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 podocyte injury in type 1 DKD mice. However, the direct molecular target of puerarin and its underlying mechanisms in DKD remain unknown. In this study, we confirmed that puerarin also improved DKD in type 2 diabetic db/db mice. Through RNA-sequencing odf 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 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. In vitro, we showed that puerarin not only interacted with Gnai1 but also increased cAMP production in human podocytes and mouse diabetic kidney in vivo. Puerarin also enhanced CREB phosphorylation, a downstream transcription factor of cAMP/PKA. Overexpression of CREB reduced high glucose-induced podocyte apoptosis. Inhibition of PKA by Rp-cAMP also diminished the effects of puerarin on 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.