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Discussion
In this study, we tested the relevance of targeting the complement system activation at the reperfusion stage in an auto-transplanted pig kidney model, using a compound with a wide range of activities, including inhibition of the complement, contact system, coagulation and fibrinolysis cascades.
RhC1INH treatment demonstrated a significant improvement of kidney function post-transplant. Measurement of serum creatinine levels showed that time to peak and height of peak were not altered, however there was a striking difference in recovery: while vehicle treated animals showed a slow recovery, rhC1INH treatment accelerated recovery with an earlier start (day 3) and sharper decreases reaching pretransplant levels within 14 days.
We investigated the chronic consequence of treatment: (i) function analysis showed that the rhC1INH- treated group recovered to pretransplant serum creatinine levels by month 1, while vehicle-treated animals never achieved levels below 250 µmol/L, threefold above baseline; (ii) IFTA exploration demonstrated that the treatment significantly reduced injury development; however rhC1INH-treated group levels were ~10 %, whereas in our experience with the same model, IFTA development could be reduced to 5 % [19]; (iii) immune response at 3 months was increased in the vehicle group, while it was lower in the rhC1INH-treated group; (iv) EMT activation was high in the vehicle group with both stainings observable in both the tubules and the interstitium of rhC1INH-treated kidneys.
Investigating further, we determined by western blot the activation of the signaling pathway emblematic of EMT and IFTA, i.e. TGF-ß. This showed that rhC1INH treatment permitted some degree of protection, with reduced activation of Smad 3 and increased levels of BMP7. However, the observed increase in TGF-ß expression in the rhC1INH group is surprising. This could be due to the fact that the treatment only partially protected against IRI. Our results are similar to another study on rhC1INH treatment and EMT [26], however the later uses a 30 min warm ischemia model in the pig, with outcomes measured after 24 h of reperfusion, hence representing a direct effect of the molecule, while herein we investigated the chronic consequences of the treatment. Hence, the treatment may have simply delayed the occurrence of fibrosis through the TGF-ß pathway. Another explanation stems from the other role of TGF-ß, namely as an anti-inflammatory cytokine [27, 28]. Indeed, the observed production of TGF-ß could have a systemic effect, reducing immune activation. This hypothesis is in compatible with our results on innate and adaptive immune cell invasion.
We characterized the impact of the treatment on transplanted kidneys. We thus tested RhC1INH impact on complement activation using immunofluorescent staining on biopsies collected 30 min after reperfusion. In healthy pigs, complement pathway effectors are absent from the kidneys [21]. In our hands, rhC1INH noticeably inhibited C4d deposition. We did not see an effect of the treatment on C1q, C3c or MASP staining, but rhC1INH also appeared to affect MBL staining, reducing its signal. However this was detected inside the cells, an atypical, although described [29], localization for this protein, and too little data is available to permit definite conclusions. Thus, rhC1INH had some effect on complement deposition, mostly C4d. This is surprising, as C4 is upstream of C3 in the complement activation cascade; however, C4d deposition is more stable, and associated to pathways beyond the cytotoxic consequences of the complement cascade, such as antibody-mediated rejection [30]. RhC1INH was injected 45 min before the kidney was biopsied. Previous studies in humans and animals showed that it distributed rapidly and reached an effective dosage within 15–30 min [24], hence its high ratio of distribution and fast effect on complement could explain the lack of effects observed on its primary targets: the primary effect would have already taken place, and we only observe their consequences. Earlier biopsies could have revealed more, however the risk associated with the procedure was contrary to the aim of the study and ethical consideration.