Effects Of Recombinant Human Erythropoietin On HK-2Cells Of RhoA/ROCK Signaling Pathway Under High Glucose In Vitro

Objective：To discuss the effect of recombinant human erythropoietin on HK-2cells ofRhoA/ROCK signaling pathway under high glucose in vitro.Methods：HK-2cells which cultured in vitro were divided into several groups randomly:blank control group, high glucose induced group(high glucose,30mmol/l), mannitolinduced group（mannitol,24.5mmol/l）, rhEPO induced group（rhEPO,20U/ml）,different concentrations of rhEPO inhibited groups(5,10,20U/ml), inhibitor of Rhokinase group(Y27632,10μmol/l). The cells were treated for24hours stimulation,The changes of HK-2cells morphology were observed under an opticalmicroscope.To evaluated HK-2cells of mRNA levels of RhoA and ROCK in eachgroup,We used reverse transcription polymerase chain reaction (RT-PCR). The levelsof E-cadherin and α-smooth muscle actin (α-SMA) proteins in each group wereassessed by immunofluorescent. ELISA was used to measure the levels of fibronectinproteins(FN)、interleukin-6(IL-6)、tumor necrosis factor–α（TNF-α）in supernatantin each group. Cell apoptosis was detected by flow cytometry.Results：The changes of HK-2cells morphology were observed under an opticalmicroscope, normal HK-2cells looked like oval or spindle, But the morphology ofHK-2cells change after giving high glucose,the cell body became longer and the cellgap became larger.Moreover,The morphology was significantly improved whengiving rhEPO.Compared with blank control group.The expression of RhoA, ROCK1mRNA in cells was significantly increased in the high glucose induced group(P<0.05). In different concentrations of rhEPO inhibited groups,The RhoAmRNA,ROCK1mRNA expressions in cells significantly decreased (P<0.05). But in theY27632group,the RhoAmRNA expression was as the same as the high glucoseinduced group,and ROCK1mRNA expression significantly decreased (P<0.05).Immunofluorescent and ELISA method showed that both the expression of α-SMA proteins and the expression of FN proteins in cells increased in the high glucoseinduced group (P <0.05), significantly decreased in the different concentrations ofrhEPO inhibited groups and inhibitor of Rho kinase group as compared to the highglucose induced group (P <0.05), and the effects were related to the concentration ofrhEPO. The expression of E-cadherin proteins increased in the high glucose inducedgroup (P <0.05), but significantly increased in the different concentrations of rhEPOinhibited groups（P<0.05）, and the effects were also related to the concentration ofrhEPO in the range of experimental rhEPO concentration. ELISA method showed thatthe expression of IL-6and TNF-αincreased in the high glucose induced group (P<0.05), significantly decreased in the rhEPO inhibited group and inhibitor of Rhokinase group as compared to the high glucose induced group (P <0.05), and theeffects were related to the concentration of rhEPO in the range of experimentalrhEPO concentration. Flow cytometry method showed that high glucose inducedapoptosis in HK-2cells, significantly inhibited in the rhEPO inhibited group andinhibitor of Rho kinase group as compared to the high glucose induced group (P<0.05). There was positive correlation between the expression of RhoA mRNA andROCK1mRNA in the high glucose induced group and the different concentrations ofrhEPO inhibited groups by pearson correlation analysis.Conclusion：High glucose can induce HK-2cells EMT by decreasing the expression ofE-cadherin proteins low and inreasing the expression of α-SMA and FN proteins,aswell as increasing the expression of RhoA mRNA,ROCK1mRNA, leading to anincrease in extracellular matrix. But rhEPO can inhibit high glucose induced EMT indose-dependent manner, finally attenuate renal fibrosis. rhEPO can also inhibitapoptosis and thus play a role in protecting the kidneys, rhEPO can also reduce theproduction of inflammatory cytokines, reduce inflammation, thus delaying theprogression of diabetic nephropathy. We guess the mechansim may be related to itsinhibition of RhoA/ROCK signaling pathway.