Beyond the Identification of Transcribed Sequences:
Functional, Evolutionary and Expression Analysis
12th International Workshop
October 25-28, 2002
Washington, DC


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The Next Step in Transcriptomics: Data Mining after Global Array Analysis and Design of a Sub-Array Specific for Cardiac Fibrosis of Renal Failure in the Rat Model

Christian Maercker1, Heidrun Ridinger1, Effi Rees1, Matthias Schick1, Christiane Rutenberg1, Eberhard Ritz2, Gerhard Mall3, Bernhard Korn1, and Kerstin Amann4
1Resource Center for Genome Research, DKFZ H0600, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany, maercker@rzpd.de, 2Dept. of Internal Medicine, University of Heidelberg, Bergheimerstr. 56a, 69115 Heidelberg, 3Dept. of Pathology, Grafenstr. 9, 64283 Darmstadt, 4Department of Pathology, University of Erlangen, Krankenhausstr. 8-10, D-91054 Erlangen
Telephone: +49 6221 424741
Fax: +49 6221 423454
Email: c.maercker@dkfz.de

Death from cardiac causes is the most common fatality in uremic patients. The cardiac alterations, to which belong changes in blood pressure, electrolyte and hormone concentration, develop very early in renal insufficiency. We follow the approach of a gene expression profiling analysis in the rat animal model to identify the pathogenesis of the lesions.

Sprague-Dawley rats were subjected to subtotal nephrectomy (SNX) or sham operation (SHAM) and followed for 2 and 12 weeks, respectively. Poly(A)+ RNA was used for expression profiling on RZPD-own global rat cDNA arrays (Rat Unigene-1, containing about 27.000 gene and EST sequences). After primary data analysis, only genes were extracted which showed a reproducible up- or down-regulation in all experiments. For data mining, genes were grouped as follows: a, All genes belonging to the group of the 10% strongest expressed genes, which were at least 2-fold up- or down-regulated, respectively; b, genes at least 5-fold up- or down-regulated, respectively; c, genes repeatedly regulated, which belong to the following gene families: 1. renin-angiotensin system (RAS) as potential participating hormone system, 2. extracellular matrix (ECM), 3. cell junctions (cell surface receptors, structural proteins), 4. signaling molecules (e.g. G-proteins, MAP/ERK cascade, second messenger, growth differentiation markers), 5. cytoskeleton (structural proteins, motor proteins), 6. growth factors. Genes and ESTs belonging to these groups were classified according to the GeneCards database (Weizmann Institute, http://bioinformatics.weizmann.ac.il/cards/) the GeneOntology database (http://www.geneontology.org/), and literature.

We identified about 150 genes strongly up- or down-regulated. Among them are not further characterized ESTs, but also genes responsible for cytoskeletal organization, energy metabolism, transport, signal transduction, etc.. Altogether, about 400 genes regulated could be classified into the selected gene families potentially involved in the cardiac lesions. In more detail, we found an up-regulation of the endothelin-receptor B (ETB), which is predominantly located on endothelial cells and which is known to up-regulated in left ventricular hypertrophy in a G protein dependent manner. Therefore, as shown for the kidney, there also might exist ETB specific answers in heart. As expected, the renin-angiotensin system (RAS) obviously is up-regulated as a inflammation reaction immediately after operation. Looking for downstream effector systems behind ETB, we found up-regulation of two Rac clones, of rhoB and ESTs similar to rhoC and rhoA. We also identified one member of the cytoplasmic mitogen-activated protein kinase/extracellular-signal regulated kinases (MAP/ERK), p38 mitogen-activated protein kinase (p38  MAPK), to be up-regulated in the 2w sample, as we would expect it in a ETB dependent manner. Up-regulated second messengers are protein kinase C and associated molecules (19 clones), other kinases (9 clones), phosphatases (9 clones), phospholipase C and associated proteins (10 clones). This is the reason, why we believe that the phosphosphatidylinositol pathway plays a major role in regulation of left ventricular hypertrophy. Expression of myosin genes (30 clones) and of genes from associated proteins, but also other motor proteins, is regulated in our experiments. Although we also find down-regulated genes, most of them are up-regulated and, most interestingly, exclusively in the 12 w sample. A number of structural cytoskeletal proteins is necessary for cell-ECM contact and ECM formation. And indeed, we also found members of this gene family in our experiments. 22 clones encoding collagen subunits or enzymes involved in collagen turnover were up-regulated, most of them after two weeks (2 w). Only single clones show changes after 12 weeks, among them collagenase (UMCase), showing, that collagen turnover might steadily be continued. Procollagen C-proteinase enhancer protein, PCOLCE) was up-regulated after 2 and 12 weeks.

Whereas the majority collagens and proteoglycans obviously is up-regulated during the first days after operation, the 12 w group is dominated laminins, together with integrins. Therefore, initiated by the activation of the renin angiotensin system (RAS) at least two pathways seem to be involved in ECM activation, one going directly via G-proteins and second messengers (short term signaling), the other via motor proteins, actins and integrin (long term signaling). Some of the profiling data already  could be confirmed by immuno-histochemical assays and in situ hybridizations (e.g. up-regulation of preproendothelin 1, endothelin 1, fibroblast growth factor). More experiments in this direction are under way. Currently, we are going for a cDNA subarray on glass slides and nylon membranes, respectively, containing about 1,000 genes potentially involved in cardiac failure and also about 100 “housekeeping” genes as controls. With this array we want to confirm the data obtained so far, but also investigate samples from other time points after operation, samples from animals treated with certain drugs before operation and other experimental setups.

Our work is supported by the BMBF, Germany.



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