Detection of pro angiogenic and inflammatory biomarkers in patients with CKD.

Cardiovascular disease (CVD) is the most common cause of death in patients with native and post-transplant chronic kidney disease (CKD). To identify new biomarkers of vascular injury and inflammation, we analyzed the proteome of plasma and circulating extracellular vesicles (EVs) in native and post-transplant CKD patients utilizing an aptamer-based assay. Proteins of angiogenesis were significantly higher in native and post-transplant CKD patients versus healthy controls. Ingenuity pathway analysis (IPA) indicated Ephrin receptor signaling, serine biosynthesis, and transforming growth factor-ß as the top pathways activated in both CKD groups. Pro-inflammatory proteins were significantly higher only in the EVs of native CKD patients. IPA indicated acute phase response signaling, insulin-like growth factor-1, tumor necrosis factor-α, and interleukin-6 pathway activation. These data indicate that pathways of angiogenesis and inflammation are activated in CKD patients' plasma and EVs, respectively. The pathways common in both native and post-transplant CKD may signal similar mechanisms of CVD.

Complement factor H-deficient mice develop spontaneous hepatic tumors.

Hepatocellular carcinoma (HCC) is difficult to detect, carries a poor prognosis, and is one of few cancers with an increasing yearly incidence. Molecular defects in complement factor H (CFH), a critical regulatory protein of the complement alternative pathway (AP), are typically associated with inflammatory diseases of the eye and kidney. Little is known regarding the role of CFH in controlling complement activation within the liver. While studying aging CFH-deficient (fH-/-) mice, we observed spontaneous hepatic tumor formation in more than 50% of aged fH-/- males. Examination of fH-/- livers (3-24 months) for evidence of complement-mediated inflammation revealed widespread deposition of complement-activation fragments throughout the sinusoids, elevated transaminase levels, increased hepatic CD8+ and F4/80+ cells, overexpression of hepatic mRNA associated with inflammatory signaling pathways, steatosis, and increased collagen deposition. Immunostaining of human HCC biopsies revealed extensive deposition of complement fragments within the tumors. Investigating the Cancer Genome Atlas also revealed that increased CFH mRNA expression is associated with improved survival in patients with HCC, whereas mutations are associated with worse survival. These results indicate that CFH is critical for controlling complement activation in the liver, and in its absence, AP activation leads to chronic inflammation and promotes hepatic carcinogenesis.

Complement fragments are biomarkers of antibody-mediated endothelial injury.

Antibody-mediated rejection (AbMR) adversely affects long-term graft survival in kidney transplantation. Currently, the diagnosis of AbMR requires a kidney biopsy, and detection of complement C4d deposition in the allograft is one of the diagnostic criteria. Complement activation also generates several soluble fragments which could potentially provide non-invasive biomarkers of the process. Furthermore, microvesicles released into the plasma from injured cells can serve as biomarkers of vascular injury. To explore whether soluble complement fragments or complement fragments bound to endothelial microvesicles can be used to non-invasively detect AbMR, we developed an in vitro model in which human endothelial cells were exposed to anti-HLA antibodies and complement sufficient serum. We found that complement fragments C4a and sC5b-9 were increased in the supernatants of cells exposed to complement-sufficient serum compared to cells treated complement-deficient serum. Furthermore, complement activation on the cell surface was associated with the release of microvesicles bearing C4 and C3 fragments. We next measured these analytes in plasma from kidney transplant recipients with biopsy-proven acute AbMR (n = 9) and compared the results with those from transplant recipients who also had impaired allograft function but who did not have AbMR (n = 30). Consistent with the in vitro results, complement fragments C4a and Ba were increased in plasma from patients with AbMR compared to control subjects (P < 0.001 and P < 0.01, respectively). Endothelial microvesicle counts were not increased in patients with AbMR, however, and the number of microvesicles with C4 and C3 bound to the surface was actually lower compared to control subjects (both P < 0.05). Our results suggest that plasma complement activation fragments may be useful as non-invasive biomarkers of antibody-mediated complement activation within the allograft. Complement-opsonized endothelial microvesicles are decreased in patients with AbMR, possibly due to enhanced clearance of microvesicles opsonized with C3 and C4 fragments.

Complement Activation in Patients with Focal Segmental Glomerulosclerosis.

