Nephrin is involved in podocyte maturation but not survival during glomerular development.

Nephrin, a major component of the glomerular slit diaphragm (SD), is both a structural protein as well as a signaling molecule influencing foot process (FP) formation and maintenance of podocyte integrity. Analyses of near-term embryonic kidneys showed normal cellular viability and no apoptosis in glomeruli from nephrin knockout mice. Moreover, expression and location of other SD or glomerular basement membrane components were similar in wild-type and mutant mice as was the location and levels of most podocyte-specific proteins. Transcriptional profiling showed that the lack of nephrin had minor impact on the expression of genes for FPs and SD proteins. Claudin 3, a tight-junction protein normally absent in glomeruli, was upregulated threefold in the knockout mice, suggesting a role of nephrin in claudin 3 gene expression within the glomeruli. Our results suggest that nephrin is expressed late in the process of podocyte differentiation and is a locus for the formation of SD and FP maintenance and physical integrity in vivo. Nephrin does not seem to have a primary role in cell survival but has a small impact on gene regulation during glomerular development.

Glomerulus proteome analysis with two-dimensional gel electrophoresis and mass spectrometry.

A proteome analysis of mammalian glomeruli was performed in mouse using two-dimensional-gel electrophoresis with separate Coomassie and silver staining and subsequent mass spectrometric identifications. Altogether, 414 protein spots were identified, revealing 232 different proteins representing a wide spectrum of activities, including enzymes (27%), cell-signalling proteins (22%), structural proteins (12%), protein folding and metabolism (13%), cell-growth-related proteins (6%), and replication, transcription and translation (4%). Only 53 of the proteins were detected in another proteome study, showing the value of analyses with different methodologies. However, 50 of the proteins were also identified in a proteome analysis of endothelial cells and 42 in one of glomerular mesangial cells, revealing distinct similarities between these tissues, but also unique differences. Finally, 80 of the proteins were not identified in a separate transcriptome analysis, while 10 of the present proteins were then indentifiable with genes implicated in glomerulus development and function, allowing direct correlation with expression data.

Glomerulus-specific mRNA transcripts and proteins identified through kidney expressed sequence tag database analysis.

The kidney glomerulus plays a crucial role in blood filtration but the molecular composition and physiology of the glomerulus is not well understood. We previously constructed and large-scale sequenced four mouse glomerular expressed sequence tag (EST) libraries from newborn and adult mouse glomeruli. Here, we compared glomerular EST profiles with whole kidney EST profiles, thereby identifying 497 transcripts corresponding to UniGene clusters that were glomerulus-enriched, that is expressed more abundantly in glomeruli than in whole kidney. These include several known protein-coding glomerulus-specific transcripts critical for glomerulus development and function, but also a large number of gene transcripts, which have not previously been shown to be expressed in the glomerulus, or implicated in glomerular functions. We used in situ hybridization to demonstrate glomerulus-specific RNA expression for six novel glomerular genes and the public Human Protein Atlas to verify glomerular protein expression for another two. The higher mRNA abundance for the eight genes in glomeruli compared with whole kidney was also verified by Taqman quantitative polymerase chain reaction. We surmise that the further characterization of these genes and proteins will increase our understanding of glomerular development and physiology.

Genome-wide scan for type 1 diabetic nephropathy in the Finnish population reveals suggestive linkage to a single locus on chromosome 3q.

Diabetic nephropathy (DN) is the primary cause of morbidity and mortality in patients with type 1 as well as type 2 diabetes, and accounts for 40% of end-stage renal disease in the Western world. Familial clustering of DN suggests importance of genetic factors in the development of the disease. In the present study, we performed a two-stage genome-wide scan to search for chromosomal loci containing susceptibility genes for nephropathy in patients with type 1 diabetes. In total, 83 discordant sib pairs (DSPs), sibs concordant for type 1 diabetes but discordant for nephropathy, were collected from Finland, a homogeneous population with one of the highest incidences of type 1 diabetes. To map loci for DN, we applied DSP analysis to detect linkage. In the initial scan, 73 DSPs were typed using 900 markers with an average intermarker distance of approximately 4 cM. Multipoint DSP analysis identified five chromosome regions (3q, 4p, 9q, 16q, and 22p) with maximum logarithm of odds (LOD) score (MLS) >or=1.0 (corresponding to a nominal P-value

Hep27, a member of the short-chain dehydrogenase/reductase family, is an NADPH-dependent dicarbonyl reductase expressed in vascular endothelial tissue.

