Oxidative stress caused by oxalate is one of the key pathogenic factors causing renal tubular injury and kidney stone formation. Recent studies have reported that ellagic acid (a natural polyphenol abundantly present in various fruits, nuts and medicinal plants with known antioxidant properties) has potential renoprotective effects, but with unclear molecular mechanisms. To investigate the cellular impact of ellagic acid, we applied quantitative proteomics, bioinformatics (protein interaction network mapping, gene ontology enrichment, and K-means clustering), and functional assays (Western blotting, OxyBlot assay, MitoTracker staining, and protein aggregation assay) to identify key pathways and functional clusters mediated by ellagic acid. Proteomic profiling identified 8 downregulated and 13 upregulated proteins induced by ellagic acid. Bioinformatic analyses identified some important central regulatory nodes within interaction networks, including ATIC, HSPH1 and TPI1 among downregulated proteins, and hnRNPK, EIF6 and HSPA8 among upregulated ones. Functional annotation indicated that stress response and mitochondrial function were involved. Functional validation revealed that ellagic acid prevented oxalate-induced protein oxidation and preserved mitochondrial integrity and membrane potential, indicating its strong antioxidant effects. However, ellagic acid did not affect protein aggregation. In conclusion, ellagic acid exerted multifaceted cytoprotective effects in renal tubular cells by modulating the expression of several proteins and their interaction networks, mitigating oxidative protein damage, and preserving mitochondrial function. These findings provide mechanistic insights into the renoprotective actions of ellagic acid and support its promise as a therapeutic agent against renal injury caused by oxalate.

Epithelial-to-mesenchymal transition (EMT) is a prominent process involved in kidney fibrosis. Calcium oxalate monohydrate (COM), a crystalline composition predominantly found in renal stones, is one of the known stimuli that can trigger the EMT process. Chlorogenic acid (CGA), a phenolic hydroxycinnamate naturally found in several plants (especially coffee beans), has several health benefits, including antifibrotic properties. However, the mechanisms underlying its preventive effects against COM-induced EMT in renal epithelial cells remain unclear. Herein, Madin-Darby canine kidney (MDCK) renal epithelial cells were exposed to COM crystals without or with CGA pre-/cotreatment and were subjected to various assays, including morphological examination, measurement of cell spindle index, transepithelial resistance (TER) measurement, immunofluorescence staining of EMT markers, and western blotting of p38 signaling proteins. Additionally, label-free quantitative proteomics using nanoLC-ESI-LTQ-Orbitrap MS/MS and functional enrichment analyses were carried out, followed by functional validation of the essential role of heat shock protein 60 (HSP60) using a small interfering RNA (siRNA)-based knockdown method. As expected, COM induced the EMT process as shown by increases in spindle index and mesenchymal marker (vimentin) but decreases in TER and epithelial marker (ZO-1). Mechanistically, these changes were accompanied with activation of p38 signaling. However, CGA pre-/cotreatment successfully prevented all these COM-induced changes. Label-free quantitative proteomics revealed significantly altered levels of 167 cellular proteins caused by COM. Among these, 55 proteins were completely preserved by CGA. Protein–protein interaction network illustrated HSP60 as a hub of these interacting proteins. While COM increased the HSP60 level, CGA successfully preserved this protein at its basal level. Moreover, knockdown of HSP60 expression by small interfering RNA could also prevent COM-induced EMT and p38 activation. These data indicate that CGA mitigates COM-induced EMT via suppression of HSP60 expression and p38 signaling activated by COM crystals.

