Simultaneous Determination of Dermatan Sulfate and Oversulfated Dermatan Sulfate in Plasma by High-Performance Liquid Chromatography with Postcolumn Fluorescence Derivatization

A chemical method for the determination of dermatan sulfate (DS) and oversulfated dermatan sulfate has been developed and applied to the pharmacokinetic study of these polysaccharides in experimental animals. The analytical procedure includes a simple preparation step of administered DS and oversulfated DS from blood plasma, HPLC for the separation and detection of DS and oversulfated DS using an Asahipak NH₂P-50 column, fluorometric reaction of the polysaccharides with guanidine in a strong alkaline medium. DS and oversulfated DS were extracted from plasma by treating it with proteinase to remove plasma proteins and recovered with endogenous plasma glycosaminoglycans by ethanol precipitation. Finally, DS and oversulfated DS were analyzed by fluorometric HPLC. The detection limits of DS and oversulfated DS were 10 and 20 ng, respectively. Furthermore, we demonstrated that artificial oversulfation of DS increased its biological half-life after intravenous administration to rats.     

Chondroitin / Dermatan Sulfate Modification Enzymes in Zebrafish Development

Chondroitin/dermatan sulfate (CS/DS) proteoglycans consist of unbranched sulfated polysaccharide chains of repeating GalNAc-GlcA/IdoA disaccharide units, attached to serine residues on specific proteins. The CS/DS proteoglycans are abundant in the extracellular matrix where they have essential functions in tissue development and homeostasis. In this report a phylogenetic analysis of vertebrate genes coding for the enzymes that modify CS/DS is presented. We identify single orthologous genes in the zebrafish genome for the sulfotransferases chst7, chst11, chst13, chst14, chst15 and ust and the epimerase dse. In contrast, two copies were found for mammalian sulfotransferases CHST3 and CHST12 and the epimerase DSEL, named chst3a and chst3b, chst12a and chst12b, dsela and dselb, respectively. Expression of CS/DS modification enzymes is spatially and temporally regulated with a large variation between different genes. We found that CS/DS 4-O-sulfotransferases and 6-O-sulfotransferases as well as CS/DS epimerases show a strong and partly overlapping expression, whereas the expression is restricted for enzymes with ability to synthesize di-sulfated disaccharides. A structural analysis further showed that CS/DS sulfation increases during embryonic development mainly due to synthesis of 4-O-sulfated GalNAc while the proportion of 6-O-sulfated GalNAc increases in later developmental stages. Di-sulfated GalNAc synthesized by Chst15 and 2-O-sulfated GlcA/IdoA synthesized by Ust are rare, in accordance with the restricted expression of these enzymes. We also compared CS/DS composition with that of heparan sulfate (HS). Notably, CS/DS biosynthesis in early zebrafish development is more dynamic than HS biosynthesis. Furthermore, HS contains disaccharides with more than one sulfate group, which are virtually absent in CS/DS.     

Dermatan Sulfate Epimerase 1-Deficient Mice Have Reduced Content and Changed Distribution of Iduronic Acids in Dermatan Sulfate and an Altered Collagen Structure in Skin

