Uric Acid Induces Inflammatory Injury In HK-2 Cells Via ROS/p38MAPK/NLRP3 Signaling Pathway

Background: Hyperuricemia(HUA)is a common metabolic syndrome.A large number of epidemiological studies have shown that elevated levels of uric acid(UA)are a major risk factor for gout,cardiovascular disease,and chronic kidney disease(CKD).At present,the pathophysiological mechanism of high uric acid-induced kidney damage has not been fully elucidated,and some studies have found that oxidative stress and inflammatory response play an important role in the occurrence and progression of uric acid-induced kidney injury.In HUA,UA promotes the production of Reactive oxygen specie(ROS).ROS and intracellular UA themselves regulate a variety of intracellular signaling pathways whose activation may lead to the development of hyperuricic nephropathy.Mitogen-activated protein kinase(MAPK)is a classical signaling pathway for many extracellular stimuli,of which p38 mitogenactivated protein kinase(p38MAPK)is at the core,which is involved in various inflammatory and autoimmune diseases.For example,activation of the NLR family pyrin domain containing 3(NLRP3)inflammasome signaling pathway is activated in the nucleotide-binding oligomerization domain(NOD)-like receptor protein 3,leading to a local or systemic inflammatory response.After activation of the NLRP3 inflammasome,it can induce the activation and maturation of the downstream proinflammatory cytokines interleukin-1β(IL-1β)and interleukin-18(IL-18),thereby causing an inflammatory response in renal tissue.Several studies have confirmed that the oxidative stress response of cells promotes the activation of p38 MAPK and aggravates tubular epithelial cell damage.However,the specific mechanism of whether high uric acid upregulates p38 MAPK to damage renal tubular epithelial cells through ROS is still unclear,so this study explored the mechanism of action of p38MAPK/NLRP3 inflammasome pathway in UA-induced renal pathology and the activation mechanism of p38MAPK/NLRP3 inflammasome through in vitro experiments.Objective: In vitro cell models were established to observe the association between high uric acid levels and human renal tubular epithelial cell(HK-2)damage.HK-2 cells were selected to screen the most suitable concentration of UA stimulation,establish a cell model of inflammatory damage,and explore the effect of UA on inflammatory injury at the cellular and molecular levels.At the same time,p38 MAPK inhibitors and ROS scavengers were used to explore the specific mechanism and relationship between UA-induced HK-2 cell damage and NLRP3-mediated cell damage.Methods: HK-2 cells were cultured in vitro,HK-2 cells were treated with multiple concentrations of soluble UA(0/10/20/30/40mg/d L)for 48 h,and the viability of cells after different concentrations was detected by inverted microscope and CCK-8 to screen out the most suitable UA treatment concentration.The cells were divided into the following six groups: control group,UA group,ROS inhibitor group,p38 MAPK inhibitor group,UA+ROS inhibitor group,UA+p38MAPK inhibitor group.In order to elucidate the mechanism of action of UA,we performed mitochondrial membrane potential determination and ROS content in HK-2 cells,and measured the expression of the inflammation-associated protein IL-1β using Western Blot technology.To better understand the mechanism of the inflammatory response,Western Blot technology was used to determine the expression of inflammasome-associated proteins of p38 MAPK and NLRP3.Results:(1)When the concentration of UA increased,the viability of HK-2 cells,the level of mitochondrial membrane potential decreased,the level of ROS and the expression level of inflammatory factor IL-1β increased significantly,and the level of p38 MAPK and downstream NLPR3 protein also increased significantly.(2)After the administration of ROS scavenger,HK-2 cell viability increased,damage decreased,and p38 MAPK pathway was significantly inhibited.(3)After the administration of p38 MAPK inhibitor,the ROS level was significantly reduced,and the expression of NLRP3 and its downstream related proteins was significantly reduced,which alleviated the damage caused by UA.Conclusion: 1.High uric acid damages HK-2 cells through oxidative stress,thereby inducing kidney function damage.2.High uric acid may activate the p38 MAPK classical inflammatory signaling pathway,resulting in HK-2 cell damage.