Volume 68, Issue 6, Pages 2651-2666 (December 2005) Plasminogen activator inhibitor-1 gene deficiency attenuates TGF-β1-induced kidney disease  Suren Krag, Carl Christian Danielsen, Peter Carmeliet, Jens Nyengaard, Lise Wogensen  Kidney International  Volume 68, Issue 6, Pages 2651-2666 (December 2005) DOI: 10.1111/j.1523-1755.2005.00737.x Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 1 (A) Real-time RT-PCR analysis of PAI-1 mRNA in 10-week-old TGF-β1+ (▴, •) and TGF-β1- (▵, ○), PAI-1 WT (▵, ▴), or PAI-1 KO (○, •) mice, (-) mean. Results are expressed as the ratio between PAI-1 and GAPDH mRNA expression. (B) Representative PAI-1 stained glomeruli from 34-week-old PAI-1 WT TGF-β1-, PAI-1 WT TGF-β1+, and PAI-1 KO TGF-β1+ mice (original magnification ×250). Arrows mark PAI-1 positive reaction in the glomeruli. Kidney International 2005 68, 2651-2666DOI: (10.1111/j.1523-1755.2005.00737.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 2 Real-time RT-PCR analysis of (A) murine TGF-β1 mRNA and (B) murine βig-h3 mRNA in 10- and 34-week-old TGF-β1+ (▴, •) and TGF-β1- (▵, ○), PAI-1 WT (▵, ▴) or PAI-1 KO (○, •) mice. Results are expressed as the ratio between TGF-β1 and GAPDH mRNA expression. (C) Ratio between βig-h3:TGF-β1 in 10- and 34-week-old TGF-β1+ (▴, •) and TGF-β1- (▵, ○), PAI-1 WT (▵, ▴) or PAI-1 KO (○, •) mice, (-) mean. #P < 0.05 vs. TGF-β1- of same PAI genotype. Kidney International 2005 68, 2651-2666DOI: (10.1111/j.1523-1755.2005.00737.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 3 (A) Representative PAS stained glomeruli from 34-week-old PAI-1 WT TGF-β1- (A), PAI-1 WT TGF-β1+ (B), and PAI-1 KO TGF-β1+ mice (C). (B) (a-d) EM photographs of glomeruli from 16-week-old mice (original magnification ×1400). (e-h) EM photographs of the basal membrane (original magnification ×7900). PAI-1 WT TGF-β1- (a, e), PAI-1 WT TGF-β1+ (b, f), PAI-1 KO TGF-β1- (c, g), and PAI-1 KO TGF-β1+ (d, h). (C) (a-h) Anti-collagen IV α1-2 (a-d) and anti-laminin β2 (e-h) immunostaining from 16-week-old mice (original magnification ×400). PAI-1 WT TGF-β1- (a, e), PAI-1 WT TGF-β1+ (b, f), PAI-1 KO TGF-β1- (c, g), and PAI-1 KO TGF-β1+ (d, h). Kidney International 2005 68, 2651-2666DOI: (10.1111/j.1523-1755.2005.00737.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 4 Real-time RT-PCR analysis of collagen type I and III and fibronectin mRNA in 10- (A) and 34-week-old (B) TGF-β1+ (▴, •) and TGF-β1- (▵, ○), PAI-1 WT (▵, ▴), or PAI-1 KO (○, •) mice, (-) mean. Results are expressed as the ratio between ECM molecules and GAPDH mRNA expression. *P < 0.05 vs. PAI-1 KO TGF-β1+. #P < 0.05 vs. TGF-β1- of same PAI genotype. Kidney International 2005 68, 2651-2666DOI: (10.1111/j.1523-1755.2005.00737.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 5 Total collagen content in 16- and 34-week-old TGF-β1+ (▴, •) and TGF-β1- (▵, ○), PAI-1 WT (▵, ▴), or PAI-1 KO (○, •) mice. Data are expressed as μg collagen/mg dried tissue, (-) mean. *P < 0.05 vs. PAI-1 KO TGF-β1+. #P < 0.05 vs. TGF-β1- of same PAI genotype. Kidney International 2005 68, 2651-2666DOI: (10.1111/j.1523-1755.2005.00737.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 6 (A) Western blot determination of fibronectin content of whole kidney extracts in 34-week-old mice: TGF-β1+ (▴, •) and TGF-β1- (▵, ○), PAI-1 WT (▵, ▴), or PAI-1 KO (○, •) mice, (-) mean.*P < 0.05 vs. PAI-1 KO TGF-β1+. #P < 0.05 vs. TGF-β1- of same PAI genotype. (B) Representative Western blot for fibronectin, lane 1: Mouse fibronectin standard. Lanes 2-5: Samples from 34-week-old mice. Kidney International 2005 68, 2651-2666DOI: (10.1111/j.1523-1755.2005.00737.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 7 Kidneys from 34-week-old mice immunostained with antibodies against collagen I (A-C) and III (D-F). PAI-1 WT TGF-β1- (A, D), PAI-1 WT TGF-β1+ (B, E), and PAI-1 KO TGF-β1+ (C, F) (original magnification ×40). Kidney International 2005 68, 2651-2666DOI: (10.1111/j.1523-1755.2005.00737.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 8 Plasmin activity in kidney extracts without (A) or with (B) addition of plasminogen to evaluate the combined biological effect of t-PA and u-PA. PAI-1 WT TGF-β1- PAI-1 WT TGF-β1+ PAI-1 KO TGF-β1- and PAI-1 KO TGF-β1+. Data are given as mean ± SD. Kidney International 2005 68, 2651-2666DOI: (10.1111/j.1523-1755.2005.00737.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 9 Gel zymography of MMP-2 (A) and MMP-9 (B) in 10-week-old TGF-β1+ and TGF-β1-, PAI-1 WT, or PAI-1 KO mice. Results are expressed as mean ± SD. (C) A representative example of a zymography gel with samples from PAI-1 WT TGF-β1+ (lanes 2, 6), PAI-1 WT TGF-β1- (lanes 3, 7), PAI-1 KO TGF-β1+ (lanes 4, 8), and PAI-1 KO TGF-β1- (lanes 5, 9) mice. Lane 1 and 10 are MMP-2 and -9 standards, respectively. Kidney International 2005 68, 2651-2666DOI: (10.1111/j.1523-1755.2005.00737.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 10 In situ zymography. Representative micrographs of glomeruli at high magnification (×400 original) (A-C) and cortex at a lower magnification (×40 original magnification) (D-G). PAI-1 WT TGF-β1- (A, D), PAI-1 WT TGF-β1+ (B, E), and PAI-1 KO TGF-β1+ (C, F) mice. Negative control (G). (A-C) Arrows mark the glomeruli, and arrowheads mark the mesangial region. (D-F) Arrowheads mark the interstitium/tubular basement membranes. Kidney International 2005 68, 2651-2666DOI: (10.1111/j.1523-1755.2005.00737.x) Copyright © 2005 International Society of Nephrology Terms and Conditions