Cyst formation following disruption of intracellular calcium signaling

Proc Natl Acad Sci U S A. 2014 Sep 30;111(39):14283-8. doi: 10.1073/pnas.1412323111. Epub 2014 Sep 16.

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.

Keywords: calcium release; polycysin 2; primary cilia.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium Signaling*
  • Cell Culture Techniques
  • Cilia / metabolism
  • Cilia / pathology
  • Culture Media
  • Gene Knockdown Techniques
  • Humans
  • Inositol 1,4,5-Trisphosphate Receptors / antagonists & inhibitors
  • Inositol 1,4,5-Trisphosphate Receptors / genetics
  • Inositol 1,4,5-Trisphosphate Receptors / metabolism
  • Kidney / metabolism
  • Kidney / pathology
  • LLC-PK1 Cells
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Mutation
  • Polycystic Kidney, Autosomal Dominant / genetics
  • Polycystic Kidney, Autosomal Dominant / metabolism*
  • Polycystic Kidney, Autosomal Dominant / pathology
  • Swine
  • TRPP Cation Channels / deficiency
  • TRPP Cation Channels / genetics
  • TRPP Cation Channels / metabolism

Substances

  • Culture Media
  • Inositol 1,4,5-Trisphosphate Receptors
  • TRPP Cation Channels
  • polycystic kidney disease 1 protein
  • polycystic kidney disease 2 protein