PROJECT SUMMARY In the proposed work, the Hunstad laboratory at Washington University will employ new models of urinary tract infection (UTI) in female, androgenized female, and male mice to determine molecular mechanisms by which host androgen exposure promotes uropathogenic Escherichia coli (UPEC) pyelonephritis as well as renal scarring, a common sequela of upper-tract UTI. Our recent findings indicate that the influence of sex, including androgen exposure, on these common bacterial infections is more complex than previously appreciated. Using newly developed and optimized models of UTI in mice, we recently demonstrated that androgen exposure is associated with increased risk for chronic cystitis and high-titer pyelonephritis, as well as formation of renal abscesses, in both male and female hosts. These findings correlate with epidemiologic data revealing higher morbidity and mortality in men who do suffer complicated UTI (compared with women), and higher incidence of UTI in women with a common hyperandrogenic condition (polycystic ovary syndrome). Moreover, we have demonstrated a separable effect of androgens on renal fibrosis (scarring), a common complication of pyelonephritis in children that can contribute to long-term sequelae such as hypertension and risk for chronic kidney disease. On the basis of these findings, we hypothesize that fundamental sex differences impact the host-pathogen interaction and cellular responses in pyelonephritis, thereby influencing pathogenicity, resolution, and subsequent renal scarring. To interrogate this hypothesis, and building on our published work, we will first comprehensively define host-sex-specific virulence requirements and sex influences on the host- pathogen interaction through single-nucleus RNA-seq, hybrid capture-enhanced bacterial RNA-seq, and insertion-site sequencing experiments on the infected kidney, in collaboration with MPI Dr. Earl and collaborator Dr. Humphreys. Candidate sex-discrepant host pathways and bacterial virulence factors will be confirmed and interrogated using germline and conditional knockout mice, bacterial mutants, and an array of bacterial pathogenesis studies. We will also define the molecular pathways underlying renal fibrosis following pyelonephritis, and how these pathways are modulated by sex and/or by androgen exposure at varying times before, during, and after UTI. In total, the proposed work will leverage new preclinical models and an array of conventional and cutting-edge experimental techniques. Our results will illuminate sex-specific host-pathogen interactions in the infected kidney and identify UPEC virulence factors important in sex-dependent outcomes of upper-tract UTI. In addition, our preclinical findings will also be translationally relevant to recurrent UTI and renal scarring in human patient populations.