Biomedical subjects

Norbert Gretz

Publications and source records attributed to Norbert Gretz.

At least 19 recordsLinked to original sources

PubMed2006

Bias and precision of estimated glomerular filtration rate in children.

Determining true glomerular filtration rate (GFR) using an exogenous marker is time-consuming and cumbersome. Therefore, creatinine-based estimates of GFR are used. Recent papers using new population-specific/local parameters in their prediction equations, standardizing creatinine determination or adding other endogenous surrogate markers of GFR, like cystatin C, could demonstrate an improvement of bias inherent in the results of the prediction equations. Precision, however, is still poor. Currently, we have to accept a precision (as defined in the so-called Bland-Altman plot) of +/-20% in adults and +/-30-40% in children. This problem of poor precision/uncertainty is especially bothering in the higher, near normal GFR range. Caution should be exercised when applying prediction equations in individuals in need of an accurate GFR determination. In that case, a real clearance procedure has to be performed. In the long run, the true clearance procedure should be simplified using new exogenous GFR markers and developing new devices, allowing GFR measurements to be performed, for example, transcutaneously. Such a procedure would be more acceptable for both patients and physicians.

Adolescent↗

PubMed2006

A truncated polycystin-2 protein causes polycystic kidney disease and retinal degeneration in transgenic rats.

The cloning of the PKD1 and PKD2 genes has led to promising new insight into the mechanisms that are responsible for cyst development in patients with autosomal dominant polycystic kidney disease. Although the dominant pattern of inheritance would argue for haploinsufficiency, a gain of function, or a dominant negative mechanism, there is good evidence that autosomal dominant polycystic kidney disease behaves like a recessive disease on a cellular level (two-hit mechanism of cystogenesis). For testing of whether other pathomechanisms in addition to the two-hit hypothesis can explain cyst formation, two transgenic rat lines that contain a truncated human polycystin-2 cDNA were generated. The protein product lacks almost the entire COOH-terminus and mimics mutations that frequently are found in patients. The transgene-encoded mRNA could be detected in multiple tissues of both transgenic lines, with the highest expression in the kidney. Both lines present with renal cysts that originate predominantly from the proximal tubule; in the tubular epithelial cells, the epitope-tagged mutant protein was detected in the brush border and in primary cilia. Further evidence of the involvement of primary cilia stems from the finding of retinal degeneration in the transgenic rats and from the fact that stably transfected LLC-PK(1) cells that inducibly produced the truncated polycystin-2 protein elaborated shorter cilia. Other experimental approaches, such as a knock-in strategy, will be necessary to validate these results, but this is the first preliminary evidence that cyst formation is due not only to somatic mutations.

Animals↗

PubMed2006

Group testing for pathway analysis improves comparability of different microarray datasets.

MOTIVATION: The wide use of DNA microarrays for the investigation of the cell transcriptome triggered the invention of numerous methods for the processing of microarray data and lead to a growing number of microarray studies that examine the same biological conditions. However, comparisons made on the level of gene lists obtained by different statistical methods or from different datasets hardly converge. We aimed at examining such discrepancies on the level of apparently affected biologically related groups of genes, e.g. metabolic or signalling pathways. This can be achieved by group testing procedures, e.g. over-representation analysis, functional class scoring (FCS), or global tests. RESULTS: Three public prostate cancer datasets obtained with the same microarray platform (HGU95A/HGU95Av2) were analyzed. Each dataset was subjected to normalization by either variance stabilizing normalization (vsn) or mixed model normalization (MMN). Then, statistical analysis of microarrays was applied to the vsn-normalized data and mixed model analysis to the data normalized by MMN. For multiple testing adjustment the false discovery rate was calculated and the threshold was set to 0.05. Gene lists from the same method applied to different datasets showed overlaps between 42 and 52%, while lists from different methods applied to the same dataset had between 63 and 85% of genes in common. A number of six gene lists obtained by the two statistical methods applied to the three datasets was then subjected to group testing by Fisher's exact test. Group testing by GSEA and global test was applied to the three datasets, as well. Fisher's exact test followed by global test showed more consistent results with respect to the concordance between analyses on gene lists obtained by different methods and different datasets than the GSEA. However, all group testing methods identified pathways that had already been described to be involved in the pathogenesis of prostate cancer. Moreover, pathways recurrently identified in these analyses are more likely to be reliable than those from a single analysis on a single dataset.

