Creating new in vitro models to examine the role of Pkd1 in cell and tubular physiology

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) results from loss of function mutations in polycystin proteins PC1 and PC2 encoded by the PKD1 and PKD2 genes. ADPKD affects 1 in 400 to 1 in 1000 people worldwide, with approximately 50% of patients progressing to end stage renal disease. The physiological function of the polycystins and the mechanisms of cystogenesis remain largely unknown. In vitro models may provide the clearest possibility to understand the immediate cell biological effects of polycystin loss, without the confounding second order injury that occurs as cysts develop. Some of the hurdles to in vitro models include clonal variation, renal segment heterogeneity, and inconsistent growth in 3D culture conditions. To address this need, we isolated renal epithelial cells from either the cortex or the medulla of Pkd1flox/flox; Pax8-rtTA; tetO-7-Cre mice crossed with the immorto-mouse expressing the temperature sensitive SV40 large T antigen. We then cloned single cells into isogenic cell lines, one cortical and one medullary. We verified the induction of Pkd1 KO by treating the cells with either 10 ug/ml doxycycline to knock out (KO) Pkd1 or vehicle control. Treatment continued for 7 days, after which Pkd1 KO was confirmed by western blot. Next, we sought to functionally characterize the two new cell lines. When the cells were grown on transwell supports, cells formed epithelial tight junctions with a trans-epithelial resistance of 2467 ohms/cm2. Resistance increased to 3130 ohms/cm2 following Pkd1 KO in the medullary clone. We next measured the rate of media acidification with or without Pkd1 KO as a proxy for glucose utilization. Pkd1 KO cells acidified faster than controls in both clones. Next, we addressed their ability to grow into tubuloids or spheroids / cysts in 3D culture using multiple approaches. Cells were either 1) suspended directly in Matrigel and plated in a 96 well plate, 2) suspended in Matrigel droplets, with multiple droplets adhering to an individual well of a 6 well plate, 3) plated within a Matrigel “sandwich”, or 4) plated within a sandwich and pulsed with glial derived neurotropic factor (GDNF) for 48 hours. All clones suspended directly in Matrigel formed spheroids, with more and larger spheroids in the Matrigel droplet cultures. Similar results were observed in the cortical clone grown in the sandwich, however the Pkd1 KO medullary clone in the same culture conditions had several giant spheroid structures at least 3 times larger than any other organoid observed in control medullary cultures. Finally, cortical cultures grown in the sandwich and pulsed with GDNF revealed larger spheroid structures in the Pkd1 KO cells than the control structures. Medullary cultures grown with GDNF revealed complicated structures with lumens and tubuloid projections in the controls, and large hollow spheroid / cystic structures in the Pkd1 KO cultures. Taken together, these data support the Matrigel sandwich with GDNF pulse may be the most useful to interrogate physiological changes in the absence of Pkd1 expression. In summary, we have created two new clonal cell lines with inducible Pkd1 KO, with isogenic controls, and the ability to differentiate into complicated tubuloids or spheroids in a genotypical manner, that may help elucidate the most proximate events occuring in renal epithelial cells after the inactivation of Pkd1. U54 DK126114. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.