Volume 56, Issue 2, Pages 571-580 (August 1999) Obstructive uropathy in the mouse: Role of osteopontin in interstitial fibrosis and apoptosis  Vuddhidej Ophascharoensuk, Cecilia M. Giachelli, Katherine Gordon, Jeremy Hughes, Raimund Pichler, Paul Brown, Lucy Liaw, Rodney Schmidt, Stuart J. Shankland, Charles E. Alpers, William G. Couser, Richard J. Johnson  Kidney International  Volume 56, Issue 2, Pages 571-580 (August 1999) DOI: 10.1046/j.1523-1755.1999.00580.x Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 1 Osteopontin (OPN) expression in normal and obstructed kidneys. In normal OPN+/+ kidneys, OPN is focally expressed in tubules (A), and is increased markedly in obstructed kidneys (C). In contrast, OPN is not expressed in normal (B) or obstructed (D) kidneys of OPN-/- mice (immunoperoxidase, ×200). Kidney International 1999 56, 571-580DOI: (10.1046/j.1523-1755.1999.00580.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 2 Interstitial macrophage accumulation in kidneys with unilateral ureteral obstruction (UUO). Kidneys from OPN+/+ mice develop a marked interstitial macrophage accumulation following obstruction (A) that was less in OPN-/- mice (B) (immunoperoxidase, ×200). The difference in macrophage accumulation was significant at days 4 and 7 when quantitated by computer assisted image analysis (C). Symbols are: (----) OPN+/+; (———) OPN-/-. Kidney International 1999 56, 571-580DOI: (10.1046/j.1523-1755.1999.00580.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 3 Types I and IV collagen immunostaining in kidneys with UUO. Both types IV (A) and type I (C) collagen deposition were increased in obstructed OPN+/+ kidneys (A), but were less in obstructed OPN-/- kidneys (B and D, respectively) (immunoperoxidase, ×200), as documented by computer assisted image analysis (E, F). Symbols are: (□) OPN+/+; (▪) OPN-/-. Kidney International 1999 56, 571-580DOI: (10.1046/j.1523-1755.1999.00580.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 4 Transforming growth factor-βbgr; (TGF-βbgr;) mRNA localization by in situ hybridization in mice with UUO. Minimal TGF-βbgr; mRNA is present with localization to occasional interstitial cells (arrow) in the renal cortex of normal mice (A). A marked increase in TGF-βbgr; mRNA (dense black granules) is noted in the tubules and interstitium of obstructed OPN+/+ mice, especially in the interstitial areas around the dilated tubules (B). Obstructed OPN-/- mice had less TGF-βbgr; mRNA despite comparable tubular dilation (C). Hybridization with sense control resulted in only background signal (D) (A, ×700; B-D, ×420). Kidney International 1999 56, 571-580DOI: (10.1046/j.1523-1755.1999.00580.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 5 Apoptosis is increased in kidneys in mice with UUO. By TUNEL assay an increase in apoptotic cells was observed in both the tubules and interstitium of OPN+/+ (A) and OPN-/- (B) mouse kidneys with UUO (magnification ×420). Obstructed kidneys of OPN-/- mice (▪) had greater tubular (C), and interstitial (D) apoptotic cells compared to obstructed kidneys of OPN+/+ (□) mice. Kidney International 1999 56, 571-580DOI: (10.1046/j.1523-1755.1999.00580.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 6 Apoptosis is increased in serum-starved renal tubular epithelial cells in which osteopontin is inhibited. Rat renal tubular epithelial cells (NRK52E) were serum-starved and the amount of apoptosis (identified by Hoechst stain) measured at 4, 8, 12 and 24 hours. Apoptosis was markedly increased in cells incubated with neutralizing anti-OPN antibody (▪; OP199) as compared with BSA (□), normal goat IgG (□; ngIgG) or cells reconstituted with 10% serum (; CS). Kidney International 1999 56, 571-580DOI: (10.1046/j.1523-1755.1999.00580.x) Copyright © 1999 International Society of Nephrology Terms and Conditions