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Zenyui Cui,
Yutaka Yoshida,
Bo Xu,
Ying Zhang,
Masaaki Nameta,
Sameh Magdeldin,
Tomoo Makiguchi,
Toshikazu Ikoma,
Hidehiko Fujinaka, Eishin Yaoita,
Tadashi Yamamoto
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ABSTRACT: BACKGROUND: The comprehensive analysis of human kidney glomerulus we previously performed using highly purified glomeruli, provided a dataset of 6,686 unique proteins representing 2,966 distinct genes. This dataset, however, contained considerable redundancy resulting from identification criteria under which all the proteins matched with the same set of peptides and its subset were reported as identified proteins. In this study we reanalyzed the raw data using the Mascot search engine and highly stringent criteria in order to select proteins with the highest scores matching peptides with scores exceeding the "Identity Threshold" and one or more unique peptides. This enabled us to exclude proteins with lower scores which only matched the same set of peptides or its subset. This approach provided a high-confidence, non-redundant dataset of identified proteins for extensive profiling, annotation, and comparison with other proteome datasets that can provide biologically relevant knowledge of glomerulus proteome. RESULTS: Protein identification using the Mascot search engine under highly stringent, computational strategy generated a non-redundant dataset of 1,817 proteins representing 1,478 genes. These proteins were represented by 2-D protein array specifying observed molecular weight and isoelectric point range of identified proteins to demonstrate differences in the observed and calculated physicochemical properties. Characteristics of glomerulus proteome could be illustrated by GO analysis and protein classification. The depth of proteomic analysis was well documented via comparison of the dynamic range of identified proteins with other proteomic analyses of human glomerulus, as well as a high coverage of biologically important pathways. Comparison of glomerulus proteome with human plasma and urine proteomes, provided by comprehensive analysis, suggested the extent and characteristics of proteins contaminated from plasma and excreted into urine, respectively. Among the latter proteins, several were demonstrated to be highly or specifically localized in the glomerulus by cross-reference analysis with the Human Protein Atlas database, and could be biomarker candidates for glomerular injury. Furthermore, comparison of ortholog proteins identified in human and mouse glomeruli suggest some biologically significant differences in glomerulus proteomes between the two species. CONCLUSIONS: A high-confidence, non-redundant dataset of proteins created by comprehensive proteomic analysis could provide a more extensive understanding of human glomerulus proteome and could be useful as a resource for the discovery of biomarkers and disease-relevant proteins.
Proteome Science 04/2013; 11(1):13. · 2.33 Impact Factor
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Zan Liu,
Bo Xu,
Masaaki Nameta,
Ying Zhang,
Sameh Magdeldin,
Yutaka Yoshida,
Keiko Yamamoto,
Hidehiko Fujinaka, Eishin Yaoita,
Masayuki Tasaki,
Yuki Nakagawa,
Kazuhide Saito,
Kota Takahashi,
Tadashi Yamamoto
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ABSTRACT: BACKGROUND: Vascular endothelial cells (VECs) play crucial roles in physiological and pathologic conditions in tissues and organs. Most of these roles are related to VEC plasma membrane proteins. In the kidney, VECs are closely associated with structures and functions; however, plasma membrane proteins in kidney VECs remain to be fully elucidated. METHODS: Rat kidneys were perfused with cationic colloidal silica nanoparticles (CCSN) to label the VEC plasma membrane. The CCSN-labeled plasma membrane fraction was collected by gradient ultracentrifugation. The VEC plasma membrane or whole-kidney lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and digested with trypsin in gels for liquid chromatography-tandem mass spectrometry. Enrichment analysis was then performed. RESULTS: The VEC plasma membrane proteins were purified by the CCSN method with high yield (approximately 20 μg from 1 g of rat kidney). By Mascot search, 582 proteins were identified in the VEC plasma membrane fraction, and 1,205 proteins were identified in the kidney lysate. In addition to 16 VEC marker proteins such as integrin beta-1 and intercellular adhesion molecule-2 (ICAM-2), 8 novel proteins such as Deltex 3-like protein and phosphatidylinositol binding clathrin assembly protein (PICALM) were identified. As expected, many key functions of plasma membranes in general and of endothelial cells in particular (i.e., leukocyte adhesion) were significantly overrepresented in the proteome of CCSN-labeled kidney VEC fraction. CONCLUSIONS: The CCSN method is a reliable technique for isolation of VEC plasma membrane from the kidney, and proteomic analysis followed by bioinformatics revealed the characteristics of in vivo VECs in the kidney.
