Norbert Gretz

Publications of Norbert Gretz

• A new transgenic rat model overexpressing the angiotensin II type 2 receptor provides evidence for inhibition of cell proliferation in the outer adrenal cortex

  Authors: Barbara Peters, Dirk Podlich, Michael Ritter, Anja Müller, Heike Wanka, Christiane Maser-Gluth, Claudia Seitz, Laura de Boni, Elena Maier, Norbert Gretz, Jörg Peters, Sigrid C. Hoffmann

  The American journal of physiology. 05/2012; 302(9):E1044-54.

  This study aimed to elucidate the role of the AT(2) receptor (AT(2)R), which is expressed and upregulated in the adrenal zona glomerulosa (ZG) under conditions of increased aldosterone production. WeThis study aimed to elucidate the role of the AT(2) receptor (AT(2)R), which is expressed and upregulated in the adrenal zona glomerulosa (ZG) under conditions of increased aldosterone production. We developed a novel transgenic rat (TGR; TGRCXmAT(2)R) that overexpresses the AT(2)R in the adrenal gland, heart, kidney, brain, skeletal muscle, testes, lung, spleen, aorta, and vein. As a consequence the total angiotensin II (Ang II) binding sites increased 7.8-fold in the kidney, 25-fold in the heart, and twofold in the adrenals. The AT(2)R number amounted to 82-98% of total Ang II binding sites. In the ZG of TGRCXmAT(2)R, the AT(2)R density was elevated threefold relative to wild-type (WT) littermates, whereas AT(1)R density remained unchanged. TGRCXmAT(2)R rats were viable and exhibited normal reproduction, blood pressure, and kidney function. Notably, a slightly but significantly reduced body weight and a moderate increase in plasma urea were observed. With respect to adrenal function, 24-h urinary and plasma aldosterone concentrations were unaffected in TGRCXmAT(2)R at baseline. Three and 14 days of Ang II infusion (300 ng·min(-1)·kg(-1)) increased plasma aldosterone levels in WT and in TGR. These changes were completely abolished by the AT(1)R blocker losartan. Of note, glomerulosa cell proliferation, as indicated by the number of Ki-67-positive glomerulosa cells, was stimulated by Ang II in TGR and WT rats; however, this increase was significantly attenuated in TGR overexpressing the AT(2)R. In conclusion, AT(2)R in the adrenal ZG inhibits Ang II-induced cell proliferation but has no obvious lasting effect on the regulation of the aldosterone production at the investigated stages.

• Online feedback-controlled renal constant infusion clearances in rats.

  Authors: Daniel Schock-Kusch, Yury Shulhevich, Qing Xie, Juergen Hesser, Dzmitry Stsepankou, Sabine Neudecker, Jochen Friedemann, Stefan Koenig, Ralf Heinrich, Friederike Hoecklin, Johannes Pill, Norbert Gretz

  Kidney international. 04/2012;

  Constant infusion clearance techniques using exogenous renal markers are considered the gold standard for assessing the glomerular filtration rate. Here we describe a constant infusion clearanceConstant infusion clearance techniques using exogenous renal markers are considered the gold standard for assessing the glomerular filtration rate. Here we describe a constant infusion clearance method in rats allowing the real-time monitoring of steady-state conditions using an automated closed-loop approach based on the transcutaneous measurement of the renal marker FITC-sinistrin. In order to optimize parameters to reach steady-state conditions as fast as possible, a Matlab-based simulation tool was established. Based on this, a real-time feedback-regulated approach for constant infusion clearance monitoring was developed. This was validated by determining hourly FITC-sinistrin plasma concentrations and the glomerular filtration rate in healthy and unilaterally nephrectomized rats. The transcutaneously assessed FITC-sinistrin fluorescence signal was found to reflect the plasma concentration. Our method allows the precise determination of the onset of steady-state marker concentration. Moreover, the steady state can be monitored and controlled in real time for several hours. This procedure is simple to perform since no urine samples and only one blood sample are required. Thus, we developed a real-time feedback-based system for optimal regulation and monitoring of a constant infusion clearance technique.Kidney International advance online publication, 18 April 2012; doi:10.1038/ki.2012.117.

• A new transgenic rat model over-expressing the angiotensin II type 2 receptor provide evidence for inhibition of cell proliferation in the outer adrenal cortex.

