Infiltration of the renal parenchyma by inflammatory cells, including lymphocytes, is a common feature of kidney diseases even in the apparent absence of exogenous immune stimuli. The mechanisms underlying T-lymphocyte activation in kidney disease are poorly understood, as are the mechanisms that prevent immune activation in the healthy kidney. This experimental protocol will examine the dynamic behavior, responses, and functions of Dendritic cells (DCs) -a migratory population of cells with specialized function in antigen uptake and presentation - within the kidney and its draining lymphoid tissue during health and explore their role in different forms of renal injury. All organs, including the kidney, have a resident population of DCs although the density, distribution and turnover vary. DC """"""""maturation"""""""" is induced by the inflammatory products of disease or injury. Mature DCs migrate from an organ to its draining lymph node where they activate T-cells to initiate cellular immune responses. In the absence of maturing stimuli, DCs may also transfer antigens to lymphoid tissue in order to activate regulatory mechanisms that prevent autoimmunity. Modulation of DC-T-cell interactions is recognized as an important therapeutic target for the prevention/treatment of immune-mediated disease. The primary hypotheses of the proposal are that: (a) trafficking of protein antigens from the kidney to the draining Iymph nodes occurs during health and actively maintains immune tolerance to renal tissue, and (b) alterations in renal DC phenotype and turnover result from diverse forms of kidney injury and contribute significantly to inflammatory renal parenchvmal damage. The experimental strategy will focus strongly on the use of in vivo techniques that directly examine DC-mediated antigen trafficking and presentation in the kidney and renal lymph nodes of mice. The first Specific Aim, to determine the biology of renal DCs during health, will employ in vivo BrdU-labeling to determine DC turnover, congenic bone marrow transfer to examine the precursor origins of renal DCs, unilateral inoculation of fluorescent particles or proteins into the kidney to track renal DC-mediated antigen trafficking, generation of transgenic mice expressing a kidney-restricted neo-antigen, and adoptive transfer of antigen-specific TCR transgenic T-cells. The second Specific Aim, to determine the role of renal DCs in the pathophysiology of diverse form of renal injury, will apply these same in vivo experimental strategies to animals subjected to one of the following four types of renal injury: (a) ischemia, (b) urinary obstruction, (c) acute glomerulonephritis, (d) genetically-based collagen deficiency (a model of Alport's nephritis). In these experiments, the disease-associated alterations to renal DC turnover, surface phenotype, and antigen trafficking as well as antigen-specific T-cell activation within the draining lymph nodes, and infiltration of the renal parenchyma by activated T-cells will be characterized. For both Specific Aims, observational studies will be followed by mechanistic studies involving blockade of specific molecular pathways, depletion of regulatory cell populations, and cross-breeding to genetically modified strains. Analytic tools will include flow cytometry, immunohistochemistry, immunofluorescence microscopy, morphometric analysis, cytokine/chemokine ELISA.
