Lupus nephritis is a severe organ manifestation of systemic lupus erythematosus (SLE), and is characterized by the production of anti-dsDNA antibodies. It is an important cause of renal failure. The mechanism through which anti-dsDNA antibodies bind to tissues and mediate kidney injury remains to be fully elucidated. Emerging evidence suggests that anti-dsDNA antibodies can bind to cells and extracellular antigens directly through cross-reactivity, independent of bridging chromatin material. Mesangial cells play an important role in normal kidney structure and functions, and its pathophysiology. Mesangial abnormalities in lupus nephritis precede more severe injuries such as lesions in the glomerular capillary loop. We previously demonstrated that the binding of human anti-dsDNA antibodies to mesangial cells (HMC) correlated with disease activity and induced inflammatory as well as fibrotic pathways. The aim of this project is to identify the cross-reactive antigen(s) on the mesangial cell surface that mediates anti-dsDNA antibody binding and the alterations in cell functions that result from this interaction. HMC plasma membrane proteins were purified. Using proteomic and biochemical approaches, we identified annexin II as the predominant cross-reactive antigen on the HMC surface that mediated human polyclonal anti-dsDNA antibody binding. Following this interaction, anti-dsDNA antibodies were internalized in a time- and temperature-dependent manner, and translocated to both the cytoplasm and nucleus within 30 min. This resulted in induction of annexin II synthesis, IL-6 secretion and cell proliferation, which was mediated through the activation of p38 MAPK, JNK and AKT. The binding activity to annexin II in the serum immunoglobulin fraction correlated with the titre of anti-dsDNA antibody. Binding activity of anti-dsDNA antibodies to annexin II correlated with clinical disease activity and circulating anti-dsDNA antibody levels. These correlations were more prominent in male patients with lupus nephritis. Glomerular annexin II expression was increased in patients with active lupus nephritis and co-localized with IgG and C3 deposition. Gene silencing of annexin II in HMC reduced anti-dsDNA antibody binding, which was accompanied by reduced IL-6 secretion and cell proliferation. Using female NZB/W F1 mice, an established murine model of lupus nephritis, we demonstrated that intra-glomerular annexin II expression increased with disease progression and was accompanied by an increase in the expression of p11, its cellular protein ligand. Our data suggest that annexin II may exist in the kidney as a heterotetramer and is involved in disease pathogenesis. At the ultrastructural level, annexin II was detected in the mesangial matrix, amongst electron dense deposits in the glomerular basement membrane, on the foot processes in podocytes and within the Bowman’s capsule. In conclusion, our data demonstrated that annexin II is the major cell surface antigen on HMC that mediates the cross-reactive binding of human anti-DNA antibodies. Through this interaction, cellular processes are triggered that contribute to the pathogenesis of lupus nephritis. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Lupus nephritis is a severe organ manifestation of systemic lupus erythematosus, characterized by the production of autoantibodies and immune-mediated injury to the kidney. Hyaluronan (HA) is a major component of the extracellular matrix, which is involved in immune-mediated renal injury. We have previously demonstrated that HA expression is increased in the glomerulus of patients with severe lupus nephritis, attributed in part to anti-dsDNA antibody-mediated induction of low molecular weight (LMW) HA and high molecular weight (HMW) HA synthesis in mesangial cells. The causal role of HA and its fragments in the pathogenesis of lupus nephritis has not been explored. In this project, two separate in vivo studies were undertaken to delineate the role of HA in disease progression in NZBWF1/J mice. In the first study, we determined the effect of 4-methylumbelliferone (MU), a specific inhibitor of HA synthesis, on renal function and histology in NZBWF1/J mice with active disease, with particular emphasis on inflammatory and fibrotic processes. In the second study, we investigated whether exogenous HA could exacerbate disease manifestations in pre-disease NZBWF1/J mice. The mechanisms underlying the effects of MU and exogenous LMW HA and HMW HA were investigated in mesangial cells isolated from NZBWF1/J mice. Female NZBWF1/J mice with established nephritis were randomized into 3 groups and treated with (1) PBS, (2) Arabic gum (Gum) or (3) MU (3g/kg/day) for 2, 4, 8 and 12 weeks. Treatment of mice with MU for 12 weeks reduced serum HA levels and abrogated intra-renal expression of HA compared to PBS and Gum treated mice. Inhibition of HA synthesis in the kidney resulted in decreased IgG and C3 deposition, reduced B cell, T cell and macrophage infiltration, matrix accumulation and TNF-, IL-6 and MCP-1 expression, which was associated