Mitochondrial Dysfunction Activates The PINK1/parkin Signaling And Mitophagy In Renal Tubular Epithelial Cells Under Albumin Overload

Objective:Albumin as a major urinary protein component presents a risk factor for chronic kidney disease progression.Mitochondrial dysfunction is one of the main reasons for albumin-induced proximal tubule cells injury.Mitophagy is considered as a pivotal protective mechanism for the elimination of dysfunctional mitochondria.The objective of this research was to determine whether albumin overload-induced mitochondrial dysfunction can activate the PINK1/Parkin mediated mitophagy in renal tubular epithelial cells(TECs).Methods: The albumin concentration with the most obvious changes in autophagy expression was 8 mg/ml.Detection index: The expression of LC3-II in HK-2 cells was detected by immunofluorescence staining and western blot technique;structural and functional changes of mitochondria were observed by electron microscopy and JC-1 fluorescent staining;cytochrome C was detected in the cytosol by western blot technique The changes in mitochondria and the mitochondria were detected by Western blot.The expression changes of PINK1 and Parkin related proteins PINK1 and Parkin in HK-2 cells and mitochondrial autophagy were detected by Western blot.The lysosome fluorescence probe Lysotracker and the mitochondrial fluorescence probe Mitotracker were used.Co-localization of lysosomes and mitochondria was observed.TUNEL assay was used to detect the apoptosis rate of HK-2 cells in each experimental group.Results:(1) Effect of Albumin Overload on Apoptosis of Renal Tubular Epithelial Cells.The effect of albumin overload on the mitochondrial structure and function of HK-2 cells.The results of JC-1 showed that the intracellular mitochondrial membrane potential decreased significantly with the increase of albumin concentration,and the difference was statistically significant(P<0.05).(2) At the same time,electron microscopy revealed that albumin overload induced mitochondrial swelling in HK-2 cells,and double-layered membrane structure was also observed.At the same time,Western blotting showed that compared with the control group,cytochrome C in the mitochondrial group decreased,and cytochrome C in the cytoplasm increased,the difference was statistically significant(P<0.05).(3) Effects of Albumin Overload on the Level of Autophagy-Related Protein LC3 in Renal Tubular Epithelial Cells,and the Effect on the Expression of mitochondrial Autophagy-associated Protein Pink1,Parkin Protein in HK-2 Cells and Changes in Mitochondrial Autophagy Levels After Albumin Treatment Immunofluorescence showed an increase in LC3-II positive spots.Immunoblotting confirmed that the expression of autophagy-related protein LC3-II increased and peaked at 8 mg/ml.The difference was statistically significant(P<0.05).After albumin treatment,compared with the control group,the immunoblotting results showed that the expression of PINK1 and Parkin increased,and the difference was statistically significant(P<0.05).The mitochondrial and lysosomal fluorescence co-localization results showed that there were fewer acidic lysosomes in the control group,and only few mitochondrial and lysosome fusions were observed,whereas in the albumin group,mitochondria and lysosomes were more fused.(4) After knocking down PARK2,the effect of albumin on the expression of Pink1,Parkin,and mitochondrial autophagy in HK-2 cells.The results showed that knocking down the expression of PARK2 could inhibit the increase of expression of PINK1 and Parkin in albumin group.At the same time,the mitochondrial and lysosome fluorescence co-localization results showed that the mitochondrial and lysosome fusion decreased,the difference was statistically significant(P<0.05).Conclusion: This study confirms that the main component of urinary protein(albumin)induces apoptosis of tubular epithelial cells and mitochondrial damage.Albumin overload damages mitochondria with autophagy activation,accompanied by an increase in the expression of mitochondrial autophagy key proteins PINK1 and Parkin.After the low level of PARK2 gene,the mitochondria-induced overload of mitochondrial autophagy key genes PINK1 and Parkin were decreased.In summary,albumin overload induces mitochondrial damage in renal tubular epithelial cells and activates PINK1/Parkin signaling pathway-mediated mitochondrial autophagy.