Projectdetails

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Isolation, characterisation and activation of human renal endothelial cells after postmortal kidney donation

Keywords:
kidney transplantation endothelial cells complement

Researchers:
Dr. J. van den Born
Prof. dr. S.P. Berger
R. Lammerts

Nature of the research:
Translational research in the field of transplantation

Fields of study:
cell biology immunology internal medicine

Background / introduction
The endothelium covers the surface of all blood vessels in our body and therefore resides at the critical interface between blood and the tissue. The endothelium plays an important role in innate and adaptive immunity and is involved in all disease states, either as primary determinant of pathophysiology or as a victim of collateral damage. 1 No endothelial cell is alike and so far there are only few universal phenotype markers known. The endothelium has different functions, corresponding to the parts of the body they are situated. For example, endothelial cells that line in the post capillary venules are primarily responsible for mediating leukocyte trafficking, while arteriolar endothelial cells regulate the vasomotor tone. This phenotypic heterogeneity is the core property of the endothelium. 2 In kidney transplantation endothelial cell activation plays an important role in immunological mediated rejection, being the first immunological barrier of the donor kidney that the recipient immune cells face. The renal endothelial cells are highly active and respond to circulating antibodies against human leukocyte antigens (HLA) of the donor and activate the complement system. Inflammation and complement deposition along the renal endothelial cells are diagnostic criteria in kidney transplant rejection, but unfortunately it is not clearly understood what exact pathophysiological role of renal endothelial cells is in rejection. 3
Research question / problem definition
In the last couple of years our group has been able to set up a reliable primary renal endothelial cell isolation method from post mortal kidney donors. Next steps will be:
1. Optimizing cell isolation techniques
2. Careful characterization of isolated endothelial cells
3. Analysis of endothelial cell activation
4. Analysis of the capacity of the endothelial cells to activate complement at various levels
This way we would have a better understanding of the role of endothelial cell activation in kidney transplantation and its role in transplant rejection.
Workplan
Various techniques will be practiced under close supervision with the possibility to develop yourself as an independent scientist working in the laboratory of experimental nephrology.
1. Endothelial cell isolation
a. Magnetic beads selection
b. Cell sorting using flow cytometry
c. Cell culture
2. Characterisation of endothelial cells
a. Immunofluoresent stainings
b. FACS stainings
c. DNA and RNA isolation
d. qRT-PCR
3. Endothelial activations assays
a. Endothelial cell activation in cell culture
b. Immunofluorescent stainings
c. FACS analysis
4. Complement activation assays
a. Immunofluorescent stainings of different complement markers
b. Complement dependent cytotocity test
c. FACS analysis on different complement markers
References
1. Aird WC. Endothelium in health and disease. Pharmacol Reports. 2008;60(1):139-143. doi:10.1055/s-2007-1002703
2. Molema G, Aird WC. Vascular Heterogeneity in the Kidney. Semin Nephrol. 2012;32(2):145-155. doi:10.1016/j.semnephrol.2012.02.001
3. Loupy A, Haas M, Solez K, et al. The Banff 2015 Kidney Meeting Report: Current Challenges in Rejection Classification and Prospects for Adopting Molecular Pathology. Am J Transplant. 2017;17(1):28-41. doi:10.1111/ajt.14107
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