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 (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.

Polycystin 2 regulates mitochondrial Ca2+ signaling, bioenergetics, and dynamics through mitofusin 2.

Mitochondria and the endoplasmic reticulum (ER) have an intimate functional relationship due to tethering proteins that bring their membranes in close (~30 nm) apposition. One function of this interorganellar junction is to increase the efficiency of Ca2+ transfer into mitochondria, thus stimulating mitochondrial respiration. Here, we showed that the ER cation-permeant channel polycystin 2 (PC2) functions to reduce mitochondria-ER contacts. In cell culture models, PC2 knockdown led to a 50% increase in mitofusin 2 (MFN2) expression, an outer mitochondrial membrane GTPase. Live-cell super-resolution and electron microscopy analyses revealed enhanced MFN2-dependent tethering between the ER and mitochondria in PC2 knockdown cells. PC2 knockdown also led to increased ER-mediated mitochondrial Ca2+ signaling, bioenergetic activation, and mitochondrial density. Mutation or deletion of the gene encoding for PC2 results in autosomal dominant polycystic kidney disease (ADPKD), a condition characterized by numerous fluid-filled cysts. In cell culture models and mice with kidney-specific PC2 knockout, knockdown of MFN2 rescued defective mitochondrial Ca2+ transfer and diminished cell proliferation in kidney cysts. Consistent with these results, cyst-lining epithelial cells from human ADPKD kidneys had a twofold increase in mitochondria and MFN2 expression. Our data suggest that PC2 normally serves to limit key mitochondrial proteins at the ER-mitochondrial interface and acts as a checkpoint for mitochondrial biogenesis and bioenergetics. Loss of this regulation may contribute to the increased oxidative metabolism and aberrant cell proliferation typical of kidney cysts in ADPKD.

Silk as a Biomaterial to Support Long-Term Three-Dimensional Tissue Cultures.

Tissue engineering has broad and diverse impacts on a variety of different applications from tissue regeneration to drug screening. While two-dimensional (2-D) cell culture platforms are suitable for tissue interfaces where planar surfaces are relevant, three dimensional (3-D) tissue models have enhanced relevance and sustainability over 2-D devices. The improvements between 2-D and 3-D functions and sustainability are related to the limitations of 2-D systems to support proper cellular morphology and signaling over time, resulting in cell overgrowth or changes in viability. For sustainable (long-term) cultures, 3-D silk protein scaffolds provide biocompatibility, porous features for transport, robust yet tunable mechanical properties, retain size and open porous structures for extended time frames due to slow proteolytic biodegradation, avoid specific cell signaling, and require no chemical cross-linking. Silk degradation can be extended for months to years without premature collapse of structures (that would result in necrosis) to support cell interactions during slow remodeling toward native tissue. Silk can also be fabricated into different material formats, such as hydrogels, tubes, sponges, composites, fibers, microspheres, and thin films, providing versatile platforms and interfaces for a variety of different applications. For sustainable tissue engineering applications, many formats have been used, including silk ionmer hydrogels that have been cultured for up to 8 weeks and porous silk scaffolds that have been cultured for up to 6 months. In this review, we highlight some of our tissue engineering work related to long-term in vitro cultures. While each tissue engineered system (adipose tissue, cortical brain tissue, intestine, kidney tissue, bone) is unique, they all use silk biomaterials as a base scaffolding material to achieve sustainable cultivation. Sustainability is important for studies that extend past a few weeks to study acute and chronic impacts of treatments, disease models, and other related applications in the field of tissue engineering.

The effects of mycoplasma contamination upon the ability to form bioengineered 3D kidney cysts.

Mycoplasma contamination of cell cultures is a pervasive, often undiagnosed and ignored problem in many laboratories that can result in reduced cell proliferation and changes in gene expression. Unless contamination is specifically suspected, it is often undetected in two dimensional (2D) cultures and the resulting effects of mycoplasma contamination are rarely appreciated and can lead to incorrect conclusions. Three dimensional (3D) tissue cultures are increasingly utilized to explore tissue development and phenotype. However, 3D cultures are more complex than 2D cell cultures and require a more controlled cellular environment in order to generate structures necessary to mimic in vivo responses and are often maintained for longer time periods. Changes to the microenvironment are assumed to have a more extreme effect upon the success of 3D tissue cultures than 2D cell cultures, but the effects of mycoplasma have not been studied. To test this hypothesis, we grew 2D cell cultures and 3D tissues from pig kidney epithelial cells (LLC-PK1) that were contaminated with mycoplasma and the same stock of cells after mycoplasma removal. We did not observe an effect of mycoplasma contamination on proliferation in 2D monolayer cell culture. However, cyst formation in 3D tissues was altered, with effects upon the number, size and structure of cysts formed. These data serve to reinforce the necessity of testing cell stocks for mycoplasma contamination.