Dermatan sulfate epimerase 1 (DS-epi1) and DS-epi2 convert glucuronic acid to iduronic acid in chondroitin/dermatan sulfate biosynthesis. Here we report on the generation of DS-epi1-null mice and the resulting alterations in the chondroitin/dermatan polysaccharide chains. The numbers of long blocks of adjacent iduronic acids are greatly decreased in skin decorin and biglycan chondroitin/dermatan sulfate, along with a parallel decrease in iduronic-2-O-sulfated-galactosamine-4-O-sulfated structures. Both iduronic acid blocks and iduronic acids surrounded by glucuronic acids are also decreased in versican-derived chains. DS-epi1-deficient mice are smaller than their wild-type littermates but otherwise have no gross macroscopic alterations. The lack of DS-epi1 affects the chondroitin/dermatan sulfate in many proteoglycans, and the consequences for skin collagen structure were initially analyzed. We found that the skin collagen architecture was altered, and electron microscopy showed that the DS-epi1-null fibrils have a larger diameter than the wild-type fibrils. The altered chondroitin/dermatan sulfate chains carried by decorin in skin are likely to affect collagen fibril formation and reduce the tensile strength of DS-epi1-null skin.Chondroitin sulfate (CS) is an unbranched polymer chain composed of alternating glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) units (36, 49). In dermatan sulfate (DS), d-glucuronic acid is converted to its epimer l-iduronic acid (IdoA) (25). The extent of this modification, which varies from a few percent of the glucuronic acid being epimerized to a predominant presence of iduronic acid, depends on the variable epimerase activity in tissues and on the core protein attached to the chain in CS/DS proteoglycans (PGs) (41, 47). The same CS/DS PG has a different iduronic acid content, depending on the cell type and tissue of origin (4, 5). The name CS/DS denotes the hybrid GlcA-IdoA nature of the chain. It has long been known that the distribution of iduronic acids within the chain is not random but follows two patterns: either they are clustered together, forming long iduronic acid blocks, or they are isolated, i.e., interspersed among surrounding glucuronic acids (11). DS epimerase 1 (DS-epi1) and DS-epi2, encoded in mouse by the Dse and Dsel (Dse-like) genes, respectively, are present in organisms ranging from Xenopus tropicalis to humans but not in worms and flies (23, 34). During DS biosynthesis, epimerization is followed by the action of eight C-specific O-sulfotranferases, which transfer a sulfate group to C-2 of both IdoA and GlcA and to C-4, C-6, and C-4/C-6 of GalNAc (18). These modification reactions, individually affecting only part of the available substrate, produce structural variability in the CS/DS chain. Considerable efforts have been made to characterize specific sequences in the CS/DS chains responsible for binding to protein and the subsequent mediation of a biological effect (28). For instance, (IdoA-2OS-GalNAc-4OS)₃- and GalNAc-4/6-diOS-containing structures bind and activate heparin cofactor II, which is the major antithrombotic system in the subendothelial layer (48). IdoA/GlcA-2OS-GalNAc-6OS-containing structures bind to pleiotrophin, mediating neuritogenic activity (3, 44). IdoA-GalNAc-4OS-containing structures bind to basic fibroblast growth factor, and the complex has been shown to be active in wound healing (46).CS/DS PGs are mainly found in the extracellular matrix. They belong to four families: lecticans, e.g., versican, aggrecan, brevican, and neurocan; collagens, e.g., collagen IX; basement membrane PGs, e.g., SMC3, collagen XV, and perlecan, containing both heparan sulfate (HS) and CS/DS; and small leucine-rich repeat PGs. Some PGs of the first three groups are referred to as CS PGs. The actual presence of iduronic acid, depending on the tissue examined and on the developmental stage, has been overlooked in many cases (37, 44). The archetypical small leucine-rich repeat PG family members decorin, biglycan, fibromodulin, and lumican bind fibrillar collagens and affect collagen fibril and scaffold formation in connective tissues (15). Decorin and biglycan are substituted with one and two CS/DS chains, respectively. Decorin is involved in collagen type I fibril formation and matrix assembly in a wide range of connective tissues and binds near the C terminus of collagen monomers, delaying their accretion to the growing fibrils. We have identified an SYIRIADTNIT sequence in decorin as essential for binding to collagen (16). The role of the decorin CS/DS chain in vivo has not been explored, although in vitro studies suggest that IdoA promotes the binding of CS/DS to collagen (31) and is required for self-association of CS/DS chains (6, 10, 22).Here the function of DS-epi1 in mice was disrupted. DS-epi1-deficient mice show CS/DS with a marked deficiency in iduronic acid-containing structures. The deletion of DS-epi1 is likely to affect many types of PGs and to result in a complex phenotype. We focus on skin alterations presumably caused by altered decorin/biglycan CS/DS chains.     

Immunohistochemical Techniques for Detection of Dermatan Sulfate Proteoglycan in Tissue Sections

Dermatan sulfate proteoglycan chains were detected in tissue sections treated with chondroitin Blyase (0.01 units/ml) in 20 mM Tris-HC1 (pH 8.0) for i hr, followed by staining with antibody 9A2 specific for unsaturated uronic acid coupled to N-acetylgdactosaa- m i n d sulfate. In contrast, after treatment with chondroitin B-lyase, no positive stpining was observed with antibodies 3B3 and 1B5 which react to the unsaturated uronic acid coupled to N-acetylgalactosamine 6-sulfate and unsaturated uronic acid coupled to N-acetylgalactospmine, respectively. The distribution of dermatan sulfate thus revealed was mnfirmed by comparison with that found by monoclonal antibody 6B6 which reacts with small pmteoglycans carrying dermatan sulfate side chains. The localization of positive staining in fib- connective tissues was almost identical with these two procedures.     