Clinical and Experimental Nephrology 12/2012; · 1.37 Impact Factor
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Sameh Magdeldin,
Yutaka Yoshida,
Huiping Li,
Yoshitaka Maeda,
Munesuke Yokoyama,
Shymaa Enany,
Ying Zhang,
Bo Xu,
Hidehiko Fujinaka, Eishin Yaoita,
Sei Sasaki,
Tadashi Yamamoto
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ABSTRACT: BACKGROUND: Most of the current proteomic researches focus on proteome alteration due to pathological disorders (i.e.: colorectal cancer) rather than normal healthy state when mentioning colon. As a result, there are lacks of information regarding normal whole tissue- colon proteome. RESULTS: We report here a detailed murine (mouse) whole tissue- colon protein reference dataset composed of 1237 confident protein (FDR < 2) with comprehensive insight on its peptide properties, cellular and subcellular localization, functional network GO annotation analysis, and its relative abundances. The presented dataset includes wide spectra of pI and Mw ranged from 3--12 and 4--600 KDa, respectively. Gravy index scoring predicted 19.5% membranous and 80.5% globularly located proteins. GO hierarchies and functional network analysis illustrated proteins function together with their relevance and implication of several candidates in malignancy such as Mitogen- activated protein kinase (Mapk8, 9) in colorectal cancer, Fibroblast growth factor receptor (Fgfr 2), Glutathione S-transferase (Gstp1) in prostate cancer, and Cell division control protein (Cdc42), Ras-related protein (Rac1,2) in pancreatic cancer. Protein abundances calculated with 3 different algorithms (NSAF, PAF and emPAI) provide a relative quantification under normal condition as guidance. CONCLUSIONS: This highly confidence colon proteome catalogue will not only serve as a useful reference for further experiments characterizing differentially expressed proteins induced from diseased conditions, but also will aid in better understanding the ontology and functional absorptive mechanism of the colon as well.
BioData Mining 08/2012; 5(1):11.
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Yutaka Yoshida,
Masaaki Nameta,
Masayoshi Kuwano,
Ying Zhang,
Xu Bo,
Sameh Magdeldin,
Zenyui Cui,
Hidehiko Fujinaka, Eishin Yaoita,
Takeshi Tomonaga,
Tadashi Yamamoto
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ABSTRACT: Abundance of blood-derived proteins in glomeruli prepared by laser microdissection from human kidney biopsy specimens has hampered in-depth proteomic analysis of glomeruli. We attempted to establish experimental platform for in-depth proteomic analysis of glomeruli by removal of blood-derived proteins from frozen biopsy samples.
Frozen sections of biopsy samples were exposed to repeated PBS washes prior to laser microdissection to remove blood-derived proteins, and glomerular dissectants were analyzed by MS. The depth of proteomic analysis was evaluated by dynamic range of identified proteins and detection of low-abundance proteins.
Two times PBS washes of frozen sections effectively eliminated blood-derived proteins in laser-microdissected glomeruli and gave an increased number of identified proteins. Analysis of glomeruli from single specimens by a linear ion trap-Orbitrap mass analyzer generated nonredundant, high-confidence datasets of more than 400 identified proteins with high reproducibility, which attained to a considerable depth of the glomerulus proteome as revealed by a wide dynamic range and identification of low-abundance proteins.
