Mesangial expansion, the principal glomerular lesion in diabetic nephropathy (DN), is preceded by a phenotypic activation and proliferation of the glomerular mesangial cells, followed by a prominent glomerular infiltration of monocytes and macrophages. The molecular mechanisms underlying glomerular infiltration by monocytes in DN are still unclear. Our new preliminary data provide compelling evidence for a causal link between glomerular hyaluronan increase and monocyte/macrophage accumulation, and that in diabetic glomeruli, hyaluronan structures mediate monocyte adhesion and activation, thereby contributing directly to the sclerotic process. We hypothesize: 1) that mesangial cells synthesize abnormal hyaluronan structures in response to hyperglycemia; 2) that specific receptors on infiltrated monocytes and macrophages adhere to these structures and, as a consequence, initiate processes of phagocytosis and activation; 3) that the hyaluronan receptor CD44 on the monocytes/macrophages is minimally necessary for phagocytosis of the hyaluronan structures; and 4) that the hyaluronan structures are only formed by dividing mesangial cells, and hence, the early phase of mesangial cell proliferation determines the numbers of infiltrated monocytes and macrophages that adhere and activate in the early stage of DN. We propose: 1a) to investigate leukocyte adhesion to cultured mesangial cells in response to elevated ambient glucose using the U937 monocytic cell line and peripheral blood mononuclear cells; and 1b) to study the molecular mechanisms that mediate monocyte binding to the adhesive hyaluronan structures; 2) to study the effects of cell cycle stages, intracellular signaling pathways, and heparin/heparan sulfate on hyaluronan synthesis, formation of adhesive hyaluronan structures, and monocyte adhesion; 3) to use sections from normal and diabetic kidneys to study the adhesion, phagocytosis and activation of monocytes in direct response to hyaluronan deposits in the mesangial matrix, and to study the effects of elevated ambient glucose, and heparin/heparan sulfate on glucose utilization, hyaluronan and sulfated glycosaminoglycan synthesis, and formation of adhesive structures by intact glomeruli isolated from normal and streptozotocin-induced diabetic rats using [3H] glucosamine and [35S]sulfate metabolic labeling, and fluorophore-assisted carbohydrate electrophoresis (FACE) analyses; and 4) to determine the effects of heparin and insulin on the synthesis of glomerular hyaluronan using FACE analysis, on formation of adhesive hyaluronan structures, and on glomerular monocyte infiltration in streptozotocin induced diabetic rats. These proposed studies would provide evidence regarding the interplay between early cellular and molecular events, particularly in the regulation and formation of monocyte adhesive hyaluronan structures, and their importance in the genesis stage of DN, and would help to develop strategies to prevent DN progression. ? ?
