Cyst formation following disruption of intracellular calcium signaling

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. primary cilia | polycysin 2 | calcium release T he commonly occurring genetic kidney disorder, autosomal dominant polycystic kidney disease (ADPKD), is the result of mutations in polycystin 1 or 2 (PC1 or PC2). The progressive cyst formation within all segments of the nephron that defines the disorder leads to renal failure requiring treatment by dialysis and/or organ transplantation (1-3). Altered Ca 2+ signaling is one of several pathways that have been implicated in the disease (4, 5). A major limitation toward elucidating the role of Ca 2+ signaling in cyst formation has been the lack of easily manipulated, physiologically relevant experimental methodologies.In the past, ADPKD research has relied largely upon data from mouse models and cells maintained in 2D cell culture. Mouse models have played a significant role in understanding the biology of cyst formation but are unable to fully recapitulate the physiology of disease progression in humans due to the inherent differences between the species including life span, genetics, and environment. Two-dimensional cell culture has the ability to provide information on signaling pathways and response to therapies in a fast, high-throughput manner, but is incapable of replicating the inherent 3D nature of cyst formation. Advances in 3D tissue culture over the past 2 decades have improved the ability to model cyst development in vitro. However, previously published 3D tissue models of ADPKD have relied upon shortter...

Rapamycin treatment dose‐dependently improves the cystic kidney in a new ADPKD mouse model via the mTORC1 and cell‐cycle‐associated CDK1/cyclin axis

“…The abnormal proliferation and apoptosis of cystic epithelial cells are hallmarks of ADPKD . Consistent with previous studies , we found that although rapamycin did not affect apoptosis in the cyst‐lining epithelia of the Vil ‐Cre; Pkd2 f3/f3 kidney (Fig.…”

PDGFRβ and oncogenic, mutant PDGFRα D842V promote disassembly of primary cilia by a PLCγ and AURKA dependent mechanism

“…Notably, chimeric mice lacking endogenous PDGFRβ display renal cysts and glomerulosclerosis (Klinghoffer et al, 2001), phenotypes associated with defective intracellular Ca 2+ signaling (Kuo et al, 2014) and ciliopathies (Hildebrandt et al, 2011). Because PDGFRα D842V and AURKA expression is linked to GIST (Corless et al, 2011;Yeh et al, 2014), and precursors of GIST cells are ciliated (Castiella et al, 2013), it will be interesting to study whether GIST cells display ciliary defects that are caused by elevated PLCγ and AURKA activity.…”

Vasopressin and disruption of calcium signalling in polycystic kidney disease

“…60 Interestingly, knockdown of IP3R1 or IP3R3, like the knockdown of PC2, induces cell proliferation, apoptosis, and cyst formation in a collagen-matrigel three-dimensional culture system using pig kidney epithelial (LLC-PK) cells. 70 Several studies have shown that store operated calcium entry is reduced in PC2 defective cells 34,51,55,57,71 and it has been suggested that the interaction between STIM1 (a transmembrane sensor for calcium concentration in the endoplasmic reticulum lumen) and ORAi (the pore-forming unit of a plasma membrane channel that permits calcium entry in response to a reduction in the endoplasmic reticulum calcium concentration) is uncoupled in these cells. 71 Although PC2 is mostly located in the endoplasmic/sarcoplasmic reticulum membrane, truncated forms lacking the endoplasmic/sarcoplasmic reticulum retention domain translocate to the plasma membrane.…”