Brandon Renner

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 H2O2 (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 H2O2 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 H2O2 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.

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.

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.

Renal Ischemia-Reperfusion Injury Amplifies the Humoral Immune Response

Renal transplant recipients who experience delayed graft function have increased risks of rejection and long-term graft failure. Ischemic damage is the most common cause of delayed graft function, and although it is known that tissue inflammation accompanies renal ischemia, it is unknown whether renal ischemia affects the production of antibodies by B lymphocytes, which may lead to chronic humoral rejection and allograft failure. Here, mice immunized with a foreign antigen 24-96 hours after renal ischemia-reperfusion injury developed increased levels of antigen-specific IgG1 compared with sham-treated controls. This amplified IgG1 response did not follow unilateral ischemia, and it did not occur in response to a T-independent antigen. To test whether innate immune activation in the kidney after ischemia affects the systemic immune response to antigen, we repeated the immunization experiment using mice deficient in factor B that lack a functional alternative pathway of complement. Renal ischemia-reperfusion injury did not cause amplification of the antigen-specific antibodies in these mice, suggesting that the increased immune response requires a functional alternative pathway of complement. Taken together, these data suggest that ischemic renal injury leads to a rise in antibody production, which may be harmful to renal allografts, possibly explaining a mechanism underlying the link between delayed graft function and long-term allograft failure.

Detection of complement activation using monoclonal antibodies against C3d

During complement activation the C3 protein is cleaved, and C3 activation fragments are covalently fixed to tissues. Tissue-bound C3 fragments are a durable biomarker of tissue inflammation, and these fragments have been exploited as addressable binding ligands for targeted therapeutics and diagnostic agents. We have generated cross-reactive murine monoclonal antibodies against human and mouse C3d, the final C3 degradation fragment generated during complement activation. We developed 3 monoclonal antibodies (3d8b, 3d9a, and 3d29) that preferentially bind to the iC3b, C3dg, and C3d fragments in solution, but do not bind to intact C3 or C3b. The same 3 clones also bind to tissue-bound C3 activation fragments when injected systemically. Using mouse models of renal and ocular disease, we confirmed that, following systemic injection, the antibodies accumulated at sites of C3 fragment deposition within the glomerulus, the renal tubulointerstitium, and the posterior pole of the eye. To detect antibodies bound within the eye, we used optical imaging and observed accumulation of the antibodies within retinal lesions in a model of choroidal neovascularization (CNV). Our results demonstrate that imaging methods that use these antibodies may provide a sensitive means of detecting and monitoring complement activation-associated tissue inflammation.

Cyclosporine Induces Endothelial Cell Release of Complement-Activating Microparticles

Defective control of the alternative pathway of complement is an important risk factor for several renal diseases, including atypical hemolytic uremic syndrome. Infections, drugs, pregnancy, and hemodynamic insults can trigger episodes of atypical hemolytic uremic syndrome in susceptible patients. Although the mechanisms linking these clinical events with disease flares are unknown, recent work has revealed that each of these clinical conditions causes cells to release microparticles. We hypothesized that microparticles released from injured endothelial cells promote intrarenal complement activation. Calcineurin inhibitors cause vascular and renal injury and can trigger hemolytic uremic syndrome. Here, we show that endothelial cells exposed to cyclosporine in vitro and in vivo release microparticles that activate the alternative pathway of complement. Cyclosporine-induced microparticles caused injury to bystander endothelial cells and are associated with complement-mediated injury of the kidneys and vasculature in cyclosporine-treated mice. Cyclosporine-induced microparticles did not bind factor H, an alternative pathway regulatory protein present in plasma, explaining their complement-activating phenotype. Finally, we found that in renal transplant patients, the number of endothelial microparticles in plasma increases 2 weeks after starting tacrolimus, and treatment with tacrolimus associated with increased C3 deposition on endothelial microparticles in the plasma of some patients. These results suggest that injury-associated release of endothelial microparticles is an important mechanism by which systemic insults trigger intravascular complement activation and complement-dependent renal diseases.

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.

IgM Contributes to Glomerular Injury in FSGS

Glomerular IgM and C3 deposits frequently accompany idiopathic FSGS and secondary glomerulosclerosis, but it is unknown whether IgM activates complement, possibly contributing to the pathogenesis of these diseases. We hypothesized that IgM natural antibody binds to neoepitopes exposed in the glomerulus after nonimmune insults, triggering activation of the complement system and further injury. We examined the effects of depleting B cells, using three different strategies, on adriamycin-induced glomerulosclerosis. First, we treated wild-type mice with an anti-murine CD20 antibody, which depletes B cells, before disease induction. Second, we evaluated adriamycin-induced glomerulosclerosis in Jh mice, a strain that lacks mature B cells. Third, we locally depleted peritoneal B cells via hypotonic shock before disease induction. All three strategies reduced deposition of IgM in the glomerulus after administration of adriamycin and attenuated the development of albuminuria. Furthermore, we found that glomerular IgM and C3 were detectable in a subset of patients with FSGS; C3 was present as an activation fragment and colocalized with glomerular IgM, suggesting that glomerular IgM may have bound a cognate ligand. Taken together, these results suggest that IgM activates the complement system within the glomerulus in an animal model of glomerulosclerosis. Strategies that reduce IgM natural antibody or that prevent complement activation may slow the progression of glomerulosclerosis.

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.

The Role of the Complement System in Acute Kidney Injury

Acute kidney injury is a common and severe clinical problem. Patients who develop acute kidney injury are at increased risk of death despite supportive measures such as hemodialysis. Research in recent years has shown that tissue inflammation is central to the pathogenesis of renal injury, even after nonimmune insults such as ischemia/reperfusion and toxins. Examination of clinical samples and preclinical models has shown that activation of the complement system is a critical cause of acute kidney injury. Furthermore, complement activation within the injured kidney is a proximal trigger of many downstream inflammatory events within the renal parenchyma that exacerbate injury to the kidney. Complement activation also may account for the systemic inflammatory events that contribute to remote organ injury and patient mortality. Complement inhibitory drugs have now entered clinical use and may provide an important new therapeutic approach for patients suffering from, or at high risk of developing, acute kidney injury.