Acute pyelonephritis, a complication of Escherichia coli bacteriuria, must represent a bacterial invasion through the kidney epithelium. To study this process, we overlaid bacterial suspensions onto monolayers of cultured human kidney proximal tubular epithelial cells and measured cytotoxicity by release of lactate dehydrogenase (LDH). Thirty-four isolates cultured from patients with acute pyelonephritis were screened for the ability to cause pyelonephritis in CBA mice by transurethral challenge. The eight most virulent strains

Tubular cells have been isolated, characterized and cultured from more than 70 adult cadaver kidneys (postmortem time less than or equal to 12 hr.). Confluent monolayers were observed at 7 days after seeding (10(6) cells/ml.) and cells demonstrating normal human karyotypes have been passaged up to 6 times. Primary isolates and monolayer cultures were negative for Factor VIII activity, and strongly positive for gamma-glutamyltransferase activity and keratin. Ultrastructurally primary isolates consisted of cells with numerous mitochondria, microvilli, cytoplasmic filaments and well-developed endocytotic apparati. Monolayer cultures examined at 7, 14, 21 and 72 days demonstrated less prominent microvilli and the additional structures of desmosomes and cell junctions. Membrane-associated and cytosolic enzyme activities were measured up to 28 days in culture. The membrane-associated enzymes gamma-glutamyltransferase and alkaline phosphatase both exhibited approximately 10-fold decreases in activity during the 1st 7 days in culture. There was an approximately 5-fold increase in pyruvate kinase activity during the same time period, while fructose-1,6-bisphosphatase activity exhibited a 5-fold decrease. Glucose-6-phosphatase activity did not change during the 28 day culture period examined. From 7 to 28 days no further changes were noted in any of the enzyme activities measured. Decreased membrane-associated enzyme activity corresponded to the ultrastructural observation of less prominent microvilli. Increases in glycolytic enzyme activity and decreases in gluconeogenic enzyme activity may reflect the presence of glucose in the culture medium. The morphologic and biochemical evidence suggests that primary isolates and cultures are proximal tubule cells which should provide a well-defined in vitro human system for future studies.

To examine the role of flagella in pathogenesis of urinary tract infection caused by Proteus mirabilis, we constructed a nonmotile, nonswarming flagellum mutant of strain WPM111 (an hpmA hemolysin mutant of strain BA6163, chosen because of its lack of in vitro cytotoxicity in renal epithelial cell internalization studies). A nonpolar mutation was introduced into the flaD gene, which encodes the flagellar cap protein. This mutation does not affect the synthesis of flagellin but rather prevents the assembly of an intact flagellar filament. In in vitro assays, the genetically characterized nonmotile mutant was found to be internalized by cultured human renal proximal tubular epithelial cells in numbers less than 1% of those of the flagellated parent strain. Internalization of the nonmotile mutant was increased significantly (14-to 21-fold) by centrifugation onto the monolayer. To assess virulence in vivo, CBA mice were challenged transurethrally with 10 7 CFU of P. mirabilis BA6163 (wild type) (n ‫؍‬ 16), WPM111 (hpmA mutant) (n ‫؍‬ 46), or BB2401 (hmpA flaD mutant) (n ‫؍‬ 46). Differences in quantitative cultures between the parent strain and the hemolysin-negative mutant were not significant. However, the hpmA flaD mutant was recovered in numbers approximately 100-fold lower than those of the hmpA mutant or the wild-type parent strain and thus was clearly attenuated. We conclude that while hemolysin does not significantly influence virulence, flagella contribute significantly to the ability of P. mirabilis to colonize the urinary tract and cause acute pyelonephritis in an experimental model of ascending urinary tract infection.

Acute pyelonephritis is a common invasive infection frequently caused by E. coli that possess P-fimbriae and secrete hemolysin. We have examined the role of P fimbriae and hemolysin in the killing of putative target cells of acute pyelonephritis, that is, human renal epithelial cells (HRPTEC). Cultures of HRPTEC were overlaid with (1) a prototypic pyelonephritogenic E. coli (CFT073) which expresses both P fimbriae and hemolysin; (2) its hemolysin-negative isogenic mutant (CFT073hlyD::TnphoA); or (3) a prototypic nonpyelonephritogenic fecal E. coli (FN414) which is negative for both P fimbriae and hemolysin. CFT073 and CFT073hlyD::TnphoA but not FN414 adhered to HRPTEC, as demonstrated by electron microscopy and direct counting. Adherence was diminished by antisera directed against P fimbriae and by a monoclonal antibody recognizing the epithelial receptor for P fimbriae. CFT073 was significantly more cytolethal for HRPTEC than its hemolysin-negative mutant. The bacteria-free filtrate of CFT073 was both hemolytic and cytolethal whereas that of CFT073hyD::TnphoA was not hemolytic and was significantly less cytolethal. Finally, we demonstrated that CFT073 passed through monolayers of HRPTEC at a higher rate than CFT073hlyD::TnphoA, indicating that hemolysin damages HRPTEC, facilitating passage of bacteria through the epithelial barrier. With HRPTEC and a pyelonephritogenic strain of E. coli we have reproduced in vitro bacterial attachment and toxin delivery by P fimbriae and hemolysin, factors epidemiologically associated with acute pyelonephritis in patients.

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