核因子κB抑制剂小白菊内酯改善2型糖尿病肾病肾小管白蛋白摄取

doi:10.24920/003573

摘要/Abstract

摘要：

目的在糖尿病肾病早期,尿白蛋白增多由肾小管重吸收功能受损所致,然而其机制尚未完全阐明。本研究旨在探究减轻炎症反应并改善胰岛素信号受损能否改善糖尿病肾病肾小管对白蛋白的摄取。
方法 8周龄雄性db/db小鼠（n=8）作为2型糖尿病肾病模型,给予核因子κB（NF-κB）抑制剂小白菊内酯（PTN）治疗,按照1 mg/kg（小鼠体重）隔日1次（连续8周）腹腔注射给药。同时给予同龄db/m组（n=5）及db/db组（n=8）小鼠等剂量的生理盐水溶解液,作为正常对照组和2型糖尿病肾病组。8周后处死小鼠,留取血和尿标本检测稳态模型胰岛素抵抗指数（HOMA-IR）和尿白蛋白肌酐比。留取肾脏组织,利用PAS染色分析肾脏组织病理,利用免疫组化和免疫印迹检测NF-κB p65、p-AKT、amnionless和cubilin表达水平,并利用免疫荧光检测肾小管摄取白蛋白情况。HKC细胞被分为胰岛素组（胰岛素干预）、TNF-α组（胰岛素和TNF-α干预）、TNF-α+PTN组（胰岛素、TNF-α和PTN干预）。对HKC细胞的白蛋白摄取水平和NF-κB p65,p-IRS-1/IRS-1,p-AKT/AKT,amnionless及cubilin表达水平进行检测。
结果与db/db组相比,db/db+PTN组HOMA-IR水平下降（36.83±14.09比31.07±28.05）,尿白蛋白肌酐比下降（190.3±7.3比143.0±97.6 mg/mmol）,但差异无统计学意义（P>0.05）。PAS染色显示PTN能够减轻db/db小鼠肾小球肥大,减少系膜区基质。与db/m组相比,db/db组小鼠肾脏NF-κB p65表达增加,p-AKT（s473）水平减低（P<0.05）。PTN能够显著降低db/db组NF-κB p65表达,改善p-AKT（s473）下降水平（P<0.05）。与db/m组相比,db/db小鼠肾脏amnionless和cubilin的表达水平降低（P<0.05）,肾小管白蛋白摄取减少,而PTN能够显著改善db/db小鼠cubilin的降低（P<0.05）,改善肾小管白蛋白摄取。胰岛素可促进HKC细胞amnionless和cubilin表达增多,促进白蛋白摄取增加（P<0.05）。TNF-α刺激HKC细胞NF-κB p65表达增加,p-IRS-1（s307）水平增高及p-AKT（s473）水平减低（P<0.05）。与TNF-α组相比,TNF-α+PTN组HKC细胞NF-κB p65表达减低,并逆转p-IRS-1（s307）和p-AKT（s473）的表达变化（P<0.05）。TNF-α减少HKC细胞amnionless和cubilin的表达水平（P<0.05）,而PTN显著改善cubilin的降低（P<0.05）。
结论炎症损伤胰岛素信号通路,进而减少amnionless-cubilin表达和白蛋白摄取。PTN能够减轻炎症反应,修复胰岛素信号通路,从而改善cubilin表达和白蛋白摄取。本研究阐述了炎症在2型糖尿病肾病中降低肾小管摄取白蛋白的作用。

关键词: 炎症, 胰岛素抵抗, 白蛋白尿, 糖尿病肾病, 小白菊内酯

Abstract:

