Genetic risk factors in typical haemolytic uraemic syndrome

Haberichter

Sadler

2012

AbstractShiga toxin (Stx) causes diarrhea-associated hemolytic uremic syndrome by damaging renal microvascular endothelium. The pentameric B subunits of Stx types 1 and 2 (Stx1B and Stx2B) are sufficient to stimulate acute VWF secretion from endothelial cells, but Stx1B and Stx2B exert distinct effects on Ca2+ and cAMP pathways. Therefore, we investigated other signaling components in StxB-induced VWF exocytosis. Incubation of HUVECs with StxB transiently increased phospholipase D (PLD) activity. Inhibition of PLD activity or shRNA-mediated PLD1 knockdown abolished StxB-induced VWF secretion. In addition, treatment with StxB triggered actin polymerization, enhanced endothelial monolayer permeability, and activated RhoA. PLD activation and VWF secretion induced by Stx1B were abolished on protein kinase Cα (PKCα) inhibition or gene silencing but were only moderately reduced by Rho or Rho kinase inhibitors. Conversely, PLD activation and VWF exocytosis induced by Stx2B were reduced by Rho/Rho kinase inhibitors and dominant-negative RhoA, whereas attenuation of PKCα did not affect either process. Another PLD1 activator, ADP-ribosylation factor 6, was involved in VWF secretion induced by Stx1B or Stx2B, but not histamine. These data indicate that Stx1B and Stx2B induce acute VWF secretion in a PLD1-dependent manner but do so by differentially modulating PKCα, RhoA, and ADP-ribosylation factor 6.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

The B subunits of Shiga-like toxins induce regulated VWF secretion in a phospholipase D1–dependent manner

Haberichter

Sadler

2012

“…6,8 Whether Stxinduced VWF secretion contributes to the pathogenesis of D ϩ HUS in humans is unknown, but such a role would be consistent with a recent linkage analysis. The risk of developing D ϩ HUS was associated (odds ratio, 3.08; P Ͻ .001) with the expression of a platelet GPIb␣ 145M variant that binds VWF with higher affinity than the more common GPIb␣ 145T variant, 22 suggesting that increased Stx-induced interactions between VWF and platelets may promote renal injury in this setting.…”

Section: Discussionmentioning

confidence: 99%

Shiga toxin (Stx)1B and Stx2B induce von Willebrand factor secretion from human umbilical vein endothelial cells through different signaling pathways

Liu

Sadler

2011

IntroductionIngestion of food contaminated with Shiga toxin (Stx)-producing Escherichia coli, such as strain O157:H7, can cause bloody diarrhea that develops into diarrhea-associated hemolytic uremic syndrome (D ϩ HUS) that is characterized by acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia. D ϩ HUS is the most common cause of acute renal failure in children and is fatal in ϳ 3%-5% of cases. 1 Several mechanisms seem to influence the course of tissue injury by Stx. Stx-producing E coli make 2 major types of Stx, each of which is composed of 1 catalytically active A subunit (ϳ 33 kDa) and 5 B subunits (ϳ 7.7 kDa) that form a homopentameric ring. Stx1 is identical to the major toxin produced by Shigella dysenteriae serotype 1. Stx2 is ϳ 50%-60% identical in sequence to Stx1 and occurs in several variants. Stx1 binds to globotriaosylceramide (Gb3) more tightly than Stx2, but Stx2 is more toxic in animal models and is more often associated with D ϩ HUS in humans than Stx1. [2][3][4] The Stx B subunits bind to Gb3 on cell surfaces and facilitate internalization of the Stx holotoxin that undergoes retrograde transport to the endoplasmic reticulum. The Stx A subunit is then translocated into the cytoplasm where it cleaves the adenine base at position 4324 of 28S ribosomal RNA, thereby blocking protein synthesis and initiating a series of responses that culminate in cell death. 5 In addition, treatment of human umbilical vein endothelial cells (HUVECs) or glomerular microvascular endothelial cells (HGMECs) with nanomolar concentrations of Stx1 or Stx2 induces rapid VWF secretion and the formation of long cellassociated VWF strings. 6 Administration of Stx2 to Adamts13 Ϫ/Ϫ mice also causes fatal microvascular thrombosis that resembles thrombotic thrombocytopenic purpura, and Adamts13 Ϫ/Ϫ mice are protected from the lethal effects of Stx if they also lack VWF. 7 We recently found that the B subunits from Stx1 or Stx2, in the absence of the A subunits, are sufficient to stimulate VWF secretion by HUVECs or HGMECs and that Stx2B subunits can induce thrombotic microangiopathy in Adamts13 Ϫ/Ϫ mice. 8 These responses indicate the existence of Stx B-induced signaling pathways that may be responsible for some of the biologic effects attributed previously to the cytotoxic Stx A subunit.At least 3 signaling pathways can contribute to agonist-induced secretion of VWF. Histamine and thrombin activate phospholipase C (PLC)-␤ that generates inositol 1,4,5-triphosphate (IP 3 ) and diacylglycerol. IP 3 increases the level of intracellular Ca 2ϩ that is required for VWF secretion induced by these agonists. In contrast, epinephrine and vasopressin induce VWF secretion by increasing intracellular cAMP and activating protein kinase A (PKA). 9 Alternatively, vascular endothelial growth factor (VEGF) stimulates VWF secretion through a pathway that depends on protein kinase C (PKC)-␦ but not on Ca 2ϩ or cAMP. 10 Whether any of these pathways contribute to Stx B-induced VWF secretion has not been determined previous...

