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. 2004 Nov;165(5):1677-88.
doi: 10.1016/S0002-9440(10)63424-4.

The mannose-binding lectin-pathway is involved in complement activation in the course of renal ischemia-reperfusion injury

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The mannose-binding lectin-pathway is involved in complement activation in the course of renal ischemia-reperfusion injury

Bart de Vries et al. Am J Pathol. 2004 Nov.

Abstract

Ischemia-reperfusion (I/R) is an important cause of acute renal failure (ARF). The complement system appears to be essentially involved in I/R injury. However, via which pathway the complement system is activated and in particular whether the mannose-binding lectin (MBL)-pathway is activated is unclear. This tempted us to study the activation and regulation of the MBL-pathway in the course of experimental renal I/R injury and in clinical post-transplant ARF. Mice subjected to renal I/R displayed evident renal MBL-depositions, depending on the duration of warm ischemia, in the early reperfusion phase. Renal deposition of C3, C6 and C9 was observed in the later reperfusion phase. The deposition of MBL-A and -C completely co-localized with the late complement factor C6, showing that MBL is involved in complement activation in the course of renal I/R injury. Moreover, the degree of early MBL-deposition correlated with complement activation, neutrophil-influx, and organ-failure observed in the later reperfusion phase. In serum of mice subjected to renal I/R MBL-A, levels increased in contrast to MBL-C levels, which dropped evidently. In line, liver mRNA levels for MBL-A increased, whereas MBL-C levels decreased. Renal MBL mRNA levels rapidly dropped in the course of renal I/R. Finally, in human biopsies, MBL-depositions were observed early after transplantation of ischemically injured kidneys. In line with our experimental data, in ischemically injured grafts displaying post-transplant organ-failure extensive MBL depositions were observed in peritubular capillaries and tubular epithelial cells. In conclusion, in experimental renal I/R injury and clinical post-transplant ARF the MBL-pathway is activated, followed by activation of the complement system. These data indicate that the MBL-pathway is involved in ischemia-induced complement activation.