Iduronic Acid in Chondroitin/Dermatan Sulfate: Biosynthesis and Biological Function

The ability of chondroitin/dermatan sulfate (CS/DS) to convey biological information is enriched by the presence of iduronic acid. DS-epimerases 1 and 2 (DS-epi1 and 2), in conjunction with DS-4-O-sulfotransferase 1, are the enzymes responsible for iduronic acid biosynthesis and will be the major focus of this review. CS/DS proteoglycans (CS/DS-PGs) are ubiquitously found in connective tissues, basement membranes, and cell surfaces or are stored intracellularly. Such wide distribution reflects the variety of biological roles in which they are involved, from extracellular matrix organization to regulation of processes such as proliferation, migration, adhesion, and differentiation. They play roles in inflammation, angiogenesis, coagulation, immunity, and wound healing. Such versatility is achieved thanks to their variable composition, both in terms of protein core and the fine structure of the CS/DS chains. Excellent reviews have been published on the collective and individual functions of each CS/DS-PG. This short review presents the biosynthesis and functions of iduronic acid-containing structures, also as revealed by the analysis of the DS-epi1- and 2-deficient mouse models.     

Characterization of Human DSPG3, a Small Dermatan Sulfate Proteoglycan

PG-Lb is a small dermatan sulfate proteoglycan that has been previously characterized in chicken. In the developing limb, chick PG-Lb appears to be exclusively expressed in the zone of flattened chondrocytes. We have cloned and sequenced the human homolog to chick PG-Lb from two human chondrocyte cDNA libraries and a human chondrocyte RNA sample. The human homolog has been named DSPG3, as it is the third member of the small dermatan sulfate proteoglycan family to be identified and characterized along with biglycan (PG-I) and decorin (PG-II). DSPG3 maps to chromosome 12q21 and is composed of 1515 nucleotides of cDNA that code for a 322-amino-acid protein. The protein contains three potential glycosaminoglycan attachment sites, two N-glycosylation sites, a poly- glutamic acid stretch, and six cysteines. By Northern analysis, we have demonstrated that DSPG3 is expressed in cartilage, as well as ligament and placental tissues.     

Cloning and Characterization of a Novel Chondroitin Sulfate/Dermatan Sulfate 4-O-Endosulfatase from a Marine Bacterium

Sulfatases are potentially useful tools for structure-function studies of glycosaminoglycans (GAGs). To date, various GAG exosulfatases have been identified in eukaryotes and prokaryotes. However, endosulfatases that act on GAGs have rarely been reported. Recently, a novel HA and CS lyase (HCLase) was identified for the first time from a marine bacterium (Han, W., Wang, W., Zhao, M., Sugahara, K., and Li, F. (2014) J. Biol. Chem. 289, 27886–27898). In this study, a putative sulfatase gene, closely linked to the hclase gene in the genome, was recombinantly expressed and characterized in detail. The recombinant protein showed a specific N-acetylgalactosamine-4-O-sulfatase activity that removes 4-O-sulfate from both disaccharides and polysaccharides of chondroitin sulfate (CS)/dermatan sulfate (DS), suggesting that this sulfatase represents a novel endosulfatase. The novel endosulfatase exhibited maximal reaction rate in a phosphate buffer (pH 8.0) at 30 °C and effectively removed 17–65% of 4-O-sulfates from various CS and DS and thus significantly inhibited the interactions of CS and DS with a positively supercharged fluorescent protein. Moreover, this endosulfatase significantly promoted the digestion of CS by HCLase, suggesting that it enhances the digestion of CS/DS by the bacterium. Therefore, this endosulfatase is a potential tool for use in CS/DS-related studies and applications.     

A Monoclonal Antibody which Recognizes a Glycosaminoglycan Epitope in Both Dermatan Sulfate and Chondroitin Sulfate Proteoglycans of Human Skin

