Rapamycin and Cl-IB-MECA were obtained from Sigma-Aldrich (Italy). Cell lines and cell culture Human normal (4/5) and human ADPKD (9.7 and 9.12) kidney epithelial cells as well as mouse PH2 (heterozygous) and PN24 (homozygous) gene knockout cell lines were generated by other laboratories [12, 13]. immunoblot and cell growth was analyzed by direct cell counting. Results The activation of A3AR by the specific agonist Cl-IB-MECA causes a marked reduction of CREB, mTOR, and ERK phosphorylation in kidney tissues of or genes which encode for polycystin-1 (PC1) or polycystin-2 (PC2), respectively . PC1 and PC2, interacting by each other, form a complex able to regulate different signalling pathways associated with cell proliferation, differentiation, and fluid secretion. Alteration of this complex leads to the dysfunction of a network of pathways, including cAMP, and mTOR signalling that may play an important role in the renal cyst development . Consistently, therapeutic interventions using compounds able to inhibit these pathways have already concluded clinical trials in ADPKD patients [3C6]. However, the treatment with mTOR inhibitors shows limited clinical success, while the use of Tolvaptan that reduces cAMP levels targeting V2 receptors, leads to significant advances in ADPKD kidney clinical picture [7, 8]. Nevertheless, the administration of Tolvaptan causes some side effects, such as polyuria and high activity of hepatic enzymes, increasing the management costs BNC105 of patients . Therefore, dialysis is still the main therapy for ADPKD patients, but it requires high healthcare and personnel costs and limits the life quality of patients . To improve benefits and reduce healthcare costs, the use of combined compounds able to target different pathways could be an attractive purpose for the treatment of ADPKD . Here, we report that this sequential treatment with rapamycin and Cl-IB-MECA causes a reduction of cell proliferation in cystic cell lines by the inhibition of mTOR, CREB, and ERK signalling. These data suggest that the combination of different BNC105 compounds could be a useful tool for the treatment of ADPKD patients. Materials and methods Media, fetal bovine serum, and plastic material for cell culture were purchased from EuroClone (Italy). Specific mTOR, P-mTOR, CREB, P-CREB, ERK, and P-ERK antibodies were obtained from Cell Signalling Technologies (EuroClone, Italy). -Actin and A3AR antibodies were acquired from Santa Cruz Technologies (DBA, Italy). Enhanced chemiluminescent substrates for Western blotting and HRP-conjugated goat anti-rabbit and anti-mouse antibodies were purchased from EuroClone (Italy). Rapamycin and Cl-IB-MECA were obtained from Sigma-Aldrich (Italy). Cell lines and cell culture Human normal (4/5) and human ADPKD (9.7 and 9.12) kidney epithelial cells as well as mouse PH2 (heterozygous) and PN24 (homozygous) gene knockout cell lines were generated by other laboratories BNC105 [12, 13]. Cell lines were produced in DMEM 50 % F12 medium supplemented with 10 %10 % FBS. Data shown in this study are in line with the 1975 declaration of Helsinki. Generation of Pkd1flox/?:Ksp-Cre mice test (unpaired analysis). Differences are considered significant at a value of < 0.05. All data are reported as mean standard deviation (SD) of at least 3 impartial experiments. Results and discussion Cl-IB-MECA reduced CREB, mTOR, and ERK activity in Pkd1flox/?:Ksp-Cre mice As previously described, adenosine type 3 receptors (A3AR) that have an inhibitory effect on adenylyl cyclase are up-regulated in human ADPKD kidney cells and tissues compared with normal controls . Consistently, A3 receptors positivity detected by immunohistochemistry is usually stronger in ADPKD kidneys where are confined to flat cells of renal cysts compared with normal kidneys (Fig. 1a). These receptors are also up-regulated in polycystic kidney tissues of vs Ct: ***< 0.001). Adenosine A3 receptors were also measured by saturation binding in cystic (tissue and PN24 cells than in Ct and PH2 (vs Ct: ***< 0.001 and PN24 vs PH2: *< 0.05). Data are expressed as mean SD of at Rabbit polyclonal to AMOTL1 least three impartial experiments Open in a separate window Fig. 2 Analysis of CREB, mTOR, and ERK activity in kidney tissuesc and renal cells treated and untreated with Cl-IB-MECA. a In basal conditions (mice than in Ct mice (mice vs Ct mice: **< 0.01). The administration of Cl-IB-MECA (0.2 mg/kg) reduces CREB activity in kidney tissues of mice (mice treated with DMSO vs mice treated with Cl-IB-MECA: **< 0.01). b Cl-IB-MECA administration does not change the kidney weight percentage calculated as ratio between kidney- and total body-weight in both control (Ct) and polycystic (< 0.05). The treatment with 100 nM Cl-IB-MECA causes a reduction of CREB activity in both 9.7 and 9.12 human cystic cells as well as in PH2 and PN24 mouse cystic cells compared with those treated with vehicle (9.7 cells treated with DMSO vs 9.7 cells treated with Cl-IB-MECA: *< 0.05; 9.12 cells treated with DMSO vs 9.12 cells treated with Cl-IB-MECA: **< 0.01; PH2 and PN24 cells treated with DMSO vs PH2 and PN24 cells treated with Cl-IB-MECA: *< 0.05). d In basal conditions (mice than in Ct mice (mice vs Ct mice: **< 0.01). The treatment with Cl-IB-MECA reduces mTOR phosphorylation in mice compared with those treated with vehicle.