Hyperosmolarity of mouse urine confounds research in urinary tract infection

lab animal Brief Communication https://doi.org/10.1038/s41684-026-01727-4 Hyperosmolarity of mouse urine confounds research in urinary tract infection Check for updates Kristian Stærk    1,2 , Janni Søvsø Hjelmager1,2, Rasmus Birkholm Grønnemose Marie Lykke Bach3, Jens Sivkær Pettersen1,2,4 & Thomas Emil Andersen    1,2    1,2, Mice have been used as models of urinary tract infection (UTI) for decades and laid the basis for the fundamental understanding of UTI pathogenesis in humans. Here we report that the high urine osmolarity of mice impacts key aspects of Escherichia coli UTI pathogenesis and represents a confounder for the translation of results to humans. Urinary tract infections (UTIs) reduce the quality of life of millions and is a considerable cause of disease and death in vulnerable individuals1. Importantly, UTI is a major driver of antibiotic consumption and antibiotic resistance, thus emphasizing the need to understand UTI pathogenesis and develop new treatment strategies2. The mouse has for decades been regarded as a critical experimental model for deciphering basic UTI pathogenesis, due to its small size, ease of maintenance, low cost and amenability to genetic manipulation3. Nevertheless, it also has shortcomings as a model of human disease. In health science, the emphasis on mice is believed to be a contributor to the failure of drug and vaccine clinical trials4,5. Awareness of the limitations of this model is therefore critical in translational medical research. Of relevance to the use of mice in UTI research is the unique capacity of mice to produce highly concentrated urine. Mice produce urine at concentrations far outside the range of humans and most other mammals6. It has remained unexplored how this fundamental physiological trait of mice impacts the transferability of UTI research to humans. We hypothesized that the highly concentrated urine of mice affects bacterial growth fitness and furthermore is an inducer of bacterial filamentation, a key step in UTI pathogenesis that has been extensively studied in mice where filamentation is abundant3,7,8. Uropathogenic Escherichia coli (UPEC) elongates and forms filaments to some extent during growth in highly concentrated human urine, and during experimental infection of human bladder epithelial cells exposed to human urine9,10. Induction of the phenotype requires human urine at concentrations above a certain threshold and is moreover affected by surface growth and pH9–11. UPEC filaments have been observed in human urine from patients with UTI12, although our experience from routine inspection of human patient urine samples is that they are relatively rare, unless the patient is treated with filament-inducing antibiotics. To assess the effect of the high osmolarity of mouse urine on UPEC morphology and infectious potential, we established a natural diuresis mouse model by allowing animals access to sweetened water (tap water supplemented with 20% (w/v) glucose) 30 h before infection. Increased 1 water intake reduces the urine concentration of mice—measured as urine specific gravity (USG, that is, density)—to levels comparable to those of humans (normal range 1.005–1.030) (Fig. 1a). Increased water intake did not significantly affect urine glucose levels, pH, the urine albumin-to-creatinine ratio or total urinary protein excretion; however, wide variability was observed in the urine albumin-to-creatinine ratio in diuretic mice (Supplementary Fig. 1). Diuresis and control mice were then experimentally infected with the prototypical UPEC isolate UTI89 transformed with the green fluorescent protein-encoding plasmid pMAN01. The mice were euthanized after 18 h—a sweet spot for observing filaments in this model (which occur 12–20 h after infection)—and bacterial morphology was examined on splayed bladders by microscopy7,13. In control mice, extensive filamentation was observed, whereas bacteria remained mainly rod-shaped in diuresis mice (mean filamentation score of 2.80 and 1.07, respectively; P = 0.0003, Mann–Whitney test) (Fig. 1b–d). In 41% of diuresis mice, rod-shaped bacteria were exclusively observed without any detectable filamentation (Fig. 1b–d). Bacterial colony-forming units (CFUs) were significantly higher in the urine (P = 0.01, Mann–Whitney) and bladders (P < 0.0001, Mann–Whitney) of diuresis mice than in controls, but not different in kidneys (Fig. 1e–g). Reverse-transcription quantitative polymerase chain reaction (RT–qPCR) analysis indicated osmotic stress in bacteria from control mice compared with diuresis mice, based on increased expression of the central osmo-responsive gene proV from the osmo-inducible proU operon and ompC (Fig. 1h, mean log2 fold change (FC) of 1.90 and 0.85, respectively)14,15. The higher bacterial burden in the diuresis mice led us to speculate that USG might also affect susceptibility to infection. Mice are intrinsically resistant to UTI and require high inocula of 108–109 CFU ml−1 for successful infection3. This dose is approximately a million times higher than that used in pigs, an alternative model animal that is naturally susceptible to UTI and produces urine with USG levels comparable to those of humans16. To test this hypothesis, groups of diuresis and control mice were inoculated with 106 and 104 CFU ml−1. At 106 CFU ml−1, only one of Department of Clinical Microbiology, Odense University Hospital, Odense, Denmark. 2Research Unit of Clinical Microbiology, University of Southern Denmark, Odense, Denmark. 3Cardiovascular and Renal Research, Department of Molecular Medicine, University of Southern Denmark, Odense, Denmark. 4 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark. e-mail: Lab Animal Brief Communication https://doi.org/10.1038/s41684-026-01727-4 a b 1.08 c **** USG 1.06 1.04 1.02 1.00 Da e f 10 4 10 Bladder CFU 3 2 1 10 3 Control Diuretic ** 0 0 –1 –1 –2 –2 * 10 10 10 Control Diuretic 7 6 10 5 4 Control Diuretic 8 10 Control Diuretic 5 10 4 10 3 10 2 10 1 10 0 10 –1 10 tr on C et ur Di tr on 8 10 Bladder CFU 1 9 10 7 ic 1 2 * 10 10 6 ol 2 3 11 10 ic 2 ol 3 4 10 4 3 C 10 i ompC 10 10 1 proV 4 0.1438 5 10 10 et h Diuretic 5 ur Control 6 10 4 10 g 10 7 6 10 0 log2FC 10 **** 8 Kidney CFU *** Di 5 20 µm Urine CFU ml–1 1 y 0 y Da Filamentation score d 20 µm 6 10 4 10 Inoculum Fig. 1 | Mouse urine is an osmotic stressor on UPEC that promotes filamentation and reduces infectious capacity. a, Promoting water intake by feeding mice with sweetened water significantly reduced USG (equals density) from day 0 (baseline) to day 1 (24 h after giving sweetened water) (n = 64, P < 0.0001, Mann–Whitney test). b–d, Significantly higher level of UPEC filamentation was observed in mice with access to regular tap water (control; b) (n = 10) compared with mice with access to sweetened wate (...truncated)