Upregulation of RIP3 promotes necroptosis via a ROS-dependent NF-κB pathway to induce chronic inflammation in HK-2 cells

Tubular atrophy/interstitial fibrosis (TA/IF) is a major cause of late allograft loss, and inflammation within areas of TA/IF is associated with adverse outcomes in kidney transplantation. However, there is currently no satisfactory method to suppress this inflammation to improve TA/IF. The present study aimed to determine the proinflammatory role of receptor-interacting protein 3 (RIP3) in TA/IF to discover a novel therapeutic target. Reverse transcription-quantitative PCR and western blotting were performed to detect the expression of RIP3 and inflammation-associated factors. Lactate dehydrogenase release assay was used to determine necroptosis. Fluorescent 2,7-dichlorodihydrofluorescein diacetate was used to detect the levels of reactive oxygen species (ROS). The results demonstrated that patients with chronic TA/IF exhibited upregulated receptor-interacting protein 3 (RIP3) expression compared with the patients who had a favorable recovery after renal transplant. Therefore, the current study used normal renal tubular epithelial cells HK-2 to establish a cellular model with a high expression level of RIP3 in order to investigate the effect of RIP3 on renal epithelial cells after transplantation. The western blotting results demonstrated that overexpression of RIP3 could significantly increase the phosphorylation level of the necroptosis executive molecule mixed lineage kinase domain-like protein. Lactate dehydrogenase release, a key feature of necroptosis, was also markedly improved by RIP3 overexpression. Moreover, a higher inflammatory response was detected in HK-2 cells with RIP3 overexpression, and this elevated inflammation could be restored by the necroptosis inhibitor necrosulfonamide. Of note, it was found that overexpression of RIP3 activated the NF-κB signaling pathway via the excessive accumulation of ROS to induce necroptosis, which ultimately led to inflammation. Collectively, these findings indicated that overexpression of RIP3 promoted necroptosis via a ROS-dependent NF-κB pathway to induce chronic inflammation, suggesting that RIP3 may have the potential to be a therapeutic target against inflammation in TA/IF.