Studies have been initiated to identify various cell surface and matrix components of normal human skin through the production and characterization of murine monoclonal antibodies. One such antibody, termed PG-4, identifies both cell surface and matrix antigens in extracts of human foetal and adult skin as the dermatan sulfate proteoglycans, decorin and biglycan, and the chondroitin sulfate proteoglycan versican. Treatment of proteoglycans with chondroitinases completely abolishes immunoreactivity for all of these antigens which suggests that the epitope resides within their glycosaminoglycan chains. Further evidence for the carbohydrate nature of the epitope derives from competition studies where protein-free chondroitin sulfate chains from shark cartilage react strongly; however, chondroitin sulfate chains from bovine tracheal cartilage fail to exhibit a significant reactivity, an indication that the epitope, although present in some chondroitin sulfate chains, does not consist of random chondroitin 4- or 6-sulfate disaccharides. The presence of the epitope on dermatan sulfate chains and on decorin was also demonstrated using competition assays. Thus, PG-4 belongs to a class of antibodies that recognize native epitopes located within glycosaminoglycan chains. It differs from previously described antibodies in this class in that it identifies both chondroitin sulfate and dermatan sulfate proteoglycans. These characteristics make PG-4 a useful monoclonal antibody probe to identify the total population of proteoglycans in human skin.     

Iduronic Acid in Chondroitin/Dermatan Sulfate Affects Directional Migration of Aortic Smooth Muscle Cells

Aortic smooth muscle cells produce chondroitin/dermatan sulfate (CS/DS) proteoglycans that regulate extracellular matrix organization and cell behavior in normal and pathological conditions. A unique feature of CS/DS proteoglycans is the presence of iduronic acid (IdoA), catalyzed by two DS epimerases. Functional ablation of DS-epi1, the main epimerase in these cells, resulted in a major reduction of IdoA both on cell surface and in secreted CS/DS proteoglycans. Downregulation of IdoA led to delayed ability to re-populate wounded areas due to loss of directional persistence of migration. DS-epi1−/− aortic smooth muscle cells, however, had not lost the general property of migration showing even increased speed of movement compared to wild type cells. Where the cell membrane adheres to the substratum, stress fibers were denser whereas focal adhesion sites were fewer. Total cellular expression of focal adhesion kinase (FAK) and phospho-FAK (pFAK) was decreased in mutant cells compared to control cells. As many pathological conditions are dependent on migration, modulation of IdoA content may point to therapeutic strategies for diseases such as cancer and atherosclerosis.     

Differential Expression of Specific Proteins during In Vitro Tomato Organogenesis1

The cotyledons of tomato (Lycopersicon esculentum L., cv. Jia Fen-10) seedlings were induced to produce calli and regenerate plants via organogenesis. Utilizing this system, the composition and content of stage-specific proteins associated with organogenesis were analyzed. Moreover, a comparison of the protein composition and content between embryogenic and nonembryogenic calli was conducted. The SDS-PAGE results and laser densitometric scanning maps showed that there were different specific proteins expressed at different stages. Among them, six proteins (61, 54, 38, 37, 35, and 23 kD) were associated with the morphogenesis of organs, and two proteins (39 and 24 kD) were related to the morphogenesis of calli. Although no distinctive difference in protein components of embryogenic calli was noted, there were different trends of changes, both for the content of the proteins 39 and 24 kD, and for the content of the total proteins, at different developmental stages of embryogenic calli. The results obtained from the embryogenic and nonembryogenic calli indicated that these two materials were distinct in the protein components as well as in its content; for example, the protein 54 kD was detected in nonembryogenic but not in embryogenic calli. The total protein content in nonembryogenic calli was lower than that in the embryogenic calli.     

Differential Expression of Immune Defences Is Associated with Specific Host-Parasite Interactions in Insects

Recent ecological studies in invertebrates show that the outcome of an infection is dependent on the specific pairing of host and parasite. Such specificity contrasts the long-held view that invertebrate innate immunity depends on a broad-spectrum recognition system. An important question is whether this specificity is due to the immune response rather than some other interplay between host and parasite genotypes. By measuring the expression of putative bumblebee homologues of antimicrobial peptides in response to infection by their gut trypanosome Crithidia bombi, we demonstrate that expression differences are associated with the specific interactions.     

Oocytes Preserve the Ability of Mouse Cumulus Cells in Culture to Synthesize Hyaluronic Acid and Dermatan Sulfate

