The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin 1 (mTORC1) signaling pathway is aberrantly activated in renal cell carcinoma (RCC). We previously demonstrated glycogen synthase kinase-3β (GSK-3β) positively regulated RCC proliferation. The aim of this study was to evaluate the role of GSK-3 in the PI3K/Akt/mTORC1 pathway and regulation of the downstream substrates, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), ribosomal protein S6 kinase (S6K), and ribosomal protein S6 (S6RP). We used human RCC cell lines (ACHN, Caki1, and A498) and, as normal controls, human renal proximal tubular epithelial cell (HRPTEpC) and non-tumorous kidney tissues that were obtained surgically for treatment of RCC patients. Rapamycin-resistant ACHN (ACHN/RR) cells were generated with chronic exposure of ACHN to rapamycin ranging from 1nM finally to 1 μM. Cell viability, cell cycling and direct interaction between GSK-3β and 4EBP1 were evaluated with MTS assay, flowcytometry and in vitro kinase assay with recombinant GSK-3β and 4EBP1products, respectively. Protein expression and phosphorylation of molecules associated with the PI3K/Akt/mTORC1 pathway were examined by immunoblotting. Effects of drug combination were determined as the combination index with CompuSyn software. Overexpression and phosphorylation of 4EBP1 and S6RP together with GSK-3 activation were observed in RCC cell lines, but not in human normal kidney cells and tissues. Cell proliferation, p4EBP1 and pS6RP were strongly suppressed by GSK-3 inhibition. Rapamycin and LY294002 sufficiently decreased pS6RP, but only moderately p4EBP1. In vitro kinase assays showed that recombinant GSK-3β phosphorylated recombinant 4EBP1, and the effect was blocked by GSK-3 inhibitors. Different from rapamycin, AR- A014418 remarkably inhibited cell proliferation, and rapidly suppressed p4EBP1 and pS6RP in ACHN and ACHN/RR (in 30 min to 1 h). AR- A014418 and rapamycin combination showed additivity at lower concentrations, but antagonism at higher concentrations. GSK-3β could directly phosphorylate 4EBP1 and activate the mTORC1 downstream signaling cascades to enhance protein biosynthesis and cell proliferation in RCC cell lines independent of rapamycin sensitivity. The direct GSK-3β/4EBP1 pathway might be an important subcellular mechanism as an inherent equipment for RCC cells to acquire clinical chemoresistance to mTORC1 inhibitors.

Ureterolithiasis is one of the most frequently diagnosed urologic diseases worldwide. Its annual incidence in Japan increased three-fold from 1965 to 2005. Ureterolithiasis incidence is affected by numerous factors, including race, sex, body weight, fluid intake, and climate. Here, we aimed to address the latter by considering the effect of seasonal variation on stone incidence and incorporating this information into a predictive model for differential diagnosis of ureteral stone from other conditions with similar presentations. We retrospectively identified 491 patients in our emergency department computer database who complained of back, flank, or lower abdominal pain during 2007–2015. Among them, 358 had stones, as confirmed by computerized tomography or plain abdominal X-ray of kidney–ureter–bladder. We also charted the mean ambient temperatures in our city for a year. The cases of ureteral stones paralleled the ambient temperatures, peaking during the hottest weather. Univariate analysis identified 13 factors associated with ureteral stones. Multivariate analysis narrowed the number to eight: age <60 years, male sex, short duration of pain (<6 h), nausea/vomiting, hydronephrosis, hematuria, history of urinary stone(s), and summer (July–September in Japan). Pain appearing during summer was nine times more likely to be due to a ureteral stone than was pain appearing during other seasons. We incorporated the eight variables identified into a predictive logistic regression model, which yielded good prediction of ureteral stones. Awareness that hot weather is associated with increased incidence of ureterolithiasis could facilitate differential diagnosis, and our prediction model could be useful for screening for ureterolithiasis.

The CHOKAI and STONE scores are prediction models for ureteral stones. The aims of the present study were to evaluate the diagnostic performance, to examine the optimal cut-off value, and to compare the diagnostic performance of each model. Patients who presented to our emergency department with renal colic were considered for this prospective study. We analyzed the predictive performance of both STONE and CHOKAI scores at their optimal cut-off values, using receiver operating characteristic (ROC) curve and area under the curve (AUC), as well as sensitivity, specificity, positive likelihood ratio (LR+), and negative likelihood ratio (LR-) at the optimal cut-off value. Of the 96 patients who met the inclusion criteria, 79 were definitively diagnosed with ureteral stones. All patients were of Japanese descent. The AUC of the CHOKAI score was 0.971 at an optimal cut-off value of 6, showing a sensitivity of 0.911, specificity of 0.941, LR+ of 15.49, and LR- of 0.094. The AUC of the STONE score was 0.873 at an optimal cut-off value of 8, showing a sensitivity of 0.823, specificity of 0.824, LR+ of 4.662, and LR- of 0.215. The AUC of the CHOKAI score was significantly higher than that of the STONE score (p=0.010). Of the 73 patients with a CHOKAI score of ≥6, 98.6% had ureteral stones, and of the 68 patients with a STONE score of ≥8, 95.6% had ureteral stones. The simplified CHOKAI score is a useful tool to screen for ureteral stones in patients with renal colic.