Activation of the Nlrp3 inflammasome by mitochondrial reactive oxygen species: A novel mechanism of albumin-induced tubulointerstitial inflammation

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Abstract

Albuminuria is not only an important marker of chronic kidney disease but also a crucial contributor to tubulointerstitial inflammation (TIF). In this study, we determined whether activation of the Nlrp3 inflammasome is involved in albuminuria induced-TIF and the underlying mechanisms of inflammasome activation by mitochondrial reactive oxygen species (mROS). We established an albumin-overload induced rat nephropathy model characterised by albuminuria, renal infiltration of inflammatory cells, tubular dilation and atrophy. The renal expression levels of the Nlrp3 inflammasome, IL-1β and IL-18 were significantly increased in this animal model. In vitro, albumin time- and dose-dependently increased the expression levels of the Nlrp3 inflammasome, IL-1β and IL18. Moreover, the silencing of the Nlrp3 gene or the use of the caspase-1 inhibitor Z-VAD-fmk significantly attenuated the albumin-induced increase in IL-1β and IL-18 expression in HK2 cells. In addition, mROS generation was elevated by albumin stimulation, whereas the ROS scavenger N-acetyl-l-cysteine (NAC) inhibited Nlrp3 expression and the release of IL-1β and IL-18. In kidney biopsy specimens obtained from patients with IgA nephropathy, Nlrp3 expression was localised to the proximal tubular epithelial cells, and this result is closely correlated with the extent of proteinuria and TIF. In summary, this study demonstrates that albuminuria may serve as an endogenous danger-associated molecular pattern (DAMP) that stimulates TIF via the mROS-mediated activation of the cytoplasmic Nlrp3 inflammasome.

Introduction

Tubulointerstitial inflammation plays a central role in the loss of renal function in chronic renal disease, but the exact mechanism remains largely unknown (Lee and Kalluri, 2010). A number of studies have demonstrated that albuminuria is not only a well-recognised hallmark of kidney disease but also a pathogenic factor involved in the development of proteinuric nephropathy, which is characterised by alterations in proximal tubular epithelial cell (PTEC) death and inflammatory cytokine production (Abbate et al., 2006, Baines and Brunskill, 2011, Eddy and Giachelli, 1995, Li et al., 2010). Albuminuria stimulates proximal tubular cells to synthesise chemokines (MCP-1 and RANTES) that recruit monocytes and T cells and contributes to the release of cytokines that attract neutrophils and fibrosis-promoting molecules (e.g., endothelin, angiotensin II, and TGF-α) through phospholipase C, MAPK, or NF-κB signal activation (Drumm et al., 2002, Gomez-Garre et al., 2001, Gorriz and Martinez-Castelao, 2012, Han et al., 2005, Liu et al., 2009, Takase et al., 2008, Wang et al., 1999). These studies raised a concern about the pathogenic role of proteinuria in the progression of chronic renal disease. However, the exact molecular mechanisms that regulate inflammation by albuminuria have not been fully elucidated.

More recent studies have focused on innate immune-sensing receptors and their role in inflammation and disease processes. The Nlrp3 inflammasome is a cytoplasmic multiprotein that contains the Nod-like receptor and ASC adaptor (Lorenz et al., 2014) and triggers the activation of caspase-1, IL-1β and IL-18 to engage innate immune defences and execute inflammatory responses (Latz et al., 2013, Schroder and Tschopp, 2010, Shi et al., 2012). Previous studies have confirmed that the inflammasome is activated by a variety of stimuli, including pathogens associated with virtually every type of pathogen-associated molecular pattern (PAMP). It is also triggered by several danger signals, including pore-forming toxins from bacteria, extracellular ATP (Mariathasan et al., 2006), ROS (Martinon et al., 2009, Zhou et al., 2010), monosodium urate crystals (Martinon et al., 2006), nucleic acids (Muruve et al., 2008), extracellular matrix components, including hyaluronan (Yamasaki et al., 2009), biglycans (Babelova et al., 2009), and environmental microparticles, such as asbestos and silica (Dostert et al., 2008, Martinon, 2012). The activation of the Nlrp3 inflammasome requires a two-step signal. Initially, the transcription and translation of immature pro-IL-1β (pro-IL-18) mRNA is induced via NF-κB activation by a primary signal, which may be derived from TLR, TNFR or IL-1R signalling. A secondary signal then activates the inflammasome and cleaves mature IL-1β/IL-18 released by inflammasome stimuli (Gross et al., 2011, Lorenz et al., 2014).

A strong association between dysregulated inflammasome activity and certain human inflammatory diseases suggests the importance of this pathway in innate immune responses, such as gouty arthritis (Martinon et al., 2006), type 2 diabetes mellitus, atherosclerosis, and inflammatory bowel disease (Strowig et al., 2012). Previous studies have demonstrated that IL-18 and caspase-1, which are two key markers of Nlrp3 inflammasome activation, are expressed in the renal tubular epithelium of patients with chronic kidney disease, suggesting that the Nlrp3 inflammasome may play an important role in regulating inflammation in kidney diseases (Gauer et al., 2007, Lonnemann et al., 2003). However, the role of albuminuria in the activation of the tubular Nlrp3 inflammasome and its subsequent contribution to tubulointerstitial inflammation remains speculative. The purpose of this study was therefore to investigate whether albuminuria directly induces mROS generation to activate the Nlrp3 inflammasome pathway in tubular cells during the genesis of tubulointerstitial inflammation.

Section snippets

Animal model

Protein-overload nephropathy (Eddy, 1989) was induced via intraperitoneal injection of bovine serum albumin (BSA) in male Wistar rats one week after right nephrectomy (initial weight 120–130 g, Academy of Military Medical Science, Animal Experiment Centre). The rats were fed standard rat chow ad libitum, given free access to water and randomly divided into two groups. In the albumin-overload group (AO, n = 10), the rats were administered a daily intraperitoneal injection of BSA (5.0 g/kg/d, fatty

Biochemical and pathological changes in experimental rats

To explore the role of albuminuria-induced renal epithelial damage and tubulointerstitial inflammation, we treated Wistar rats with daily injections of 5 g/kg bovine serum albumin (BSA) or normal saline for nine weeks. It was found that rats with albumin overload (AO) presented significantly increased protein excretion and urine albumin levels starting on week 2 (Fig. 1A and B). In addition, N-acetyl-β-d-glycosaminidase (NAG), an accepted marker of tubular injury, was also overtly increased in

Discussion

The presence of persistent amounts of albumin in urine is a sign of kidney damage and a predictor of disease progression. The potential mechanism underlying how albumin impacts the progression to CKD remains unknown. Many studies have demonstrated a positive correlation between the degree of albuminuria and disease progression and confirmed the tubular toxic nature of albuminuria (Abbate et al., 2006, Baines and Brunskill, 2011, Eddy, 1989, Eddy and Giachelli, 1995, Thomas et al., 1999). To

Conflict of interest statements

All co-authors have seen and agree with the contents of the manuscript and there is no financial interest to report.

Acknowledgments

This study was supported by grants from the National Natural Scientific Foundation (No. 81130010), the Clinic Research Center of Jiangsu Province (No. BL2014080). No part of this manuscript has been published elsewhere or has been submitted to another journal. All of the co-authors have read and agree with the contents of this manuscript, and there are no financial interests to report.

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