Biomarkers, Tumor↗

PubMed2006

Distinct immunohistochemical expression of osteopontin in the adult rat major salivary glands.

Osteopontin is a multifunctional protein secreted by epithelial cells of various tissues. Its expression in the adult rat major salivary glands has not yet been studied. We examined osteopontin expression by immunohistochemistry using a well characterized monoclonal antibody. Submandibular glands of young adult male rats (70-100 days old) showed specific expression in secretion granules of granular duct cells but also in cells of the striated ducts and excretory duct. In the major sublingual as well as the parotid gland expression was found solely in the duct system. In addition, a few interstitial-like cells exhibiting very strong immunostaining for osteopontin could be found in either organ. Expression could neither be seen in acinar cells nor in cells of the intercalated ducts. Moreover, in submandibular glands of more aged rats (6- to 7-month old) which show well developed granular convoluted tubules, there was almost exclusive expression of osteopontin in granular duct cells as well as in some interstitial-like cells, but barely in the striated/excretory duct system. Western blot analysis of the submandibular gland showed a specific band migrating at approximately 74 kDa, detectable at both age stages. Osteopontin secreted fom granular duct cells may influence the composition of the saliva, e.g. thereby modulating pathways affecting sialolithiasis. Its expression in striated duct cells may also hint to roles such as cell-cell attachment or cell differentiation. The cell-specific expression detected in the rat major salivary glands differs in part from that reported in mice, human and monkey.

Aging↗

PubMed2006

Pharmacological profile and toxicity of fluorescein-labelled sinistrin, a novel marker for GFR measurements.

There is an evident and growing medical need for an accurate determination of kidney function for a broad spectrum of indications. The glomerular filtration rate (GFR) is the most accepted indicator of renal function. Due to difficulties in performing the test, GFR is currently determined rarely in clinical practice. A procedure for such GFR determination has to be safe, accurate and easy to handle. By using the new compound fluorescein isothiocyanate-sinistrin (FS) these requirements are met. The pharmacological profile and tolerability of FS, selected from among various newly synthesized, labelled compounds intended for use as GFR markers, was characterized in male Sprague-Dawley rats following i.v. application. Using the newly described fluorometric method, FS can be determined much more easily in serum and urine than with the established enzymatic method. After i.v. dosing, FS concentrations in serum declined rapidly in various experimental groups to a comparable extent (t (1/2), mean+/-SD: 22.4+/-8.3 to 26.2+/-5.4 min). Its increase after unilateral nephrectomy reflects the loss of filtration capacity. Comparable concentration-time curves of FS in serum measured fluorometrically and enzymatically suggest no relevant alteration of pharmacokinetic behaviour by the labelling. This notion is supported by the high urinary excretion rate and absence of biliary excretion. The higher sensitivity of the fluorometric method suggests a dose of FS of 100 mg in humans compared with 5 g of sinistrin or inulin. FS was well tolerated after single and multiple applications. On the basis of these results, the kinetics of FS are comparable with the gold standard inulin or sinistrin, but FS is superior in handling. Providing the data can be transferred from rat to human, determination of GFR using the new method should result in an improvement of acceptance by both physicians and patients.

Animals↗

PubMed2006

Argonaute--a database for gene regulation by mammalian microRNAs.

MicroRNAs (miRNAs) constitute a recently discovered class of small non-coding RNAs that regulate expression of target genes either by decreasing the stability of the target mRNA or by translational inhibition. They are involved in diverse processes, including cellular differentiation, proliferation and apoptosis. Recent evidence also suggests their importance for cancerogenesis. By far the most important model systems in cancer research are mammalian organisms. Thus, we decided to compile comprehensive information on mammalian miRNAs, their origin and regulated target genes in an exhaustive, curated database called Argonaute (http://www.ma.uni-heidelberg.de/apps/zmf/argonaute/interface). Argonaute collects latest information from both literature and other databases. In contrast to current databases on miRNAs like miRBase::Sequences, NONCODE or RNAdb, Argonaute hosts additional information on the origin of an miRNA, i.e. in which host gene it is encoded, its expression in different tissues and its known or proposed function, its potential target genes including Gene Ontology annotation, as well as miRNA families and proteins known to be involved in miRNA processing. Additionally, target genes are linked to an information retrieval system that provides comprehensive information from sequence databases and a simultaneous search of MEDLINE with all synonyms of a given gene. The web interface allows the user to get information for a single or multiple miRNAs, either selected or uploaded through a text file. Argonaute currently has information on 839 miRNAs from human, mouse and rat.