  Authors: Barbara Peters, Dirk Podlich, Michael Ritter, Anja Müller, Heike Wanka, Christiane Maser-Gluth, Claudia Seitz, Laura de Boni, Elena Maier, Norbert Gretz, Jörg Peters, Sigrid C Hoffmann

  American journal of physiology. Endocrinology and metabolism. 02/2012;

  This study aimed to elucidate the role of the AT(2)-receptor (AT(2)R), which is expressed and up-regulated in the adrenal zona glomerulosa (ZG) under conditions of increased aldosterone production.This study aimed to elucidate the role of the AT(2)-receptor (AT(2)R), which is expressed and up-regulated in the adrenal zona glomerulosa (ZG) under conditions of increased aldosterone production. We developed a novel transgenic rat (TGRCXmAT(2)R) that over-expresses the AT(2)R in the adrenal gland, heart, kidney, brain, skeletal muscle, testes, lung, spleen, aorta and vein. As a consequence the total Angiotensin II (AngII) binding sites increased by 7.8-fold in the kidney, by 25-fold in the heart, by 2-fold in the adrenals. The AT(2)R number amounted to 98 - 82% of total AngII binding sites. In the ZG of TGRCXmAT(2)R the AT(2)R density was elevated by 3-fold relative to wild-type littermates (WT), whereas AT(1)R density remained unchanged. TGRCXmAT(2)R rats were viable, exhibited normal reproduction, blood pressure and kidney function. Notably, a slightly but significantly reduced body weight and a moderate increase in plasma urea were observed. With respect to adrenal function, 24h-urinary and plasma aldosterone concentrations were unaffected in TGRCXmAT(2)R at baseline. AngII infusion (300ng/min/kg) for 3- and 14-days, respectively, increased plasma aldosterone levels in both, WT and TGR. These changes were completely abolished by the AT(1)R blocker losartan. Of note, glomerulosa cell proliferation, as indicated by the number of Ki-67 positive glomerulosa cells, was stimulated by AngII in TGR and WT rats, however, this increase was significantly attenuated in TGR overexpressing the AT(2)R. In conclusion, AT(2)R in the adrenal ZG inhibits AngII induced cells proliferation, but has no obvious lasting effect on the regulation of the aldosterone production at the investigated stages.

• A miR-1207-5p binding site polymorphism abolishes regulation of HBEGF and is associated with disease severity in CFHR5 nephropathy.

  Authors: Gregory Papagregoriou, Kamil Erguler, Harsh Dweep, Konstantinos Voskarides, Panayiota Koupepidou, Yiannis Athanasiou, Alkis Pierides, Norbert Gretz, Kyriacos N Felekkis, Constantinos Deltas

  PloS one. 01/2012; 7(2):e31021.

  Heparin binding epidermal growth factor (HBEGF) is expressed in podocytes and was shown to play a role in glomerular physiology. MicroRNA binding sites on the 3'UTR of HBEGF were predicted usingHeparin binding epidermal growth factor (HBEGF) is expressed in podocytes and was shown to play a role in glomerular physiology. MicroRNA binding sites on the 3'UTR of HBEGF were predicted using miRWalk algorithm and followed by DNA sequencing in 103 patients diagnosed with mild or severe glomerulopathy. A single nucleotide polymorphism, miRSNP C1936T (rs13385), was identified at the 3'UTR of HBEGF that corresponds to the second base of the hsa-miR-1207-5p seed region. When AB8/13 undifferentiated podocytes were transfected with miRNA mimics of hsa-miR-1207-5p, the HBEGF protein levels were reduced by about 50%. A DNA fragment containing the miRSNP allele-1936C was cloned into the pMIR-Report Luciferase vector and co-transfected with miRNA mimics of hsa-miR-1207-5p into AB8/13 podocytes. In agreement with western blot data, this resulted in reduced luciferase expression demonstrating the ability of hsa-miR-1207-5p to directly regulate HBEGF expression. On the contrary, in the presence of the miRSNP 1936T allele, this regulation was abolished. Collectively, these results demonstrate that variant 1936T of this miRSNP prevents hsa-miR-1207-5p from down-regulating HBEGF in podocytes. We hypothesized that this variant has a functional role as a genetic modifier. To this end, we showed that in a cohort of 78 patients diagnosed with CFHR5 nephropathy (also known as C3-glomerulopathy), inheritance of miRSNP 1936T allele was significantly increased in the group demonstrating progression to chronic renal failure on long follow-up. No similar association was detected in a cohort of patients with thin basement membrane nephropathy. This is the first report associating a miRSNP as genetic modifier to a monogenic renal disorder.

• New insights into the influence of cigarette smoking on urothelial carcinogenesis: Smoking-induced gene expression in tumor-free urothelium might discriminate muscle-invasive from nonmuscle-invasive urothelial bladder cancer.

  Authors: Ute Gabriel, Li Li, Christian Bolenz, Annette Steidler, Bettina Kränzlin, Maria Saile, Norbert Gretz, Lutz Trojan, Maurice Stephan Michel

  Molecular carcinogenesis. 10/2011;

