Research On The Mechanism Of PACS-2 And GADD45B In Diabetic Renal Tubular Injury

ObjectiveDiabetic kidney disease（DKD）is one of the most common chronic microvascular complications of diabetes and the main cause of end-stage renal disease requiring dialysis treatment.Preventing and delaying the progress of diabetic kidney disease has become a global public health issue,and more targeted treatment strategies need to be explored.So far,a large number of studies have reported that renal tubule pathology plays a central role in the pathogenesis of diabetic kidney disease.In the occurrence and development of diabetic kidney disease,the damage of renal tubular epithelial cells includes endoplasmic reticulum stress,mitochondrial dysfunction,tubular epithelial-mesenchymal transition（EMT）and apoptosis,etc.,and finally tubular interstitial fibrosis.Therefore,this study is divided into two parts to explore the mechanism of diabetic renal tubular injury.MethodsPart Ⅰ:The mechanism of phosphofurin acidic cluster sorting protein 2（PACS-2）in diabetic renal tubular injury.1.STZ-induced type 1 diabetes and spontaneous type 2 diabetes db/db mice were used as the research subjects.HE,Picrosirius red,PAS staining,and transmission electron microscopy were used to observe the pathological changes of kidney tissues and the mitochondrial-associated endoplasmic reticulum membrane（MAM）structure of renal tubular cells in diabetic mice;The total,cytoplasm,mitochondria,MAM,and endoplasmic reticulum proteins were isolated and extracted,and the expression of PACS-2 in each component protein was detected by immunohistochemistry,RT-PCR and Western blot.2.PACS-2 knockout and overexpression mouse models were established,respectively,to detect the liver and kidney function,blood lipid,and urinary biochemical indexes of mice in each group.HE,Picrosirius red,PAS staining,and transmission electron microscopy were used to observe the pathological changes of kidney tissues and the MAM structure of renal tubular cells in diabetic mice.Immunohistochemistry and Western blot were used to detect the markers of endoplasmic reticulum stress,mitochondrial dysfunction,apoptosis,and fibrosis in mice kidneys.3.Human proximal renal tubular epithelial cells（HK-2）were used as the research subjects.After the treatment with high glucose,and the MAM structure of HK-2 cells was observed by transmission electron microscopy and proximity ligation assay.The total,cytoplasm,mitochondria,MAM and endoplasmic reticulum proteins were isolated and extracted,and the expression of PACS-2 in each component protein was detected by immunofluorescence,RT-PCR,and Western blot.After PACS-2or/and FATE1（MAM uncoupler）overexpression,endoplasmic reticulum and mitochondrial co-localization staining and proximity ligation assay were used to observe the MAM structure of HK-2 cells.The markers of endoplasmic reticulum stress,mitochondrial dysfunction,apoptosis,and fibrosis were detected by immunofluorescence and Western blot.Part Ⅱ:The mechanism of growth arrest and DNA damage-inducible 45B（GADD45B）in diabetic renal tubular injury.1.Spontaneous type 2 diabetes db/db mice and HK-2 cells were used as the research subjects.HE,Masson,PAS staining,and biochemical analysis were used to observe the pathological changes of kidney tissues and urinary protein levels.Total RNA was extracted from mice for RNA sequencing to observe the genomes differentially expressed in the two groups.Immunohistochemistry,RT-PCR,and Western blot were used to verify the differential expression of GADD45B in vivo and in vitro.After GADD45B knockdown and overexpression,the markers of EMT and apoptosis were observed by immunofluorescence and Western blot.2.KEGG pathway analysis was performed to identify enriched pathways in the differentially expressed genes between groups.Western blot was used to observe the effects of GADD45B knockdown and overexpression on the inhibition and activation of mitogen-activated protein kinase（MAPK）pathways（including p38 MAPK,JNK,and ERK pathways）.Immunohistochemistry and Western blot were used to verify the levels of EMT,apoptosis,and activity of MAPK pathway in mice kidneys.3.SB203580 and SP600125 were used to inhibit p38 MAPK and JNK pathways,and Western blot was used to observe whether inhibition of p38 MAPK and JNK pathways could reverse