Implementation of washing of frozen section with PBS successfully removed blood-derived proteins and resulted in an in-depth proteomic analysis of laser-microdissected glomeruli, suggesting applicability to clinical study.
PROTEOMICS - CLINICAL APPLICATIONS 08/2012; 6(7-8):412-7. · 1.81 Impact Factor
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ABSTRACT: We have previously constructed an in-depth human glomerulus proteome database from a large amount of sample for understanding renal disease pathogenesis and aiding the biomarker exploration. However, it is usually a challenge for clinical research to get enough tissues for large-scale proteomic characterization. Therefore, in this study, we focused on high-confidence proteomics analysis on small amounts of human glomeruli comparable to those obtained from biopsies using different mass spectrometers and compared these results to the comprehensive database.
One microgram of human glomerular protein digest was analyzed each on five LC- combined mass spectrometers (LIT-TOF, LTQ-Orbitrap, Q-TOF, LIT and MALDI-TOF/TOF) yielding 139, 185, 94, 255 and 108 proteins respectively identified with strict criteria to ensure high confidence (> 99%) and low false discovery rate (FDR) (< 1%). An integrated profile of 332 distinct glomerular proteins was subsequently generated without discerned bias due to protein physicochemical properties (pI and MW), of which around 60% were detected commonly by more than two LC-MS/MS platforms. Comparative analysis with the comprehensive database demonstrated 14 proteins uniquely identified in this study and more than 70% of identified proteins in small datasets were concentrated to the top abundant 500 in the comprehensive database which consists of 2775 non-redundant proteins.
This study showed representative human glomerulus proteomic profiles obtained from biopsies through analysis of comparable amounts of samples by different mass spectrometry. Our results implicated that high abundant proteins are more likely to be reproducibly identified in multiple mass spectrometers runs and different mass spectrometers. Furthermore, many podocyte essential proteins such as nephrin, podocin, podocalyxin and synaptopodin were also identified from the small samples in this study. Bioinformatic enrichment analysis results extended our understanding of the major glomerular proteins about their subcellular distributions and functions. The present study indicated that the proteins localized in certain cellular compartments, such as actin cytoskeleton, mitochondrial matrix, cell surface, basolateral plasma membrane, contractile fiber, proteinaceous extracellular matrix and adherens junction, represent high abundant glomerular proteins and these subcellular structures are also highly significantly over-represented in the glomerulus compared to the whole human background.
Proteome Science 08/2011; 9(1):47. · 2.33 Impact Factor
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ABSTRACT: In the field of bottom-up proteomics, heavy contamination of human keratins could hinder the comprehensive protein identification, especially for the detection of low abundance proteins. In this study, we examined the keratin contamination in the four major experimental procedures in gel-based proteomic analysis including gel preparation, gel electrophoresis, gel staining, and in-gel digestion. We found that in-gel digestion procedure might be of importance corresponding to keratin contaminants compared to the other three ones. The human keratin contamination was reduced significantly by using an electrostatic eliminator during in-gel digestion, suggesting that static electricity built up on insulated experimental materials might be one of the essential causes of keratin contamination. We herein proposed a series of methods for improving experimental conditions and sample treatment in order to minimize the keratin contamination in an economical and practical way.
