Nocturnin在糖尿病肾病近端肾小管损伤中的作用及机制研究

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease, but its pathogenesis is not yet fully understood. Renal tubular injury plays an important role in the development of DN. Our preliminary data found that the expression of Nocturnin was increased in renal proximal tubules of diabetic patients and mice, knocking down of Nocturnin could protect proximal tubular cells from damage caused by high glucose and palmitic acid. In high glucose and palmitic acid treated proximal tubular cells, a series of key transcription factors and enzymes involved in fatty acid β oxidation were decreased. As Nocturnin was reported being able to regulate lipid metabolism, we speculate that Nocturnin may disturb fatty acid β oxidation in diabetic renal proximal tubule，thus participating the development of DN. We planned to investigate the changes in the rhythm of Noturnin expression i n renal tubules of DN，further comfirm the role of Nocturnin in the development of DN and its association with fatty acid β oxidation using the established proximal tubule specific Nocturnin KO mice.This study will give new theories to pathogenesis of DN. The result may provide a possible new target for the prevention and treatment of DN.

糖尿病肾病（DN）是导致终末期肾病的主要疾病之一，其发病机制并不完全明了。肾小管损伤是DN发生发展的重要病理基础。我们的前期研究发现：碳分解代谢物阻遏4样蛋白Nocturnin在糖尿病患者及小鼠的肾脏近端小管显著高表达；在近端肾小管上皮细胞（HK2）中抑制Nocturnin能抵抗高糖、高脂引起的细胞损伤。高糖高脂干预的HK2细胞中，调控脂肪酸β氧化的多个关键转录因子和酶低表达；而既往研究发现Nocturnin调控脂代谢相关蛋白。因此，我们推测Nocturnin可能导致近端肾小管脂肪酸代谢障碍，参与糖尿病肾病发生发展。我们将研究糖尿病肾病肾小管上皮细胞中Noturnin的表达节律变化，利用自主建立的近端肾小管特异性Nocturnin敲除小鼠模型明确Nocturnin是否通过影响肾小管上皮细胞脂肪酸β氧化参与DN发生发展。本研究将为DN发生机制提供新的理论基础，有望为DN防治提供新的靶点。

糖尿病肾脏病（Diabetic kidney disease，DKD）是糖尿病主要的微血管并发症，已成为全球终末期肾病的主要病因。然而其发病机制尚不十分清楚。近端肾小管损伤是DKD发生发展的重要病理生理机制。我们利用Cre-loxp系统成功构建了近端肾小管特异性Nocturnin敲除小鼠模型。利用STZ诱导糖尿病小鼠模型并饲养12周，分为四组：野生型非糖尿病组（WT-NDM），野生型糖尿病组（WT-DM），近端肾小管Nocturnin敲除非糖尿病组（KO-NDM）及敲除糖尿病组（KO-DM）。与WT-NDM小鼠相比，WT-DM小鼠血清肌酐、尿白蛋白/肌酐（UAlb/Cr）、尿NGAL/Cr显著升高；WT-DM小鼠肾脏KIM-1表达显著升高，电镜显示肾小球基底膜增厚，足突融合明显，近端肾小管线粒体明显肿胀，肾脏炎症、纤维化、上皮-间质转分化和氧化应激明显加重。与WT-DM组相比，KO-DM小鼠血清肌酐、UAlb/Cr、尿NGAL/Cr水平显著降低；KO-DM组肾脏KIM-1表达显著降低，肾脏超微结构明显改善，炎症、纤维化、上皮-间质转分化和氧化应激减轻。我们进一步研究了Nocturnin敲除减轻糖尿病肾脏病变的机制，我们发现，与WT-NDM组相比，WT-DM小鼠肾脏NADPH水平降低，SGLT2表达明显升高；而在KO-DM小鼠肾脏中NADPH水平升高，SGLT2表达明显降低。体外培养人近端肾小管上皮（HK-2）细胞，在高糖条件下抑制Nocturnin，HK-2细胞炎症、纤维化和氧化应激指标显著改善，SGLT2表达显著降低，NADPH水平升高，NADH/NADPH降低；NADPH合成关键酶ME1的mRNA水平显著升高。综上所述，糖尿病状态下，Nocturnin可能通过上调SGLT2表达、降低NADPH合成及增加NADPH降解而诱导氧化应激失衡，导致近端肾小管损伤。本研究提示Nocturnin参与DKD的发生，有望为DKD的发生机制提供新的理论依据，并可能为DKD的治疗提供新的潜在药物靶点。