The Possible Mechanisms Of Effects Of NF-κB Inhibitor Parthenolide On Promoting Renal Tubules Albumin Uptake In Type 2 Diabetic Nephropathy

Background and objective:Diabetic nephropathy is one of the most severe microvascular complications of diabetes mellitus.The progress of diabetic nephropathy could be indirectly reflected by the amount of albuminuria.The increase of albuminuria could accelerate glomerulosclerosis,tubulointerstitial fibrosis and the progress of renal dysfunction.In addition,albuminuria is closely related to renal events,cardiovascular events and all-cause mortality in patients with diabetes mellitus.Recently,increased albuminuria caused by the reduction of albumin reabsorption in renal tubules is highlighted as the cause of early stage of diabetic nephropathy.The underlying mechanism has not been fully figured out.Current studies have shown that type 2 diabetes mellitus is featured by insulin resistance,which is mainly induced by chronic inflammation.Clinical studies suggest that urinary albumin is closely related to insulin resistance,but the mechanism has not been fully elucidated.In this study,we supposed that inflammatory damage to insulin signaling pathway promoted the increase of albuminuria.We aimed to observe and to explore whether reducing inflammation and remodeling insulin signaling could improve albumin endocytosis of renal tubules in diabetic nephropathy.Methods:(1)The diabetic nephropathy mice model was established and Parthenolide(PTN)was used in the treatment group.8-weeks db/m(C57BLKS/J-LepRdb/+)mice and db/db(C57BLKS/J-LepRdb/LepRdb)mice were used.Mice are divided into 3 groups:db/m group(db/m mice group,control group),db/db group(db/db mice group,DN group)and db/db+PTN group(db/db mice+PTN group,PTN treatment group).NF-κB inhibitor PTN(1 mg/kg,body weight)were used intraperitoneally every other day to the db/db mice and other mice were treated with the drug solution by the same way.After 8-weeks administration of drugs,16-weeks mice were sacrificed and blood,urine and kidney samples of mice were collected.Expressions of NF-κB p65,insulin signaling pathway protein p-AKT/AKT and albumin receptor complex amnionless-cubilin in the kidneys were tested.(2)HKC cells were cultured in vitro.HKC cells were challenged with insulin(100nM).The expressions of albumin receptors and albumin uptake in HKC cells were detected.The pathological state of type 2 dibetic nephropathy were mimicked in vitro.HKC cells were challenged with tumor necrosis factor-a(TNF-a 25ng/ml)and PTN(5μM).Albumin uptake and expressions of NF-κB p65,insulin signaling pathway proteins and albumin receptors of HKC cells were measured by fluorometric detection and immunoblotting.Results:(1)The levels of HOMA-IR and URCA in 16 weeks-old db/m mice were higher than those in db/m mice(P<0.05).Compared with db/db mice,db/db+PTN group demonstrated slightly decreased systemic insulin resistance index(homeostatic model assessment for insulin resistance,HOMA-IR)levels and reduced UACR levels,however the difference in HOMA-IR and UACR levels between db/db group and db/db+PTN group was not statistically significant(P>0.05).(2)Under PAS staining,the db/db mice kidney demonstrated the glomerular hypertrophy and the increase of mesangial matrix.PTN could partially alleviate the above pathological changes in kidneys of db/db mice.(3)Expression of NF-κB p65 is increased and phosphorylation of AKT(Ser473)is decreased in db/db mice compared with db/m mice(P<0.05).PTN significantly reduced expression of NF-κB p65 and ameliorate the decline of phosphorylation of AKT(Ser473)(P<0.05).Compared with db/m mice,the expression of amnionless and cubilin decreased in db/db mice(P<0.05),and PTN could partly reverse this change and significantly attenuate the reduction of cubilin expression(P<0.05).(4)Insulin promoted expressions of amnionless and cubilin and albumin uptake of HKC cells(P<0.05).In TNF-a+insulin group,expression of NF-κB p65 increased,and decreased phosphorylation of AKT(Ser473)and increased phosphorylation of insulin receptor substrate-1(Ser307)were also found in HKC cells compared with insulin group(P<0.05).In TNF-a+PTN group,PTN significantly reduced expression of NF-κB p65 and phosphorylation of AKT(Ser473)and promoted phosphorylation of I insulin receptor substrate-1(Ser307)of HKC cells(P<0.05).Expressions of amnionless and cubilin obviously decreased in TNF-a+insulin group(P<0.05).PTN could significantly attenuate the reduction of cubilin expression compared to TNF-a+insulin group(P<0.05).Conclusions:The study found that inflammation impaired insulin signaling and reduced expressiong of amnionless and cubilin in renal tubules,which caused reduction of albumin uptake and albuminuria.PTN could reduce inflammation,remodel impaired insulin signaling,and then increase albumin uptake of renal tubules by ameliorating expression of albumin receptors in diabetic nephropathy.