Monitoring Complement Activation

For most nephrologists, the complement system remains a complex and rather daunting cascade. Its implication in a wide range of autoimmune kidney diseases has long been acknowledged, but the complement system was not, until recently, a specific therapeutic target.

The identification of the dysregulation of the complement alternative pathway as a major risk factor for atypical hemolytic uremic syndrome (aHUS) and the subsequent availability of the anti-C5 antibody (eculizumab) have sparked among nephrologists a new interest for complement. This has opened new perspectives and generated new clinical and experimental questions in the field of thrombotic microangiopathies (TMAs) as illustrated by the article by Palomo et al. (1) in this issue of CJASN.

For instance, although the benefit of eculizumab in complement-mediated aHUS is currently clear (2), there is an ongoing debate around the monitoring of optimal complement blockade and treatment duration. Furthermore, in the heterogeneous group of kidney TMA distinct from aHUS (secondary hemolytic uremic syndrome, Hemolysis, Elevated Liver enzymes and Low Platelet count (HELLP) syndrome, etc.), it has become crucial to determine whether complement activation contributes to kidney damage and thus, whether complement inhibition would be beneficial.

Unfortunately, complement component assays in serum (C3, C4, soluble C5b-9, AP50, and CH50) are not reliable biomarkers for the diagnosis of complement activation in any form of TMA, including aHUS (3). Alternative tests have been developed, including the Ham modified test of complement-induced cytotoxicity (4) and assays of complement activation products deposited on endothelial cells in vitro (5).

The second subset of tests aims to reproduce in vitro the complement activation in the microcirculation that is central to the pathogenesis of complement-mediated aHUS. It aims to assess the capacity of the serum or plasma from an individual to induce the deposition of complement activation products at the surface of endothelial cells and hence, induce complement-mediated vascular damage.

In this issue of CJASN, Palomo et al. (1) report on their experience with such in vitro test using human microvascular endothelial cells, a slightly modified version of the previous test designed by Galbusera et al. (5). To circumvent the high variability of the test performed with serum, Palomo et al. (1) reverted to the use of citrated plasma activated with the addition of normal serum.

They show that activated plasma from eight patients with aHUS in the acute phase induced intense deposition of C5b-9 at the surface of microvascular endothelial cells (1). Complement deposition normalizes during remission under eculizumab (at standard or reduced dosing) and most interestingly, during remission in the absence of any treatment.

The authors also tested activated plasma from a small number of patients with TMA, in which the implication of complement is still debated: HELLP syndrome (n=3), severe preeclampsia (n=7), and malignant hypertension not related to aHUS (n=5) (1). Indeed, the frequency of complement gene rare variants is significantly lower in patients with HELLP syndrome (3%–24%) (6,7) and patients with preeclampsia (18% in one series) (8) compared with patients with aHUS, and it is rather similar to the one found in healthy individuals (9). Finally, there are some uncertainties concerning the overlap between malignant hypertension and complement-mediated TMA.

However, in the setting of HELLP and preeclampsia, the absence of complement constitutional dysregulation does not exclude a transient complement activation that may contribute to endothelial damage as the results of Palomo et al. (1) seem to suggest. Activated plasma from patients with acute HELLP and preeclampsia induced a significant deposition of C5b-9 at the surface of endothelial cells. In contrast, plasma from patients with malignant hypertension clinically not attributed to aHUS did not induce complement deposition, whereas plasma from patients with acute aHUS and malignant hypertension did. Therefore, is the ultimate test to determine whether, in a given setting, complement is activated or not finally at hand?

The work by Palomo et al. (1) is clearly a step forward. It is always interesting to see how a new test (albeit with modifications compared with the technique previously used by Galbusera et al. [5]) performs in different laboratories. Their results may have direct clinical implications as illustrated by two patients from the series by Palomo et al. (1) for whom in vitro results translated into modification of treatment with eculizumab. They may also provide a tool to distinguish aHUS from other causes of malignant hypertension, a challenging clinical situation.

More generally, these tests may help to exclude complement activation in some clinical situations with a potentially good negative predictive value. However, enthusiasm does preclude caution. First, complement activation frequently occurs in physiologic situations, is not necessarily harmful in all conditions (including probably some TMA), and is most frequently self-limiting.

For instance, the results of Palomo et al. (1) show that complement deposition on endothelial cells normalizes in patients with hemolytic uremic syndrome during remission, even in the absence of eculizumab. Furthermore, in patients with HELLP syndrome and patients with preeclampsia, complement deposition on endothelial cells persists up to 40 days postpartum when clinical remission has already occurred and then, abates without any specific treatment.

Additionally, the occurrence of HELLP syndrome and preeclampsia in patients with aHUS (10) treated with eculizumab pleads against a central role of complement in the pathogenesis of these two pregnancy complications. Second, endothelial cells (dermal and not kidney one has to keep in mind) assays are relatively highly specialized techniques, which cannot be easily performed and reproduced in all laboratories. They also yield inconsistent results depending on the type of cell used (i.e., Ham modified test [4]) and their state of activation.

The addition of normal serum to plasma is a potential confounding factor, because there is an ongoing debate around a thrombin-mediated activation of C5 (the results of Palomo et al. [1] argue for such occurrence). Additionally, it is surprising that plasma from patients with membrane cofactor protein variants induces complement deposition on endothelial cells in vitro when these patients carry a defect in a membrane-bound and not a circulating complement regulator. One cannot exclude that hemolysis by itself (and the liberation of heme), a common feature to all forms of TMAs, potentially contributes to complement deposition on endothelial cells.

Finally, the authors state that, in two patients with aHUS, in vitro test enabled the identification of partial remission in one and disease relapse in another (1). However, partial remission as well as relapse had been already diagnosed on the basis of clinical assessment and simple biomarkers (creatinine, platelets, lactate dehydrogenase, and haptoglobin). Moreover, complement genes variants have been previously identified as a major predictor of aHUS relapse after eculizumab discontinuation (11).

The design of new in vitro tests to apprehend complement activation (and inhibition under treatment) in TMA and other kidney diseases is clearly needed. The work by Palomo et al. (1) is thus a valuable contribution to this topic. Nevertheless, in our quest for new biomarkers, we should not forget the old and rather reliable ones.

Disclosures

Dr. Fakhouri reports consultancy fees, lecture fees, and/or travel support from Achillion, Alexion Pharmaceuticals, Apellis, and Roche. Dr. Frémeaux-Bacchi has received consultancy, lecture fees, and/or travel support from Alexion Pharmaceuticals, Apellis, and Roche.