  Smoking is the main risk factor for urothelial bladder cancer. In former smokers the risk decreases but does not reach the low level of never smokers. This indicates reversible and permanentSmoking is the main risk factor for urothelial bladder cancer. In former smokers the risk decreases but does not reach the low level of never smokers. This indicates reversible and permanent smoking-derived genetic alterations. Transcriptional changes may point to mechanisms, how smoking promotes urothelial bladder cancer. To identify smoking-derived transcriptional changes we performed gene expression profiling in current, former, and never smokers, using tumor and tumor-free urothelium from patients with nonmuscle-invasive urothelial bladder cancer (NMIBC) or muscle-invasive urothelial bladder cancer (MIBC). Smoking turned out to influence gene expression much less than tumor stage (NMIBC or MIBC) and tumor transformation (tumor-free or tumor). Smoking seemed to influence gene expression in patients with MIBC more strongly compared to those with NMIBC. The least irreversible changes after smoking cessation were proposed in tumor-free urothelium from patients with NMIBC. Growth factors and oncogenes were up-regulated in tumor-free urothelium from smokers with MIBC but not from smokers with NMIBC. A panel of genes up-regulated in smokers have potential for early detection and distinction of MIBC from NMIBC using tumor-free tissue. © 2011 Wiley Periodicals, Inc.

• Selective downregulation of retinoic acid-inducible gene I within the intestinal epithelial compartment in Crohn's disease.

  Authors: Benjamin Funke, Felix Lasitschka, Wilfried Roth, Roland Penzel, Stefan Meuer, Maria Saile, Norbert Gretz, Bernd Sido, Peter Schirmacher, Frank Autschbach

  Inflammatory bowel diseases. 09/2011; 17(9):1943-54.

  A defective innate immune response may contribute to the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC). Employing a global gene expression analysis, this study was aimed atA defective innate immune response may contribute to the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC). Employing a global gene expression analysis, this study was aimed at identifying specifically regulated genes within the epithelial compartment in inflammatory bowel disease (IBD). The epithelial fraction of human ileal mucosa samples from surgical specimens was obtained by laser microdissection. Gene expression was examined by global expression profiling (n = 18, Affymetrix), quantitative reverse-transcription polymerase chain reaction (RT-PCR) (n = 35), immunoblot analysis (n = 9), and immunohistochemistry (n = 25). Global expression profiling revealed a pronounced downregulation of the retinoic acid-inducible gene I (RIG-I) within the epithelial layer of the ileum in patients with CD but not with UC. The downregulation of RIG-I was confirmed by quantitative RT-PCR, immunoblot analysis, and immunohistochemistry. Epithelial downregulation of RIG-I, a known pattern recognition receptor for viral components, might contribute to alterations of the innate mucosal immune response, particularly in CD.

• The Raf kinase inhibitor PLX5568 slows cyst proliferation in rat polycystic kidney disease but promotes renal and hepatic fibrosis.

  Authors: Bjoern Buchholz, Bernd Klanke, Gunnar Schley, Gideon Bollag, James Tsai, Sven Kroening, Daisuke Yoshihara, Darren P Wallace, Bettina Kraenzlin, Norbert Gretz, Peter Hirth, Kai-Uwe Eckardt, Wanja M Bernhardt

  Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association. 07/2011; 26(11):3458-65.

  BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of renal failure. Aberrant epithelial cell proliferation is a major cause of progressive cyst enlargement in ADPKD.BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of renal failure. Aberrant epithelial cell proliferation is a major cause of progressive cyst enlargement in ADPKD. Since activation of the Ras/Raf signaling system has been detected in cyst-lining epithelia, inhibition of Raf kinase has been proposed as an approach to retard the progression of ADPKD. Methods and results. PLX5568, a novel selective small molecule inhibitor of Raf kinases, attenuated proliferation of human ADPKD cyst epithelial cells. It reduced in vitro cyst growth of Madin-Darby Canine Kidney cells and of human ADPKD cells within a collagen gel. In male cy/+ rats with polycystic kidneys, PLX5568 inhibited renal cyst growth along with a significant reduction in the number of proliferating cell nuclear antigen- and phosphorylated extracellular signal-regulated kinase-positive cyst-lining epithelial cells. Furthermore, treated animals showed increased capacity to concentrate urine. However, PLX5568 did not lead to a consistent improvement of renal function. Moreover, although relative cyst volume was decreased, total kidney-to-body weight ratio was not significantly reduced by PLX5568. Further analyses revealed a 2-fold increase of renal and hepatic fibrosis in animals treated with PLX5568. CONCLUSIONS: PLX5568 attenuated cyst enlargement in vitro and in a rat model of ADPKD without improving kidney function, presumably due to increased renal fibrosis. These data suggest that effective therapies for the treatment of ADPKD will need to target fibrosis as well as the growth of cysts.

• Division of labor by dual feedback regulators controls JAK2/STAT5 signaling over broad ligand range.

  Authors: Julie Bachmann, Andreas Raue, Marcel Schilling, Martin E Böhm, Clemens Kreutz, Daniel Kaschek, Hauke Busch, Norbert Gretz, Wolf D Lehmann, Jens Timmer, Ursula Klingmüller

  Molecular systems biology. 07/2011; 7:516.

