The Role And Mechanism Of REDD1 In Diabetic Nephropathy

Objectives:This study investigated the role and possible mechanism of REDD1（Developmental and DNA injury response regulatory gene 1）in renal tubular cell injury in diabetic nephropathy（DN）patients,diabetic nephropathy model mice and human tubular cells.Knockdown REDD1 inhibit the improvement of renal tubular cell apoptosis,EMT and disorder of lipid metabolism in diabetic nephropathy,and provide experimental basis for further elucidating the etiology of diabetic nephropathy and exploring the potential therapeutic targets of diabetic nephropathy.Method:1.The expression and significance of REDD1 in diabetic nephropathy patients,mice,and HK-2 cells.（1）The expression and significance of REDD1 in the renal tissue of diabetic nephropathy patients.From January 2017 to October 2019,40 patients with diabetic nephropathy were hospitalized in the Department of Nephrology of the second hospital of Hebei Medical University.The control group consisted of 10patients with normal blood glucose.The detailed clinical data of all patients were collected,including age,gender,height,weight,course of disease,blood pressure,medication history.Meanwhile,blood samples were also retained to detect the clinical indicators of the patients.All renal tissue samples were dehydrated,embedded in paraffin,sectioned and stained by routine pathology,and the expression of REDD1 was detected by immunohistochemistry.Fat droplet deposition in renal tissue was observed by oil red O staining.Apoptosis in the renal tissue was visualized by TUNEL staining.To analyze the relationship between REDD 1 expression and apoptosis and lipid deposition in renal tissues of patients with diabetic nephropathy.（2）The expression and significance of REDD1 in kidney of diabetic nephropathy mice.Using 16w db/db mice with the C57BL/ks background and littermate control db/m mice with the same background.The expression of REDD1 in kidney tissues of each group was examined by immunohistochemistry.Lipid droplet deposition in mouse kidney tissues was observed by oil red O staining.Apoptosis in mouse kidney tissues was visualized by TUNEL staining.To analyze the relationship between REDD 1 expression and apoptosis and lipid deposition in renal tissues of db/db mice.2.Knockdown of the REDD1 gene in C57BL/ks mice by using adenovirus injection techniques.The experiment group was:8 db/m mice and24 db/db mice were randomly divided into three groups:db/db group,db/db+no-load virus control group（db/db+B）,db/db+REDD1sh RNA adenovirus AAV9 injection group（db/db+AV）.Western blot measured the expression levels of apoptosis-related proteins（Bax,Bcl-2 and Cleaved-Caspase 3）,oxidative stress levels（Nox4）,fatty acid synthesis related proteins（SREBP-1,ACC and FASN）,related proteins of fatty acid oxidation（PPAR,ACOX1 and CPT1）,and transdifferentiation-related proteins（α-SMA and E-cadherin）.The localization and expression of Bax,Bcl-2,Cleaved-Caspase 3,SREBP-1,FASN,ACC,PPARα,ACOX1,CPT1 in kidneys of mice by immunohistochemistry.Lipid deposition in each group was determined by oil red O staining.3.Using human HK-2 cells as the study subjects,and HK-2 cells were stimulated with high glucose concentrations for 6,12,24,48 and 72 h.Cells were divided into three groups:normal concentration glucose（NG）,high permeability（M）,and high concentration glucose（HG）.The expression level and localization of REDD1 in HK-2 cells was determined by Western Blot,RT-q PCR and cellular immunofluorescence.The experiments were performed in REDD1si RNA transfected with high glucose stimulated cells for 48 hours.The experimental groups were:normal concentration glucose（NG）,high concentration glucose（HG）,high concentration glucose+no-load plasmid control group（HG+C）and high concentration glucose+REDD1si RNA transfection group（HG+si REDD1）.Apoptosis in each group was determined by TUNEL.DCFH-DA and Mito SOX staining were examined for the levels of ROS by laser confocal microscopy and flow cytometry.Intracellular