Browsing by Author "Phuangkham S."

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    Comprehensive identification of stone-promoting proteins in the urine of kidney stone formers
    (2025-05-01) Hadpech S.; Peerapen P.; Rattananinsruang P.; Detsangiamsak S.; Phuangkham S.; Chotikawanich E.; Sritippayawan S.; Thongboonkerd V.; Hadpech S.; Mahidol University
    Urinary proteins have crucial roles in modulating kidney stone formation. While stone-inhibiting urinary proteins are well characterized, stone-promoting urinary proteins are insufficiently explored. This knowledge gap limits our ability to fully comprehend the pathogenic mechanisms underlying nephrolithiasis and hampers the development of targeted therapeutic/preventive interventions. Therefore, we systematically identified stone-promoting proteins from the urine of 30 calcium oxalate (CaOx) nephrolithiatic patients (stone formers). Urinary proteins were fractionated by anion exchange and size-exclusion chromatography. A total of 15 protein fractions (SF1-SF15) were tested for their modulating activities on CaOx crystals by various assays compared with the control. The fractions with net CaOx-promoting activities were then identified by nanoLC-ESI-Qq-TOF MS/MS. From 15 fractions, 9 had net CaOx-promoting activities in all crystal assays. Among 3–99 proteins identified from these fractions, alpha-1acid glycoprotein 2, alpha-1-antitrypsin, apolipoprotein D, CD44 antigen, endosialin, fibrinogen alpha chain, interleukin-18-binding protein, kallikrein-1, retinol-binding protein 4, and titin have been found to increase in the urine of stone formers compared with controls, reinforcing their potential roles as CaOx stone promoters. This study offers the largest collection of CaOx stone-promoting proteins that will shed light on pathogenic mechanisms of nephrolithiasis and may allow further development of new drug targets to treat/prevent nephrolithiasis.
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    Neutrophil gelatinase-associated lipocalin (NGAL) promotes calcium oxalate crystallization, growth, aggregation, adhesion to renal cells and invasion through extracellular matrix via its oxalate-binding capability
    (2025-08-01) Yoodee S.; Peerapen P.; Hadpech S.; Suebsuk Y.; Detsangiamsak S.; Chantarasaka S.; Phuangkham S.; Noonin C.; Thongboonkerd V.; Yoodee S.; Mahidol University
    Neutrophil gelatinase-associated lipocalin (NGAL) is commonly found in the urine of patients with kidney diseases including nephrolithiasis. Nevertheless, its role in stone formation was unknown. Herein, we systematically examined the effects of NGAL on multiple calcium oxalate (CaOx) stone-development processes. Recombinant human NGAL was produced, purified and subjected to multiple crystal assays. The analyses demonstrated that all NGAL concentrations (0.01–10 μg/ml) concentration-dependently increased the size of CaOx crystals after initial crystallization. At later processes, crystal size expansion, aggregation and adhesion to renal cells were also concentration-dependently induced by NGAL. Additionally, NGAL at all concentrations enhanced extracellular matrix (ECM) invasion by crystals. Mechanistic examinations revealed that NGAL had an affinity to bind oxalate ions without affinity with calcium ions, and the binding of NGAL to CaOx crystal surfaces was confirmed by an immunofluorescence method. Finally, neutralization of the crystal-NGAL complex using a specific anti-NGAL antibody prior to crystal-cell adhesion assay revealed that NGAL functioned as an adhesive medium to enhance crystal-cell adhesion. In conclusion, our findings indicate that NGAL promotes CaOx stone development by enhancing CaOx crystallization, growth, aggregation, adhesion to renal cells and ECM invasion via its oxalate-binding capability.
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    Quercetin inhibits calcium oxalate crystallization and growth but promotes crystal aggregation and invasion
    (2024-01-01) Chaiyarit S.; Phuangkham S.; Thongboonkerd V.; Chaiyarit S.; Mahidol University
    Recent evidence has shown an association between kidney stone pathogenesis and oxidative stress. Many anti-oxidants have been studied with an aim for stone prevention. Quercetin, a natural flavonol, is one among those eminent anti-oxidants with satisfactory anti-inflammatory property to cope with renal tissue injury in kidney stone disease. Nevertheless, its direct effect (if any) on calcium oxalate (CaOx) crystals and the stone formation mechanism had not been previously explored. This study has addressed the ability of quercetin at various concentrations (2.5, 5, 10, 20, 40, 80 and 160 μM) to directly modulate CaOx crystallization, growth, aggregation, adhesion on kidney cells, and invasion through the matrix. The data have shown that quercetin significantly inhibits CaOx crystallization and crystal growth but promotes crystal aggregation in concentration-dependent manner. However, quercetin at all these concentrations do not affect CaOx adhesion on kidney cells. For the invasion, quercetin at all concentrations constantly promotes CaOx invasion through the matrix without concentration-dependent pattern. These discoveries have demonstrated for the first time that quercetin has direct but dual modulatory effects on CaOx crystals. While quercetin inhibits CaOx crystallization and growth, on the other hand, it promotes CaOx crystal aggregation and invasion through the matrix. These data highlight the role for quercetin in direct modulation of the CaOx crystals that may intervene the stone pathogenesis.
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    The modulatory effects of large and small extracellular vesicles from normal human urine on calcium oxalate crystallization, growth, aggregation, adhesion on renal cells, and invasion through extracellular matrix: An in vitro study
    (2024-04-01) Hadpech S.; Chaiyarit S.; Phuangkham S.; Sukphan S.; Thongboonkerd V.; Hadpech S.; Mahidol University
    Urinary extracellular vesicles (uEVs) play important roles in physiologic condition and various renal/urological disorders. However, their roles in kidney stone disease remain unclear. This study aimed to examine modulatory effects of large and small uEVs derived from normal human urine on calcium oxalate (CaOx) crystals (the main component in kidney stones). After isolation, large uEVs, small uEVs and total urinary proteins (TUPs) with equal (protein equivalent) concentration were added into various crystal assays to compare with the control (without uEVs or TUPs). TUPs strongly inhibited CaOx crystallization, growth, aggregation and crystal-cell adhesion. Large uEVs had lesser degree of inhibition against crystallization, growth and crystal-cell adhesion, and comparable degree of aggregation inhibition compared with TUPs. Small uEVs had comparable inhibitory effects as of TUPs for all these crystal assays. However, TUPs and large uEVs slightly promoted CaOx invasion through extracellular matrix, whereas small uEVs did not affect this. Matching of the proteins reported in six uEVs datasets with those in the kidney stone modulator (StoneMod) database revealed that uEVs contained 18 known CaOx stone modulators (mainly inhibitors). These findings suggest that uEVs derived from normal human urine serve as CaOx stone inhibitors to prevent healthy individuals from kidney stone formation.