“…The results of a recent linkage analysis are consistent with this hypothesis: the risk of developing D ϩ HUS was associated (odds ratio 3.08, P Ͻ .001) with expression of the platelet GPIb␣ 145M polymorphic variant, which has greater affinity for VWF than the more common GPIb␣ 145T variant. 22 Stx1 and Stx2 both bind Gb3 and use the same mechanism to inactivate ribosomes. However, Stx2 causes D ϩ HUS more often than does Stx1, and this difference has not been explained.…”

Section: Discussionmentioning

confidence: 99%

Shiga toxin B subunits induce VWF secretion by human endothelial cells and thrombotic microangiopathy in ADAMTS13-deficient mice

Motto

Bundle

et al. 2010

IntroductionShiga toxin-producing E coli (STEC) are responsible for a life-threatening food borne illness consisting of bloody diarrhea that may develop into the hemolytic uremic syndrome (HUS), which is characterized histologically by glomerular microvascular platelet adhesion/aggregation and fibrin polymer formation. Classic clinical features of diarrhea-associated HUS (D ϩ HUS) include acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia. D ϩ HUS is the most common cause of renal failure in children in the United States and is fatal in approximately 3%-5% of cases. 1,2 STEC produce 2 main serogroups of Shiga toxins, Stx1 and Stx2. Shiga toxins (Stx) are complex holotoxins with an AB 5 structure. The active subunit (A) has N-glycosidase activity that cleaves the adenine from position 4324 of the 28S rRNA of the 60S ribosomal subunit. The B subunits form a noncovalently linked pentamer that mediates toxin attachment to membrane globotriaosylceramide receptors (Gb3 or CD77). After receptor binding and internalization, Stx travel in a retrograde direction through early/recycling endosomes to the trans-Golgi network, Golgi apparatus, and endoplasmic reticulum (ER). The toxins are transported across the ER membrane to the cytosol where the A subunit cleaves adenine 4324 from the 28S rRNA of the 60S ribosomal subunit, inhibiting protein synthesis and initiating a cascade of reactions termed the ribotoxic stress response, which ultimately leads to cell death. 3 It is worth noting that at least a few hours are required before these effects occur.Recently, Nolasco and colleagues 4 reported that human umbilical vein endothelial cells (HUVECs) and human glomerular microvascular endothelial cells (HGMECs) secreted large amount of ultralarge von Willebrand factor (VWF) after treatment with Stx1 or Stx2 holotoxins. The amount of VWF secreted in response to 1-10nM Stx was comparable to that released by 0.1-20mM histamine. VWF secretion occurred within 10 minutes, which appears to be too fast to attribute to the ribotoxic stress response. In addition, challenge of Adamts13 Ϫ/Ϫ mice with Stx causes systemic microvascular thrombosis that resembles human thrombotic thrombocytopenic purpura, 5 and the absence of VWF completely protects against Stx-induced thrombocytopenia and death. 6 Whether these responses require the catalytically active Stx A subunit was not assessed.We now find that pentameric B subunits of Stx are sufficient to induce acute secretion of ultralarge VWF from cultured human endothelial cells and cause thrombotic microangiopathy in a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS13)-deficient mice. These activities are independent of the ribotoxic effect of Stx A subunits. Binding and clustering of Gb3 receptor is necessary but not sufficient for Stx B-induced VWF secretion. In addition, Gb3 localization in membrane rafts is required. These results uncover a previously unsuspected mechanism for Stx to cause endothelial activation and VWF-dependent t...