Discovery of polypodiside as a Keap1-dependent Nrf2 activator attenuating oxidative stress and accumulation of extracellular matrix in glomerular mesangial cells under high glucose

Abstract

Diabetic nephropathy (DN) is a severe microvascular complication of diabetes mellitus. High glucose has resulted in oxidative stress and following renal fibrosis as the crucial nodes of this disease. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor regulating transcription of many antioxidant genes and suppressing synthesis of extracellular matrix. To discover Nrf2 activators targeting DN, we have evaluated polypodiside using cell-based assays. The results showed polypodiside inhibited the high glucose-induced self-limited proliferation of glomerular meangial cells. Activation of Nrf2 and enhanced transcription to antioxidant response elements were observed in the presence of polypodiside. Oxidative stress and accumulation of extracellular matrix induced by high glucose in glomerular meangial cells have been ameliorated by polypodiside. Further investigations revealed the effects of polypodiside on glomerular meangial cells were associated with activation of Nrf2. Co-immunoprecipitation of Nrf2 disclosed polypodiside disrupted the Kelch-like ECH-associated protein-1 (Keap1)-Nrf2 interaction. Molecular docking elucidated polypodiside could enter the Nrf2 binding cavity of Keap1 via interacting with the residues encompassing that cavity. These findings indicate polypodiside is a Keap1-dependent Nrf2 activator affording the catabatic effects against oxidative stress and accumulation of extracellular matrix in glomerular meangial cells under high glucose.

Introduction

Diabetic nephropathy (DN) is one of severe microvascular complications of diabetes mellitus, which represents the leading cause of end stage renal disease.¹ In clinic, the typical character of DN is proteinuria followed by progressive injury of renal function.² Histopathological observation under microscope has found the glomerular basement membrane thickening and mesangial expansion in kidney of early DN patients, which are caused by mesangial cells proliferation and accumulation of excessive extracellular matrix.³ Without timely prevention, glomerular hypertrophy and glomerulosclerosis and renal fibrosis will occur, which will cause proteinuria and renal dysfunction.⁴ In clinical practice, anti-hyperglycemic and anti-hypertensive agents were employed to control the blood glucose and blood pressure of DN patients.⁵ Recently, sodium glucose co-transporter 2 (SGLT2) inhibitors such as canagliflozin, dapagliflozin, empagliflozin and ipragliflozin showed potential to attenuate progression of DN through blocking renal glucose reabsorption, and especially canagliflozin were approved by FDA for the treatment of DN in 2019.⁶ However, these agents are universal to complications of diabetes mellitus and indicate the uncertain adverse effects.6, 7 Up to date, though some other agents are effective to mitigate DN via diverse mechanisms, there are no drugs treating DN which directly target glomerulosclerosis and renal fibrosis in clinical application, and dialysis and transplantation are employed to improve renal failure.4, 8, 9 Therefore, discovery of novel drugs targeting DN is imperative.

The pathogenesis of DN has shown transforming growth factor-β₁ (TGF-β₁) is tightly linked to the development of DN, since it is the most potent promoter for extracellular matrix accumulation in all the three isoforms of TGF-β.⁸ Under high glucose, TGF-β₁ is produced in glomerular mesangial cells and regulates synthesis of extracellular matrix including collagen IV, fibronectin, and laminin.¹⁰ Meanwhile, high glucose has resulted in the overproduction of reactive oxygen species (ROS), which is the major effector for oxidative stress.¹¹ In addition to the lipid peroxidation as well as DNA and protein oxidation, ROS can induce the secretion of extracellular matrix through up-regulating the expression of TGF-β₁.¹²

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor regulating the expression of target genes including cytoprotective and defensive enzymes which protect cells against oxidative stress.¹³ In basal condition, Kelch-like ECH-associated protein-1 (Keap1) sequesters Nrf2 in cytosol and facilitates its degradation by 26S proteasome.¹⁴ However, in the presence of oxidants, electrophiles or activators, the Keap1-Nrf2 interaction is disrupted and Nrf2 will be dissociated from the complex and translocate into nucleus. And then Nrf2 binds to the antioxidant response elements (ARE) sequence in the promoter region of Nrf2-regulated genes to enhance the transcription of these genes.¹⁵ In the progression of DN, persistent high glucose has led to oxidative stress and fibrosis.¹⁶ Therefore, activating Nrf2 can give a promising approach for the therapy of DN.

There are six functional domains (Neh1 to Neh6) in the structure of Nrf2. And Keap1 has a BTB domain at its N-terminus, an IVR domain and a Kelch domain.¹⁷ Structural analysis of Keap1-Nrf2 system has shown two Keap1 monomers form a homodimer through respective BTB domains together with Cul3 and Rbx proteins, which promotes the ubiquitination of Nrf2 as an E3 ubiquitin ligase.¹⁸ At the same time, two Kelch domains of the homodimer interact with DLG and ETGE motifs in Neh2 domain of Nrf2 to make up the “hinge and latch” model due to the low affinity of DLG and high affinity of ETGE.¹⁹ Oxidants or electrophiles can modify cysteine residues in BTB and IVR domains of Keap1, which results in the dissociation of Kelch domain from DLG motif.²⁰ Therefore, targeting the Kelch domain of Keap1 directly will block the formation of Keap1-Nrf2 complex and lead to the activation of Nrf2.²¹

In the discovery of Nrf2 activators, natural products play a pivotal role.²² Many natural compounds with potential electrophilic moieties such as sulforaphane, curcumin, and resveratrol, have shown cytoprotective effects via activating Nrf2.²³ Polypodiside is a novel heterodimer of coumaric acid glucosides previously identified from the Chinese fern Polypodium hastatum.²⁴ This compound possesses similar electrophilic moieties in structure compared to above natural Nrf2 activators (Fig. 1), which inspired us to explore polypodiside as a natural Nrf2 activator and possible mechanisms using cell-base assays. Herein, we report polypodiside activates Nrf2 and attenuates oxidative stress and accumulation of extracellular matrix in glomerular mesangial cells under high glucose.