Long-term administration of advanced glycation end-product stimulates the activation of NLRP3 inflammasome and sparking the development of renal injury

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Abstract

The accumulation of advanced glycation end-products (AGEs) and the enhanced interaction of AGE with their cellular receptor (RAGE) have been implicated in the progression of chronic kidney disease. The purpose of this study was to examine whether the AGE/RAGE-induced nephrotoxic effects are associated with inflammasome activation and endothelial dysfunction. Chronic renal injury was examined in BALB/c mice by the long-term administration of carbonyl-AGE for 16 weeks. Endothelial dysfunction was detected by measuring the number of circulating endothelial progenitor cells (EPCs) and the levels of nitric oxide synthase (eNOS) and nitric oxide (NO) in kidneys. Results showed that administration of methylglyoxal–bovine serum albumin (MG-BSA) AGE accelerated renal MG, carboxyethyl lysine, carboxymethyl lysine and malondialdehyde formation and, in parallel, the levels of serum creatinine and blood urea nitrogen (BUN) were significantly increased. Expression of RAGE and NLRP3 inflammasome-related proteins (TXNIP, NLRP3, procaspase-1 and caspase-1) and IL (interleukin)-1β secretion were upregulated, whereas the levels of EPCs, eNOS and NO were lower in MG-BSA-treated mice. This induction by MG-BSA was significantly inhibited by RAGE antagonist. Our results firstly reveal a possible mechanism of AGE-mediated renal dysfunction upon NLRP3 inflammasome activation. Therapeutic blockade of RAGE may ameliorate renal and endothelial functions in subjects under high AGE burden.

Introduction

Advanced glycation end-products (AGEs) are the stable and complex substances produce by Maillard reactions that occur in foodstuffs during heating, processing and storage, and it can lead to a wide variety of food-associated phenomena, including flavors, aromas and colors. The Maillard reaction usually takes several days to several weeks to complete; the reaction rate is extraordinarily low because no enzymatic catalysis is involved. A more succinct descriptive term for AGE is glycotoxins, because once AGEs are formed, they are nearly irreversible. [1]. Therefore, the accumulation AGE in vivo has been implicated as a critical factor in the pathogenesis of chronic kidney disease, aging and other health disorders [2], [3].

The receptor for AGE (RAGE) is a member of the immunoglobulin superfamily of receptors, which engages a number of ligands, including AGE, and has been regarded as the bridge association between oxidative stress, inflammation and kidney diseases [4]. Both the accumulation of AGE and RAGE expression play important roles in the pathogenesis of diabetic nephropathy [5]. Similarly, upregulation of RAGE occurs on cells such as endothelial cells and vessel segments in patients with inflammatory renal disease [6]. Activation of RAGE is known to induce NADPH oxidase and cellular ROS production within the renal cortex [7], [8]. In addition, AGE-bound RAGE causes upregulation of the transcription factor nuclear factor-κB and its target inflammatory genes such as IL-1β and TNF (tumor necrosis factor)-α [9].

Increasing knowledge has implicated that inflammation may contribute to the development of kidney diseases. The inflammasome is a multiprotein complex consisting of an NOD-like receptor (NLR) protein, an adaptor protein, and pro-caspase-1; the NACHT, LRR and PYD domain-containing protein 3 (NLRP3). NLRP3 mRNA expression is higher in the kidney than in other tissues in BALB/c mice, and the distribution of NLRP3 exhibits strongly positive staining in renal tubular cells by immunohistochemistry (IHC) analysis [10]. Thus, NLRP3 may exert crucial role in renal inflammatory responses. The inflammatory reaction caused by inflammasome activation is largely due to the binding of NLRP3 to adaptor proteins that convert inactivated pro-caspase-1 into active caspase-1. Activated caspase-1 converts pro-IL-1β into IL-1β to induce an inflammatory reaction. The roles of the NLRP3 inflammasome in chronic kidney disease and acute kidney disease have been extensively discussed [11]; the NLRP3 inflammasome has been shown to be an important factor for the development of kidney inflammation and fibrosis [12]. In addition, calcium oxalate crystal-induced oxidative damage in the rat kidney has been associated with NLRP3 activation [13].

Globally, the NLRP3 inflammasome is the best characterized inflammasome; the production of ROS induced by NADPH oxidase results in activation of NLRP3 by thioredoxin-interacting protein (TXNIP) [14], leading to podocyte and mesangial cell damage [15], [16]. Administration of an NADPH oxidase inhibitor to rats inhibits TXNIP and inflammasome activation by reducing ROS production in the kidney [13]. Therefore, reducing NADPH oxidase-related ROS production may decrease inflammasome activation and IL-1β production; however, whether the AGE–RAGE interaction influences NLRP3 activation remains unclear. Therefore, this study investigated whether long-term AGE administration results in RAGE expression that promotes ROS generation, NLRP3 inflammasome activation and IL-1β secretion, resulting in kidney injury. This study also assessed whether RAGE antagonist (RA) administration has a kidney-protective effect.

Section snippets

Chemicals

Bovine serum albumin (BSA), glyoxal (GO), glycolaldehyde (GOA), glucose (GLU), methylglyoxal (MG) and glyceraldehyde (GA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). An RA was purchased from Millipore (Bedford, MA, USA). The primary antibodies used for Western blotting and IHC included antibodies against RAGE (Millipore), NADPH oxidase 4 (NOX4; Abcam, USA), TXNIP (Abcam, USA), NLRP3 (Novus, USA), pro-caspase and caspase-1 (Santa Cruz Biotech, USA), and endothelial nitric oxide

The effects of different carbonyl-derived AGE on renal function

As shown in Fig. 1, after intraperitoneal administration with different types of carbonyl-derived AGE at 240 mg/kg BW for 24 weeks, the serum Cr levels at 16 weeks were significantly higher in the groups administered MG-BSA than in the other groups. These effects persisted until week 24. The urinary total protein excretion at the end of the experiment, which was normalized by the Cr level, was significantly higher in the MG-BSA group than in the BSA group (Fig. 1a and b). HE staining revealed

Discussion

This study provides the first demonstration of the MG-BSA-mediated activation of the NLRP3 inflammasome via RAGE in the kidney. The results showed that blockade of RAGE inhibited AGE–RAGE-induced ROS production and attenuated TXNIP–NLRP3 activation, thus reducing the levels of the pro-inflammatory cytokine IL-1β and consequently attenuating abnormal kidney function and vascular endothelial dysfunction.

Proinflammatory cytokine overproduction is widely known to lead to kidney injury [23].

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