Endothelial Microparticles and Systemic Complement Activation in Patients With Chronic Kidney Disease

Diana Jalal, Brandon Renner, Jennifer Laskowski, Erik Stites, James Cooper, Karissa Valente, Zhiying You, Loni Perrenoud, Moglie Le Quintrec, Ismaeel Muhamed, Uwe Christians, Jelena Klawitter, Margaret A Lindorfer, Ronald P Taylor, V Michael Holers, Joshua M Thurman, Diana Jalal, Brandon Renner, Jennifer Laskowski, Erik Stites, James Cooper, Karissa Valente, Zhiying You, Loni Perrenoud, Moglie Le Quintrec, Ismaeel Muhamed, Uwe Christians, Jelena Klawitter, Margaret A Lindorfer, Ronald P Taylor, V Michael Holers, Joshua M Thurman

Abstract

Background: Endothelial microparticles are associated with chronic kidney disease (CKD) and complement activation. We hypothesized that the complement pathway is activated in patients with CKD via endothelial microparticles and that complement activation correlates with endothelial dysfunction in CKD.

Methods and results: We analyzed complement data of 30 healthy subjects, 30 patients with stage III/IV CKD, and 30 renal transplant recipients with stage III/IV CKD, evaluating the potential correlation of complement fragments with brachial artery flow-mediated dilation, Chronic Kidney Disease Epidemiology Collaboration glomerular filtration rate, and urinary albumin/creatinine ratio. Endothelial microparticles were characterized via proteomic analysis and compared between study groups. Complement fragment Ba was significantly increased in CKD and post-kidney transplant CKD. Plasma Ba levels correlated significantly with lower brachial artery flow-mediated dilation, lower Chronic Kidney Disease Epidemiology Collaboration glomerular filtration rate, and higher urinary albumin/creatinine ratio. Factor D levels were significantly higher in the plasma microparticles of patients with CKD versus healthy controls. Plasma microparticles isolated from patients with CKD and containing factor D activated the alternative pathway in vitro.

Conclusion: The alternative complement pathway is activated in CKD and correlates with endothelial dysfunction and markers of CKD. Future studies are needed to evaluate whether endothelial microparticles with increased factor D play a pathologic role in CKD-associated vascular disease.

Clinical trial registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02230202.

Keywords: Microparticles complement activation; chronic kidney disease.

© 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Figures

Figure 1
Figure 1
Levels of complement activation fragments are higher in patients with stage III/IV CKD compared with healthy controls. A, Plasma Ba and C5b‐9 levels were significantly higher in stage III/IV CKD and transplant patients compared with healthy controls. There was also a trend toward higher levels of the other complement fragments in CKD patients, but these levels were not significantly higher than in healthy controls. B, Urine Ba levels were higher in patients with stage III/IV CKD compared with healthy controls. ***P<0.001, *P<0.05.
Figure 2
Figure 2
Plasma levels of complement fragment Ba are significantly and inversely correlated with BA‐FMD in all of the recruited participants (correlation coefficient −0.26, P=0.016) suggesting that complement activation is related to endothelial dysfunction.
Figure 3
Figure 3
Complement proteins in microparticles from patients with CKD are altered compared with microparticles in healthy controls. We used an aptamer‐based assay (SOMAscan) to measure microparticle proteins with high sensitivity. A, Factor D levels were significantly higher in the microparticles of patients with CKD and patients with transplants with CKD compared with healthy controls. Factor B levels were lower in these groups compared with healthy controls. B, Levels of C3, iC3b, and C3d were not significantly different among the patient groups. C, Levels of the complement inhibitory protein CD59 was higher in patients with CKD than in healthy controls. Levels of factor H and decay accelerating factor were not statistically different among the 3 groups. ***P<0.001, *P<0.05.
Figure 4
Figure 4
Endothelial microparticle number and bound C3 fragments are not significantly increased in patients with CKD. A, We used sizing beads to identify 0.2 to 1.0 μm by forward scatter and size scatter. Counting beads were added to the plasma before microparticle purification, and were used to compare the number of microparticles isolated from each sample. Endothelial microparticles were analyzed by gating on the CD105+CD41a‐ microparticle population. We also analyzed C3 fragment deposition on the endothelial microparticles. B, The size of the microparticles purified from plasma was slightly larger than those in the original plasma sample when compared by forward scatter (FSC). *P<0.05. Data points from healthy control samples are white, and data points from CKD samples are solid black. C, The size of the purified microparticles was the same in the 3 different patient groups studied. D, The number of microparticles in patients with CKD and patients with transplants was not significantly higher than in healthy controls. E, The amounts of C3 fragments deposited on the endothelial microparticles in patients with CKD and patients with transplants was not significantly different than in healthy controls.
Figure 5
Figure 5
Microparticle‐associated factor D activates plasma complement proteins. To test whether FD in plasma microparticles is catalytically active, we examined whether it could restore complement activity to (FDS. A, Using an alternative pathway assay, we confirmed that purified FD restored activity to FDS. We also confirmed that an anti‐FD reduced complement activity in the FDS reconstituted with FD. B, Similarly, (CKD MPs restored activity to the FDS. The addition of an inhibitory antibody to factor D prevented this response, however, confirming that the effect was due to factor D protein contained within the isolated microparticles. C, Staining of the microparticles for factor D confirmed that it was present on the surface. Anti‐FD indicates anti‐factor D antibody; CKD MPs, microparticles purified from the plasma of a patient with CKD; FD, factor D; and FDS, factor D–depleted serum. ***P<0.001, **P<0.01, *P<0.05.

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