The Effect Of Myeloid-specific Depletion Of CUL4B On The Progression Of Diabetic Kidney Disease And The Underlying Mechanism

Background and objectivesIn recent years,the incidence and prevalence of diabetes have increased significantly worldwide.Diabetes has become a global health burden.China has the highest number of people with diabetes in the world.Diabetic kidney disease(DKD),a common complication of diabetes,is directly related to the clinical prognosis of diabetic patients.Its clinical manifestations are mainly proteinuria and impaired renal function.Its pathological manifestations are mainly glomerulonodular sclerosis and interstitial fibrosis of the kidney.Macrophages play an important role in the development of DKD.Prolonged hyperglycemia leads to the accumulation of advanced glycation end products(AGE),which stimulate the release of inflammatory mediators from renal parenchymal cells and macrophages to recruit inflammatory cells to cause renal injury and promote the development of DKD.Inhibition of macrophage activation or reduction of macrophage infiltration into the kidney slows the progression of DKD.Cullin 4B(CUL4B)serves as a scaffold protein in the CUL4B-RING ubiquitin ligase complex(CRL4B).CRL4B is involved in the regulation of a variety of biological processes,mainly through polyubiquitination of substrate proteins for proteasomal degradation or catalyzing monoubiquitination of H2AK119 for epigenetic inhibition.Previous studies have shown that specific knockout of Cul4b in the haematopoietic system leads to abnormal accumulation of myeloid-derived suppressor cells(MDSCs)in mice.Myeloid-specific Cul4b deficiency exacerbates lipopolysaccharides(LPS)-induced peritonitis and LPS or gram-negative bacteria-induced septic shock.Little is known about the role of myeloid CUL4B in chronic inflammation such as DKD.Therefore,in this study the myeloid-specific Cul4b knockout mice were used to investigate the effect of myeloid CUL4B deletion on the progression of DKD and the underlying mechanism.Methods and Results1.High glucose induces CUL4B upregulation in macrophages.In order to investigate the role of CUL4B in the progression of DKD,mouse peritoneal macrophages(PM)and bone marrow-derived macrophages(BMDM)were treated with glucose or AGE.CUL4B protein was upregulated in a time-and dose-dependent manner upon glucose or AGE treatment.2.Myeloid-specific knockout of Cul4b alleviates DKD.1)Construct myeloid-specific Cul4b knockout mice.To clarify the role of myeloid CUL4B in the development of DKD,we generated mice with Cul4b specifically knocked out in myeloid cells(LysM-Cre+/-Cul4bflox/flox mice,Cul4bMKO mice)by crossing LysM-Cre+/transgenic mice to the Cul4bflox/flox mice previously constructed by our group.The LysM-Cre-/Cul4bflox/flox(Cul4bcon)littemates were used as control.Western analysis confirmed the knockout efficiency.2)Myeloid-specific knockout of Cul4b alleviates DKD phenotype.Using Cul4bMKO mice and Cul4bcon mice,we established a db/db spontaneous diabetes model as well as an STZ/HFD-induced diabetic mouse model.Through histological analysis(H&E staining,PAS staining and Masson staining)of the kidneys and immunohistochemistry or immunofluoresent staining and Western blot analysis of markers for podocytes and fibrosis,we found that myeloid deletion of Cul4b gene significantly attenuated proteinuria,glomerular hypertrophy,mesangial matrix expansion,podocyte and tubular injury and renal fibrosis in DKD mice in both models.3.Cul4b deletion reduces macrophage infiltration in the kidneys of DKD mice.To investigate the mechanism underlying the attenuated DKD caused by myeloid-specific Cul4b deficiency,we immunostained the macrophage marker CD68 in the kidneys and found that myeloid-specific knockout of Cul4b resulted in significantly reduced number of macrophages in the kidneys of both DKD mice.To determine whether myeloid-specific knockout of Cul4b prevented macrophage infiltration from the peripheral circulation into the kidney,we conducted adoptive transfer assays.We depleted macrophages in db/db mice using chlorophosphate liposomes,and then injected these mice with bone marrow cells isolated from Cul4bcon or Cul4MKO mice.48 hours later,kidney cells were isolated and applied to flow cytometry analysis.Mice injected with Cul4bMKO bone marrow cells had a significantly lower proportion of macrophages in the kidney than mice injected with Cul4bcon bone marrow cells,indicating that myeloid-specific knockout of Cul4b inhibits macrophage infiltration into diabetic kidneys.4.Deficiency of Cul4b inhibits macrophage migration and adhesion.To explore the mechanism of reduced macrophage infiltration in the kidneys of DKD mice caused by myeloid-specific knockout of Cul4b,we compared the migration and adhesion ability of Cul4bcon and Cul4bMKO BMDMs in vitro using Transwell and Fibronectin adhesion assays.Cul4b