Selective protein enrichment in calcium oxalate stone matrix: a window to pathogenesis?
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Urine proteins are thought to control calcium oxalate stone formation, but over 1000 proteins have been reported in stone matrix obscuring their relative importance. Proteins critical to stone formation should be present at increased relative abundance in stone matrix compared to urine, so quantitative protein distribution data were obtained for stone matrix compared to prior urine proteome data. Matrix proteins were isolated from eight stones (> 90% calcium oxalate content) by crystal dissolution and further purified by ultradiafiltration (> 10 kDa membrane). Proteomic analyses were performed using label-free spectral counting tandem mass spectrometry, followed by stringent filtering. The average matrix proteome was compared to the average urine proteome observed in random urine samples from 25 calcium oxalate stone formers reported previously. Five proteins were prominently enriched in matrix, accounting for a mass fraction of > 30% of matrix protein, but only 3% of urine protein. Many highly abundant urinary proteins, like albumin and uromodulin, were present in matrix at reduced relative abundance compared to urine, likely indicating non-selective inclusion in matrix. Furthermore, grouping proteins by isoelectric point demonstrated that the stone matrix proteome was highly enriched in both strongly anionic (i.e., osteopontin) and strongly cationic (i.e., histone) proteins, most of which are normally found in intracellular or nuclear compartments. The fact that highly anionic and highly cationic proteins aggregate at low concentrations and these aggregates can induce crystal aggregation suggests that protein aggregation may facilitate calcium oxalate stone formation, while cell injury processes are implicated by the presence of many intracellular proteins.
KeywordsNephrolithiasis Urine proteome Calcium oxalate Kidney calculi
We gratefully acknowledge the primary financial support provided in part with resources and the use of facilities at the Clement J. Zablocki Department of Veterans Affairs Medical Center, Milwaukee, WI, and in part by the National Institutes of Health/National Institute for Diabetes, Digestive, and Kidney Diseases (DK 82550) (JAW). Additional financial support was provided by the Froedtert Foundation-Storey Fund and the Medical College of Wisconsin. We also gratefully acknowledge the technical support of MIS.MAC (Mandel International Stone and Molecular Analysis Center), Milwaukee, WI, for stone analysis, as well as and the technical support of Brian Halligan, PhD, for proteomic data analysis and Sergey Tarima, PhD, for statistical analysis. We also gratefully acknowledge additional technical support from Andrew Vallejos from the Clinical and Translational Studies Institute at the Medical College of Wisconsin in performing the WebGestalt searches that were added in response to initial review.
This study was primarily funded with resources and the use of facilities at the Clement J. Zablocki Department of Veterans Affairs Medical Center, Milwaukee, WI, and in part by a grant from the National Institutes of Health (NIDDK, DK 82550—JAW). Additional financial support was provided by the Froedtert Foundation-Storey Fund and the Medical College of Wisconsin.
Compliance with ethical standards
Conflict of interest
There were no conflicts of interest for any of the authors of this work.
JAW has a consulting agreement with Merck Pharmaceuticals, unrelated to this work.
Four CaOx kidney stones in this study were obtained from de-identified, pathological waste specimens previously characterized at the Mandel International Stone and Molecular Analysis Center (MIS.MAC, Milwaukee, WI, USA) or the National VA Crystal Identification Center (Milwaukee, WI, USA) and were used without obtaining IRB approval. The use of these samples for publication was reviewed with the VA IRB. While the VA IRB could not grant retrospective approval for studying these samples, they did agree that the data could be published with appropriate acknowledgment of their origin and lack of IRB approval. An additional four stones were obtained from newly recruited patients following their presentation for stone removal surgery at Froedtert Hospital under IRB approval (protocol number PRO21952), and these samples were identified as S1 through S4. All procedures performed in these studies were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments.
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