Coronin-1A serves as a serum biomarker for supportive diagnosis of Mycobacterium tuberculosis infection.

Introduction: The severity and spread of tuberculosis, a major burden, can be prevented by more rapid and accurate laboratory diagnosis. The purpose of this study is to systematically explore candidate serum proteins in patients with Mycobacterium tuberculosis infection for further application as novel biomarkers. Methods: Our study was performed in two major steps: screening of the literature for potential biomarkers, and then validation of their levels in patients and controls. Many serum/plasma proteins previously reported to be abnormally expressed in patients with tuberculosis between 2012 and 2021 were comprehensively assembled. The biological role in tuberculosis was also predicted for each using the bioinformatics tool STRING. Candidate proteins found to have the same expression in other related diseases were excluded. Subsequently, the serum level of the candidate serum/plasma protein that met the aforementioned criteria was validated by sandwich ELISA; diagnostic performance was analysed by the area under the curve (AUC) of the receiver operating characteristic (ROC). Results: From 103 collected serum/plasma proteins, coronin-1A was found to have abnormal expression only in patients with tuberculosis and was associated with tuberculosis. In addition, the validation of coronin-1A in the serum of patients with pulmonary tuberculosis revealed a higher level than in that of healthy individuals. Furthermore, the area under the ROC curve for diagnostic power of coronin-1A was 0.866, with high sensitivity and specificity at a cut-point of approximately 52.7 ng/mL. Conclusions: We concluded that the level of serum coronin-1A might serve as a novel biomarker for alternative laboratory examination to effectively distinguish patients with tuberculosis from those with other related diseases and healthy individuals.

Serum alpha-1-antitrypsin level in the severity prognosis of systemic lupus erythematosus patients: Systematic exploration of novel biomarker.

Background: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affecting multi-organs injury and failure. The rapid, precision, and specificity prognosis by laboratory investigations could prevent active stage and severity in the disease. Aims: To systematically explore and investigate the candidate serum protein for development into the novel biomarker for severity prognosis of SLE patients. Methods: The proteins previously reported in abnormal level in serum/plasma of SLE patients since 2014-2020 were comprehensive collected. Thereafter, these serum proteins that found in other diseases were excluded. The association with molecules related to SLE severity of these candidate proteins were then predicted using bioinformatics STIRNG tool. The level of altered protein, which had the strong interaction to the severity molecules in serumof SLE patients was validated by Western blot, analyzed the correlation with anti-nuclear antigen (ANA) and performance of diagnosis, respectively. Results: From 26 collected serum/plasma proteins, alpha-1-antitrypsin protein was found the abnormal level in only SLE patients and strongly associated with severity molecules including C-reactive protein (CRP), complement C3, and C4. Additionally, the validation of serum alpha-1-antitrypsin in SLE patients exhibited the higher level than healthy controls and also had the positive correlation with ANA titer (r = 0.710). Furthermore, the area under ROC curve for diagnostic power of alpha-1-antitrypsin was 0.970 with 100% sensitivity and 90% specificity at cut-off 0.131/total serum protein. Conclusions: The higher level of alpha-1-antitrypsin in serum samples of SLE patients indicated as the novel biomarker for reliable and specific prognosis of disease severity.

Systematic Evaluation of Urinary VCAM1 as Novel Biomarker for Prognosis of Lupus Nephritis.

BACKGROUND: Lupus nephritis was established as the major cause of morbidity and mortality in systemic lupus erythematosus (SLE) patients. This severity could be monitored by laboratory prognosis with accuracy and specificity for the disease. Recently, various SLE laboratory investigations still have had improper prognosis and also possibly causing tissue injury in the patients. The present study thus aimed to systematically explore a novel urinary protein for further development into a prognosis biomarker in patients. METHODS: Several urinary proteins that previously were reported with abnormal expression in SLE patients between 2014 - 2019 were comprehensively collected. These proteins were also analyzed for functional category and sites of expression using STIRNG, FUNRICH, and Uniport database. Thereafter, the level of altered protein candidate was then validated by western blotting, correlation analysis, and diagnostic performance. RESULTS: Vascular cell adhesion molecule-1 (VCAM1) protein was found in higher levels and also specific in SLE patients with nephritis. Moreover, VCAM1 has a role in immune response, inflammation, is expressed on plasma membrane of renal cells, and has the ability to secret into urine. The level of VCAM1 in urine of SLE patients was significantly higher than in healthy controls. The area under ROC curve (AUC) was also 0.891. Furthermore, the level of VCAM1 was dramatically associated with the severity of renal insufficiency (r = 0.738). CONCLUSIONS: VCAM1 may be a novel urinary biomarker for reliable prognosis of nephritis severity in SLE patients.

