葉酸營養狀況對脂質代謝和慢性腎病變的影響之研究

全球肥胖和末期腎臟病的盛行率逐年攀升，肥胖致腎臟病變(obesity-related nephropathy)的發生率亦大幅增加。過多的內臟脂肪囤積是導致糖尿病、高血壓和高瘦素血症的主要原因，更是促進慢性發炎反應與慢性腎臟病的危險因子。隨著現代化飲食傾向高油脂、含糖飲料與少蔬果的攝食，易導致肥胖和葉酸攝取不足。已知，身體質量指數和內臟脂肪量與血液葉酸濃度呈負相關，暗示葉酸營養狀況與肥胖的發生具有關聯性。本研究的假說為葉酸營養狀況與腎功能有關，長期缺葉酸可能影響脂質代謝，促進慢性發炎反應和影響免疫調節功能，加劇慢性腎病變的病程。在氟化鈉誘導腎炎小鼠實驗中，使用12週齡的C57BL/6背景Vhlh+/+小鼠，分別以含葉酸(f1)或缺葉酸(f0)的正常油脂(normal-fat，NF-f0或NF-f1)飼料餵飼14週，於犧牲前4週，每天管餵生理食鹽水(對照組)或氟化鈉(誘導組)。結果顯示，葉酸缺乏與氟化鈉不影響小鼠的尿蛋白和血液尿素氮，但顯著增加血清肌酸酐、TGF-β1、IL-6和MCP-1，顯示葉酸缺乏加劇氟化鈉誘導的腎功能異常與慢性發炎反應。為探討葉酸營養狀況對脂質代謝的影響，6週齡的C57BL/6小鼠分別以f1或f0的高油(high-fat，HF-f1或HF-f0)、高油高果糖(high-fat high-fructose，HFF-f1或HFF-f0)、NF-f1或NF-f0飼料餵飼12個月後犧牲。結果顯示，攝食缺葉酸飼料顯著增加小鼠的脂肪組織重量、脂肪細胞大小、血清瘦素濃度和肝臟脂質含量，又以高油高果糖缺葉酸小鼠最為顯著，且增加體重、肝臟脂質合成相關基因的表現量、血清轉胺酶活性和非酒精性脂肪肝病變程度。續以3T3-L1脂肪細胞株驗證缺葉酸培養顯著增加脂質堆積、瘦素分泌量、脂質合成相關基因的表現量，如：Cebpα、Acc1、Fasn和Fabp4；也增加脂肪細胞在氯化鈷誘導缺氧反應下HIF-1α的蛋白質表現量，缺氧相關基因的表現量，如：Hif1α、Glut1和Il6，且測得培養液中葡萄糖殘餘量較低。顯示，葉酸缺乏促進脂肪細胞增大，進而增加瘦素分泌，其機制可能與缺葉酸促進脂肪細胞缺氧壓力有關。因脂肪細胞增大後可能分泌較多發炎性細胞激素，以不同葉酸含量的培養液培養3T3-L1前脂肪細胞和分化成熟的脂肪細胞來驗證。結果發現，缺葉酸培養增加沒有或有添加LPS刺激下的NF-κB轉錄活性，MCP-1和IL-6分泌量。另以缺葉酸的脂肪細胞條件培養液和肥胖小鼠血清、leptin、LPS或LPS+IFN-γ培養刺激RAW264.7巨噬細胞株或初代腹腔巨噬細胞，發現葉酸缺乏顯著增加NO、TNF-α、MCP-1和IL-6的分泌量，顯示缺葉酸增加巨噬細胞的促發炎活性。進一步分析初代脾臟細胞分泌細胞激素的能力，發現缺葉酸會顯著降低抗發炎的IL-10，顯示葉酸缺乏影響免疫調節功能。葉酸缺乏也影響MES-13腎小球間質細胞株與人類腎小管上皮細胞株在沒有或有添加LPS或LPS+leptin刺激下MCP-1、IL-6和TGF-β1的分泌量，回補葉酸則可降低細胞激素的生成。在飲食誘發肥胖致慢性腎病變小鼠實驗中，高油或高油高果糖飲食增加尿蛋白和尿液嗜中性白血球明膠酶相關運載蛋白，也增加腎臟中MCP-1、IL-6、TNF-α、TGF-β1和瘦素的含量，以及膠原蛋白沉積，進而促進慢性腎衰竭的程度。葉酸缺乏則進一步增加促發炎與促纖維化的細胞激素和瘦素，以及Hif1α基因表現；高油高果糖又缺葉酸更顯著增加腎纖維化訊息路徑分子STAT3和Smad2/3的磷酸化，從而加劇腎纖維化。最後，以MES-13細胞培養實驗證明缺葉酸可能藉由mTORc1/HIF-1α促進腎細胞的缺氧壓力，進而增加下游基因膠原蛋白Col1a1的表現。綜合上述，微量營養素葉酸不足影響脂質代謝與免疫調節功能並促進慢性發炎反應，進而加劇慢性腎病變。

