CAUSE OF DEATH

1. Histopathological findings and analysis of the oxidative and nitrosative stress in lung and kidney tissue from Pandemic 2009 Influenza A (H1N1) infection23rd 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

2. CAUSE OF DEATH Epidemiologic multicenter study of 100 H1N1 patients in the ICU. Nin et al. J Critical Care 2010 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.

3. 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

4. (mg/dl) 4 3.5 3 2.5 Non AKI 2 Early AKI 1.5 Late AKI 1 0.5 0 0 1 2 3 4 7 14 Blood levels of creatinine in the course of the disease % DAYS MORTALITY RATE N. Nin et al.ACUTE RENAL FAILURE IN CRITICALLY ILL MECHANICALLY VENTILATED PATIENTS WITH INFLUENZA A (H1N1) VIRAL PNEUMONIA. ICM SUMMITED

5. 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

6. Materials and Methods • Routine histological and histochemical analysis. • Double immunofluorescence and confocal microscopic analysis for • Specificmarkers 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): antibodiesafter rabbit immunization with purified recombinant NP.

7. Results in pulmonary pathology I • Diffuse alveolar damage: exudative and proliferative patterns with alveolar and interstitial edema, reactive pneumocytes, fibrinousexudate, hyaline membranes and mild inflammation. • Pulmonary hemorrhage. • Necrotizing bronchiolitiswith destruction of bronchiolar wall and severe acute inflammation. • Fibrosis in one patient (45 days of clinical course).

8. 26 yo female who died with severe hypoxemia 2 h after admission Extensive exudate of fibrin-rich edema fluid in the alveolar space

9. 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

10. Necrotizing bronchiolitis with desquamation and necrosis of bronchial epithelium

11. A 37 yo male with H1N1 infection who died 16 days after ICU admission Type II pneumocytes Type I pneumocytes

12. A 32 yo female dead 45 days after hospital admission for H1N1 viral infection Interstitial fibrosis with thickening of the muscular artery wall

13. Results in pulmonarypathology IINitro-oxidative stress • Increased oxidative and nitrosative stress measured by IF in lung tissue by • oxidyzed dihydroethidium (DHE) • iNOS protein • protein nitration (Nitrotyrosine)

14. 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

15. Results in pulmonarypathology III H1N1 influenza virus detection in lungtissue Immunofluorescence for influenza A virus nucleoprotein (in red) and DAPI, a nuclear marker (in blue). A linear immunoreactivity for viral nucleoprotein is observed, suggesting protein presence in alveolar lining pneumocytes

16. Doublestainingcolocalizing H1N1 virus in thelung A B * * • 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).

17. Results in kidneypathology 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.

18. Histopathological findings Focal acute tubular necrosis of distal tubules in 2 of 4 cases: epithelial cell swelling, mitoses, necrosis and intratubular cell shedding.

19. ATN

20. Normal glomeruli Focal ischemic signs

21. 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.

22. Nitric Oxide inducingenzyme (iNOS) IF in kidneyfrominfectedpatients No renal failure Anti-iNOS 100x Renal failure

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

24. NP CD10

25. VIRUS IN RENAL DISTAL TUBULE (AQP1 +NP) Red: Viral NP Blue: nuclei (DAPI) Green: AQP1 4663-09

26. 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.

27. Gracias Centro Nacional de Biotecnología, CSIC, Madrid, Spain. Juan Ortín, Lorena Ver