Research On The Function Of Transcription Factor KLF14 In HFD-induced Renal Injury

Objective:The transcription factor Kruppel-like factor 14(KLF14)has been associated with type 2 diabetes and high-density lipoprotein-cholesterol(HDL-c)through genome-wide association studies.There is also research shows that the deletion of the Kruppel-like factor 14(KLF14)gene in mice causes aneuploidy,centrosome amplification,and spontaneous tumorigenesis.But its role in kidney still remains totally unknown.In this study,we generated the KLF 14-deficient mice and report the role of KLF14 in kidney.Methods and Results:KLF 14-deficient Mice was generated using the Transcription Activator-Like Effector Nuclease(TALEN)technique.The blood biochemical index analysis showed that,compared with the Wildtype(KLF14+/+)mice,the level of high-density lipoprotein cholesterol in KLF 14-deficient Mice significantly decreased.The histological changes in glomeruli were examined by PAS staining(Periodic Acid-Schiff staining),and cell proliferation was detected by PCNA immunohistochemistry staining.We observed that the disruption of the Kruppel-like factor 14 resulted in aggravated accumulation of extracellular matrix and cell proliferation,and KLF 14-/-mice expressed a higher percentage of PCNA positive cells than KLF14+/+ mice.By using RNA-seq technology,38 differentially expressed genes were found in kidneys of KLF 14-deficient mice,among which 12 genes were up-regulated and 26 genes were down-regulated.Differentially expressed genes are mainly involved in metabolism,proliferation,and signal transduction.In order to clarify the target gene of transcription factor KLF 14 in kidney,2621 peak related genes were obtained through ChIP-Seq technology.These genes mainly involve metabolism,proliferation,inflammation.The combined analysis of RNA-Seq and ChIP-Seq results showed that there were 5 intersecting genes,which were Socs2,Btg2,Hdc,Ier2,and Akrlb3.Dual luciferase reporter assay and electrophoretic mobility shift assay(EMSA)showed that Btg2 is the target gene of KLF 14.Furthermore,the EDU assay also proved that KLF 14 had an anti-proliferation effect on primary mouse renal mesangial cells(MRMC)by targeting Btg2.Then KLF14+/+ mice and KLF14-/-mice were used to construct a HFD-induced renal injury model,and we performed IPGTT and IPITT tests to evaluate the glucose tolerance and insulin sensitivity,the results showed that the degree of glucose intolerance in the KLF14-/-HFD group was significantly higher than that in the KLF14+/+ HFD group,and the insulin sensitivity of the KLF14-/-HFD group was less than that of the KLF14+/+ HFD group.The renal pathological results showed that there was a significant increase in the mesangial matrix and the proliferation of mesangial cells in the HFD group,and the increase of the KLF14-/-HFD group was more obvious than that of the KLF14+/+ HFD group.The results of electron microscopy showed that the renal structure in the STD group was basically normal,and the HFD group had obvious lipid vacuoles and increase of the mesangial matrix compared with the STD group.Oil red staining was used to observe the accumulation of lipid in four groups,the results showed that the lipid accumulation in the HFD group was significantly heavier than that of the STD group and the deletion of KLF14 in the HFD group could aggravate the lipid deposition.Then renal inflammatory indicators were detected,the results showed that the expression level of TLR2 and TLR4 increased in the HFD group and the increase was more obvious in the KLF14-/-HFD mice.The results of NF-?B pathway showed that HFD could increase the expression of p-NF-KB and decrease the expression of p-I?B?,and the trend was more obvious in the KLF14-/-HFD mice.We further used suspension array to detect the expression level of inflammatory cytokines in the kidney,the results showed that there was no significant difference in the levels of inflammatory cytokines between the KLF14-/-STD group and KLF14+/+ STD group,while in the HFD group,the level of IL-1? and IL-6 in the KLF14-/-group were higher than those in the KLF14+/+ group.Conclusion:RNA-Seq and ChIP-Seq results showed KLF14 is mainly involved in Lipid metabolism,proliferation and inflammation response.KLF14 had an anti-proliferation effect on primary mouse renal mesangial cells(MRMC)by targeting Btg2.Deletion of KLF14 can aggravate the accumulation of lipid in the kidney and the level of inflammation,and increase the proliferation of mesangial cells.