with improved renal functions. To confirm that HA influenced pathogenesis of lupus nephritis, pre-disease NZBWF1/J mice were randomized to receive (1) PBS, (2) LMW HA or (3) HMW HA for periods up to 24 weeks. Administration of LMW HA and HMW HA into NZBWF1/J mice by tail-vein injection induced intra-glomerular deposition of IgG and C3, B cell infiltration, glomerular hypercellularity and tubular atrophy, which was accompanied by induction of MAPK signaling pathways, enhanced MCP-1 expression, and increased matrix deposition in the glomerular and tubular basement membranes. In vitro studies showed that exogenous IL-6, IL-1, TGF-1 and TNF- induced HA synthesis in murine mesangial cells (MMC), with over 80% secreted into the conditioned medium. This was accompanied by an increase in pro-inflammatory cytokine secretion and synthesis of fibronectin and laminin. Inhibition of HA synthesis with MU significantly decreased cytokine secretion and fibronectin synthesis. The ability of HA to induce inflammatory and fibrotic processes in mesangial cells was confirmed in separate studies in which MMC were incubated with exogenous LMW HA and HMW HA. In summary, these original findings provide evidence of a direct effect of HA on intra-renal inflammation and fibrosis in lupus nephritis. Approaches to inhibit HA synthesis may offer novel therapeutic strategies to delay disease progression. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Lu, Jianxin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 218-241). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Lupus nephritis is characterized by the production of anti-dsDNA antibodies, deposition of immune complexes within the kidney parenchyma, proliferation of resident renal cells and induction of inflammatory and fibrotic processes. Approximately 70% of patients with lupus nephritis show immune aggregates along the tubular basement membrane, which is accompanied by an influx of infiltrating cells and increased intra-renal expression of IL-6. Much attention has focused on the inflammatory processes in the kidney during pathogenesis of lupus nephritis whereas mechanisms of fibrogenesis are less well characterized. Tubulo-interstitial injury is a key indicator of poor prognosis of renal function. Given that the tubulo-interstitium occupies over 80% of the kidney volume, injury to this compartment will have a major impact on renal function. There is evidence to show that proximal tubular epithelial cells (PTEC) undergo epithelial-to-mesenchymal transition (EMT) during pathological disorders and adopt a fibroblastic morphology with increased fibrogenic potential. We have previously demonstrated that anti-dsDNA antibodies bound directly to the surface of PTEC through cross-reactive proteins, which were subsequently internalized and translocated to the nucleus where they induced functional changes. Using a proteomic approach, this study identified the cross-reactive antigens that mediated anti-dsDNA antibody binding and intracellular localization in PTEC and the functional consequences thereafter, focusing on EMT and fibrogenic events. Human polyclonal anti-dsDNA antibodies isolated from patients with lupus nephritis bound to Ku70 in plasma membrane extracts isolated from PTEC, and to Ku70, Ku80 and major vault protein in cytosolic and nuclear fractions. Anti-dsDNA antibodies increased synthesis of Ku70, Ku80 and major vault protein in PTEC in a time-dependent manner. Expression of these proteins was localized to proximal tubules especially those undergoing atrophy, and staining was more prominent in renal biopsies from patients with lupus nephritis compared to non-lupus renal disease or control specimens. Binding of anti-dsDNA antibodies to PTEC increased phosphorylation of MAPK and PKC signaling pathways that was accompanied by a concomitant increase in IL-6, IL-8 and TGF-1 secretion and synthesis of β-catenin, fibroblast specific protein-1, fibronectin and laminin. Inhibition of MAPK and PKC signaling pathways with specific inhibitors revealed differential regulation of inflammatory and fibrotic processes by these signaling pathways. In this respect, increased ERK, p38 MAPK, JNK and PKC phosphorylation in PTEC following anti-dsDNA antibody stimulation enhanced IL-6, IL-8 and fibronectin synthesis, whereas increased ERK and JNK phosphorylation upregulated TGF-β1 secretion. Increased β-catenin synthesis was mediated through JNK and PKC phosphorylation. Taken together, our data suggest that PTEC contribute to the pathogenesis of renal inflammation and fibrosis in lupus nephritis. We hypothesize that anti-dsDNA antibodies bind to Ku70 on the plasma membrane of PTEC to mediate inflammation, cell activation and increased fibrogenesis. Although synthesis of EMT markers was increased in PTEC after anti-dsDNA antibody stimulation, transition to a fibroblastic morphology was not observed under our experimental setting suggesting that induction of the EMT cascade is an early event before phenotypic alterations. / published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy