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Title: Effects of immune complexes on glomerular endothelial cells : implications for lupus nephritis
Authors: Wang, Linlin
Degree: Ph.D.
Issue Date: 2017
Abstract: Lupus nephritis (LN) is a common and severe complication of systemic lupus erythematosus, with poor prognosis. Lupus nephritis can lead to permanent renal damage and is the major cause of morbidity and mortality in lupus patients. Although multi-factors have been proved to be involved in disease pathogenesis, including the genetic, environmental, and immunological factors, the precise mechanisms of renal impairments are still not thoroughly understood. Glomerular endothelial cells (GECs) is one type of the renal parenchymal cells and plays important roles in maintaining the integrity of the tripartite renal filtration barrier, which is the structural foundation of renal function. GEC injury and dysfunction will unavoidably affect renal physiology. There is accumulating evidence revealing vascular lesions and endothelial cell injury in LN. However, GECs has been little studied and the underlying pathways for GEC injury in LN remain unknown. Immune complex (IC) formation is one characteristic of LN. Detection of specific autoantibodies and ICs is among the criteria for disease diagnosis. Immune complexes can exist in the circulation or be deposited in tissues. In the kidney, ICs are mainly deposited in subendothelial, subepithelial, or mesangial areas. Since the kidney is a vessel-rich organ and receives approximately 1/5 of the resting cardiac output, GECs are constantly exposed to the circulating ICs or GECs can maintain contact with ICs that deposit in the subendothelial areas. This IC challenge may impair GEC function. Thus, we aimed to investigate the effects of ICs on GECs in vitro and try to understand the details of GEC injury in LN. In the present project, we used heat-aggregated gamma globulin (HAGG) to substitute the ICs. TNF-alpha was also used to simulate the inflammatory microenvironments in the kidney in LN. Cultured human GECs were incubated with HAGG alone or in combination with TNF-alpha. Different aspects of endothelial cell functions were evaluated.
We first investigated the effect of HAGG on autophagy in GECs. Autophagy is a highly conserved catabolic process to degrade cytoplasmic contents through lysosomes. Autophagy presents at basal level and can be activated in response to different stresses. Autophagy is required for maintaining cell homeostasis and exhibits cytoprotective roles in many cell types. Impaired autophagy is reported in many diseases and pathological conditions. Our results showed that HAGG incubation led to decreased LC3 conversion, increased p62 expression, and decreased fluorescence intensity of LC3 puncta staining. Incubation with HAGG also significantly increased the phosphorylations of the key nodes in the mTOR-dependent pathways, including mTOR, p70s6k, 4E-BP-1, and Akt. Therefore, our results indicated that autophagy was suppressed by HAGG in GECs, through an Akt/mTOR-dependent pathway. Next, we evaluated the characteristic cell functions in GECs under HAGG stimulation. Results showed that HAGG treatment changed GEC morphology, induced apoptosis and suppressed cell viability. For tube formation assay, HAGG led to decreased number of junctions and number of meshes, suggesting inhibited angiogenesis. Moreover, HAGG induced intracellular NO productions in GECs, partly through the Akt-eNOS pathway. Mimicking the inflammatory condition, combination of HAGG and TNF-alpha led to obvious cell morphological changes, further decreased cell viability, increased apoptosis, increased NO production, and decreased ability for tube formation. To investigate the possible relationship between autophagy and endothelial cell functions, we used autophagic regulators, rapamycin and 3-Methyladenine, to stimulate and inhibit autophagy respectively. Akt was a key molecule when considering the interaction between autophagy and NO production. Incubation with HAGG activated Akt, which could subsequently activate eNOS and induce NO production in GECs. Meanwhile, activated Akt phosphorylated mTOR and eventually suppressed autophagy. In conclusion, HAGG incubation can affect endothelial cells functions, including autophagy, cell morphology, cell viability, NO production, and angiogenesis. These effects of HAGG on GECs functions, especially under inflammatory microenvironment, are implicated in the renal damage in LN patients. Since renal injury is the most important predictor of mortality in SLE patients, it is important to translate these findings to drug development and treatment strategies to improve the prognosis of LN.
Subjects: Hong Kong Polytechnic University -- Dissertations
Lupus nephritis
Systemic lupus erythematosus
Pages: xxi, 207 pages : illustrations
Appears in Collections:Thesis

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