BackgroundIndoleamine 2, 3-dioxygenase (IDO) is an immunomodulatory molecule that has been implicated in several biological processes. Although IDO has been linked with some renal diseases, its role in renal fibrosis is still unclear. Because IDO may be modulated by TGF-β1, a potent fibrogenic molecule, we hypothesized that IDO could be involved in renal fibrosis, especially acting in the TGF-β1-induced tubular epithelial-mesenchymal transition (EMT). We analyzed the IDO expression and activity in a model of renal fibrogenesis, and the effect of the IDO inhibitor 1-methyl-tryptophan (MT) on TGF-β1-induced EMT using tubular cell culture.MethodsMale Wistar rats where submited to 7 days of UUO. Non-obstructed kidneys (CL) and kidneys from SHAM rats were used as controls. Masson’s Tricrome and macrophages counting were used to chatacterize the tissue fibrosis. The EMT was analysed though immunohistochemistry and qRT-PCR. Immunohistochemestry in tissue has used to show IDO expression.MDCK cells were incubated with TGF- β1 to analyse IDO expression. Additionally, effects of TGF- β1 and the inhibition of IDO over the EMT process was acessed by immunoessays and scrath wound essay.ResultsIDO was markedly expressed in cortical and medular tubules of the UUO kidneys. Similarly to the immunolocalizaton of TGF- β1, accompanied by loss of e-cadherin expression and an increase of mesenchymal markers. Results in vitro with MDCK cells, showed that IDO was increased after stimulus with TGF-β1, and treatment with MT potentiated its expression. MDCK stimulated with TGF-β1 had higher migratory activity (scratch-wound assay), which was exacerbated by MT treatment.ConclusionsIDO is constitutively expressed in tubular cells and increases during renal fibrogenesis. Although IDO is induced by TGF-β1 in tubular cells, its chemical inhibitor acts as a profibrotic agent.

Immune escape and metastasis are the hallmarks of several types of cancer including bladder cancer. One of the mechanisms involved in these processes has been linked to indoleamine 2,3-dioxygenase (IDO). Although IDO is classically recognized for its immunomodulatory property, it has presented nonimmunological effects in some tumors. TGF-β1 is believed to contribute to carcinoma development by modulating immunossupressive molecules, including IDO. In addition, TGF-β1 induces the epithelial-mesenchymal transition (EMT), which is a critical step in the tumor invasiveness and metastasis. We investigated the role of MT and IDO modulation in the induction of EMT by TGF-β1 in T24 human bladder carcinoma cells. When T24 cells were incubated with the IDO inhibitor (MT, 1-methyl-D-tryptophan), with TGF-β1, and with MT+TGF-β1, a significant decrease of IDO expression and activity was observed. In addition, downregulation of e-cadherin and upregulation of n-cadherin and EMT transcription factors were induced by the treatments, confirming the induction of EMT. siRNA-mediated knockdown of IDO decreased e-cadherin expression, but had no effect on EMT transcription factors. In the scratch-wound assay, the heightened migration process was intensified when the cells were incubated with MT+TGF-β1. These effects were associated with a robust inhibition of Akt activation. After inoculation of T24 cells under the kidney capsule of Balb/c nude, the cells were positive for IDO in the center of the cell infiltrate, being negative in the periphery, where EMT is high. In conclusion, inhibition of IDO by TGF-β1 and MT is associated with EMT in T24 human bladder carcinoma cells. MT has potentiating effect in TGF-β1-induced EMT, independently of IDO. This nonimmunological effect of MT should be considered if IDO is the target to avoid immune escape in bladder cancer.

Angiogenesis is an essential process for the establishment, development, and dissemination of several malignant tumors including bladder cancer. The hypoxic condition promotes the stabilization of hypoxia-inducible factor 1 alpha (HIF-1α), which translocates to the nucleus to mediate angiogenic factors including the vascular endothelial growth factor A (VEGF-A). AnaeroGen system was developed for microbiology area to create a low oxygen tension required to the growth of anaerobic bacteria. Here, we hypothesized the use of AnaeroGen system to induce hypoxia in T24 human bladder carcinoma cells, in order to promote the overexpression of VEGF-A. T24 cells were cultured in six-well plates containing McCoy medium. Exposures of T24 cells to hypoxia for 1, 8, 24, and 48 h were performed using the Oxoid AnaeroGen system, while T24 cells under normoxia were used as control. The expression of VEGF-A and HIF-1α was analyzed by real-time PCR. ELISA for HIF-1α was carried out. The VEGF-A expression increased significantly by Oxoid AnaeroGen-induced hypoxia in a time-depending manner, reaching the peak in 48 h of hypoxia. Although HIF-1α mRNA was not changed, HIF-1α protein was increased in the presence of hypoxia, reaching a peak at 8 h. These results demonstrated that the Oxoid AnaeroGen system is a simple method to expose T24 cells to hypoxia and efficiently to upregulate VEGF expression in T24 cells.

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