MicroRNA-218 promotes high glucose-induced apoptosis in podocytes by targeting heme oxygenase-1

https://doi.org/10.1016/j.bbrc.2016.02.028Get rights and content

Highlights

  • miR-218 is up-regulated in HG-treated podocytes.

  • miR-218 promotes podocyte apoptosis.

  • HO-1 is a target of miR-218.

  • Knockdown of HO-1 blocks the anti-apoptotic effect of anti-miR-218 in podocytes.

  • miR-218 regulates p38-MAPK phosphorylation.

Abstract

Emerging evidence has demonstrated that microRNAs (miRNAs) play a mediatory role in the pathogenesis of diabetic nephropathy. In this study, we found that miR-218 was upregulated in high glucose (HG) treated podocytes, which are essential components of the glomerular filtration barrier and a major prognostic determinant in diabetic nephropathy. Additionally, up-regulation of miR-218 was accompanied by an increased rate of podocyte death and down-regulation in the level of nephrin, a key marker of podocytes. However, inhibition of miR-218 exerted the opposite effect. In addition, the dual-luciferase reporter assay showed that miR-218 directly targeted the 3′-untranslated region of heme oxygenase-1 (HO-1), and further study confirmed an increase of HO-1 in HG-treated podocytes transfected with anti-miR-218. Knockdown of HO-1 blocked the anti-apoptotic effect of anti-miR-218. Furthermore, inhibition of miR-218 was associated with decreased expression of the known pro-apoptotic molecule p38-mitogen-activated protein kinase (p38-MAPK) activation. Following preconditioning with SB203580, an inhibitor of p38-MAPK, the stimulatory effect of HG on podocyte apoptosis was strikingly ameliorated. These findings suggested that miR-218 accelerated HG-induced podocyte apoptosis through directly down-regulating HO-1 and facilitating p38-MAPK activation.

Introduction

Diabetic nephropathy (DN) is a major microvascular complication of diabetes and the leading cause of end-stage renal disease (ESRD) [1]. In the early stages of DN, the main clinical feature is development of persistent microalbuminuria, which is usually followed by a progressive decline in renal function [2]. Therefore, there is an imperative need to intervene in the early stages of DN. Recently, increasing evidence indicated that podocyte loss is a hallmark of early DN [3]. Podocytes constitute the glomerular filtration barrier together with endothelial cells and the glomerular basement membrane. Besides, podocytes are highly differentiated cells with poor proliferation ability, and the extent of their injury is a major prognostic determinant in DN as their loss is accompanied by increased proteinuria, progressively declining glomerular filtration rate (GFR) and glomerular dysfunction [3], [4]. Therefore, there has been a great deal of interest in developing podocyte-based therapeutics for preventing DN.

MicroRNAs (miRNAs) are a class of approximately 22 nucleotide, small, noncoding RNAs that negatively regulate gene expression by binding to the 3′-UTR of their target messenger RNAs [5]. Emerging evidence demonstrated that a number of miRNAs have ectopic expression in the kidney and are involved in the progression of kidney disease. MicroRNA-29c was shown to be up-regulated in the kidney glomeruli of db/db diabetic mice and its knockdown remarkably reduced albuminuria and kidney mesangial matrix accumulation [6]. Another report showed that miR-216a and miR-217 were upregulated in TGF-β-treated mouse glomerular mesangial cells, and activated Akt kinase by downregulating phosphatase and tensin homologue (PTEN), an Ebox regulator [7]. In addition, miRNAs also play vital roles in podocyte homeostasis. Mice with podocyte-specific deletions of Dicer or Drosha, two critical RNAase III enzymes in the miRNA biogenesis pathway, sustained podocyte dysregulation, proteinuria defects and collapsing glomerulopathy [8], [9], [10]. However, the roles of miRNAs in DN pathogenesis need to be further elucidated.

Heme oxygenase-1 (HMOX1 or HO-1) enzyme, a 32 kDa, rate-limiting enzyme in heme catabolism, is an antioxidant defense, and key cytoprotective, enzyme [11]. The first human case of HO-1 deficiency revealed that in patients with severe growth retardation, HO-1 deficiency leads to oxidative stress and enhanced endothelial cell injury [12]. Interestingly, various reports indicate that HO-1 is upregulated in the kidney under negative conditions and exerts a renoprotective effect against renal injury [13], [14]. In vivo and in vitro studies also demonstrated that in glomeruli of streptozotocin (STZ) treated rats and in mouse podocytes treated with high glucose, inhibition of HO-1 promoted podocyte apoptosis [15]. In addition, through two miRNA target prediction algorithms (miRanda and TargetScan), we found miR-218, a pro-apoptosis related molecule, could directly target HO-1, leading us to hypothesize that miR-218 may participate in the pathologic process of DN through regulating HO-1.

In this study, we show that miR-218 is upregulated in high glucose (HG)-induced podocytes, which accelerates cell apoptosis by suppressing its direct target gene, HO-1. We also found that HO-1 inhibited phosphorylation of p38-MAPK in HG-stimulated podocytes. Taken together, our study suggests that miR-218 may be a potential therapeutic target for DN treatment.

Section snippets

Podocyte culture

Conditionally immortalized mouse podocytes were obtained from the Cell Resource Center of Peking Union Medical College (Beijing, China). Podocytes were cultured as follows: firstly, podocytes were cultured in RPMI 1640 medium (Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Sigma–Aldrich) and 100 U/ml of penicillin/streptomycin (Solarbio, Beijing, China) in the presence of 10 U/ml γ-interferon (γ-IFN) (Sangon Biotech, Shanghai, China) at 33 °C in a humidified

miR-218 is upregulated in HG-treated podocytes

To investigate the potential role of miR-218 in podocytes, we analyzed the expression of miR-218 at different time points (0 h, 24 h, 48 h and 72 h) following stimulation with HG (25 mM) by RT-qPCR. The results demonstrated that miR-218 levels were up-regulated following HG treatment in podocytes in a time-dependent manner (Fig. 1A), suggesting that miR-218 might be involved in the response to hyperglycemic conditions.

Down-regulation of miR-218 suppresses apoptosis of podocytes

Because DN is characterized by increased apoptosis in podocytes [3], [16],

Discussion

In this study, we determined that miR-218 expression increased in cultured podocytes exposed to HG. Our results also suggested that through directly targeting HO-1, miR-218 promoted p38-MAPK activity, thus facilitating podocyte apoptosis. We concluded that miR-218 may act as a potential therapeutic target for DN treatment.

Previous studies showed that miR-218 plays important roles in cancers. In patients with medulloblastoma, miR-218 was downregulated and acted as a tumor suppressor in

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