Recent research has highlighted the converging step of diabetic kidney disease (DKD) and chronic kidney disease (CKD) at cellular senescence and fibrotic processes. Therefore, therapeutic interventions targeting cellular senescence may ameliorate or slow the progression of DKD and CKD. This study evaluated the anti-senescence properties of the three most abundant phytochemicals in green tea, including epigallocatechin-3-gallate (EGCG), l-theanine and theophylline, in proximal tubular epithelial cells (PTECs) exposed to high-glucose. Additionally, their anti-fibrotic activities were examined in distal tubular epithelial cells (DTECs) and renal fibroblasts treated with the conditioned medium containing senescence-associated secretory phenotypes (SASPs) secreted from high-glucose-treated PTECs. The results showed that high-glucose triggered various senescence markers in PTECs, including cell enlargement, increased senescence-associated β-galactosidase activity, increased p21 level, S-phase cell-cycle arrest, suppressed cellular metabolic activity, and elevated SASPs levels. While phosphorylated (p)-p38, p-glycogen synthase kinase-3β (p-GSK-3β) and β-catenin remained unchanged, sirtuin-1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) were suppressed by high-glucose. Moreover, the conditioned medium containing SASPs secreted from high-glucose-treated PTECs induced epithelial-mesenchymal transition (EMT) (shown by decreased E-cadherin and zonula occludens-1 (ZO-1), and increased vimentin and fibronectin) in DTECs and renal fibroblast activation (shown by increased fibronectin, collagen 1α, vimentin and actin stress fiber). However, EGCG, l-theanine and theophylline effectively abolished all these high-glucose-induced effects on senescence and SIRT1/PGC-1α axis in PTECs, EMT in DTECs, and renal fibroblast activation. In summary, this study highlights the anti-senescence, anti-EMT and anti-fibrotic properties of these green tea bioactive compounds via SIRT1/PGC-1α axis to counteract the high-glucose-induced effects in DKD.

GDP-L-fucose synthase (or FX protein) has recently been identified in the urine from subjects with calcium oxalate (CaOx) kidney stones, but not in the normal urine. However, its role in kidney stone formation was unknown. We, therefore, produced and purified recombinant human FX protein and examined its stone-modulating effects using various assays compared with blank and negative controls. Crystal assays revealed that the FX protein promoted CaOx crystallization, crystal aggregation and crystal invasion through extracellular matrix (ECM). On the other hand, FX inhibited CaOx crystal growth and crystal-cell adhesion. Potential mechanisms underlying its crystal modulation were investigated. Ca2+- and Ox2--binding affinity assays demonstrated that FX effectively bound both Ca2+ and Ox2- ions. Immunofluorescence staining using a monoclonal antibody specific to the human FX protein revealed the direct binding of this protein to CaOx crystal surfaces. In conclusion, our findings indicate that the human FX protein is a novel dual CaOx stone modulator. While it promotes crystallization, aggregation and ECM invasion of CaOx crystals, it inhibits the crystal growth and adhesion to renal cells via the binding with free Ca2+ and Ox2- ions as well as the crystal surfaces.

A recent study has identified several urinary proteins from calcium oxalate (CaOx) stone formers (patients with CaOx kidney stones) that promote stone development. Nevertheless, the physicochemical properties responsible for their prolithogenic effects remained undefined. In this study, stone formers' urinary proteins were fractionated into 15 fractions (SF1-SF15) using chromatographic methods and analyzed through a series of CaOx crystal assays, followed by protein identification via tandem mass spectrometry. Physicochemical properties of all proteins identified in individual fractions, along with their crystal-promoting activities, were then analyzed and subjected to the Pearson correlation test. The majority of protein fractions exhibited crystal-promoting effects. Crystallization-promoting activity positively correlated with the proportion of oxalate-binding proteins, while aggregation-promoting activity positively correlated with the number of oxalate-binding sites/protein and isoelectric point (pI). Crystal adhesion-promoting activity negatively correlated with the number of oxalate-binding sites/protein, pI, proportion of basic proteins, and proportion of alpha-helix structure but positively correlated with the proportion of acidic proteins and proportion of random-coil structure. None of the physicochemical properties correlated with crystal growth-promoting activity. In conclusion, some physicochemical properties of urinary proteins from stone formers are associated with their CaOx stone-promoting activities and may serve as predictors for their prolithogenic potential.

