The Significance Of Inhibiting Pyroptosis Of Renal Tubular Epithelial Cells Via Inhibiting AKT In The Treatment Of Gouty Nephropathy

Background:Gouty nephropathy is an inflammatory disease characterized by deposition of uric acid in the renal tubules and renal interstitium and secondary renal damage caused by the increased uric acid in the body.Involvement of the renal tubules and renal interstitium is the main pathological change.The pathogenesis of gouty nephropathy is associated with the local deposition of urate in the kidney,but the specific mechanism is not completely clear.Therefore,it is significant to explore the pathogenesis of gouty nephropathy for exploring new therapeutic targets.Many previous studies have shown that monosodium urate(MSU)can activate NLRP3 inflammasome,which in turn activates Caspase-1.Then Caspase-1 further cleaves GSDMD,and the N-terminal of GSDMD forms the pore on cell membrane,causing cell pyroptosis.As mentioned above,the local deposition of urate crystals in the kidney especially in the renal tubules contributes to renal damage.However,whether the renal damage caused by urate crystals is related to GSDMD-mediated pyroptosis of renal tubular epithelial cells has not been reported yet.According to other and our previous studies,the activation of AKT can lead to pyroptosis and neutrophil necrosis(NETosis).Moreover,inhibition of AKT phosphorylation can inhibit Caspase-1 activation.Objectives:This study aimed to investigate:(1)Can MSU activate Caspase-1 and cause GSDMD-mediated pyroptosis in renal tubular epithelial cells,leading to the occurrence of gouty nephropathy?(2)Whether inhibiting AKT can block renal tubular epithelial cells pyroptosis,thereby alleviating the disease condition in the mouse model of gouty nephropathy?Methods:We first constructed and selected the most suitable mouse model of gouty nephropathy through induction with different dose combination of "potassium oxazine and hypoxanthine".In in vivo experiment,the effects of AKT inhibitor on uric acid,renal function and renal pathology in the mouse model of gouty nephropathy were observed.Then immunohistochemistry and immunofluorescence were used to determine whether AKT inhibitor could inhibit the expression of Caspase-1 and GSDMD of renal tubular epithelial cells in the mouse model of gouty nephropathy.In in vitro study,human renal tubular epithelial cell line was stimulated with LPS and MSU to observe whether LPS and MSU could induce renal tubular epithelial cell necrosis and IL-1β release,and whether AKT inhibitor could inhibit LPS and MSUinduced renal tubular epithelial cell necrosis and IL-1β release.Results:(1)“150mg/kg potassium oxazinate and 300mg/kg hypoxanthine" is the best dose combination for inducing gouty nephropathy in this experiment;(2)The levels of serum uric acid,urea nitrogen and creatinine of the model group were higher than those of the control group,which were significantly decreased by AKT inhibitor;(3)HE staining showed a large number of inflammatory cell infiltrate around the renal tubules in the model group,and AKT inhibitor could significantly reduce inflammatory cell infiltration.Compared with the control group,the MASSON staining of kidney tissue was increased in the model group,suggesting that renal fibrosis was increased.And AKT inhibitor could significantly inhibit renal fibrosis of the mouse model;(4)The results of immunohistochemistry and immunofluorescence both showed that in model group,the expression of Caspase-1 and GSDMD in the renal tubules was higher than that in the control group,and AKT inhibitor could significantly reduce the expression of Caspase-1 and GSDMD in the kidney of the mouse model;(5)The in vitro experiment showed that LPS and MSU could increase the release of LDH and IL1β by human renal tubular epithelial cell line,and AKT inhibitor could reduce the release of LDH and IL-1β by human renal tubular epithelial cell line.Conclusions:In mice with gouty nephropathy,Caspase-1 of renal tubular epithelial cells is activated,and GSDMD-mediated pyroptosis occurs.AKT inhibitor can block the activation of Caspase-1-GSDMD and alleviate the disease condition in the mouse model of gouty nephropathy.AKT inhibitor can reduce the release of LDH and IL-1β by renal tubular epithelial cells.These results suggest that inhibition of AKT phosphorylation may be a potential new target for the treatment of gouty nephropathy.