Journal of proteomics 03/2011; 74(7):1022-9. · 5.07 Impact Factor
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Ryo Koda,
Linning Zhao, Eishin Yaoita,
Yutaka Yoshida,
Sachiko Tsukita,
Atsushi Tamura,
Masaaki Nameta,
Ying Zhang,
Hidehiko Fujinaka,
Sameh Magdeldin,
Bo Xu,
Ichiei Narita,
Tadashi Yamamoto
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ABSTRACT: Tight junctions are the main intercellular junctions of podocytes of the renal glomerulus under nephrotic conditions. Their requisite components, claudins, still remain to be identified. We have measured the mRNA levels of claudin subtypes by quantitative real-time PCR using isolated rat glomeruli. Claudin-5 was found to be expressed most abundantly in glomeruli. Mass spectrometric analysis of membrane preparation from isolated glomeruli also confirmed only claudin-5 expression without any detection of other claudin subtypes. In situ hybridization and immunolocalization studies revealed that claudin-5 was localized mainly in glomeruli where podocytes were the only cells expressing claudin-5. Claudin-5 protein was observed on the entire surface of podocytes including apical and basal domains of the plasma membrane in the normal condition and was inclined to be concentrated on tight junctions in puromycin aminonucleoside nephrosis. Total protein levels of claudin-5 in isolated glomeruli were not significantly upregulated in the nephrosis. These findings suggest that claudin-5 is a main claudin expressed in podocytes and that the formation of tight junctions in the nephrosis may be due to local recruitment of claudin-5 rather than due to total upregulation of the claudin protein levels.
Cell and Tissue Research 01/2011; 343(3):637-48. · 3.11 Impact Factor
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ABSTRACT: Insulin-like growth factor I (IGF-I) acts on target cells in an endocrine and/or local manner through the IGF-I receptor (IGF-IR), and its actions are modulated by multiple IGF binding proteins (IGFBP). To elucidate the roles of local IGFBP in kidney glomeruli, the expression and localization of their genes were examined and compared with normal and proteinuric kidney glomeruli.
A cDNA microarray database (MAd-761) was constructed using human kidney glomeruli and cortices. The gene expression levels of IGF-I, IGF-1R and IGFBP (1-10) were examined in glomeruli and cortices by polymerase chain reaction (PCR) and in situ hybridization (ISH), and the expression levels of IGFBP that were abundantly found in the glomerulus were compared between normal and proteinuric kidneys in rats and humans.
IGFBP-2, -7 and -8 were demonstrated to be abundantly and preferentially expressed in the glomerulus. In PCR, the expression levels of the IGFBP-2, -7, -8 and -10 genes in glomeruli were shown to have more than doubled compared with their levels in the cortices. In ISH, the IGFBP-2, -7, -8 and -10 genes were found to be localized in glomerular cells including podocytes, and their increased expression was observed in inflammatory glomeruli. IGF-I gene expression was localized in glomerular podocytes, whereas the IGF-IR gene was expressed in glomerular podocytes and cortical tubular cells. In nephrotic rats, the expression of the IGFBP-10 gene was increased in glomerular podocytes; however, the expression levels of IGFBP-2, -7 and -8 did not change.
IGFBP-2, -7, -8 and -10 are produced by normal and injured glomerular podocytes and may regulate local IGF-I actions in podocytes and/or cortical tubular cells in the kidney.
Nephrology 10/2010; 15(7):700-9. · 1.31 Impact Factor
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Sameh Magdeldin,
Huiping Li,
Yutaka Yoshida,
Ichiro Satokata,
Yoshitaka Maeda,
Munesuke Yokoyama,
Shymaa Enany,
Ying Zhang,
Bo Xu,
Hidehiko Fujinaka, Eishin Yaoita,
Tadashi Yamamoto
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ABSTRACT: Aquaporin (AQP) family plays a pivotal role in fluid secretion and absorption, especially in the digestive system and secretory glands. Within this family, AQP8 was reported to be widely expressed in the epithelia of the digestive tract, liver, and pancreas. In two parallel experimental platforms with different analytical and comparative approaches, in-gel tryptic digestion with macro-embedded spreadsheet analysis and in-solution tryptic digestion with LC-MS alignment based approach, we compared wild-type and AQP8 knockout mice colon proteomes. Shared result between both experiments revealed down-regulation of α-amylase 2 in AQP8-deleted mice model. Verification on both transcriptional and translational levels confirmed the involvement of AQP8 in α-amylase 2 regulation. Given the profound role of AQP8 as a water and solutes transporter, it might be important in modulating α-amylase 2 synthesis by colonic epithelial cells as well. Here, we also proved the capability of our coupled approaches for selecting the most reliable and significant candidates, an applicable process for initial screening of biological biomarkers in complex specimens and tissue extracts.
