The Effect And Mechanism Of Ca CaMKII Inhibitory Peptide AIP In Alleviating Renal Fibrosis

Renal fibrosis is a pathological change in the kidney tissue that occurs in response to long-term external stimuli and is characterized by the massive activation of myofibroblasts and excessive deposition of extracellular matrix（ECM）that destroys the functional renal parenchyma.Renal fibrosis is closely associated with the continuous development of acute kidney injury and chronic kidney disease（CKD）.The development of CKD eventually leads to end-stage renal disease,which has high morbidity and mortality and ultimately leads to organ failure.The diagnosis of renal fibrosis is difficult in the early stages and mainly relies on native kidney biopsy in the later period.At present,there is no ideal drug to treat renal fibrosis in the clinic,but efforts have been made to intervene in primary diseases,such as diabetic nephropathy,hypertensive nephropathy,and glomerulonephritis,and in rejection reactions after kidney transplantation.Noninvasive diagnostic techniques and effective treatment measures for renal fibrosis are limited,and the pathological mechanisms have not been fully elucidated.Therefore,novel drugs urgently need to be developed,and new molecules that can be targeted by drugs to treat or delay renal fibrosis urgently need to be explored.A large number of cytokines and other factors are produced in the process of renal fibrosis,which is caused by long-term injury or persistent inflammatory stimulation.These cytokines can influence renal intrinsic cells,cause organelle dysfunction,result in endoplasmic reticulum stress,lead to the release of Ca²⁺stored in the endoplasmic reticulum and cause a dramatic increase in the intracellular Ca²⁺concentration.Ca²⁺is an important intracellular signaling molecule,and deregulation of Ca²⁺homeostasis can cause abnormal activation of downstream signaling pathways.CaMKII is an intracellular Ca²⁺-sensing protein that plays a critical role in intracellular Ca²⁺signal transmission.Upregulation of CaMKII increases proinflammatory signaling,amplifies inflammatory responses,and induces a variety of diseases.Pharmacological inhibition of CaMKII is a potential therapeutic strategy for the prevention of myocardial injury and heart disease.The activation of CaMKII can also mediate the proliferation and phenotypic transformation of fibroblasts,promote the production of ECM and is associated with the development of various kidney diseases.In addition,CaMKII can participate in the activation and regulation of many signaling pathways,such as those related to oxidative stress,NF-κB,TGF-β/Smad,MAPK,Wnt,Akt and other factors,leading to renal fibrosis.However,there are no reports on treatments aiming to alleviate renal fibrosis by targeting CaMKII.Inhibiting excessive activation of CaMKII could theoretically protect renal function and inhibit the progression of renal fibrosis.Therefore,targeting the Ca²⁺/CaMKII signaling pathway,especially targeting the overactivation of CaMKII,could be a new strategy for the treatment of renal fibrosis and organ fibrosis.Peptide drugs have attracted significant attention because they integrate the advantages of small molecule drugs and protein drugs.Based on the possible aberrant remodeling of calcium signaling pathways and the overactivation of CaMKII that occur during the development of renal fibrosis,we selected AIP,a specific inhibitory peptide of CaMKII.We used the UUO model to evaluate the ability of AIP to ameliorate renal fibrosis in vivo as well as NIH-3T3,HK2 and NRK-52E cells exposed to TGF-β1 as in vitro models to further explore the possible mechanism of AIP.The results showed that AIP could reverse the abnormal protein and m RNA expression levels of major fibrosis markers,alleviate the kidney injury caused by UUO and significantly attenuate the degree of renal tubulointerstitial fibrosis in vivo and in vitro.To determine the mechanism,we examined the effect of AIP on epithelial-mesenchymal transition（EMT）,as well as its inhibitory effects on CaMKII and downstream signaling pathways in vivo and in vitro.We found that AIP could inhibit the expression of various EMT-related markers,significantly inhibit the phosphorylation of CaMKII and affect the transduction of the TGF-β/Smad and Raf/ERK signaling pathways.From these results,we concluded that AIP can inhibit the activation of fibroblasts and the phenotypic transformation of epithelial cells,reduce the production of ECM,and ultimately alleviate renal fibrosis by downregulating the activation of CaMKII and blocking transduction of the TGF-β/Smad and Raf/ERK pathways in vitro and in vivo.In conclusion,AIP could be a potential therapeutic drug for renal fibrosis,CaMKII could be a new pharmacologic target for treating renal fibrosis,and targeting the Ca²⁺signaling pathway could be a new strategy for drug development in renal fibrosis.