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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
  • 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

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
  • 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.
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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.
results i
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.
results ii

Results II

NITRATION AND OXIDATION

InflammatoryMediators

serum levels
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

protein nitration by western blot
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

tissue levels by rt pcr
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

thrombotic microangiopathy tma
Thrombotic Microangiopathy (TMA)
  • Hemolytic Uremic Syndrome
  • Thrombotic Thrombocytopenic Purpura (TTP)
  • Preeclampsia and Eclampsia
  • Antiphospholipid Antibody Syndrome
  • Disseminated Intravascular Coagulation
  • Lupus
  • Esclerodermia
  • Severe Hypertension
  • HIV
conclusions
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.
conclusions1
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.
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Gracias

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

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