Upload
dwayne-logan
View
220
Download
0
Tags:
Embed Size (px)
Citation preview
Histopathological findings and analysis of the oxidative and nitrosative stress in lung
and kidney tissue from Pandemic 2009 Influenza A (H1N1) infection
23rd European Congress of PathologyHelsinki, 2011
R Granados, P Fernández-Segoviano, N Nin, JA Lorente, C Sánchez-Rodríguez, L S, L Soto, J
Hidalgo, J Ortín, A Esteban.
Hospital Universitario de Getafe, Madrid
• Influenza A virus (H1N1) may elicit severe respiratory dysfunction and acute kidney injury (AKI) leading to death.
• The specific cell target for the infection has not been found.
CAUSE OF DEATH
Epidemiologic multicenter study of 100 H1N1 patients in the ICU. Nin et al. J Critical Care 2010
HYPOXIA
• All patients who died, mantained refractary hypoxia during the entire course of the disease.
• They developed ARDS.
• Viral continuous replication is supossed to be the cause of refractary hypoxia.
5350
47
4037
30
47
20
10
20
6
40
0
10
20
30
40
50
60
First week Second week Third week > Third week
Hypoxia
MOF
Shock
%
ARDS
0
0.5
1
1.5
2
2.5
3
3.5
4
0 1 2 3 4 7 14
(mg/dl)
Non AKI
Early AKI
Late AKI
DAYS
N. Nin et al. ACUTE RENAL FAILURE IN CRITICALLY ILL MECHANICALLY VENTILATED PATIENTS WITH INFLUENZA A (H1N1) VIRAL PNEUMONIA. ICM SUMMITED
Blood levels of creatinine in the course of the disease
4456
92
0
20
40
60
80
100
Non Aki Early AKI Late AKI
MORTALITY RATE
%
Aims of the study
• In 11 fatal cases of H1N1 infection • Postmortem lung tissue from 7 patients• Kidney biopsies from 4 patients
• To analize:
– Histopathological findings
– Oxidative and nitrosative stress
– Localization of viral particles in lung and kidney
Materials and Methods
• Routine histological and histochemical analysis. • Double immunofluorescence and confocal microscopic analysis for
– Specific markers of nefron segments and alveolar cells:• aquaporin 1 and CD10: proximal tubules• Nefrin: podocytes• CK7: distal tubules• Aquaporin 5: pneumocytes type 1• Surfactant protein: pneumocytes type 2• CD68: macrophages
– Oxidative and nitrosative stress markers:• oxidized dihydroethydium (DHE): presence of oxygen free radicals.• inducible NO synthase (iNOS): increased NO.• nitrotyrosine (NT): protein nitration, superoxide anion and NO.
– Human influenza nucleoprotein (NP): antibodies after rabbit immunization with purified recombinant NP.
Results in pulmonary pathology I
• Diffuse alveolar damage: exudative and proliferative patterns with alveolar and interstitial edema, reactive pneumocytes, fibrinous exudate, hyaline membranes and mild inflammation.
• Pulmonary hemorrhage.
• Necrotizing bronchiolitis with destruction of bronchiolar wall and severe acute inflammation.
• Fibrosis in one patient (45 days of clinical course).
26 yo female who died with severe hypoxemia 2 h after admission
Extensive exudate of fibrin-rich edema fluid in the alveolar space
A postmortem sample from a 16 yo male 8 days after ICU admission
Diffuse alveolar damage: hyaline membranes lining the alveolar spaces and inflammatory infiltrates
Necrotizing bronchiolitis with desquamation and necrosis of bronchial epithelium
Type II pneumocytes
Type I pneumocytes
A 37 yo male with H1N1 infection who died 16 days after ICU admission
A 32 yo female dead 45 days after hospital admission for H1N1 viral infection
Interstitial fibrosis with thickening of the muscular artery wall
Results in pulmonary pathology IINitro-oxidative stress
• Increased oxidative and nitrosative stress measured by IF in lung tissue by
– oxidyzed dihydroethidium (DHE)– iNOS protein – protein nitration (Nitrotyrosine)
A B
C D
Nitrosative and oxidative stress markers in lung tissue
Oxidation (DHE)
iNOS
Nitration (NT)
Control DiseaseControl Disease
Blue: DAPI in nucleiRed: marker
Results in pulmonary pathology III H1N1 influenza virus detection in lung tissue
Double staining colocalizing H1N1 virus in the lung
A) Immunofluorescence for type I pneumocytes (aquoporin 5 positive cells in green with asterisks) and viral nucleoprotein (in pink). A type I pneumocyte containing viral nucleoprotein is observed (arrow)(confocal scanning microscopy,original magnification x 63).
B) Immunofluorescence for macrophages (CD68 in green) and viral nucleoprotein (in red). Macrophages containing viral nucleoprotein are identified (confocal scanning microscopy, original magnification x 63).
**
A B
Results in kidney pathology I
• The histology from the 2 patients with AKI showed acute tubular necrosis (ATN) in distal tubules.
• There was increased nitrosative and oxidative stress markers (DHE, iNOS and NT) in the renal cortex of patients with kidney failure, but not in those with normal renal function.
• Cases with AKI selectively showed viral NP immunoreactivity in distal tubules and in parietal Bowman´s capsule epithelium.
Histopathological findings
• Focal acute tubular necrosis of distal tubules in 2 of 4 cases: epithelial cell swelling, mitoses, necrosis and intratubular cell shedding.
ATN
Normal glomeruli Focal ischemic signs
Superoxide levels by dihydroethydium probe in renal tissue from Influenza A patients
A 60 yo male with a creatinine increasing to 4.7 mg/dl.
A 23 yo female without renal failure
Anti-iNOS100x
Nitric Oxide inducing enzyme (iNOS) IF in kidney from infected patientsNitric Oxide inducing enzyme (iNOS) IF in kidney from infected patients
No renal failure
Renal failure
Anti-NP40x
Anti-NP100x
Bowman’s capsule
Renal tubule
Virus localization with NP antibody IF
NPNP
CD10CD10
4663-09
VIRUS IN RENAL DISTAL TUBULE (AQP1 +NP)
Red: Viral NPBlue: nuclei (DAPI)Green: AQP1
Conclusions
• Fatal H1N1 viral infection causes ARDS and acute tubular necrosis in distal tubules.
• The disease courses with prolonged oxidative and nitrosative stress in lung and renal cortical tissue.
• Viral particles are seen in distal tubules, Bowman´s capsule, type 1 pneumocytes and alveolar macrophages.
• These findings suggest persistent viral replication despite antiviral treatment.
Gracias
ARGENTINAARGENTINA CHILECHILE ESPAÑAESPAÑA URUGUAYURUGUAY
Hospital PosadasHospital Posadas Hospital Instituto Hospital Instituto Nacional del TóraxNacional del Tórax
Hospital de Hospital de GetafeGetafe Hospital MacielHospital Maciel
Hospital AustralHospital Austral Univ. CatolicaUniv. Catolica Hospital MilitarHospital Militar
Hospital SantojaniniHospital Santojanini Clínica IndisaClínica Indisa Hospital Hospital EspañolEspañol
Sanatorio VelezSanatorio Velez Hospital de Hospital de ConcepciónConcepción
Sanatorio Sanatorio CASMUCASMU
Sanatorio Sanatorio LegomagioreLegomagiore
Sanatorio Sanatorio CUDAMCUDAM
Centro Nacional de Biotecnología, CSIC, Madrid, Spain. Juan Ortín, Lorena Ver