Study On Kidney Protection Of Nuciferine And Its Mechanisms In Hyperuricemia Of Animals

With the improvement of people's living standards as well as diet structure changes,incidence rate of hyperuricemia has increased gradually.A long-lasting process of hyperuricemia is one of the important risk factors for metabolic syndrome and kidney injury.Therefore,the study on the prevention and treatment of hyperuricemia and early renal damage is particularly important.Nuciferine is a major constituent of aromatic ring-containing alkaloids in lotus leaf.This compound shows insulin secretion promotion,anti-hyperlipidemia,anti-oxidation and anti-inflammation.Clinical study observes that He-ye Jiang-zhi decoction,mainly composed of lotus leaf,effectively reduces blood uric acid levels in the patients with metabolic syndrome and hyperuricemia,However,the hypouricemic and nephroprotective effects of nuciferine from lotus leaf and the possible molecular mechanisms are still not clear.Therefore,based on our previous results,nephroprotective action of nuciferine and its molecular mechanism were investigated in potassium oxonate-induced hyperuricemia of mice,fructose-induced metabolic syndrome of rats,as well as high uric acid or fructose-induced injury of human renal proximal tubule(HK-2)cells.Male Kunming mice were given 250 mg/kg potassium oxonate via intragastric administration,then followed by intragastric administration with 10,20 and 40 mg/kg nuciferine,as well as 5 mg/kg allopurinol(positive control)1 h later for seven consecutive days.Control group were given equal volume of saline.In potassium oxonate-induced hyperuricemic mice,nuciferine was confirmed to have hypouricemic action,anti-inflammation and renal function improvement,evidenced by reduction of serum uric acid,creatinine and blood urea nitrogen(BUN)levels,elevation of fraction of excretion of uric acid(FEUA),and alleviation of renal inflammatory pathology.Nuciferine down-regulated renal protein levels of urate transporter 1(URAT1)and glucose transporter 9(GLUT9)to reduce urate reabsorption,and up-regulated renal protein levels of ATP-binding cassette,subfamily G,membrane 2(ABCG2)and organic anion transporter 1(OAT1)to increase urate secretion,resulting in the enhancement of kidney uric acid excretion to reduce serum uric acid levels in hyperuricemic mice.These data suggest that nuciferine exhibit uricosuric action in hyperuricemia.Moreover,nuciferine was found to up-regulate renal protein levels of organic cation transporter 1(OCT1),and organic cation/carnitine transporters 1/2(OCTN1/2)in hyperuricemic mice,being consistent with its improvement of kidney function.Furthermore,renal protein levels of Toll-like receptor 4(TLR4)and myeloid differentiation factor 88(MyD88)were up-regulated in hyperuricemic mice,with the activation of nuclear factor-?B(NF-?B)pathway and NOD-like receptor pyrin domain containing 3(NLRP3)inflammasome(the increased protein levels of NLRP3,apoptosis-associated speck-like protein containing(ASC)and Caspase-1).The activated NLRP3 inflammasome promoted the cleavage and maturation of pro-interleukin-1? to excessively secret IL-1?,causing renal inflammation in this animal model.More importantly,nuciferine down-regulated renal protein levels of TLR4 and MyD88,and suppressed the activation of NF-?B pathway and NLRP3 inflammasome in hyperuricemic mice.Consistently,nuciferine-treated hyperuricemic mice showed reduction of serum and kidney IL-1? levels and alleviation of inflammatory pathology.Furthermore,we investigated the hypouricmic and nephroprotective effects of nuciferine and its mechanism in fructose-induced metabolic syndrome of rats.Male Sprague-Dawley rats had 10%fructose solution in drinking water for 6 weeks,and then were given nuciferine at 10,20 and 40 mg/kg,and allopurinol at 5 mg/kg(positive control)per day by intragastric administration for an additional 6 weeks.Control group was given equal volume of saline.High fructose-fed rats developed metabolic syndrome including hyperuricemia with renal dysfunction(elevation of serum uric acid,creatinine and BUN levels,and reduction of FEUA),insulin resistance,dyslipidemia and systemic inflammation.Fructose feeding