Journal of Proteome Research 10/2010; 9(12):6635-46. · 5.11 Impact Factor
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ABSTRACT: Aquaporin (AQP) family plays a fundamental role in transmembrane water and small solutes movement. Within this family, aquaporin 8 (AQP8), showed to be widely distributed in the digestive system especially colon. To investigate the possible protein alterations involved in AQP8 regulation and trafficking, we extensively compared between wild type and AQP8 knockout mouse colon using semi-quantitative fluorescence- stained two dimensional gel electrophoresis (2-DE) coupled with nano LC-Ms/Ms. Our analysis revealed identification and regulation of 21 proteins, most notably, actin-related family which suggests its possible involvement in regulating AQP8 secretory vesicles migration to be integrated as a cell membrane protein.
Journal of proteomics 09/2010; 73(10):2031-40. · 5.07 Impact Factor
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Ying Zhang,
Yutaka Yoshida,
Masaaki Nameta,
Bo Xu,
Izumi Taguchi,
Takako Ikeda,
Hidehiko Fujinaka,
Sameh Magdeldin,
Sameh Magdeldin Mohamed,
Hiroyasu Tsukaguchi,
Yutaka Harita, Eishin Yaoita,
Tadashi Yamamoto
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ABSTRACT: Tyrosine phosphorylation of proteins has been a focus of extensive studies since it plays crucial roles in regulation of diverse biological reactions. To understand the involvement of tyrosine phosphorylation in kidney functions, a comprehensive proteomic study for tyrosine-phosphorylated proteins was performed in the normal rat kidney.
Two-dimensional gel electrophoresis and immunoprecipitation using anti-phosphotyrosine antibodies were employed to detect tyrosine-phosphorylated proteins. The proteins were analysed by mass spectrometry and validated by immunological analyses using specific antibodies.
Most of tyrosine-phosphorylated proteins were confined to the glomerulus and predominantly localized along the glomerular capillary wall, especially in the foot processes of podocytes. Our systematic proteomic analysis identified nephrin, SHPS-1 (tyrosine-protein phosphatase non-receptor-type substrate 1), FAK1 and paxillin as major tyrosine-phosphorylated proteins and Neph1, talin and vinculin as minor tyrosine-phosphorylated proteins. In the present study, SHPS-1 was identified as a novel tyrosine-phosphorylated protein in the glomerulus and was also predominantly localized at the foot processes. Mass spectrometric analysis identified in vivo phosphorylation sites of SHPS-1 on Y460, Y477 and Y501.
This study identified tyrosine-phosphorylated proteins in normal rat kidney, which were prominently rich in the glomerulus and localized at the podocyte foot processes. These proteins were categorized as cell-to-cell or cell-to-matrix adhesion complex-related molecules, suggesting their pivotal roles in the glomerular ultrafiltration.