Dual modulatory effects of diosmin on calcium oxalate kidney stone formation processes: Crystallization, growth, aggregation, crystal-cell adhesion, internalization into renal tubular cells, and invasion through extracellular matrix.

Diosmin is a natural flavone glycoside (bioflavonoid) found in fruits and plants with several pharmacological activities. It has been widely used as a dietary supplement or therapeutic agent in various diseases/disorders. Although recommended, evidence of its protective mechanisms against kidney stone disease (nephrolithiasis/urolithiasis), especially calcium oxalate (CaOx) monohydrate (COM) that is the most common type, remained unclear. In this study, we thus systematically evaluated the effects of diosmin (at 2.5-160 nM) on various stages of kidney stone formation processes, including COM crystallization, crystal growth, aggregation, crystal-cell adhesion, internalization into renal tubular cells and invasion through extracellular matrix (ECM). The results showed that diosmin had dose-dependent modulatory effects on all the mentioned COM kidney stone processes. Diosmin significantly increased COM crystal number and mass during crystallization, but reduced crystal size and growth. While diosmin promoted crystal aggregation, it inhibited crystal-cell adhesion and internalization into renal tubular cells. Finally, diosmin promoted crystal invasion through the ECM. Our data provide evidence demonstrating both inhibiting and promoting effects of diosmin on COM kidney stone formation processes. Based on these dual modulatory activities of diosmin, its anti-urolithiasis role is doubtful and cautions should be made for its use in kidney stone disease.

Anti-Inflammatory Effect of Pineapple Rhizome Bromelain through Downregulation of the NF-κB- and MAPKs-Signaling Pathways in Lipopolysaccharide (LPS)-Stimulated RAW264.7 Cells.

Bromelain is a mixture of proteolytic enzymes derived from pineapple (Ananas comosus) fruit and stem possessing several beneficial properties, particularly anti-inflammatory activity. However, the molecular mechanisms underlying the anti-inflammatory effects of bromelain are unclear. This study investigated the anti-inflammatory effects and inhibitory molecular mechanisms of crude and purified rhizome bromelains on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells. RAW264.7 cells were pre-treated with various concentrations of crude bromelain (CB) or purified bromelain (PB), and then treated with LPS. The production levels of pro-inflammatory cytokines and mediators, including nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-α were determined by Griess and ELISA assays. The expressions of inducible nitric oxide synthetase (iNOS), cyclooxygenase (COX)-2, nuclear factor kappa B (NF-κB), and mitogen-activated protein kinases (MAPKs)-signaling pathway-related proteins were examined by western blot analysis. The pre-treatment of bromelain dose-dependently reduced LPS-induced pro-inflammatory cytokines and mediators, which correlated with downregulation of iNOS and COX-2 expressions. The inhibitory potency of PB was stronger than that of CB. PB also suppressed phosphorylated NF-κB (p65), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, extracellular signal-regulated kinases, c-Jun amino-terminal kinases, and p38 proteins in LPS-treated cells. PB then exhibited potent anti-inflammatory effects on LPS-induced inflammatory responses in RAW264.7 cells by inhibiting the NF-κB and MAPKs-signaling pathways.

Modulatory effects of fibronectin on calcium oxalate crystallization, growth, aggregation, adhesion on renal tubular cells, and invasion through extracellular matrix.

