Endothelial Damage Arising From High Salt Hypertension Is Elucidated by Vascular Bed Systematic Profiling.

BACKGROUND: Considerable evidence links dietary salt intake with the development of hypertension, left ventricular hypertrophy, and increased risk of stroke and coronary heart disease. Despite extensive epidemiological and basic science interrogation of the relationship between high salt (HS) intake and blood pressure, it remains unclear how HS impacts endothelial cell (EC) and vascular structure in vivo. This study aims to elucidate HS-induced vascular pathology using a differential systemic decellularization in vivo approach. METHODS: We performed systematic molecular characterization of the endothelial glycocalyx and EC proteomes in mice with HS (8%) diet-induced hypertension versus healthy control animals. Isolation of eGC and EC compartments was achieved using differential systemic decellularization in vivo methodology. Altered protein expression in hypertensive compared to normal mice was characterized by liquid chromatography tandem mass spectrometry. Proteomic results were validated using functional assays, microscopic imaging, and histopathologic evaluation. RESULTS: Proteomic analysis revealed a significant downregulation of eGC and associated proteins in HS diet-induced hypertensive mice (among 1696 proteins identified in this group, 723 were markedly decreased in abundance, while only 168 were increased in abundance. Bioinformatic analysis indicated substantial derangement of the eGC layer, which was subsequently confirmed by fluorescent and electron microscopy assessment of vessel damage ex vivo. In the EC fraction, HS-induced hypertension significantly altered protein mediators of contractility, metabolism, mechanotransduction, renal function, and the coagulation cascade. In particular, we observed dysregulation of integrin subunits α2, α2b, and α5, which was associated with arterial wall inflammation and substantial infiltration of CD68+ monocyte-macrophages. Consequently, HS-induced hypertensive mice also displayed reduced vascular integrity of multiple organs including lungs, kidneys, and heart. CONCLUSIONS: These findings provide novel molecular insight into HS-induced structural changes in eGC and EC composition that may increase cardiovascular risk and potentially guide the development of new diagnostics and therapeutic interventions.

Characterizations of PMCA2-interacting complex and its role as a calcium oxalate crystal-binding protein.

Three isoforms of plasma membrane Ca2+-ATPase (PMCA) are expressed in the kidney. While PMCA1 and PMCA4 play major role in regulating Ca2+ reabsorption, the role for PMCA2 remains vaguely defined. To define PMCA2 function, PMCA2-interacting complex was characterized by immunoprecipitation followed by nanoLC-ESI-Qq-TripleTOF MS/MS (IP-MS). After subtracting non-specific binders using isotype-controlled IP-MS, 474 proteins were identified as PMCA2-interacting partners. Among these, eight were known and 20 were potential PMCA2-interacting partners based on bioinformatic prediction, whereas other 446 were novel and had not been previously reported/predicted. Quantitative immuno-co-localization assay confirmed the association of PMCA2 with these partners. Gene ontology analysis revealed binding activity as the major molecular function of PMCA2-interacting complex. Functional validation using calcium oxalate monohydrate (COM) crystal-protein binding, crystal-cell adhesion, and crystal internalization assays together with neutralization by anti-PMCA2 antibody compared to isotype-controlled IgG and blank control, revealed a novel role of PMCA2 as a COM crystal-binding protein that was crucial for crystal retention and uptake. In summary, a large number of novel PMCA2-interacting proteins have been defined and a novel function of PMCA2 as a COM crystal-binding protein sheds light onto its involvement, at least in part, in kidney stone pathogenesis.

Chaperonomics in leptospirosis.

INTRODUCTION: Knowledge of the function of molecular chaperones is required for a better understanding of cellular proteostasis. Nevertheless, such information is currently dispersed as most of previous studies investigated chaperones on a single-angle basis. Recently, a new subdiscipline of chaperonology, namely 'chaperonomics' (defined as 'systematic analysis of chaperone genes, transcripts, proteins, or their interaction networks using omics technologies'), has been emerging to better understand biological, physiological, and pathological roles of chaperones. Areas covered: This review provides broad overviews of bacterial chaperones, heat shock proteins (HSPs), and leptospirosis, and then focuses on recent progress of chaperonomics applied to define roles of HSPs in various pathogenic and saprophytic leptospiral species and serovars. Expert commentary: Comprehensive analysis of leptospiral chaperones/HSPs using a chaperonomics approach holds great promise for better understanding of functional roles of chaperones/HSPs in bacterial survival and disease pathogenesis. Moreover, this new approach may also lead to further development of chaperones/HSPs-based diagnostics and/or vaccine discovery for leptospirosis.

Microvillar injury in renal tubular epithelial cells induced by calcium oxalate crystal and the protective role of epigallocatechin-3-gallate.

