Presentation Authors: Victor Hugo Canela*, Sharon Bledsoe, Daria Barwinska, James E. Lingeman, Tarek M. Ashkar, James C. Williams, Jr., Indianapolis, IN
Introduction: Urolithiasis affects approximately 12% of the world population, and more than half of affected individuals will have recurrence within 10 years of the first episode. A detailed hypothesis of the mechanisms of stone disease etiology remains elusive and thus makes stone disease difficult to treat and to prevent. The present study assessed the feasibility of histologically sectioning human stones and using laser microdissection (LMD) to obtain samples for proteomic analysis.
Methods: Stones were imaged by micro-Computed Tomography, demineralized, embedded in paraffin, histologically sectioned and stained. Regions of the stone-matrix layers were dissected via LMD (Leica LMD 6) and collected in phosphate buffer. Extracted proteins were digested using Trypsin/Lysine C mix proteases and run in a Q Exactive Orbitrap High Field Liquid Chromatography (LC)/Mass Spectrometry (MS) system. Data were searched using MaxQuant (version 126.96.36.199) and filtered using False Discovery Rate for alpha=0.01.
Results: Histological sections of stones showed tightly packed layers of matrix that were clearly visible using bright field LMD microscopy (Figure 1A). Average area dissected by LMD for LC/MS was 1.64x106 Âµm2, and these revealed and yielded an average of 777 distinct proteins each. Initial analysis shows proteins to be similar to those previously reported for kidney stones, including proteins involved in cell injury and repair as well as important mediators of the immune system.
Conclusions: Human kidney stones can be histologically sectioned and laser microdissected. A relatively small region of sectioned stone yields significant proteomic data. Utilization and optimizing of these novel methods and techniques in the future will pave the way into a deeper understanding of stone genesis. Future investigation of the stone matrix proteome should provide insight into the mechanisms underlying the pathogenesis of urolithiasis in an effort to develop effective therapies and prevention treatments.
Source of Funding: P01 DK056788