Comparative Characterization of Shiga Toxin Type 2 and Subtilase Cytotoxin Effects on Human Renal Epithelial and Endothelial Cells Grown in Monolayer and Bilayer Conditions

Abstract

Postdiarrheal hemolytic uremic syndrome (HUS) affects children under 5 years old and is responsible for the development of acute and chronic renal failure, particularly in Argentina. This pathology is a complication of Shiga toxin (Stx)-producing Escherichia coli infection and renal damage is attributed to Stx types 1 and 2 (Stx1, Stx2) produced by Escherichia coli O157:H7 and many other STEC serotypes. It has been reported the production of Subtilase cytotoxin (SubAB) by non-O157 STEC isolated from cases of childhood diarrhea. Therefore, it is proposed that SubAB may contribute to HUS pathogenesis. The human kidney is the most affected organ because very Stx-sensitive cells express high amounts of biologically active receptor. In this study, we investigated the effects of Stx2 and SubAB on primary cultures of human glomerular endothelial cells (HGEC) and on a human tubular epithelial cell line (HK-2) in monoculture and coculture conditions. We have established the coculture as a human renal proximal tubule model to study water absorption and cytotoxicity in the presence of Stx2 and SubAB. We obtained and characterized cocultures of HGEC and HK-2. Under basal conditions, HGEC monolayers exhibited the lowest electrical resistance (TEER) and the highest water permeability, while the HGEC/HK-2 bilayers showed the highest TEER and the lowest water permeability. In addition, at times as short as 20-30 minutes, Stx2 and SubAB caused the inhibition of water absorption across HK-2 and HGEC monolayers and this effect was not related to a decrease in cell viability. However, toxins did not have inhibitory effects on water movement across HGEC/HK-2 bilayers. After 72 h, Stx2 inhibited the cell viability of HGEC and HK-2 monolayers, but these effects were attenuated in HGEC/HK-2 bilayers. On the other hand, SubAB cytotoxicity shows a tendency to be attenuated by the bilayers. Our data provide evidence about the different effects of these toxins on the bilayers respect to the monolayers. This in vitro model of communication between human renal microvascular endothelial cells and human proximal tubular epithelial cells is a representative model of the human proximal tubule to study the effects of Stx2 and SubAB related to the development of HUS.

Fig 1. Schematic description of cell monoculture and coculture systems.

HGEC cells were seeded on the lower side of a Millicell support (0.4 μm membrane pore size), and HK-2 cells into the upper side. Cells were either cultured in monoculture (A and B) or in coculture (C). To evaluate the effects of Stx2 and SubAB on monoculture and coculture, Stx2 or SubAB were added to the lower side.

Fig 2. Morphology of HGEC/HK-2 bilayer.

Human glomerular endothelial cells (HGEC) and human proximal tubular epithelial cell line (HK-2) were grown in Millicell inserts as described. After confluence (7 days of culture), the filters were fixed, sectioned and stained with H&E (A) or Hoechst (B) to be observed by optical and fluorescence microscopy, respectively. A and B (×400); insert in panel A (×1000 magnification).

Fig 3. Integrity of endothelial and epithelial monolayers and bilayer.

(A) The electrical resistance (TEER, Ω.cm²) across monolayers and bilayers was measured during the development of cell culture. (B) Differences in TEER values between HGEC and HK-2 monlayers and HGEC/HK-2 bilayer at confluence (7 days). Each value represents mean ± SEM, of six experiments. *P <0.05, **P <0.001, ***P <0.0001.

Fig 4. Functional characterization of monolayers and bilayers.

Under basal conditions, the net absorptive water transport (Jw, μl/min.cm2) was recorded in HGEC, HK-2 monolayers and HGEC/HK-2 bilayer. Each value represents mean ± SEM, of six experiments. *P 0.05, **P< 0.001.

Fig 5. Effects of Stx2 and SubAB on the net absorptive water transport (Jw).

Data represent the time course of the Jw across HGEC (A), HK-2 (B) and HGEC/HK-2 (C) incubated with PBS (Ctrl) or Stx2 (10 ng/ml) or SubAB (1500 ng/ml) (time = 0) on the lower side. A time-dependent Jw inhibition was observed in the case of monolayers but not in the bilayer. Each value represents mean ± SEM, of three experiments. Stx2 or SubAB vs Ctrl, *P <0.05, **P <0.001, ***P <0.0001.

Fig 6. Inhibition of cell viability in monolayers and bilayer by Stx2 and SubAB.

HGEC and HK-2 monolayers and HGEC/HK-2 bilayer were exposed to 1 ng/ml Stx2 or 150 ng/ml SubAB in growth-arrested conditions for 72 h. Then, cells were incubated with neutral red for an additional 1 h at 37°C in 5% CO₂. Absorbance of each well was read at 540 nm. One hundred percent represents cells incubated under identical conditions but without toxin treatment (Ctrl). Results are expressed as means ± SEM of nine experiments, Stx2 or SubAB vs Ctrl, *P <0.05 and HGEC/HK-2 vs HGEC or HK-2, #P <0.05.