During the last funding cycle, the PPG made significant advances in our understanding of the mechanisms of stone formation and growth. Project 3 has made a comprehensive and essential histopathological characterization of the papillae in human stone disease, and distinguished two main phenotypes that are essential to the pathogenesis of stone disease: plaque deposition and intratubular crystal plugging. A grasp of the contribution of each of these phenotypes to the type of stone disease is essential to understand the underlying pathology, and eventually developing targeted therapies. Achieving this objective requires the performance of unprecedented, detailed, studies of the various microenvironments within the human papillae of stone forming patients. The aims in the current proposal are designed to perform such in-depth investigations of the cellular and molecular mechanisms underlying these key events, using state-of-the-art methodology and skills of the research team. The role of interstitial calcium concentration in the formation of plaque is a fundamental concept. Aim 1 will firmly establish the importance of increased interstitial calcium in the papilla on the pathogenesis of plaque. Aim 2 will investigate the role of innate immune activation in plaque formation. Key downstream effects of innate immunity, such as oxidative stress, inflammatory signaling and crystal modulator deposition will be carefully studied. This aim is expected to define with high confidence, the role of inflammation in the pathogenesis of plaque deposition in human stone disease. Aim 3 will investigate a key mechanism of how plugging disease propagates in the papilla, by determining if ducts with plugs can stimulate oxidative stress and inflammatory signaling beyond the area contiguous to plugging. This cross -talk could lead to upregulation of adhesion molecules that facilitate more plugging in neighboring ducts. Finally, Aim 4 will investigate the rarely-studied papillary urothelium, by establishing if overgrowth of new stones over plaque is preceded by dysfunction of the overlying papillary urothelium. Methods used in these investigations will include: laser micro-dissection, 3 dimensional imaging of the papillae using confocal microscopy, tissue cytometry, state-of the art protein analysis and redox proteomic assays. Combined together, and in synergy with aims from project 1 and 2, these new aims, will greatly advance our understanding of the precise mechanisms of stone formation and growth, which will hopefully translate into more effective clinical treatments for stone disease.