Animals↗

PubMed2005

Missense mutation in sterile alpha motif of novel protein SamCystin is associated with polycystic kidney disease in (cy/+) rat.

Autosomal dominant polycystic kidney disease (PKD) is the most common genetic disease that leads to kidney failure in humans. In addition to the known causative genes PKD1 and PKD2, there are mutations that result in cystic changes in the kidney, such as nephronophthisis, autosomal recessive polycystic kidney disease, or medullary cystic kidney disease. Recent efforts to improve the understanding of renal cystogenesis have been greatly enhanced by studies in rodent models of PKD. Genetic studies in the (cy/+) rat showed that PKD spontaneously develops as a consequence of a mutation in a gene different from the rat orthologs of PKD1 and PKD2 or other genes that are known to be involved in human cystic kidney diseases. This article reports the positional cloning and mutation analysis of the rat PKD gene, which revealed a C to T transition that replaces an arginine by a tryptophan at amino acid 823 in the protein sequence. It was determined that Pkdr1 is specifically expressed in renal proximal tubules and encodes a novel protein, SamCystin, that contains ankyrin repeats and a sterile alpha motif. The characterization of this protein, which does not share structural homologies with known polycystins, may give new insights into the pathophysiology of renal cyst development in patients.

Animals↗

PubMed2005

Cell-specific deletion of glucosylceramide synthase in brain leads to severe neural defects after birth.

Sialic acid-containing glycosphingolipids, i.e., gangliosides, constitute a major component of neuronal cells and are thought to be essential for brain function. UDP-glucose:ceramide glucosyltransferase (Ugcg) catalyzes the initial step of glycosphingolipid (GSL) biosynthesis. To gain insight into the role of GSLs in brain development and function, a cell-specific disruption of Ugcg was performed as indicated by the absence of virtually all glucosylceramide-based GSLs. Shortly after birth, mice showed dysfunction of cerebellum and peripheral nerves, associated with structural defects. Axon branching of Purkinje cells was significantly reduced. In primary cultures of neurons, dendritic complexity was clearly diminished, and pruning occurred early. Myelin sheaths of peripheral nerves were broadened and focally severely disorganized. GSL deficiency also led to a down-regulation of gene expression sets involved in brain development and homeostasis. Mice died approximately 3 weeks after birth. These results imply that GSLs are essential for brain maturation.

Animals↗

PubMed2005

Bedside estimation of the glomerular filtration rate in hospitalized elderly patients.

AIMS: To evaluate the quality of bedside estimation of glomerular filtration rate (GFR) in hospitalized elderly patients. METHODS: We evaluated common estimators of GFR in 29 women and 32 men aged 60 and older hospitalized in a geriatric ward: creatinine clearance (CCR), the Cockcroft-Gault formula (CG), the modification of diet in renal disease formula (MDRD), Baracskay formula (BAR), and a newly developed formula derived recently by us (GCM). Inulin clearance (CINU) was used to assess GFR. Exclusion criteria were mental illness and urinary incontinence. RESULTS: According to Bland and Altman accuracy and precision of all estimators were low and there was an underestimation of actual GFR: CCR 38.9 ml/min; CG 39.7 ml/min; MDRD 19.8 ml/min; BAR 34.0 ml/min, and GCM: 24.7 ml/min. The accuracy and precision of all methods were even lower in patients with a GFR of >90 ml/min and in patients with diabetes. In receiver-operating characteristics (ROC analysis) all formulas were superior to serum creatinine and overall MDRD disclosed the best results in detecting both a GFR of <90 ml/min and <60 ml/min. CONCLUSIONS: In general, estimation errors are large in an acute care setting. However, formula estimation is clearly superior to serum creatinine and CCR. MDRD gave the best results but may be replaced by the more simple CG and GCM formulas, whereas BAR was inferior.

Aged↗

PubMed2005

Direct fluorometric analysis of a newly synthesised fluorescein-labelled marker for glomerular filtration rate.