A soluble factor(s) produced by fully grown oocytes is essential, together with follicle stimulating hormone (FSH), to stimulate in vitro hyaluronic acid (HA) synthesis by mouse cumulus cells (CCs). The stability of the response to this stimulus by CCs in culture was investigated. The data showed that preculture for 8 hr in basal medium reduced to approximately 30% the ability of CCs to synthesize HA in response to FSH or dibutyryl cyclic AMP (Bt₂cAMP) and soluble oocyte factor(s). However, if CCs were precultured for the same period of time as intact cumulus cell-oocyte complexes, or in the presence of fully grown oocytes, or in medium conditioned by fully grown oocytes, their ability to synthesize HA was 75-95% preserved. In vitro stimulation of dermatan sulfate (DS) synthesis by CCs does not require oocyte factors and is induced by FSH or Bt₂cAMP treatment alone. However, the preservation of such activity, like that of HA synthesis, depended on the presence of a soluble oocyte factor(s) during preculture. The presence of isolated oocytes or of oocyte-conditioned medium also prevented the spreading of CCs in culture. However, inhibiting CC spreading by culture on agar-coated plates or in serum-free medium did not preserve their HA or DS synthetic activity, thus suggesting that the two oocyte actions on CCs are independent. Growing oocytes were unable both to induce HA synthesis in freshly isolated CCs stimulated with FSH and to preserve the ability to synthesize HA and DS in 8-hr precultured CCs. The results suggest that the stability of the differentiated state of mouse CCs in vitro depends upon continued exposure to a soluble factor(s) produced by fully grown oocytes.     

Coordinated Expression Domains in Mammalian Genomes

Background

Gene order in eukaryotic genomes is not random. Genes showing similar expression (coexpression) patterns are often clustered along the genome. The goal of this study is to characterize coexpression clustering in mammalian genomes and to investigate the underlying mechanisms.

Methodology/Principal Findings

We detect clustering of coexpressed genes across multiple scales, from neighboring genes to chromosomal domains that span tens of megabases and, in some cases, entire chromosomes. Coexpression domains may be positively or negatively correlated with other domains, within and between chromosomes. We find that long-range expression domains are associated with gene density, which in turn is related to physical organization of the chromosomes within the nucleus. We show that gene expression changes between healthy and diseased tissue samples occur in a gene density-dependent manner.

Conclusions/Significance

We demonstrate that coexpression domains exist across multiple scales. We identify potential mechanisms for short-range as well as long-range coexpression domains. We provide evidence that the three-dimensional architecture of the chromosomes may underlie long-range coexpression domains. Chromosome territory reorganization may play a role in common human diseases such as Alzheimer''s disease and psoriasis.     

Specific Chaperones and Regulatory Domains in Control of Amyloid Formation

Many proteins can form amyloid-like fibrils in vitro, but only about 30 amyloids are linked to disease, whereas some proteins form physiological amyloid-like assemblies. This raises questions of how the formation of toxic protein species during amyloidogenesis is prevented or contained in vivo. Intrinsic chaperoning or regulatory factors can control the aggregation in different protein systems, thereby preventing unwanted aggregation and enabling the biological use of amyloidogenic proteins. The molecular actions of these chaperones and regulators provide clues to the prevention of amyloid disease, as well as to the harnessing of amyloidogenic proteins in medicine and biotechnology.     

Spatial Representations Are Specific to Different Domains of Knowledge

There is evidence that many abstract concepts are represented cognitively in a spatial format. However, it is unknown whether similar spatial processes are employed in different knowledge domains, or whether individuals exhibit similar spatial profiles within and across domains. This research investigated similarities in spatial representation in two knowledge domains – mathematics and music. Sixty-one adults completed analogous number magnitude and pitch discrimination tasks: the Spatial-Numerical Association of Response Codes and Spatial-Musical Association of Response Codes tasks. Subgroups of individuals with different response patterns were identified through cluster analyses. For both the mathematical and musical tasks, approximately half of the participants showed the expected spatial judgment effect when explicitly cued to focus on the spatial properties of the stimuli. Despite this, performances on the two tasks were largely independent. Consistent with previous research, the study provides evidence for the spatial representation of number and pitch in the majority of individuals. However, there was little evidence to support the claim that the same spatial representation processes underpin mathematical and musical judgments.     

Differential Expression of Proteoglycans in Tissue Remodeling and Lymphangiogenesis after Experimental Renal Transplantation in Rats

Background

Chronic transplant dysfunction explains the majority of late renal allograft loss and is accompanied by extensive tissue remodeling leading to transplant vasculopathy, glomerulosclerosis and interstitial fibrosis. Matrix proteoglycans mediate cell-cell and cell-matrix interactions and play key roles in tissue remodeling. The aim of this study was to characterize differential heparan sulfate proteoglycan and chondroitin sulfate proteoglycan expression in transplant vasculopathy, glomerulosclerosis and interstitial fibrosis in renal allografts with chronic transplant dysfunction.