Human Hep27 was originally isolated from growth-arrested HepG2 cells and identified as a member of the superfamily of short-chain dehydrogenases/reductases (SDR). Its substrate specificity has not been determined, but a cross-species comparison suggests that it occurs in widely divergent species, such as human, Cenorhabditis elegans, Drosophila and Arabidopsis thaliana. In this study, Hep27 was expressed as a His(6) fusion protein, and subjected to a substrate screen, using a compound library of SDR substrates, comprising steroids, retinoids, sugars and carbonyl compounds. Whereas no steroid dehydrogenase or retinoid activity was detected, it was found that Hep27 catalyzed the NADPH-dependent reduction of dicarbonyl compounds, like 3,4-hexanedione and 1-phenyl-1,2-propanedione with similar turnover numbers as DCXR (a mitochondrial dicarbonyl reductase/xylulose reductase). In contrast, Hep27 does not convert sugar substrates like xylulose or threose. Based on its substrate specificity and expression in endothelial tissues, it is suggested that Hep27 functions as a dicarbonyl reductase in enzymatic inactivation of reactive carbonyls, involved in covalent modification of cellular components.

The effect of dexamethasone on defective nephrin transport caused by ER stress: a potential mechanism for the therapeutic action of glucocorticoids in the acquired glomerular diseases.

The mechanism by which glucocorticoids govern antiproteinuric effect in nephrotic syndrome remains unknown. Present study examined the protective role of dexamethasone (DEX) in the intracellular trafficking of nephrin under endoplasmic reticulum (ER) stress. Human embryonic kidney-293 cell line expressing a full-length human nephrin was cultured in mediums containing 5.5 or 25 mM glucose with or without DEX. The result revealed that glucose starvation evoked a rapid ER stress leading to formation of underglycosylated nephrin that was remained in the ER as a complex with calreticulin/calnexin. DEX rescued this interfered trafficking through binding to its receptor and stimulating the mitochondrial transcripts and adenosine 5' triphosphate (ATP) production, leading to synthesis of fully glycosylated nephrin. These results suggest that ER-stress in podocytes may cause alteration of nephrin N-glycosylation, which may be an underlying factor in the pathomechanism of the proteinuria in nephrotic syndrome. DEX may restore this imbalance by stimulating expression of mitochondrial genes, resulted in the production of ATP that is essential factor for proper folding machinery aided by the ER chaperones.

Differential expression of a gene signature for scavenger/lectin receptors by endothelial cells and macrophages in human lymph node sinuses, the primary sites of regional metastasis.

Sentinel lymph node biopsy for several cancers has shown that metastatic tumour cells are preferentially arrested in the lymph node sinuses. To study the molecular components of this sinusoidal trap, gene profiling of lymph node (sinuses) versus tonsil (no sinuses) was performed. Among other groups of molecules, an intriguing gene signature of scavenger and lectin-like receptors was identified. Nine of the 13 genes were preferentially expressed in sinusoidal cells by immunohistochemistry. Using stabilin-2 and monoclonal antibody 3A5 as exclusive endothelial cell (EC) and macrophage (Mvarphi) markers, respectively, lymph node sinusoidal ECs (stabilin-2+, LYVE-1+, DC-SIGNR+, MARCO+, stabilin-1+, MMR+) and sinusoidal Mvarphi (MMR+, DC-SIGN+, sialoadhesin+, CD163+, stabilin-1+ ) showed distinct, but overlapping expression patterns of the signature molecules by double labelling immunofluorescence. The number of stabilin-1+ sinusoidal Mvarphi, however, varied considerably between samples, indicating turnover/differentiation dynamics in this sinusoidal cell population. In the hepatic sinuses, LYVE-1 and CD36 were strongly up-regulated on both sinusoidal ECs and Mvarphi, while DC-SIGNR and DC-SIGN were strongly down-regulated; in contrast to lymph node sinusoidal ECs, MARCO was confined to Mvarphi (Kupffer cells) in the liver sinuses. As Mvarphi are not present in the wall and lumen of splenic sinuses, splenic sinuses expressed a considerably reduced repertoire of scavenger/lectin receptors lacking sialoadhesin, CD36, CD163, and MARCO; in addition, DC-SIGNR was absent from splenic sinusoidal ECs, while DC-SIGN and thrombomodulin were strongly expressed. Interestingly, most of the signature molecules are known to mediate tumour cell adhesion in addition to their functions as scavenger or pattern recognition receptors. This study establishes a gene and tissue database platform to test the hypothesis that additive expression of the lymph node sinus signature genes in sinusoidal ECs and Mvarphi may contribute to selective tumour cell metastasis in lymph nodes and liver including organ-specific mechanisms, such as intraluminal retention or transmigration, while sparing the spleen.

