. Transcriptomic analysis of bladder urothelial cells revealed sex-specific gene expression changes with the shared activation of focal adhesion and extracellular matrix pathways. Western blotting and immunostaining confirmed focal adhesion kinase activation, a central component of the focal adhesion pathway, in the bladders of obese female and male mice. In primary human urothelial cells, focal adhesion kinase overexpression promoted UPEC invasion. These findings indicate that obesity enhances urinary tract infection susceptibility and identify focal adhesion kinase as a conserved pathway deregulated by obesity that contributes to increased UPEC vulnerability.
© 2025 The Author(s).

ABSTRACT Obesity is a significant public health concern that is associated with numerous health risks. Infections are a major complication of obesity, but the mechanisms responsible for increased infection risk are poorly defined. Here, we use a diet induced obesity mouse model and investigate how obesity impacts urinary tract infection (UTI) susceptibility and bladder immune defenses. Our results show that high-fat diet fed female and male mice exhibit increased susceptibility to uropathogenic E. coli (UPEC) following experimental UTI. Transcriptomic analysis of bladder urothelial cells shows that obesity alters gene expression in a sex-specific manner, with distinct differentially expressed genes in male and female mice, but shared activation of focal adhesion and extracellular matrix signaling. Western blot and immunostaining confirm activation of focal adhesion kinase, a central component of the focal adhesion pathway, in the bladders of obese female and male mice. Mechanistically, experiments using primary human urothelial cells demonstrate that focal adhesion kinase overexpression promotes UPEC invasion. These findings demonstrate that obesity enhances UTI susceptibility by activating focal adhesion kinase and promoting bacterial invasion of the urothelium. Together, they explain how obesity promotes UTI vulnerability and identify modifiable targets for managing obesity-associated UTI. Significance Statement Obesity is associated with an increased risk of urinary tract infections (UTIs), but the underlying mechanisms promoting infection susceptibility remain poorly understood. Here, we show that diet-induced obesity drives sex-specific changes in bladder urothelial gene expression, including distinct immune responses in male and female mice. Despite these differences, both sexes exhibit activation of focal adhesion kinase (FAK). FAK overexpression promotes bacterial invasion into human bladder cells. These findings provide a mechanistic explanation for obesity-associated UTI susceptibility and suggest that targeting FAK signaling could offer a therapeutic strategy to prevent UTIs, with implications for personalized interventions in obesity.

Seven novel and potent Raf small molecule kinase inhibitors (C1-7) were evaluated in seven-day oral repeat dose rat toxicity studies. All compounds tested induced hyperplasia in multiple tissues. Consistently affected was stratified squamous epithelium at a number of sites and transitional epithelium of urinary bladder and kidney. A seven-day time course study in rats showed morphologic evidence of epithelial proliferation in the nonglandular stomach within four to five hours after a single dose of C-1. Similar indications of cellular proliferation were observed in the urinary bladder by day 2 and in the heart, kidney, and liver by day 3. Transcriptional evidence of proliferation in the urinary bladder was detected within four to five hours after a single dose consistent with activation of the PI3K/AKT and ERK/MAPK pathways. In a twenty-eight-day rat toxicity study of C-1, hyperplasia was observed in the esophagus, nonglandular stomach, skin, urinary bladder, kidney, and heart. Hyperplasia of transitional epithelium of the urinary bladder was particularly severe and in one female rat was accompanied by the presence of a transitional cell carcinoma. These results suggest that these Raf inhibitors induce early transcriptional changes driving unchecked cell proliferation, resulting in marked tissue hyperplasia that can progress to carcinoma within a short time frame.