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The risk potential of biotechnology-derived polypeptides causing nephrotoxicity based on assessment in human and animal renal cell lines using molecular and cellular biology techniques

Objective

To develop a spectrum of immortalized renal cell lines tailored to combine characteristics that make them sensitive to biomolecules in vitro. These will be used to screen novel verterinary and therapeutic biotechnology products to document change that underly adverse effects in vivo. The molecular basis of cellular changes will be used for risk assessment to maximise the economic benefits of development.
The project objectives were the optimization of culture conditions, functional characterization of cell systems, response of cells to biotechnology products, and validation of new cell systems. A major emphasis is on the design of novel human and animal renal cell lines. Current objectives include tailoring new cells, fully characterizing them, and developing sensitive methods to document cell modulation by potentially toxic molecules.

The first tailored cells and cells with improved phenotypic expression are being characterized biochemically and morphologically. The combined use of electrophysiology and fluorescent probes and specialized biochemical and molecular biological techniques are giving new insight into the effects of chemicals and biotechnology products on cells.

Several techniques and cell culture systems have been developed which should allow for design of safer biotechnology derived products and earlier detection of nephrotoxicity leading to more effective treatment.
We are developing an array of sensitive and selective in vitro methods for assessing the potential nephrotoxicity of biotechnology polypeptides and novel molecules that may affect the kidney in the clinical situation by :

Tailoring cells by fusion, transfection, nuclear injection and optimizing culture conditions (varying media, changing substrata, etc.), using primary rat, rabbit and pig cells; and continuous cell lines derived from the rat, pig, dog, man that represent each of the different regions of the kidney.
Defining functional and structural characteristics of these cells by molecular and cell biology techniques, including immunochemistry, fluorescent probes, hormone responsiveness, handling of macromolecules and ultrastructural assessment.
Establishing a full profile of response to biotechnology products by monitoring the secondary messenger systems, ion fluxes and membrane function, secretory proteins, electrophysiology, ultrastructure and immunohistochemistry. Polypeptide handling will be characterised by radio-, fluorescent or immuno-morphological and receptor binding ligand techniques. Polypeptides will include those with known nephrotoxic potential (eg, interferons), those affecting the kidney (ANF, Vassopressin), recombinant products, immunomodulators, neuropeptides, or appropriate biotechnology products will be compared.
New cells will be made available to all participants. Validation will include multicentre evaluation of cell culture methods and the correlative evaluation performed using higher order in vitro systems, such as the isolated defined nephron segment and the isolated perfused kidney.
Transfer technology and use the knowledge base for veterinary and human clinical safety and risk assessment of biotechnology produced polypeptides and novel molecules.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

University of East London
Address
Romford Road
E15 4LZ London
United Kingdom

Participants (5)

HANNOVER MEDICAL SCHOOL
Germany
Address
Carl-neuberg-strasse 1
30625 Hannover
LEOPOLD-FRANZENS-UNIVERSITAET INNSBRUCK
Austria
Address
3,Fritz-pregl-strasse 3
6020 Innsbruck
University College Dublin
Ireland
Address
Fosters Avenue Blackrock
4 Dublin
Université de Lausanne
Switzerland
Address
27,Rue Du Bugnon
1005 Lausanne
Université de Rouen - Haute Normandie
France
Address
Avenue De L'université
76800 Saint-etienne-du-rouvray