Supplementary Material for: IL-37 Ameliorates Renal Fibrosis by Restoring CPT1A Mediated Fatty Acid Oxidation in Diabetic Kidney Disease

Introduction: Diabetic kidney disease (DKD) is a major source of chronic kidney disease (CKD) and end-stage renal disease (ESRD). The injury of glomerular in DKD is the primary focus, however proximal tubulopathy also is an indispensable factor in the progression of DKD. Interleukin-37 (IL-37), an anti-inflammatory cytokine of IL-1 family member, has been demonstrated to be associated with diabetes and its relative complications in recent years, but the effect of IL-37 on renal fibrosis in DKD is unclear. Methods: We established streptozotocin plus high fat diet (STZ/HFD)-induced DKD mice model with wild type (WT) or IL-37 transgenic (IL-37tg) mice. Masson and HE staining, immunostaining, and Western blot were used to observe renal fibrosis. In addition, RNA-sequencing was applied to explore the potential mechanisms of IL-37. In vitro, treatment of human proximal tubular (HK-2) cells with 30 mmol/L high glucose or 300 ng/ml recombinant IL-37 further elucidated the possible mechanism of IL-37 inhibition of DKD renal fibrosis. Results: In this work, we first verified the decreased expression of IL-37 in kidney of DKD patient and its correlation with clinical features of renal impairment. Moreover, IL-37 expression markedly attenuated proteinuria and renal fibrosis in DKD mice. Using RNA-sequencing, we found and confirmed a novel role of IL-37 in ameliorating fatty-acid oxidation (FAO) reduction of renal tubular epithelial cells both in vivo and in intro. In addition, further mechanistic studies showed that IL-37 alleviated the FAO reduction in HK-2 cells and renal fibrosis in DKD mice through upregulating carnitine palmitoyl-transferase 1A (CPT1A), an important catalyzer for FAO pathway. Conclusion: These data suggest that IL-37 attenuates renal fibrosis via regulating FAO in renal epithelial cells. Upregulation of IL-37 levels might be an effective therapeutic avenue for DKD.

引言：糖尿病肾病（Diabetic kidney disease, DKD）是慢性肾脏病（chronic kidney disease, CKD）与终末期肾病（end-stage renal disease, ESRD）的主要诱因。DKD中肾小球损伤曾是研究的核心关注点，但近端肾小管病变同样是DKD进展中不可或缺的关键环节。白细胞介素-37（Interleukin-37, IL-37）作为IL-1家族的抗炎细胞因子，近年来被证实与糖尿病及其相关并发症密切相关，但IL-37对DKD肾纤维化的调控作用仍有待阐明。方法：本研究以野生型（wild type, WT）及IL-37转基因（IL-37 transgenic, IL-37tg）小鼠为模型对象，采用链脲佐菌素联合高脂饮食（streptozotocin plus high fat diet, STZ/HFD）构建DKD小鼠模型。通过Masson染色、苏木精-伊红（HE）染色、免疫染色及蛋白质印迹（Western blot）检测肾纤维化程度；此外，借助RNA测序（RNA-sequencing）技术探究IL-37发挥调控作用的潜在分子机制。体外实验中，以30 mmol/L高糖或300 ng/ml重组IL-37处理人近端肾小管上皮HK-2细胞，进一步明确IL-37抑制DKD肾纤维化的潜在机制。结果：本研究首先证实，DKD患者肾脏组织中IL-37的表达水平显著降低，且其表达与肾功能损害的临床特征密切相关。此外，IL-37可显著减轻DKD小鼠的蛋白尿症状与肾纤维化程度。通过RNA测序分析，我们发现并证实了IL-37在体内（in vivo）及体外（in vitro）均可改善肾小管上皮细胞的脂肪酸氧化（fatty-acid oxidation, FAO）功能损伤这一新的生物学作用。进一步的机制研究显示，IL-37可通过上调脂肪酸氧化通路的关键催化酶肉碱棕榈酰转移酶1A（carnitine palmitoyl-transferase 1A, CPT1A），缓解HK-2细胞的FAO功能损伤，并改善DKD小鼠的肾纤维化。结论：上述研究结果表明，IL-37可通过调控肾上皮细胞的脂肪酸氧化通路减轻肾纤维化；上调IL-37的表达水平可能成为DKD的有效治疗策略。