Introduction

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.

6. 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.

7. 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.

9. Diffuse glomerular damage (> 50%)

10. Global glomerular damage

11. Segmental glomerular damage

12. Congestión

13. Mesangiolysis

14. Focal Hyalinosis

15. Ischemic changes

17. Acute Tubular Necrosis

18. PAS positive granules in proximal tubules

20. Absence of vascular damage

21. Results II NITRATION AND OXIDATION InflammatoryMediators

22. 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

23. 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

24. 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

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

26. 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.

27. 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.

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