Department of Nephrology, Peking Union Medical College Hospital, Chinese Academy of Medicine Sciences & Peking Union Medical College, Beijing 100730, China.
Chin Med Sci J. 2020 Mar 31;35(1):31-42. doi: 10.24920/003573.
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 (=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 (=5) and db/db mice (=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 . 31.07±28.05) and urine albumin creatinine ratio (190.3±7.3 . 143.0±97.6 mg/mmol); however, the differences were not statistically significant (>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 (<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 (<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 (<0.05), and PTN could significantly increase the expression of cubilin (<0.05), and improve albumin uptake in tubules. Insulin promoted albumin uptake and the expression of amnionless and cubilin in HKC cells (<0.05). TNF-α stimulated the expression of NF-κB p65, increased p-IRS-1 (s307) and reduced p-AKT (s473) in HKC cells (<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 (<0.05). The expression of amnionless and cubilin decreased in the TNF-α group (<0.05), and PTN could significantly increase the expression of cubilin (<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.
目的 肾小管损伤性重吸收被认为是糖尿病肾病早期白蛋白尿增加的原因之一，但其中的机制尚未完全阐明。本研究旨在探讨减轻炎症和重塑胰岛素信号通路是否能改善肾小管对白蛋白的摄取。
方法 给予 8 周龄雄性 db/db 小鼠（=8），即 2 型糖尿病肾病模型，每隔一天腹腔内注射核因子 kappa-B（NF-κB）抑制剂角鲨烯（PTN，1mg/kg）8 周，作为治疗组。同时，给予年龄匹配的雄性 db/m 小鼠（=5）和 db/db 小鼠（=8）生理盐水作为对照组和 2 型糖尿病肾病组。处死小鼠后，收集血液和尿液检测稳态模型评估的胰岛素抵抗（HOMA-IR）和尿白蛋白肌酐比，并对肾组织进行病理分析，用过碘酸希夫（PAS）染色、NF-κB p65、磷酸化 AKT（p-AKT）、amnionless 和 cubilin 免疫组化和 Western blot 分析，以及用免疫荧光法分析肾小管对白蛋白的摄取。此外，将 HKC 细胞分为单独用胰岛素处理的胰岛素组、同时用胰岛素和肿瘤坏死因子（TNF-α）处理的 TNF-α 组以及用 TNF-α、胰岛素和角鲨烯处理的 TNF-α+PTN 组。测量 HKC 细胞中白蛋白摄取和 NF-κB p65、p-IRS-1/IRS-1、p-AKT/AKT、amnionless 和 cubilin 的表达水平。
结果 与 db/db 组相比，db/db+PTN 组的 HOMA-IR（36.83±14.09 vs. 31.07±28.05）和尿白蛋白肌酐比（190.3±7.3 vs. 143.0±97.6mg/mmol）水平降低，但差异无统计学意义（>0.05）。PAS 染色显示，PTN 可减轻肾小球肥大，减少系膜区基质。与 db/m 组相比，db/db 组 NF-κB p65 肾表达增加，p-AKT（s473）减少（<0.05）。与 db/db 组相比，PTN 可显著降低 NF-κB p65 肾表达，并改善 p-AKT（s473）下降（<0.05）。与 db/m 组相比，db/db 组 amnionless 和 cubilin 表达减少，肾小管对白蛋白的摄取减少（<0.05），PTN 可显著增加 cubilin 的表达（<0.05），并改善肾小管对白蛋白的摄取。胰岛素促进 HKC 细胞中 amnionless 和 cubilin 的表达和白蛋白摄取（<0.05）。TNF-α 刺激 NF-κB p65 的表达，增加 p-IRS-1（s307）并减少 p-AKT（s473）在 HKC 细胞中（<0.05）。在 TNF-α+PTN 组中，与 TNF-α 组相比，NF-κB p65 的表达下降，p-IRS-1（s307）和 p-AKT（s473）得到恢复（<0.05）。TNF-α 组 amnionless 和 cubilin 的表达减少（<0.05），PTN 可显著增加 cubilin 的表达（<0.05）。
结论 炎症导致胰岛素信号受损，降低了 amnionless-cubilin 的表达和白蛋白摄取。PTN 可减轻炎症并重塑受损的胰岛素信号通路，从而促进 cubilin 的表达和白蛋白摄取。我们的研究可以阐明炎症在 2 型糖尿病肾病中肾小管白蛋白摄取减少中的作用。