EMT and apoptosis caused by high glucose or GADD45B overexpression in HK-2 cells.ResultsPart Ⅰ The mechanism of PACS-2 in diabetic renal tubular injury1.Compared with non-diabetic mice,both type 1 and type 2 diabetic mice showed pathological manifestations of diabetic kidney disease and reduced MAM area of renal tubular cells.The expressions of PACS-2 in total,cytoplasm,mitochondria,MAM,and endoplasmic reticulum proteins from kidneys of type 1 and type 2 diabetic mice were all down-regulated.2.Compared with wild-type diabetic mice,the levels of kidney weight/body weight（KW/BW）,urinary albumin excretion（UAE）,urinary albumin to creatinine ratio（ACR）,N-acetyl-β-D-glucosaminidase（NAG）,serum creatinine（Scr）,and blood urea nitrogen（BUN）were increased in Pacs-2^-/-diabetic mice.Diabetes-related pathological changes of renal tubule and renal interstitial fibrosis in Pacs-2^-/-mice were more serious than those in wild-type mice,and the decrease of MAM area was more obvious.Pacs-2 knockout increased renal endoplasmic reticulum stress,mitochondrial dysfunction,apoptosis,and fibrosis in diabetic mice.The overexpression of PACS-2 decreased the levels of KW/BW,UAE,ACR,NAG,Scr,and BUN in diabetic mice,alleviated the diabetes-related pathological changes of renal tubule and renal interstitial fibrosis,increased the MAM area,and inhibited renal endoplasmic reticulum stress,mitochondrial dysfunction,apoptosis and fibrosis in diabetic mice.The change of PACS-2 expression did not affect the blood glucose,body weight,liver and kidney function,and blood lipid levels of mice.3.Compared with normal glucose,MAM area of HK-2 cells were decreased under high glucose condition,and the expressions of PACS-2 in total,cytoplasm,mitochondria,MAM and endoplasmic reticulum proteins in HK-2 cells were all down-regulated.PACS-2 overexpression under high glucose conditions restored the structural integrity of MAM.PACS-2 overexpression alleviated endoplasmic reticulum stress,apoptosis,and fibrosis induced by high glucose,and improved mitochondrial function.FATE1 transfection can uncouple the interaction between endoplasmic reticulum and mitochondria,and weaken the protective effect of PACS-2 overexpression on HK-2 cells under high glucose condition.Part Ⅱ The mechanism of GADD45B in diabetic renal tubular injury1.The levels of UAE,Scr,and BUN in db/db mice were significantly increased,which showed the pathological characteristics of diabetic kidney disease.Compared with db/m mice,renal RNA sequencing results showed a total of 3642 differentially expressed genes in db/m mice,among which the relative expression of GADD45B was significantly higher than that of db/m mice,with a Log₂ ^{fold change} of 2.364.In vitro and in vivo experiments confirmed that GADD45B expression was increased in renal tubules of diabetic mice and high glucose-stimulated HK-2 cells.GADD45B knockdown reduced the high glucose-induced EMT and apoptosis of HK-2 cells,and the GADD45B overexpression exacerbated the EMT and apoptosis levels of HK-2cells.2.KEGG Pathway analysis of differential genes revealed 39 related signaling pathways,including the MAPK pathway.In the MAPK pathway,the expression of 63related genes including GADD45B was significantly changed.In vitro validation showed increased activation of p38 MAPK,JNK,and ERK pathways in HK-2 cells treated with high glucose.GADD45B knockdown inhibited the activities of p38 and JNK pathways,while GADD45B overexpression increased the activities of p38MAPK and JNK pathways.GADD45B expression did not affect the activity of the ERK pathway.In vivo experiments confirmed that the levels of renal EMT and apoptosis were increased in diabetic mice,and the activities of p38 MAPK,JNK,and ERK pathways were significantly up-regulated.3.SB203580（phosphorylation inhibitor of p38 MAPK）and SP600125（phosphorylation inhibitor of JNK）could inhibit the activation of p38 MAPK and JNK pathways caused by high glucose or GADD45B overexpression,respectively,and reverse the high glucose or GADD45B overexpression-induced EMT and apoptosis of HK-2 cells.Conclusions1.PACS-2 alleviates diabetic renal tubular injury by maintaining MAM integrity.2.GADD45B regulates renal diabetic renal tubular injury by activating p38MAPK and JNK signaling pathways,while GADD45B has no significant effect on the ERK pathway.