Effect of Arsenate and p-Phenylenediamine on the Expression of Aquaporins in Cultured Human Urothelial Cells

Abstract

Background: Exposure to arsenic (As) or p‑phenylenediamine (PPD) can lead to dysfunction, or even cancer, in various types of organs, including the urinary bladder, yet the underlying mechanisms remain unclear. Aquaporins (AQPs) are widely expressed small water channel proteins that provide the major route for the transport of water and other small molecules across plasma membranes in diverse cell types. Altered expression of AQPs has been associated with pathologies in all major organs, including the urinary bladder.

Objective: The present in vitro study was performed as a first step towards exploring the possible involvement of AQPs in As- and PPD‑induced bladder diseases.

Methods: An immortalized normal human urothelial cell line was employed. Cells were exposed to different concentrations of sodium arsenate (0‑20 μM) or PPD (0‑200 μM) for 48 h. Cell viability was subsequently assessed. The mRNA and protein expression levels of AQPs (specifically, AQP3, 4, 7, 9, and 11) were analyzed using reverse transcription‑quantitative polymerase chain reaction and Western blot analyses, respectively.

Results: The viability of the cells was decreased in a concentration-dependent manner upon exposure to arsenate. The mRNA and protein expression levels of AQP3, 4, 7, and 9 were substantially reduced, whereas the expression of AQP11 was largely unchanged. As for the experiments with PPD, treatment with increasing concentrations of PPD induced a gradual decrease in cell viability. The mRNA and protein expression levels of AQP3, 4, and 11 were generally unaltered; however, a marked reduction in the expression levels of AQP7 was observed, contrasting with a gradual concentration-dependent decrease in the expression of AQP9.

Conclusion: The importance of the differential expression profiles of the AQPs induced by arsenate and PPD requires further investigation; nevertheless, the findings of the present study suggest that AQPs have a role in As‑ and PPD‑induced bladder diseases.

Keywords: aquaporin; arsenate; p-phenylenediamine; urinary bladder; urothelial cell.

Figure 1. Effect of sodium arsenate on the cell viability and expression levels of AQP3 and AQP4 in SV-HUC1 cells.

(A) Cell viability. The data are presented as the mean ± SEM for three independent experiments. (B, D) RT‑qPCR analysis was used to examine the effect of arsenate on the mRNA expression levels of AQP3 and AQP4. The results are shown as the mean ± SEM for data from four different experiments, and the expression levels were normalized against that of the housekeeping gene, GAPDH. (C, E) Western blot analysis was used to detect the effect of arsenate on the protein expression levels of AQP3 and AQP4. The expression levels were normalized against that of β‑actin. *P<0.05 compared with control (0 µM As). AQP: aquaporin, SEM: standard error of the mean, RT-qPCR: reverse transcription-quantitative polymerase chain reaction

Figure 2. Effect of sodium arsenate on the expression levels of AQP7, 9, and 11 in SV-HUC1 cells.

(A, C, E) RT‑qPCR analysis of AQP7, AQP9, and AQP11. The results are shown as the mean ± SEM for data from four different experiments, and the expression levels were normalized against that of the housekeeping gene, GAPDH. (B, D, F) Western blot analysis was used to detect the protein expression levels of AQP7, AQP9, and AQP11. The expression level was normalized against that of β‑actin. *P<0.05 vs. control (0 µM As); #P<0.05 vs. 10 µM. AQP: aquaporin, SEM: standard error of the mean, RT-qPCR: reverse transcription-quantitative polymerase chain reaction

Figure 3. Effect of PPD on the cell viability and expression levels of AQP3 and AQP4 in SV-HUC1 cells.

(A) Cell viability. Data are shown as the mean ± SEM of three independent experiments. (B, D) RT‑qPCR analysis was used to examine the effect of PPD on the mRNA expression levels of AQP3 and AQP4. The results shown are the mean ± SEM for data from four different experiments, and the expression levels were normalized against that of the housekeeping gene, GAPDH. (C, E) Western blot analysis was used to detect the effect of PPD on the protein expression levels of AQP3 and AQP4. The expression levels were normalized against that of β‑actin. *P<0.05 vs. control (0 µM PPD). AQP: aquaporin, SEM: standard error of the mean, RT-qPCR: reverse transcription-quantitative polymerase chain reaction, PPD: p-phenylenediamine

Figure 4. Effect of PPD on the expression levels of AQP7, 9, and 11 in SV-HUC1 cells.

(A, C, E) RT‑qPCR analysis of AQP7, AQP9, and AQP11. The results are shown as the mean ± SEM for data from four different experiments, and the expression levels were normalized against that of the housekeeping gene, GAPDH. (B, D, F) Western blot analysis was used to detect the protein expression levels of AQP7, AQP9, and AQP11. The expression levels were normalized against that of β‑actin. *P<0.05 vs. control (0 µM PPD); #P<0.05 vs. 50 µM PPD. AQP: aquaporin, SEM: standard error of the mean, RT-qPCR: reverse transcription-quantitative polymerase chain reaction, PPD: p-phenylenediamine