Understanding the Pathogenesis of Human Kidney Stone Disease by Spatially Mapping its Proteome

Abstract

BackgroundUrolithiasis affects approximately one‐in‐eleven people in the United States, and more than half of affected individuals will have recurrence within ten years of the first episode. A detailed hypothesis of the mechanisms of stone disease etiology remains elusive, and thus difficult to treat and prevent. The present study aims to advance the understanding of the pathogenesis of stone disease by determining the pattern of protein organization within the matrix of human kidney stones.MethodsThe methodological approach of this work relies on an innovative technique to perform histological sectioning of calcium oxalate (CaOx) stones following demineralization. Multi‐photon imaging and label‐free proteomics were used on fluorescently‐identified, laser micro‐dissected specific regions to assess proteome identity and signaling across spatial coordinates within the stone‐matrix.ResultsThe average area of laser micro‐dissected samples for proteomic analysis was 1.64×106μm2, and these samples yielded an average of 629 distinct proteins. Dissection of broad regions of CaOx stone by LMD yielded similar proteins as found in larger specimens of pulverized CaOx stone powder. Proteins identified in LMD and pulverized samples included those involved in cell injury and repair as well as important mediators of the immune system. Fatty acid synthase, uromodulin, myosin‐9, osteopontin‐D, and complement C3 are examples of proteins abundant in the stone‐matrix that have been linked to inflammation and regulation of the immune system. More recent results show brilliant autofluorescence of decalcified CaOx stones, which will allow LMD of distinctive regions of the stone without staining.ConclusionThis is the first demonstration of laser microdissection applied to kidney stones. Proteomic analysis in the different regions along with the interface region will provide invaluable information in understanding the growth of CaOx. These studies will further insight into the underlying signaling events that could become therapeutic targets to prevent stone growth.Support or Funding InformationSource of Funding: P01 DK056788LMD of human kidney stones.Demineralized stone‐matrix on LMD slides were stained with methylene blue (before). Laser microdissection of layers in the stone matrix were successfully collected in phosphate buffer for protein extraction and proteomic analysis (after). Dissection of broad regions of CaOx stone by LMD yielded similar proteins as found in larger specimens of pulverized CaOx stone powder.Figure 1