Biomedical subjects

K Tryggvason

Publications and source records attributed to K Tryggvason.

At least 19 recordsLinked to original sources

PubMed2006

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 <or=0.015). In the second stage, additional 43 markers flanking these five loci were genotyped in all 83 DSPs. Using simulations, we determined the empirical threshold with LOD score of 1.76 and 3.12 for suggestive and significant linkage, respectively. No locus reached the genome-wide significance of 5%. However, one locus on 3q reached suggestive linkage with MLS of 2.67 (P=4.4 x 10(-4)). These results, together with data from others, suggest that the locus on 3q most likely has a susceptibility gene for DN.

Adolescent↗

PubMed2006

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.

Biomarkers↗

PubMed2006

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.

Alcohol Dehydrogenase↗

PubMed2006

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.

Adenosine Triphosphate↗

PubMed2004

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.

Amino Acid Sequence↗

PubMed2003

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.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2003

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.

Adenoma↗

PubMed2003

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.

Cell Adhesion Molecules↗

PubMed2001

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.

Biological Transport↗

PubMed2001

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.

Alcohol Oxidoreductases↗

PubMed2001

Biosynthesis of 9-cis-retinoic acid in vivo. The roles of different retinol dehydrogenases and a structure-activity analysis of microsomal retinol dehydrogenases.

Retinoic acid is generated by a two-step mechanism. First, retinol is converted into retinal by a retinol dehydrogenase, and, subsequently, retinoic acid is formed by a retinal dehydrogenase. In vitro, several enzymes are suggested to act in this metabolic pathway. However, little is known regarding their capacity to contribute to retinoic acid biosynthesis in vivo. We have developed a versatile cell reporter system to analyze the role of several of these enzymes in 9-cis-retinoic acid biosynthesis in vivo. Using a Gal4-retinoid X receptor fusion protein-based luciferase reporter assay, the formation of 9-cis-retinoic acid from 9-cis-retinol was measured in cells transfected with expression plasmids encoding different combinations of retinol and retinal dehydrogenases. The results suggested that efficient formation of 9-cis-retinoic acid required co-expression of retinol and retinal dehydrogenases. Interestingly, the cytosolic alcohol dehydrogenase 4 failed to efficiently catalyze 9-cis-retinol oxidation. A structure-activity analysis showed that mutants of two retinol dehydrogenases, devoid of the carboxyl-terminal cytoplasmic tails, displayed greatly reduced enzymatic activities in vivo, but were active in vitro. The cytoplasmic tails mediate efficient endoplasmic reticulum localization of the enzymes, suggesting that the unique milieu in the endoplasmic reticulum compartment is necessary for in vivo activity of microsomal retinol dehydrogenases.

Alcohol Dehydrogenase↗

PubMed2001

The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death.

A mouse model for congenital nephrotic syndrome (NPHS1) was generated by inactivating the nephrin gene (Nphs1) in embryonic stem cells by homologous recombination. The targeting construct contained the Escherichia coli lacZ gene as a reporter for the Nphs1 promoter. Mice homozygous for inactivated Nphs1 were born at an expected frequency of 25%. Although seemingly normal at birth, they immediately developed massive proteinuria and edema and died within 24 h. The kidneys of null mice exhibited enlarged Bowman's spaces, dilated tubuli, effacement of podocyte foot processes and absence of the slit diaphragm, essentially as found in human NPHS1 patients. In addition to expression in glomerular podocytes, the reporter gene was expressed in the brain and pancreas of (+/-) and (-/-) mice. In the brain, expression was localized to the ventricular zone of the fourth ventricle, the developing spinal cord, cerebellum, hippocampus and olfactory bulb. In the cerebellum, the expression was seen in radial glial cells. Neither anatomical nor morphological abnormalities were observed in the brains of null mice.

Alleles↗

PubMed2001

Mutation spectrum in the nephrin gene (NPHS1) in congenital nephrotic syndrome.

Congenital nephrotic syndrome, Finnish type (CNF or NPHS1), is an autosomal recessive disease characterized by massive proteinuria and development of nephrotic syndrome shortly after birth. The disease is most common in Finland, but many patients have been identified in other populations. The disease is caused by mutations in the gene for nephrin which is a key component of the glomerual ultrafilter, the podocyte slit diaphragm. A total of 30 mutations have been reported in the nephrin gene in patients with congenital nephrotic syndrome worldwide. In the Finnish population, two main mutations have been found. These two nonsense mutations account for over 94% of all mutations in Finland. Most mutations found in non-Finnish patients are missense mutations, but they include also nonsense and splice site mutations, as well as deletions and insertions. This mutation update summarizes the nature of all previously reported nephrin mutations and, additionally, describes 20 novel mutations recently identified in our laboratory.

Codon, Nonsense↗

PubMed2001

Two novel alternatively spliced 9-bp exons in the COL4A5 gene.

