RIG-I,a novel DAMPs sensor for myoglobin, activates NF-κB/caspase-3 signaling in CS-AKI model

Background: Acute kidney injury(AKI) is the main life-threatening complication of crush syndrome(CS), and myoglobin is accepted as the main pathogenic factor. The pattern recognition receptor retinoicacid-inducible gene I(RIG-I) has been reported to exert anti-viral effects function in the innate immune response. However, it is not clear whether RIG-I plays a role in CS-AKI. The present research was carried out to explore the role of RIG-I in CS-AKI.Methods: Sprague-Dawley rats were randomly divided into two groups: the sham and CS groups(n=12). After administration of anesthesia, the double hind limbs of rats in the CS group were put under a pressure of 3 kg for 16 h to mimic crush conditions. The rats in both groups were denied access to food and water. Rats were sacrificed at 12 h or 36 h after pressure was relieved. The successful establishment of the CS-AKI model was confirmed by serum biochemical analysis and renal histological examination. In addition, RNA sequencing was performed on rat kidney tissue to identify molecular pathways involved in CS-AKI. Furthermore, NRK-52 E cells were treated with 200 μmol/L ferrous myoglobin to mimic CS-AKI at the cellular level. The cells and cell supernatant samples were collected at 6 h or 24 h. Small interfering RNAs(siRNA) was used to knock down RIG-I expression. The relative expression levels of molecules involved in the RIG-I pathway in rat kidney or cells samples were measured by quantitative real-time PCR(qPCR), Western blotting analysis, and immunohistochemistry(IHC) staining. Tumor necrosis factor-α(TNF-α) was d etected by ELISA. Co-immunoprecipitation(Co-IP) assays were used to detect the interaction between RIG-I and myoglobin.Results: RNA sequencing of CS-AKI rat kidney tissue revealed that the different expression of RIG-I signaling pathway. qPCR, Western blotting, and IHC assays showed that RIG-I, nuclear factor kappa-B(NF-κB) P65, p-P65, and the a poptotic marker caspase-3 and cleaved caspase-3 were up-regulated in the CS group(P<0.05). However, the levels of interferon regulatory factor 3(IRF3), p-IRF3 and the antiviral factor interferon-beta(IFN-β) showed no significant c hanges between the sham and CS groups. Co-IP assays showed the interaction between RIG-I and myoglobin in the kidneys of the CS group. Depletion of RIG-I could alleviate the myoglobin induced expression of apoptosis-associated molecules via the NF-κB/caspase-3 axis.C onclusions: RIG-I is a novel damage-associated molecular patterns(DAMPs) sensor for myoglobin and participates in the NF-κB/caspase-3 signaling pathway in CS-AKI. In the development of CS-AKI, specific intervention in the RIG-I p athway might be a potential therapeutic strategy for CS-AKI.