Icariin modulates mitochondrial function and apoptosis in high glucose-induced glomerular podocytes through G protein-coupled estrogen receptors

Highlights

• The inhibition of icariin on podocyte apoptosis is proposed.

• Icariin inhibits apoptosis by protecting the integrity of mitochondrial membranes in podocytes.

• Icariin increases Bcl-2 mitochondrial translocation via the GPER pathway in podocytes.

Abstract

Podocyte apoptosis in glomerular lesions has been found to have a dominant role in the progression of diabetic nephropathy. The present research aimed to explore the beneficial effect of icariin on diabetic podocytes by interfering in the process of apoptosis. Podocyte apoptosis was significantly exacerbated after high glucose treatment, with the level of reactive oxygen species (ROS) increasing simultaneously. Here, we demonstrated that icariin, which is a G protein-coupled estrogen receptor 1 (GPER) agonist, inhibited podocyte apoptosis by reducing ROS, maintaining the integrity of mitochondrial membranes. Moreover, the stabilization of mitochondria by icariin was reversed when GPER was knocked down in podocytes. Meanwhile, icariin inhibited the caspase cascade in podocyte apoptosis by promoting Bcl-2 expression and mitochondrial translocation. The above findings at least partly elucidated the mechanism by which icariin stabilized podocytes by inducing the mitochondrial Bcl-2 translocation and therefore preventing downstream apoptosis.

Introduction

Diabetic nephropathy (DN) is the leading cause of end-stage renal failure throughout the world (Ahmad, 2015). Glomeruli and their intrinsic cells undergo functional and morphological changes in the development of DN, of which podocytes are considered critical cells (Li et al., 2007). Reduction of podocytes mediated by apoptosis has been observed in patients at early and late stages of DN (Burlaka et al., 2016, Lu et al., 2016). Podocyte loss plays a central role in the initiation of glomerular lesions, triggering a sequence of events, such as glomerulosclerosis and tubule-interstitial fibrosis (Menini et al., 2007), which may compromise the integrity of the glomerular filtration barrier (Xu et al., 2016). Some drugs can delay the progression of DN by improving podocyte injuries, such as renin-angiotensin-aldosterone system inhibitors, statins, active vitamin D, corticosteroids, and phosphatase inhibitors (Blacklock et al., 2011).

The Bcl-2 family of proteins plays a central role in the intrinsic apoptotic mechanism (Gao et al., 2013). This family consists of both pro- (Bax, Bad, Bak, and Bid) and anti-apoptotic (Bcl-2 and Bcl-xl) proteins that modulate the execution phase of the cell death pathway (Mao et al., 2013). Studies have confirmed that Bcl-2 can inhibit apoptosis (Albanito et al., 2007, Lin et al., 2009). The G protein-coupled estrogen receptor, a seven-transmembrane spanning receptor, was demonstrated to be capable of mediating estrogen actions. Recent studies have shown that GPER is expressed in cancer, and its activation is associated with a drastic reduction of cell proliferation through the initiation of mitochondria-dependent apoptotic pathway (Wang et al., 2017, Xu et al., 2016). Chimento et al. found that GPER and its agonists, such as G-1 can be considered as a potentially new pharmacological tool to reduce the growth of Leydig cell tumors through regulating the expression of Bcl-2 (Chimento et al., 2013).

Icariin, flavonoids extracted from Epimedium species, have been shown to have a wide range of pharmacological and biological activities, including estrogenic activity, antioxidant activity, antitumor activity, immunoregulation, neuroprotection, and improved sexual function (Li et al., 2010, Pan et al., 2007, Urano and Tohda, 2010, Wang et al., 2009, Zeng et al., 2010b). Early studies have shown that icariin treatment for 8 weeks improved the cardiac function in significantly diabetic rats, which may be related to the attenuation of mitochondrial oxidative stress (Bao and Chen, 2011). Several studies have also shown that icariin had a protective effect on the early stage of experimental DN induced by streptozotocin (Qi et al., 2011a). We have previously shown that icariin can inhibit the expression of transforming growth factor-β (TGF-β) via a GPER pathway and inhibit the downstream extracellular signal-related kinase (ERK) and Smad signaling pathways to attenuate high glucose-induced renal mesangial cell fibrosis (Li et al., 2013, Li et al., 2014). Based on previous studies, we hypothesized that icariin could potentially protect DN by modulating podocyte apoptosis.

As a GPER agonist, however, whether icariin has a direct effect on podocyte apoptosis remains unknown. The aim of this study was mainly to explore the regulation of icariin on the mitochondrial apoptosis pathway through GPER by detecting Bcl-2 mitochondrial translocation.