葉酸與果糖對腺嘌呤誘發腎損傷小鼠免疫反應的影響

國人末期腎臟病的盛行率是世界第一，且罹患率逐年上升。現代生活型態的外食比例高，傾向高油脂、含糖飲料與少蔬果的攝食習慣，易導致肥胖與葉酸攝取不足。本研究室先前研究指出，葉酸缺乏會促進高油高果糖飲食小鼠的發炎介質分泌，進而加劇慢性腎病變。因此，本研究欲探討葉酸不足與果糖對腺嘌呤誘發腎損傷小鼠的免疫反應之影響。實驗一以 7 週齡 C57BL/6 小鼠分 4 組：正常飼料組 (Ctrl)、高果糖組 (Hfru(ade))、高果糖+腺嘌呤組(Hfru+ade)、高果糖葉酸不足+腺嘌呤組 (Hfru-f+ade)。22 週齡時，以 0.1% (w/w) 腺嘌呤加入飼料誘發小鼠腎損傷共 22 週。44 週齡時犧牲小鼠，分析相關指標。結果顯示，腺嘌呤誘發組 (+ade) 的免疫細胞浸潤、腎纖維化面積、腎臟 MCP-1 和TGF-β 的含量，均顯著較 Ctrl 組高，確認腺嘌呤誘發腎損傷和腎炎。Hfru(ade) 組比 Ctrl 組腎臟有顯著較高的 MCP-1 和 TGF-β、顯著較低的 IL-10，顯示高果糖可能不利於腎發炎的調控。Hfru-f+ade 組脾臟細胞 TNF-α、IL-6、IL-2 的分泌量顯著高於 Hfru+ade 組。此外，脾臟 IL-2 與腎臟 MCP-1、TGF-β 呈正相關，腎臟 MCP-1 與 TGF-β亦呈正相關，可能葉酸不足使 T 細胞出現代償性反應，促進脾臟 IL-2 分泌，增加腎臟促發炎 MCP-1 分泌並促進具抗發炎功能的 TGF-β 分泌，然而，過多的 TGF-β 則促進腎臟走向纖維化。實驗二以 8 週齡 C57BL/6 小鼠分 6 組： Ctrl、Hfru、Ctrl +ade、Hfru+ade、高果糖低葉酸+腺嘌呤組 (Hfru-f+ade)、高果糖葉酸補充+腺嘌呤組 (Hfru+f10+ade)。18 週齡時，以 0.1~0.2% (w/w) 腺嘌呤誘發小鼠腎損傷共 7 週，於 25 週齡時犧牲小鼠，分析相關指標。結果顯示，腺嘌呤誘發組 (+ade) 的免疫細胞浸潤、腎纖維化面積、腎臟 MCP-1 和 TGF-β 的含量，均顯著較 Ctrl 組高，確認腺嘌呤誘發腎損傷和腎炎。Hfru 組與 Ctrl 組的腎臟和脾臟細胞激素分泌量均無顯著差異，可能是餵予高果糖飲食的時間不夠久所導致。Ctrl+ade 組比 Ctrl 組腎臟有顯著較高的 MCP-1、IL-6 和 TGF-β、顯著較低的IL-1β 和 IL-10，顯示腺嘌呤會促進腎臟發炎，降低抗發炎能力，導致腎臟纖維化。Hfru-f+ade 組脾臟重量和細胞數目為組間最低，TNF-α、IL-6、IL-10、IFN-γ 和 IL-2分泌量均低於 Hfru+ade 組，顯示較高劑量的腺嘌呤可能會降低攝取低葉酸飲食小鼠的免疫反應。Hfru+f10+ade 組比 Hfru+ade 組腎臟有顯著較低MCP-1、趨勢性較低 TNF-α、趨勢性較高 IL-6和IL-10，顯示葉酸補充有利於抗發炎細胞激素的分泌和調控發炎反應。綜合上述結果，葉酸不足可能使體內處於較高的發炎傾向，在誘發腎損傷下更易引起腎臟發炎和纖維化，加劇小鼠腎損傷。

關鍵字

葉酸；果糖；腺嘌呤；腎損傷；免疫反應

並列摘要

Taiwan has the highest global prevalence of end-stage renal disease (ESRD), with an increasing incidence annually. Western diet and sweetened beverages may result in obesity and inadequate folate intake. Previous studies have demonstrated that folate deficiency promotes the secretion of inflammatory mediators and exacerbates chronic nephropathy in high-fat high-fructose diet-fed mice. Therefore, this study aims to investigate the effects of fructose and folate on immune response in adenine-induced kidney injury murine model. In experiment 1, 7-week-old C57BL/6 mice were divided into four groups: AIN-93 diet (Ctrl), high-fructose diet (Hfru(ade)), Hfru with adenine (Hfru+ade), Hfru+ade with 0.2 mg/kg folate (Hfru-f+ade). Mice were treated with 0.1% (w/w) adenine on 22-week-old to induce kidney injury, and were sacrificed on 44-week-old. Adenine-induced groups significantly increased immune cell infiltration, renal fibrosis area, the secretion of renal MCP-1 and TGF-β compared to Ctrl, confirming adenine-induced kidney injury and nephritis. Hfru(ade) group showed significantly increased secretion of renal MCP-1 and TGF-β, but decreased IL-10 level, indicating high fructose is detrimental to renal inflammation regulation. Hfru-f+ade group increased secretion of TNF-α, IL-6, and IL-2 in splenocytes compared to Hfru+ade. In addition, splenic IL-2 level was positively correlated with renal MCP-1 and TGF-β, while renal TGF-β level was positively correlated with MCP-1. It shows that folate insufficiency triggers a compensatory T cell response, resulting in secretion of IL-2 in the spleen and pro-inflammatory MCP-1 in the kidney, and promoting the secretion of TGF-β. However, excessive TGF-β can lead to renal fibrosis. In experiment 2, 8-week-old C57BL/6 mice were divided into six groups: Ctrl, Hfru, Ctrl+ade, Hfru+ade, Hfru+ade with 0.2 mg/kg folate (Hfru-f+ade), Hfru+ade with 20 mg/kg folate (Hfru+f10+ade). Mice were treated with 0.1%-0.2% (w/w) adenine on 18-week-old and were sacrificed on 25-week-old. Adenine-induced groups significantly increased immune cell infiltration, renal fibrosis area, the secretion of renal MCP-1 and TGF-β compared to Ctrl, confirming adenine-induced kidney injury and nephritis. The Hfru group did not significantly differ from Ctrl, possibly due to insufficient duration. Ctrl+ade group showed increased secretion of renal MCP-1, IL-6 and TGF-β, but decreased IL-1β and IL-10 levels to Ctrl group, indicating that adenine can promote kidney inflammation and reduce anti-inflammatory ability, further leading to renal fibrosis. The spleen weight and cell number of the mice in Hfru-f+ade group were the lowest among the groups, and the secretion of TNF-α, IL-6, IL-10, IFN-γ and IL-2 were all lower than those in the Hfru+ade group, showing higher doses of adenine may reduce immune responses in mice fed a low-folate diet. Compared with Hfru+ade group, Hfru+f10+ade group had lower MCP-1, trending lower TNF-α, and higher IL-6 and IL-10, indicating folate supplement stimulates anti-inflammatory cytokine secretion to regulate inflammatory responses. Based on the above results, folate insufficiency may predispose to a pro-inflammatory state, and enhance renal inflammation and fibrosis during kidney injury, thereby worsening kidney damage in mice.