A PAI-1 mutant, PAI-1R, slows progression of diabetic nephropathy

Abstract

Plasminogen activator inhibitor-1 (PAI-1) has been implicated in renal fibrosis. In vitro, PAI-1 inhibits plasmin generation, and this decreases mesangial extracellular matrix turnover. PAI-1R, a mutant PAI-1, increases glomerular plasmin generation, reverses PAI-1 inhibition of matrix degradation, and reduces disease in experimental glomerulonephritis. This study sought to determine whether short-term administration of PAI-1R could slow the progression of glomerulosclerosis in the db/db mouse, a model of type 2 diabetes in which mesangial matrix accumulation is evident by 20 wk of age. Untreated uninephrectomized db/db mice developed progressive albuminuria and mesangial matrix expansion between weeks 20 and 22, associated with increased renal mRNA encoding alpha1(I) and (IV) collagens and fibronectin. Treatment with PAI-1R prevented these changes without affecting body weight, blood glucose, glycosylated hemoglobin, creatinine, or creatinine clearance; therefore, PAI-1R may prevent progression of glomerulosclerosis in type 2 diabetes.

Figure 1.

Time course of glomerular Vn and endogenous PAI-1 staining in diabetic db/db mice. Representative photomicrographs of glomeruli were from three mice at different ages. Polyclonal rabbit anti-mouse Vn antibody (1:300 dilution; provided by Emile de Heer, Department of Nephrology and Pathology, Leiden University Medical Center, Leiden, Netherlands) and rabbit anti-rat PAI-1 antibody (400 μg/ml dilution; American Diagnostica, Greenwich, CT) were used as the primary antibodies. FITC-conjugated swine anti-rabbit IgG (Dako Corp., Carpinteria, CA) was used as secondary antibody. Control slides treated with PBS instead of primary antibodies showed no staining. Data are from three mice at each group. *P < 0.05 versus uninephrectomized nondiabetic db/m control at 22 wk of age; **P < 0.05 versus uninephrectomized diabetic db/db mice at 18 wk of age. Magnification, ×400.

Figure 2.

(A) Time course of disappearance of injected PAI-1R from diabetic glomeruli in db/db mice at week 20. Representative photomicrographs of glomeruli from three mice at each time point that were administered an injection of PAI-1R at 100 μg/mouse intraperitoneally. A goat anti-human PAI-1 antibody (1:100; American Diagnostica) was used as the primary antibody, which was specific for human PAI-1 and did not stain mouse PAI-1. FITC-conjugated donkey anti-goat IgG (1:200 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) was applied as secondary antibody. (B) Co-localization of PAI-1R and Vn in diabetic glomeruli. A glomerulus from a db/db mouse at week 20 that was killed 3 h after PAI-1R injection. Staining for human PAI-1R (green) and mouse Vn (red). Double staining for PAI-1R and Vn (yellow). Injected PAI-1R co-localized with endogenous mouse Vn in the mesangium. Without PAI-1R injection, no staining for human PAI-1 was seen in the kidney. Magnification, ×400.

Figure 3.

Dosage effect of PAI-1R on glucose levels (A), albuminuria (B), and glomerular histology (C) in diabetic db/db mice. *P < 0.05 versus uninephrectomized nondiabetic control db/m mice at 22 wk of age (NC; n = 9); #P < 0.05 versus uninephrectomized diabetic db/db mice at 20 wk of age (DC20; n = 5); §P < 0.05 versus uninephrectomized diabetic db/db mice at 22 wk of age, treated with PBS for 2 wk (DC22; n = 5). PAI-1R25, 22-wk diabetic mice treated with PAI-1R at 25 μg/mouse from weeks 20 to 22 (n = 7); PAI-1R100, 22-wk diabetic mice treated with PAI-1R at 100 μg/mouse from weeks 20 to 22 (n = 7).

Figure 4.

Effect of PAI-1R on albuminuria in diabetic db/db mice from weeks 20 to 22. *P < 0.05 versus NC22; #P < 0.05 versus DC20; §P < 0.05 versus DC22. PAI-1R22, 22-wk diabetic mice treated with PAI-1R at 0.5 mg/kg body wt, intraperitoneally, twice daily from weeks 20 to 22.

Figure 5.

Effect of PAI-1R on glomerular matrix protein accumulation in diabetic db/db mice from weeks 20 to 22. For immunofluorescence staining, rabbit anti–type IV collagen (Rockland Immunochemicals, Gilbertsville, PA) and rabbit anti-rat FN (Chemicon, CA) were used as the primary antibodies. FITC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories) was used as the secondary antibody. FITC-conjugated rabbit anti-human fibrinogen/fibrin (Dako Corp.) was used directly to detect fibrinogen/fibrin. *P < 0.05 versus NC22; #P < 0.05 versus DC20; §P < 0.05 versus DC22. Magnification, ×400.

Figure 6.

Effect of PAI-1R on collagens and FN production in renal cortex tissue in diabetic db/db mice from weeks 20 to 22. (A and B) Representative Western blots illustrate type IV collagen (Col IV; A) and type I collagen (Col I; B) protein expression. The immunostaining band was visualized by enhanced chemiluminescence (ECL) Western blotting detection reagents (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). The densitometric intensity of β-actin staining detected by monoclonal mouse anti–β-actin IgG was adjusted for the protein loading equality. For comparison, this ratio was set at unity for normal control samples, and other lanes on the same gel were expressed as fold increase over this value. The respective graphs summarize the results of band density measurements. (C) FN content in renal cortex was detected by ELISA. *P < 0.05 versus NC22; #P < 0.05 versus DC20; §P < 0.05 versus DC22.

Figure 7.

Effect of PAI-1R on TGF-β1, PAI-1, and matrix proteins mRNA expression in renal cortex tissue in diabetic db/db mice from weeks 20 to 22. Expression of mRNA was determined by real-time reverse transcriptase–PCR. Changes in mRNA levels were determined by first correcting the amplification of β-actin for each sample. For comparison, this ratio was set at unity for normal control samples, and other groups were expressed as fold increase over this value. (A) Expression of TGF-β1 mRNA. (B) Expression of PAI-1 mRNA. (C) Expression of FN mRNA. (D) Expression of α1(I) collagen mRNA. (E) Expression of type α1(IV) collagen mRNA. *P < 0.05 versus NC22; #P < 0.05 versus DC20; §P < 0.05 versus DC22.

Figure 8.

Effects of PAI-1R on plasmin activity in renal cortex tissue in diabetic db/db mice. Results were expressed as 10⁻⁵ U/mg tissue. *P < 0.05 versus NC22; #P < 0.05 versus DC20; §P < 0.05 versus DC22.

Figure 9.

Effect of PAI-1R on TGF-β1 protein levels and Smad2 signaling in renal cortex tissue in diabetic db/db mice. (A) Total TGF-β1 protein levels were measured by ELISA. (B) Representative Western blots illustrate phosphorylated Smad2 (p-Smad2), total Smad2, and β-actin protein expression. The immunostaining band was visualized by ECL Western blotting detection reagents. The densitometric intensity of total Smad2 staining was adjusted for the protein loading equality. For comparison, this ratio was set at unity for normal control samples, and other lanes on the same gel were expressed as fold increase over this value. The respective graph summarizes the results of band density measurements. *P < 0.05 versus NC22; #P < 0.05 versus DC20; §P < 0.05 versus DC22.