  Cellular signal transduction is governed by multiple feedback mechanisms to elicit robust cellular decisions. The specific contributions of individual feedback regulators, however, remain unclear.Cellular signal transduction is governed by multiple feedback mechanisms to elicit robust cellular decisions. The specific contributions of individual feedback regulators, however, remain unclear. Based on extensive time-resolved data sets in primary erythroid progenitor cells, we established a dynamic pathway model to dissect the roles of the two transcriptional negative feedback regulators of the suppressor of cytokine signaling (SOCS) family, CIS and SOCS3, in JAK2/STAT5 signaling. Facilitated by the model, we calculated the STAT5 response for experimentally unobservable Epo concentrations and provide a quantitative link between cell survival and the integrated response of STAT5 in the nucleus. Model predictions show that the two feedbacks CIS and SOCS3 are most effective at different ligand concentration ranges due to their distinct inhibitory mechanisms. This divided function of dual feedback regulation enables control of STAT5 responses for Epo concentrations that can vary 1000-fold in vivo. Our modeling approach reveals dose-dependent feedback control as key property to regulate STAT5-mediated survival decisions over a broad range of ligand concentrations.

• Suppression of chronic damage in renal allografts by Liver X receptor (LXR) activation relevant contribution of macrophage LXRα.

  Authors: Eva Kiss, Zoran Popovic, Jens Bedke, Shijun Wang, Mahnaz Bonrouhi, Norbert Gretz, Paula Stettner, Daniel Teupser, Joachim Thiery, Stefan Porubsky, Judith Adams, Hermann-Josef Gröne

  The American journal of pathology. 07/2011; 179(1):92-103.

  Liver X receptors (LXR)-α,β regulate intracellular cholesterol homeostasis and inhibit inflammatory gene expression. We studied the effects of the LXRα,β-agonist GW3965 on acute and chronic organLiver X receptors (LXR)-α,β regulate intracellular cholesterol homeostasis and inhibit inflammatory gene expression. We studied the effects of the LXRα,β-agonist GW3965 on acute and chronic organ damage in the F344-LEW rat kidney transplantation model. In addition, to gain LXR isoform and cell-specific insights BALB/c kidneys were transplanted into mice with macrophage overexpression of LXRα (mLXRα-tg) and evaluated 7 and 42 days after transplantation. After 56 days GW3965 improved significantly function and morphology of rat kidney allografts by substantial reduction of mononuclear cell infiltrate and fibrosis; in vitro GW3965 reduced inflammatory activity of bone marrow-derived macrophages (BMDMs) and alloreactivity of T cells. Kidneys transplanted into mLXRα-tg mice were also protected from development of chronic allograft dysfunction. Similarly to GW3965-activated BMDMs, mLXRα-tg macrophages secreted significantly less monocyte chemoattractant protein 1 and macrophage inflammatory protein 1β. Interestingly, 7 days after transplantation, when the total number of intragraft macrophages did not differ, evidently more arginase 1- and mannose receptor C type 1-positive cells were found in LXR rat and mice kidney allografts; in vitro both LXR activation by GW3965 and mLXRα overexpression accentuated the induction of alternative activation of BMDMs by IL-4/IL-13, suggesting an additional mechanism by LXRs to prevent graft damage. The results highlight the relevance of macrophage LXRα in allograft rejection and prevention of fibrosis.

• Quantification of glomerular number and size distribution in normal rat kidneys using magnetic resonance imaging.

  Authors: Melanie Heilmann, Sabine Neudecker, Ivo Wolf, Lina Gubhaju, Carsten Sticht, Daniel Schock-Kusch, Wilhelm Kriz, John F Bertram, Lothar R Schad, Norbert Gretz

  Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association. 06/2011; 27(1):100-7.

  Glomerular number and size are important risk factors for chronic kidney disease (CKD) and cardiovascular disease and have traditionally been estimated using invasive techniques. Here, we report aGlomerular number and size are important risk factors for chronic kidney disease (CKD) and cardiovascular disease and have traditionally been estimated using invasive techniques. Here, we report a novel technique to count and size every glomerulus in the rat kidney using magnetic resonance imaging (MRI). The ferromagnetic nature of cationized ferritin allowed visualization of single glomeruli in high-resolution susceptibility-weighted MRI. A segmentation algorithm was used to identify and count all glomeruli within the whole kidney. To prove our concept, we estimated total glomerular number and mean glomerular volume of each kidney using design-based stereology. The glomerular counts obtained with MRI agreed well with estimates obtained using traditional methods [MRI, 32 785 (3117); stereology, 35 132 (3123)]. For the first time, the glomerular volume distribution for the entire kidney is shown. Additionally, the method is substantially faster than the current methods. MRI provides a new method for measuring these important microanatomical markers of disease risk and leads the way to in vivo analysis of these parameters, including longitudinal studies of animal models of CKD.

• Validation of a new non-invasive blood pressure measurement method on mice via pulse wave propagation time measurement on a cuff.