Nephrology Dialysis Transplantation 06/2010; 25(6):1785-95. · 3.40 Impact Factor
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Sameh Magdeldin,
Yaser Elewa,
Takako Ikeda,
Junko Ikei,
Ying Zhang,
Bo Xu,
Masaaki Nameta,
Hidehiko Fujinaka,
Yutaka Yoshida, Eishin Yaoita,
Tadashi Yamamoto
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ABSTRACT: In order to investigate the effects of dietary supplementation rich in omega 3 and omega 6 fatty acids, we set up an experiment of twenty four C57BL/6J male mice segregated into 3 groups: normal diet (ND), omega 3 polyunsaturated fatty acid (n-3 PUFA,) and omega 6 (n-6 PUFA). At the end of the experiment that lasted for 1 month, food consumption of ND and n-3 PUFA were similar while it decreased in n-6 PUFA group. Total cholesterol, triglycerides, free fatty acids, and phospholipids profiles were increased in n-6 PUFA. LDL decreased in n-3 PUFA while increased in n-6 PUFA fed mice comparing to control group. On the other hand, there was no difference between treatments in HDL and glucose levels. Expression of leptin (ob) gene transcripts in epididymal fat were significantly elevated in n-6 PUFA mice compared to ND and n-3 PUFA groups while hypothalamic ob receptor A (obRa) mRNA did not changed in response to diet regimes. Transmission and scanning electron microscopy showed different degrees in fatty changes in the liver of both PUFA groups including lipid droplet infiltration and Ito cells with over accumulated lipids. In conclusion, under PUFA dietary supplementation, the hyperlipidemic status and elevated ob expression of n-6 PUFA but not n-3 PUFA fed mice suggests altered lipid metabolism between PUFA groups and/or different endocrine involvement. Moreover, the coincidently structural changes observed in liver of this group direct us to call for further studies to investigate the anti-obesity effect and safety of these PUFA under high supplementation condition.
General Physiology and Biophysics 09/2009; 28(3):266-75. · 1.19 Impact Factor
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ABSTRACT: A chemokine fractalkine (FKN/CX3CL1) is induced primarily by endothelial cells and accumulates inflammatory cells via its receptor CX3CR1. Since glomerular preferential expression of FKN/CX3CL1 gene was reported in normal human kidney, we presumed FKN/CX3CL1 might play some roles in glomerular physiology. The purpose of this study is to examine the expression and localization of FKN/CX3CL1 in normal and proteinuric glomeruli.
Normal and proteinuric rat kidneys were studied. The gene and protein expressions of FKN/CX3CL1 and CX3CR1 were examined by real-time RT-PCR, in situ hybridization and immunohistochemistry, Western blotting.
By real-time RT-PCR, glomerular preferential expression of FKN/CX3CL1 was confirmed, whereas CX3CR1 was detected in glomeruli and cortices. The localization of FKN/CX3CL1 gene and protein were demonstrated in glomerular cells including podocytes. In nephrotic puromycin aminonucleoside (PAN) nephrosis glomeruli, increased expression of FKN/CX3CL1 in podocyte was shown by immunohistochemistry. Western blotting showed that in nephrotic glomeruli, the membrane-anchored form of FKN/CX3CL1 was increased while the soluble form was decreased.
The expression of FKN/CX3CL1 in normal podocytes and the increased expression of the membrane-anchored form in nephrotic glomeruli strongly suggest that FKN/CX3CL1 may play roles in glomerular physiology such as maintaining glomerular filtration barrier.
Nephron Experimental Nephrology 08/2009; 113(2):e45-56. · 1.86 Impact Factor
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ABSTRACT: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) expressing Panton-Valentine leukocidin (PVL) have been characterized worldwide, but it has not been done in Egypt. In this study, we analyzed the molecular characteristics of PVL+CA-MRSA in Egypt, compared their genetic patterns with that of PVL+ methicillin-susceptible S. aureus and PVL+CA-MRSA from different countries, and investigated the allelic variations among their lukS-PV and lukF-PV gene sequences. The prevalence of PVL+MRSA was 19.04%. They belonged to different genetic clones with multi-locus sequence types (MLST) 30, 80, and the novel type 1010. ST80 strains showed unique antibiotic resistance profile that was distinguishable from the European ST80 clone: they had no resistance to tetracycline and fusidic acid. Two nonsynonymous substitutions in the lukS-PV gene region and two haplotype variants among these isolates were detected. We revealed that PVL is a good marker for CA-MRSA infections and that PVL+ isolates belonged to different genotypes.
