ESPN 51th Annual Meeting

ESPN 2018


 
Development of cell models derived from early and late stage ADPKD patients
CAROLINE WEYDERT 1 JEAN-PAUL DECUYPERE 1 PETER JANSSENS 2 ELKE BEHAEGHEL 1 STEPHANIE DE RECHTER 1 BERT BAMMENS 3 Rudi Vennekes 4 DJALILA MEKAHLI 3

1- Department of development and regeneration, KU LEUVEN, Leuven, Belgium
2- UZ BRUSSEL, Brussels, Belgium
3- UZ LEUVEN, Leuven, Belgium
4- Department of Molecular and Cellular Medicine, KU Leuven, Leuven, Belgium
 
Introduction:

Despite several research efforts, the molecular mechanisms behind cyst formation in Autosomal Dominant Polycystic Kidney Disease are still unclear. Nephrectomised kidneys currently form the main source of human-derived cystic cells and cell lines. However, as they represent an advanced disease stage with many possible compensatory cellular alterations, it will be difficult to unravel cyst-initiating factors in these cell models.  Therefore, we aim to establish a molecularly and functionally well-characterized novel renal cell model, collected non-invasively via urine from paediatric ADPKD patients. 

Material and methods:

First, we generated proximal tubules epithelial cells (PTEC) collected from urine of genotyped ADPKD children with signs of early disease and adult patients with end-stage renal disease. Primary cells were cultured and subsequently immortalised. Next, monoclonal cell lines were obtained and validated using a panel of molecular PTEC markers. Functional characterization included proliferation measurements (IncuCyte), Ca2+-imaging using FURA 2-AM, detection of phosphorylation of downstream mTOR targets using Western Blotting analysis and cAMP-metabolism measurements (HTRF).

Results:

Cell lines were obtained from urine (5 controls + 5 patients). These cell lines express the PTEC markers aquaporin-1, multidrug resistance protein 1 and the epithelial marker EpCAM, as measured by Western Blotting analysis and RT-qPCR. The absence of cadherin-1 confirms the PTEC origin, as cadherin-1 is strongly expressed in all nephron segments except in proximal tubules. In addition, our cells express cilia, as shown by Arl13b staining. Furthermore, the cell lines from ADPKD patients with PKD1 mutation exhibited a lower PC1 expression compared to control cells. Currently, these cell lines are further validated for differences in proliferation, cAMP response, Ca2+ and mTOR metabolism between disease and control.

Conclusions:

We present a novel human-derived cell model, collected non-invasively from patients with signs of early ADPKD. As such, this model can be exploited to identify cyst-initiating factors in ADPKD.