  Authors: Xuan P Nguyen, Ralf Kronemayer, Peter Herrmann, Atila Mejía, Zamira Daw, Xuan D Nguyen, Bettina Kränzlin, Norbert Gretz

  Biomedizinische Technik. Biomedical engineering. 06/2011; 56(3):153-8.

  In the present article, we describe the validation of a new non-invasive method for measuring blood pressure (BP) which also enables to determine the three BP values: systolic, diastolic and meanIn the present article, we describe the validation of a new non-invasive method for measuring blood pressure (BP) which also enables to determine the three BP values: systolic, diastolic and mean value. Our method is based on the pulse transit time (PTT) measurement along an artery directly at the BP cuff. The accuracy of this method was evaluated by comparison with the direct simultaneous measurement of blood pressure from 40 anesthetized female mice. Close correlation between the gained data from these two methods was observed.

• miRWalk--database: prediction of possible miRNA binding sites by "walking" the genes of three genomes.

  Authors: Harsh Dweep, Carsten Sticht, Priyanka Pandey, Norbert Gretz

  Journal of biomedical informatics. 05/2011; 44(5):839-47.

  MicroRNAs are small, non-coding RNA molecules that can complementarily bind to the mRNA 3'-UTR region to regulate the gene expression by transcriptional repression or induction of mRNA degradation.MicroRNAs are small, non-coding RNA molecules that can complementarily bind to the mRNA 3'-UTR region to regulate the gene expression by transcriptional repression or induction of mRNA degradation. Increasing evidence suggests a new mechanism by which miRNAs may regulate target gene expression by binding in promoter and amino acid coding regions. Most of the existing databases on miRNAs are restricted to mRNA 3'-UTR region. To address this issue, we present miRWalk, a comprehensive database on miRNAs, which hosts predicted as well as validated miRNA binding sites, information on all known genes of human, mouse and rat. All mRNAs, mitochondrial genes and 10 kb upstream flanking regions of all known genes of human, mouse and rat were analyzed by using a newly developed algorithm named 'miRWalk' as well as with eight already established programs for putative miRNA binding sites. An automated and extensive text-mining search was performed on PubMed database to extract validated information on miRNAs. Combined information was put into a MySQL database. miRWalk presents predicted and validated information on miRNA-target interaction. Such a resource enables researchers to validate new targets of miRNA not only on 3'-UTR, but also on the other regions of all known genes. The 'Validated Target module' is updated every month and the 'Predicted Target module' is updated every 6 months. miRWalk is freely available at http://mirwalk.uni-hd.de/.

• Hepatic deficiency in transcriptional cofactor TBL1 promotes liver steatosis and hypertriglyceridemia.

  Authors: Philipp Kulozik, Allan Jones, Frits Mattijssen, Adam J Rose, Anja Reimann, Daniela Strzoda, Stefan Kleinsorg, Christina Raupp, Jürgen Kleinschmidt, Karin Müller-Decker [......] Carsten Sticht, Norbert Gretz, Christian von Loeffelholz, Martin Stockmann, Andreas Pfeiffer, Sigrid Stöhr, Geesje M Dallinga-Thie, Peter P Nawroth, Mauricio Berriel Diaz, Stephan Herzig

  Cell metabolism. 04/2011; 13(4):389-400.

  The aberrant accumulation of lipids in the liver ("fatty liver") is tightly associated with several components of the metabolic syndrome, including type 2 diabetes, coronary heart disease, andThe aberrant accumulation of lipids in the liver ("fatty liver") is tightly associated with several components of the metabolic syndrome, including type 2 diabetes, coronary heart disease, and atherosclerosis. Here we show that the impaired hepatic expression of transcriptional cofactor transducin beta-like (TBL) 1 represents a common feature of mono- and multigenic fatty liver mouse models. Indeed, the liver-specific ablation of TBL1 gene expression in healthy mice promoted hypertriglyceridemia and hepatic steatosis under both normal and high-fat dietary conditions. TBL1 deficiency resulted in inhibition of fatty acid oxidation due to impaired functional cooperation with its heterodimerization partner TBL-related (TBLR) 1 and the nuclear receptor peroxisome proliferator-activated receptor (PPAR) α. As TBL1 expression levels were found to also inversely correlate with liver fat content in human patients, the lack of hepatic TBL1/TBLR1 cofactor activity may represent a molecular rationale for hepatic steatosis in subjects with obesity and the metabolic syndrome.

• Increased number of microRNA target sites in genes encoded in CNV regions. Evidence for an evolutionary genomic interaction.

  Authors: Kyriakos Felekkis, Konstantinos Voskarides, Harsh Dweep, Carsten Sticht, Norbert Gretz, Constantinos Deltas

  Molecular biology and evolution. 03/2011; 28(9):2421-4.