Methods

Renal allografts were transplanted in the Dark Agouti-to-Wistar Furth rat strain combination. Dark Agouti-to-Dark Agouti isografts and non-transplanted Dark Agouti kidneys served as controls. Allograft and isograft recipients were sacrificed 66 and 81 days (mean) after transplantation, respectively. Heparan sulfate proteoglycan (collXVIII, perlecan and agrin) and chondroitin sulfate proteoglycan (versican) expression, as well as CD31 and LYVE-1 (vascular and lymphatic endothelium, respectively) expression were (semi-) quantitatively analyzed using immunofluorescence.

Findings

Arteries with transplant vasculopathy and sclerotic glomeruli in allografts displayed pronounced neo-expression of collXVIII and perlecan. In contrast, in interstitial fibrosis expression of the chondroitin sulfate proteoglycan versican dominated. In the cortical tubular basement membranes in both iso- and allografts, induction of collXVIII was detected. Allografts presented extensive lymphangiogenesis (p<0.01 compared to isografts and non-transplanted controls), which was associated with induced perlecan expression underneath the lymphatic endothelium (p<0.05 and p<0.01 compared to isografts and non-transplanted controls, respectively). Both the magnitude of lymphangiogenesis and perlecan expression correlated with severity of interstitial fibrosis and impaired graft function.

Interpretation

Our results reveal that changes in the extent of expression and the type of proteoglycans being expressed are tightly associated with tissue remodeling after renal transplantation. Therefore, proteoglycans might be potential targets for clinical intervention in renal chronic transplant dysfunction.     

Identification of Specific Chondroitin Sulfate Species in Cutaneous Autoimmune Disease

Cutaneous lupus erythematosus and dermatomyositis (DM) are chronic inflammatory diseases of the skin with accumulated dermal mucin. Earlier work has shown chondroitin sulfate (CS) accumulation within the dermis of discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE), and DM lesions compared with control skin. Immunohistochemistry for C4S revealed a greater density in DLE and DM lesions, whereas SCLE lesions did not differ from controls. Scleredema and scleromyxedema are attributed to increased hyaluronic acid, and lesional samples from these diseases also demonstrated accumulated dermal C4S. Interferon-γ and interleukin-1α, but not interferon-α, treatment of cultured dermal fibroblasts induced mRNA expression of CHST-11, which attaches sulfates to the 4-position of unsulfated chondroitin. These studies on possible CS core proteins revealed that serglycin, known to have C6S side chains in endothelial cells, had greater density within DM dermal endothelia but not in DLE or SCLE, following the pattern of C6S overexpression reported previously. CD44 variants expand the CS binding repertoire of the glycoprotein; CD44v7 co-localized to the distribution of C4S in DLE lesions, a finding not observed in DM, SCLE lesions, or controls. Because C4S and C6S have immunologic effects, their dysregulation in cutaneous mucinoses may contribute to the pathogenesis of these disorders.     

紧密连接蛋白claudin-4 在肾透明细胞癌中的表达及预后意义

目的:探讨claudin-4在肾透明细胞癌中的表达及与临床病理特征及预后的相关性。方法:应用免疫组织化学技术检测50例肾透明细胞癌组织及10例正常肾组织标本中claudin-4的表达情况,并分析与临床病理参数的相关性;随机选取其中4例肾透明细胞癌患者癌组织及4例正常肾新鲜组织,Western blot检测claudin-4的表达情况,并与免疫组化检测结果进行相关性分析;结合随访结果分析claudin-4的表达水平与患者无疾病生存期的关系。结果:Claudin-4在肾透明细胞癌组织中的表达显著高于肾正常组织(P<0.05),表达水平与肿瘤的直径、TNM分期、临床分期及淋巴结转移相关;Western blot结果也表明claudin-4在肾透明细胞癌组织中的表达显著高于肾正常组织(P<0.01),与免疫组化结果呈显著正相关(r2=0.748,P<0.01);Kaplan-Meier分析显示claudin-4高表达组患者的患者无疾病生存期为20.7个月,显著低于低表达组的30.8个月。COX多因素比例风险模型分析显示,claudin-4的表达不是肾透明细胞癌的独立预后因素(RR值=1.686,95%CI 0.174~16.311,P=0.652)。结论:claudin-4蛋白表达上调可能促进肾透明细胞癌的发生和发展,有可能作为肾透明细胞癌潜在的治疗靶点及影响不良预后的指标。