published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Microbiology / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Master / Master 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 / Biochemistry / Doctoral / Doctor of Philosophy

Not available / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

No description available.

Clonal cell lines were isolated from mammary gland tissue epithelial cell cultures of lactating cows. Early passage clonal bovine mammary epithelial cells (clone LMH17) gave rise to several established cell lines (MAC-T lines) after being cotransfected with plasmids containing the temperature sensitive mutant SV40 large T antigen gene (pBAPSV40TtsA58) and the bacterial phosphotransferase gene (pSV2-neo). Unlike other cell types which were transformed after being transfected with SV40, MAC-T cells maintained many characteristics of non-transformed cells: MAC-T cells were serum and anchorage dependent, showed contact inhibition, and were not tumorigenic in immunodeficient mice. However, Southern transfer analysis revealed an integrated SV40 gene and cells showed no senescence after 50 passages. These cells are morphologically indistinguishable from parental LMH17 cells and retain the typical morphology of mammary epithelial cells. Positive cytokeratin immunostaining and the absence of vimentin staining indicated that these cells were epithelial in origin. / MAC-T cells grew rapidly on plastic substratum with a doubling time of approximately 17 hours and became differentiated when grown on floating collagen gels in the presence of prolactin. The differentiated phenotype was characterized to include (1) the ability to form secretory domes with a lumen from a pavement of columnar cells; (2) increased casein mRNA abundance; (3) increased alpha S and beta casein secretion; (4) increased number and size of casein secretory vesicles; and (5) increased lactose synthesis and secretion.