Shiga Toxin Type 2 Aggravates G1/S Phase Cell Cycle Arrest, Mediating Caspase-Independent Cell Death under Hyperosmotic Conditions in the Kidney.

Shiga toxin (Stx) is a virulence factor produced by Shigella dysenteriae serotype 1 and Stx-producing Escherichia coli (STEC). It causes severe renal damage, leading to hemolytic uremic syndrome (HUS). The main target organ of Stx, the kidney, plays a role in maintaining water homeostasis in the body by increasing an osmotic gradient from the cortex to the medulla. However, the activity of Stxs under kidney hyperosmotic conditions is not well understood. In this study, to investigate the Stx effects under hyperosmotic interstitial fluid, kidney epithelial cells and 3D spheroids were treated with Stx2 and NaCl. Stx2 treatment without NaCl addition increased ER stress, mitochondrial damage, reactive oxygen species, and cleaved caspase 3, 7, while co-treatment with Stx2 and NaCl showed reduced ER-mediated apoptosis. Significantly, NaCl treatment increased HSP70 expression, leading to reduced ER stress by Stx2. Furthermore, cell toxicity by Stx2 and NaCl treatment was increased by the HSP70 inhibitor. Contrariwise, DNA repair gene expression level was decreased, and G1/S phase cell cycle arrest was aggravated after being treated with Stx2 and NaCl than the Stx2 group. Importantly, lactate dehydrogenase and early/late apoptotic cell death were promoted by Stx2 under NaCl treatment. Changes in Stx2 activity at NaCl conditions were confirmed through transcriptome analysis. In conclusion, a hyperosmotic environment changes the cell death mechanism and accelerates the cytotoxicity of Stx, which suggests the importance of studying environmental factors from a pathological perspective and a therapeutic perspective at HUS.