Project details

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The effect of human cytomegalovirus-encoded viral chemokine receptors on renal fibrogenesis in renal transplantation

Keywords:
virus kidney transplantation fibrosis

Researchers:
Dr. J. van den Born
prof. dr. W.J. van Son

Nature of the research:
Experimental and translational research in the field of kidney transplantation

Fields of study:
cell biology internal medicine nephrology

Background / introduction
50-60% of the Dutch population is seropositive for HCMV, with an age-dependent increase to over 90%. Infection is a challenge after renal transplantation, when immunosuppression is applied to prevent rejection of the transplant1. Patients with persistent intragraft HCMV demonstrated reduced graft survival and renal filtration function post-transplantation, and higher prevalence of transplant dysfunction2,3. HCMV encodes viral chemokine receptors (amongst others US28 and UL33), which demonstrate constitutive activity and can module intracellular signalling4. US28 regulates signalling implied in epithelial-to-mesenchymal transition (EMT) and interstitial fibrosis (eg. Src, TCF-LEF, STAT3).
Research question / problem definition
Our overall objective is to characterize the role of viral chemokine receptors US28 and UL33 in renal fibrogenesis, a key feature in human cytomegalovirus (HCMV)-mediated renal transplant dysfunction. We determine how chemokine receptors influence cell phenotype transition and its secretion profile through transfection and HCMV infection. This will result in better understanding of HCMV pathogenesis and potential for intervention on the molecular level.
We recently discovered expression of US28 in vascular smooth muscle and tubular epithelial cells in human recipient renal transplant biopsies; two renal compartments displaying key histopathological features during transplant dysfunction5. This is seen during active and latent infection, primary infection and reactivation and at various moments after transplantation. Initial infections confirm susceptibility of both cell types for HCMV and suggest a phenotype transition due to infection. We therefore investigate how viral chemokine expression during HCMV infection contributes to renal transplant dysfunction by initiating or instigating renal fibrogenesis. We hypothesize US28 to be involved in key features of fibrogenesis during transplant dysfunction; more specifically inducing loss of polarity, epithelial-to-mesenchymal transition and extracellular matrix production.
Workplan
(1) Vascular smooth muscle cells and tubular epithelial cells will be infected with wild type, UV-inactivated and various mutant HCMV strains; US28-, UL33-, US28+UL33-deficient and signalling-mute US28.
- Determine optimal HCMV titer, incubation time, infection development
- Infection will be validated using:
- Morphology under light microscope
- Immunohistochemical staining, western blot and qPCR on HCMV antigens (a.o. IEA, US28, UL33, UL54)

(2) vSMCs and TECs will be transfected with US28-, UL33-, signalling-mute US28 and UL33 expression constructs. Transfection will be validated using:
- qPCR for US28, UL33
- Luciferase reporter assays for US28 downstream targets

We will subsequently determine US28 functional effects;
(3) Effect on loss of polarity, contractile-to-synthetic in vascular smooth muscle cells and epithelial-to-mesenchymal transition tubular epithelial cells;
- Functional assays:
- Collagen contraction assay
- Migration assay (silicone inserts and transwell assay)
- Luciferase reporter assay

- Immunohistochemistry, immunofluorescence, western blot and qPCR for epithelial resp. contractile and synthetic markers
- Immunohistochemistry and immunofluorescence for production of extracellular matrix (eg. collagen)

(4) Effect on fibrotic cytokine and extracellular matrix production;
- ELISA for fibrotic cytokines; TGF-b1, CTGF, PDGF
- Immunohistochemistry, immunofluorescence, western blot and qPCR for epithelial resp. contractile and synthetic markers
- Co-culture with fibroblasts to observe phenotype change
References
1. van Son WJ, The TH. Cytomegalovirus infection after organ transplantation: An update with special emphasis on renal transplantation. Transpl Int 1989 Oct;2(3):147-64.
2. Helantera I, Koskinen P, Finne P, Loginov R, Kyllonen L, Salmela K, Gronhagen-Riska C, Lautenschlager I. Persistent cytomegalovirus infection in kidney allografts is associated with inferior graft function and survival. Transpl Int 2006 Nov;19(11):893-900.
3. Dzabic M, Rahbar A, Yaiw KC, Naghibi M, Religa P, Fellstrom B, Larsson E, Soderberg-Naucler C. Intragraft cytomegalovirus protein expression is associated with reduced renal allograft survival. Clin Infect Dis 2011 Nov;53(10):969-76.
4. Smit M, Lira S, Leurs R. Chemokine receptors as drug targets. Methods and Principles in Medicinal Chemistry 2011.
5. Strutz F. Pathogenesis of tubulointerstitial fibrosis in chronic allograft dysfunction. Clin Transplant 2009 Dec;23 Suppl 21:26-32.
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