Gain of chromosome 3q is an early and consistent genetic aberration in carcinomas of the vulva.

The aim was to determine whether specific gains of chromosome 3q and laminin-5gamma2-chain expression can improve early detection of invasive capacity in precancerous and squamous cell carcinoma of the vulva (VSCC). Six VSCC and three precancerous lesions were studied. Multicolor fluorescence in situ hybridization (FISH) probe sets were applied to nuclei suspensions prepared from archival material using the Hedley method. The probe panel consists of the centromers of chromosome 7, chromosome 3, and the TERC gene residing on the long arm of chromosome 3. Laminin-5gamma2-chain immunohistochemical analysis was performed on corresponding specimens and was expressed only in the VSCC. The genome-specific FISH analysis revealed 3q amplification in 43% of the nuclei analyzed for the VSCC and 22% of the nuclei for the precancerous lesions. Low-level 3q amplifications were found in precancerous lesions with an average fold increase of 1.15 for 3q. The invasive lesions showed higher average fold increases for 3q, averaging 1.32. Laminin-5gamma2-chain protein was expressed only in VSCC, whereas 3q gains were observed both in precancerous lesions and in VSCC, indicating that gain of chromosome 3q is an early and consistent event during carcinogenesis of VSCC.

Degradation of proinsulin C-peptide in kidney and placenta extracts by a specific endoprotease activity.

Degradation of proinsulin C-peptide in mouse kidney and human placenta extracts was studied using reverse-phase high-performance liquid chromatography and nano-electrospray mass spectrometry. In total, 15 proteolytic cleavage sites were identified in human and mouse C-peptides. Early sites included the peptide bonds N-terminal of Val/Leu10, Leu12, Leu21, Leu24 and Leu26 in different combinations for the two tissues and two peptides. Notably, these cleavages were N-terminal of a hydrophobic residue, and all but one N-terminal of Leu. A late degradation product of the human peptide detected in the kidney extract was the C-terminal hexapeptide, containing just one residue more than the biologically active C-terminal pentapeptide of C-peptide. We conclude that the degradation of C-peptide in kidney and placenta follows similar patterns, dominated by endopeptidase cleavages N-terminal of Leu.

Laminin 5 gamma 2 chain expression: a marker of early invasiveness in colorectal adenomas.

AIM: Polyps of the colon and rectum are considered to be premalignant lesions in the development of colorectal cancer. However, knowledge of how normal epithelial cells gain invasive properties is limited. Laminin 5 gamma 2 chain expression was investigated to determine the role of laminin 5 as a marker of potential invasiveness in colorectal polyps. MATERIAL/METHODS: Sixty seven polyps of different types (15 hyperplastic polyps, 12 serrated adenomas, 16 tubular adenomas, and 24 adenomas with a villous component) were assessed for gamma 2 chain expression of laminin 5 by immunohistochemistry on archival, paraffin wax embedded sections. RESULTS: Ten polyps stained positive and the number of polyps expressing the laminin 5 gamma 2 chain increased significantly as the phenotype of the adenomas became more atypical: none of the 15 hyperplastic polyps, two of the 16 tubular adenomas (12.5%), and six of the 24 adenomas with a villous component (25%) were positive. Two of 12 (17%) serrated adenomas, regarded as a distinct form of colorectal neoplasia, showed gamma 2 chain expression. Furthermore, laminin 5 gamma 2 chain expression correlated with lesion size. Polyps smaller than 10 mm expressed the gamma 2 chain less frequently than did those equal to or larger than 10 mm. CONCLUSION: Laminin 5 gamma 2 chain expression was found to increase progressively towards a more atypical phenotype of adenoma. The results suggest that, in the future, laminin 5 gamma 2 chain expression may be used as an indicator of incipient malignant transformation of a benign colorectal adenoma.