  MicroRNAs (miRNAs) and copy number variations (CNVs) are two newly discovered genetic elements that have revolutionized the field of molecular biology and genetics. By performing in silico wholeMicroRNAs (miRNAs) and copy number variations (CNVs) are two newly discovered genetic elements that have revolutionized the field of molecular biology and genetics. By performing in silico whole genome analysis, we demonstrate that both the number of miRNAs that target genes found in CNV regions as well as the number of miRNA-binding sites are significantly higher than those of genes found in non-CNV regions. This suggests that miRNAs may have acted as equilibrators of gene expression during evolution in an attempt to regulate aberrant gene expression and to increase the tolerance to genome plasticity.

• First multimodal embolization particles visible on x-ray/computed tomography and magnetic resonance imaging.

  Authors: Soenke H Bartling, Johannes Budjan, Hagit Aviv, Stefan Haneder, Bettina Kraenzlin, Henrik Michaely, Shlomo Margel, Steffen Diehl, Wolfhard Semmler, Norbert Gretz, Stefan O Schönberg, Maliha Sadick

  Investigative radiology. 03/2011; 46(3):178-86.

  Embolization therapy is gaining importance in the treatment of malignant lesions, and even more in benign lesions. Current embolization materials are not visible in imaging modalities. However, it isEmbolization therapy is gaining importance in the treatment of malignant lesions, and even more in benign lesions. Current embolization materials are not visible in imaging modalities. However, it is assumed that directly visible embolization material may provide several advantages over current embolization agents, ranging from particle shunt and reflux prevention to improved therapy control and follow-up assessment. X-ray- as well as magnetic resonance imaging (MRI)-visible embolization materials have been demonstrated in experiments. In this study, we present an embolization material with the property of being visible in more than one imaging modality, namely MRI and x-ray/computed tomography (CT). Characterization and testing of the substance in animal models was performed. To reduce the chance of adverse reactions and to facilitate clinical approval, materials have been applied that are similar to those that are approved and being used on a routine basis in diagnostic imaging. Therefore, x-ray-visible Iodine was combined with MRI-visible Iron (Fe3O4) in a macroparticle (diameter, 40-200 μm). Its core, consisting of a copolymerized monomer MAOETIB (2-methacryloyloxyethyl [2,3,5-triiodobenzoate]), was coated with ultra-small paramagnetic iron oxide nanoparticles (150 nm). After in vitro testing, including signal to noise measurements in CT and MRI (n = 5), its ability to embolize tissue was tested in an established tumor embolization model in rabbits (n = 6). Digital subtraction angiography (DSA) (Integris, Philips), CT (Definition, Siemens Healthcare Section, Forchheim, Germany), and MRI (3 Tesla Magnetom Tim Trio MRI, Siemens Healthcare Section, Forchheim, Germany) were performed before, during, and after embolization. Imaging signal changes that could be attributed to embolization particles were assessed by visual inspection and rated on an ordinal scale by 3 radiologists, from 1 to 3. Histologic analysis of organs was performed. Particles provided a sufficient image contrast on DSA, CT (signal to noise [SNR], 13 ± 2.5), and MRI (SNR, 35 ± 1) in in vitro scans. Successful embolization of renal tissue was confirmed by catheter angiography, revealing at least partial perfusion stop in all kidneys. Signal changes that were attributed to particles residing within the kidney were found in all cases in all the 3 imaging modalities. Localization distribution of particles corresponded well in all imaging modalities. Dynamic imaging during embolization provided real-time monitoring of the inflow of embolization particles within DSA, CT, and MRI. Histologic visualization of the residing particles as well as associated thrombosis in renal arteries could be performed. Visual assessment of the likelihood of embolization particle presence received full rating scores (153/153) after embolization. Multimodal-visible embolization particles have been developed, characterized, and tested in vivo in an animal model. Their implementation in clinical radiology may provide optimization of embolization procedures with regard to prevention of particle misplacement and direct intraprocedural visualization, at the same time improving follow-up examinations by utilizing the complementary characteristics of CT and MRI. Radiation dose savings can also be considered. All these advantages could contribute to future refinements and improvements in embolization therapy. Additionally, new approaches in embolization research may open up.

• Transcutaneous assessment of renal function in conscious rats with a device for measuring FITC-sinistrin disappearance curves.

  Authors: Daniel Schock-Kusch, Qing Xie, Yury Shulhevich, Juergen Hesser, Dzmitry Stsepankou, Maliha Sadick, Stefan Koenig, Friederike Hoecklin, Johannes Pill, Norbert Gretz

  Kidney international. 03/2011; 79(11):1254-8.

