Abstract revised Leukocyte cell-derived chemotaxin-2 (LECT2) amyloidosis (ALECT2) is the third most common type of kidney amyloidosis in the United States. ALECT2 results from misfolding of the LECT2 protein into amyloid fibrils, which accumulate in the kidneys and lead to their failure. ALECT2 is believed to be caused by the Ile40→Val (I40V) mutation in LECT2 in combination with a second, as-yet unidentified condition. Preliminary data demonstrated that loss of LECT2’s single bound zinc ion, in combination with vigorous stirring, was sufficient to cause the protein to form amyloid on its own. This project will address two main concerns: whether the proposed microfluidic shear devices (designed to mimic blood flow through the vasculature of the kidney) can induce amyloid formation, and whether structures of LECT2 fibrils and soluble oligomers can be determined by cryoEM. Aim 1 will test two new microfluidic chip designs that have been engineered to maximize flow shear and protein aggregation while recapitulating the branched and parallel structures of kidney vessels. Prototype chips have been fabricated, and these will be evaluated using LECT2 samples in conditions that test the hypothesized ‘second-hit’ condition for ALECT2 (loss of zinc, partial reduction of disulfide bonds). Fibrillization will be monitored by microscopy under flow conditions, and aggregated proteins will be recovered from the chips and subjected to negative stain EM analysis. Aim 2 will seek to obtain high-quality cryoEM images of WT and I40V LECT2 fibrils, as well as soluble oligomers. These species will initially be generated by conventional stirring, and later by the microfluidic devices in Aim 1 if possible. Efforts will be directed toward obtaining individual fibrils, as existing fibrillization protocols tend to produce large clumps, and on purifying soluble oligomers to a monodisperse population. Together, these aims will fill in critical gaps in the hypotheses that link flow shear and the I40V mutation to the development of ALECT2.