Liver Regeneration Intervention Renal Tubular Cell Injury In Hypoxic Effect Of Inflammatory Signaling Pathways

Objective: To observe the expression of augmenter of liver regeneration (Augmenter of liver regeneration, ALR)in the renal tubular epithelial cells which were cultured in hypoxia and hypoxia/reoxygenation and to investigate the influence on on the p38MAPK signaling pathway of hypoxia and hypoxia/reoxygenation, so as to explore the role of ALR in the inflammatory mechanism.Methods: NRK-52E cells with 10% FBS RPMI1640 culture medium were cultured in 37℃, 5% CO₂ incubator for 24 hours.The NRK-52E cells were divided into (1) normal group: with 10% FBS, RPMI1640 in 37℃, 5% CO₂ incubator,saturated humid incubator; (2) hypoxia at different time (15min, 30min, 60min, 2h, 4h, 8h, 12h, 18h, 24h) group: when cells grew to 80～90%, changed to serum-free medium , three gas incubator (1% O2, 5% CO₂, 94% N2) for correspongding time ; (3) hypoxia/reoxygenation (4h/12h, 4h/24h) group:when cells grew to 80～90% , changed to serum-free medium, three gas incubator (1% O2, 5% CO₂, 94% N2) after 4 hours, changed the cells to normal medium and incubatored for correspongding times (12h , 24h). Under the microscope, observed the cell's morphological changes ; the ALR mRNA expression of the NRK-52E cells were detected with RT-PCR method; ALR protein expression was detected with Western blot , the expression of the phosphorylated p38 and ATF-2 were detected with Western blot, ELISA detected the expression of ICAM-1 and TNF-αin supernatant. 15min hypoxic cells were invited into the control group, the rALR intervention group (20μg, 100μg of rALR) and the inhibitor treated group, detected the expression of p38MAPK with Western blot.Results:1.Normal control NRK-52E cells were cuboidal or polygonal cells with typical cobblestone morphology under inverted microscope. Cells intervented by hypoxia and hypoxia/reoxygenation in different time. The suspension cells increased with the hypoxia time prolonged. The morphology of the intervented cells had no significant difference with the control group under the light microscope.2. RT-PCR results showed: The mRNA expression of ALR was low in normal cells, the mRNA expression of ALR in hypoxia groups began to increase at 4h. The difference was significantly compared with 0, 2h hypoxia group (F = 167.558 P = 0.005 <0.01). The mRNA expression of ALR in 8,12,18,24h hypoxia group were significantly increased than 0, 2, 4 h hypoxia group (F = 89.761 P = 0.009 <0.01), however compared with each other among 8,12,18,24h hypoxia group the mRNA expression of ALR had no significant difference (F = 0.102 P = 0.957> 0.05).3. Western blot analysis showed: normal cells had a small amount of ALR protein expression, the protein expression of ALR in hypoxia groups began to increase at 8h. However the difference was not significant compared with 0, 2, 4 h hypoxia group (F = 5.693, P = 0.051> 0.05).The protein expression of ALR in 12h hypoxia group was significantly increased compared with the other groups (F = 311.612, P = 0.001 <0.01).The protein expression of ALR in 18 h and 24h hypoxia groups had no significant difference (P = 0.254> 0.05).4. Western blot analysis showed: hypoxia 4h, reoxygenation 12, 24h, ALR protein was significantly increased compared with the normal group, the difference was statistically significant (F = 81.56, P = 0.0003 <0.01), but there were no statistically significant differences between the two hypoxia/ reoxygenation groups. (P = 0.661> 0.05).5.Western blot analysis showed: the expression changes of phosphorylated p38 protein at 0, 15, 30, 60 min after anoxia : compared to normal group, the expression of phosphorylated p38 protein was significantly increased at 15,30,60 min.,the expression was most significant at 15min,and the levels of phosphorylated p38 protein decreased with time,but the difference was statistically significant (F = 212.467, P <0.01) compared with normal group.6.Western blot analysis showed:the expression changes of phosphorylated ATF-2 protein at 0, 15, 30, 60 min, 2h after anoxia: the levels of phosphorylated ATF-2 increased significantly compared with normal group, and the expression was most obviously at 2h, the difference was statistically significant ( P <0.01) compared to normal group.7. ELISA results showed: the levels of ICAM-1 in supernatant with hypoxia 30,60,120 min were significantly increased with time than normal control group (P <0.01). The levels of TNF-αin supernatant with hypoxia from 15 min to 120 min were significantly higher than the negative control group ( P <0.01).However, differences between the hypoxia groups was not significant ( P > 0.05).8. Western blot analysis showed: 20μg rALR significantly inhibited the activation of phosphorylated p38 compared with no intervention (P<0.01) and SB203580 intervention (P = 0.008 <0.01). Low doses of rALR (20μg) could inhibit the protein expression of activated p-p38 (P <0.01). However,the effect of inhibition was weaker when intervented with increased rALR doses(100μg).Conclusions:1. On nucleic acid and protein level, only a small amount of ALR expressed in general cell culture conditions,however hypoxia can increase the expression of ALR in renal tubular epithelial cells,and hypoxia / reoxygenation can induce the expression of ALR in earlier stage compared with the hypoxia. 2. The activation of p38MAPK signaling pathway was induced by hypoxia in rat proximal tubular epithelial cells, resulting in phosphorylation of p38 protein,phosphorylation of ATF-2 protein,inflammatory mediators ICAM-1,TNF-αprotein production increasing.3.A small doses of exogenous rALR can significantly inhibit the activation of p38MAPK significantly , suggesting that ALR may play a protective role.4. By inhibiting the activation of p38MAPK in renal tubular epithelial cells,thereby reducing the production of inflammatory mediators from injury,ALR may play a role of protection.