BACKGROUND: Recent pre-clinical studies have shown that complement activation contributes to glomerular and tubular injury in experimental FSGS. Although complement proteins are detected in the glomeruli of some patients with FSGS, it is not known whether this is due to complement activation or whether the proteins are simply trapped in sclerotic glomeruli. We measured complement activation fragments in the plasma and urine of patients with primary FSGS to determine whether complement activation is part of the disease process. STUDY DESIGN: Plasma and urine samples from patients with biopsy-proven FSGS who participated in the FSGS Clinical Trial were analyzed. SETTING AND PARTICIPANTS: We identified 19 patients for whom samples were available from weeks 0, 26, 52 and 78. The results for these FSGS patients were compared to results in samples from 10 healthy controls, 10 patients with chronic kidney disease (CKD), 20 patients with vasculitis, and 23 patients with lupus nephritis. OUTCOMES: Longitudinal control of proteinuria and estimated glomerular filtration rate (eGFR). MEASUREMENTS: Levels of the complement fragments Ba, Bb, C4a, and sC5b-9 in plasma and urine. RESULTS: Plasma and urine Ba, C4a, sC5b-9 were significantly higher in FSGS patients at the time of diagnosis than in the control groups. Plasma Ba levels inversely correlated with the eGFR at the time of diagnosis and at the end of the study. Plasma and urine Ba levels at the end of the study positively correlated with the level of proteinuria, the primary outcome of the study. LIMITATIONS: Limited number of patients with samples from all time-points. CONCLUSIONS: The complement system is activated in patients with primary FSGS, and elevated levels of plasma Ba correlate with more severe disease. Measurement of complement fragments may identify a subset of patients in whom the complement system is activated. Further investigations are needed to confirm our findings and to determine the prognostic significance of complement activation in patients with FSGS.

Complement activation and toll-like receptor-2 signaling contribute to cytokine production after renal ischemia/reperfusion.

The innate immune system causes tissue inflammation and injury after renal ischemia/reperfusion (I/R). The complement system is activated on ischemic tubular epithelial cells (TECs) and induces the cells to produce pro-inflammatory chemokines. TECs also express toll-like receptors (TLRs)-2 and -4. Signaling through the TLRs induces TECs to produce a variety of chemokines, some of which can also be induced by complement activation fragments. We sought to determine whether the effects of complement activation and TLR signaling in TECs are redundant, or whether additive protection can be achieved by blocking both of these innate immune systems. To confirm that the complement system, TLR-2 signaling, and TLR-4 signaling induce production of a similar repertoire of inflammatory chemokines, we stimulated TECs with complement sufficient serum or with TLR-2 and TLR-4 ligands in vitro. We found that all three of these stimuli induce TECs to produce KC, MIP-2, IL-6, and TNF-α, and that there was a trend toward greater production of KC in cells exposed to two stimuli. Based upon these results, we hypothesized that mice deficient in both complement activation and TLR-2 signaling would demonstrate greater protection from I/R than mice deficient only in the complement system. To test this hypothesis we induced ischemic acute kidney injury (AKI) in wild-type mice, mice with targeted deletion of complement factor B (fB(-/-) mice), or mice with targeted deletion of factor B and TLR-2 (fB(-/-)TLR2(-/-) mice). Surprisingly, we found that fB(-/-)TLR2(-/-) mice developed more severe injury than those with single deficiency of factor B. Our results indicate that blockade of the complement system may be more protective than simultaneous blockade of both the complement system and TLR-2 in ischemic AKI.

Binding of factor H to tubular epithelial cells limits interstitial complement activation in ischemic injury.

Factor H is a regulator of the alternative pathway of complement, and genetic studies have shown that patients with mutations in factor H are at increased risk for several types of renal disease. Pathogenic activation of the alternative pathway in acquired diseases, such as ischemic acute kidney injury, suggests that native factor H has a limited capacity to control the alternative pathway in the kidney. Here we found that an absolute deficiency of factor H produced by gene deletion prevented complement activation on tubulointerstitial cells after ischemia/reperfusion (I/R) injury, likely because alternative pathway proteins were consumed in the fluid phase. In contrast, when fluid-phase regulation by factor H was maintained while the interaction of factor H with cell surfaces was blocked by a recombinant inhibitor protein, complement activation after renal I/R increased. Finally, a recombinant form of factor H, specifically targeted to sites of C3 deposition, reduced complement activation in the tubulointerstitium after ischemic injury. Thus, although factor H does not fully prevent activation of the alternative pathway of complement on ischemic tubules, its interaction with the tubule epithelial cell surface is critical for limiting complement activation and attenuating renal injury after ischemia.

