HUC-MSCs Increased The Activity Of AOPPs-inhibited Autophagy In HK-2 Cells

BackgroundChronic kidney disease(CKD)has become a global health problem.Recent studies demonstrated that injury in tubulointerstitial rather than glomeruli had a greater effect on renal function.Nephrotoxic substances caused reduced metabolic activity,hypertrophy and other pathological changes of HK-2 cells,resulting in renal tubulointerstitial fibrosis.Autophagy is a common intracellular lysosome-dependent degradation approach in eukaryotic cells.Our previous study discovered that advanced oxidation protein products(AOPPs)induced endoplasmic reticulum stress(ERS),an early stress event exposed to cell injury,and inhibited autophagy activity in HK-2 cells.However,the underlying mechanism remains unknown.Although emerging studies indicated a direct link between ERS and autophagy,whether AOPPs induced ERS is associated with the reduction in autophagy activity and the underlying mechanism of the association remains to be elucidated.Renal repair is determent to be a prospective therapy to slow down the progression of CKD.Human umbilical cord mesenchymal stem cells(hUC-MSCs),a hot spot applied in renal repair,were accepted to have a protective effect on repairing cell and tissue damage in CKD.The membrane microcapsules derived from hUC-MSCs induce other cells to migrate into the injured position and promote cell proliferation and differentiation via transmission of genetic information,contributing to repair injured cell and tissue.Nevertheless,whether hUC-MSCs increased the activity of AOPPs-inhibited autophagy in HK-2 cells and its potential mechanism is unclear.ObjectivesHK-2 cells were the main object in the study.We firstly examine whether AOPPs induced ERS inhibits autophagy activity in HK-2 cells and its underlying mechanism.Further,we explored whether hUC-MSCs alleviate the inhibiting effect of AOPPs on autophagy activity in HK-2 cells and its underlying mechanism.Methods1.AOPPs preparation and content determination.2.Cell culture of HK-2 cells.3.To investigate the role of AMPK/mTOR signal pathway in AOPPs induced reduction in autophagy activity in HK-2 cells,we firstly examined the expression of AMPK/mTOR signal pathway in AOPPs stimulated HK-2 cells by Western blot.Further,we added AMPK inhibitor(Compoun C)and AMPK inducer(AICAR)respectively prior to AOPPs in HK-2 cells,and examined the autophagy activity as determined by LC3II/LC3I,Beclinl,and p62,and the expression of AMPK/mTOR signal pathway by Western blot.4.To address whether ERS mediates AOPPs-induced inhibition of autophagy via AMPK/mTOR pathway in HK-2 cells,we treated HK-2 cells with ERS inducer(Thapsigargin)and ERS inhibitor(Salubrinal)respectively prior to AOPPs and examined the expression levels of ERS,autophagy,and AMPK/mTOR signal pathway related proteins by Western blot.5.To explore whether hUC-MSCs increase the reduced autophagy activity and decrease the elevated ERS in AOPPs stimulated HK-2 cells,hUC-MSCs were co-cultured with HK-2 cells followed AOPPs stimulation.Western blot was used to determine the expression of autophagy and ERS related6.To address whether hUC-MSCs increase the autophagy activity via inhibiting ERS,hUC-MSCs were co-cultured with HK-2 cells followed by ERS inducer in AOPPs stimulated HK-2 cells.Western blot and RT-qPCR were used to determine the expression of autophagy and ERS related proteins.Results1.AOPPs inhibited autophagy of HK-2 cells through the activation of AMPK/mTOR signal pathway.Western blot analyses showed that AOPPs reduced the level of p-AMPK and increased the level of p-mTOR in HK-2 cells,resulting in activation of the AMPK/mTOR signaling pathway.Further,AMPK inhibitor alone exhibited a similar negative effect of AOPPs on autophagy activity as indicated by decreased p-AMPK,increased p-mTOR,and inactive autophagy in HK-2 cells,whereas AMPK inducer partly reversed the effect of AOPPs on autophagy activity.in AOPPs treated HK-2 cells.2.Endoplasmic reticulum stress mediated AOPPs-induced autophagy inhibition by activation of the AMPK/mTOR signal pathway in HK-2 cells.Western blot analyses showed that ERS activator alone increased the expression of GRP78(an ERS biomarker protein),activated the AMPK/mTOR signal pathway and inhibited autophagy,consistent with the effect of AOPPs on HK-2 cells.In contrast,ERS inhibitor led to a reduction in GRP78 and suppressed the activity of AMPK/mTOR signal pathway and increased autophagy in HK-2 cells exposed to AOPPs.3.AOPPs-induced autophagy inhibition partly revesed by hUC-MSCs.And hUC-MSCs inhibited AOPPs-induced endoplasmic reticulum stress in HK-2 cells.After stimulation of AOPPs for 48h,HK-2 cells were co-cultured with hUC-MSCs for 48h.Western blot analysis showed that hUC-MSCs increased autophagy activity in HK-2 cells stimulated with AOPPs as indicated by elevated expression of LC3-II/LC3-I,Beclinl and decreased expression of p62.Further,RT-qPCR and Western blot analyses revealed that hUC-MSCs decreased the expression of GRP78 and CHOP at either mRNA or protein level,resulting in reduction in the level of AOPPs-induced ERS in HK-2 cells.4.HUC-MSCs increased autophagy activity by inhibiting endoplasmic reticulum stress in HK-2 cellsRT-qPCR and Western blot analyses found that ERS activator significantly increased the level of ERS as indicated by GRP78 and CHOP and reduced the autophagy activity determined by reduction in the expression of LC3II/LC3I and Beclinl and elevation in the expression of p62 in co-cultured hUC-MSCs and HK-2 cells.ConclusionERS mediated AOPPs-induced inhibition of autophagy via activating AMPK/mTOR signal pathway in HK-2 cells.hUC-MSCs increased the activation of AOPPs-inhibited autophagy by inhibiting ERS in HK-2 cells.