Molecular and functional characterization of water transport in human renal tubular epithelial cells (HRTEC): Effects of Shiga toxin type 2 (Stx2) and its B subunit (Stx2B).

PISTONE CREYDT V, SILBERSTEIN C, NÚÑEZ P, IBARRA C.

Melbourne, Australia

Workshop; VTEC2006 International Symposium and Workshop on Shiga toxin (verocytotoxin)-producing Escherichia coli infections.; 2006

Australian Society for Microbiology

Stx2 secreted by Escherichia coli is able to cause Hemolytic Uremic Syndrome (HUS). One of the most important consequences of HUS is the toxic effect of Stx2 on renal tubular epithelial cells that contributes to the development of acute renal insufficiency and its evolution towards a chronic condition. The goals of this work were to characterize water transport through HRTEC and to analyze functional changes in the presence or absence of Stx2 and Stx2B. HRTEC primary cell cultures were obtained from human renal cortex epithelial cells. Expression of AQP1 water channels, αENaC and APXL sodium channels, and NHE3 sodium-proton-exchanger was observed using RT-PCR and Western blot analysis. Functionality was evaluated by measuring water absorption (Jw) through HRTEC monolayers grown on permeable supports. Cell viability was measured by neutral red uptake. Protein synthesis inhibition was determined by 32S-methionine uptake. Hoescht staining was used to measure apoptosis. Results indicate that the Jw was significantly decreased (0.29 ± 0.01 vs 0.19 ± 0.01 μl.min-1.cm-2, n=3) when the HRTEC monolayers were incubated for 1h with 10 pg/ml of Stx2. Jw inhibition was time and toxin concentration dependent. At higher toxin concentrations (100 pg/ml –100 ng/ml) and/or longer incubation times (1-6 h) a stimulation of apoptosis together with a decrease in viability and protein synthesis were detected. Jw was also inhibited by Stx2B but at a higher concentration (100 ng/ml, for 1h) than those used for Stx2 holotoxin. Under these conditions, Stx2B did not modify viability, protein synthesis or apoptosis. We suggest that while Stx2 holotoxin affects the water transport as a consequence of a general protein synthesis inhibition, Stx2B modulates specifically the transport mechanisms involved in water absorption. These findings also suggest that the Stx2B could contribute to the development of acute renal failure observed in patients with HUS.