葉酸缺乏促進HK-2腎小管細胞株發炎介質分泌與代謝重整

Abstract

台灣末期腎臟病盛行率、洗腎人口高居世界之冠，其增幅亦名列前茅；又，國人葉酸邊緣缺乏盛行率漸增。本研究室已發表葉酸缺乏加劇高果糖高油誘發之小鼠腎損傷，以及補充 γ-aminobutyric acid (GABA) 延緩自發性腎炎小鼠之腎損傷之研究，故本研究欲探討葉酸、果糖和 GABA 飲食因子對腎小管細胞激素分泌及代謝之影響。研究方法為以人類腎小管上皮細胞株 HK-2 細胞在各濃度的葉酸、果糖和 GABA 以及有無 LPS 刺激、有無 LDH 抑制劑 oxamate 等不同條件下，分別測定促發炎細胞激素 IL-6、MCP-1、抗發炎/纖維化細胞激素 TGF-β1 分泌量、腎損傷相關基因 Vegf, Kim-1, Caspase-3 mRNA 表現量、乳酸生成和其生成酵素 lactate dehydrogenase (LDH) 活性，以及脂肪酸氧化酵素 carnitine-palmitoyltransferase 1 (CPT1A)、參與能量代謝與粒線體生合成之轉錄活化因子 PPARγ coactivator (PGC)-1α、纖維化指標 α-smooth muscle actin (α-SMA) 之表現量，並以海馬生物能量儀測定HK-2細胞在葉酸充足或缺乏時之耗氧率代表其粒線體功能。結果顯示，2 mM 果糖增加 HK-2 細胞 Vegf, Kim-1, Caspase-3 mRNA 表現量，5 mM 果糖促進其 IL-6、MCP-1 分泌；添加 GABA 則可減緩果糖處理或葉酸缺乏下之促發炎細胞激素分泌。葉酸缺乏的 HK-2 細胞內 ROS 累積、LDH 活性上升，造成胞內外乳酸含量上升。雖然 CPT1A 表現量上升，但粒線體基礎耗氧率下降，電子傳遞鏈去耦合且ATP產量下降。若以 oxamate 抑制葉酸缺乏 HK-2 細胞的乳酸生成，則使其 IL-6 分泌下降，而 MCP-1 分泌上升；反之，添加乳酸使 IL-6、TGF-β1 分泌量上升，但 MCP-1 分泌下降。由以上結果可知，葉酸缺乏的 HK-2 細胞粒線體 ATP 生成功能受損、乳酸生成增加，進而影響促發炎激素分泌；同時，葉酸缺乏亦加劇 LPS 刺激下的乳酸生成，顯示葉酸缺乏可能影響腎小管細胞的能量代謝傾向、促進乳酸生成，並增加促發炎細胞激素的分泌。

Taiwan has the highest prevalence of end-stage renal disease and hemodialysis globally, with a growing population suffered from folate insufficiency. Our previous studies have shown that folate deficiency worsened renal damage in mice fed with high-fructose, high-fat diet and that γ-aminobutyric acid (GABA) mitigated kidney injury in mice with spontaneous nephritis. Building on this, the present study aims to investigate how folate, fructose, and GABA might influence cytokine secretion and metabolism in renal tubular cells. HK-2 cells (human kidney proximal tubular epithelial cell line) were cultured with varying concentrations of folate, fructose, and GABA, with or without LPS stimulation, with or without oxamate (LDH inhibitor) for determination of IL-6, MCP-1 (pro-inflammatory cytokines), TGF-β1 (anti-inflammatory/profibrotic cytokine) secretion, Vegf, Kim-1, Caspase-3 (renal injury-related genes) expression, lactate production, and CPT1A (a key enzyme in fatty acid oxidation), PGC-1α (a key transcription factor in regulating mitochondrial biogenesis), α-SMA (a pro-fibrotic marker) expression, alongside oxygen consumption rate (mitochondrial function marker) using Seahorse Analyzer. Results showed that 2 mM fructose increased mRNA expression of Vegf, Kim-1, Caspase-3; 5 mM fructose increased IL-6, MCP-1 secretion, while GABA alleviated pro-inflammatory cytokine secretion induced by fructose or folate deficiency. Notably, folate deficiency led to ROS accumulation, elevated LDH activity, and lactate buildup. Folate deficient HK-2 cells showed decreased mitochondrial basal respiration, decoupling in ATP production, despite a rise in CPT1A expression. Inhibiting lactate production in folate-deficient HK-2 cells increased MCP-1 secretion, with suppressed IL-6 secretion; lactate addition increased IL-6 and TGF-β1 secretion, with reduced MCP-1 secretion. These findings suggest that folate deficiency impairs mitochondrial ATP synthesis, elevates lactate production, and exacerbates pro-inflammatory cytokine secretion and LPS-induced lactate production in HK-2 cells, highlighting its critical role in metabolic preference, lactate production, and proinflammatory cytokine secretion.