Role of mitochondrial dysfunction in renal fibrosis promoted by hypochlorite-modified albumin in a remnant kidney model and protective effects of antioxidant peptide SS-31

Abstract

Oxidative stress aggravates renal fibrosis, a pathway involved in almost all forms of chronic kidney disease (CKD). However, the underlying mechanism involved in the pathogenesis of renal oxidative stress has not been completely elucidated. In this study, we explored the role and mechanism of hypochlorite-modified albumin (HOCl-alb) in mediating oxidative stress and fibrotic response in a remnant-kidney rat model. Five-sixths nephrectomy (5/6 NX) was performed on the rats and then the animals were randomly assigned to intravenous treatment with either vehicle alone, or HOCl-rat serum albumin (RSA) in the presence or absence of SS-31 (administered intraperitoneally). A sham-operation control group was set up concurrently. Compared with the control group, 5/6 NX animals displayed marked mitochondrial (mt) dysfunction, as evidenced by decrease of mitochondrial membrane potential (MMP), ATP production, mtDNA copy number alterations and manganese superoxide dismutase (MnSOD) activity, release of cytochrome C (Cyto C) from mitochondria to the cytoplasm, and increase of mitochondrial reactive oxygen species in renal tissues. They also displayed increased levels of HOCl-alb in both plasma and renal tissues. These changes were accompanied by accumulation of extracellular matrix, worsened proteinuria, deteriorated renal function, and a marked increase of macrophage infiltration along with up-regulation of monocyte chemoattractant protein (MCP)−1 and transforming growth factor (TGF)-β1 expression. HOCl-alb challenge further exacerbated the above biological effects in 5/6 NX animals, but these adverse effects were prevented by administration of SS-31, a mitochondrial targeted antioxidant peptide. These data suggest that accumulation of HOCl-alb may promote renal inflammation and fibrosis, probably related to mitochondrial oxidative stress and dysfunction and that the mitochondrial targeted peptide SS-31 might be a novel therapy for renal fibrosis and chronic renal failure (CRF).

Introduction

Chronic renal failure (CRF) is a global public health problem commonly associated with high morbidity and mortality (García-García and Jha, 2015). CRF is defined by the development of glomerulosclerosis and interstitial fibrosis (Shimamura and Morrison, 1975), a common final pathway involved in almost all forms of chronic kidney disease (CKD) (Boor et al., 2010, Duffield, 2014). By far, therapies in CRF are inadequate and unable to keep pace with the progression of the disease.

Oxidative stress is defined as disequilibrium between the generation and scavenging of reactive oxygen species. Abnormal reactive oxygen species production primarily results from mitochondrial dysfunction, NADPH oxidase activation and endoplasmic reticulum stress (Cao et al., 2014). Mitochondria are not only the main source of reactive oxygen species but also the organelles most sensitive to oxidative stress (Indo et al., 2015). In vitro, mitochondrial dysfunction mediates epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells (Yuan et al., 2012). In vivo, mitochondrial protection restores interstitial fibrosis in experimental models of unilateral ureteral occlusion (UUO) (Sun et al., 2014) and five-sixths nephrectomy (5/6 NX) (Chen et al., 2013, Yu et al., 2016), indicating a potential association between mitochondrial dysfunction and renal fibrosis.

Hypochlorite-modified albumin (HOCl-alb) is formed during the reaction between proteins (mainly albumin) and the hypochlorite (HOCl) that originates via the action of the myeloperoxidase (MPO) of active neutrophils, a process concurrent with oxidative stress (Witko-Sarsat et al., 1996). Levels of HOCl-alb are much higher in CKD patients with advanced CRF who are on or not yet on dialysis (Cao et al., 2014). MPO deficiency could ameliorate the progression of chronic kidney disease in mice (Lehners et al., 2014). However, over-loading of HOCl-alb results in activation of a redox-sensitive pathway and induces renal macrophage infiltration in the remnant kidneys (Li et al., 2007). HOCl-alb overload also induces the overexpression of monocyte chemoattractant protein (MCP)−1 and transforming growth factor (TGF)-β1 in diabetes rats (Shi et al., 2008), and perturbs renal cell functions, including vascular endothelial dysfunction (Tang et al., 2016), podocyte depletion and apoptosis (Zhou et al., 2009), overexpression of fibronectin and collagen IV in mesangial cells (Wei et al., 2008), and EMT in tubular cells (Tang et al., 2015) in vitro. However, the mechanisms invoked by HOCl-alb in renal fibrosis, and the involvement of mitochondrial dysfunction in mediating the detrimental effects of HOCl-alb, remain largely unknown.

A new cell-permeable tetrapeptide named SS-31 was reported to target and accumulate in the inner membrane of mitochondria in a membrane potential-independent manner and to scavenge reactive oxygen species, thereby preventing mitochondrial depolarization, mitochondrial permeability transition, and Cyto C release from mitochondria to cytoplasm (Zhao et al., 2004). SS-31 normalized mitochondrial potential (△Ψ_m) and ATP alterations and inhibited Cyto C release and reactive oxygen species generation in mouse mesangial cells (Hou et al., 2016) in vitro and alleviated renal damage in diabetic kidneys (Hou et al., 2016) and in UUO rat models (Mizuguchi et al., 2008) in vivo.

Here, we hypothesize that mitochondrial oxidative stress and dysfunction is involved in both glomerulosclerosis and interstitial fibrosis, both of which are promoted by HOCl-alb, and that these effects can be attenuated by the SS-31 antioxidant peptide. We used a 5/6 NX rat model as a surrogate for human CRF to examine our hypothesis.