Pathogenic mechanisms of kidney stone disease remained unclear. This study investigated its initial cellular/molecular mechanisms when calcium oxalate monohydrate (COM) crystal adhered to renal tubular cells. Transmission electron microscopy revealed decreased length and density of microvilli, whereas Western blot analysis showed that whole-cell ezrin (a microvillus-stabilizing protein), not ß-actin, was decreased in COM-treated cells. Immunofluorescence staining, followed by laser-scanning confocal microscopy and subcellular fractionations, revealed decreases in both ezrin and F-/ß-actin at apical membrane. Cytoskeletal extraction by Triton X-100 showed reduced cytoskeleton-associated ezrin, consistent with colocalization data of ezrin/F-actin. Thr567-phosphorylated ezrin and RhoA increased in COM-treated cells. A protein oxidation blot assay showed an increase in oxidized proteins in COM-treated cells that could be prevented by epigallocatechin-3-gallate (EGCG), which also preserved the whole-cell ezrin level, stabilized apical membrane ezrin/F-actin colocalization, and maintained microvillar structure in COM-treated and H2O2-treated cells. Our data clearly demonstrated the reduction of ezrin and actin expression at the apical membrane of COM-treated cells, most likely because of oxidative stress, which could be prevented by EGCG. These findings provide a novel approach to better understanding of the pathogenesis of kidney stone disease in its initial phase and offer potential preventive strategy against microvillar injury induced by COM crystals in patients with kidney stones.-Fong-ngern, K., Vinaiphat, A., Thongboonkerd, V. Microvillar injury in renal tubular epithelial cells induced by calcium oxalate crystal and the protective role of epigallocatechin-3-gallate.

Response of renal tubular cells to differential types and doses of calcium oxalate crystals: Integrative proteome network analysis and functional investigations.

We have previously identified changes in the cellular proteome of renal tubular cells induced by low-dose (100 µg/mL) and high-dose (1000 µg/mL) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals. However, the functional significance of such expression data remained unclear. In this study, we performed comparative analyses and functional investigations of four proteomic datasets to define potential mechanisms by which renal tubular cells responded to differential crystal types and doses. The data showed that high-dose induced greater changes than low-dose, whereas COM induced more changes than COD. Luciferin-luciferase ATP assay revealed increased intracellular ATP level by high-dose of both COM and COD. OxyBlot assay and Western blotting showed accumulated intracellular oxidized proteins but decreased ubiquitinated proteins by high-dose of both crystals. Flow cytometric analysis of cell death showed that high-dose of both crystals, particularly COM, significantly increased cell death. Also, crystal adhesion assay showed higher degree of cell-crystal adhesion in high-dose and COM when compared to low-dose and COD, respectively. Finally, pretreatment of epigallocatechin-3-gallate revealed a protective effect on COM/COD crystals-induced oxidative stress and cell-crystal adhesion. Collectively, these data may provide a better understanding of cellular responses of renal tubular cells to COM/COD crystals in kidney stone disease.

Lamin A/C in renal tubular cells is important for tissue repair, cell proliferation, and calcium oxalate crystal adhesion, and is associated with potential crystal receptors.

A previous study reported that lamin A/C (LMNA) expression was increased in renal tubular cells adhered with calcium oxalate monohydrate (COM) crystals; however, its functional significance in kidney stone disease remained unknown. In the present study, increased levels of LMNA and its partner, nesprin-1 (SYNE1), in Madin-Darby canine kidney cells upon COM crystal adhesion were confirmed by Western blotting and immunofluorescence staining. LMNA was then knocked down by small interfering RNA. Immunofluorescence staining confirmed the efficiency of small interfering RNA of LMNA (si-LMNA), which also reduced expression of its partner, SYNE1. Scratch assay and total cell count revealed defects in tissue repair and cell proliferation, respectively, whereas cell death quantitation showed no cytotoxicity in si-LMNA-transfected cells. Crystal-binding assay highlighted the role of LMNA in crystal adhesion, whereas protein network analysis revealed interactions between LMNA and potential COM crystal receptors. Their associations were confirmed by reduced levels of these proteins, including vimentin, tubulin, enolase, S100, and annexin A2, in si-LMNA-transfected cells. These data have demonstrated for the first time, to our knowledge, that LMNA in renal tubular cells is important for tissue repair, cell proliferation, and COM crystal adhesion and is associated with potential COM crystal receptors. Therefore, LMNA may serve as a potential target for prevention of kidney stone disease and its recurrence.-Pongsakul, N., Vinaiphat, A., Chanchaem, P., Fong-ngern, K., Thongboonkerd, V. Lamin A/C in renal tubular cells is important for tissue repair, cell proliferation, and calcium oxalate crystal adhesion, and is associated with potential crystal receptors.