B cell subsets contribute to renal injury and renal protection after ischemia/reperfusion.

Ischemia/reperfusion (I/R) triggers a robust inflammatory response within the kidney. Numerous components of the immune system contribute to the resultant renal injury, including the complement system. We sought to identify whether natural Abs bind to the postischemic kidney and contribute to complement activation after I/R. We depleted peritoneal B cells in mice by hypotonic shock. Depletion of the peritoneal B cells prevented the deposition of IgM within the glomeruli after renal I/R and attenuated renal injury after I/R. We found that glomerular IgM activates the classical pathway of complement, but it does not cause substantial deposition of C3 within the kidney. Furthermore, mice deficient in classical pathway proteins were not protected from injury, indicating that glomerular IgM does not cause injury through activation of the classical pathway. We also subjected mice deficient in all mature B cells (µMT mice) to renal I/R and found that they sustained worse renal injury than wild-type controls. Serum IL-10 levels were lower in the µMT mice. Taken together, these results indicate that natural Ab produced by peritoneal B cells binds within the glomerulus after renal I/R and contributes to functional renal injury. However, nonperitoneal B cells attenuate renal injury after I/R, possibly through the production of IL-10.

The complement inhibitors Crry and factor H are critical for preventing autologous complement activation on renal tubular epithelial cells.

Congenital and acquired deficiencies of complement regulatory proteins are associated with pathologic complement activation in several renal diseases. To elucidate the mechanisms by which renal tubular epithelial cells (TECs) control the complement system, we examined the expression of complement regulatory proteins by the cells. We found that Crry is the only membrane-bound complement regulator expressed by murine TECs, and its expression is concentrated on the basolateral surface. Consistent with the polarized localization of Crry, less complement activation was observed when the basolateral surface of TECs was exposed to serum than when the apical surface was exposed. Furthermore, greater complement activation occurred when the basolateral surface of TECs from Crry(-/-)fB(-/-) mice was exposed to normal serum compared with TECs from wild-type mice. Complement activation on the apical and basolateral surfaces was also greater when factor H, an alternative pathway regulatory protein found in serum, was blocked from interacting with the cells. Finally, we injected Crry(-/-)fB(-/-) and Crry(+/+)fB(-/-) mice with purified factor B (an essential protein of the alternative pathway). Spontaneous complement activation was seen on the tubules of Crry(-/-)fB(-/-) mice after injection with factor B, and the mice developed acute tubular injury. These studies indicate that factor H and Crry regulate complement activation on the basolateral surface of TECs and that factor H regulates complement activation on the apical surface. However, congenital deficiency of Crry or reduced expression of the protein on the basolateral surface of injured cells permits spontaneous complement activation and tubular injury.

A targeted inhibitor of the complement alternative pathway reduces RPE injury and angiogenesis in models of age-related macular degeneration.

Genetic variations in complement factor H (fH), an inhibitor of the complement alternative pathway (CAP), and oxidative stress are associated with age-related macular degeneration (AMD). Recently, novel complement therapeutics have been created with the capacity to be "targeted" to sites of complement activation. One example is our recombinant form of fH, CR2-fH, which consists of the N-terminus of mouse fH that contains the CAP-inhibitory domain, linked to a complement receptor 2 (CR2) targeting fragment that binds complement activation products. CR2-fH was investigated in vivo in the mouse model of choroidal neovascularization (CNV) and in vitro in oxidatively stressed RPE cell monolayers. RPE deterioration and CNV development were found to require CAP activation, and specific CAP inhibition by CR2-fH reduced the loss of RPE integrity and angiogenesis in CNV. In both the in vivo and in vitro paradigm of RPE damage, a model requiring molecular events known to be involved in AMD, complement-dependent VEGF production, was confirmed. These data may open new avenues for AMD treatment strategies.

Aseptic injury to epithelial cells alters cell surface complement regulation in a tissue specific fashion.