knockout significantly inhibited the migration and adhesion of macrophages,while overexpression of CUL4B in Cul4bcon BMDMs significantly enhanced migration and adhesion.Of note,expression of CUL4B in Cul4bMKO BMDMs rescued the decreased migration and adhesion.Alteration of CUL4B expression in mouse macrophage cell line RAW264.7 also affected their migration and adhesion.5.CUL4B promotes macrophage migration and adhesion through upregulation of ITGA9.1)Regulation of macrophage migration and adhesion by CUL4B requires the ubiquitination activity of CRL4B complex.To investigate the molecular mechanisms by which CUL4B regulates macrophage migration and adhesion,we first analyzed whether the regulation of macrophage migration and adhesion by CUL4B relies on the ubiquitination activity of the CRL4B complex.The activity of CRL4B complex requires neddylation of CUL4B and the Cullin domain of CUL4B.Using Cul4bcon and Cul4bMKO BMDMs,we found that treatment with the neddylation inhibitor MLN4924 significantly inhibited macrophage migration and adhesion.On the other hand,expression of wild type Cul4b rescued migration and adhesion defects caused by CUL4B deletion,while expression of the Cullin-deleted Cul4b mutant did not.These results suggest that the regulation of CUL4B on macrophage migration and adhesion is dependent on the ubiquitination activity of CRL4B complex.2)CUL4B promotes macrophage migration and adhesion through upregulation of ITGA9.To identify the downstream molecules mediating the regulation of migration and adhesion by CUL4B,we performed RNA sequencing on Cul4bcon and Cul4bMKO BMDMs after 24 h AGE or control treatment.We found that the expression of Itga9,which encodes integrin α9(ITGA9),was upregulated by AGE treatment and downregulated by Cul4b knockout.Western blot further revealed that ITGA9 protein was upregulated in macrophages treated with high glucose or overexpressing Cul4b and downregulated in Cul4b knockdown or knockout macrophages,suggesting that CUL4B positively regulates ITGA9.Migration and adhesion assays showed that knockdown of Itga9 inhibited migration and adhesion of RAW264.7 and BMDMs,while overexpression of Itga9 enhanced their migration and adhesion.More importantly,overexpression of Itga9 rescued the reduced migratory and adhesive capacity of Cul4bMKO BMDM.Cul4bMKO bone marrow cells overexpressing Itga9 exhibited enhanced renal infiltration when injected into the db/db mice depleted of endogenous macrophages.These data together suggest that CUL4B promotes macrophage migration,adhesion and infiltration into the kidneys of diabetic mice through upregulation of ITGA9.6.CUL4B upregulates ITGA9 by repressing miR-194-5p.Through online miRNA target databases prediction,RT-qPCR,Western blot and luciferase reporter assays,we found that CUL4B negatively regulated microRNA-194-5p(miR-194-5p)and that miR-94-5p bound to and inhibited Itga9 mRNA.Analysis of renal immune cells from mice with DKD and peripheral blood mononuclear cells from patients with DKD revealed that in vivo CUL4B negatively correlated with miR-194-5p and positively correlated with ITGA9.miR-194-5p mimics inhibited high glucose-induced upregulation of ITGA9 and enhanced macrophage migration and adhesion,while miR-194-5p inhibitors rescued downregulation of ITGA9 and reduction in migration and adhesion in Cul4b knockdown or knockout macrophages,suggesting that CUL4B upregulates ITGA9 and promotes macrophage migration and adhesion through inhibiting miR-194-5p.7.The CRL4B complex cooperates with the PRC2 complex and histone deacetylase to repress miR-194-5p transcription.To determine whether the CRL4B complex directly represses transcription of miR-194-5p,we performed chromatin immunoprecipitation assays(ChIP).We found enrichment of CUL4B in the-2.9kb region of the miR-194-5p promoter.In the meanwhile,we found that enhancer of zeste homolog 2(EZH2),the core molecule of the polycomb repressive complex 2(PRC2),and histone deacetylase 1(HDAC1)were also enriched in the same region.Knockdown of Cul4b significantly reduced the enrichment of EZH2 and HDAC1 in the miR-194-5p promoter region as well as altering the enrichment of the corresponding histone modifications,suggesting that the enrichment of the PRC2 complex and HD AC in the miR-194-5p promoter region relies on CUL4B.Inhibition of EZH2 or HD AC suppressed miR-194-5p downregulation,ITGA9 upregulation and enhanced macrophage migration and adhesion induced by high glucose or CUL4B overexpression,suggesting that cooperation with PRC2 and HD AC is essential for repression of miR-194-5p by CRL4B.Conclusions1.Myeloid-specific knockout of Cul4b ameliorates disease phenotypes and renal infiltration of macrophages in DKD mice.2.High glucose upreguates CUL4B in macrophages,which increases ITGA9 through inhibiting transcription of miR-194-5p,leading to enhanced cell migration and adhesion.