PROJECT SUMMARY/ ABSTRACT. This Equipment Supplement project seeks to acquire a high-resolution fluorescent microscope to advance the goals of my R35 MIRA project. The MIRA supported research program focuses on understanding the cellular and molecular mechanisms underlying the progression of tissue injury mediated by meprin metalloproteases. Two goals in the project rely heavily on microscopic imaging technology with complementary software for quantitation of the staining intensity as a measure of protein expression levels. Meprins comprise of two subunits, α and β, which form two protein isoforms, meprin A (α-α or α-β) and meprin B (β-β) with distinct and overlapping substrates. Meprins are most abundantly expressed in the brush- border membranes of proximal kidney tubules and small intestines. Meprins are also expressed in leukocytes (monocytes and macrophages), podocytes, skin, endothelial cells, and cancer cells. Meprins have been implicated in the pathophysiology of inflammatory- and fibrosis-associated diseases that include kidney disease, inflammatory bowel disease, lung fibrosis, neurodegenerative disease (e.g. Alzheimer’s disease), and cancer. Single nucleotide polymorphisms (SNPs) in the meprin β gene were shown to associate with severity of certain diseases such as diabetic kidney disease and cancer. My research group uses a combination of molecular biology and proteomic approaches to identify meprin substrates and characterize the interactions between meprin isoforms and their substrates. These are coupled with in vivo studies with meprin knockout mouse models to determine how meprin activity impacts the progression of disease. Known meprin substrates include extracellular matrix (ECM) proteins, modulators of inflammation (e.g. proinflammatory cytokines [IL-1β, IL-6, IL-18, MCP-1; and anti-inflammatory proteins Ac-SDKP), cell signaling proteins (e.g. protein kinase A and protein kinase C), mediators of the hypoxia response (e.g. osteosarcoma-9), tight junction proteins (e.g. claudin 5, occludin, E-cadherin, and Z0-1) cytoskeletal proteins (e.g. villin and actin) and proteins that contribute to plaques in AD (e.g. amyloid precursor protein and triggering receptor expressed on myeloid cells 2). The diversity of meprins substrates suggests that complex mechanisms are involved under different conditions and in different organs. It’s important to gain understanding of these mechanisms to facilitate development of diagnostic and therapeutic tools. For the MIRA proposal, we are conducting studies in three areas; (i) to determine how SNPS in the meprin β gene impact its interactions with substrates and physiological sheddases, (ii) determine how meprin interactions with substrates modulate signaling pathways and impact responses in hypoxia, inflammation, and ECM metabolism, and (iii) to evaluate the use of meprin and meprin cleavage products as biomarkers for development of diagnostic tools for early detection of disease. The proposed researc...