We have recently shown that oxidative stress of ARPE-19 cells alters the expression of the cell surface complement regulatory proteins DAF and CD59, and permits increased activation of complement when the cells are subsequently exposed to serum. Based upon these results, we hypothesized that RPE cells respond to cellular stress as if it is infection, and reduce their surface expression of complement regulatory proteins to foster the local immune response. To test this hypothesis, we examined whether cellular hypoxia would produce a similar change in ARPE-19 cells. In addition, we asked whether this response to oxidative stress is universal in all epithelial cells, by examining the expression of complement regulatory proteins on the surface of the renal and pulmonary epithelial cells. We found that the expression of complement regulatory proteins is altered by aseptic cellular stressors such as hypoxia and oxidative stress, but the response to these conditions differs from tissue to tissue. In RPE cells oxidative stress reduces the expression of the cell surface complement regulators and sensitizes the cells to complement mediated injury. This specific response is not seen in epithelial cells from the lung or kidney, and is not induced by hypoxia. These studies help explain the unique mechanisms by which uncontrolled complement activation may contribute to the development of AMD.

Renal inflammation: targeted iron oxide nanoparticles for molecular MR imaging in mice.

PURPOSE: To determine the feasibility of T2-weighted magnetic resonance (MR) imaging in the noninvasive quantification of renal inflammation by using superparamagnetic iron oxide (SPIO) nanoparticles targeted to tissue-bound C3 activation fragments in a mouse model of lupus nephritis. MATERIALS AND METHODS: All animal procedures were approved by the University of Colorado-Denver animal care and use committee. SPIO nanoparticles were encapsulated by using amine-functionalized phospholipids. A recombinant protein containing the C3d-binding region of complement receptor type 2 (CR2) was then conjugated to the surface of the SPIO nanoparticle. Five MRL/lpr mice (a model of lupus nephritis) and six C57BL/6 wild-type mice were assessed with T2-weighted MR imaging at baseline and after SPIO injection. The same five MRL/lpr mice and three C57BL/6 mice also underwent MR imaging after injection of CR2-targeted SPIO. A series of T2-weighted pulses with 16 echo times was used to enable precise T2 mapping and calculation of T2 relaxation times in the cortex and outer and inner medulla of the kidneys, as well as in the spleen, muscle, and fat. The effects of treatment and animal genotype on T2 relaxation times were analyzed with repeated-measures analysis of variance. RESULTS: At baseline, the T2-weighted signal intensity in the kidneys of MRL/lpr mice was higher than that in the kidneys of wild-type mice. Injection of untargeted SPIO did not alter the T2-weighted signal in the kidneys in either strain of mice. Injection of CR2-targeted SPIO in MRL/lpr mice, however, caused a significant accumulation of targeted iron oxide with a subsequent decrease in T2 relaxation times in the cortex and outer and inner medulla of the kidneys. No changes in T2 relaxation time were observed in the wild-type mice after injection of targeted SPIO. CONCLUSION: Injection of CR2-conjugated SPIO caused a significant reduction in T2-weighted MR imaging signal and T2 relaxation time in nephritic kidneys.

Oxidative stress renders retinal pigment epithelial cells susceptible to complement-mediated injury.

Uncontrolled activation of the alternative pathway of complement is thought to be associated with age-related macular degeneration (AMD). The alternative pathway is continuously activated in the fluid phase, and tissue surfaces require continuous complement inhibition to prevent spontaneous autologous tissue injury. Here, we examined the effects of oxidative stress on the ability of immortalized human retinal pigment epithelial cells (ARPE-19) to regulate complement activation on their cell surface. Combined treatment with H(2)O(2) (to induce oxidative stress) and complement-sufficient serum was found to disrupt the barrier function of stable ARPE-19 monolayers as determined by transepithelial resistance (TER) measurements. Neither treatment alone had any effect. TER reduction was correlated with increased cell surface deposition of C3, and could be prevented by using C7-depleted serum, an essential component of the terminal complement pathway. Treatment with H(2)O(2) reduced surface expression of the complement inhibitors DAF, CD55, and CD59, and impaired regulation at the cell surface by factor H present within the serum. Combined treatment of the monolayers with H(2)O(2) and serum elicited polarized secretion of vascular epidermal growth factor (VEGF). Both, secretion of VEGF and TER reduction could be attenuated using either an alternative pathway inhibitor or by blocking VEGF receptor-1/2 signaling. Regarded together, these studies demonstrate that oxidative stress reduces regulation of complement on the surface of ARPE-19 cells, increasing complement activation. This sublytic activation results in VEGF release, which mediates disruption of the cell monolayer. These findings link oxidative stress, complement activation, and apical VEGF release, which have all been associated with the pathogenesis of AMD.