The Function And Mechanism Of Palmitoyl Acyltransferase ZDHHC6 In Kidney

Chapter oneMolecular discovery of ZDHHC6 based on transcriptomics and clinical phenotype of DN patientsBackground and objectiveDiabetic nephropathy(DN)is one of the most common microvascular complications of diabetes mellitus,which seriously endangers human health.There is an urgent need to have a deeper understanding of its pathogenesis and progression mechanisms.In recent years,great progress has been made in the area of protein palmitoylation and its catalytic enzymes,the ZDHHC family,and their role in various pathological and physiological processes has received more and more attention.However,the potential relevance of palmitoylation and ZDHHC family in DN has not been reported util now.Here,we characterized the importance of ZDHHC in DN based on our DN glomerular transcriptomics data,and emphasized the role of ZDHHC6 in the pathogenesis and progression mechanism of DN.MethodsWe first analyzed the transcriptome-clinical phenotype data,through the weighted gene co-expression network analysis(WGCNA),finding clusters of highly related genes,which were then correlated with the estimated glomerular filtration rate(eGFR)and proteinuria.And then,we learned the expression and location of the ZDHHC family,especially the discovered ZDHHC6 by analyzing our DN transcriptome data and mining the databases of Nephroseq and KIT,an online analyzer for kidney single cell datasets.After that,we observed the expression of ZDHHC6 protein by immunohistochemistry(IHC).Finally,we predicted the involvement of ZDHHC6 in the mechanism of DN podocyte damage by enrichment analysis of the podocyte markers that can be palmitoylated.ResultsZDHHC6 has the highest expression level among ZDHHC family in the glomerulus.WGCNA found that the three modules of turquoise,brown and purple were closely related to DN proteinuria and eGFR.ZDHHC6 is the hub gene of the turquoise module,whose expression at transcription level is positively related to eGFR(r=0.59)and inversely related to proteinuria(r=-0.71).Transcriptome data showed that ZDHHC6 was highly co-expressed with podocyte markers,and KIT sequencing data showed that it was mainly expressed in glomerular podocytes.Additionally,IHC staining showed that the expression of ZDHHC6 protein in DN glomeruli was significantly reduced,which was consistent with the analysis results of our chip data.Pathway analysis of podocyte proteins that can be palmitoylated suggests that ZDHHC is likely involved in the mechanism of cytoskeletal regulation.ConclusionsZDHHC6 is mainly expressed in glomerular podocytes and is the member with the highest expression in the kidney.With reduced expression in DN glomeruli,ZDHHC6 has a close relationship with DN clinical phenotype.And it is predicted that ZDHHC6 is involved in the pathogenesis and progression mechanism of DN by regulating the podocyte cytoskeleton system.Chapter twoResearch on ZDHHC6 and glomerular function in zebrafish modelObjectiveTo study the role of zdhhc6 in the maintenance of normal glomerular function with zebrafish model.MethodsMegAlign software was used to compare the zdhhc6 amino acid sequence similarity.The zebrafish glomeruli were isolated and sent for sequencing analysis to learn the expression of the zdhhc family.Zdhhc6 knockout(KO)zebrafish G0 was constructed by CRISPR/Cas9 technology,and its phenotype was statistically analyzed,also,its glomerular ultrastructure was examined by transmission electron microscopy.Double color immunofluorescence was used to learn the expression location of zdhhc6 protein.The transgenic reporter zebrafish Tg(pod:eGFP)expressing green fluorescent in podocytes were used to visualize the effect of zdhhc6 KO on podocyte The whole mount in situ hybridization(WISH)was used to detect the expression changes of podocyte and their progenitor cell markers as well as renal tubular markers.ResultsThe amino acid sequence similarity of zdhhc6 between zebrafish and human is as high as 73.4%identity.The sequencing data of zebrafish glomeruli show that zdhhc6 is a relatively high and stable expressed member in the zdhhc family.Zdhhc6 KO zebrafish constructed by CRISPR/Cas9 technology developed pericardial edema and systemic edema,and injection of human or zebrafish zdhhc6 mRNA could effectively rescue the phenotype.And transmission electron microscopy showed glomerular developmental defect in these zdhhc6 KO zebrafish,mainly characterized by podocytes process effacement.The double color immunofluorescence showed that zdhhc6 protein co-localized with the podocyte marker synaptopodin.Knockout of zdhhc6 could significantly reduce the fluorescence expression of GFP in podocytes in Tg(pod:eGFP)embryos.The results of whole-mount in situ hybridization(WISH)showed that in zdhhc6 KO zebrafish,the hybridization signals of podocyte markers(podocin and nephrin)and podocyte progenitor cell markers(pax2a and lhx1a)were reduced,while that of the renal tubule markers(Cdh17 and megalin)was not obviously changed.ConclusionsKnockout of zdhhc6 mainly destroys the structure and function of podocytes,but has no obvious effect on renal tubules.Zdhhc6 is essential for the normal function of zebrafish glomerulus.Its loss can lead to extensive fusion of the foot processes,renal dysfunction,and pericardial or systemic edema in zebrafish.Chapter threeThe protection effect and the underlying mechanism of ZDHHC6 on PodocyteObjectiveBoth the analysis of DN transcriptome data and results of our above studies of zdhhc6 in zebrafish suggest that ZDHHC6 is mainly expressed in glomerular podocytes and has an important role in the stability of podocyte functions.As podocyte injury is vital in the pathogenesis of glomerular diseases,we explored the effect of ZDHHC6 on podocyte and the underlying mechanism.MethodsA phylogenetic tree of the ZDHHC protein family was constructed with the MP method to find other family members close to ZDHHC6.And ZDHHC6 was knockdown in human podocytes with ZDHHC6 siRNA.Based on the acyl-biotin exchange(ABE)technology,we detected ZDHHC6 target palmitoylated proteins by label-free quantitative proteomics and bioinformatics analyses.According to the enrichment analysis results,we assessed the effect of ZDHHC6 on podocytes through the following in vitro experiments:(1)analysis of podocyte apoptosis with Annexin V-FITC/PI,(2)observe of podocyte cytoskeleton with phalloidin staining,(3)detect of cellular reactive oxygen species with DCFH-DA labeling,(4)detect of mitochondrial membrane potential(MMP)by JC-1 staining.ResultsZDHHC6 siRNA can specifically inhibit the expression of ZDHHC6,while with no effect on the expression of ZDHHC1,ZDHHC5 and ZDHHC8.Through bioinformatics analysis of the differential palmitoylated proteins detected between the ZDHHC6 siRNA transfection group(C6siRNA)and the control group(CTL),the Ferroptosis pathway and the Cell cycle pathway,as well as the three pathways close related with cytoskeleton regulation,e.g.the Leukocyte transendothelial migration pathway,the Regulation of actin cytoskeleton pathway and the Cell adhesion molecules pathway,were all significantly enriched.The above results indicated that ZDHHC6 might contribute to podocyte cytoskeleton regulation and cell survival through palmitoylation of its target proteins.This is consistent with the results of the bioinformatics analysis in the chapter one study.The results of in vitro experiments showed that both ZDHHC6 inhibition and TGF-β1 treatment could induce podocyte apoptosis,cytoskeletal structure rearrangement,reactive oxygen generation and mitochondrial depolymerization.Moreover,inhibition of ZDHHC6 could further aggravate TGF-β1 induced podocyte damage.ConclusionsZDHHC6 plays an important role in the podocyte homeostasis.Knockdown of ZDHHC6 could cause an increase of apoptotic podocytes,which might be related to cytoskeleton change,increased production of reactive oxygen species and the decrease of mitochondrial membrane potential.