Physiologic changes of urinary proteome by caffeine and excessive water intake.

BACKGROUND: Diurnal variations and physiologic changes of urinary proteome have been suggested in the urinary proteomics field. However, no clear evidence has been demonstrated. The present study thus aimed to define changes in urinary proteome by physiological stimuli, i.e. caffeine intake and excessive water drinking, both of which cause physiologic diuresis. METHODS: Urine samples were collected from 30 healthy individuals under three different conditions: (i) morning void as the control; (ii) after drinking a cup of coffee; and (iii) after drinking 1 L of water within 20 min. Thereafter, differentially excreted proteins were analyzed by 2-DE proteomics approach and validated by Western blotting and ELISA. RESULTS: Spot matching, quantitative intensity analysis, and ANOVA followed by Tukey's post-hoc multiple comparisons and the Bonferroni correction revealed significant differences in levels of five protein spots among three different conditions. These proteins were identified by quadrupole time-of-flight mass spectrometry (Q-TOF MS) and/or MS/MS analyses as kininogen 1 isoform 3, ß-actin, prostaglandin D synthase (PGDS), fibrinogen α-chain and immunoglobulin light chain. Among these, the decreased level of immunoglobulin was successfully validated by Western blotting and ELISA. CONCLUSIONS: These data indicated that caffeine intake and excessive water drinking could affect urinary excretion of some proteins and may affect urinary proteome analysis.

Proteomics for comprehensive characterization of extracellular vesicles in neurodegenerative disease.

Extracellular vesicles (EVs) are small lipid bilayer particles ubiquitously released by almost every cell type. A specific and selective constituents of EVs loaded with variety of proteins, lipids, small noncoding RNAs, and long non-coding RNAs are reflective of cellular events, type, and physiologic/pathophysiologic status of the cell of origin. Moreover, these molecular contents carry information from the cell of origin to recipient cells, modulating intercellular communication. Recent studies demonstrated that EVs not only play a neuroprotective role by mediating the removal of toxic proteins, but also emerge as an important player in various neurodegenerative disease onset and progression through facilitating of misfolded proteins propagation. For this reason, neurodegenerative disease-associated differences in EV proteome relative to normal EVs can be used to fulfil diagnostic, prognostic, and therapeutic purposes. Nonetheless, characterizing EV proteome obtained from biological samples (brain tissue and body fluids, including urea, blood, saliva, and CSF) is a challenging task. Herein, we review the status of EV proteome profiling and the updated discovery of potential biomarkers for the diagnosis of neurodegenerative disease with an emphasis on the integration of high-throughput advanced mass spectrometry (MS) technologies for both qualitative and quantitative analysis of EVs in different clinical tissue/body fluid samples in past five years.

Application of Advanced Mass Spectrometry-Based Proteomics to Study Hypoxia Driven Cancer Progression.

Cancer is one of the largest contributors to the burden of chronic disease in the world and is the second leading cause of death globally. It is associated with episodes of low-oxygen stress (hypoxia or ischemia/reperfusion) that promotes cancer progression and therapeutic resistance. Efforts have been made in the past using traditional proteomic approaches to decipher oxygen deprivation stress-related mechanisms of the disease initiation and progression and to identify key proteins as a therapeutic target for the treatment and prevention. Despite the potential benefits of proteomic in translational research for the discovery of new drugs, the therapeutic outcome with this approach has not met expectations in clinical trials. This is mainly due to the disease complexity which possess a multifaceted molecular pathology. Therefore, novel strategies to identify and characterize clinically important sets of modulators and molecular events for multi-target drug discovery are needed. Here, we review important past and current studies on proteomics in cancer with an emphasis on recent pioneered labeling approaches in mass spectrometry (MS)-based systematic quantitative analysis to improve clinical success. We also discuss the results of the selected innovative publications that integrate advanced proteomic technologies (e.g. MALDI-MSI, pSILAC/SILAC/iTRAQ/TMT-LC-MS/MS, MRM-MS) for comprehensive analysis of proteome dynamics in different biosystems, including cell type, cell species, and subcellular proteome (i.e. secretome and chromatome). Finally, we discuss the future direction and challenges in the application of these technological advancements in mass spectrometry within the context of cancer and hypoxia.

Advances in extracellular vesicles analysis.

Extracellular vesicles (EVs) play an important role in intercellular communication in normal cellular process and pathological conditions by facilitating the transport of cellular content from one cell to another. EVs as conveyors of various biological molecules with their ability to redirect effects on a target cell physiological function in cell type-specific manner makes EVs an excellent candidate for drug delivery vehicle in disease therapy. Moreover, unique characteristics and contents of EVs which differ depends on cellular origin and physiological state make them a valuable source of diagnostic biomarker. Herein, we review the current progress in extracellular vesicle (EV) analysis, its transition from biomedical research to advancing therapy, and recent pioneered approaches to characterize and quantify EVs' subclasses with an emphasis on the integration of advanced technologies for both qualitative and quantitative analysis of EVs in different clinical tissue/body fluid samples.

Clinical implications of extracellular vesicles in neurodegenerative diseases.

Introduction: Extracellular vesicles (EVs) released by neural cells play a crucial role in intracellular communication in both physiological and pathological states. Recent studies have shown that the neuropathogenic manifestation of many progressive nervous system diseases including Parkinson's disease (PD), Alzheimer's diseases (AD), and amyotrophic lateral sclerosis (ALS). These diseases are frequently found to be associated with the accumulation of misfolded proteins, exploit EVs for the spread of aggregates to naive cells in a prion-like mechanism. Therefore, characterization of EVs and understanding their mechanism of action could open a window of opportunity to discover biomarkers and therapeutic targets in a disease-specific manner. Areas covered: In this review, we discuss the role of neural cells-derived EVs in normal and disease states. We also highlight their biomedical potential in modern medicine, including the use of circulating EVs as biomarkers for diagnosis with a special focus on newly-identified potential biomarkers in neurodegenerative disease, and novel methodologies in EVs isolation. Expert opinion: Systematic and comprehensive analysis of EVs in different biofluid sources is needed. Considering the potential for tremendous clinical benefits of EVs research in neurodegenerative disease, there is also an urgent need to standardize neural cells-derived EV enrichment protocols for consensus results.

Discrimination of urinary exosomes from microvesicles by lipidomics using thin layer liquid chromatography (TLC) coupled with MALDI-TOF mass spectrometry.

Urinary extracellular vesicles (EVs), including microvesicles and exosomes, play several important roles in cell biology and serve as potential biomarkers in various kidney diseases. Although they have differential biophysical properties, specific biomarkers are required to discriminate these EVs during isolation/purification. The present study aimed to define differential lipidome profiles of urinary microvesicles vs. exosomes. Urine samples collected from eight healthy individuals were pooled and underwent lipid extraction using 2:1(v/v) chloroform/methanol. The recovered lipids were resolved by thin layer liquid chromatography (TLC) and analyzed by MALDI-TOF MS. From three and five TLC bands observed in microvesicles and exosomes, respectively, several fatty acids, glycerolipids and phospholipids were identified from both EVs without clear differential patterns. However, their sphingolipid profiles were unique. Ceramide phosphates (CerP), hexosyl sphingoid bases (HexSph), lactosyl ceramides (LacCer), mannosyl di-PI-ceramides (M(IP)2 C), sulfatides hexosyl ceramide (SHexCer) and sulfatides hexoxyl sphingoid bases (SHexSph) were detectable only in urinary exosomes, whereas phosphatidylinositol ceramides (PI-Cer) were detectable only in urinary microvesicles. The presence of CerP only in urinary exosomes was successfully validated by dot blot analysis. Our extensive lipidome analyses of urinary microvesicles vs. exosomes provide potential lipidome markers to discriminate exosomes from microvesicles and may lead to better understanding of EVs biogenesis.

More complete polarization of renal tubular epithelial cells by artificial urine.

Cell polarization using Transwell is a common method employed to study renal tubular epithelial cells. However, this conventional protocol does not precisely recapitulate renal tubular epithelial cell phenotypes. In this study, we simulated renal physiological microenvironment by replacing serum-containing culture medium in upper chamber of the Transwell with physiologic artificial urine (AU) (to mimic renal tubular fluid), whereas the lower chamber still contained serum-containing medium (to mimic plasma-enriched renal interstitium). Comparing to the conventional protocol (control), the AU-assisted protocol offered more complete polarization of MDCK renal tubular cells as indicated by higher transepithelial electrical resistance (TER) and greater levels of tight junction (TJ) proteins (ZO-1 and occludin). Transmission electron microscopy (TEM) showed greater densities of TJ and desmosome, narrower intercellular spaces, greater cell height, and longer microvilli in the AU-treated cells. Secretome analysis revealed that the AU-treated cells secreted greater proportion of the proteins matched to normal human urinary proteome via both classical and non-classical secretory pathways. Finally, modifying/omitting each component of AU (one at a time) followed by validation revealed that urea was responsible for such property of AU to improve cell polarization. These data indicate that replacing AU on the upper chamber of Transwell can improve or optimize renal cell polarization for more precise investigations of renal physiology and cell biology in vitro.

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.