Department of Nephrology, Faculty of Medicine, University of Thessaly, Biopolis, Mezourlo Hill, 41110, Larissa, Greece.
Int Urol Nephrol. 2020 Jun;52(6):1179-1189. doi: 10.1007/s11255-020-02481-3. Epub 2020 May 2.
Glucotoxicity in renal tubular epithelial cells (RPTECs) contributes to the pathogenesis of diabetic nephropathy. Sodium-glucose cotransporter 2 (SGLT2) inhibitors may exert their renoprotective effect by preventing glucotoxicity. We tested whether the confirmed in capillary endothelial cells unifying model of glucotoxicity can be applied in RPTECs and the impact of dapagliflozin.
In primary human RPTECs cultured in normal or high glucose medium in the presence or not of dapagliflozin, we assessed glucose consumption, SCLT2 expression, reactive oxygen species (ROS) production, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity, D-sorbitol and methylglyoxal cell content, O-linked β-N-acetyl glucosamine (O-Glc-NAc)-modified proteins, protein kinase C (PKC) activity, transforming growth factor-β1 (TGF-β1), interleukin-8 (IL-8), cell necrosis, and cell apoptosis using colorimetric and immunoenzymatic assays, and western blotting.
High glucose increases SGLT2 expression and glucose consumption. ROS are overproduced, and GAPDH is inhibited. The accumulation due to GAPDH inhibition glycolytic products are diverted into four noxious pathways. The polyol pathway assessed by D-sorbitol, the hexosamine pathway determined by O-GlcNAc-modified proteins, the lipid synthesis pathway assessed by PKC activity, and the advanced glycation end-products (AGEs) formation assessed by methylglyoxal. Eventually, these paths lead to overproduction of TGF-β1 and IL-8, as well as to cell necrosis and apoptosis. Dapagliflozin ameliorates all the above cascade of events.
Our results support a unifying model for glucotoxicity in RPTECs. Dapagliflozin by decreasing the elevated glucose influx into the RPTECs under high glucose conditions ameliorates glucotoxicity.
肾近端小管上皮细胞（RPTEC）中的糖毒性会导致糖尿病肾病的发病机制。钠-葡萄糖共转运蛋白 2（SGLT2）抑制剂通过防止糖毒性可能发挥其肾脏保护作用。我们测试了在毛细血管内皮细胞中证实的统一糖毒性模型是否可应用于 RPTEC 以及达格列净的影响。
在正常或高葡萄糖培养基中培养的原代人 RPTEC 中，存在或不存在达格列净的情况下，我们使用比色法和免疫酶法测定葡萄糖消耗，SCLT2 表达，活性氧（ROS）产生，甘油醛 3-磷酸脱氢酶（GAPDH）活性，D-山梨醇和甲基乙二醛细胞含量，O-连接的β-N-乙酰葡萄糖胺（O-Glc-NAc）修饰的蛋白质，蛋白激酶 C（PKC）活性，转化生长因子-β1（TGF-β1），白细胞介素-8（IL-8），细胞坏死和细胞凋亡，Western印迹。
高葡萄糖增加 SGLT2 的表达和葡萄糖消耗。ROS 过度产生，GAPDH 受到抑制。由于 GAPDH 抑制导致糖酵解产物的积累而转向四条有害途径。通过 D-山梨醇评估的多元醇途径，通过 O-GlcNAc 修饰的蛋白质确定的己糖胺途径，通过 PKC 活性评估的脂质合成途径以及通过甲基乙二醛评估的晚期糖基化终产物（AGEs）形成途径。最终，这些途径导致 TGF-β1 和 IL-8 的过度产生，以及细胞坏死和凋亡。达格列净改善了上述所有级联事件。
我们的结果支持了 RPTEC 中糖毒性的统一模型。达格列净通过降低高糖条件下进入 RPTEC 的升高的葡萄糖内流，改善了糖毒性。