  Determination of the urinary or plasma clearance of exogenous renal markers, such as inulin or iohexol, is considered to be the gold standard for glomerular filtration rate (GFR) measurement. Here,Determination of the urinary or plasma clearance of exogenous renal markers, such as inulin or iohexol, is considered to be the gold standard for glomerular filtration rate (GFR) measurement. Here, we describe a technique allowing determination of renal function based on transcutaneously measured elimination kinetics of fluorescein isothiocyanate (FITC)-sinistrin, the FITC-labeled active pharmaceutical ingredient of a commercially available marker of GFR. A low cost device transcutaneously excites FITC-sinistrin at 480  nm and detects the emitted light through the skin at 520  nm. A radio-frequency transmission allows remote monitoring and real-time analysis of FITC-sinistrin excretion as a marker of renal function. Due to miniaturization, the whole device fits on the back of freely moving rats, and requires neither blood sampling nor laboratory assays. As proof of principle, comparative measurements of transcutaneous and plasma elimination kinetics of FITC-sinistrin were compared in freely moving healthy rats, rats showing reduced kidney function due to unilateral nephrectomy and PKD/Mhm rats with cystic kidney disease. Results show highly comparable elimination half-lives and GFR values in all animal groups. Bland-Altman analysis of enzymatically compared with transcutaneously measured GFR found a mean difference (bias) of 0.01 and a -0.30 to 0.33 ml/min per 100 g body weight with 95% limit of agreement. Thus, with this device, renal function can be reliably measured in freely moving rats eliminating the need for and influence of anesthesia on renal function.

• Inducible renal principal cell-specific mineralocorticoid receptor gene inactivation in mice.

  Authors: Caroline Ronzaud, Johannes Loffing, Norbert Gretz, Günther Schütz, Stefan Berger

  American journal of physiology. Renal physiology. 03/2011; 300(3):F756-60.

  To investigate the role of the mineralocorticoid receptor (MR) in renal ENaC-mediated sodium reabsorption, we have previously used the Cre-loxP system to generate mice with principal-cell specific MRTo investigate the role of the mineralocorticoid receptor (MR) in renal ENaC-mediated sodium reabsorption, we have previously used the Cre-loxP system to generate mice with principal-cell specific MR ablation (MR(AQP2Cre) mice). To restrict Cre expression to principal cells, we have used the regulatory elements of the mouse aquaporin-2 (AQP2) gene to drive Cre expression. Since AQP2 is already expressed during renal development, MR ablation took place long before the analysis performed at the adult stage. To investigate whether the early onset of MR ablation affected the adult renal sodium handling, we developed a transgene expressing the CreER(T2) fusion protein under control of the regulatory elements of the AQP2 gene (AQP2CreER(T2)). Immunofluorescence revealed MR loss in the collecting duct (CD) and late connecting tubule after induction of MR ablation by tamoxifen in MR(AQP2CreERT2) mice that equals the MR loss in MR(AQP2Cre) mice. Surprisingly, tamoxifen-independent MR loss is observed in CDs of noninduced mutants without affecting circulating aldosterone levels. Under a low-salt diet, the induced ablation of MR at the adult stage recapitulates the renal sodium wasting observed in mice with constitutive early-onset MR ablation. The AQP2CreER(T2) transgene is a new tool for investigating in vivo the function of genes downstream of MR in renal ENaC-mediated sodium reabsorption by inducible somatic gene inactivation.

• Comparative transcriptome profiling of amyloid precursor protein family members in the adult cortex.

  Authors: Dorothee Aydin, Mikhail A Filippov, Jakob-Andreas Tschäpe, Norbert Gretz, Marco Prinz, Roland Eils, Benedikt Brors, Ulrike C Müller

  BMC genomics. 03/2011; 12:160.

  The β-amyloid precursor protein (APP) and the related β-amyloid precursor-like proteins (APLPs) undergo complex proteolytic processing giving rise to several fragments. Whereas it is well establishedThe β-amyloid precursor protein (APP) and the related β-amyloid precursor-like proteins (APLPs) undergo complex proteolytic processing giving rise to several fragments. Whereas it is well established that Aβ accumulation is a central trigger for Alzheimer's disease, the physiological role of APP family members and their diverse proteolytic products is still largely unknown. The secreted APPsα ectodomain has been shown to be involved in neuroprotection and synaptic plasticity. The γ-secretase-generated APP intracellular domain (AICD) functions as a transcriptional regulator in heterologous reporter assays although its role for endogenous gene regulation has remained controversial. To gain further insight into the molecular changes associated with knockout phenotypes and to elucidate the physiological functions of APP family members including their proposed role as transcriptional regulators, we performed DNA microarray transcriptome profiling of prefrontal cortex of adult wild-type (WT), APP knockout (APP-/-), APLP2 knockout (APLP2-/-) and APPsα knockin mice (APPα/α) expressing solely the secreted APPsα ectodomain. Biological pathways affected by the lack of APP family members included neurogenesis, transcription, and kinase activity. Comparative analysis of transcriptome changes between mutant and wild-type mice, followed by qPCR validation, identified co-regulated gene sets. Interestingly, these included heat shock proteins and plasticity-related genes that were both down-regulated in knockout cortices. In contrast, we failed to detect significant differences in expression of previously proposed AICD target genes including Bace1, Kai1, Gsk3b, p53, Tip60, and Vglut2. Only Egfr was slightly up-regulated in APLP2-/- mice. Comparison of APP-/- and APPα/α with wild-type mice revealed a high proportion of co-regulated genes indicating an important role of the C-terminus for cellular signaling. Finally, comparison of APLP2-/- on different genetic backgrounds revealed that background-related transcriptome changes may dominate over changes due to the knockout of a single gene. Shared transcriptome profiles corroborated closely related physiological functions of APP family members in the adult central nervous system. As expression of proposed AICD target genes was not altered in adult cortex, this may indicate that these genes are not affected by lack of APP under resting conditions or only in a small subset of cells.