Microbiological Research 08/2009; 165(2):152-62. · 2.31 Impact Factor
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ABSTRACT: Mesangial cells bear the tensional forces generated in the glomerular capillary wall. Not only mesangial cells per se, but also their intercellular junctions should be physically stable against the tensional forces; this prompted the search for actin filament-reinforced adherens junctions of mesangial cells. We previously reported alpha- and beta-catenins localized at the cell-cell contact sites of mesangial cells in the rat. Classical cadherin expressed by mesangial cells, however, remains to be elucidated.
Expression of classical cadherins, especially N-cadherin, was examined in rat glomeruli by ribonuclease protection assay, Western blot analysis and immunofluorescence and immunoelectron microscopy.
Ribonuclease protection assay detected significant expression of N-cadherin in rat glomeruli. Western blot analysis showed that rabbit and murine antibodies against N-cadherin reacted with a specific band in isolated glomeruli. Immunofluorescence microscopy revealed that both antibodies reacted only with the mesangium in glomeruli. Immunoelectron microscopy demonstrated that the immunogold particles for N-cadherin were found predominantly at cell-cell contact sites of mesangial cells where actin filaments concentrated.
N-cadherin interconnects mesangial cells, suggesting that the cadherin-catenin-actin filament system in the mesangium may play a role in the counteraction of the hydraulic pressure gradient across the capillary wall.
Nephron Experimental Nephrology 07/2009; 112(4):e92-8. · 1.86 Impact Factor
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Masayuki Tasaki,
Yutaka Yoshida,
Masahito Miyamoto,
Masaaki Nameta,
Lino M Cuellar,
Bo Xu,
Ying Zhang, Eishin Yaoita,
Yuki Nakagawa,
Kazuhide Saito,
Tadashi Yamamoto,
Kota Takahashi
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ABSTRACT: It is generally admitted that ABO(H) blood group antigens are linked to lipids and proteins. Although glycolipids carrying ABO antigens have been well characterized in human kidneys, glycoproteins carrying ABO antigens are largely unknown, and their molecular properties remain to be elucidated.
All the blood group A antigen-linked proteins in human kidney could be solubilized and captured on immobilized Helix pomatia lectin that recognizes A antigens. These proteins were separated on SDS-PAGE gels. The gel pieces containing protein bands immunoreactive with anti-A antibody were excised, in-gel digested with trypsin, and analyzed by nanoLC tandem mass spectrometer. Protein candidates that carry ABO antigens were confirmed by immunoprecipitation and double-labeled immunofluorescense microscopy.
All the glycoproteins carrying ABO antigens were found to be Asn-linked glycoproteins, and presented as multiple bands on SDS-PAGE with molecular masses ranging from 60 to 270 kDa. The protein bands were subjected for mass spectrometric analysis, which identified 121 distinct proteins with high confidence. Of the identified proteins, 55 N-glycosylated, membrane proteins were selected as glycoprotein candidates that carry ABO antigens. Among them, most abundantly expressed proteins as estimated by the number of peptide matches in the MS spectrometric analysis, such as platelet endothelial cell adhesion molecule 1, plasmalemmal vesicle-associated protein, and von Willebrand factor, were further characterized.
Several glycoproteins were identified that represented major glycoproteins carrying ABO antigens in the human kidney, which exhibited distinct features in localization to most of vascular endothelial cells.
Transplantation 05/2009; 87(8):1125-33. · 4.00 Impact Factor
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Clinical Proteomics. 01/2009; 1:90.
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ABSTRACT: To find novel genes abundantly and preferentially expressed in human glomerulus, we constructed a glomerular cDNA library and verified the reliability of our database by comparison with the Stanford Microarray Database (SMD), followed by reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization (ISH).
