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pathophysiology of the hemolytic uremic syndrome using in vitro and in vivo models of Shiga toxin mediated renal damage
x, 123 leaves : illustrations (some color) ; 28 cm.
ABSTRACT: Shiga toxins (Stxs) are expressed by the enteric pathogens Shigella dysenteriae 1 and enterohemorrhagic Escherichia coli (EHEC). Stx-producing organisms cause bloody diarrhea with the potential to progress to acute renal failure. Stxs are potent protein synthesis inhibitors and are believed to be largely responsible for the renal damage seen in post diarrheal hemolytic uremic syndrome (D+HUS). Mice have been extensively employed as an animal model of renal damage caused by Shiga toxins. In this study, we examined the role of the proinflammatory cytokine tumor necrosis factor-[alpha] (TNF-[alpha]) in development of toxin-mediated renal disease in mice. Mice pre-treated with TNF-[alpha] adminstration and challenged with Shiga toxintype 1 (Stx1) showed increased survival compared to mice treated with Stx1 alone. Conversely, mice treated with Stx1 before TNF-[alpha] administration succumbed quicker than mice given Stx1 alone. Increased lethality in mice treated with Stx1 followed by TNF-[alpha] was associated with evidence of glomerular damage and loss of renal function. No differences in renal histopathology were noted between animals treated with Stx1 alone versus the TNF-[alpha] pre-treatment group, although we noted a sparing of renal function when TNF-[alpha] was administered before toxin. Compared to treatment with Stx1 alone, treatment with TNF-[alpha] after toxin altered the renal cytokine profile so that expression of proinflammatory cytokines TNF-[alpha] and Interluekin-1 beta (IL-1[beta]) increased, and expression of the anti-inflammatory cytokine Interleukin-10 (IL-10) decreased. Increased lethality in mice treated with Stx1 followed by TNF-[alpha] was associated with higher numbers of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive renal tubule cells suggesting that increased lethality involved enhanced apoptosis. These data suggest that the early administration of TNF-[alpha] may be a candidate interventional strategy blocking disease progression, while TNF-[alpha] production after intoxication may exacerbate disease. Additionally, these data show that glomerular endothelial are not the sole target for Shiga toxin mediated damage. Numerous studies have demonstrated the importance of the epithelium of the kidney. Therefore, using the immortalized human proximal tubular epithelial cell line, HK-2, we show these cells to express an abundance of the membrane bound toxin receptor, globotriaosylceramide (Gb₃) and to be differentially susceptible to the cytotoxic action of Stx1 and Stx2. Hk-2 cells are extremely sensitive to Stx1 when comparted to Stx2. Additionally, at early timepoints (24h) HK-2 cells are significantly more sensitive to Stxs than the prototypical Vero cells, however by 72 h Vero cell monolayers were completely destroyed while some HD-2 cells survived. This finding suggests that a subpopulation of HK-2 cells may survive Stx2 challenge. Furthermore, HK-2 cells traffic labeled Stx1 B-subunit to both the lysosomal and endoplasmic reticulum (ER) cellular compartments suggesting that intracellular trafficking may play a role in the cells susceptibilty to Shiga toxin mediated damage. Interestingly, even though cytokines are not produced, Stx2 induced the expression of two key chemokines, Marcophage Inflammatory Protein-1 alpha and Macrophage Inflammatory Protein-1 beta (Mip1-[alpha] and Mip-1[beta]). Additionally, we show that Stx1 and Stx2 differentially activate components of the ER stress response in HK-2 cells. Finally, we demonstrate significant Poly ADP ribose Polymerase (PARP) cleavage after exposure to both Stx1 and Stx2, however procaspase-3 cleavage was undetectable, suggesting that HK-2 cells may undergo apoptosis in a caspase-3 independent manner.
"Submitted to the Office of Research and Graduate Studies at Texas A&M Health Science Center in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2011."
Approved as to style and content by: Vernon L. Tesh, Alan Parrish, Jon Skare, Helene Andrews-Polymenis, James E. Samuel.
Ph. D. Texas A&M University System Health Science Center 2011.
Includes bibliographical references (leaves 102-121).