Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals

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 (orpkTg737) 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 Ca2+ 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. Cilium-competent monolayers responded to flow with an increase in cell Ca2+ derived from both extracellular and intracellular stores. This flow-induced Ca2+ signal was less robust in cilium-deficient monolayers. Flow-induced Ca2+ 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 Ca2+. 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

Defect in recruiting effector memory CD8+ T-cells in malignant pleural effusions compared to normal pleural fluid

International audienceBackgroundMalignant pleural effusions (MPE) are a common and fatal complication in cancers including lung or breast cancers, or malignant pleural mesothelioma (MPM). MPE animal models and immunotherapy trials in MPM patients previously suggested defects of the cellular immunity in MPE. However only few observational studies of the immune response were done in MPM patients, using questionable control groups (transudate...).MethodsWe compared T cell populations evaluated by flow cytometry from blood and pleural effusion of untreated patients with MPM (n = 58), pleural metastasis of adenocarcinoma (n = 30) or with benign pleural lesions associated with asbestos exposure (n = 23). Blood and pleural fluid were also obtained from healthy subjects, providing normal values for T cell populations.ResultsBlood CD4+ or CD8+ T cells percentages were similar in all groups of patients or healthy subjects. Whereas pleural fluid from healthy controls contained mainly CD8+ T cells, benign or malignant pleural effusions included mainly CD4+ T cells. Effector memory T cells were the main T cell subpopulation in pleural fluid from healthy subjects. In contrast, there was a striking and selective recruitment of central memory CD4+ T cells in MPE, but not of effector cells CD8+ T cells or NK cells in the pleural fluid as one would expect in order to obtain an efficient immune response.ConclusionsComparing for the first time MPE to pleural fluid from healthy subjects, we found a local defect in recruiting effector CD8+ T cells, which may be involved in the escape of tumor cells from immune response. Further studies are needed to characterize which subtypes of effector CD8+ T cells are involved, opening prospects for cell therapy in MPE and MPM