RNA was extracted from normal human glomeruli, and the cDNA library was constructed by plasmid cloning. Out of 5 x 10(3) clones from the library, 91 UniGene clusters of more than three clones were identified as 'glomerular-abundant genes'. All these genes were referred to the SMD, and 18 genes were defined as 'glomerular preferential genes'. Four unknown genes -IFI27, CRHBP, FLJ10154 and SEMA5B- were selected for RT-PCR to compare expression in the glomerulus with that in the cortex and medulla, and for ISH to examine glomerular localization. Also, three unknown genes that were glomerular abundant but not listed in the SMD -DDX5, HSPC138, and MGC10940- were selected for RT-PCR and ISH. Finally, a kidney biopsy specimen of crescentic glomerulonephritis was used for ISH to examine glomerular expression for CRHBP mRNA.
Among the selected seven glomerular-abundant genes, six were confirmed as 'glomerular preferential genes' by RT-PCR. By ISH, all these genes were demonstrated in podocytes. The expression of CRHBP mRNA in a single living podocyte was not changed between normal and crescentic glomerulus.
Glomerular preferential expression and podocyte localization of these novel genes have been demonstrated for the first time. Because some of these genes were not listed in SMD, our database can be a useful tool to find novel human glomerular genes.
Nephrology 12/2008; 14(1):94-104. · 1.31 Impact Factor
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ABSTRACT: Tight junctions rarely exist in podocytes of the normal renal glomerulus, whereas they are the main intercellular junctions of podocytes in nephrosis and in the early stage of development. Claudins have been identified as tight junction-specific integral membrane proteins. Those of podocytes, however, remain to be elucidated. In the present study, we investigated the expression and localization of claudin-6 in the rat kidney, especially in podocytes. Western blot analysis and RT-PCR revealed that the neonatal kidney expressed much higher levels of claudin-6 than the adult kidney. Immunofluorescence microscopy showed intense claudin-6 staining in most of the tubules and glomeruli in neonates. The staining in tubules declined distinctly in adults, whereas staining in glomeruli was well preserved during development. Claudin-6 in glomeruli was distributed along the glomerular capillary wall and colocalized with zonula occludens-1. The staining became conspicuous after kidney perfusion with protamine sulfate (PS) to increase tight junctions in podocytes. Immunoelectron microscopy showed that immunogold particles for claudin-6 were accumulated at close cell-cell contact sites of podocytes in PS-perfused kidneys, whereas a very limited number of immunogold particles were detected, mainly on the basal cell membrane and occasionally at the slit diaphragm and close cell-cell contact sites in normal control kidneys. In puromycin aminonucleoside nephrosis, immunogold particles were also found mainly at cell-contact sites of podocytes. These findings indicate that claudin-6 is a transmembrane protein of tight junctions in podocytes during development and under pathological conditions.
AJP Regulatory Integrative and Comparative Physiology 07/2008; 294(6):R1856-62. · 3.34 Impact Factor
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ABSTRACT: The kidney glomerulus is the site of plasma filtration and production of primary urine in the kidney. The structure not only plays a pivotal role in ultrafiltration of plasma into urine but also is the locus of kidney diseases progressing to chronic renal failure. Patients afflicted with these glomerular diseases frequently progress to irreversible loss of renal function and inevitably require replacement therapies. The diagnosis and treatment of glomerular diseases are now based on clinical manifestations, urinary protein excretion level, and renal pathology of needle biopsy specimens. The molecular mechanisms underlying the progression of glomerular diseases are still obscure despite a great number of clinical and experimental studies. Proteomics is a particularly promising approach for the discovery of proteins relevant to physiological and pathophysiological processes, and has been recently employed in nephrology. Although until now most efforts of proteomic analysis have been conducted with urine, the biological fluid that is easily collected without invasive procedures, proteomic analysis of the glomerulus, the tissue most proximal to the disease loci, is the most straightforward approach. In this review, we attempt to outline the current status of clinical proteomics of the glomerulus and provide a perspective of protein biomarker discovery of glomerular diseases.
Proteomics. Clinical applications 03/2008; 2(3):420-7. · 1.97 Impact Factor
