The Role And Mechanism Of CAMP-CREB Pathway In ADPKD

Background:Autosomal dominant polycystic kidney disease(ADPKD)is the most common inherited disorder of the kidneys,and one of the leading causes of end-stage renal disease.Polycystic kidney disease is characterized by the progressive formation of bilateral renal cysts.These fluid-filled cysts damage the surrounding tissue and eventually lead to kidney failure.However,the molecular mechanism underlying cyst formation in ADPKD is currently unclear.And there is presently no effective treatment for ADPKD.Abnormal activation of the c AMP-PKA signaling pathway promotes cystogenesis in ADPKD.CREB is an important transcription factor downstream of the c AMP-PKA pathway.At present,the pathophysiological role of CREB in ADPKD is unknown.We aimed to clarify the function and molecular mechanism of CREB in ADPKD,and to explore the possibility of using CREB as a target to treat ADPKD.Methods: 1.The expression levels of CREB and phosphorylated CREB in mouse kidney tissues and primary cells were detected by Western blotting.The distribution of CREB in ADPKD kidney tissue was determined by immunofluorescence staining.And the correlation between CREB status and ADPKD was further clarified in patient samples.2.We used an A-CREB transgenic mouse model to investigate the effect of inhibiting CREB transcriptional activity on the progression of ADPKD.We evaluated the effects of A-CREB in ADPKD by measuring kidney weight and body weight ratio,renal cystic index,and blood urea nitrogen in ADPKD mice.3.We used 666-15,a small molecular inhibitor of CREB to treat ADPKD mouse model.We evaluated the therapeutic effect of targeting CREB transcriptional activity on ADPKD.We explored whether long-term injection of 666-15 has toxicity and side effects in mice.4.We employed chromatin immunoprecipitation sequencing(Ch IP-seq)technology to clarify the genomic distribution of phosphorylated CREB on the cystic cell.We used Ch IP-seq combined with RNA sequencing(RNA-seq)analysis to explore the molecular mechanism underlying CREB promoting ADPKD.In order to understand the transcriptional regulation mechanism of CREB,a motif analysis was performed to find the co-associated transcription factors related to CREB.Results:First,we found that phosphorylated CREB levels were significantly increased in renal cyst cells.And the transcription level of the classic targets of CREB was also upregulated.Subsequently,we used A-CREB,a dominant negative inhibitor of CREB,to suppress the CREB transcriptional activity in vivo,which showed a significant alleviating effect on ADPKD disease progression.These results suggest the upregulation of CREB targets is associated with cyst formation and expansion.We selected 666-15 as a small molecular inhibitor of CREB for drug intervention in ADPKD mouse models.The results indicate that 666-15 can effectively alleviate ADPKD progression and improve kidney function in mice.At the same time,no significant toxicity and side effects of 666-15 were observed.Finally,we employed RNA-seq and Ch IP-seq technology,combined with bioinformatics analysis,to identify genes regulated by phosphorylated CREB in ADPKD.We also identified transcription factors that related to CREB.Conclusion:Phosphorylated CREB promotes cyst formation and expansion by activating the transcription of cystogenic genes.We provide a novel strategy based on targeting CREB transcriptional activity to reduce cyst growth in ADPKD.