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Thrombotic Microangiopathy in Sepsis Animal Model: Oxidative Stress Study

Investigating renal dysfunction in septic patients, this study explores oxidative and nitrosative stress in an induced sepsis model using E.Coli in pigs. Results show increased inflammatory mediators and thrombotic microangiopathy. The study concludes that TMA is the histological expression of vascular damage in sepsis.

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Thrombotic Microangiopathy in Sepsis Animal Model: Oxidative Stress Study

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  1. Thrombotic Microangiopathy and Increased Nitrosative and Oxidative Stress in an Animal Model of Induced Sepsis23rd European Congress of PathologyHelsinki, 2011 R Granados, L El Bouayadi, N Nin, A Ferruelo, C Sánchez, Y Rojas, P Cardinal, A Esteban, M de Paula, JA Lorente Hospital Universitario de Getafe, Madrid

  2. Introduction • Renal dysfunction in severely ill septic patients correlates with a very high mortality. • Sepsis is associated to an increase in oxidative stress causing impairment of systemic blood flow, damage of microvasculature and tissue hypoxia. • Since the physiopathological events are not fully understood, animal models are of great interest.

  3. Objectives 1.-To study the morphology of the lesions associated to sepsis. 2.-To measure the genetic expresion of some mediators of oxidative and nitrosative stress. - Inducible Nitric Oxyde Synthase (iNOS) - Tumor Necrosis factor (TNF) - Nitrotyrosine (NT) - Interleukine 6 (IL-6) 3.-To quantify the degree of protein nitration and oxydation.

  4. Materials and Methods I • An experimental (n=16) intravenous 1.5x109 UFC/ml E. coli solution or control (n=9) sterile saline was injected in pigs. • E. Coli strain serogroup O101, negative for enterotoxins LT and Sta, verotoxins VT1 and VT2 or necrotizing cytotoxic factors CNF1 and CNF2. • Vital signs and renal blood flow was monitorized.

  5. Materials and Methods II • Histologicalanalysis: • Semiquantitativeanalysis of 24 glomerular, tubulointerstitial and vascular damagecriteria. • Nitrotyrosine and iNOSlocationby IF in renal cortex. • Serumlevels of cytokines and NGAL. • Gen expresionanalysisof iNOS, TNF, NT and IL-6 by RT-PCR or Western blot.

  6. Results I • All animals inoculated with E. Coli developed a hypodynamic pattern with low cardiac output and decreseased renal blood flow similar to that seen in septic patients. • There was acute glomerular damage with a thrombotic microangiopathy (TMA) pattern in 10 out of 16 cases (62,5%) of induced sepsis. • None of the control cases had TMA.

  7. Diffuse glomerular damage (> 50%)

  8. Global glomerular damage

  9. Segmental glomerular damage

  10. Congestión

  11. Mesangiolysis

  12. Focal Hyalinosis

  13. Ischemic changes

  14. Acute Tubular Necrosis

  15. PAS positive granules in proximal tubules

  16. Absence of vascular damage

  17. Results II NITRATION AND OXIDATION InflammatoryMediators

  18. Serum levels TNF, IL-6 and NGAL were significantly elevated in septic animals TNF: 5109,9/0,13 ng/ug protein IL6: 1296/0,27 ng/ug protein NGAL: 1121,15/172,98 ng/ml

  19. Protein Nitration by Western Blot kDa Significant elevation of NT protein and iNOS was seen in the renal cortex of septic animals. NT: control: 3688, sepsis: 8900 iNOS: control:7628, sepsis: 10776

  20. Tissue levels by RT-PCR RQ Increased gene expression of TNF, IL-6 and iNOS, was seen in renal cortex of septic animals TNF: 4,25 IL6: 58,75 iNOS: 6,17

  21. Thrombotic Microangiopathy (TMA) • Hemolytic Uremic Syndrome • Thrombotic Thrombocytopenic Purpura (TTP) • Preeclampsia and Eclampsia • Antiphospholipid Antibody Syndrome • Disseminated Intravascular Coagulation • Lupus • Esclerodermia • Severe Hypertension • HIV

  22. Conclusions • Our sepsis-induced animal model reproduces the hemodynamic compromise and renal failure of septic patients. • Thrombotic microangiopathy (TMA) is the histological expression of the vascular damage caused by sepsis in this model.

  23. Conclusions • Increased oxidative and nitrosative activity and elevated inflammatory mediators are seen in serum and in renal cortical tissue. • The development of TMA is most probably the result of an increased thrombogenic effect of a damaged glomerular endothelium.

  24. Gracias Centro Nacional de Biotecnología, CSIC, Madrid, Spain. Juan Ortín, Lorena Ver

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