Fibronectin, an extracellular matrix (ECM) protein, has been thought to be involved in pathogenic mechanisms of kidney stone disease, especially calcium oxalate (CaOx) type. Nevertheless, its precise roles in modulation of CaOx crystal remained unclear. We thus performed a systematic evaluation of effects of fibronectin on CaOx monohydrate (COM) crystal (the major causative chemical crystal in kidney stone formation) in various stages of kidney stone pathogenesis, including crystallization, crystal growth, aggregation, adhesion onto renal tubular cells, and invasion through ECM in renal interstitium. The data showed that fibronectin significantly decreased crystallization, growth and adhesive capability of COM crystals in a dose-dependent manner. In contrast, COM crystal aggregation and invasion through ECM migration chamber were significantly enhanced by fibronectin in a dose-dependent fashion. Sequence analysis revealed three calcium-binding and six oxalate-binding domains in fibronectin. Immunofluorescence study confirmed binding of fibronectin to COM crystals. Additionally, calcium- and oxalate-affinity assays confirmed depletion of both calcium and oxalate ions after incubation with fibronectin. Moreover, calcium-saturated and oxalate-saturated forms of fibronectin markedly reduced the modulatory activities of fibronectin on COM crystallization, crystal growth, aggregation, and adhesion onto the cells. These data strongly indicate the dual functions of fibronectin, which serves as an inhibitor for COM crystallization, crystal growth and adhesion onto renal tubular cells, but on the other hand, acts as a promoter for COM crystal aggregation and invasion through ECM. Finally, its COM crystal modulatory activities are most likely mediated through binding with calcium and oxalate ions on the crystals and in their environment.

Cell cycle shift from G0/G1 to S and G2/M phases is responsible for increased adhesion of calcium oxalate crystals on repairing renal tubular cells at injured site.

Renal tubular cell injury can enhance calcium oxalate monohydrate (COM) crystal adhesion at the injured site and thus may increase the stone risk. Nevertheless, underlying mechanism of such enhancement remained unclear. In the present study, confluent MDCK renal tubular cell monolayers were scratched to allow cells to proliferate and repair the injured site. At 12-h post-scratch, the repairing cells had significant increases in crystal adhesion capacity and cell proliferation as compared to the control. Cell cycle analysis using flow cytometry demonstrated that the repairing cells underwent cell cycle shift from G0/G1 to S and G2/M phases. Cyclosporin A (CsA) and hydroxyurea (HU) at sub-toxic doses caused cell cycle shift mimicking that observed in the repairing cells. Crystal-cell adhesion assay confirmed the increased crystal adhesion capacity of the CsA-treated and HU-treated cells similar to that of the repairing cells. These findings provide evidence indicating that cell cycle shift from G0/G1 to S and G2/M phases is responsible, at least in part, for the increased adhesion of COM crystals on repairing renal tubular cells at the injured site.

Targeted functional investigations guided by integrative proteome network analysis revealed significant perturbations of renal tubular cell functions induced by high glucose.

Recently, several studies employed various proteomic approaches to define diabetes-induced changes in renal proteins. However, functional significance of those datasets in diabetic nephropathy remained unclear. We thus performed integrative proteome network analysis of such datasets followed by various targeted functional studies in distal renal tubular cells treated with high glucose (HG) (25 mM) compared to normal glucose (NG) (5.5 mM) and NG + mannitol (M) (5.5 + 19.5 mM). The data showed that at 96 h when cell proliferation/death, tight junction protein and ß-/F-actin expression and organization, and transepithelial resistance remained unchanged, only HG caused increased levels of HSP90, HSP70, and HSP60, and increased accumulation of intracellular protein aggregates. In addition, HG also induced overproduction of intracellular ROS, decreased catalase level, increased level of oxidatively modified proteins, increased intracellular ATP level, and defective transepithelial Ca2+ transport. However, both HG and M increased the levels of ubiquitinated proteins. Taken together, this study demonstrated significant perturbations of distal renal tubular cells induced by HG based on targeted functional studies guided by integrative proteome network analysis. These data may, at least in part, lead to better understanding of the pathogenic mechanisms of diabetic nephropathy.

Protective effect of epigallocatechin-3-gallate (EGCG) via Nrf2 pathway against oxalate-induced epithelial mesenchymal transition (EMT) of renal tubular cells.