Kidney stone disease (KSD), particularly calcium oxalate (CaOx) type, remains a global health problem. Many efforts have been made to prevent KSD, including the use of some dietary supplements. However, mechanisms underlying their anti-KSD properties have remained poorly understood, and their relative anti-KSD properties have previously been unknown. Herein, we systematically evaluated and compared the inhibitory effects of five well-known dietary supplements on CaOx crystals. Caffeine (CAF), epigallocatechin-3-gallate (EGCG), N-acetylcysteine (NAC), resveratrol (RES) and trigonelline (TRIG) (at 1, 10 and 100 μM, which are within their physiologic levels in the urine) were subjected to CaOx crystallization, growth and aggregation assays. Degrees of their CaOx crystal-inhibitory activities were then compared. CAF inhibited crystal formation, EGCG inhibited crystal formation and growth, NAC inhibited crystal aggregation, RES inhibited crystal growth, and TRIG inhibited crystal formation and growth. However, RES promoted crystal aggregation and thus served as a dual modulator (acting as an inhibitor and promoter at different steps of stone formation). Almost all of these inhibitory effects were concentration-dependent. Comparing the CaOx-inhibitory activities of these compounds revealed that EGCG was the most potent inhibitor against CaOx crystal formation (with the crystal abundance-inhibitory activity of 85.61 ± 5.12 %), whereas RES was the most potent inhibitor against CaOx crystal growth (with the crystal growth-inhibitory activity of 92.99 ± 1.67 %). NAC was the only inhibitor against CaOx crystal aggregation (with the crystal aggregation-inhibitory activity of 22.97 ± 0.75 %). These data indicate the direct inhibitory effects of various dietary supplements against CaOx crystal formation, growth and aggregation, supporting their roles in KSD prevention.

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 anion-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.

The development of calcium oxalate (CaOx) stones in the kidney comprises multiple processes involving sophisticated interactions between stone minerals and other molecules, especially urinary proteins. Prosaposin (PSAP) has recently been identified in the urine from healthy (non-stone) subjects, not in the urine from CaOx stone subjects. Consequently, PSAP may serve as a CaOx stone inhibitor, but without experimental evidence. This study thus aimed to systematically investigate the stone-modulatory effects of PSAP on four critical CaOx stone-forming processes. Recombinant human PSAP protein was successfully produced and purified in a soluble form and then subjected to all crystal assays at final concentrations of 0.001, 0.01, 0.1, 1 and 10 μg/ml. The results showed that PSAP concentration-dependently inhibited CaOx crystallization, crystal growth and crystal aggregation. Also, PSAP at all concentrations reduced crystal adhesion on the surfaces of tubular cells to the same degree. To address mechanisms underlying its crystal-inhibitory effects, we further investigated the binding abilities of PSAP with Ca2+ and Ox2- ions, and CaOx crystal surfaces. Ca2+- and Ox2--consumption assays revealed that PSAP concentration-dependently bound with both Ca2+ and Ox2- ions. Immunofluorescence study revealed that PSAP effectively bound to the crystal surfaces. These findings indicate that PSAP serves as a novel inhibitor of CaOx stone formation at crystallization, crystal growth, aggregation and crystal-cell interactions phases via its binding abilities with Ca2+ and Ox2- ions and CaOx crystal surfaces.

It is generally known that kidney stone disease (KSD) is associated with alterations in urinary microbiome, but the roles of the urinary microbiome in KSD pathogenesis remain unclear. This study addressed the impact of Lactobacillus acidophilus (a commensal bacterium found in normal urine) on renal epithelial integrity, calcium oxalate monohydrate (COM) crystal-cell adhesion, expression of membrane receptors of COM crystals, and oxalate degradation under oxalate-induced stress (a known inducer of KSD). Inner medullary collecting duct cells (mIMCD-3) were cultured for 24 h under control or oxalate-induced (by 0.6 mM sodium oxalate; NaOx) conditions without or with L. acidophilus (at 1 × 103 colony-forming unit (CFU)/ml) co-incubation. NaOx reduced transepithelial resistance (TER) of the mIMCD-3 monolayer and downregulated ZO-1, a tight junction (TJ) protein. Additionally, NaOx enhanced the COM crystal-binding capability of mIMCD-3 cells by upregulating a COM crystal receptor, annexin A2, on cell membranes. Such harmful effects of NaOx were abolished when mIMCD-3 cells were co-cultured with L. acidophilus. Moreover, culturing L. acidophilus in artificial urine (AU) supplemented with NaOx for 24 h revealed that the oxalate level in AU decreased, suggesting the oxalate-degrading activity of the bacterium in an AU environment. L. acidophilus prevented oxalate-mediated renal epithelial barrier disruption and COM crystal adhesion to renal epithelial cells by preserving ZO-1 and annexin A2 expression at their basal levels, at least in part, via its oxalate-degrading property. Not applicable (This is not a clinical trial).