up-regulated renal protein levels of GLUT9 and renal specific transporter(RST),down-regulated renal protein levels of ABCG2,OAT1,urate transporter(UAT),OCT1/2 and OCTN1 in rats.These results suggest that dysregulation of these renal organic ion transporters may contribute to fructose-induced hyperurecemia and renal dysfunction in rats.Treatment of nuciferine attenuated metabolic syndrome,and lowered serum uric acid,creatinine and BUN levels,resulting in FEUA elevation in fructose-fed rats.Nuciferine also reversed fructose-induced protein level alteration of these renal organic ion transporters in rats.Additionally,renal tubular epithelial cell injury,renal interstitial inflammatory cell infiltration,podocyte injury and proteinuria were observed in fructose-fed rats.High fructose feeding up-regulated TLR4 and MyD88 protein levels,inhibited superoxide dismutase(SOD)and catalase(CAT)activity,increased reactive oxygen species(ROS)production,causing oxidative stress in the kidney of rats Moreover,NF-?B pathway and NLRP3 inflammasome were activated to increase the secretion of IL-1? and inflammatory response in the kidney of fructose-fed rats Nuciferine restored fructose-induced these disturbances to alleviate renal inflammation in rats.Furthermore,excessive fructose feeding increased renal protein levels of intergrin-linked kinases(ILK),monocyte chemotactic protein-1(MCP-1),interleukin-6(IL-6),tumor necrosis factor-?(TNF-?)in rats.Nuciferine down-regulated renal protein levels of ILK,MCP-1,IL-6 and TNF-? to alleviate podocyte injury and albuminuria,and improve kidney pathological changes in fructose-induced metabolic syndrome of ratsTo further verify the above findings,we investigated the effects of nuciferine on high uric acid or fructose-induced inflammation and injury in HK-2 cells.HK-2 cells were incubated with 4 mg/dL uric acid or 5 mM fructose combined with 5-40 ?M nuciferine for 24 h,respectively.First,4 mg/dL uric acid stimulation for 24 h remarkably increased IL-1? levels in culture supernatant,as well as protein levels of TLR4,MyD88,NF-?B,NLRP3,ASC and Caspase-1 in HK-2 cells,indicating that high uric acid stimulation induces inflammatory response.Nuciferine(10-40 ?M)down-regulated TLR4,MyD88,NF-?B,NLRP3,ASC and Caspase-1 protein levels,and reduced IL-1? secretion in this cell model,indicating that nuciferine may directly suppress the TLR4/MyD88/NF-?B pathway and NLRP3 inflammasome activation to inhibit inflammatory response in high uric acid-exposed HK-2 cells.Next,5 mM fructose stimulation for 6 h remarkably increased intracellular uric acid levels in HK-2 cells,accompanied by the elevated protein levels of URAT1 and GLUT9,and reduced protein levels of ABCG2,OAT1,UAT,OCT1/2 and OCTN1(24 h).Nuciferine(5-40 ?M)reversed high fructose-induced protein level alteration of these organic ion transporters in HK-2 cells.These data may further demonstrate hypouricemic action and renal function improvement of nuciferine by regulating renal protein levels of organic ion transporters in hyperuricemia of animals.Moreover,5 mM fructose stimulation for 24 h remarkably up-regulated TLR4,MyD88,NF-?B,NLRP3,ASC,Caspase-1,ILK and MCP-1 protein levels in HK-2 cells,as well as increased IL-1? levels in culture supernatant,which were restored by nuciferine(5-40?M),respectively.These results may further prove the mechanism that nuciferine may inhibite TLR4/MyD88/NF-?B pathway and NLRP3 inflammasome activation,reduce IL-1? secretion,regulate ILK and MCP-1 to improve renal inflammation and injury in hyperuricemia.In conclusion,nuciferine reduced serum uric acid levels and improved renal function by regulating renal protein levels of organic ion transporters in hyperuricemia of animals.Nuciferine was found to alleviate kidney inflammation by suppressing TLR4/MyD88/NF-?B pathway and NLRP3 inflammasome activation in hyperuricemia of animals.In addition,nuciferine improved fructose-induced metabolic syndrome,inhibited oxidative stress,and down-regulated ILK and MCP-1 expression to alleviate kidney podocyte injury and proteinuria in rats.These results provide the experimental evidence for nuciferine in the prevention and treatment of renal injury in hyperuricemia.