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Thrombotic Microangiopathy and Increased Nitrosative and Oxidative Stress in an Animal Model of Induced Sepsis 23rd European Congress of Pathology Helsinki, 2011. R Granados, L El Bouayadi, N Nin, A Ferruelo, C Sánchez, Y Rojas, P Cardinal, A Esteban, M de Paula, JA Lorente

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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


Introduction
Introduction Oxidative Stress in an Animal Model of Induced Sepsis

  • 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.


Objectives
Objectives Oxidative Stress in an Animal Model of Induced Sepsis

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.


Materials and methods i
Materials and Methods I Oxidative Stress in an Animal Model of Induced Sepsis

  • 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.


  • Materials Oxidative Stress in an Animal Model of Induced Sepsis 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.


  • Results i
    Results I Oxidative Stress in an Animal Model of Induced Sepsis

    • 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.


    Diffuse glomerular damage 50
    Diffuse glomerular damage (> 50%) Oxidative Stress in an Animal Model of Induced Sepsis


    Global glomerular damage Oxidative Stress in an Animal Model of Induced Sepsis


    Segmental glomerular damage Oxidative Stress in an Animal Model of Induced Sepsis


    Congesti n
    Congestión Oxidative Stress in an Animal Model of Induced Sepsis


    Mesangiolysis
    Mesangiolysis Oxidative Stress in an Animal Model of Induced Sepsis


    Focal Hyalinosis Oxidative Stress in an Animal Model of Induced Sepsis


    Ischemic changes Oxidative Stress in an Animal Model of Induced Sepsis


    Acute Tubular Necrosis Oxidative Stress in an Animal Model of Induced Sepsis


    PAS positive granules in proximal tubules Oxidative Stress in an Animal Model of Induced Sepsis


    Absence of vascular damage Oxidative Stress in an Animal Model of Induced Sepsis


    Results ii

    Results II Oxidative Stress in an Animal Model of Induced Sepsis

    NITRATION AND OXIDATION

    InflammatoryMediators


    Serum levels
    Serum levels Oxidative Stress in an Animal Model of Induced Sepsis

    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


    Protein nitration by western blot
    Protein Nitration by Western Blot Oxidative Stress in an Animal Model of Induced Sepsis

    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


    Tissue levels by rt pcr
    Tissue levels by RT-PCR Oxidative Stress in an Animal Model of Induced Sepsis

    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


    Thrombotic microangiopathy tma
    Thrombotic Microangiopathy (TMA) Oxidative Stress in an Animal Model of Induced Sepsis

    • Hemolytic Uremic Syndrome

    • Thrombotic Thrombocytopenic Purpura (TTP)

    • Preeclampsia and Eclampsia

    • Antiphospholipid Antibody Syndrome

    • Disseminated Intravascular Coagulation

    • Lupus

    • Esclerodermia

    • Severe Hypertension

    • HIV


    Conclusions
    Conclusions Oxidative Stress in an Animal Model of Induced Sepsis

    • 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.


    Conclusions1
    Conclusions Oxidative Stress in an Animal Model of Induced Sepsis

    • 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.


    Gracias Oxidative Stress in an Animal Model of Induced Sepsis

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


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