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 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. ARDS

(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 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 II Nitro-oxidative stress Increased oxidative and nitrosative stress measured by IF in lung tissue by – oxidyzed dihydroethidium (DHE) – iNOS protein – protein nitration (Nitrotyrosine)

A B CD Nitrosative and oxidative stress markers in lung tissue Oxidation (DHE) iNOS Nitration (NT) Control Disease 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)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). * * AB

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-iNOS 100x Nitric Oxide inducing enzyme (iNOS) IF in kidney from infected patients No renal failure Renal failure

Anti-NP 40x Anti-NP 100x Bowman’s capsule Renal tubule Virus localization with NP antibody IF

NP CD10

VIRUS IN RENAL DISTAL TUBULE (AQP1 +NP) 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.

GraciasARGENTINACHILEESPAÑAURUGUAY Hospital Posadas Hospital Instituto Nacional del Tórax Hospital de Getafe Hospital Maciel Hospital Austral Univ. Catolica Hospital Militar Hospital Santojanini Clínica Indisa Hospital Español Sanatorio Velez Hospital de Concepción Sanatorio CASMU Sanatorio Legomagiore Sanatorio CUDAM Centro Nacional de Biotecnología, CSIC, Madrid, Spain. Juan Ortín, Lorena Ver