There is an obvious and growing medical need for an accurate determination of kidney function in the diagnosis and management of renal diseases. The glomerular filtration rate (GFR) is the accepted gold standard measurement of kidney function. Several approaches to estimate the GFR are available, but most of them are inconvenient and, therefore, of limited acceptance. A new method of quantification with fluorescein-isothiocyanate (FITC) sinistrin (FS), a novel GFR marker, has been evaluated. The method is based on the fluorescence label of FS and can be performed with a standard fluorometer. To control the interference of protein with the fluorescence signal, a calibration function was developed. The accuracy of the fluorometric method established is comparable to the so-called "gold standard" of enzymatic determination of polyfructosan. Moreover, FS is easy to handle and requires low-cost instruments. Our results demonstrate the potential of the direct fluorometric analysis of the new FITC-labelled marker of being a precise, simple, rapid and cost-effective method for diagnosing disturbed kidney function and monitoring its treatment efficacy. The dramatic decrease in analytical effort will result in a significantly higher acceptability of GFR determination.

Animals↗

PubMed2005

Fluorescein-labeled sinistrin as marker of glomerular filtration rate.

There is an obvious and growing medical need for an accurate and easy to handle determination of glomerular filtration rate (GFR) for a broad spectrum of indications. Newly synthesized fluorescein-isothiocyanate (FITC)-sinistrin (FS) with various degrees of labeling was selected by its physicochemical properties and good tolerability out of a number of dye-labeled compounds intended for use as GFR markers for characterization of its pharmacological profile. With respect to solubility FS is more convenient in handling compared to FITC-inulin (FI). Up to 100 mg ml(-1) of FS can be dissolved in aqueous solvents at room temperature, whereas FI can only be solubilized after warming up to 55 degrees C. This reveals a considerable advantage of FS over FI in preparation of galenical formulations for intended i.v. application. A fluorometric method was established to determine FS concentration in blood serum with a comparable accuracy to the established enzymatic method for polyfructosanes. Similar concentration time curves in blood serum of FS measured fluorometrically and enzymatically suggest no relevant change of pharmacokinetic behavior by dye labeling. This notion is supported by the rapid renal and missing of biliary excretion. On the basis of these results, FS is superior in handling to the available GFR markers and makes it highly interesting as a novel diagnostic drug.

Animals↗

PubMed2005

13-cis retinoic acid inhibits development and progression of chronic allograft nephropathy.

Chronic allograft nephropathy is characterized by chronic inflammation and fibrosis. Because retinoids exhibit anti-proliferative, anti-inflammatory, and anti-fibrotic functions, the effects of low and high doses of 13-cis-retinoic acid (13cRA) were studied in a chronic Fisher344-->Lewis transplantation model. In 13cRA animals, independent of dose (2 or 20 mg/kg body weight/day) and start (0 or 14 days after transplantation) of 13cRA administration, serum creatinine was significantly lower and chronic rejection damage was dramatically reduced, including subendothelial fibrosis of preglomerular vessels and chronic tubulointerstitial damage. The number of infiltrating mononuclear cells and their proliferative activity were significantly diminished. The mRNA expression of chemokines (MCP-1/CCL2, MIP-1alpha/CCL3, IP-10/CXCL10, RANTES/CCL5) and proteins associated with fibrosis (plasminogen activator inhibitor-1, transforming growth factor-beta1, and collagens I and III) were strikingly lower in treated allografts. In vitro, activated peritoneal macrophages of 13cRA-treated rats showed a pronounced decrease in protein secretion of inflammatory cytokines (eg, tumor necrosis factor-alpha, interleukin-6). The suppression of the proinflammatory chemokine RANTES/CCL5 x 13cRA in fibroblasts could be mapped to a promoter module comprising IRF-1 and nuclear factor-kappaB binding elements, but direct binding of retinoid receptors to promoter elements could be excluded. In summary, 13cRA acted as a potent immunosuppressive and anti-fibrotic agent able to prevent and inhibit progression of chronic allograft nephropathy.

Animals↗

PubMed2004

Angiotensin II type 1 receptor overexpression in podocytes induces glomerulosclerosis in transgenic rats.

