以HK-2腎小管細胞株探討葉酸與高葡萄糖或果糖對腎的影響

Abstract

糖尿病腎病變是糖尿病的嚴重併發症，也是導致末期腎臟病最主要原因。現 代化飲食易引起肥胖及葉酸營養不足，近年研究觀察到葉酸不足會增加慢性腎臟 病的風險，因此，葉酸與腎臟疾病的關係值得探討。已知高葡萄糖與果糖皆會誘 導腎細胞發炎與纖維化，進而導致腎損傷，而本研究室先前也觀察到葉酸缺乏會 加劇高豬油高果糖飲食小鼠的腎炎與纖維化，但高糖與葉酸對於腎細胞的影響尚 不清楚，故本研究欲使用近端腎小管上皮細胞株 HK-2 細胞，探討葉酸營養狀況 與高糖對腎細胞分泌促發炎和纖維化因子的影響。將 HK-2 細胞培養於不同濃度 葡萄糖或果糖，結果發現葡萄糖或果糖會增加 HK-2 細胞分泌 IL-6、MCP-1 與 TGF-β1。低濃度 LPS 與葡萄糖共同刺激增加 MCP-1，而高濃度 LPS 與葡萄糖或 果糖共同刺激則增加 IL-6 與 TGF-β1。瘦體素與葡萄糖共同刺激 HK-2 細胞增加 IL-6、MCP-1 與 TGF-β1，瘦體素與果糖共同刺激則增加 IL-6。以葡萄糖或果糖刺 激 HK-2 細胞皆導致 p53 基因表現量顯著增加，表示葡萄糖或果糖會增加細胞凋亡 基因表現。此外，以不同葉酸濃度 (f0、f1 與 f5) 培養 HK-2 細胞，發現 f0 顯著降 低 IL-6，但增加 MCP-1 與 TGF-β1。不同葉酸與高糖培養下，f0 降低葡萄糖刺激 的 IL-6，但增加 MCP-1 與 TGF-β1，而 f5 則降低葡萄糖或果糖刺激所分泌的 MCP- 1。最後，不同葉酸濃度培養下，f0 顯著增加 kim-1、 p53 與 caspase-3 基因表現。 總結，葡萄糖與果糖加劇 LPS 或瘦體素刺激下腎細胞分泌促發炎與纖維化因子， 也會增加細胞凋亡基因表現。葉酸缺乏加劇腎細胞在高葡萄糖與果糖下 MCP-1 與 TGF-β1 的分泌，補充葉酸則降低 MCP-1 的分泌。缺葉酸也顯著增加腎損傷與細 胞凋亡相關基因，顯示葉酸缺乏可能為促進腎臟疾病的危險因子。

Diabetic kidney disease is the major cause of end-stage kidney disease. Modern diets are closely associated with the development of obesity and folate deficiency. Previous studies have shown that low folate levels showed a significant association with the higher risk of chronic kidney disease. However, whether folate deficiency and high sugar could result in renal inflammation and fibrosis is still unclear. The aim of this study was to investigate the effects of folate and sugar on the proinflammatory and fibrotic factors by using renal tube cell line (HK-2). HK-2 cells were cultured in different concentrations of glucose or fructose. The results demonstrated that glucose or fructose had a concentration- dependent effect on the secretion of IL-6, MCP-1 and TGF-β1. Co-stimulation of low concentrations of LPS with glucose increased MCP-1, while co-stimulation of high concentrations of LPS with glucose or fructose increased IL-6 and TGF-β1.Further, co- stimulation of leptin and glucose increased IL-6, MCP-1 and TGF-β1 in HK-2 cells, and co-stimulation of leptin and fructose increased IL-6. Stimulation of HK-2 cells with glucose or fructose resulted in a significant increase in p53 gene expression. In addition, HK-2 cells were cultured with different folate medium (f0, f1, and f5). f0 was found to significantly reduce IL-6, but increase MCP-1 and TGF-β1. Under different folate status and high sugar cultures, f0 decreased glucose-stimulated IL-6, but increased MCP-1 and TGF-β1, while f5 decreased glucose- or fructose-stimulated MCP-1 secretion. Finally, f0 significantly increased the expression of kim-1, p53 and caspase-3 genes. In conclusion, high sugar increased proinflammatory and fibrotic cytokines from HK-2 cells stimulated by LPS or leptin. Folate deficiency aggravates the secretion of MCP-1 and TGF-β1 from HK-2 cells under high sugar status, while supplementation of folate reduces MCP-1. Folate deficiency also increases genes related to renal injury and apoptosis, and thus, inadequate folate status might be one of the risk factors for kidney disease.