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Figures

Figure 1
Figure 1
Ischemia followed by reperfusion leads to renal deposition of MBL-A and MBL-C. Positive staining for MBL-A and -C in kidneys from healthy mice is only observed in glomeruli (A and F, respectively, right top corner). Renal ischemia for 15 minutes followed by 2 hours of reperfusion did not induce MBL-deposition (B and G). After 30 to 45 minutes of renal ischemia, MBL-A depositions were already evident after 2 hours of reperfusion, mainly localized to tubular epithelium in the medullar region (C and D, respectively). MBL-C depositions were already evident after 1 to 2 hours of reperfusion after 30 minutes and most pronounced after 45 minutes of renal ischemia (H and I, respectively) and localized to peritubular capillaries (H, right top corner) and interstitium (I, right top corner). At the late phase (24 hours of reperfusion) evident depositions of MBL-A were observed in the cortico-medullary region (E) and the medulla (E, right top corner). Most pronounced, depositions of MBL-C were observed in the vasculature (J, right bottom corner) and on tubular epithelial cells in the cortico-medullary region (J) and the medulla (J, right bottom corner). Magnification, ×200 (×600 for the insets in A, C, F, H, I and bottom corner, J.
Figure 2
Figure 2
Ischemia-induced MBL-deposition precedes C6-deposition. Kidneys of healthy animals (A and D) showed only glomerular MBL-A and MBL-C staining (in red) and no deposition of C6 (in green). Renal ischemia for 45 minutes followed by 1 hour of reperfusion (B and E) induced evident tubular epithelial deposition of MBL-C (E) but no MBL-A deposition (B). However, no C6-deposition is observed in the early reperfusion phase (B and E). At the late reperfusion phase (24 hours of reperfusion) both MBL-A and MBL-C depositions are observed (C and F, respectively). Interestingly, after 24 hours of reperfusion evident C6-staining is present which appears to co-localize to both MBL-A and -C (C and F). Red staining, MBL-A (A–C) and MBL-C (D–F) (Texas-red); green staining, C6 (FITC); blue staining, nuclei (DAPI). Magnification, ×200 and ×600 for right top corners.
Figure 3
Figure 3
Ischemia-induced MBL-deposition co-localizes with C6-deposition. Kidneys subjected to 45 minutes ischemia followed by 24 hours reperfusion showed intense MBL-A and MBL-C depositions (left). At the same time, evident C6 deposition was observed (middle panel). The overlay pictures show that both MBL-A and MBL-C co-localize with C6 deposition (right). Interestingly, whereas C6 completely co-localizes with MBL-A, deposition of C6 does also occur without evident MBL-C deposition. Red staining, MBL-A and MBL-C (Texas-red); green staining, C6 (FITC); blue staining, nuclei (DAPI). Magnification, ×200 and ×600 for the bottom.
Figure 4
Figure 4
Serum MBL-A and -C levels in the course of renal I/R. Serum MBL-levels were determined after 45 minutes of ischemia followed by various reperfusion times using a sandwich ELISA. As compared to control mice, serum MBL-A levels increased twofold after 24 hours of reperfusion (A). In contrast, serum MBL-C levels dropped gradually during reperfusion resulting in a 50% reduction after 24 hours of reperfusion (B). Statistical significance as compared to control-treated animals was denoted at P < 0.05 (*). Data are expressed as median serum concentration of MBL (μg/ml) with interquartile ranges.
Figure 5
Figure 5
Renal ischemia-reperfusion (I/R) differentially regulates renal and hepatic MBL-A and -C mRNA levels. Renal I/R induced a rapid down-regulation of renal MBL-A and -C mRNA levels as compared to healthy control tissue (A). Renal ischemia led to an evident up-regulation of hepatic MBL-A mRNA levels at 24 hours of reperfusion in contrast to MBL-C expression levels (B). Shown are representative samples (n = 4 per group) calibrated against equal amounts of β-actin mRNA.
Figure 6
Figure 6
Neutrophil-influx (A) and renal function loss (B) induced by renal warm ischemia. A: Renal neutrophil-influx was assessed quantitatively by determination of MPO increase in mice subjected to various periods of warm ischemia followed by 24 hours of reperfusion. Values are presented relative to the amount of MPO present in the contralateral kidney harvested immediately after reperfusion and normalized with respect to the MPO increase at 24 hours in mice subjected to 45 minutes of ischemia. Statistical significance as compared to control animals was denoted at P < 0.05 (*) and P < 0.01 (**). The presented data are means ± SEM. B: Renal function after 15, 30, and 45 minutes of ischemia and 24 hours of reperfusion as reflected by blood urea nitrogen (BUN) content. Statistical significance as compared to control animals was denoted at P < 0.05 (*) and P < 0.01 (**). The presented data are means ± SEM.
Figure 7
Figure 7
Renal deposition of MBL in early post-transplant biopsies of transplanted human kidneys. In pre-transplant renal biopsies no MBL could be detected (A0–F0). After transplantation of kidneys derived from heart-beating (HB) donors no MBL deposition is present (A1). In contrast, transplantation of ischemically injured non-heart-beating (NHB) donor kidneys induced rapid glomerular MBL deposition (B1 and C1). Moreover, in NHB grafts that displayed primary non-function extensive vascular and tubular MBL-depositions are observed (D1, interlobular artery and tubular casts; E1 peritubular capillary and tubular epithelium; F1 and G, peritubular capillary). A double-staining for MBL (green) and von Willebrand Factor (red) shows MBL deposition in peritubular capillaries (G). Red staining, filamentous actin (Texas-red), except for G, von Willebrand Factor (Texas-Red); green staining, MBL (FITC); blue staining, nuclei (DAPI). Magnification, ×200 for A–D and ×600 for E–G. Each letter represents a kidney (pre-transplant biopsy marked with 0, post-transplant biopsy marked with 1).

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