Cyst formation following disruption of intracellular calcium signaling

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 (Ca²⁺) signaling is a known consequence of PC2 mutations. PC2 functions as a Ca²⁺-activated Ca²⁺ channel of the endoplasmic reticulum. We hypothesize that Ca²⁺ signaling through PC2, or other intracellular Ca²⁺ channels such as the inositol 1,4,5-trisphosphate receptor (InsP3R), is necessary to maintain renal epithelial cell function and that disruption of the Ca²⁺ signaling leads to renal cyst development. The cell line LLC-PK1 has traditionally been used for studying PKD-causing mutations and Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ signaling lead to ADPKD cysts.The 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 Ca²⁺ signaling is one of several pathways that have been implicated in the disease (4, 5). A major limitation toward elucidating the role of Ca²⁺ 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 short-term culture of Madin-Darby canine kidney (MDCK) cells (6–12) or cells from patients (13–18) or PC1-null mice (19, 20; for review, see ref. 21). Recently, 3D tissues have been developed that incorporate mouse cells containing a shRNA-mediated knockdown of PC1 (9, 19). The benefits of this system include the use of a cell line, thus eliminating the need to isolate primary cells, and the use of cells with a stable genetic background.Ca²⁺ signaling underpins many cellular processes ranging from cell proliferation to cell death. Intracellular Ca²⁺ levels can be modified by opening of the inositol 1, 4, 5-trisphosphate receptor (InsP3R) or other intracellular Ca²⁺ release channels, including PC2. Over 99% of PC2 resides on the endoplasmic reticulum (22), where it is known to act as a modulator of the InsP3R and the ryanodine receptor (RyR) (23), with the remainder on the primary cilia. PC2 itself can function as a Ca²⁺-activated Ca²⁺ release channel (22, 24).Although it was demonstrated in 3D cultures that the knockdown of PC1 leads to cyst development (25), the effect of knocking down PC2 or other Ca²⁺-signaling proteins has not been shown. It has been hypothesized that the disruption of PC2, or the proteins that it interacts with, will result in cyst growth, as Ca²⁺ is a major signaling molecule (26, 27). Cells with decreased PC2 have been linked with decreased Ca²⁺ signaling (28), and overexpression of PC2 has been shown to act as an inhibitor of cell proliferation (29). Changing PC2 expression levels alters the uptake of Ca²⁺ into the endoplasmic reticulum, leading to liver cyst formation (30), but no direct link involving the release of Ca²⁺ from the endoplasmic reticulum has been implicated in renal cyst development. Similarly, changes in the expression of the InsP3R have been correlated with various disease conditions; for example, the InsP3R is upregulated in colorectal cancer (31), but downregulated in bile duct obstruction and cholestasis (32, 33).Here, we demonstrate that cyst formation can be followed for several weeks using a 3D culture system and that the disruption of intracellular Ca²⁺ signaling, through the knockdown of either InsP3R or PC2, leads to cyst development.