application-retinoic-acid-to-obtain-osteocytes-cultures-from-primary

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico

The mucoactive drug S-carbocysteine lysine salt monohydrate (S-CMC-Lys) stimulates glutathione (GSH) efflux from respiratory cells. Since GSH is one of the most important redox regulatory mechanisms, the aim of this study was to evaluate the S-CMC-Lys effects on GSH efflux and intracellular concentration during an oxidative stress induced by the hydroxyl radical (xOH). Experiments were performed on cultured human respiratory WI-26VA4 cells by means of patch-clamp experiments in whole-cell configuration and of fluorimetric analyses at confocal microscope. xOH exposure induced an irreversible inhibition of the GSH and chloride currents that was prevented if the cells were incubated with S-CMC-Lys. In this instance, the currents were inhibited by the specific blocker CFTR(inh)-172. CFT1-C2 cells, which lack a functional CFTR channel, were not responsive to S-CMC-Lys, but the stimulatory effect of the drug was restored in LCFSN-infected CFT1 cells, functionally corrected to express CFTR. Fluorimetric measurements performed on the S-CMC-Lys-incubated cells revealed a significant increase of the GSH concentration that was completely hindered after oxidative stress and abolished by CFTR(inh)-172. The cellular content of reactive oxygen species was significantly lower in the S-CMC-Lys-treated cells either before or after xOH exposure. As a conclusion, S-CMC-Lys could exert a protective function during oxidative stress, therefore preventing or reducing the ROS-mediated inflammatory response.

S-CMC-Lys protective effects on human respiratory cells during oxidative stress.

[Synaptic-like signals at the filtration barrier: the role of nephrin].

The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) activity and localization are influenced by the cytoskeleton, in particular by actin and its polymerization state. In this study we investigated whether the expression of the hypertensive mutations of α-adducin (G460W-S586C in humans, F316Y in rats), an actin capping protein, led to a functional modification of CFTR activity and surface expression. The experiments were performed on HEK293 T cells cotransfected with CFTR and the human wild type (WT) or G460W mutated α-adducin. In whole-cell patch-clamp experiments, both the CFTR chloride current and the slope of current activation after forskolin addition were significantly higher in HEK cells overexpressing the G460W adducin. A higher plasma membrane density of active CFTR channels was confirmed by cell-attached patch-clamp experiments, both in HEK cells and in cultured primary DCT cells, isolated from MHS (Milan Hypertensive Strain, a Wistar rat (Rattus norvegicus) hypertensive model carrying the F316Y adducin mutation), compared to MNS (Milan Normotensive Strain) rats. Western blot experiments demonstrated an increase of the plasma membrane CFTR protein expression, with a modification of the channel glycosylation state, in the presence of the mutated adducin. A higher retention of CFTR protein in the plasma membrane was confirmed both by FRAP (Fluorescence Recovery After Photobleaching) and photoactivation experiments. The present data indicate that in HEK cells and in isolated DCT cells the presence of the G460W-S586C hypertensive variant of adducin increases CFTR channel activity, possibly by altering its membrane turnover and inducing a retention of the channel in the plasmamembrane. Since CFTR is known to modulate the activity of many others transport systems, the increased surface expression of the channel could have consequences on the whole network of transport in kidney cells.

Hypertension-linked mutation of α-adducin increases CFTR surface expression and activity in HEK and cultured rat distal convoluted tubule cells.

Segmental glomerulosclerosis is the end-point of a series of processes with have podocyte damage as a common denominator. This review summarizes the important advances that have been made in the past 2 years leading to the comprehension of several molecular mechanisms of regulation of podocyte physiology and pathology.

Anna Mondini

Renal Research Laboratory

Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts