Effects of metalloproteinase inhibition in a murine model of renal ischemia-reperfusion injury

Abstract

Ischemia-reperfusion injury (IRI) is a leading cause of acute tubular necrosis (ATN) and delayed graft function in transplanted organs. Up-regulation of matrix metalloproteinases (MMPs) propagates the microinflammatory response that drives IRI. This study sought to determine the specific effects of Marimastat (Vernalis, BB-2516), a broad spectrum MMP and TNF-alpha-converting enzyme inhibitor, on IRI-induced ATN. Mice were pretreated with Marimastat or methylcellulose vehicle for 4 d before surgery. Renal pedicles were bilaterally occluded for 30 min and allowed to reperfuse for 24 h. Baseline creatinine levels were consistent between experimental groups; however, post-IRI creatinine levels were 4-fold higher in control mice (p < 0.0001). The mean difference between the post-IRI histology grades of Marimastat-treated and control kidneys was 1.57 (p = 0.003), demonstrating more severe damage to control kidneys. Post-IRI mean (+/-SEM) MMP-2 activity rose from baseline levels in control mice (3.62 +/- 0.99); however, pretreated mice presented only a slight increase in mean MMP-2 activity (1.57 +/- 0.72) (p < 0.001). In conclusion, these data demonstrate that MMP inhibition is associated with a reduction of IRI in a murine model.

Figure 1. Histological scoring criteria

(A-O) Representative PAS stained kidney sections, unless otherwise stated, obtained 24hr after IR. Representative grade denoted at the top left corner of each frame. (A-B) Grade 0, normal kidney. (C) Grade 1, normal kidney (H&E) with focal apoptosis (denoted by arrow). (D-F) Grade 2, focal tubular necrosis (TN) (<50% of CMJ). (G-I) Grade 3, TN in the CMJ. (J-L) Grade 4, TN in the CMJ with focal extension into the cortex. (M-O) Grade 5, TN extending through the cortex to the surface of the kidney. Necrosis is labeled with a star (*).

Figure 2. Distribution of Histological injury scores

(A) Vehicle (100 μl of 0.45% methylcellulose), denoted in black, and Marimastat (100 mg/kg in 100 μl of vehicle), denoted in grey, were administered twice daily. Scores were determined 24hr post-IRI by a blinded pathologist.

Figure 3. Creatinine levels 24hr post-IRI

In vivo creatinine levels were measured prior to injury and prior to euthanasia (24 hours post-IRI). Vehicle (100 μl of 0.45% methylcellulose) and Marimastat (100 mg/kg in 100 μl of vehicle) were administered 96 hours prior to surgery and was continued 24 h post operatively for a total of 10 doses. *P= 0.003 compared with baseline levels.

Figure 4. Matrix metalloproteinase activity

Gelatin zymography was performed on mouse urine collected prior to and following IRI; both proenzyme and activated proteinases appear as zones of substrate clearing. (A) Graph of MMP-2 and (B) MMP-9 activity prior to injury and prior to euthanasia (24hr post-IRI) in control and Marimastat-treated mice.

Figure 5. Western Blots of tissue TACE, MMP-2 and MMP-9

Western Blots were performed on sham, control and Marimastat treated (MT) kidneys. TACE expressed as 120 kDa and 85 kDa bands in sham kidney; 85 kDa and 45 kDa bands in untreated (control) kidney; and 85 kDa and 45 kDa bands in MT kidney (A). MMP-2 was expressed as a 65 kDa band in the sham, control and MT kidneys (B). MMP-9 was not expressed in sham kidney but expressed as 90 kDa in control and MT kidneys (C).