ER Stress-autophagic Response Alleviates Protein Overload-induced Cell Injury In NRE-52E

The pathologic and pathophysiologic changes of many forms of proteinuricrenal diseases is a complicated and chronic process, the final changes of which arecharacterized by glomerular sclerosis or renal tubularinterstitial fibrosis.However, therecent studies showed a more intimate relationship between tubulointerstitial lesionsand renal impairment. It has been proven that the degree of proteinuria correlatesclosely with the progression of chronic renal failure. A great numbers of animalmodels as well as clinical observations of protein-overload indicated that urinaryproteins directly resulted in tubulointerstitial damages.However,the mechanismsremain not fully understood.In addition,there are no effective therapeutic strategies ontubuloinierstitial injury resulting from proteinuria at present.To study the mechanisms by which urinary proteins led by ADM anddelipidated endotoxin-free BSA damage tubular epithelial cells (NRK-52E), thefollowing events were hypothesized: the accumulation of intracellular proteins causeendoplasmic reticulum stress; simultaneously, accumulation of protein congestlysosomal degradation pathway, inhibiting autophagy reaction; due to the relevance ofendoplasmic reticulum stress and autophagy, the unfolded protein response intensifies,causing cell damage and even death. Therefore, we design the following ideas:Objective:Through albumin overload NRK-52E cells induced by ADM and delipidatedendotoxin-free BSA,this study simulates the environment in vivo to study themechanism of protein overload-induced tubular epithelial cell apoptosis. Inaddition,we use an autophagy inhibitor chloroquine (CQ) to inhibit autophagyend-stage and observe renal apoptosis of tubular epithelial cells, laying the technicaland theoretical basis to the rapeutic target of progressive renal damage.Methods:1.MTT detection by ADM, BSA and combined effects of CQ on NRK-52E cellviability;2.Observe morphological changes of cells by an inverted microscope;3.After double staining of Annexin V/propidium iodide, flow cytometry(FCM) observed apoptosis of NRK-52E cells;4.western blot was used to observe theexpression of GRP78, CHOP, LC3, Beclin1and p-PERK.Results:MTT assay showed that NRK-52E cell damage caused by ADM and BSA in atime-and dose-dependent; while observed under the inverted microscope, cellsbecame round and cell gap increases; flow cytometry showed ADM, BSA andcombined of CQ significantly increased apoptosis of NRK-52E cells; western blotshowed the expression of GRP78, CHOP and LC3in ADM+BSA group was higherthan the separate ADM or BSA group, while the relative expression of Beclin1reduced; with CQ inhibiting autophagy,the expression of endoplasmic reticulumstress-associated protein GRP78, CHOP and the apoptotic factor p-PERK wasincreased.Conclusions:1. Protein overload caused by ADM and BSA causes NRK-52E cells to occurendoplasmic reticulum stress and induce apoptosis of cells.2. Inhibition of autophagy leads to enhance endoplasmic reticulum stress,increasingsignificantly the injury of renal tubular epithelial cells.It can provide a theoreticalbasis of finding effective targets for the treatment of proteinuria to renal tubularinjury.