lipid deposition was measured by oil red O.Western blot detects the expression of apoptosis-related proteins（Bax,Bcl-2 and Cleaved-Caspase 3）,oxidative stress levels（Nox4）,fatty acid synthesis related proteins（SREBP-1,ACC and FASN）,related proteins of fatty acid oxidation（PPAR,ACOX1 and CPT1）,and transdifferentiation-related proteins（α-SMA and E-cadherin）.The expression levels of REDD1,SREBP-1,CPT1,α-SMA,and E-cadherin were determined by immunofluorescence.The expression levels of REDD1,Bax,Bcl-2,Cleaved-Caspase 3,α-SMA,and E-cadherin m RNA were determined by RT-q PCR.The morphology of HK-2 cells in each group was observed under an inverted microscope.Result:1.The expression and significance of REDD1 in diabetic nephropathy patients,mice,and HK-2 cells.（1）Immunohistochemistry showed that the expression REDD1 was increased in the renal tubular cells of diabetic nephropathy patients and db/db mice.Western blot and RT-q PCR results showed that high glucose can stimulate the expression of REDD1 in a time-dependent manner.（2）Increased expression of REDD1 was positively correlated with blood Scr,TC and TG in diabetic nephropathy patients and db/db mice.Increasing expression of REDD1 was positively correlated with bloodβ₂MG and cystatin-C in diabetic nephropathy.（3）Increased expression of REDD1 was associated with apoptosis,fibrosis,and lipid deposition in renal tissues of diabetic nephropathy patients and db/db mice.Increased expression of REDD1 was associated with apoptosis,EMT,and lipid deposition in HK-2 cells.2.Effects and mechanism of knockdown REDD1 on apoptosis and EMT of DN mice and HK-2 cells induced by HG（1）Immunohistochemistry and Western blot showed that knockdown of REDD1 attenuated the downregulation of Bax and Cleaved-Caspase 3 and Bcl-2 in mice with DN.In HK-2 cells stimulated at HG,transfection with REDD1 si RNA reduced the number of apoptosis,and reduced the Bax/Bcl-2ratio and down-regulated the expression level of Cleaved-Caspase 3.（2）Western blot and immunohistochemistry showed decreased expression ofα-SMA and elevated E-cadherin in knockdown of REDD1 in DN mice compared to db/db mice.The results obtained for HK-2 cells were consistent with those obtained in mice with DN.（3）REDD1si RNA significantly reduced Nox4 expression and ROS production in HK-2 cells stimulated with HG.（4）Antioxidant drugs（NAC or Tempol）can reduce the increase in REDD1expression in HK-2 cells.3.Role and mechanism of REDD1 in lipid metabolism disorder in renal cells of DN（1）Lipid droplet accumulation was reduced in mice with REDD1knockdown diabetic nephropathy and showed decreased expression of SREBP-1,ACC and FASN.（2）PPAR,ACOX1,and CPT1 was elevated in the kidneys of REDD1-knockdown DN mice.（3）REDD1 si RNA reduces HG-stimulated lipid droplet aggregation and downregulates the levels of SREBP-1,ACC and FASN.（4）REDD1 si RNA can increase the cellular PPARα,ACOX1,and CPT1 levels in HK-2 cells stimulated with HG.（5）REDD1 knockdown affected the production of MAMs and ATP within the mitochondria in mice with DN,in addition to the impaired mitochondrial function upon REDD1 si RNA transfection with HG-induced HK-2 cells.（6）REDD1 si RNA can inhibit the expression of ChREBP in podocytes.Knockdown of the ChREBP gene in podocytes can downregulate the levels of ACC and FASN,increased PPARα,ACOX1,and CPT1 levels.Conclusion:1.The expression of REDD1 was increased in the renal tubular epithelial cells of both diabetic nephropathy patients and mice,and REDD1 was significantly increased in HK-2 cells cultured with high glucose.REDD1 may be involved in the process of renal tubular epithelial cell injury in diabetic nephropathy.2.REDD1 knockdown alleviated renal tubular cell apoptosis and transdifferentiation in diabetic nephropathy.This process may be caused by the activation of oxidative stress induced by REDD 1 through complex formation by binding with TXNIP.3.REDD1 may cause lipid deposition in diabetic kidney cells by changing MAMs and mitochondrial function,and by regulating ChREBP.