cytochalasin-d and Pyelonephritis

Proteus mirabilis, a common agent of bacteriuria in humans, causes acute pyelonephritis and bacteremia. Renal epithelium provides a barrier between luminal organisms and the renal interstitium. We have hypothesized that P. mirabilis may be internalized into renal epithelium. To test this hypothesis, we added suspensions of three P. mirabilis strains (10(8) CFU) to confluent monolayers of primary cultures of human renal proximal tubular epithelial cells (HRPTEC) and, after 3 h, found the bacteria internalized within membrane-bound vacuoles by light and electron microscopy. Internalization of bacteria by HRPTEC was corroborated by using the gentamicin protection assay. Cytolysis of HRPTEC by the HpmA hemolysin, however, was a confounding factor in this assay, and therefore a hemolysin-negative hpmA mutant was used in subsequent experiments. The nonhemolytic mutant WPM111 did not disrupt the monolayer and was recovered in numbers that were 10- to 100-fold higher than those of the hemolytic parent BA6163. Cytochalasin D (20 micrograms/ml) inhibited internalization of Salmonella typhimurium but not that of P. mirabilis, suggesting that the latter species enters HRPTEC by a mechanism that is not dependent on actin polymerization. We suggest that HpmA hemolysin-mediated cytotoxicity and internalization of bacteria by HRPTEC may play a role in the development of Proteus pyelonephritis.

Topics: Cells, Cultured; Cytochalasin D; Epithelium; Humans; Kidney Tubules, Proximal; Microscopy, Electron; Proteus mirabilis; Pyelonephritis; Vacuoles

A gentamicin survival assay, using primary human renal epithelial cells and Escherichia coli strains isolated from the feces of asymptomatic individuals and from the urine or blood of patients with acute pyelonephritis, was used to investigate bacterial internalization as a model for renal parenchymal invasion in pyelonephritis. E. coli strains, regardless of their origin, efficiently entered into human renal epithelial cells, a process inhibited by cytochalasin D. While the percentage of survival of nonhemolytic pyelonephritis isolates did not differ from that of fecal isolates, survival of hemolytic pyelonephritis strains was lower than that of nonhemolytic strains, perhaps as a consequence of the greater cytotoxicity of hemolytic strains. There was no evidence of intracellular multiplication of E. coli. These results demonstrate that human renal epithelial cells are capable of efficient uptake of E. coli regardless of the source of the bacteria.

Topics: Cells, Cultured; Cytochalasin D; Dose-Response Relationship, Drug; Epithelial Cells; Epithelium; Escherichia coli; Escherichia coli Infections; Feces; Female; Gentamicins; Humans; Kidney; Kidney Tubules, Collecting; Kidney Tubules, Proximal; Microbial Sensitivity Tests; Pyelonephritis