This study evaluated effect of oxalate on epithelial mesenchymal transition (EMT) and potential anti-fibrotic property of epigallocatechin-3-gallate (EGCG). MDCK renal tubular cells were incubated with 0.5 mM sodium oxalate for 24-h with/without 1-h pretreatment with 25 µM EGCG. Microscopic examination, immunoblotting and immunofluorescence staining revealed that oxalate-treated cells gained mesenchymal phenotypes by fibroblast-like morphological change and increasing expression of vimentin and fibronectin, while levels of epithelial markers (E-cadherin, occludin, cytokeratin and ZO-1) were decreased. EGCG pretreatment could prevent all these changes and molecular mechanisms underlying the prevention by EGCG were most likely due to reduced production of intracellular ROS through activation of Nrf2 signaling and increased catalase anti-oxidant enzyme. Knockdown of Nrf2 by small interfering RNA (siRNA) abrogated all the effects of EGCG, confirming that the EGCG protection against oxalate-induced EMT was mediated via Nrf2. Taken together, our data indicate that oxalate turned on EMT of renal tubular cells that could be prevented by EGCG via Nrf2 pathway. These findings also shed light onto development of novel therapeutics or preventive strategies of renal fibrosis in the future.

Alpha-tubulin enhanced renal tubular cell proliferation and tissue repair but reduced cell death and cell-crystal adhesion.

Adhesion of calcium oxalate (CaOx) crystals on renal tubular epithelial cells is a critical event for kidney stone disease that triggers many cascades of cellular response. Our previous expression proteomics study identified several altered proteins in MDCK renal tubular cells induced by CaOx crystals. However, functional significance of those changes had not been investigated. The present study thus aimed to define functional roles of such proteome data. Global protein network analysis using STRING software revealed α-tubulin, which was decreased, as one of central nodes of protein-protein interactions. Overexpression of α-tubulin (pcDNA6.2-TUBA1A) was then performed and its efficacy was confirmed. pcDNA6.2-TUBA1A could maintain levels of α-tubulin and its direct interacting partner, vimentin, after crystal exposure. Also, pcDNA6.2-TUBA1A successfully reduced cell death to almost the basal level and increased cell proliferation after crystal exposure. Additionally, tissue repair capacity was improved in pcDNA6.2-TUBA1A cells. Moreover, cell-crystal adhesion was reduced by pcDNA6.2-TUBA1A. Finally, levels of potential crystal receptors (HSP90, HSP70, and α-enolase) on apical membrane were dramatically reduced to basal levels by pcDNA6.2-TUBA1A. These findings implicate that α-tubulin has protective roles in kidney stone disease by preventing cell death and cell-crystal adhesion, but on the other hand, enhancing cell proliferation and tissue repair function.

In vitro evidence of the promoting effect of testosterone in kidney stone disease: A proteomics approach and functional validation.

UNLABELLED: Incidence of kidney stone disease in males is 2- to 4-fold greater than in females. This study aimed to determine effects of testosterone on kidney stone disease using a proteomics approach. MDCK renal tubular cells were treated with or without 20nM testosterone for 7days. Cellular proteins were extracted, resolved by 2-DE, and stained with Deep Purple fluorescence dye (n=5 gels derived from 5 independent samples/group). Spot matching, quantitative intensity analysis, and statistics revealed significant changes in levels of nine protein spots after testosterone treatment. These proteins were then identified by nanoLC-ESI-Qq-TOF MS/MS. Global protein network analysis using STRING software revealed α-enolase as the central node of protein-protein interactions. The increased level of α-enolase was then confirmed by Western blotting analysis, whereas immunofluorescence study revealed the increased α-enolase on cell surface and intracellularly. Functional analysis confirmed the potential role of the increased α-enolase in enhanced calcium oxalate monohydrate (COM) crystal-cell adhesion induced by testosterone. Finally, neutralization of surface α-enolase using anti-α-enolase antibody successfully reduced the enhanced COM crystal-cell adhesion to the basal level. Our data provided in vitro evidence of promoting effect of testosterone on kidney stone disease via enhanced COM crystal-cell adhesion by the increased surface α-enolase. BIOLOGICAL SIGNIFICANCE: The incidence of kidney stone disease in male is 2- to 4-fold greater than in female. One of the possible factors of the male preference is the higher testosterone hormone level. However, precise molecular mechanisms that testosterone plays in kidney stone disease remained unclear. Our present study is the first exploratory investigation on such aspect using a proteomics approach. Our data also provide a novel mechanistic aspect of how testosterone can impact the risk of kidney stone formation (i.e. the discovery that testosterone increases alpha-enolase expression on the surface of renal tubular cells that is responsible, at least in part, for crystal-cell adhesion).