Stress alters the expression of aquaporins in cultured rat intestinal epithelial cells

Abstract

Aquaporins (AQPs) are widely-expressed small water channel proteins that provide the major route for water transport across plasma membranes in various cell types. Although the quantity of water transported in the intestinal tract is second only to that in the kidney, the precise role of AQPs in this organ remains largely uncertain. The present study reports the effects of hypertonic stress and ischemia/reperfusion injury on the expression of AQPs in intestinal epithelial cells. Cultured rat intestinal epithelial cells were incubated in 300 mM mannitol-containing, hypertonic culture medium or subjected to simulated ischemia/reperfusion treatment. The cell viability was evaluated by MTT assay, and the expression of AQPs was determined by semi-quantitative reverse transcription polymerase chain reaction and western blotting. Despite reduced viability, the cells exposed to hypertonic stress for 16 h demonstrated enhanced expression of AQP1 mRNA and protein. AQP9 and glycosylated AQP11 proteins were also markedly upregulated. Ischemia alone did not affect the cell viability, but subsequent reperfusion significantly reduced viability. The mRNA expression levels of all the tested AQPs were not altered by ischemia alone or by ischemia/reperfusion; however, AQP8 protein was markedly reduced by ischemic injury. In addition, treatment with ischemia alone eradicated the normally-expressed, non-glycosylated AQP11 protein whilst inducing pronounced expression of the glycosylated form. These observations may indicate that AQPs function in the intestinal epithelia in response to stress.

Keywords: aquaporin; hypertonic stress; intestinal epithelial cells; ischemia/reperfusion; mannitol.

Figure 1.

Effect of hypertonic stress on the viability of IEC-6 cells. Values presented are the mean ± standard error of the mean from 3 experiments. *P<0.05 vs. isotonic control. OD, optical density.

Figure 2.

Reverse transcription polymerase chain reaction analysis of AQP1 mRNA expression in IEC-6 cells in response to hypertonic stress. The expression of AQP1 was normalized against that of the housekeeper gene, β-actin. (A) Representative gel; (B) densitometric analysis of AQP1 mRNA expression. The expression in the isotonic control condition is arbitrarily normalized to 1. Results presented are the mean ± standard error of the mean from 3 experiments. *P<0.05 vs. control. AQP, aquaporin.

Figure 3.

Western blot analysis of AQP1 protein expression in IEC-6 cells in response to 16 h of hypertonic exposure. The expression of AQP1 was normalized against that of β-actin. (A) Representative immunoblot; (B) densitometric analysis of AQP1 protein expression. Results presented are the mean ± standard error of the mean from 3 experiments. *P<0.05 vs. isotonic control. AQP, aquaporin.

Figure 4.

Western blot analysis of AQP9 protein expression in IEC-6 cells in response to 16 h of hypertonic exposure. The expression of AQP9 was normalized against that of β-actin. (A) Representative immunoblot; (B) densitometric analysis of AQP9 protein expression. Results presented are the mean ± standard error of the mean from 3 experiments. *P<0.05 vs. isotonic control. AQP, aquaporin.

Figure 5.

Western blot analysis of AQP11 protein expression in IEC-6 cells in response to 16 h of hypertonic exposure. The expression of AQP11 was normalized against that of β-actin. (A) Representative immunoblot; (B) densitometric analysis of AQP11 protein expression. Results presented are the mean ± standard error of the mean from 3 experiments. *P<0.05 vs. isotonic control. AQP, aquaporin.

Figure 6.

Effect of ischemia and ischemia/reperfusion on the viability of IEC-6 cells. Values presented are the mean ± standard error of the mean of at least 3 experiments. *P<0.05 vs. isotonic control. OD, optical density.

Figure 7.

Western blot analysis of AQP8 protein expression in IEC-6 cells in response to ischemia, ischemia-reperfusion. The expression of AQP8 was normalized against that of β-actin. (A) Representative immunoblot; (B) densitometric analysis of AQP11 protein expression. Values presented are the mean ± standard error of the mean from 3 experiments. *P<0.05 vs. isotonic control. AQP, aquaporin.

Figure 8.

Western blot analysis of AQP11 protein expression in IEC-6 cells in response to ischemia and ischemia-reperfusion. The expression of AQP11 was normalized against that of β-actin. (A) Representative immunoblot; (B) densitometric analysis of AQP11 protein expression. Values presented are the mean ± standard error of the mean of 3 experiments. *P<0.05 vs. isotonic control. AQP, aquaporin; C, control; I, ischemia; I/R, ischemia-reperfusion.