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; NÚÑEZ P; SILBERSTEIN C; IBARRA C

Melbourne, Australia

Congreso; 6th International Symposium on Shiga Toxin (Verocytotoxin) – producing Escherichia coli infections; 2006

Stx2 secreted by E. 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. Presence of water channels (AQPs), sodium channels (ENaC and APXL) and sodium-proton-exchange (NHEs) was observed using of RT-PCR and WB. Functionality was evaluated by measuring water absorption (Jw) through monolayers of HRTEC grown on permeable supports. Cell viability was measured by incorporation of neutral red, protein synthesis inhibition by 32S-methionine incorporation, and cell apoptosis by means of Hoescht staining. Results indicate that HRTEC express AQP1, aENaC and APXL, and have a Jw value of 0.26±0.02 ml.min-1.cm-2. The Jw value decreased significantly (p<0.05) with 10pg/ml of Stx2 after an hour of incubation. Jw inhibition was time and toxin concentration dependent. Nevertheless, when higher concentrations and incubation times with Stx2 were employed, not only an inhibition of Jw was observed, but also a decrease in viability and protein synthesis, together with a stimulation of apoptosis. Stx2B also inhibited Jw in a dose and time dependent manner, but at a higher concentration. Under these conditions, Stx2B did not modified viability, protein synthesis or apoptosis. In conclusion, the results suggest that Stx2 and Stx2B inhibit the ion and water mechanism implied in water transport. This effect could be consequence of general protein inhibition or may directly be involved the sodium and water transport systems.