Table_1_Ketogenic diet alleviates renal fibrosis in mice by enhancing fatty acid oxidation through the free fatty acid receptor 3 pathway.DOCX

IntroductionThe ketogenic diet (KD), as a dietary intervention, has gained importance in the treatment of solid organ structural remodeling, but its role in renal fibrosis has not been explored.
MethodsMale C57BL/6 mice were fed a normal diet or a KD for 6 weeks prior to unilateral ureteral obstruction (UUO), a well-established in vivo model of renal fibrosis in rodents. Seven days after UUO, serum and kidney samples were collected. Serum β-hydroxybutyrate (β-OHB) concentrations and renal fibrosis were assessed. NRK52E cells were treated with TGFβ1, a fibrosis-inducing cytokine, and with or without β-OHB, a ketone body metabolized by KD, to investigate the mechanism underlying renal fibrosis.
ResultsKD significantly enhanced serum β-OHB levels in mice. Histological analysis revealed that KD alleviated structural destruction and fibrosis in obstructed kidneys and reduced the expression of the fibrosis protein markers α-SMA, Col1a1, and Col3a1. Expression of the rate-limiting enzymes involved in fatty acid oxidation (FAO), Cpt1a and Acox1, significantly decreased after UUO and were upregulated by KD. However, the protective effect of KD was abolished by etomoxir (a Cpt1a inhibitor). Besides, our study observed that KD significantly suppressed UUO-induced macrophage infiltration and the expression of IL-6 in the obstructive kidneys. In NRK52E cells, fibrosis-related signaling was increased by TGFβ1 and reduced by β-OHB. β-OHB treatment restored the impaired expression of Cpt1a. The effect of β-OHB was blocked by siRNA targeting free fatty acid receptor 3 (FFAR3), suggesting that β-OHB might function through the FFAR3-dependent pathway.
DiscussionOur results highlight that KD attenuates UUO-induced renal fibrosis by enhancing FAO via the FFAR3-dependent pathway, which provides a promising dietary therapy for renal fibrosis.

**引言** 生酮饮食（ketogenic diet, KD）作为一种膳食干预手段，在实体器官结构重塑的治疗中已受到广泛重视，但其在肾纤维化中的作用尚未得到探索。

**方法** 本研究中，雄性C57BL/6小鼠在构建单侧输尿管梗阻（unilateral ureteral obstruction, UUO）——一种经典的啮齿类动物肾纤维化体内模型——前，分别喂食正常饲料或生酮饮食，持续6周。单侧输尿管梗阻造模7天后，采集小鼠血清与肾脏组织样本，检测血清β-羟基丁酸（β-hydroxybutyrate, β-OHB）浓度与肾纤维化程度。另外，用促纤维化细胞因子转化生长因子β1（transforming growth factor β1, TGFβ1）处理NRK52E细胞，并添加或不添加生酮饮食代谢产生的酮体β-羟基丁酸，以探究肾纤维化的潜在分子机制。

**结果** 生酮饮食可显著提升小鼠血清β-羟基丁酸水平。组织学分析显示，生酮饮食可减轻梗阻侧肾脏的结构破坏与纤维化程度，并降低纤维化标志物蛋白α-平滑肌肌动蛋白（α-SMA）、胶原蛋白1a1（Col1a1）及胶原蛋白3a1（Col3a1）的表达水平。单侧输尿管梗阻造模后，脂肪酸氧化（fatty acid oxidation, FAO）的限速酶Cpt1a与Acox1的表达显著下调，而生酮饮食可上调这两种酶的表达。然而，依托莫司（一种Cpt1a抑制剂）可抵消生酮饮食的保护作用。此外，本研究还观察到，生酮饮食可显著抑制单侧输尿管梗阻诱导的梗阻侧肾脏巨噬细胞浸润与白细胞介素6（IL-6）表达。在NRK52E细胞中，转化生长因子β1可激活纤维化相关信号通路，而β-羟基丁酸可逆转这一过程。β-羟基丁酸处理可恢复被抑制的Cpt1a表达，且该作用可被靶向游离脂肪酸受体3（free fatty acid receptor 3, FFAR3）的小干扰RNA（siRNA）阻断，提示β-羟基丁酸可能通过依赖游离脂肪酸受体3的通路发挥功能。

**讨论** 本研究结果表明，生酮饮食可通过依赖游离脂肪酸受体3的通路增强脂肪酸氧化，从而减轻单侧输尿管梗阻诱导的肾纤维化，为肾纤维化的膳食治疗提供了一种极具潜力的方案。