State Key Laboratory of Natural Medicines, Department of TCMs Pharmaceuticals, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 210009, China.
Division of Nephrology and Hypertension, School of Medicine, University of California Irvine, Irvine, CA, USA.
Acta Pharmacol Sin. 2022 Oct;43(10):2609-2623. doi: 10.1038/s41401-022-00898-3. Epub 2022 Mar 28.
Renal fibrosis is an unavoidable end result of all forms of progressive chronic kidney diseases (CKD). Discovery of efficacious drugs against renal fibrosis is in crucial need. In a preliminary study we found that a derivative of artemisinin, dihydroartemisinin (DHA), exerted strong renoprotection, and reversed renal fibrosis in adenine-induced CKD mouse model. In this study we investigated the anti-fibrotic mechanisms of DHA, particularly its specific target in renal cells. Renal fibrosis was induced in mice by unilateral ureteral obstruction (UUO) or oral administration of adenine (80 mg · kg), the mice received DHA (30 mg · kg · d, i.g.) for 14 or 21 days, respectively. We showed that DHA administration markedly attenuated the inflammation and fibrotic responses in the kidneys and significantly improved the renal function in both the renal fibrosis mouse models. In adenine-treated mice, DHA was more effective than 5-azacytidine against renal fibrosis. The anti-fibrotic effects of DHA were also observed in TGF-β1-treated HK-2 cells. In order to determine the target protein of DHA, we conducted pull-down technology coupled with shotgun proteomics using a small-molecule probe based on the structure of DHA (biotin-DHA). As a results, DNA methyltransferase 1 (DNMT1) was identified as the anti-fibrotic target of DHA in 3 different types of renal cell lines (HK-2, HEK293 and 3T3). We demonstrated that DHA directly bound to Asn 1529 and Thr 1528 of DNMT1 with a Kd value of 8.18 μM. In primary mouse renal tubular cells, we showed that DHA (10 μM) promoted DNMT1 degradation via the ubiquitin-proteasome pathway. DHA-reduced DNMT1 expression effectively reversed Klotho promoter hypermethylation, which led to the reversal of Klotho protein loss in the kidney of UUO mice. This subsequently resulted in inhibition of the Wnt/β-catenin and TGF-β/Smad signaling pathways and consequently conferred renoprotection in the animals. Knockdown of Klotho abolished the renoprotective effect of DHA in UUO mice. Our study reveals a novel pharmacological activity for DHA, i.e., renoprotection. DHA exhibits this effect by targeting DNMT1 to reverse Klotho repression. This study provides an evidence for the possible clinical application of DHA in the treatment of renal fibrosis.
肾纤维化是所有形式的进行性慢性肾脏病（CKD）不可避免的终末结果。迫切需要发现针对肾纤维化的有效药物。在一项初步研究中，我们发现青蒿素的一种衍生物，双氢青蒿素（DHA），具有很强的肾保护作用，并在腺嘌呤诱导的 CKD 小鼠模型中逆转了肾纤维化。在这项研究中，我们研究了 DHA 的抗纤维化机制，特别是其在肾细胞中的特定靶标。单侧输尿管梗阻（UUO）或口服腺嘌呤（80mg·kg）诱导小鼠肾纤维化，分别给予 DHA（30mg·kg·d，ig）14 或 21 天。结果表明，DHA 给药可显著减轻肾脏的炎症和纤维化反应，并显著改善两种肾纤维化小鼠模型的肾功能。在腺嘌呤处理的小鼠中，DHA 比 5-氮杂胞苷更有效对抗肾纤维化。DHA 的抗纤维化作用也在 TGF-β1 处理的 HK-2 细胞中观察到。为了确定 DHA 的靶蛋白，我们使用基于 DHA 结构的小分子探针（生物素-DHA）进行了下拉技术和鸟枪法蛋白质组学。结果，在 3 种不同类型的肾细胞系（HK-2、HEK293 和 3T3）中，鉴定出 DNA 甲基转移酶 1（DNMT1）是 DHA 的抗纤维化靶标。我们证明 DHA 与 DNMT1 的 Asn1529 和 Thr1528 直接结合，Kd 值为 8.18μM。在原代小鼠肾小管细胞中，我们表明 DHA（10μM）通过泛素-蛋白酶体途径促进 DNMT1 降解。DHA 降低 DNMT1 表达可有效逆转 Klotho 启动子过度甲基化，导致 UUO 小鼠肾脏中 Klotho 蛋白丢失的逆转。这继而抑制了 Wnt/β-catenin 和 TGF-β/Smad 信号通路，从而在动物中发挥了肾保护作用。Klotho 的敲低消除了 DHA 在 UUO 小鼠中的肾保护作用。我们的研究揭示了 DHA 的一种新的药理学活性，即肾保护作用。DHA 通过靶向 DNMT1 来逆转 Klotho 抑制，从而发挥这种作用。这项研究为 DHA 在治疗肾纤维化中的临床应用提供了证据。
