Pyelonephritis represents a subset of urinary tract infections that occur from bacteria ascending from the lower to the upper reaches of the genitourinary system, such as the kidney. The renal system contains a range of hydrodynamically and immunologically challenging, interconnected microenvironments where the invading pathogen may populate during the course of the infection. The situation at the infection foci changes dynamically, vacillating between bacterial colonization and clearance, to which the outcome is a summation of all host-pathogen elements in play. A selection of important determinants includes factors of microbial origin, effects of eukaryotic cell signaling, physiological facets of the infected organ, and signals from distal organs. Improved understanding of the multifactorial aspects of molecular pathogenesis of infection requires intravital, cross-disciplinary approaches with high spatio-temporal resolution. The advancement of such approaches promises to eventually provide a comprehensive understanding of the integrated pathophysiology of pyelonephritis.