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Selective protein enrichment in calcium oxalate stone matrix: a window to pathogenesis?

ABSTRACT: 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.

SUBMITTER: Wesson JA

PROVIDER: S-EPMC8496971 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Introduction: About 1 in 11 Americans will experience a kidney stone, but underlying causes remain obscure. The objective of the present study was to separate idiopathic calcium oxalate stone formers by whether or not they showed positive evidence of forming a stone on Randall's plaque (RP). Materials and Methods: In patients undergoing either percutaneous or ureteroscopic procedures for kidney stone removal, all stone material was extracted and analyzed using micro-CT imaging to identify those attached to RP. Twenty-four-hour urine samples were collected weeks after the stone removal procedure and patients were off of medications that would affect urine composition. The endoscopic video was analyzed for papillary pathology (RP, pitting, plugging, dilated ducts, and loss of papillary shape) by an observer blinded to the data on stone type. The percent papillary area occupied by RP and ductal plugging was quantified using image analysis software. Results: Patients having even one stone on RP (N = 36) did not differ from non-RP patients (N = 37) in age, sex, BMI, or other clinical characteristics. Compared with the non-RP group, RP stone formers had more numerous, but smaller, stones, more abundant papillary RP formation, and fewer ductal plugs, both by quantitative measurement of surface area (on average, three times more plaque area, but only 41% as much plug area as in non-RP patients) and by semiquantitative visual grading. Serum and blood values did not differ between RP and non-RP stone formers by any measure. Conclusions: Growth of many small stones on plaque seems the pathogenetic scheme for the RP stone-forming phenotype, whereas the non-RP phenotype stone pathogenesis pathway is less obvious. Higher papillary plugging in non-RP patients suggests that plugs play a role in stone formation and that these patients have a greater degree of papillary damage. Underlying mechanisms that create these distinctive phenotypes are presently unknown.

Project description:BACKGROUND: The 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. METHODS: Proteins 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. RESULTS: Five 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. CONCLUSIONS: We 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.

Project description:BackgroundTo explore long-non-coding RNA (lncRNA), microRNA (miRNA) and messenger RNA (mRNA) expression profiles and their biological functions in the urine samples in calcium oxalate (CaOx) patients.MethodsFive CaOx kidney stone patients were recruited in CaOx stone group and six healthy people were included as control group, whose midstream morning urine was collected before the patients were given any medicine on admission. After total RNA was extracted from urine, microarray of miRNA, mRNA and lncRNA were applied to explore their expression variation. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to reveal the gene functions of the dysregulated lncRNA-associated competing endogenous RNA (ceRNA) network. Quantitative real-time PCR were performed on HK-2 cells treated with sodium oxalate (NaOx) to further screen out the differentially expression profiles of these RNAs.ResultsA total of nine miRNAs, 883 mRNAs and 1002 lncRNAs were differentially expressed in urine of CaOx patients compared with normal population. GO analysis revealed that most of mRNAs from ceRNA network were enriched in terms of respiratory burst, regulation of mitophagy, and protein kinase regulator activity. KEGG pathway analysis of these genes related to ceRNA network highlight their critical role in pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, and Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling pathway. Five miRNAs (miR-6796-3p, miR-30d-5p, miR-3192-3p, miR-518b and miR-6776-3p), four mRNAs (NT5E, CDH4, CLEC14A, CCNL1) and six lncRNAs (lnc-TIGD1L2-3, lnc-KIN-1, lnc-FAM72B-4, lnc-EVI5L-1, lnc-SERPINI1-2, lnc-MB-6) from the HK-2 cells induced by NaOx were consistent with the expression changes of microarray results.ConclusionThe differential expressed miRNAs, mRNAs and lncRNAs may be associated with numerous variations of the signaling pathways or regulation of metabolism and kinase activity, providing potential biomarkers for early diagnosis of urolithiasis and new basis for further research of urolithiasis mechanism.

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Selective protein enrichment in calcium oxalate stone matrix: a window to pathogenesis?

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