Thrombotic Microangiopathy and Increased Nitrosative and Oxidative Stress in an Animal Model of...

Thrombotic Microangiopathy and Increased Nitrosative and Oxidative Stress in an Animal Model of Induced

Sepsis

23rd 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

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

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

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

0

1000

2000

3000

4000

5000

6000

TNF

(ng/μg)

IL6 (ng/μg) NGAL

(mg/ml)

Control

Sepsis

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

Control

Sepsis

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