The Role And Regulation Of Decorin In The Progression Of Autosomal Dominant Polycystic Kidney Disease

Objective: ADPKD is a common hereditary nephropathy caused mainly by mutations in the PKD1 or PKD2 gene,leading to the formation and expansion of renal cysts.Over the past 40 years,great progress has been made in ADPKD on the development,diagnosis and treatment,but effective intervention and treatment in ADPKD is still absent.Mutations in the PKD1 or PKD2 gene can cause a series of abnormalities in signal transduction,and a complex network system is established by these aberrant signaling pathways.Bioinformatics analysis of the renal tissue expression profile datasets of two ADPKD patients revealed that the core node gene DCN is abnormally expressed in polycystic kidney disease,and is biologically associated with various differentially expressed genes(DEGs),participating in various signal pathways.The DCN gene encoding protein decorin is one of the components of the extracellular matrix.Decorin(DCN),the most widely studied member of the SLRP family,participates in extracellular matrix(ECM)assembly,influences cell adhesion,proliferation,differentiation,and apoptosis.Small leucine-rich proteoglycans(SLRPs),named for their small size(up to 42 kD)and the leucine-rich repeats of the protein core,are biologically active components of ECM.Decorin protein plays an anti-inflammatory and anti-fibrotic role in the progression of chronic kidney disease.In-depth exploration of the biological functions of DCN in ADPKD will provide a new research field for the prevention and treatment in ADPKD.Methods:(1)The kidney tissue microarray data of ADPKD patients and normal individuals were searched and obtained from NCBI Gene Expression Omnibus(NCBI-GEO).DEGs were identified,and enriched pathways and central node genes were elucidated.Six DEGs were verified at the mRNA level using early induction of kidney tissue in mice with polycystic kidney disease and kidney tissue of ADPKD patients.Abnormal expression of the DCN gene as a core node gene in polycystic kidney disease was confirmed.(2)Using different biological models of polycystic kidney disease(Pkd2-MO zebrafish,early and late induced polycystic kidney disease mouse model),the expression of decorin protein was detected at mRNA and protein levels,and abnormally high expression of decorin in the progression stage of polycystic kidney disease was identified.(3)Using single-cell RNA sequencing technology and late-induced polycystic kidney disease mice model,to determine the localization and expression of DCN gene in different single kidney cells,and to explore the possible biological functions of DCN gene in polycystic kidney disease.(4)Cultured cyst-lining epithelial cells(CLECs)in vitro,using RNA interference technology to clarify the regulation of DCN gene on the growth of cyst-lining epithelial cells,and explore the mechanism of decorin regulating the growth of CLECs.Results:(1)Bioinformatics analysis on GSE7869 and GSE35831 revealed 561 DEGs in ADPKD patients renal tissue.The protein-protein interaction(PPI)analysis of DEGs was used to obtain the top 10 node genes.Using the early-induced Pkd1 mouse and ADPKD patients renal tissue,six DEGs were identified at the mRNA level.The abnormal expression of DCN gene as a core node gene in polycystic kidney disease was verified.(2)Abnormal expression of the DCN gene and its encoded protein decorin were observed at different stages on polycystic kidney disease.In the Pkd2-MO zebrafish model of polycystic kidney disease,the DCN gene and its encoded decorin were lowly expressed in the stage of cyst formation in polycystic kidney disease compared with the negative control.In the early-induced and late-induced Pkd1 mice at the stage of cyst development and ADPKD patients renal tissue,the DCN protein was highly expressed,and immunohistochemistry(IHC)showed that decorin mainly distributed in the CLECs and the interstitial area of the renal tissue.Therefore,the expression level of DCN protein is different in the stages of cysts formation and development.It is confirmed that DCN protein was highly expressed in the stage of cysts development.(3)Single cell RNA sequencing(scRNA-seq)results showed that DCN gene was up-regulated in some proximal tubule epithelial cells and T lymphocytes in the renal tissues at the advanced stage of polycystic kidney disease.At the same time,the DCN in all kidney cells was positively correlated with the expression of cell cycle regulatory genes Mcm2 and Pcna,and negatively correlated with the expression of cell apoptosis regulatory gene Bcl2.(4)In vitro experiments,using RNA interference technology,knockdown of DCN gene expression in CLECs,we found that CLECs cell cycle was arrested in G2 / M phase,while apoptosis increased.The cell growth curve confirmed that the proliferation of CLECs was reduced.The expression of TNF-? and MCP-1 in DCN-siRNA-interfering CLECs was reduced.Conclusions:(1)Bioinformatics analysis of ADPKD renal tissue RNA profiles can provide new research targets.The abnormal expression of DCN gene as a core node gene in polycystic kidney disease was verified.(2)In the early-induced and late-induced Pkd1 mice at the stage of cyst development and ADPKD patients renal tissue,the DCN protein was highly expressed,and immunohistochemistry(IHC)showed that decorin mainly distributed in the CLECs and the interstitial area of the renal tissue.The DCN gene and its encoded protein show different expression at stages of the development of polycystic kidney disease.(3)Single cell RNA sequencing(scRNA-seq)results showed that DCN gene was up-regulated in some proximal tubule epithelial cells and T lymphocytes in the renal tissues at the advanced stage of polycystic kidney disease.DCN was positively correlated with the expression of cell cycle regulatory genes Mcm2 and Pcna,and negatively correlated with the expression of cell apoptosis regulatory gene Bcl2.(4)In vitro experiments,using RNA interference technology,knockdown(KD)of DCN gene expression in CLECs,we found that CLECs cell cycle was arrested in G2 / M phase,while apoptosis increased.The cell growth curve confirmed that the proliferation of CLECs was reduced.The expression of TNF-? and MCP-1 in DCN-KD CLECs was inhibited.