• Two non-invasive GFR-estimation methods in rat models of polycystic kidney disease: 3.0 Tesla dynamic contrast-enhanced MRI and optical imaging.

  Authors: Maliha Sadick, Ulrike Attenberger, Bettina Kraenzlin, Hany Kayed, Stefan O Schoenberg, Norbert Gretz, Daniel Schock-Kusch

  Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association. 03/2011; 26(10):3101-8.

  The aim of this study was the assessment of kidney morphology and glomerular filtration rate (GFR) in rat models of polycystic kidney disease and a healthy control group of Sprague-Dawley rats (SDThe aim of this study was the assessment of kidney morphology and glomerular filtration rate (GFR) in rat models of polycystic kidney disease and a healthy control group of Sprague-Dawley rats (SD rats). The performance of two non-invasive GFR estimation methods-3.0 Tesla magnetic resonance imaging (MRI) and optical imaging were investigated. Data of GFR assessment was compared to surrogate markers of kidney function and renal histology. Optical imaging of GFR was performed transcutaneously in a small animal imaging system with the fluorescent renal marker fluorescein-isothiocyanate-labelled-sinistrin. Morphologic and dynamic renal imaging was done on a clinical 3.0T MR scanner. Renal perfusion analysis was performed with a two-compartment filtration model. The healthy SD rats showed physiological levels of creatinine and urea, indicating normal kidney function. These parameters were elevated in the small animal groups of polycystic kidney disease. For the calculation of perfusion and filtration parameters of kidney function in MRI, a 2D turbo FLASH sequence was performed and allowed to distinguish between normal GFR of healthy rats and reduced GFR of rats with polycystic kidney disease. Also, MRI GFR varied among two different rat strains of polycystic kidney disease, according to their status of renal function impairment. Optical imaging GFR confirmed higher GFR values in healthy rats compared to ill rats but did not show different results among the two rat strains of polycystic kidney disease. For this reason, MRI and optical imaging GFR estimation presented an intra-method bias. Both non-invasive estimation methods of GFR, MRI and optical imaging, can differentiate between healthy rats and animals with limited kidney function. Furthermore, optical imaging, unlike MRI, seems to consider that disease progression with increase of renal polycystic deterioration does not correlate with decrease of GFR in the initial stage of compensatory hyperfiltration.

• Dynamic mathematical modeling of IL13-induced signaling in Hodgkin and primary mediastinal B-cell lymphoma allows prediction of therapeutic targets.

  Authors: Valentina Raia, Marcel Schilling, Martin Böhm, Bettina Hahn, Andreas Kowarsch, Andreas Raue, Carsten Sticht, Sebastian Bohl, Maria Saile, Peter Möller, Norbert Gretz, Jens Timmer, Fabian Theis, Wolf-Dieter Lehmann, Peter Lichter, Ursula Klingmüller

  Cancer research. 02/2011; 71(3):693-704.

  Primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) share a frequent constitutive activation of JAK (Janus kinase)/STAT signaling pathway. Because of complex, nonlinearPrimary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) share a frequent constitutive activation of JAK (Janus kinase)/STAT signaling pathway. Because of complex, nonlinear relations within the pathway, key dynamic properties remained to be identified to predict possible strategies for intervention. We report the development of dynamic pathway models based on quantitative data collected on signaling components of JAK/STAT pathway in two lymphoma-derived cell lines, MedB-1 and L1236, representative of PMBL and cHL, respectively. We show that the amounts of STAT5 and STAT6 are higher whereas those of SHP1 are lower in the two lymphoma cell lines than in normal B cells. Distinctively, L1236 cells harbor more JAK2 and less SHP1 molecules per cell than MedB-1 or control cells. In both lymphoma cell lines, we observe interleukin-13 (IL13)-induced activation of IL4 receptor α, JAK2, and STAT5, but not of STAT6. Genome-wide, 11 early and 16 sustained genes are upregulated by IL13 in both lymphoma cell lines. Specifically, the known STAT-inducible negative regulators CISH and SOCS3 are upregulated within 2 hours in MedB-1 but not in L1236 cells. On the basis of this detailed quantitative information, we established two mathematical models, MedB-1 and L1236 model, able to describe the respective experimental data. Most of the model parameters are identifiable and therefore the models are predictive. Sensitivity analysis of the model identifies six possible therapeutic targets able to reduce gene expression levels in L1236 cells and three in MedB-1. We experimentally confirm reduction in target gene expression in response to inhibition of STAT5 phosphorylation, thereby validating one of the predicted targets.