Correction: Hydronephrotic Urine in the Obstructed Kidney Promotes Urothelial Carcinoma Cell Proliferation, Migration, Invasion through the Activation of mTORC2-AKT and ERK Signaling Pathways

<p>Correction: Hydronephrotic Urine in the Obstructed Kidney Promotes Urothelial Carcinoma Cell Proliferation, Migration, Invasion through the Activation of mTORC2-AKT and ERK Signaling Pathways</p

Hydronephrotic Urine in the Obstructed Kidney Promotes Urothelial Carcinoma Cell Proliferation, Migration, Invasion through the Activation of mTORC2-AKT and ERK Signaling Pathways

<div><p>Obstructive nephropathy is the most common presentation of urothelial carcinoma. The role of the urine in the obstructed kidney namely “hydronephrotic urine” in urothelial carcinoma has not been extensively explored. This study aims to evaluate whether hydronephrotic urine in the obstructed kidney could promote urothelial carcinoma. The hydronephrotic urine was collected from the obstructed kidneys of Sprague-Dawley rats induced by different periods of unilateral ureteral obstruction (UUO). By the inhibition of LY294002 and PD184352, we confirm that hydronephrotic urine promotes urothelial carcinoma cell (T24) and immortalized normal urothelial cells (E6) proliferation, migration and invasion in a dose-dependent manner through the activation of the mTORC2-AKT and ERK signaling pathways. Hydronephrotic urine also increases the expression of cyclin-D2, cyclin-B and CDK2. It also decreases the expression of p27 and p21 in both urothelial carcinoma cells and normal urothelial cells. By the protein array study, we demonstrate that many growth factors which promote tumor cell survival and metastasis are over-expressed in a time-dependent manner in the hydronephrotic urine, including beta-FGF, IFN-γ, PDGF-BB, PIGF, TGF-β, VEGF-A, VEGF-D and EGF. These results suggest that hydronephrotic urine promotes normal and malignant urothelial cells proliferation, migration and invasion, through the activation of the mTORC2-AKT and ERK signaling pathways. Further investigation using live animal models of tumor growth may be needed to clarify aspects of these statements.</p> </div

Hydronephrotic urine induced the phosphorylation of mTOR-Ser2481 and AKT-Ser473 in rat kidney after 3 weeks of UUO.

<p>The phosphorylation of mTOR-Ser2481 and AKT-Ser473 in normal rat kidney and rat kidney after 3 weeks of UUO was analyzed by immunohistochemistry stain (200X). Arrows indicate the urothelial cell layer in kidney.</p

Inhibition of mTORC2-AKT and ERK signaling pathway reduced T24 cells and E6 cells cell viability, migration and invasion in hydronephrotic urine treatment.

<p>To confirm the importance of mTORC2-AKT and ERK signaling pathway in T24 and E6 cells after the treatment of hydronephrotic urine, we used LY294002 (PI3K inhibitor) and PD184352 (ERK inhibitor) to analysis the cell function. (A) T24 cells and E6 cells were cultured in serum free medium and treated with LY294002 (20µM) and PD184352 (10µM) for 60 min and stimulated with 20µl/ml hydronephrotic urine from UUO 3 weeks for 30 min. The phosphorylation of mTOR-Ser2481, AKT-Ser473 and ERK were detected by western blotting (D: DMSO, LY: LY294002, PD: PD184352). (B, C) T24 cells and E6 cells were cultured in serum free medium and treated with LY294002 (20µM) and PD184352 (10µM) and stimulated with 20µl/ml hydronephrotic urine from UUO 3 weeks. The cells would be counted the cell number after treatment for 0, 24 and 48 hrs, respectively (Control: without hydronephrotic urine treatment, LY: LY294002, ERKi: PD184352). (D, E) T24 cells and E6 cells were cultured in serum free medium and treated with LY294002 (20µM) and PD184352 (10µM) and stimulated with 20µl/ml hydronephrotic urine from UUO 3 weeks for 24 hrs. The cells would be analyzed the migration capability by wound healing assay (Control: without hydronephrotic urine treatment, LY: LY294002, ERKi: PD184352). (F, G) T24 cells and E6 cells were cultured in serum free medium and treated with LY294002 (20µM) and PD184352 (10µM) and stimulated with 20µl/ml hydronephrotic urine from UUO 3 weeks for 12 hrs. The cells would be analyzed the invasion capability by transwell motility assay (Control: without hydronephrotic urine treatment, LY: LY294002, ERKi: PD184352).</p

Hydronephrotic urine promoted invasive capability of T24 cells and E6 cells.

