Therapeutic targeting of classical and lectin pathways of complement protects from ischemia-reperfusion-induced renal damage
Giuseppe Castellano, Rita Melchiorre, Antonia Loverre, Pasquale Ditonno, Vincenzo Montinaro, Michele Rossini, Chiara Divella, Michele Battaglia, Giuseppe Lucarelli, Gennaro Annunziata, Silvano Palazzo, Francesco Paolo Selvaggi, Francesco Staffieri, Antonio Crovace, Mohamed R Daha, Maurice Mannesse, Sandra van Wetering, Francesco Paolo Schena, Giuseppe Grandaliano, Giuseppe Castellano, Rita Melchiorre, Antonia Loverre, Pasquale Ditonno, Vincenzo Montinaro, Michele Rossini, Chiara Divella, Michele Battaglia, Giuseppe Lucarelli, Gennaro Annunziata, Silvano Palazzo, Francesco Paolo Selvaggi, Francesco Staffieri, Antonio Crovace, Mohamed R Daha, Maurice Mannesse, Sandra van Wetering, Francesco Paolo Schena, Giuseppe Grandaliano
Abstract
Ischemia-reperfusion injury is the major cause of delayed graft function in transplanted kidneys, an early event significantly affecting long-term graft function and survival. Several studies in rodents suggest that the alternative pathway of the complement system plays a pivotal role in renal ischemia-reperfusion injury. However, limited information is currently available from humans and larger animals. Here we demonstrated that 30 minutes of ischemia resulted in the induction of C4d/C1q, C4d/MLB, and MBL/MASP-2 deposits in a swine model of ischemia-reperfusion injury. The infusion of C1-inhibitor led to a significant reduction in peritubular capillary and glomerular C4d and C5b-9 deposition. Moreover, complement-inhibiting treatment significantly reduced the numbers of infiltrating CD163(+), SWC3a(+), CD4a(+), and CD8a(+) cells. C1-inhibitor administration led to significant inhibition of tubular damage and tubular epithelial cells apoptosis. Interestingly, we report that focal C4d-deposition colocalizes with C1q and MBL at the peritubular and glomerular capillary levels also in patients with delayed graft function. In conclusion, we demonstrated the activation and a pathogenic role of classical and lectin pathways of complement in a swine model of ischemia-reperfusion-induced renal damage. Therefore, inhibition of these two pathways might represent a novel therapeutic approach in the prevention of delayed graft function in kidney transplant recipients.
Figures
Classical, alternative, and lectin pathway activation in a pig model of ischemia-reperfusion injury. Frozen pig kidney sections were examined for expression of the membrane attack complex (C5b-9) by immunofluorescence and confocal analysis (A−D). C5b-9-deposition, completely absent before ischemia (A), was significantly up-regulated 30 minutes after reperfusion (B), in particular at the peritubular and capillary level (C, arrow) and disappeared 24 hours after reperfusion (D). C4d deposition was evaluated by immunohistochemistry on paraffin-embedded kidney sections (E−H). Specific C4d staining was not observed at T0 (E). Thirty min of reperfusion induced a diffuse deposition of C4d (F) with a specific localization at the peritubular capillaries (G, arrows) that disappeared at 24 hours (H). Factor B deposition was investigated by immunofluorescence and confocal analysis (I−L). Factor B deposition, completely absent before ischemia (I), was significantly up-regulated 30 minutes after reperfusion (J), particularly at the interstitial and tubular level (K), and disappeared 24 hours after reperfusion (L). Complement C3 deposition was investigated by immunofluorescence and confocal analysis (M−P). Complement C3 deposition, absent before ischemia (M), was significantly increased 30 minutes after reperfusion (N−O; N, peritubular capillaries indicated by arrows), and disappeared 24 hours after reperfusion (P). In confocal microscopy images nuclei were stained with TO-PRO-3 (blue).
