Puerarin attenuates diabetic kidney injury through interaction with Guanidine nucleotide-binding protein Gi subunit alpha-1 (Gnai1) subunit

Radix puerariae, a traditional Chinese herbal medication, has been used to treat patients with diabetic kidney disease (DKD). Our previous studies demonstrated that puerarin, the active compound of radix puerariae, improves diabetic podocyte injury in type 1 DKD mice through attenuation of oxidative stress. However, the direct molecular target of puerarin and its underlined mechanisms in DKD remains unknown. In this study, we first confirmed that puerarin also improved DKD in type 2 diabetic mice (db/db). Through RNA-sequencing of isolated glomeruli, we found that differentially expressed genes (DEGs) that were altered in the glomeruli of these diabetic mice but reversed by puerarin treatment were involved mostly in oxidative stress, inflammatory, and fibrosis. Further analysis of these reversed DEGs revealed protein kinase A (PKA) was among the top pathways. By utilizing the drug affinity responsive target stability (DARTS) method combined with mass spectrometry analysis we identified guanine nucleotide-binding protein Gi alpha-1 (Gnai1) as the direct binding partner of puerarin. Gnai1 is an inhibitor of cAMP production which is known to have protection against podocyte injury. Searching Nephroseq datasets revealed that Gnai1 expression increased in the glomeruli of human DKD. In vitro, we showed that puerarin not only interacted with Gnai1 but also increased cAMP production in human podocytes and kidney cortex of mice treated with peurarin. Puerarin also enhanced CREB phosphorylation, a downstream transcription factor of cAMP/PKA. Overexpression of CREB reduced high glucose-induced podocyte apoptosis. We conclude that the renal protective effects of puerarin are likely through inhibiting Gnai1 to activate cAMP/PKA/CREB pathway in podocytes. Overall design: We used unbiased approach to further dissect the molecular mechanisms of puerarin in DKD by focusing on its direct binding partners. We were able to identify Gnai1 as a direct interacting molecule of puerarin in podocytes and our data suggest that puerarin may improve podocyte injury through inhibition of Gnai1 and activation of cAMP/PKA pathway.

葛根（Radix puerariae）作为一味传统中药，已被用于治疗糖尿病肾病（diabetic kidney disease, DKD）患者。本团队前期研究证实，葛根的活性成分葛根素（puerarin）可通过减轻氧化应激，改善1型糖尿病肾病小鼠的糖尿病足细胞损伤。然而，葛根素在糖尿病肾病中的直接分子靶点及其潜在机制仍未明确。本研究首先证实，葛根素同样可改善2型糖尿病db/db小鼠的糖尿病肾病表型。通过对分离的肾小球进行RNA测序（RNA-sequencing）分析，我们发现糖尿病小鼠肾小球中发生表达改变、且经葛根素治疗后可被逆转的差异表达基因（differentially expressed genes, DEGs），主要富集于氧化应激、炎症反应及纤维化相关通路。对这些被逆转的差异表达基因进行进一步分析后发现，蛋白激酶A（protein kinase A, PKA）通路位列核心通路之中。我们采用药物亲和力响应靶标稳定性（drug affinity responsive target stability, DARTS）技术结合质谱分析，鉴定出鸟苷酸结合蛋白Giα1（guanine nucleotide-binding protein Gi alpha-1, Gnai1）为葛根素的直接结合伴侣。Gnai1是环磷酸腺苷（cAMP）生成的抑制剂，已知其可对足细胞损伤发挥保护作用。检索Nephroseq数据集发现，人糖尿病肾病患者肾小球内Gnai1的表达水平显著升高。体外实验结果显示，葛根素不仅可与Gnai1发生相互作用，还可在人足细胞及葛根素干预的小鼠肾皮质组织中提升cAMP的生成水平。此外，葛根素可增强cAMP/PKA通路下游转录因子CREB的磷酸化水平。过表达CREB可减轻高糖诱导的足细胞凋亡。综上，我们认为葛根素的肾脏保护作用可能通过抑制Gnai1，从而激活足细胞内的cAMP/PKA/CREB通路得以实现。整体实验设计：本研究采用无偏倚研究策略，聚焦葛根素的直接结合靶点，以进一步解析其在糖尿病肾病中的分子机制。我们成功鉴定出Gnai1为葛根素在足细胞中的直接互作分子，实验数据表明葛根素或可通过抑制Gnai1、激活cAMP/PKA通路，改善糖尿病足细胞损伤。