背景:糖尿病肾病(DKD)是糖尿病(DM)的严重并发症。有人提出,在DKD发作期间,近端小管上皮细胞(PTEC)功能的修饰先于肾小球损伤。本研究旨在确定DM早期肾钠处理的修饰及其分子机制。
方法:链脲佐菌素(STZ)诱导的糖尿病BALB/c小鼠(STZ组)和LLC-PK1细胞,PTEC的模型,被使用。在初始注射STZ后第4周评估所有参数。
结果:早期DKD以超滤和PTEC功能障碍为特征。STZ组由于肾小管钠重吸收受损,尿钠排泄增加。这与皮质(NaK)ATPase(NKA)α1亚基表达和酶活性的降低以及O-GlcNAci化的增加有关。对DKD患者的RNAseq分析显示谷氨酰胺-果糖转氨酶(GFAT)基因的表达增加,己糖胺生物合成途径的限速步骤,NKA表达减少。用10μMthiametG孵育LLC-PK1细胞,O-GlcNAcase的抑制剂,降低NKA的表达和活性并增加O-GlcNAcylation。此外,6-重氮-5-氧代-L-正亮氨酸(DON),GFAT抑制剂,或者dapagliflozin,SGLT2抑制剂,避免了HG对与O-GlcNAcylation降低相关的NKA表达和活性的抑制作用。
结论:我们的结果表明,肾小管钠重吸收的损害,在DM的早期阶段,是由于SGLT2介导的HG流入PTEC,O-GlcNAcylation增加,NKA表达和活性降低。
Diabetic kidney disease (DKD) is a severe complication of diabetes mellitus (DM). It has been proposed that modifications in the function of proximal tubule epithelial cells (PTECs) precede glomerular damage during the onset of DKD. This study aimed to identify modifications in renal sodium handling in the early stage of DM and its molecular mechanism.
Streptozotocin (STZ)-induced diabetic BALB/c mice (STZ group) and LLC-PK1 cells, a model of PTECs, were used. All parameters were assessed in the 4th week after an initial injection of STZ.
Early stage of DKD was characterized by hyperfiltration and PTEC dysfunction. STZ group exhibited increased urinary sodium excretion due to impairment of tubular sodium reabsorption. This was correlated to a decrease in cortical (Na++K+)ATPase (NKA) α1 subunit expression and enzyme activity and an increase in O-GlcNAcylation. RNAseq analysis of patients with DKD revealed an increase in expression of the glutamine-fructose aminotransferase (GFAT) gene, a rate-limiting step of hexosamine biosynthetic pathway, and a decrease in NKA expression. Incubation of LLC-PK1 cells with 10 μM thiamet G, an inhibitor of O-GlcNAcase, reduced the expression and activity of NKA and increased O-GlcNAcylation. Furthermore, 6-diazo-5-oxo-L-norleucine (DON), a GFAT inhibitor, or dapagliflozin, an SGLT2 inhibitor, avoided the inhibitory effect of HG on expression and activity of NKA associated with the decrease in O-GlcNAcylation.
Our results show that the impairment of tubular sodium reabsorption, in the early stage of DM, is due to SGLT2-mediated HG influx in PTECs, increase in O-GlcNAcylation and reduction in NKA expression and activity.