| ObjectiveThe former study have confirmed that in rat model of obstructive jaundice,the most important pathophysiological course is the damage of collecting duct cells which mainly perform down-expression of aquaporin 2(AQP2). The main mechanism of renal injury is the endotoxin damage. We take the follow-up study to research the effect of endotoxin to mouse inner medullary collecting duct cells (mIMCD-3) and corresponding changes of AQP2.MethodsTrained and passage mIMCD-3 cells to produce climb films. Immunohistochemical staining was used to test the expression and localization of AQP2. Cells were seeded in 96-well plates and incubated for 4-24h with different concentration of LPS. The CCK-8 was used to determine the inhibitory effect. Selecting the final concentration of 10μmol/L of LPS to be cultured with cells for 4-24h, flow cytometry was used to calculate inhibitory rate. The expression of AQP2 was detected by immunohistochemistry。ResultsmIMCD-3 expressed AQP2. After immunohistochemical staining of climb film, a large number of brown-red particles piled up like plates could be seen in the cytoplasm. It proved that APQ2 localized in the cytoplasm in the basic state. At the same time, negative control cells which didnot add first antibody, showed none of brown-red particles generated in the cytoplasm. The entire cytoplasm was stained light blue.LPS inhibited mIMCD-3 proliferation. one-way ANOVA was used to analysis the significance of concentration,the results had statistic significance(P<0.05).Doing inter-group analysis,4 hours 0.5μg/L and 1μg/L,5μg/L and 10μg/L; 8 hours 0.5μg /L and 1μg/L; 12 hours 0.1μg/L and 0.5μg/L,0.5μg/L and 24 hours 1μg/L,1μg/ L and 5μg/L,5μg/L and 10μg/L comparison, the results did not have statistical difference (P> 0.05). It proved that when the concentration was less than 5μg/L, the inhibition of cell proliferation was not very apparent. When the concentration gradient increased, the inhibitory effect increased with dose-dependent manner. With the concentration of 0.1μg/L the cells did not inhibit, but proliferated. When the time was up to 24 hours, the adjacent inter-group comparison had non-statistical differences.It had statistical difference(P<0.05). However, 0.1μg/L group 4 hours and 8 hours,0.5μg/L group 8 hours and 12 hours,12 hours and 24 hours compared, 1μg/L group 4 hours and 8-hour comparison, they had no significant difference (P>0.05). It Proved that when the concentration was less than 1μg/L, the inhibitory rate changed little over time. Once greater than or equal to the concentration of 5μg/L, the contrasts between any two groups were significantly different. It showed a time-dependent manner.LPS-induced apoptosis. Flow cytometry results showed that the inhibition of LPS on mIMCD-3 was mainly expressed in the manner of apoptosis. When time extended, apoptosis rate increased (P<0.01).AQP2 expressed mainly in the cytoplasm as evidenced large piles of brown-red particles. The expression of AQP 2 reduced when LPS joined. The granules became sparse brown, lighter in color by immunohistochemistry. By analyzing average optical density, the results had statistical difference (P<0.01). However, it had no statistic significance between 4-hour group and 0-hour group (P=0.897). But it had obviously statistically different between 24-hour group and 4-hour group,24-hour group and the 8-hour group (P<0.01). It proved in a short period of effect time, less than four hours, the expression of AQP2 did not apparently change. When time extended, the expression of AQP2 decreased.ConclusionmIMCD-3 expresses AQP2 which mainly localizes in the cytoplasm in the basic state. LPS inhibits the proliferation of mIMCD-3 with a time-dependent and dose-dependent manner. The inhibitory effect of LPS on cell proliferation is mainly expressed in the induction of apoptosis. With the time extending, the apoptosis rate increases which has a linear correlation. AQP2 plays an important role in LPS-mediated apoptosis of mouse inner medullary collecting duct cells.
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