<p>Hydronephrotic urine from 1 to 5 weeks after UUO promoted the invasion of T24 cells and E6 cells. (A, B) T24 cells and E6 cells were cultured in hydronephrotic urine from 1 to 5 weeks after UUO for 12 hrs and plated in the upper compartment of an 8-µm pore-size transwell invasion chamber which was coated with growth factor-reduced materigel and cultured in serum-free medium for 24 hrs. The numbers of invasive cells were determined by Giemsa staining (Control: without hydronephrotic urine treatment).</p

EGF induces proliferation, migration, invasion and activation of mTORC2-AKT and ERK signaling pathway in T24 and E6 cells.

<p>To identify whether ERK and mTORC2-AKT signaling pathway were activated by the EGF in the urothelial carcinoma cell, we analyzed the phosphorylation of AKT-Ser473 and ERK in T24 and E6 cells after treatment with EGF. (A, B) T24 cells and E6 cells were cultured in EGF (100ng/ml) for 0, 0.5, 1, 6, 12, 24 hrs, respectively. The phosphorylation of AKT-Ser473 and ERK was detected by western blotting. (C, D) T24 cells and E6 cells were cultured in serum free medium and stimulated with EGF (100ng/ml). The cells would be counted the cell number after treatment for 0 and 48 hrs, respectively. (E) T24 cells and E6 cells were cultured in EGF (100ng/ml) for 0, 24 and 48 hrs, respectively. The expressions of cyclin-B and cyclin-D1/2 were detected by western blotting. (F, G) T24 cells and E6 cells were cultured in serum free medium stimulated with EGF (100ng/ml) for 24 hrs. The cells would be analyzed the migration capability by wound healing assay. (H, I) T24 cells and E6 cells were cultured in serum free medium and stimulated with EGF (100ng/ml) for 12 hrs. The cells would be analyzed the invasion capability by transwell motility assay.</p

The expression of angiogenetic growth factors and cytokines in the hydronephrotic urine after UUO were higher than those in normal urine.

<p>In Human Angiogenesis Antibody Array analysis, the expressions of several growth factors were increased in the hydronephrotic urine from kidneys subjected to unilateral ureteral obstruction. (A) The Human Angiogenesis Antibody Array membrane was incubated with normal rat urine and hydronephrotic urine after 3 weeks UUO for 24 hrs. (B) The expression of bFGF, IFN-γ, PDGF-BB, PIGF, TGF-β, VEGF-A, VEGF-D and EGF in hydronephrotic urine were higher than those in normal urine. The relative intensity indicates the increase and decrease of the intensity of growth factors in hydronephrotic urine minus the intensity of growth factors in normal urine.</p

Hydronephrotic urine promoted the migratory capability of T24 cells and E6 cells.

<p>A wound healing assay was used to assess the migratory capability of T24 cells and E6 cells. (A, B) T24 cells and E6 cells were cultured in hydronephrotic urine from 1 to 5 weeks after UUO for 24 hrs. The ratio of gap healing was defined as the gap distance at 0h divided by the gap distance at 24h (0h/24h) (Control: without hydronephrotic urine treatment).</p

Hydronephrotic urine induced the expression of the cell cycle-regulated proteins and promoted the proliferation of T24 cells and E6 cells.

<p>(A) T24 cells and E6 cells were cultured in the hydronephrotic urine (10, 20, 50 µl/ml) at 3 weeks after UUO following serum starvation for 24 hrs. The expressions of cell cycle regulated proteins were analyzed by western blotting. (B) T24 cells were cultured in the hydronephrotic urine from 1, 2, 3, 4 and 5 weeks after UUO under serum starvation. Hydronephrotic urine at 2, 3, 4 and 5 weeks after UUO promoted cell proliferation but hydronephrotic urine did not promote T24 cells proliferation at 1 week after UUO (Control: without hydronephrotic urine treatment). (C) E6 cells were cultured in the hydronephrotic urine from 1, 2, 3, 4 and 5 weeks after UUO. Hydronephrotic urine at 1 to 5 weeks after UUO promoted proliferation of E6 cells (Control: without hydronephrotic urine treatment).</p

Incidence rate and hazard ratios of dementia in different severity and visiting numbers of hyponatremia.

<p>Incidence rate and hazard ratios of dementia in different severity and visiting numbers of hyponatremia.</p