Kidney stone disease (KSD) is a worldwide social and economic burden. Potassium citrate (KCitrate) has been used for preventing KSD recurrence, but many patients develop gastrointestinal symptoms and subsequently discontinue therapy. Alkyl citrate esters have been thought to be safer but with no clear evidence of their efficacy and renal toxicity. This study compared the dissolution efficacies of various alkyl citrate esters (trimethyl citrate or TMC, triethyl citrate or TEC, and isopropyl citrate or IPC) and inorganic citrate salts (NaCitrate, KCitrate, and MgCitrate) on calcium oxalate monohydrate (COM) crystals and their cytotoxic effects on renal cells. At 50 mM (utilizing KCl and EDTA as negative and positive controls, respectively), IPC was the most effective alkyl citrate ester, whereas NaCitrate and KCitrate were the most effective inorganic citrate salts to dissolve COM crystals. These potent compounds were further investigated, whereas others with much fewer dissolution effects were not pursued. Cytotoxic analysis of the three most potent citrate forms revealed that NaCitrate and KCitrate at ≤12.5 mM did not affect renal cell viability, whereas IPC showed obvious cytotoxic effects at all concentrations tested. A time-course study utilizing a nontoxic concentration (12.5 mM) revealed that both NaCitrate and KCitrate reduced COM crystal size to less than a quarter at the end of the assay. Their dissolution capability was confirmed by measuring [Ca2+] of the remaining crystalline materials. From these results, NaCitrate and KCitrate are the most favorable for direct dissolution of COM crystals and renal cell tolerance. While alkyl citrate esters may still have a therapeutic role in KSD, their poor direct efficacies and potential renal toxicity must be considered.

Urinary proteins from stone formers/patients have been hypothesized to promote the formation of kidney stones, but with unclear information. In this study, urinary proteins derived from patients with calcium oxalate (CaOx) kidney stones were fractionated based on their isoelectric points using fast protein liquid chromatography with a GigaCap Q-650M column. Individual protein fractions were then purified and examined by multiple CaOx crystal assays, which simulate the stone-forming events, and identified by tandem mass spectrometry. Their physicochemical properties were then analyzed to find their correlations with the crystal-promoting activities. From all 9 fractions (SFQ1 to SFQ9), almost all of them promoted CaOx crystallization, growth, aggregation, and crystal-cell adhesion, and 10, 12, 71, 71, 60, 55, 25, 38, and 6 were identified from SFQ1 to SFQ9, respectively. Among several abundance-weighted physicochemical parameters, molecular weight, instability index, amino acid composition, and secondary structure positively correlated with abundance-weighted crystal aggregation-promoting activity and crystal adhesion-promoting activity. The findings elucidated the roles of urinary proteins from stone formers in kidney stone promotion, contributing to a better understanding of disease mechanisms.

A COVID-19 booster dose has been found to be effective in our fight against SARS-CoV-2 infection. However, their long-term beneficial or adverse effects among healthy individuals are not fully understood. We investigated the impact of the Pfizer-BioNTech-(BNT162b2) booster dose on plasma proteome profiles of fully vaccinated healthy individuals in a mimic of reinfection to understand the disease mechanisms and to identify novel diagnostic and prognostic biomarkers. In contrast to prebooster, postbooster recipients exhibited a distinct proteomic signature following SARS-CoV-2 spike (S) protein stimulation. The gene ontology (GO) terms of biological processes revealed the five most significant functions enriched in stress and immune responses, especially via complement and blood coagulation systems. Likewise, the Reactome pathway demonstrated significant activation of complement cascade, platelet degranulation, and innate immune systems. Moreover, the protein-protein interaction network exhibited regulation of body fluid levels and acute inflammatory response. In summary, our study identified abundant dysregulated signatures predominantly associated with the complement, the innate immune system, and platelet degranulation. Besides eliciting humoral immunity, our study also found key proteins involved in blood coagulation pathways that could perhaps shed light on individuals exhibiting comorbidities associated with COVID-19 vaccination. Therefore, factors dysregulated following SARS-CoV-2 spike (S) protein stimulation may provide insights into pathways potentially implicated in post-vaccination reactions.

