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 Summary of overall project objectives
EuReGene pursues specific objectives in four areas that are most relevant to functional genomic
research in human kidney disease:
- Functional genomics technologies (Topic 1)
- Renal development (Topic 2)
- Pathophysiology (Topic 3)
- Complex genetics (Topic 4)
The generation of novel tools, methodologies and resources for genome research (Topic 1) are technological
objectives that apply to and overarch all scientific objectives in the disease-related projects on Renal
development (Topic 2), Pathophysiology of primary and acquired renal disorders (Topic 3) and Complex
genetics of kidney diseases (Topic 4).
Specific objectives
and results achieved in month 1 month 12
i) Functional genomics technologies (Topic 1)
Specific objectives:
Methods for automated high throughput in situ hybridization (ISH) on kidney sections and optical projection
tomography (OPT) are used to establish a three-dimensional map of the embryonic and the adult kidney transcriptome
in mouse and Xenopus. All data produced will be incorporated in specific databases. Bioinformatic tools will be
developed to link all EuReGene databases and to establish a kidney atlas where mechanisms of renal development and
disease can be studied in a spatio-temporal framework.
Progress:
The work is proceeding well. New methods, including 10 micron paraffin sectioning have been developed and the original
aim of having 400 genes analysed by month 18 is still on target. 78% of solute carrier (SLC) genes have already been
studied at embryonic day 14.5 and uploaded into databases. Development of EuReGene’s bioinformatics resources has
involved intense negotiation of standards and practices between EuReGene partners and outside collaborators.
Ontologies for the developing and adult kidney have been created and subjected to peer-review, and a Virtual Curation
Centre was implemented to facilitate exchange of information amongst partners.
ii) Renal development (Topic 2)
Specific objectives:
Developmentally expressed genes will be identified on a large scale using microarray and in situ hybridization in cell
lines, organ cultures and embryos, and their expression mapped back onto the developing kidney. Candidate genes will
be followed up by gene targeting or transgenic studies. Cell lineage and organ culture studies will be performed to
understand the origin of cellular components of the kidney. Differentiation pathways from renal stem cells to components
of the kidney will be explored. All data will be incorporated in a 3D atlas of the renal transcriptome.
Progress:
During the first year we have generated several transgenic mice that express markers under control of kidney specific
promoters and crossed them with a line that allows conditional immortalization of isolated cells. We are in the process
of establishing first cell isolates that will be characterized in detail. We have been using microarrays to map transcription
during metanephric kidney development from E10.5 to E17.5 and in the adult kidney and we have established a protocol for
manual microdissection of defined nephron segments. A panel of nephron segment-specific markers has been compiled and validation
is in progress. We started to map the full complement of solute carrier (SLC) genes expressed in the developing and adult
kidney. To date, we have generated gene expression data for >180 Xenopus SLC genes during pronephric kidney development and
>220 mouse SLC genes in the E14.5 embryo.
iii) Pathophysiology (Topic 3)
Specific objectives:
Rat and mouse models with perturbations in key renal genes will be established using knockout and transgene technologies.
Focus will be on genes associated with human renal disease. Sophisticated methods for characterization of renal (dys)function
in laboratory animals including morphological and pathophysiological tests, expression profiling, and imaging will be applied
to define disease processes and to uncover novel regulatory networks in renal (patho)physiology.
Progress:
During this first year it has been established that nephrin, an essential component of the glomerular ultra filtration system,
is a downstream target of Wt1. It has also been shown that ectopic activation of Pax2 in podocytes results in renal disease. A
renal expression map of the family of sortilin sorting receptors (5 isoforms, 4 being expressed in the kidney) was established,
at the mRNA and protein levels. In parallel, mice genetically deficient in sortilin-1 and sortilin-4 have been generated. Renal
stress models have been developed in rats to study perturbations in renal ion and phosphate transport caused by abnormal
distribution of NaPi transporter. A complete, description of the 3-dimensional organization of the mouse nephron was obtained.
iv) Complex genetics (Topic 4)
Specific objectives:
Systematic random ENU mutagenesis approaches in zebrafish and mouse
will be applied to generate new models of impaired renal development and
function, which will be further characterized by pathophysiological and
positional cloning approaches. Established rodent models of diabetic nephropathy,
glomerulosclerosis, proteinuria and renal stone disease will be used to
map modifier gene loci that affect disease progression and to identify new
disease genes by linkage/positional cloning.
Progress:
Progress in the first year of the project has been good with all projects underway and producing results. Several putative
functional mutations in Wt1 homologues have been identified, meeting the objective of generating new fish models. The blood
and urine screen has been established in ENU mice and at least 300 mice have been through the screen with the data being analyzed.
During these first 12 months of the program, candidate regions for genes underlying kidney stones in 2 ENU mice lines have been
confirmed and refined, and 3 candidate genes mapping to these regions are being tested. Similar progress has been made for mapping
gene(s) responsible for proteinuria in rats, with identification of 5 new genomic regions and the testing of one candidate gene.
Public dissemination of knowledge
Following intellectual property rights protection, research information generated in EuReGene will be made available through
freely accessible websites and databases. A number of measures have been implemented at this stage to ascertain timely information
of the public. In the project period reported here, the following activities have been carried out:
- implementation of a website with information for the scientific community, stakeholders in health politics, and the interested public
www.euregene.org
- strategic partnership with patent advocacy groups in Germany and UK, and their support in organization of several
workshops/seminars for patients and their families
- a summer school for European students working in the field renal disease research
- a public program for short-term fellowships
- an international symposium on “Renal Development” with experts from Europe and the US
Long term expected results and achievements
Our integrated research approach will have a fundamental impact
on our understanding of renal development and disease. After 4 years,
we expect to deliver the following measurable results:
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a comprehensive kidney atlas of renal developmental and pathophysiological processes (as data and as 3D image reconstructions)
- novel discovery tools including zebrafish, Xenopus, mouse and rat models, as well as new cell and organ cultures for kidney research
- repositories (models, cell lines, biopsies, DNA) and databases (expression maps) that are accessible throughout the scientific community
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a list of candidate genes responsible for (i) developmental, (ii) complex genetic and (iii) acquired renal diseases (diabetic nephropathy,
glomerulosclerosis, nephrotoxicity, proteinuria, end-organ damage) representing major new targets for diagnosis, drug development, and
therapeutic intervention
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a patent portfolio that protects the intellectual property rights of the EuReGene consortium and forms the basis for commercial exploitation
and funding beyond the FP6 period
- websites that inform stakeholders (patient advocacy groups, health care providers, scientists) about latest developments in renal disease research
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