C4d deposition in the swine model of ischemia-reperfusion-induced renal injury is due to both classical and lectin pathways activation. Co-localization of C4d with C1q (A−C), MBL (D−F), and MBL/MASP-2 (G−I) was investigated on frozen kidney sections by immunofluorescence/confocal microscopy. A: Interstitial and capillary deposition of C4d (green), (B) interstitial deposition of C1q (red); (C) co-deposition of C1q-C4d at interstitial level (merge). D: Interstitial and capillary deposition of C4d (green), (E) interstitial and capillary deposition of MBL (red); (F) co-deposition of MBL-C4d at interstitial and capillary level (merge). The same pattern of co-deposition was also observed for MASP-2/MBL (G−I). The complement cascade was predominantly activated by the lectin and alternative pathways at 15 minutes of reperfusion. MBL, factor B and C1q deposits were investigated by immunofluorescence/confocal microscopy. The extent of MBL (J) and factor B (K) deposition was significantly higher than the one observed for C1q (L). Nuclei were stained with TO-PRO-3 (blue).
Assessment of functional activity of classical, alternative, and lectin pathways in pig sera. A: Increasing concentrations of rhC1INH were incubated with sera from healthy pigs to evaluate the capacity to inhibit the classical and lectin pathways (Wielisa, Wieslab). *P < 0.05 versus positive control (pig sera with vehicle). B: Three healthy pigs received 500 U/kg of rhC1INH; then, serum samples were collected at different time points and evaluated for complement activation by Wielisa. *P < 0.05 versus positive control.
Inhibition of C4d deposition by rhC1INH treatment in a swine model of ischemia-reperfusion-induced renal injury. C4d deposits were evaluated by immunohistochemistry on paraffin-embedded kidney sections from animals treated either with rhC1INH (500 UI/kg, A−C) or vehicle (D−F). The C4d deposits, absent before ischemia induction (A, D), remained significantly lower in the treated group both at the tubulointerstitial (B) and glomerular (C) level compared with untreated animals (tubulointerstitial: E; glomerular: F) Original magnification ×400x. G: Quantification of peritubular capillary C4d+ at the different times of reperfusion in treated (n = 5) and control animals (n = 5). Results are expressed as the mean ± SD of C4d+ capillary/high power field (hpf). *P < 0.05 versus time-matched vehicle-treated animals. H: Quantification of glomeruli C4d+. Results are expressed as the mean ± SD of glomeruli C4d+. *P < 0.05 vs. time-matched vehicle-treated animals.
Inhibition of C5b-9 (A−C) and MASP-2 (D−F) deposition by rhC1INH treatment in a swine model of ischemia-reperfusion−induced renal injury. C5b-9 and MASP-2 deposits were investigated by confocal microscopy. Both were absent before ischemia (A and D, respectively) and were up-regulated 30 minutes after reperfusion (B and E, respectively) in vehicle-treated animals. C1INH infusion caused a marked reduction in the deposition of both, C5b-9 (C) and MASP-2 (F).
Modulation of monocyte and dendritic cell infiltration by rhC1INH treatment in a swine model of ischemia-reperfusion-induced renal injury. CD163 (a monocyte/macrophage marker) expression was evaluated in kidney tissue cryosection by immunohistochemistry. A low number of infiltrating CD163+ cells was observed before ischemia within the interstitial compartment (A). Twenty-four hours of reperfusion induced a significant recruitment of CD163+ cells (B) in vehicle-treated animals, whereas rhC1INH administration caused a marked reduction in the number of infiltrating monocytes (C). D: Quantification of CD163+ cells at different time points after induction of ischemia-reperfusion. Results are expressed as mean ± s.d of CD163+cells/high power field (hpf). *P < 0.05 versus T0 and versus T24 hours of rhC1INH-treated animals. Original magnification ×400. The protein expression of SWC3 (a marker of dendritic cells) was investigated by immunofluorescence/confocal microscopy. Very few infiltrating SWC3A+ cells were observed before ischemia within the interstitial compartment (E). Twenty-four hours of reperfusion induced a significant recruitment of SWC3A+ cells (F) in vehicle-treated animals, whereas rhC1INH infusion caused a marked reduction in the number of infiltrating dendritic cells (G). TO-PRO-3 (blue) was used to counterstain nuclei. Original magnification ×630. H: Quantification of SWC3a+ cells during ischemia-reperfusion. Results are expressed as mean ± s.d of SWC3A+ cells/high power field (hpf). *P < 0.05 versus T0 and versus T24 hours of rhC1INH-treated animals.