Neutrophil secretory proteins are frequently found in calcium oxalate (CaOx) kidney stone matrix, suggesting their involvement in stone pathogenesis, but with unclear mechanisms. We therefore investigated the effects of secretome (a set of secretory proteins) from CaOx monohydrate (COM)-exposed versus control dHL-60 (neutrophil-like) cells on crystal nucleation (crystallisation), growth, aggregation and invasion. Quantitative proteomics was also performed to identify significantly altered secretory proteins, followed by analyses of their physicochemical properties and biological relevance. The data demonstrated that both COM-treated and control secretomes inhibited crystallisation and crystal growth, but the inhibitory effects from the COM-treated secretome were slightly weaker. By contrast, both of them promoted crystal aggregation, with the more potent effect from the COM-treated secretome. However, neither of them had a modulatory effect on crystal invasion. Quantitative proteomics revealed 20 decreased and 9 increased proteins in the COM-treated secretome compared with the control. Analyses of physicochemical properties showed that the increased secretory proteins tended to have a lower instability index and a smaller number of oxalate-binding motifs/protein. Main molecular functions of the increased group were catalytic, hydrolase and transporter activities, whereas those of the decreased group included RNA binding, molecular adaptor activity and catalytic activity. These data indicate that the neutrophil secretome inhibits crystallisation and crystal growth but promotes crystal aggregation. The COM-treated secretome exerts weaker inhibitory effects on crystallisation and growth but has a stronger promoting effect on crystal aggregation. These findings enhance our understanding of the roles of neutrophils in kidney stone pathogenesis.

Kidney stone formation is a sophisticated pathogenic process regulated by several stone-modulatory proteins, namely inhibitors and promoters. However, the characteristics of these proteins that determine their modulatory activities have remained largely unknown. Herein, we investigated potential characteristics of inhibitors and promoters involved in the stone-forming process by comparing amino acid contents, various physico-chemical properties, and calcium- and oxalate-binding properties of all proteins identified from normal urine (NU), stone former urine (SFU), and stone matrix (SM). Simple comparisons among the three groups showed that SM proteins apparently differed from NU and SFU proteins for almost all parameters, whereas no significant differences were detected between NU and SFU proteins. We then compared the proteins that were uniquely found in NU&SM with those uniquely found in SFU&SM, as it was hypothesized that they preferentially acted as stone inhibitors and promoters, respectively. The analyses revealed that proteins rich in amino acids with negative charges, normalized van der Waals volume of 2.95-4.0 and exposed solvent accessibility, acidic proteins, high molecular weight (MW) proteins, stable proteins, and hydrophilic proteins tended to be the stone inhibitors. On the other hand, proteins rich in aromatic and polar amino acids, basic proteins, low-MW proteins, unstable proteins, and hydrophobic proteins tended to be the stone promoters. These findings may help predict the stone-modulatory activities of proteins in urine and SM, and may inform the molecular design of kidney stone therapies.

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.

Introduction Various urinary parameters are used for determining kidney stone risk. However, almost all of the widely used lithogenic indices rely on urinary concentrations of small molecules/ions and pH. Objective To address whether urinary macromolecules (especially oxidatively modified proteins) also play a critical role in determining the stone risk. Methods Complexed urinary proteins (proteome) were purified from healthy individuals and calcium oxalate (CaOx) stone formers and performed various crystal assays and quantitative proteomics to compare them. Bioinformatic analyses were performed to gain additional insights, and the obtained data were verified by ELISA. Results While the normal urinary proteome inhibited CaOx stone-forming mechanisms (i.e., crystallization, growth and aggregation), the stone formers’ urinary proteome promoted all these CaOx crystal parameters. Descriptive proteomics by nanoLC-ESI-LTQ-Orbitrap-MS/MS analysis identified 203 and 381 proteins in the urine of healthy individuals and stone formers, respectively. Analyses of physicochemical properties revealed only molecular mass and isoelectric point that slightly increased in the stone formers’ urine, whereas instability index, grand average of hydrophathicity (GRAVY) and amino acid composition were comparable. Interestingly, proportion of oxidatively modified proteins (particularly those with methionine oxidation, methionine dioxidation and cysteine trioxidation) markedly increased (∼2.5-fold) in the stone formers’ urine. Quantitative proteomics revealed 89 increased and 56 decreased proteins in the stone formers’ urine. The oxidized proteins had a greater proportion (>3-fold) in the increased proteins (77 %) compared with the decreased ones (23 %), whereas the non-oxidized proteins showed comparable proportions (54 % and 46 %, respectively). Functional enrichment analyses revealed a correlation between the increased proteins and oxidative stress biological processes and molecular functions. Finally, ELISA confirmed the significantly increased levels of oxidized proteins in the stone formers’ urine compared with that of healthy individuals. Conclusion These data implicate that oxidatively modified proteome serves as a key pathogenic factor or risk for CaOx kidney stone formation.