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The effect of kaempferol on renal calcium oxalate crystal formation and calcium oxalate crystal-induced kidney injuryObjective Renal tubular epithelial cell injury and calcium oxalate crystal deposition play an important role in the process of renal calcium oxalate crystal formation.The purpose of this study aimed to investigate the effect of kaempferol on calcium oxalate crystal-induced kidney injury,as well as renal calcium oxalate crystal deposition and formation.Methods In the mice experiments,we first established the renal calcium oxalate crystal model by intraperitoneal injection of glyoxylic acid.Mice were randomly divided into five groups: normal control group,crystal model group,solvent control group,kaempferol(25mg/kg)treatment group and kaempferol(50mg/kg)treatment group.The formation of calcium oxalate crystals in mice kidneys was observed by HE staining and Pizzolato staining on paraffin sections.Renal tubular damages were examined by PAS staining and TUNEL fluorescence staining.The expression levels of OPN and CD44 in renal tubular epithelial cells were detected by IHC staining.Additionally,serum BUN,Cr and NGAL were tested.In followed HK-2 cell experiments,cells were stimulated with calcium oxalate monohydrate(COM)crystals and kaempferol at different concentrations.Cells were divided into five groups according to different treatments: normal control group,COM crystals group,COM crystals+10?M kaempferol group,COM crystals+20?M kaempferol group,as well as COM crystals+40?M kaempferol group.Afterwards,intracellular LDH,cell apoptosis,crystal adhesion,and the expression levels of Caspase-1 protein were assessed.Results The mice renal calcium oxalate crystal model was successfully established.The number of calcium oxalate crystals,the tubular damages,the expression levels of OPN and CD44 proteins,the levels of BUN,Cr and NGAL were significantly higher in the crystal model group when compared to those in the normal control group.However,the levels of above indicators were significantly lower in the kaempferol treatment group than those in the crystal model group.And the higher the concentration of kaempferol,the greater the declined degree.On the other hand,in the HK-2 cell experiments,the results showed no inhibitory effect of 10,20,and 40 ?M kaempferol on cell proliferative activity.In addition,LDH levels,the levels of cell apoptosis and necrosis,adhesive amount of crystals,as well as the expression levels of Caspase-1 protein were significantly higher in the COM crystals group than the normal control group.But after the treatment with kaempferol,the levels of these indicators were significantly decreased in a concentration-dependent manner.Conclusions Kaempferol is able to inhibit calcium oxalate crystal adhesion and deposition,as well as to alleviate calcium oxalate crystal-associated renal injury.Kaempferol benefits to reduce the formation of calcium oxalate crystals in the kidney.Kaempferol may have potential pharmacological activity and applicative value in inhibiting the formation of calcium oxalate nephrolithiasis.Part ? The role of kaempferol in inhibiting renal calcium oxalate crystal formation and kidney damage by regulating the oxidative stress and inflammationObjective Oxidative stress and inflammation have been found to fundamentally contribute to renal calcium oxalate crystal-induced injury as well as calcium oxalate crystal formation.It aimed to study the role and mechanism of kaempferol in reducing calcium oxalate crystal formation and crystal-induced injury in the kidney by modulating the oxidative stress and inflammation.Methods Firstly,the expression levels of NOX2,i NOS,NFk B-P65,MCP-1 and F4/80 in mice renal tissues were evaluated by IHC staining.Whereafter,ROS,H2O2,MDA,GSH and SOD were measured.Real-time quantitative PCR was used to detect the expression levels of inflammation-associated cytokines.And in the HK-2 cell experiments,ROS,H2O2,MDA,GSH,SOD and inflammation-associated cytokines were examined in cells after different treatments.Moreover,the expression levels of NOX2 and its homologues were evaluated after treatment with COM crystals and kaempferol.Furthermore,MCP-1,i NOS and NFk B-P65 proteins were assessed by WB.Results In the mice experiments,the results demonstrated that the expression levels of NOX2,i NOS,NFk B-P65,MCP-1 and F4/80 in the kidney tissues were significantly increased in the crystal model group compared with the normal control group.However,compared with the crystal model group,the expression