PACS-2 Ameliorates Tubular Injury In Diabetic Kidney Disease By Facilitating ER-phagy

Diabetic kidney disease（DKD）is the major microvascular complication of diabetes and the most critical pathogenic factor of end-stage renal disease.Recent evidence shows that the autophagy of the endoplasmic reticulum（ER）,referred to as ER-phagy,is conducive to alleviating ER stress and reconstructing ER homeostasis by selectively recognizing and degrading redundant and damaged ER,thus playing a protective role in a variety of diseases.However,whether and how ER-phagy is related to the occurrence and development of DKD is unclear.Phosphofurin acidic cluster sorting protein-2（PACS-2）is a protein with membrane trafficking function.It can bind and transport cargo molecules that contain acidic clusters to subcellular organelles like the nucleus,thus regulating the functions of these cargo molecules.Recent studies found that PACS-2 ameliorated the tubular injury of DKD by maintaining ER homeostasis,and it is reported that suppressing PACS-2 expression will inhibit the occurrence of autophagy,suggesting that PACS-2 may participate in ER-phagy.However,whether or not PACS-2 can improve DKD tubular injury by regulating ER-phagy is unclear.Family with sequence similarity 134,member B（FAM134B）is an ER membrane located ER-phagy receptor that plays a critical role in ER-phagy.The latest study reported that the expression of FAM134B was transcriptionally regulated by transcription factor EB（TFEB）.Interestingly,it is estimated that the amino acid sequence of TFEB also contains acidic cluster sequences that can be recognized by PACS-2,suggesting that TFEB may be a cargo molecule of PACS-2.Based on the above analysis,we propose the following hypothesis:PACS-2 may play a renal protective role in DKD by maintaining the occurrence of ER-phagy through transporting TFEB into the nucleus and then promoting FAM134B transcription.Objective:To investigate whether PACS-2 can improve DKD tubular injury by regulating TFEB/FAM134B/ER-phagy axis.Methods:1.Mice with PACS-2 gene specific knockout in proximal renal tubular epithelial cells and the control littermates were divided into four groups:（1）control group(PACS-2^Ctrl),（2）gene knockout group(PACS-2^{pt KO}),（3）streptozocin（STZ）induced DKD model group(PACS-2^Ctrl+STZ),and（4）gene knockout DKD model group(PACS-2^{pt KO}+STZ).The model mice were given an intraperitoneal injection of STZ at the dose of 50mg/kg for 5 days,and then fed for 12weeks.Blood glucose and body weight were monitored every 2 weeks.The urine samples were obtained before the mice were sacrificed.Then urinary albumin and creatinine were detected.And pathological staining of renal tissue was applied to estimate renal pathological injury of the mice.Meanwhile,the level of ER-phagy,and the expression of PACS-2,inflammatory cytokines,fibrosis-related molecules and FAM134B in the renal cortex were observed by transmission electron microscope,immunohistochemical staining,immunofluorescence staining,western blot,and real-time fluorescence quantitative PCR.2.HK-2 cells were divided into five groups:（1）control group（5m M glucose）,（2）high glucose group（30m M glucose）,（3）high glucose+PACS-2 overexpression group,（4）high glucose+FAM134B si RNA group,and（5）high glucose+PACS-2 overexpression+FAM134B si RNA group.The expression of PACS-2 and FAM134B,the level of ER-phagy,and the expression of inflammatory cytokines and fibrosis-related molecules were detected by western blot,cellular immunofluorescence staining,and real-time fluorescence quantitative PCR.3.Cell nucleocytoplasmic separation technology and subsequent western blot were applied to investigate the effects of high glucose and PACS-2 overexpression on the level of PACS-2 and TFEB in the nucleus of HK-2 cells.And a protein-protein interaction prediction website was used to predict the interaction between PACS-2 and TFEB,which was further verified by immune-coprecipitation technology.Results:1.Compared with PACS-2^Ctrlgroup,mice in the PACS-2^Ctrl+STZ group had elevated blood glucose,reduced body weight,increased urinary albumin/creatinine ratio,decreased expression of PACS-2 and FAM134B,inhibited ER-phagy,significant macrophage infiltration,elevated expression of inflammatory cytokines and extracellular matrix protein,and significant tubular injury.No significant changes were found for blood glucose or body weight when compared the PACS-2^{pt KO}+STZ group to the PACS-2^Ctrl+STZ group,while the other changes were further aggravated in PACS-2^{pt KO}+STZ mice.2.After cultured with high glucose,the expressions of PACS-2 and FAM134B in HK-2 cells were decreased,accompanied by inhibited ER-phagy and the aggravation of inflammatory cytokines and fibrosis-related proteins.These alterations were partially reversed by transfection of PACS-2 plasmid.While suppression of FAM134B expression by si RNA further aggravated the inhibition of ER-phagy and the elevated expression of inflammatory cytokines and fibrosis-related molecules caused by high glucose.Moreover,overexpression of PACS-2could not improve the above changes caused by FAM134B si RNA.3.When compared to the control group,the abundance of PACS-2and TFEB in the nucleus were significantly downregulated in the high glucose group,while overexpression of PACS-2 could promote the relocation of TFEB in the nucleus.Besides,the protein-protein interaction prediction website predicted interaction models between PACS-2 and TFEB.And the interaction between PACS-2 and TFEB was further validated by immune-coprecipitation technology.Conclusion:The deficiency of PACS-2 and destroyed ER-phagy are critical contributors to DKD renal tubular injury,and PACS-2ameliorates DKD tubular injury by promoting ER-phagy through promoting TFEB nuclear translocation and inducing the expression of FAM134B.