Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by hamartomatous tumours of the brain, heart, skin, lung and kidney. Patients with TSC show a diverse range of neurological features (including seizures, cognitive disability and autism) and renal manifestations (including angiomyolipomas, epithelial cysts and renal cell carcinoma (RCC)). TSC is caused by inactivating mutations in TSC1 and TSC2, which encode hamartin and tuberin, respectively. These two proteins form a complex that negatively regulates mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of cellular growth and metabolism. In clinical trials, allosteric inhibitors of mTORC1 decrease angiomyolipoma size, but the tumours regrow after treatment cessation. Therefore, the development of strategies to eliminate rather than suppress angiomyolipomas remains a high priority. This Review describes important advances in the TSC field and highlights several remaining critical knowledge gaps: the factors that promote aggressive behaviour by a subset of TSC-associated RCCs; the molecular mechanisms underlying early-onset cystogenesis in TSC2-PKD1 contiguous gene deletion syndrome; the effect of early, long-term mTORC1 inhibition on the development of TSC renal disease; and the identification of the cell or cells of origin of angiomyolipomas.

. Loss of primary cilia results in deregulated and unabated apical calcium entry in ARPKD collecting duct cells.

Renal epithelial cells release ATP constitutively under basal conditions and release higher quantities of purine nucleotide in response to stimuli. ATP filtered at the glomerulus, secreted by epithelial cells along the nephron, and released serosally by macula densa cells for feedback signaling to afferent arterioles within the glomerulus has important physiological signaling roles within kidneys. In autosomal recessive polycystic kidney disease (ARPKD) mice and humans, collecting duct epithelial cells lack an apical central cilium or express dysfunctional proteins within that monocilium. Collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpk Tg737 ) mouse model of ARPKD lack a well-formed apical central cilium, thought to be a sensory organelle. We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation. Constitutive ATP release under basal conditions was low and not different in mutant versus rescued monolayers. However, genetically rescued principal cell monolayers released ATP three-to fivefold more robustly in response to ionomycin. Principal cell monolayers with fully formed apical monocilia responded three-to fivefold greater to hypotonicity than mutant monolayers lacking monocilia. In support of the idea that monocilia are sensory organelles, intentionally harsh pipetting of medium directly onto the center of the monolayer induced ATP release in genetically rescued monolayers that possessed apical monocilia. Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia. Our data also show that an increase in cytosolic free Ca 2+ primes the ATP pool that is released in response to mechanical stimuli. It also appears that hypotonic cell swelling and mechanical pipetting stimuli trigger release of a common ATP pool. Ciliumcompetent monolayers responded to flow with an increase in cell Ca 2+ derived from both extracellular and intracellular stores. This flow-induced Ca 2+ signal was less robust in cilium-deficient monolayers. Flow-induced Ca 2+ signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca 2+ . Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na + ) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases.Purinergic Signalling (...

Primary cilia are dynamic, complex structures that contain Ͼ500 proteins, including several related to polycystic kidney disease. How these proteins target to cilia and assemble is unknown. We previously identified Cys1 as the gene responsible for disease in Cys1 cpk mice, a mouse model of autosomal recessive polycystic kidney disease; this gene encodes cystin, a 145-amino acid cilium-associated protein. Here, we characterized the localization of cystin in the embryonic kidney and liver, in isolated renal collecting ducts, and in an inner medullary collecting duct mouse cell line. Because endogenous levels of cystin expression are low, we generated inner medullary collecting duct cell lines that stably express enhanced green fluorescence protein-tagged constructs of wild-type cystin or various truncation mutants. We determined that cystin is myristoylated at its G2 residue and that N-myristoylated cystin fractionates with membrane microdomains. Furthermore, the N-myristoylation signal is necessary but not sufficient to target cystin to the primary cilium. Analysis of deletion and chimeric constructs identified an AxEGG motif that is necessary to target and retain cystin in the cilium. Derangement of these localization motifs may lead to cystic kidney disease.

Primary cilia dysfunction alters renal tubular cell proliferation and differentiation and associates with accelerated cyst formation in polycystic kidney disease. However, the mechanism leading from primary ciliary dysfunction to renal cyst formation is unknown. We hypothesize that primary cilia prevent renal cyst formation by suppressing pathologic tubular cell hypertrophy and proliferation. Unilateral nephrectomy initiates tubular cell hypertrophy and proliferation in the contralateral kidney and provides a tool to examine primary cilia regulation of renal hypertrophy. Conditional knockout of the primary cilia ift88 gene leads to delayed, adult-onset renal cystic disease, which provides a window of opportunity to conduct unilateral nephrectomy and examine downstream kinetics of renal hypertrophy and cyst formation. In wild-type animals, unilateral nephrectomy activated the mTOR pathway and produced appropriate structural and functional hypertrophy without renal cyst formation. However, in ift88 conditional knockout animals, unilateral nephrectomy triggered increased renal hypertrophy and accelerated renal cyst formation, leading to renal dysfunction. mTOR signaling also increased compared with wild-type animals, suggesting a mechanistic cascade starting with primary ciliary dysfunction, leading to excessive mTOR signaling and renal hypertrophic signaling and culminating in cyst formation. These data suggest that events initiating hypertrophic signaling, such as structural or functional loss of renal mass, may accelerate progression of adult polycystic kidney disease toward end-stage renal disease.