levels of these proteins in the kaempferol treatment group were significantly decreased.And the higher the concentration of kaempferol,the greater the changed degree.In addition,when compared to the normal control group,the levels of ROS,H2O2,MDA,IL-1?,IL-6 and TNF-? were significantly higher,while the levels of GSH,SOD,IL-10,IL-4 and Arg1 were significantly lower in the crystal model group.But in the kaempferol treatment group,the levels of these above indicators were reversed in a dose–dependent fashion.On the other hand,in the HK-2 cell experiments,it was indicated that the levels of ROS,H2O2 and MDA were increased after COM crystals stimulation,while the levels of GSH and SOD were reduced.However,these effects were reversible after the treatment with kaempferol in a concentration-dependent manner.Furthermore,it was confirmed that the expression level of NOX2 was most significantly decreased among all homologues after the treatment with kaempferol.Moreover,the levels of IL-1?,IL-6,TNF-?,NFk B-P65,MCP-1 and i NOS were significantly reduced after the treatment with kaempferol,while the levels of IL-10,IL-4 and Arg1 were significantly increased in a concentration-dependent manner.Conclusions Kaempferol can relieve calcium oxalate crystal-induced oxidative stress and inflammation in the kidney,and further suppress calcium oxalate crystal-induced renal injury,as well as renal calcium oxalate crystal formation.Part ? The effect of kaempferol on renal calcium oxalate crystal formation and renal oxidative and inflammatory injury via the AR/NOX2 signaling pathwayObjective Androgen receptor(AR)mediated pathways play an important role in the formative process of calcium oxalate nephrolithiasis.This study aimed to investigate the effect and mechanism of kaempferol in ameliorating renal calcium oxalate crystal formation,as well as crystal-associated renal oxidative and inflammatory injury by regulating the AR/NOX2 signaling pathway.Methods Over-expression of AR in the kidney was induced by subcutaneous injection of testosterone propionate in the mice experiments.In the meanwhile,the high expression of intracellular AR was induced by dihydrotestosterone(DHT)stimulation in the HK-2 cell experiments.The effects of kaempferol on AR expression in tissues and cells were further evaluated.Afterwards,the expression levels of AR and NOX2 were assessed by fluorescence staining,and the intracellular interaction between AR and NOX2,as well as the detailed mechanism of AR regulating NOX2 expression were evaluated by chromatin immunopr-ecipitation(Ch IP)and luciferase reporter assay in HK-2 cells after treatment with COM crystals and kaempferol.Finally,by a series of in vivo and in vitro experiments,the effects of kaempferol on calcium oxalate crystal adhesion and deposition,as well as crystal-induced renal injury,oxidative stress and inflammation by regulating the AR/NOX2 pathway were evaluated.Results First of all,the results of in vivo and in vitro experiments have confirmed that kaempferol functioned on the inhibition of AR expression in renal tubular epithelial cells.Moreover,the results of fluorescence staining revealed that intracellular expression of AR and NOX2 were synchronously increased in HK-2 cells after the stimulation of COM crystals and DHT,while both were decreased after simultaneous treatment with kaempferol.Moreover,by followed Ch IP and luciferase reporter experiments,it was corroborated that AR could directly bind to the promoter regions within the upstream of NOX2 and then promote the transcription of NOX2.But kaempferol would reduce the transcriptional expression of NOX2 by the down-regulation of AR.In addition,the further results of in vivo and in vitro experiments verified that after the over-expression of AR,the amounts of calcium oxalate crystal adhesion and deposition,tissular or intracellular injury,the expression levels of OPN and CD44,the levels of oxidative stress and inflammation were significantly increased.Nevertheless,all the above effects were changed in the opposite way in a concentration-dependent fashion after the treatment with kaempferol.Conclusions Kaempferol is able to repress the expression of AR in renal tubular epithelial cells.Kaempferol can decrease calcium oxalate crystal-associated renal injury,as well as crystal deposition and formation by mitigating oxidative stress and inflammation in the kidney via the AR/NOX2 pathway.Kaempferol has potential value in the treatment and prevention of calcium oxalate nephrolithiasis. |
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