Causes and consequences of proteinuria: the kidney filtration barrier and progressive renal failure.

The past few years have witnessed a major breakthrough in the understanding of the molecular mechanisms and ultrastructural changes behind the development of proteinuria. The discovery of several proteins in the glomerular podocyte and slit diaphragm, where mutations lead to disease, has revealed the importance of this cell with its diaphragm as the major filtration barrier as opposed to the glomerular basement membrane (GBM) previously ascribed this function. Furthermore, accumulating clinical as well as experimental evidence points to the harmful effects of proteinuria, irrespective of the original damage. The purpose of this review is to shed light on what we know today about the two sides of this 'coin', the causes and the consequences of proteinuria.

Laminin-5 gamma2-chain expression and DNA ploidy as predictors of prognosis in endometrial carcinoma.

Expression of the laminin-5 gamma2-chain in carcinoma cells has been implicated in tumor invasion. The aim was to investigate the expression and prognostic significance of the ln-5 gamma2-chain compared with clinicopathological factors and tumor cell DNA ploidy in endometrial carcinoma. Histological specimens from 80 endometrial carcinomas were examined with respect to immunohistochemical ln-5 gamma2-chain expression and correlated to the clinicopathological characteristics, DNA ploidy, and survival. Sixty-eight of 80 investigated cases were judged to be positive for the ln-5 gamma2-chain. Ln-5 gamma2-chain did not show any correlation to stage, histopathological subtype, grade, and DNA ploidy. In univariate analyses, advanced stage (p < 0.001), nonendometrioid carcinoma (p = 0.030), low grade (p < 0.001), aneuploid tumors (p < 0.001), and ln-5 gamma2-chain expression (p = 0.017) were highly associated with poor survival. Aneuploid tumors in combination with strong ln-5 gamma2-chain expression were significant predictors (p < 0.001) of poor prognosis. In multivariate analyses including stage, histopathological subgroup, grade, DNA ploidy, and ln-5 gamma2-chain expression, all lost their significant prognostic information except for stage (p < 0.001) and grade (p < 0.05). Ln-5 gamma2-chain expression and DNA ploidy both as a single parameter and in combination were demonstrated to be signifi- cant prognostic factors in univariate analysis. However, stage and grade provided more useful clinical information beyond histopathological subgroup, DNA ploidy, and ln-5 gamma2-chain expression. The results also indicate that ln-5 gamma2-chain expression is upregulated during the progression of endometrial carcinoma.

Laminin-5 gamma 2 chain as an invasivity marker for uni- and multifocal lesions in the lower anogenital tract.

During recent decades it has become apparent that there are two types of vulvar disease: the classic type found in elderly women with unicentric and unifocal lesions, and the type found in younger women, in which precancerous and invasive changes develop in the anogenital lower tract in a multicentric and multifocal fashion, often over a long period of observation. The laminin-5 gamma 2 chain is an extracellular protein that is a component of the basement membrane. Recently its expression has been recognized as a marker in cervical cancer that permits identification of invasive capacity. The aim of our study was to determine if laminin-5 gamma 2 chain antibody can act as a sensitivity marker of invasive capacity in precancerous and invasive carcinoma in women with uni- and multifocal changes in the anogenital tract. The result showed that all patients in the older group of women with invasive carcinoma of the vulva had moderate to high positive expression of the laminin-5 gamma 2 chain. In the group of younger patients with multifocal precancerous changes observed over long periods, most of the patients with vulva intraepithelial neoplasia (VIN) 3 showed laminin-5 gamma 2 chain positivity already in the precancerous changes, and all of them developed invasivity during the period of observation. Normal epithelium without atypia was mostly negative or of low immunoreactivity of laminin-5. In conclusion, positive laminin-5 gamma 2 chain expression seems to indicate the invasiveness potential of precancerous lesions and is also expressed in all investigated invasive carcinomas of the anogenital tract.

