Protection of Human Podocytes from Shiga Toxin 2-Induced Phosphorylation of Mitogen-Activated Protein Kinases and Apoptosis by Human Serum Amyloid P Component

Abstract

jHemolytic uremic syndrome (HUS) is mainly induced by Shiga toxin 2 (Stx2)-producing Escherichia coli. Proteinuria can occur in the early phase of the disease, and its persistence determines the renal prognosis. Stx2 may injure podocytes and induce proteinuria. Human serum amyloid P component (SAP), a member of the pentraxin family, has been shown to protect against Stx2-induced lethality in mice in vivo, presumably by specific binding to the toxin. We therefore tested the hypothesis that SAP can protect against Stx2-induced injury of human podocytes. To elucidate the mechanisms underlying podocyte injury in HUS-associated proteinuria, we assessed Stx2-induced activation of mitogen-activated protein kinases (MAPKs) and apoptosis in immortalized human podocytes and evaluated the impact of SAP on Stx2-induced damage. Human podocytes express Stx2-binding globotriaosylceramide 3. Stx2 applied to cultured podocytes was internalized and then activated p38␣ MAPK and c-Jun N-terminal kinase (JNK), important signaling steps in cell differentiation and apoptosis. Stx2 also activated caspase 3, resulting in an increased level of apoptosis. Coincubation of podocytes with SAP and Stx2 mitigated the effects of Stx2 and induced upregulation of antiapoptotic Bcl2. These data suggest that podocytes are a target of Stx2 and that SAP protects podocytes against Stx2-induced injury. SAP may therefore be a useful therapeutic option.

Shiga Toxin Mediated Neurologic Changes in Murine Model of Disease

“…In some cells, protein synthesis inhibition activates apoptotic death pathways, resulting in death of the Stx-treated cells (Jones et al, 2000 ; Ching et al, 2002 ; Fujii et al, 2003 ). Several cell types in the kidney are known to be killed by Stx (van Setten et al, 1997 ; Hughes et al, 1998 ; Fuller et al, 2011 ; Dettmar et al, 2014 ), and we observed extensive cellular necrosis and damage to the kidneys of the Stx2a-treated mice. No hyperintensities were observed by MRI in the brains of living mice, indicating that unlike the kidney, massive cellular death is not the mechanism by which Stx-induces brain damage.…”

Differentiation of human iPSCs into functional podocytes

“…Conditionally immortalized human podocyte-like cells, generously provided by Prof. M. Saleem, Bristol, UK [ 15 ] served as reference controls. Cells were cultured on collagen type I-coated dishes (BioCoat ™ , BD Biosciences) at the permissive temperature of 33 °C in RPMI 1640 medium, supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, glutamine, and insulin, transferrin, sodium selenite mixture (ITS) (Sigma-Aldrich), and 10% fetal bovine serum (GIBCO) as recently described [ 62 ]. At 70–80% confluence, cultures were switched to 37 °C to induce growth arrest and subsequent differentiation (non-permissive condition), respectively, and were further maintained for 2 to 3 weeks.…”

Shiga toxin type-2 (Stx2) induces glutamate release via phosphoinositide 3-kinase (PI3K) pathway in murine neurons

“…High calcium influx induced by an excess glutamate is known to promote excitotoxicity by mechanisms including apoptosis (Yang et al, 2014 ). Also, we and others have shown in other cell types in vivo and in vitro that Stx2 can promote apoptosis (Kiyokawa et al, 1998 ; Jones et al, 2000 ; Kojio et al, 2000 ; Fujii et al, 2003 , 2008 ; Wilson et al, 2005 ; Creydt et al, 2006 ; Stone et al, 2012 ; Dettmar et al, 2014 ), and therefore, we investigated in vitro and in vivo , whether Stx2 induced apoptosis in neurons. Stx2-induced apoptosis was measured by TUNEL stain.…”