Project description:Formation of salivary stones, a rare pathological condition, currently lacks non-surgical treatment options. The underlying causes remain unknown, although one hypothesis suggests bacterial infections may play a role. To investigate these theories and identify proteins whose altered levels might impact salivary gland homeostasis, leading to stone formation, optimal sample processing protocols were developed. The objective was to maximise protein extraction efficiency, evaluating sonication enhancement for this purpose. To establish a universal protocol applicable to various biological materials, different sample types—saliva, salivary stones, kidney stones, salivary glands, and Raoultella ornithinolytica—were processed and analysed. Since sonication increased the number of detected proteins, the subsequent step involved assessing the feasibility of quantitative analysis. Proteins identified in saliva and salivary stones were compared, revealing seventy-four overlapping proteins primarily associated with bacterial infections and extracellular regions, aligning with previous findings from salivary stone analysis alone. It suggests the potential for selecting high-confidence biomarkers responsible for stone formation, particularly by analysing saliva from healthy donors as a control group. Furthermore, 56 proteins were detected in both salivary stones and kidney stones, suggesting the potential to identify proteins crucial for stone formation across various diseases or pathological states.
Project description:Knowledge of natural stone promoters in the urine was previously limited, but is essential to develop new targets for better management of calcium oxalate (CaOx) kidney stones. This work, therefore, unveils such information in the urine of CaOx stone formers (patients with stones). Urinary proteins were fractionated by DEAE/GigaQ exchange chromatography, and individual fractions were subjected to multiple crystal assays. The fractions with the summed crystal-promoting score ≥3 were subjected to proteomic analysis using nanoLC-ESI-Qq-TOF tandem mass spectrometry (MS/MS). Almost all of the chromatographic fractions (SFQ1-SFQ9) showed promoting effects on CaOx crystallization, growth, aggregation and crystal-cell adhesion. Among them, SFQ2, SFQ8 and SFQ9 provided the greatest summed crystal-promoting score, implicating their roles in stone promotion. MS/MS successfully identified 12, 38 and 6 proteins in fractions SFQ2, SFQ8 and SFQ9, respectively. Among all proteins identified, CD44 antigen, galectin-3-binding protein, kallikrein-1, and protein AMBP were found in more than one fraction, suggesting that they might serve as candidates for the stone promoters. These findings narrow the gap to better understand the pathogenesis and offer opportunities to define new therapeutic targets for better management of CaOx kidney stones.
Project description:To investigate the transcriptome differences between wild-type and Vhl mutant mice in kidney stones process, we established CaOx-induced nephrolithiasis mouse model and performed RNA-sequencing.
Project description:Cystinuria is a rare renal genetic disease caused by mutations in cystine transporter genes and characterized by defective cystine reabsorption leading to kidney stones. In 14% of cases patients undergo nephrectomy, but given the difficulty to predict the evolution of the disease, the identification of markers of kidney damage would improve the follow up of patients with a higher risk. The aim of the present study is to develop a robust, reproducible and non-invasive methodology for proteomic analysis of urinary exosomes using high resolution mass spectrometry. A clinical pilot study, conducted on 8 cystinuria patients vs. 10 controls, highlighted 165 proteins, of which 38 were up-regulated, that separate cystinuria patients from controls, and further discriminate between severe and moderate forms of the disease. These proteins include markers of kidney injury, circulating proteins and a neutrophil signature. Analysis of selected proteins by immunobloting, performed on six additional cystinuria patients, validated the mass spectrometry data. To our knowledge, this is the first successful proteomic study in cystinuria unmasking potential role of inflammation in this disease. The workflow we have developed is applicable to investigate urinanry exosomes in different renal diseases and to search for diagnostic/prognostic markers.
Project description:Abnormal N-glycosylation of uromodulin (Umod, also known as Tamm-Horsfall protein) has been implicated in the pathogenesis of various urinary system diseases. However, the site-specific N-glycosylation patterns of urinary Umod in healthy controls (HCs) versus those with kidney stones (KSs) have yet to be detailed. In this study, we isolated and purified Umod from the urine of 24 patients with calcium oxalate (CaOx) KSs and 24 HCs using a refined diatomaceous earth adsorption method. After digestion with trypsin and Glu-C, intact N-glycopeptides (IGPs) were enriched via hydrophilic interaction liquid chromatography (HILIC) and analyzed via EThcD-sceHCD-MS/MS. Data processing was conducted using Byonic and PANDA software. A total of 700 IGPs, 8 N-glycosites, and 145 N-glycans were identified, marking the most extensive identification of N-glycosylation in Umod to date. Through quantitative and site-specific N-glycosylation analyses, the study found 39 IGPs to be up-regulated (p<0.05, FC>1.5) and 60 down-regulated IGPs (p<0.05, FC<0.67) in KS groups. Additionally, the analysis of N-glycan composition and site-specific N-glycosylation highlighted KS-specific N-glycosylation modifications. Overall, these results suggest that variations in Umod N-glycosylation may critically contribute to the formation of CaOx kidney stones by modulating its function.