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PAI-1 deficiency attenuates the fibrogenic response to ureteral obstruction
Takashi Oda, Young Ok Jung, Heung Soo Kim, Xiaohe Cai, Jesús M López-Guisa, Yukiko Ikeda and Allison A Eddy
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Figure 1.
Sirius red staining of interstitial collagen fibrils. (A–D) These are from plasminogen-activator inhibitor-1 (PAI-1) wild-type (+/+) mice that were sham-operated (sham), and after 3, 7 and 14 days (3D, 7D, and 14D) of unilateral ureteral obstruction (UUO), respectively. (E–H) These are -/- sham, 3D, 7D, and 14D UUO, respectively (
400). (I) Data are mean 
1 SD % of the interstitial area stained. Symbols are: (
) ++ mice; (
) -/- mice; *P < 0.05 compared with sham controls of the same genotype.
Figure 2.
Total kidney collagen in () +/+ and () -/- mice. Results are mean 1 SD. *P < 0.05 compared with sham controls of the same genotype.
Figure 3.
(A) Representative Northern blot autoradiograph of renal mRNA levels for plasminogen-activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), and 
2-antiplasmin (2-AP). The graph shows the results of PAI-1 Northern blotting when RNA from each experimental animal was loaded into separate wells, expressed in arbitrary units as mean 1 SD (B). No PAI-1 mRNA band was detected in the kidneys of the -/- mice. *P < 0.05 compared with sham controls of the same genotype. Similar analyses (data not shown) found a significant 50% increase in uPA only at 7D after UUO and a 300 to 400% increase in tPA at 3D, 7D, and 14D after UUO compared with sham kidneys, and a significant 20 to 60% decrease in 
2-AP at all time points. There were no differences in the tPA, uPA, and 2-AP responses between the +/+ and -/- mice.
Figure 4.
PAI-1 Western blotting of kidney supernatants identified a definite PAI-1 protein band (53 kD) only in the +/+ mice at 7D and 14D. Amido black staining shown in the lower portion of this figure confirmed equivalency of protein loading and transfer.
Full figure and legend (65K)
Figure 5.
(A) Representative plasminogen gel zymogram. The graph illustrates the mean relative activity after analysis of each experimental animal (B). Symbols are: () ++ mice; () -/- mice; *P < 0.05 compared with sham controls of the same genotype. Abbreviation is: H.uPA, human urokinase-type plasminogen activator.
Figure 6.
Renal plasmin activity expressed as mean relative activity 1 SD. Symbols are: () ++ mice; () -/- mice; *P < 0.05 compared with sham controls of the same genotype.
Figure 7.
(A) Representative Northern blot autoradiograph of renal mRNA levels for 1(I) procollagen (Col I) and 1(III) procollagen (Col III) and transforming growth factor-
(TGF-). The graphs show the results when RNA from each experimental animal was loaded into separate wells and probed, expressed in arbitrary units as mean 1 SD (B). Symbols are: () ++ mice; () -/- mice; *P < 0.05 compared with sham controls of the same genotype.
Figure 8.
F4/80+ interstitial macrophages. (A–D) These are +/+ sham, 3D, 7D, and 14D UUO, respectively. (E–H) These are -/- sham, 3D, 7D, and 14D UUO, respectively (400). (I) The graph is % interstitial area stained expressed as mean 1 SD. *P < 0.05 compared with sham controls of the same genotype.
Figure 9.
-Smooth muscle actin (-SMA)-positive interstitial myofibroblasts. (A–D) These are +/+ sham, 3D, 7D, and 14D UUO respectively. (E–H) These are -/- sham, 3D, 7D, and 14D UUO, respectively. (I) The graph is % interstitial area stained expressed as mean 1 SD. *P < 0.05 compared with sham controls of the same genotype (400).
Figure 10.
Murine macrophage chemotactic response to murine PAI-1 and uPA. Symbols are: (
) PAI-1; () uPA; (#) PAI-1 + urokinase receptor macrophages (uPAR -/- M
) Results are the mean values of five separate experiments. The (+) control line indicates the mean number of macrophages recruited in response to 10-8 mol/L f-met-leu-phe (42 7 in 5 experiments). The (-) control line indicates the mean number of macrophages recruited in response to media alone (18 6 in 5 experiments). In three separate experiments using peritoneal M from mice with a genetic deficiency of the urokinase receptor (uPAR-/-), a similar chemotactic response to PAI-1 was observed (# linked with a broken line illustrate mean values). *P < 0.05 compared with the negative control.