關鍵字

葉酸缺乏；肥胖；瘦素；缺氧；慢性腎病變；腎纖維化

並列摘要

The global prevalence of obesity and end-stage renal disease is increasing simultaneously and rapidly by the year, and the incidence of obesity-related nephropathy (ORN) has also increased significantly. Excessive visceral fat accumulation is the main cause of diabetes, hypertension, and hyperleptinemia, and it is also a risk factor for chronic inflammation and chronic kidney disease. As modern diets tend to be high in fat, sugar-sweetened beverages, and low in fruits and vegetables, which results in obesity and inadequate folate intake. Body mass index and visceral fat mass are known to be inversely correlated with sera folate levels, implying a link between folate nutritional status and the development of ORN. Therefore, the hypothesis of this study is that folate insufficiency promotes ORN in mice by affecting lipid metabolism, inflammatory response, and immune regulation. To investigate the effects of folate nutritional status on renal function, twelve-week-old C57BL/6 background Vhlh+/+ mice were fed a normal-fat (NF) diet with folate (NF-f1) or without folate (NF-f0) for 14 weeks. Four weeks before sacrifice, the mice were oral-gavaged with PBS or sodium fluoride (NaF). As a result, folate deficiency and NaF did not affect the urine protein and blood urea nitrogen of mice, but significantly increased serum creatinine, TGF-β1, IL-6, and MCP-1 levels, showing that folate deficiency exacerbates NaF-induced kidney dysfunction and chronic inflammation. Then, six-week-old male C57BL/6 mice were fed with a diet with f1 or f0 in a high-fat (HF) diet (HF-f1 or HF-f0), or a high-fat high-fructose (HFF) diet (HFF-f1 or HFF-f0) for 12 months, compared with NF-f1 and NF-f0. The results showed that folate-deficient diet significantly increased white adipose tissue weight, adipocyte size, serum leptin level, and hepatic triglyceride (TG) in mice, among which the HFF-f0 mice were the most significant, and increased body weight, the expression hepatic lipogenesis-related genes, serum transaminase activity, and degree of non-alcoholic fatty liver disease. Increased intracellular TG, leptin secretion, and the expression of lipogenesis-related genes Cebpα, Acc1, Fasn, and Fabp4, higher HIF-1α protein and hypoxia-related genes Hif1α, Glut1, and Il6 expression, lower residual glucose in the culture medium also detected in folate-deficient 3T3-L1 adipocytes. It has been shown that folate deficiency promotes the hypertrophy of adipocytes, thereby increasing the secretion of leptin, and the mechanism may be related to the fact that folate deficiency promotes the hypoxic stress of adipocytes. Since adipocytes may secrete more inflammatory cytokines after hypertrophy, 3T3-L1 preadipocytes and differentiated mature adipocytes were cultured in a medium with different folic acid content to verify. It was found that folate-deficient culture increased NF-κB transcriptional activity, MCP-1 and IL-6 secretions without or with LPS stimulation. Then, stimulate the RAW264.7 macrophages or primary peritoneal macrophages by culturing folate-deficient adipocyte conditioned-media, obese mouse serum, leptin, LPS, or LPS+IFN-γ, and found that folate deficiency significantly increased nitric oxide, TNF-α, MCP-1, and IL-6 secretions, showing that folate deficiency increased the pro-inflammatory activity of macrophages. Further analysis of the ability of primary splenocytes to secrete cytokines, it is interesting that folate deficiency can significantly reduce anti-inflammatory IL-10, showing that folate status affects immune regulation. Folate deficiency also increased the secretion of MCP-1, IL-6, and TGF-β1 in glomerular mesangial cells (MES-13) and human renal tubular epithelial cells without or with LPS or LPS+leptin stimulation, after replenishment of folic acid showing reduction of cytokine productions. Finally, in the obesity-induced chronic nephropathy experiment, HFF diet increased urinary protein and neutrophil gelatinase-associated lipocalin excretions, renal MCP-1, IL-6, TNF-α, TGF-β1, and leptin contents, as well as collagen deposition, thereby promoting the chronic renal failure. Folate deficiency further increased pro-inflammatory and pro-fibrotic cytokines and leptin, as well as Hif1α gene expression. HFF diet together with folate deficiency significantly increased the phosphorylation of renal fibrosis signaling pathway molecules STAT3 and Smad2/3, thus aggravating renal fibrosis. Though the in vitro experiments proved that folate deficiency might promote the hypoxic stress of MES-13 cells through mTORc1/HIF-1α, thereby increasing the expression of the downstream gene collagen Col1a1. Based on the above, the insufficiency of micronutrient folic acid affecting lipid metabolism and immune regulation might further promote chronic inflammation, which in turn exacerbates chronic nephropathy.