Ouabain Protects Human Renal Cells against the Cytotoxic Effects of Shiga Toxin Type 2 and Subtilase Cytotoxin

Abstract

Hemolytic uremic syndrome (HUS) is one of the most common causes of acute renal failure in children. The majority of cases are associated with Shiga toxin (Stx)-producing Escherichia coli (STEC). In Argentina, HUS is endemic and presents the highest incidence rate in the world. STEC strains expressing Stx type 2 (Stx2) are responsible for the most severe cases of this pathology. Subtilase cytotoxin (SubAB) is another STEC virulence factor that may contribute to HUS pathogenesis. To date, neither a licensed vaccine nor effective therapy for HUS is available for humans. Considering that Ouabain (OUA) may prevent the apoptosis process, in this study we evaluated if OUA is able to avoid the damage caused by Stx2 and SubAB on human glomerular endothelial cells (HGEC) and the human proximal tubule epithelial cell (HK-2) line. HGEC and HK-2 were pretreated with OUA and then incubated with the toxins. OUA protected the HGEC viability from Stx2 and SubAB cytotoxic effects, and also prevented the HK-2 viability from Stx2 effects. The protective action of OUA on HGEC and HK-2 was associated with a decrease in apoptosis and an increase in cell proliferation. Our data provide evidence that OUA could be considered as a therapeutic strategy to avoid the renal damage that precedes HUS.

Keywords: Hemolytic Uremic Syndrome; Ouabain; prevention; shiga toxin; subtilase cytotoxin.

Figure 1

Oubain (OUA) non-cytotoxic concentrations. Human glomerular endothelial cells (HGEC) and human proximal tubule epithelial cells (HK-2) placed in 96-well plates were exposed to between 5 nM and 50 nM OUA 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 OUA treatment (Ctrl). Results are expressed as means ± SEM of five experiments, OUA concentrations >30 nM vs. Ctrl, * p < 0.05; HK-2: OUA (10–30 nM) vs. Ctrl, # p < 0.05.

Figure 2

OUA protection on HGEC and HK-2 exposed to toxins. HGEC (A) and HK-2 (B) placed in 96-well plates were pre-treated with between 5 nM and 30 nM OUA for 24 h, and then incubated with Stx type 2 (Stx2) (Stx2 (10 ng/mL for HGEC or 20 ng/mL for HK-2) or subtilase cytotoxin (SubAB) (100 ng/mL) in the presence of OUA for other 48 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 OUA or toxins treatment (Ctrl). Results are expressed as means ± SEM of five experiments, OUA + Stx2 vs. Stx2 or OUA + SubAB vs. SubAB, * p < 0.05.

Figure 3

HGEC are protected from toxins morphologic disturbances. HGEC seeded on gelatin-coated glass coverslips were pre-treated with 20 nM OUA for 24 h and then incubated with 10 ng/mL Stx2 or 100 ng/mL SubAB in the presence of OUA for another 48 h. Finally, cells were stained with H&E and the morphology (A) and number of HGEC (B) were analyzed by light microscopy (×200 and ×400). Results are expressed as means ± SEM of three experiments. One hundred percent represents values of controls. OUA vs. Ctrl ns, OUA + Stx2 vs. Stx2 or OUA + SubAB vs. SubAB, * p < 0.05. Thick arrows: intracellular edema. Thin arrows: elongated shape.

Figure 4

HK-2 are protected from Stx2 morphologic disturbances. HK-2 seeded on glass coverslips were pre-treated with 20 nM OUA for 24 h and then incubated with 20 ng/mL Stx2 and in the presence of OUA for an additional 48 h. Finally, cells were stained with H&E and the morphology (A) and number of HK-2 (B) were analyzed by light microscopy (×200 and ×400). Results are expressed as means ± SEM of three experiments. One hundred percent represents values of cells control. OUA vs. Ctrl ns, OUA + Stx2 vs. Stx2, * p < 0.05. Thick arrows: intracellular edema.

Figure 5

OUA (20 nM) did not modify the electric current across HGEC and HK-2. HGEC (A) and HK-2 (B) were grown to confluence on Millicell cell culture inserts. Then, TEER and V were measured and values were recorded every 5 min. Following this, the current (I = mA·cm²) was calculated before and after treatment with OUA at 20 nM or 3 mM. Results are expressed as means ± SEM of three experiments. 3 mM OUA vs. 20 nM OUA, * p < 0.05.

Figure 6

Prevention of apoptosis and necrosis by OUA. Annexin V-FITC/IP double staining assay was used to quantify necrosis and apoptosis by flow cytometry. HGEC (A) and HK-2 (B) cells were pre-treated with 20 nM OUA for 24 h and then incubated with Stx2 (10 ng/mL for HGEC or 20 ng/mL for HK-2) or SubAB (100 ng/mL for HGEC) and in the presence of OUA for an additional 48 h. Then, cells were labeled with Annexin V-FITC/IP for 10 min. Results are expressed as means ± SEM of three experiments. OUA vs. Ctrl ns, OUA + Stx2 vs. Stx2 or OUA + SubAB vs. SubAB, * p < 0.05. A representative experiment is shown in panels (C,D).

Figure 7

OUA increases the number of viable HGEC and HK-2. HGEC and HK-2 were grown to confluence in 6-well plates and then treated with OUA (20 nM) for 72 h. After that, cells were trypsinized, centrifuged and resuspended with 0.4% trypan blue. Finally, cells were counted into a Neubauer chamber. Results are expressed means ± SEM of three experiments. OUA vs. Ctrl * p < 0.05.

Figure 8

OUA stimulated HGEC and HK-2 proliferation. HGEC (A) and HK-2 (C) were pre-treated with 20 nM OUA for 24 h. Then, cells were incubated with 10 ng/mL Stx2 or 100 ng/mL SubAB (for HGEC) or 20 ng/mL Stx2 (for HK-2), and in the presence of OUA for an additional 48 h. After treatment, cells were fixed with 70% ethanol, incubated with PI (50 μg/mL) for 30 min and then analyzed by flow cytometry. The cell cycle phases were analyzed by Cyflogic software. Results are expressed as means ± SEM of three experiments. OUA vs. Ctrl or OUA + Stx2 vs. Stx2, * p < 0.05. OUA + SubAB vs. SubAB, ns. Stx2 vs. Ctrl or SubAB vs. Ctrl, # p < 0.05. A representative experiment is shown in panels (B,D).