The existence of an alternatively used 18-bp sequence has been described in type IV collagen alpha 5 chain mRNA from human kidney (Guo et al., Kidney Int 44, 1993). In this study we have shown that this sequence is encoded by two exons, termed 41A and 41B, that are located in a 9-kb intron 41 that was sequenced in its entirety. The sequences of exon 41A and 41B were shown to be present in mRNAs from a variety of tissues.

Alternative Splicing↗

PubMed2001

Podocyte proteins in Galloway-Mowat syndrome.

Galloway-Mowat syndrome is an autosomal recessive disorder characterized by early onset nephrotic syndrome and central nervous system anomalies. Mutations in podocyte proteins, such as nephrin, alpha-actinin 4, and podocin, are associated with proteinuria and nephrotic syndrome. The genetic defect in Galloway-Mowat syndrome is as yet unknown. We postulated that in Galloway-Mowat syndrome the mutation would be in a protein that is expressed both in podocytes and neurons, such as synaptopodin, GLEPP1, or nephrin. We therefore analyzed kidney tissue from normal children (n=3), children with congenital nephrotic syndrome of the Finnish type (CNF, n=3), minimal change disease (MCD, n=3), focal segmental glomerulosclerosis (FSGS, n=3), and Galloway-Mowat syndrome (n=4) by immunohistochemistry for expression of synaptopodin, GLEPP1, intracellular domain of nephrin (nephrin-I), and extracellular domain of nephrin (nephrin-E). Synaptopodin, GLEPP1, and nephrin were strongly expressed in normal kidney tissue. Nephrin was absent, and synaptopodin and GLEPP1 expression were decreased in CNF. The expression of all three proteins was reduced in MCD and FSGS; the decrease in expression being more marked in FSGS. Synaptopodin, GLEPP1, and nephrin expression was present, although reduced in Galloway-Mowat syndrome. We conclude that the reduced expression of synaptopodin, GLEPP1, and nephrin in Galloway- Mowat syndrome is a secondary phenomenon related to the proteinuria, and hence synaptopodin, GLEPP1, and nephrin are probably not the proteins mutated in Galloway-Mowat syndrome.

Central Nervous System↗

PubMed2001

Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome.

We investigated the distribution of nephrin by immunofluorescence microscopy in renal biopsies of patients with nephrotic syndrome: 13 with membranous glomerulonephritis (GN), 10 with minimal change GN, and seven with focal segmental glomerulosclerosis. As control, six patients with IgA GN without nephrotic syndrome and 10 normal controls were studied. We found an extensive loss of staining for nephrin and a shift from a podocyte-staining pattern to a granular pattern in patients with nephrotic syndrome, irrespective of the primary disease. In membranous GN, nephrin was co-localized with IgG immune deposits. In the attempt to explain these results, we investigated in vitro whether stimuli acting on the cell cytoskeleton, known to be involved in the pathogenesis of GN, may induce redistribution of nephrin on the surface of human cultured podocytes. Aggregated but not disaggregated human IgG(4), plasmalemmal insertion of membrane attack complex of complement, tumor necrosis factor-alpha, and puromycin, induced the shedding of nephrin with a loss of surface expression. This phenomenon was abrogated by cytochalasin and sodium azide. These results suggest that the activation of cell cytoskeleton may modify surface expression of nephrin allowing a dislocation from plasma membrane to an extracellular site.

Adolescent↗

PubMed2001

Congenital nephrotic syndromes.

Many acquired and familial renal diseases in man lead to kidney dysfunction and nephrotic syndrome. These diseases share a common pathological fate in the form of glomerular dysfunction and proteinuria. Classification of the disease is difficult because the onset of pathological appearance in congenital nephrotic syndrome (CNS) varies considerably. Recently, classification has been aided by applying molecular genetics to identify genes involved in the pathogenesis of proteinuria. Light has also been shed on the biology and mechanisms of glomerular filtration and the molecular pathogenesis of CNS.

Animals↗

PubMed2001

Properly formed but improperly localized synaptic specializations in the absence of laminin alpha4.

Precise apposition of pre- to postsynaptic specializations is required for optimal function of chemical synapses, but little is known about how it is achieved. At the skeletal neuromuscular junction, active zones (transmitter release sites) in the nerve terminal lie directly opposite junctional folds in the postsynaptic membrane. Few active zones or junctional folds form in mice lacking the laminin beta2 chain, which is normally concentrated in the synaptic cleft. beta2 and the broadly expressed gamma1 chain form heterotrimers with alpha chains, three of which, alpha2, alpha4 and alpha5, are present in the synaptic cleft. Thus, alpha2beta2gamma1, alpha4beta2gamma1 and alpha5beta2gamma1 heterotrimers are all lost in beta2 mutants. In mice lacking laminin alpha4, active zones and junctional folds form in normal numbers, but are not precisely apposed to each other. Thus, formation and localization of synaptic specializations are regulated separately, and alpha4beta2gamma1 (called laminin-9) is critical in the latter process.

Animals↗