Klotho Preservation By Rhein Suppresses Toll-like Receptor 4 And Attenuates Lipopolysaccharide-induced Acute Kidney Injury

Background:Acute kidney injury(AKI)incurred by pathogenic microorganism infection or excessive inflammatory response is a common clinical illness with high incidence and may develop into chronic kidney disease.Anti-infection treatments by antibiotics are effective for AKI of known pathogens.However,the effective treatment for AKI of unknown etiology is lacking.Recent studies suggest that endogenous anti-inflammatory proteins/mediators play important roles in the development of AKI.Therefore,targeting endogenous anti-inflammatory protein,beneficially modulaing endogenous protection system and strengthening the anti-inflammatory potency have become a new hot spot of AKI therapy.Klotho is an anti-aging gene discovered in 1997 and has significant anti-inflammatory function.Klotho includes a membrane and a secretory form,mainly expressed in renal tubular epithelial cells.Secretory type Klotho exhibits glycosidase activity,which can regulate the stability and function of many mineral receptors and ion channels/transporters through its deglycosylation ability.Klotho can inhibit toll-like receptor(TLR)signaling pathways and show anti-inflammatory effects,but its intracellular targets are not clear.Klotho level drops in AKI and its anti-inflammatory effect are suppressed.Maintaining endogenous Klotho level has been proven to alleviate the pathogenesis of acute and chronic kidney injury effectively.Our labratory previously found that Rhein,a lipotropic anthraquinonoid compound isolated from medicinal plant rhubarbwidely used in clinical treatments of inflammatory disorders,can effectively reverse the decline of Klotho in animal models of acute and chronic kidney injury,but the precise molecular mechanism is unclear.TLRs are the first line of host defenses that sense the pathogen-associated molecular patterns(PAMPs)or danger-associated molecular patterns(DAMPs)and activate signal transduction and expression of inflammatory cytokines.There are at least 11 members in TLR famil in human,among them TLR4 can specifically recognize gram-negative bacteria lipopolysaccharide(LPS)and the inflammation-related damaged cellular components.TLR4 is heavily glycosylated and its glycosylation status dictates its membrane transport/expression and its stability.Objects:In this study we explore the significance of Rhein restoration of Klotho in acute inflammation and AKI as well as the underlying mechanismsin order to provide the mechanistic explanations for the anti-inflammtion effects of Klotho preservation by Rhein and other natural or synthetic materials in clinical treatments of AKI and inflammatory renal diseases.Methods and Results:Methods:1.in vivo experiments:we studied the anti-inflammatory and renal protective effects of Rhein by reversing Klotho in acute inflammation and AKI induced by LPS.By silencing Klotho via small interference RNA technology,we confirmed the specific role of Klotho in mediating Rhein anti-inflammatory and renal protection.2.In vitro cell experiments:we examined the anti-inflammatory effects of Rhein by preserving depressed Klotho that inhibited TLR4 abundance,we further explored the deglycosylating capacity of Klotho that suppressed TLR4 and caused TLR4 degradation.Results:1.In vivo experiments:Rhein alleviated the acute inflammation and tissue pathological injury induced by LPS in mouse kidney,which were accompanied by Klotho preservtion and TLR4 inhibition.In vitro experiments:Klotho,highly presented in mononuclear macrophages,is downregulated after LPS stimulation.Rhein reversed Klotho repression and decreased TLR4 levels and inhibited the expression of inflammatory cytokines in LPS-activated macrophages.2.Rhein up-regulated Klotho and downregulated TLR4,which substantially relied on TLR4 in dumpping inflammation induced by LPS.Through its glycosidase activity,Klotho targeted TLR4 and reduced its stability,leading to the accelerated TLR4 degradation via autophagy-lysosome pathway.3.In Klotho-knockdown cells,Rhein failed to suppress LPS-activated NF-?B pathway and the expression of inflammatory cytokines.In Klotho-knockdown mice,Rhein inhibitions of the renal inflammation and kidney damage induced by LPS were largely abolished,suggesting that Klotho is essential for Rhein anti-inflammation and renoprotection.Conclusions:Klotho is highly expressed in macrophages and directly involved in regulating renal local and systemic inflammations.Klotho inhibits TLR4-associated inflammatory responses through deglycosylating TLR4 and causing its lysosomal degradation.Rhein preservation of Klotho inhibits Toll-like receptor 4 and contributes significantly to its anti-inflammatory and renal protective functions.