ESPN 53rd Annual Meeting

ESPN 2021


 
Use of epithelial models to study cyst formation in PKD
FATIMA HASSAN 1 THOMAS POKRANT 1 JAN FAIX 1 DIETER HAFFNER 1 WOLFGANG H ZIEGLER 1

1- HANNOVER MEDICAL SCHOOL
 
Introduction:

Hereditary cystic kidney disease is associated with defective epithelial morphogenesis and/or homeostasis. To address molecular aspects of genetically induced epithelial defects, canine renal tubular epithelial cells, MDCK, appear to provide a well-established model. In 3D culture, MDCK cells form monolayered epithelial spheroids with apicobasal polarity and controlled water and ion transport. These structures can be employed to study consequences of pharmacological stimulation on cell morphology and epithelial barrier. Here, we study loss of fibrocystin, the cause of ARPKD, on the behavior of epithelial spheroids to evaluate usefulness of the genetic model in two closely related MDCK cell lines.

Material and methods:

Fibrocystin function was suppressed by Pkhd1-silencing and CRISPR/Cas9-based genetic knockout in MDCKII and pl-MDCK, sub-cloned principal-like cells, respectively. Cells were grown in matrigel to allow spheroid formation, within 4 days, and treated with forskolin (FSK) to stimulate cAMP-induced cystic growth, as reported for PKD conditions. Lumen and cyst size were determined based on apical and basolateral markers using ImageJ/FIJI. Cyst formation was correlated to induction of STAT3 signaling.

Results:

Our data show that MDCKII and pl-MDCK respond differently to FSK. Lumen shrinkage was observed in MDCKII spheroids upon treatment, while Pkhd1-silencing led to mild lumen expansion with no further impact of FSK. In contrast, pl-MDCK spheroids show enhanced lumen size for Pkhd1-knockout lines and strong expansion upon FSK stimulation of control and knockouts, mimicking PKD behavior. As proposed for loss of fibrocystin, pSTAT3 was induced in stimulated Pkhd1-knockout cells but not controls.

Conclusions:

3D-culture models require functional evaluation of epithelial behavior to adequately reproduce tissue properties in vivo. Despite formation of well-established, polarized epithelial sheet and spheroids, MDCKII appear less suitable for PKD disease modeling, while pI-MDCK may constitute a useful cell model for pharmacological treatment and comparison of different disease genetics.