Volume 13, Issue 1, Pages 118-126 (January 2006) Adenovirus-mediated gene transfer and lipoprotein-mediated protein delivery of plasma PAF-AH ameliorates proteinuria in rat model of glomerulosclerosis  Naoyuki Iso-O, Hiroshi Noto, Masumi Hara, Masako Togo, Ken Karasawa, Noriko Ohashi, Eisei Noiri, Yoshiaki Hashimoto, Takashi Kadowaki, Satoshi Kimura, Tsuyoshi Watanabe, Kazuhisa Tsukamoto  Molecular Therapy  Volume 13, Issue 1, Pages 118-126 (January 2006) DOI: 10.1016/j.ymthe.2005.08.011 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Plasma PAF-AH activity, plasma 8,12-iso-iPF(2α)-VI (8-epi PGF2α) levels, and urinary 8-OHdG levels after adenovirus-mediated gene transfer. (A) Changes in plasma PAF-AH activity after injection of AdPAFAH (⋄) (n = 9 until day 14 and n = 6 after day 21), AdLacZ (□) (n = 9 until day 14 and n = 6 after day 21), or PBS (○) (n = 4). (B) Distribution of PAF-AH activity and PAF-AH protein on lipoprotein particles 3 days after injection of AdPAFAH. Each fraction fractionated by FPLC was subjected to measurement of total cholesterol (□) or PAF-AH activity (♦). WB, Western blot analysis of the fractionated samples for PAF-AH protein. (C, D) Time-course changes in plasma 8-epi PGF2α levels (C) and urinary 8-OHdG levels (D) after injection of AdPAFAH (⋄) (n = 9 until day 14 and n = 6 after day 21), AdLacZ (□) (n = 9 until day 14 and n = 6 after day 21), or PBS (○) (n = 4). **P < 0.001 and *P < 0.05 compared with the other groups on the same day. Molecular Therapy 2006 13, 118-126DOI: (10.1016/j.ymthe.2005.08.011) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Changes in daily urine protein levels after injection of adenoviruses. (◇) AdPAFAH (n = 9 until day 14 and n = 6 after day 21); (□) AdLacZ (n = 9 until day 14 and n = 6 after day 21); (○) PBS (n = 4). †P < 0.01 and *P < 0.05 compared to other groups on the same day. Molecular Therapy 2006 13, 118-126DOI: (10.1016/j.ymthe.2005.08.011) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Representative immunohistological analysis of liver and kidney samples of Imai rats. Tissue samples were subjected to hPAFAH immunostaining. (A–D) Liver specimens from rats injected with AdPAFAH on day 0 (A), day 3 (B), day 7 (C), and day 28 (D). (E–H) Kidney specimens from rats injected with AdPAFAH on day 0 (E), day 3 (F), day 7 (G), and day 28 (H). Molecular Therapy 2006 13, 118-126DOI: (10.1016/j.ymthe.2005.08.011) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Immunostaining of kidney and aorta samples. (A–E) Samples subjected to hPAFAH immunostaining. (A and B) Kidney specimens from Imai rats injected with AdLacZ (A) or AdPAFAH (B) on day 7 (low magnification). Black arrowheads indicate glomeruli, and red arrowheads indicate small arterioles. (C) Kidney specimen from Imai rats injected with HDL particles abundant in hPAFAH. (D and E) Thoracic aorta samples from Imai rats injected with AdLacZ (D) or AdPAFAH (E) on day 7. (F, G) Double staining (utilizing color detection) of kidney samples on day 7 for PAF-AH (brown) and RECA-1 (purple) (F) or PAF-AH (brown) and Thy-1 (purple) (G). (H–J) Double staining of kidney samples on day 7 utilizing the fluorescence method. Sample was stained for Thy-1 (H, green) and hPAFAH (I, red), and the images were merged. Molecular Therapy 2006 13, 118-126DOI: (10.1016/j.ymthe.2005.08.011) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Analysis of liver and kidney samples of Imai rats by in situ hybridization. (A) Liver specimen of a rat 3 days post-injection of AdLacZ. (B) Liver specimen of a rat 3 days post-injection of AdPAFAH. (C, D) Kidney specimens of rats injected with AdPAFAH on day 3 (C) and day 7 (D). Arrowheads indicate glomeruli. Molecular Therapy 2006 13, 118-126DOI: (10.1016/j.ymthe.2005.08.011) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 6 HNE immunostaining and PAS staining of kidney samples. (A, B) HNE immunostaining of specimens from Imai rats injected with AdLacZ on day 14 (A) or AdPAFAH on day 14 (B). Arrowheads indicate glomeruli. (C, D) PAS staining of specimens from rats injected with AdLacZ on day 28 (C) or AdPAFAH on day 28 (D). (E) Semiquantitative estimation of glomerular sclerosis on day 28. The x axis represents the urinary protein levels before injection, and the y axis represents the glomerulosclerotic score. (♦) AdPAFAH (n = 6); (□) AdLacZ (n = 6); (○) PBS (n = 4). Molecular Therapy 2006 13, 118-126DOI: (10.1016/j.ymthe.2005.08.011) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions