Related Experiment Video
Updated: Apr 23, 2026
Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry
Published on: October 28, 2014
Cyst formation following disruption of intracellular calcium signaling
Ivana Y Kuo1, Teresa M DesRochers2, Erica P Kimmerling2
1Departments of Pharmacology and.
Abstract:
Mutations in polycystin 1 and 2 (PC1 and PC2) cause the common genetic kidney disorder autosomal dominant polycystic kidney disease (ADPKD). It is unknown how these mutations result in renal cysts, but dysregulation of calcium (Ca(2+)) signaling is a known consequence of PC2 mutations. PC2 functions as a Ca(2+)-activated Ca(2+) channel of the endoplasmic reticulum. We hypothesize that Ca(2+) signaling through PC2, or other intracellular Ca(2+) channels such as the inositol 1,4,5-trisphosphate receptor (InsP3R), is necessary to maintain renal epithelial cell function and that disruption of the Ca(2+) signaling leads to renal cyst development. The cell line LLC-PK1 has traditionally been used for studying PKD-causing mutations and Ca(2+) signaling in 2D culture systems. We demonstrate that this cell line can be used in long-term (8 wk) 3D tissue culture systems. In 2D systems, knockdown of InsP3R results in decreased Ca(2+) transient signals that are rescued by overexpression of PC2. In 3D systems, knockdown of either PC2 or InsP3R leads to cyst formation, but knockdown of InsP3R type 1 (InsP3R1) generated the largest cysts. InsP3R1 and InsP3R3 are differentially localized in both mouse and human kidney, suggesting that regional disruption of Ca(2+) signaling contributes to cystogenesis. All cysts had intact cilia 2 wk after starting 3D culture, but the cells with InsP3R1 knockdown lost cilia as the cysts grew. Studies combining 2D and 3D cell culture systems will assist in understanding how mutations in PC2 that confer altered Ca(2+) signaling lead to ADPKD cysts.
Insights
Calcium signaling disruption via polycystin 2 (PC2) or inositol trisphosphate receptors (InsP3R) causes kidney cysts in autosomal dominant polycystic kidney disease (ADPKD). InsP3R1 knockdown led to the largest cysts and cilia loss, highlighting calcium signaling
Area of Science:
- Nephrology
- Cell Biology
- Genetics
Background:
- Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in polycystin 1 (PC1) and polycystin 2 (PC2).
- Dysregulated calcium (Ca2+) signaling is a known outcome of PC2 mutations, impacting renal epithelial cell function.
- PC2 acts as a Ca2+-activated Ca2+ channel in the endoplasmic reticulum.
Purpose of the Study:
- To investigate the role of Ca2+ signaling through PC2 and inositol 1,4,5-trisphosphate receptors (InsP3Rs) in maintaining renal epithelial cell function.
- To determine if disruption of Ca2+ signaling contributes to renal cyst development in ADPKD.
- To validate the use of LLC-PK1 cells in 3D culture for studying ADPKD.
Main Methods:
- Utilized 2D and long-term 3D cell culture systems with LLC-PK1 cells.
- Employed knockdown techniques for PC2 and InsP3R (specifically InsP3R1 and InsP3R3).
- Assessed Ca2+ transient signals, cyst formation, and cilia integrity in cultured cells.
Main Results:
- Knockdown of InsP3R in 2D systems reduced Ca2+ signals, which were rescued by PC2 overexpression.
- Knockdown of PC2 or InsP3R in 3D systems induced cyst formation.
- InsP3R1 knockdown resulted in the largest cysts and subsequent cilia loss in growing cysts.
Conclusions:
- Disruption of Ca2+ signaling via PC2 or InsP3Rs is implicated in ADPKD cystogenesis.
- InsP3R1 plays a critical role in maintaining renal epithelial cell structure and function.
- Differential localization of InsP3R isoforms suggests regional Ca2+ signaling dysregulation contributes to cyst development.
- Combined 2D and 3D culture models are valuable for understanding ADPKD pathogenesis related to Ca2+ signaling alterations.
Related Concept Videos
01:27Feedback Regulation of Calcium Concentration
Various transmembrane receptors, such as G protein-coupled receptors (GPCRs), elicit a response to extracellular signals by increasing cytosolic calcium. Activated GPCRs...
01:24Intracellular Signaling Cascades
01:24Intracellular Signaling Cascades
01:16Calmodulin-dependent Signaling
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
01:16Cell Motility through Blebbing
Blebbing Through the Matrix
In multicellular...
01:16Cellular Injury IV: Necrosis