Angiotensin II (AngII) is a critical determinant of glomerular function involving both hemodynamic and pressure-independent effects that are insufficiently understood. A novel transgenic rat (TGR) model with overexpression of the human AngII type 1 receptor (hAT1) in podocytes was developed to study the consequences of an increased AT1 signaling on the structure and function of the glomerular filter. Use of the nephrin promoter to target the podocytes resulted in an expression of the hAT1 at a level roughly two times higher than the endogenous AT1 throughout life. All male TGR developed significant albuminuria starting at 8 to 15 wk of age; systolic BP was not elevated. More or less concurrently, structural changes at the glomerulus were encountered, starting with ubiquitous formation of pseudocysts at podocytes, followed by foot process effacement and local detachments. This damage progressed to nephron loss via the well known pathway typical for classic focal segmental glomerulosclerosis. The structural changes significantly correlated with age (r(2) = 0.76) and urinary albumin excretion (r(2) = 0.70). The data provide direct evidence that increased AT1 signaling in podocytes leads to protein leakage and structural podocyte damage progressing to focal segmental glomerulosclerosis.

Albuminuria↗

PubMed2004

Liver regeneration in FGF-2-deficient mice: VEGF acts as potential functional substitute for FGF-2.

BACKGROUND/AIMS: The angiogenic properties, its role in mesoderm differentiation and cell culture studies implicate an important role of fibroblast growth factor (FGF-2) in liver regeneration. The aim of the study was to evaluate this role in a FGF-2 knockout mouse model. METHODS: Liver regeneration after left hemihepatectomy (partial hepatectomy, PH) was evaluated in homozygous FGF-2 deficient (-/-) mice (male C57BL/6J) and their FGF-2 competent (+/+) littermates (controls) (day 0-10). RESULTS: FGF-2-(-/-) mice displayed normal dynamics in liver regeneration. FGF-2 protein was overexpressed 4 days post PH in controls. BrdU incorporation showed a biphasic pattern in FGF-2-(-/-) mice, whereas it decreased continuously after one peak (day 2) in controls. In FGF-2-(-/-) livers hepatic growth factor mRNA post PH was 1 day longer decreased and markedly less elevated thereafter compared with control. Vascular endothelial growth factor (VEGF) mRNA levels were clearly increased in FGF-2-(-/-) mice pre- and postoperatively in contrast to controls. VEGF protein levels in livers of FGF-2-(-/-) mice were elevated preoperatively, but similar in both groups after PH. With SU5416, a VEGF-receptor inhibitor, liver regeneration in FGF-2-(-/-) mice was reduced significantly, whereas it remained unchanged in controls. CONCLUSIONS: Liver regeneration dynamics in FGF-2-(-/-) mice were comparable with controls, potentially due to a functional substitution of FGF-2 by VEGF.

Angiogenesis Inhibitors↗

PubMed2004

Immunohistochemical and mRNA localization of the angiotensin II receptor subtype 2 (AT2) in follicular granulosa cells of the rat ovary.

A local renin-angiotensin system (RAS), including specific angiotensin II receptor subtypes, is present in the rat ovary. Immunohistochemistry using a polyclonal antibody and mRNA in situ hybridization were performed on perfusion-fixed, paraffin-embedded ovaries obtained from untreated sexually mature, normally cycling rats. Immunofluorescent staining revealed an exclusive and distinct labeling of follicular granulosa cells showing a plaque-like expression pattern at the cell borders, being detectable in different stages of atretic degeneration. On adjacent sections the expression of the respective mRNA could be shown in granulosa cells of the same follicle. The AT2 receptor may be implicated in the ovarian atretic process by influencing follicular cell-cell communication.

Animals↗

PubMed2003

Enhanced green fluorescent protein-transfection of murine colon carcinoma cells: key for early tumor detection and quantification.

Many animal models for metastatic colorectal cancer represent clinical manifestations just inaccurately. We introduce a novel mouse model for metastastatic colorectal cancer. In order to remain close to the clinical disease a syngenic murine colon carcinoma cell line (colon 26 cells) was transfected with enhanced green fluorescent protein (EGFP). The transfected cells maintain the highly malignant attributes of the wild-type cells. Following injection into the portal circulation of Balb/c-mice, liver metastases occur in the same time span. Using the fluorescent attributes of the transfected cells, an approximation of the tumor load in liver tissue can be achieved by fluorescence activated cell sorting (FACS) and fluoroscan analysis. Tumor cell load in liver tissue can be accurately measured by Northern blot and Western blot analysis of liver tissue containing EGFP-transfected colon cancer metastases (1250 cells/mg liver tissue and 1000 cells/mg liver tissue) respectively. Confocal microscopy and intravital microscopy confirmed the growth of tumor metastases, originating from the intravascular compartments. The presented animal model using EGFP-transfected colon 26 cells allows the detecting of tumor growth in vivo and post mortem, as well as an accurate quantification of the tumor load in the liver tissue.

