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 Pathology
Helsinki, 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
Causa de Muerte
Shock
17
DOM
52
Hipoxia
refractaria
72
0
20
40
60
80
Epidemiologic multicenter study of 100 H1N1
patients in the ICU. Nin et al. J Critical Care 2010
Hypoxia
HYPOXIA
MOF
60
Shock
53
50
47 47
50
%
• All patients who died,
mantained refractary
hypoxia during the entire
course of the disease.
40
40
37
30
30
20
20
20
10
6
10
0
First week
Second week
Third week
ARDS
• They developed ARDS.
• Viral continuous replication
is supossed to be the cause
of refractary hypoxia.
40
> Third week
(mg/dl)
4
Blood levels of creatinine in the course of the disease
3.5
3
2.5
Non AKI
2
1.5
Early AKI
1
Late AKI
0.5
0
0
1
2
3
4
7
14
DAYS
MORTALITY RATE
92
100
%
80
60
56
44
40
20
0
Non Aki
Early AKI
Late AKI
N. Nin et al. ACUTE RENAL FAILURE IN CRITICALLY ILL MECHANICALLY VENTILATED PATIENTS WITH INFLUENZA A (H1N1) VIRAL PNEUMONIA. ICM SUMMITED
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
A 37 yo male with H1N1 infection who died 16 days after ICU admission
Type I
pneumocytes
Type II
pneumocytes
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 II
Nitro-oxidative stress
• Increased oxidative and nitrosative stress measured by IF
in lung tissue by
–
–
–
oxidyzed dihydroethidium (DHE)
iNOS protein
protein nitration (Nitrotyrosine)
Nitrosative and oxidative stress markers in lung tissue
Control
Disease
Oxidation (DHE)
A
B
iNOS
C
D
Nitration (NT)
Blue: DAPI in nuclei
Red: marker
Results in pulmonary pathology III
H1N1 influenza virus detection in lung tissue
Double staining colocalizing H1N1 virus in the lung
A
B
*
*
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).
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 23 yo female without renal failure
A 60 yo male with a creatinine
increasing to 4.7 mg/dl.
Nitric Oxide inducing enzyme (iNOS) IF in kidney from infected patients
No renal failure
Anti-iNOS
100x
Renal failure
Virus localization with NP antibody IF
Bowman’s capsule
Anti-NP
40x
Renal tubule
Anti-NP
100x
NP
CD10
VIRUS IN RENAL DISTAL TUBULE (AQP1 +NP)
4663-09
Red: Viral NP
Blue: 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
Centro Nacional de Biotecnología,
CSIC, Madrid, Spain. Juan Ortín,
Lorena Ver
ARGENTINA
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