Objective Injured tubular reabsorption is highlighted as one of the causes of increased albuminuria in the early stage of diabetic nephropathy; however, the underlying mechanism has not been fully elucidated. In this study, we aimed to explore whether reducing inflammation and remodeling the insulin signaling pathway could improve albumin uptake of renal tubules.
Methods 8-week-old male db/db mice (n=8), a type 2 diabetic nephropathy model, administered with nuclear factor kappa-B (NF-κB) inhibitor parthenolide (PTN, 1 mg/kg) intraperitoneally every other day for 8 weeks, were as the treatment group. Meanwhile, the age-matched male db/m mice (n=5) and db/db mice (n=8) were treated with saline as the control group and type 2 diabetic nephropathy group. When the mice were sacrificed, blood and urine were collected to examine homeostasis model assessment of insulin resistance (HOMA-IR) and urine albumin creatinine ratio, and kidney samples were used to analyze histopathologic changes with periodic acid-Schiff (PAS) staining, NF-κB p65, phosphorylation of AKT (p-AKT), amnionless and cubilin expressions with immunohistochemistry as well as western blot, and the albumin uptake of renal tubules by using immunofluorescence. In addition, HKC cells were divided into the insulin group treated with insulin alone, the TNF-α group treated with insulin and tumor necrosis factor (TNF-α), and the TNF-α+PTN group exposed to PTN, insulin and TNF-α. The levels of albumin uptake and expression levels of NF-κB p65, p-IRS-1/IRS-1, p-AKT/AKT, amnionless and cubilin in HKC cells were measured.
Results Compared with the db/db group, the db/db+PTN group demonstrated decreased levels of HOMA-IR (36.83±14.09 vs. 31.07±28.05) and urine albumin creatinine ratio (190.3±7.3 vs. 143.0±97.6 mg/mmol); however, the differences were not statistically significant (P>0.05). Periodic acid-Schiff staining showed PTN could alleviate the glomerular hypertrophy and reduce the matrix in mesangial areas of db/db mice. The renal expression of NF-κB p65 was increased and p-AKT (s473) decreased in the db/db group compared with the db/m group (P<0.05). PTN significantly reduced the renal expression of NF-κB p65 and ameliorated the decline of p-AKT (s473) compared with the db/db group (P<0.05). Compared with the db/m group, the expression of amnionless and cubilin decreased and albumin uptake in tubules were reduced in the db/db group (P<0.05), and PTN could significantly increase the expression of cubilin (P<0.05), and improve albumin uptake in tubules. Insulin promoted albumin uptake and the expression of amnionless and cubilin in HKC cells (P<0.05). TNF-α stimulated the expression of NF-κB p65, increased p-IRS-1 (s307) and reduced p-AKT (s473) in HKC cells (P<0.05). In the TNF-α+PTN group, the expression of NF-κB p65 declined and p-IRS-1 (s307) and p-AKT (s473) were restored, compared with the TNF-α group (P<0.05). The expression of amnionless and cubilin decreased in the TNF-α group (P<0.05), and PTN could significantly increase the expression of cubilin (P<0.05).
Conclusions Inflammation caused damage to insulin signaling, which reduced amnionless-cubilin expression and albumin uptake. PTN could reduce inflammation and remodel the impaired insulin signaling pathway, which promoted the expression of cubilin and albumin uptake. Our study can shed light on the role of inflammation in the reduction of albumin uptake of renal tubules in type 2 diabetic nephropathy.

Key words: inflammation, insulin resistance, albuminuria, diabetic nephropathy, parthenolide

Hao Qiufa, Wang Baobao, Zhang Wei, Qiu Wei, Liu Qianling, Li Xuemei. NF-κB Inhibitor Parthenolide Promotes Renal Tubules Albumin Uptake in Type 2 Diabetic Nephropathy[J].Chinese Medical Sciences Journal, 2020, 35(1): 31-42.

图/表 6

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Groups	n	Blood glucose (mmol/L)	Serum insulin (mIU/L)	HOMA-IR	Serum creatinine (μmol/L)	UACR (mg/mmol)
db/m	5	10.01±1.73	14.93±4.00	6.60±1.70	9.56±0.98	19.5±14.5
db/db	8	41.19±7.31^*	21.02±9.95	36.83±14.09^*	7.20±2.82	190.3±7.3^*
db/db+PTN	8	41.00±14.78	21.30±7.30	31.07±28.05	7.56±2.42	143.0±97.6
F value		17.323	0.277	3.881	1.655	2.419
P value		<0.001	0.761	0.042	0.222	0.121

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