Modulation of lymphocytes infiltration by rhC1INH treatment in a swine model of ischemia-reperfusion−induced renal injury. CD4a expression was evaluated in kidney tissue cryosection by immunohistochemistry. Very few infiltrating CD4a+ cells were observed before ischemia within the interstitial compartment (A). Twenty-four hours of reperfusion induced a significant recruitment of CD4a+ cells (B) in vehicle-treated animals, whereas rhC1INH administration caused a marked reduction in the number of infiltrating CD4a+ lymphocytes (C). D: Quantification of CD4a+ cells at different time points after induction of ischemia-reperfusion. Results are expressed as the mean±s.d of CD4a+cells/high power field (hpf). *P < 0.05 versus T0 and versus T24 hours of rhC1INH-treated animals. CD8a expression was investigated in kidney tissue cryosection by immunohistochemistry. Very few infiltrating CD8a+ cells were observed before ischemia within the interstitial compartment (E). Twenty-four hours of reperfusion induced the recruitment of CD8a+ cells (F) in vehicle-treated animals, whereas rhC1INH administration caused a marked reduction in the number of infiltrating CD8a+ T cells (G). H: Quantification of CD8a+ cells at different time points after induction of ischemia-reperfusion. Nuclei were counterstained by hematoxylin. Original magnification ×400. *P < .05 versus T0 and versus T24 hours of rhC1INH-treated animals.
Modulation of tubulointerstitial damage and tubular cell apoptosis by rhC1INH treatment in a swine model of ischemia-reperfusion−induced renal injury. Compared with basal conditions (A) ischemia-reperfusion induced an evident vacuolization and an early cellular infiltrate associated with interstitial edema at the tubulointerstitial level (B: 60 minutes and C: 24 hours of reperfusion). Significantly less edema and tubular damage were present in pigs treated with rhC1INH (D: baseline, E: 60 minutes, and F: 24 hours of reperfusion). Nuclear protein expression of caspase-3 (a specific marker of late cell apoptosis) was evaluated by immunohistochemistry on paraffin-embedded tissue sections. Very few caspase-3+ cells were observed before ischemia (G). An increase in the number of apoptotic tubular cells was observed after 60 minutes (H) and 24 hours (I) of reperfusion in vehicle-treated animals. RhC1INH infusion caused a dramatic reduction in caspase-3+ cells at both time points (J: baseline, K: 60 minutes, and L: 24 hour). M: Quantification of caspase-3+ cells after renal ischemia-reperfusion injury. Results are expressed as mean ± SD of caspase-3+ cells/high power fields (hpf). *P < 0.05 versus vehicle-treated animals (magnification ×400).
C4d deposition in graft biopsies of patients with DGF. C4d deposits were evaluated by immunohistochemistry on paraffin-embedded graft tissue as described in the methods section. C4d deposits were observed in patients with DGF (A, B, and C) at interstitial (A), peri-tubular capillary (B), and glomerular capillary (C) level, whereas no C4d was found in control grafts (D). Co-localization of C4d with C1q (E–G–I) and MBL (F–H–J) was investigated by immunofluorescence/confocal microscopy. C4d (green, E) co-localized with C1q (red, G) at peri-tubular capillary sites (merge, I). C4d deposits (green, F) co-localized with MBL (red, H) at the same site (merge, J). TO-PRO-3 (blue) was used to stain nuclei. Original magnifications were either (A–D) ×400 or (E–J) ×630.
Source: PubMed