Myeloperoxidase is critically involved in the induction of organ damage after renal ischemia reperfusion

Abstract

In this study the role of myeloperoxidase (MPO) in a murine (C57BL/6) model of ischemia and reperfusion (I/R)-induced renal failure was investigated. The renal function after I/R was analyzed in MPO-deficient (Mpo(-/-)) mice and compared with wild-type (WT) controls. A significant reduction in renal function loss (blood urea nitrogen) was observed after 24 hours of reperfusion of ischemically damaged kidneys in Mpo(-/-) mice compared with I/R WT controls (I/R Mpo(-/-) = 31.3 +/- 1.7 mmol/L versus I/R WT = 42.8 +/- 2.1 mmol/L, sham = 7.0 +/- 0.5 mmol/L; P = 0.003). The early reperfusion phase (2 hours of reperfusion) was characterized by a substantial increase in apoptosis and early complement activation, surprisingly similar in Mpo(-/-) and WT mice. Improved renal function in Mpo(-/-) mice after extended reperfusion was accompanied by a reduced neutrophil influx (P = 0.017) compared with WT controls. Activation and deposition of complement was not significantly reduced in Mpo(-/-) mice compared with WT controls after 24 hours of reperfusion, indicating no specific in vivo role for MPO in activating complement after renal I/R. Taken together, these results demonstrated an important contribution of MPO in the induction of organ damage after renal I/R by influencing critical factors such as neutrophil extravasation but not complement activation.

Figure 1

Similar renal morphology of ischemically damaged WT and KO kidneys after 24 hours of reperfusion. Microphotographs showing periodic acid-Schiff staining of kidney sections of sham-treated (A) as well as WT (B) and Mpo^−/− (C) mice subjected to 40 minutes of ischemia followed by 24 hours of reperfusion. Shown are representative microphotographs of all groups. Original magnifications, ×100.

Figure 2

MPO deficiency significantly reduces renal function loss after renal I/R. Renal function after 24 hours of reperfusion as reflected by BUN concentration. Statistical significance of renal function in Mpo^−/− mice as compared with WT animals was denoted at P = 0.003 (*).

Figure 3

MPO deficiency does not affect I/R-induced early apoptosis. Shown are two sham-treated animals as well as two representatives of each experimental group. Loading controls are given in the inset.

Figure 4

Two hours of reperfusion of ischemically damaged kidneys results in early MBL-C deposition, similar in Mpo^−/− and WT mice. MBL-C binding in WT sham-treated (not shown) and KO sham-treated (A) animals was only observed in glomeruli. MBL-C deposition was evident in peritubular capillaries, the interstitium, and along the epithelial brush border of damaged tubules in both WT (B) and Mpo^−/− (C) mice subjected to renal I/R. Shown are representative microphotographs of all groups. Original magnifications: ×200; ×600 (inset).

Figure 5

MPO plasma levels, representing total neutrophil activation, are increased 24 hours after reperfusion of ischemically damaged WT kidneys. No MPO was detected in Mpo^−/− mice. Statistical significance of WT MPO levels as compared with sham-treated animals was denoted at P < 0.0001.

Figure 6

Immunofluorescent staining showing PMN sequestration (red, Alexa 568) and MPO presence (green, Alexa 488) in kidneys of WT (A–C) and Mpo^−/− (E–G) mice subjected to I/R. Overlapping PMN and MPO data depict the fact that MPO is predominantly located around NIMP-R14-positive PMNs. D (WT) and H (Mpo^−/−) show PMN infiltration in relation to structural changes. Shown are representative microphotographs of all groups. Original magnifications, ×200.

Figure 7

MPO deficiency reduces neutrophil infiltration after renal I/R. Statistical significance as compared with WT animals was denoted at P = 0.017.

Figure 8

MBL-C and C3 (insets) deposition in WT and Mpo^−/− mice after 24 hours of reperfusion. MBL-C and C3 deposition was not different in WT (B) or Mpo^−/− (C) mice. A: Virtually no MBL-C or C3 staining was detected in sham-treated WT (not shown) and Mpo^−/− mice. Corresponding data were obtained for MBL-A deposition (data not shown). Shown are representative microphotographs of all groups. Original magnifications: ×200; ×100 (insets).

Figure 9

Common pathway activation as determined by immunohistochemical and Western blot analyses of C6 deposition in WT and Mpo^−/− mice after 24 hours of reperfusion. C6 deposition in Mpo^−/− mice (C) was similar as compared with WT (B) controls. A: No C6 staining was detected between WT sham (not shown) and Mpo^−/− sham-treated mice. Shown are representative microphotographs of all groups. Western blot results for C6 (sham in A; WT in B; KO in C) under reducing conditions are shown in the insets. Original magnifications, ×100.

Figure 10

Similar common pathway activation between Mpo^−/− (C) and WT (B) mice after 24 hours of reperfusion was confirmed by C9 immunostaining. WT sham (not shown) and Mpo^−/− sham-treated mice (A) did not demonstrate any C9 staining. Original magnifications: ×100; ×200 (insets).