Abstract
BackgroundThe increasing number of patients suffering from urolithiasis represents one of the major challenges which nephrologists face worldwide today. For enhancing therapeutic outcomes of this disease, the pathogenic basis for the formation of renal stones is the need of hour. Proteins are found as major component in human renal stone matrix and are considered to have a potential role in crystal–membrane interaction, crystal growth and stone formation but their role in urolithiasis still remains obscure.MethodsProteins were isolated from the matrix of human CaOx containing kidney stones. Proteins having MW>3 kDa were subjected to anion exchange chromatography followed by molecular-sieve chromatography. The effect of these purified proteins was tested against CaOx nucleation and growth and on oxalate injured Madin–Darby Canine Kidney (MDCK) renal epithelial cells for their activity. Proteins were identified by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF MS) followed by database search with MASCOT server. In silico molecular interaction studies with CaOx crystals were also investigated.ResultsFive proteins were identified from the matrix of calcium oxalate kidney stones by MALDI-TOF MS followed by database search with MASCOT server with the competence to control the stone formation process. Out of which two proteins were promoters, two were inhibitors and one protein had a dual activity of both inhibition and promotion towards CaOx nucleation and growth. Further molecular modelling calculations revealed the mode of interaction of these proteins with CaOx at the molecular level.ConclusionsWe identified and characterized Ethanolamine-phosphate cytidylyltransferase, Ras GTPase-activating-like protein, UDP-glucose:glycoprotein glucosyltransferase 2, RIMS-binding protein 3A, Macrophage-capping protein as novel proteins from the matrix of human calcium oxalate stone which play a critical role in kidney stone formation. Thus, these proteins having potential to modulate calcium oxalate crystallization will throw light on understanding and controlling urolithiasis in humans.
Highlights
Human renal stones are composed of crystalline and noncrystalline phases; 80% of stones are composed of calcium oxalate (CaOx) and the supporting structure i.e. the organic matrix accounts for 2–5% of the total stone weight [1,2] and is distributed throughout the architecture of all stones [3]
We identified and characterized five novel proteins: Ethanolamine-phosphate cytidylyltransferase, Ras GTPase-activating-like protein, UDP-glucose:glycoprotein glucosyltransferase 2, RIMS-binding protein and Macrophage-capping protein 3A which play a significant role in stone formation
Of all types of renal stones, calcium oxalate (CaOx) is the most common composition found by chemical analysis and till date the pathogenic mechanisms of stone formation remain unclear
Summary
Human renal stones are composed of crystalline and noncrystalline phases; 80% of stones are composed of calcium oxalate (CaOx) and the supporting structure i.e. the organic matrix accounts for 2–5% of the total stone weight [1,2] and is distributed throughout the architecture of all stones [3]. Proteins constitute a major portion of the matrix and the organic matrix is considered to be important in stone formation and growth [4]. Proteins that can bind to oxalate would be mediators of such pathologic expression Identification of such proteins can throw light on stone pathogenesis. Present studies were conducted to isolate proteins from the human renal stone matrix and to assess their influence on different stages of CaOx formation. We present evidence for the presence of five novel proteins from human kidney stone matrix which play a critical role in influencing stone formation. Proteins are found as major component in human renal stone matrix and are considered to have a potential role in crystal–membrane interaction, crystal growth and stone formation but their role in urolithiasis still remains obscure
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