Animals↗

PubMed2003

Mutations in a novel gene, NPHP3, cause adolescent nephronophthisis, tapeto-retinal degeneration and hepatic fibrosis.

Nephronophthisis (NPHP), a group of autosomal recessive cystic kidney disorders, is the most common genetic cause of progressive renal failure in children and young adults. NPHP may be associated with Leber congenital amaurosis, tapeto-retinal degeneration, cerebellar ataxia, cone-shaped epiphyses, congenital oculomotor apraxia and hepatic fibrosis. Loci associated with an infantile type of NPHP on 9q22-q31 (NPHP2), juvenile types of NPHP on chromosomes 2q12-q13 (NPHP1) and 1p36 (NPHP4) and an adolescent type of NPHP on 3q21-q22 (NPHP3) have been mapped. NPHP1 and NPHP4 have been identified, and interaction of the respective encoded proteins nephrocystin and nephrocystin-4 has been shown. Here we report the identification of NPHP3, encoding a novel 1,330-amino acid protein that interacts with nephrocystin. We describe mutations in NPHP3 in families with isolated NPHP and in families with NPHP with associated hepatic fibrosis or tapeto-retinal degeneration. We show that the mouse ortholog Nphp3 is expressed in the node, kidney tubules, retina, respiratory epithelium, liver, biliary tract and neural tissues. In addition, we show that a homozygous missense mutation in Nphp3 is probably responsible for the polycystic kidney disease (pcy) mouse phenotype. Interventional studies in the pcy mouse have shown beneficial effects by modification of protein intake and administration of methylprednisolone, suggesting therapeutic strategies for treating individuals with NPHP3.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Supplementation and inhibition of nitric oxide synthesis influences bacterial transit time during bacterial translocation in rats.

In the obstructed gut, nitric oxide (NO) may influence intestinal barrier function and translocation of bacteria. By using a novel experimental approach, we investigated the effect of supplementation and inhibition of NO synthesis on the time interval necessary for translocation of green fluorescent protein-transfected Escherichia coli (GFP-uv E. coli) in a rat model of small bowel obstruction. In anesthetized Wistar rats, 4 x 10(8) GFP-uv E. coli were administered into a reservoir of terminal ileum formed by ligature. Animals were randomized to receive either i.v. arginine (10 mg/kg), aminoguanidine (300 mg/kg), L-NAME (25 mg/kg), or saline (control). Translocation of GFP-uv E. coli was assessed using intravital video microscopy. Minimal transit time of translocation was measured as time from injection of GFP-uv E. coli into the gut lumen until bacteria were observed in the lamina submucosa and as time from injection of bacteria into the gut lumen until bacteria were observed in the lamina muscularis propria. Minimal transit times were expressed as mean +/- SD. Bacterial translocation into the submucosa and muscularis propria took 36 +/- 7 min and 81 +/- 9 min, respectively in control animals receiving saline. Aminoguanidine and L-NAME caused a marked delay of minimal transit time into the submucosa (63 +/- 5 min and 61 +/- 7 min, respectively; P < 0.05). Arginine significantly accelerated bacterial translocation into the muscularis propria (61 +/- 9 min, P < 0.05). GFP-uv E. coli were detected on frozen sections of small bowel, mesentery, liver, and spleen 2 h after GFP-uv E. coli administration in all animals. A marked upregulation of inducible NO synthase (NOS) in the obstructed bowel segment was demonstrated on immunohistochemistry. The assessment of a newly defined parameter, minimal bacterial transit time, may serve as an additional functional aspect of intestinal barrier function for pathophysiological and pharmacological studies. Aminoguanidine, L-NAME, and arginine were effective in influencing minimal transit time of E. coli during small bowel obstruction.

Animals↗