Thrombotic Microangiopathy and Increased Nitrosative and Oxidative Stress in an Animal Model of Induced
Sepsis23rd 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
Hospital Universitario de Getafe, Madrid
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.
Objectives1.-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 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 and Methods II
Histological analysis:•Semiquantitative analysis of 24 glomerular,
tubulointerstitial and vascular damage criteria. •Nitrotyrosine and iNOS location by IF in renal cortex.
Serum levels of cytokines and NGAL.
Gen expresion analysis of iNOS, TNF, NT and IL-6 by RT-PCR or Western blot.
• 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 I
Diffuse glomerular damage (> 50%)
Global glomerular damage
Segmental glomerular damage
Congestión
Mesangiolysis
Focal Hyalinosis
Ischemic changes
Acute Tubular Necrosis
PAS positive granules in proximal tubules
Absence of vascular damage
Results II
NITRATION AND OXIDATIONInflammatory Mediators
Serum levels
0100020003000400050006000
TNF(ng/μg)
IL6 (ng/μg) NGAL(mg/ml)
ControlSepsis
TNF, IL-6 and NGAL were significantly elevated in septic animals
TNF: 5109,9/0,13 ng/ug proteinIL6: 1296/0,27 ng/ug proteinNGAL: 1121,15/172,98 ng/ml
Protein Nitration by Western Blot
Significant elevation of NT protein and iNOS was seen in the renal cortex of septic animals.
NT: control: 3688, sepsis: 8900iNOS: control:7628, sepsis: 10776
kDa
Tissue levels by RT-PCR
0
10
20
30
40
50
60
TNF IL6 iNOS
ControlSepsis
Increased gene expression of TNF, IL-6 and iNOS, was seen in renal cortex of septic animals
TNF: 4,25IL6: 58,75iNOS: 6,17
RQ
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
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.
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.
Gracias
Centro Nacional de Biotecnología, CSIC, Madrid, Spain. Juan Ortín, Lorena Ver