Defective nephrin trafficking caused by missense mutations in the NPHS1 gene: insight into the mechanisms of congenital nephrotic syndrome.

Congenital nephrotic syndrome of the Finnish type (CNF or NPHS1) is an autosomal recessive kidney disorder resulting in severe proteinurea and renal dysfunction. Although the disease occurs predominantly in the Finnish population, many cases in other populations have also been reported. The disease gene (NPHS1) encodes nephrin, a podocyte transmembrane protein that is an essential component of the podocyte slit diaphragm, the renal ultrafilter. Since the discovery of the gene, many mutations have been reported in the NPHS1 gene in patients with diverse ethnic background. A surprisingly large number of these mutations are missense mutations resulting in single amino acid substitutions. In order to study the pathomechanism of these missense mutations, we have investigated the fate of 21 such mutations hitherto identified in NPHS1 patients. Immunostaining of stable transfected cells expressing the nephrin mutants demonstrated that most of the mutants showed only endoplasmic reticulum (ER) staining and no detectable cell surface localization. Immunoelectron microscopy of cells expressing the wild-type and a mutant nephrin further confirmed that the mutant nephrin could be abundantly found in the ER but not on the plasma membrane. Subcellular fractionation of wild-type and a mutant cell line clearly showed an altered subcellular distribution and molecular mobility of the mutant nephrin. In summary, our data indicate that a defective intracellular nephrin transport, most likely due to misfolding, is the most common consequence of missense mutations in NPHS1.

Genetic kidney diseases disclose the pathogenesis of proteinuria.

The sieving of plasma components occurs in the kidney through the glomerular capillary wall. This filter is composed of three layers: endothelium, glomerular basement membrane (GBM), and podocyte foot processes connected by slit diaphragms. Defects in this barrier lead to proteinuria and nephrotic syndrome. Previously, defective GBM was regarded to be responsible for proteinuria. However, recent work on genetic diseases has indicated that podocytes and the slit diaphragm are crucial in restricting protein leakage. Congenital nephrotic syndrome of the Finnish type (NPHS1) is caused by mutations in a novel NPHS1 gene, which encodes for a cell adhesion protein, nephrin. This protein is synthesized by podocytes, and seems to be a major component of the slit diaphragm. In severe NPHS1, lack of nephrin leads to missing slit diaphragm. The role of nephrin in acquired kidney diseases remains unknown. In addition to nephrin, other podocyte proteins (podocin, alpha-actinin-4, CD2AP, FAT) have recently been identified and associated with the development of proteinuria. It seems that the slit diaphragm and its interplay with the podocyte cytoskeleton is critical for the normal sieving process, and defects in one of these components easily lead to proteinuria.

Unraveling the molecular make-up of the glomerular podocyte slit diaphragm.

Recent discoveries in podocyte proteins involved in the renal filtration barrier have shed new light on the ultrastructure of the kidney filter and pathogenesis of proteinuria. The identification of nephrin, a component of the slit diaphragm, and the intracellular slit diaphragm associated proteins CD2AP and podocin has demonstrated the existence of proteins that directly contribute to a functional kidney filter. Mutations in the genes for these three proteins result in proteinuria and nephrotic syndrome, and these proteins are also likely to be involved more generally in the pathomechanisms of proteinuria. This new knowledge has been promoted particularly through the powerful methods of molecular genetics and molecular biology. In this minireview, we present the recent progress in research of the podocyte slit diaphragm.

Biochemical defects in 11-cis-retinol dehydrogenase mutants associated with fundus albipunctatus.

Mutations in the gene encoding 11-cis-retinol dehydrogenase (RDH5; EC ) are associated with fundus albipunctatus, an autosomal recessive eye disease characterized by stationary night blindness and accumulation of white spots in the retina. In addition, some mutated alleles are associated with development of cone dystrophy, especially in elderly patients. The numbers of identified RDH5 mutations linked to fundus albipunctatus have increased considerably during recent years. In this work, we have characterized the biochemical and cell biological properties of 11 mutants of RDH5 to understand the molecular pathology of the disease. All RDH5 mutants showed decreased protein stability and subcellular mislocalization and, in most cases, loss of enzymatic activity in vitro and in vivo. Surprisingly, mutant A294P displays significant enzymatic activity. Cross-linking studies and molecular modeling showed that RDH5 is dimeric, and co-expression analyses of wild-type and mutated alleles showed that the mutated enzymes, in a trans-dominant-negative manner, influenced the in vivo enzymatic properties of functional variants of the enzyme, particularly the A294P mutant. Thus, under certain conditions, nonfunctional alleles act in a dominant-negative way on functional but relatively unstable mutated alleles. However, in heterozygous individuals carrying one wild-type allele, the disease is recessive, probably due to the stability of the wild-type enzyme.

Laminin-5 gamma 2 chain expression correlates with unfavorable prognosis in colon carcinomas.

Expression of the gamma 2 chain at the invasive front of different tumors has indicated an important role for laminin-5 in cell migration during tumor invasion and tissue remodeling. As there is considerable need for reliable invasion and prognostic markers we evaluated the correlation of laminin-5 gamma 2 chain expression with clinicopathologic parameters and patient survival in 93 primary colon carcinomas. Epithelial cells of normal mucosa were consistently negative for staining. In contrast, positive cytoplasmic staining was observed in 89 tumors (96%). Twenty-four (26%) cases were scored as sparse, 34 (37%) as moderate, and 31 (33%) as frequent gamma 2 chain expression. There was a significant association of laminin-5 gamma 2 chain expression and local invasiveness of colon carcinomas according to Dukes stage (A-C) (p=0.001) and tumor budding (p<0.001). A statistical significance could also be noted in decreasing tumor differentiation (p<0.001) and correlation to tumor size (p=0.032). No correlation was observed to tumor site. Univariate analysis identified laminin-5 (p=0.010), tumor differentiation (p=0.006) and Dukes grade (p<0.001) as significant variables in predicting prognosis. However, by multivariate analyses, this study could not demonstrate that laminin-5 gamma 2 chain expression is an independent predictive factor for survival. The results indicate that laminin-5 gamma 2 chain expression is up-regulated during the progression of human colon cancer and that it plays a role in the aggressiveness of these tumors. Demonstration of laminin-5 gamma 2 chain positivity also facilitates detection of individual cells or minor cell clusters invading the surrounding stroma. Figures on http://www.esacp.org/acp/2001/22-4/lenander.htm.

Ulcerative colitis and colorectal carcinoma: DNA-profile, laminin-5 gamma2 chain and cyclin A expression as early markers for risk assessment.

BACKGROUND: Ulcerative colitis patients are at increased risk for developing colorectal carcinomas. Despite expensive surveillance programmes, clinical practice reflects an uncertainty in individual risk assessment. The aim of the study was to evaluate independent cellular features with possible predictive value. METHODS: Two patient groups were selected: group A comprised 8 patients with ulcerative colitis-associated colorectal carcinomas, group B comprised 16 ulcerative colitis patients with risk factors (duration of disease, extent of inflammation, epithelial dysplasias). A total of 683 paraffin-embedded mucosal biopsies were retrospectively evaluated for inflammatory activity, grade of dysplasia, ploidy status, laminin-5 gamma2 chain and cyclin A expression. RESULTS: Mild or moderate inflammatory activity was present in 78% of all biopsies, low- or high-grade dysplasia in 5.5%. There was no difference in inflammatory activity and dysplasia between patient groups. In group A, 75% of the biopsies exhibited aneuploid DNA distribution patterns. Group B showed mainly proliferative-diploid cell populations (85% / P = 0.006). Laminin-5 gamma2 chain was expressed in 13% of all biopsies, with a higher frequency in group A (P = 0.002). Cyclin A expression was found in 98% of all biopsies, with a higher number of immunopositive cells in group A biopsies (P = 0.014). CONCLUSIONS: Combined nuclear DNA assessment, laminin-5 gamma2 chain and cyclin A expression may help to identify ulcerative colitis patients with an increased risk for cancer development.