Introduction
Hyperlipidemia is one of the most prevalent diseases in both developing and developed countries. Hyperlipidemia has been hypothesized to play an important role in the progression of kidney injury [
1], at least partially, due to deleterious renal lipid accumulation. Lipids may cause both glomerular and tubular cell injury and promote renal disease progression [
2]. Several molecular mechanisms mediating cellular dysfunction and injury caused by lipid accumulation in renal tubular segments have been investigated, including the generation of reactive oxygen species (ROS), damages of multiple organelles, release of proinflammatory and profibrotic factors, and lipid-induced apoptosis [
3]. Our recent study demonstrated that kidney collecting duct cell injuries induced by lipid accumulation may cause retention of water, presumably leading to an increase in preload and blood pressure [
4].
In physiological condition, ROS plays an important role in proliferation, differentiation, and apoptosis of various cells, including renal collecting duct cells [
5]. Oxidative stress during various metabolic disturbances (e.g. hyperlipidemia) may cause damages of kidney epithelial cells by affecting several signaling pathways, leading to end stage renal disease (ESRD) [
6]. Generally, ROS generation is mediated by two pathways, enzymatic and nonenzymatic pathways. One of critical enzymatic pathways is nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase, NOX) composed of two isoforms NOX4 and NOX2, NOX4 is highly expressed in kidney tubular epithelial cells which constitutively produce hydrogen peroxide (H
2O
2), a prevalent ROS detected [
7]. As the major NADPH isoform in the kidney, NOX4 contributes to redox processes in diabetic and obese kidneys [
8]. Conversely, mitochondrial electron transport chain (mETC) deficiencies, advanced glycation end products (AGEs), glucose auto-oxidation, etc. were identified as nonenzymatic pathways [
9].
Renal oxidative stress is often a consequence of pro-oxidant enzyme-induced ROS production and concomitant depletion of antioxidants. Among the enzymatic systems implicated in ROS generation in the kidney, the NOXs appear to be the key contributors. Besides, mitochondrial dysfunction (MtD) is also involved in cholesterol- induced kidney damage [
10], since intracellular levels of ROS can be induced by the electron leakage from mitochondrial respiratory chain. Cholesterol in excess can cause injuries in glomerular, tubular and tubulointerstitial cells through multiple mechanisms, one of which is increased production of ROS through both mitochondria and NADPH oxidases pathways.
HMG-CoA reductase inhibitors statins clearly reduce the risk of cardiovascular disease and mortality; they attenuated proteinuria and preserved renal function independent of other variables. Clinical evidence has shown that statins treatment is beneficial to the kidney of patients with hyperlipidemia [
11]. HMG-CoA reductase was found expressed in collecting ducts and proximal tubular epithelial cells, which was greatly increased by high-fat diet, indicating a potential role of tubular epithelial cells in cholesterol regulation in the kidney. Statins may inhibit NADPH oxidase stability on the plasma membrane [
12] and protect vascular damage in diabetic patients by their antioxidant properties [
13]. Our recent data demonstrated that hyperlipidemia induced NLRP3 activation and exacerbated kidney injury in animals with high-fat diet, which was at least partially prevented by statins treatment [
4]. NLRP3 inflammasome may sense directly the presence of ROS produced by damaged mitochondria [
5] and assembly could be triggered by extracellular ATP and ROS [
14]. In our study, the protective effect of statins is at least partially attributed to inhibition of NLRP3 components [
4], however, whether statins prevent lipid-induced kidney injuries through inhibiting ROS was not investigated.
The purpose of the present study is to examine whether statins prevents ROS production induced by cholesterol and potential molecular mechanisms in rats with chronic kidney disease and in cultured collecting duct cells.
Discussion
In the current study, we found an anti-oxidative stress effect of statins in the kidney. Statins downregulated protein and mRNA expression of NOX2/NOX4 and improved mitochondrial function, by which statins suppressed ROS production and prevented cholesterol-induced kidney injuries. The present data supported our previous findings [
4] that statins may protect kidney injury from cholesterol overload, independent of its property of lowering lipid.
Consequent ROS production can trigger abnormal signaling pathways involving diverse signaling mediators such as transcription factors, inflammatory cytokines, chemokines, and vasoactive substances [
17]. Persistently, increased expression and activation of these signaling molecules contribute to the functional and structural changes in the kidney. ROS-induced kidney injuries have been recognized in diabetic and obese nephropathy. High glucose induced mitochondrial damage in renal tubular cells which was associated with ROS generation. ROS, acting as a key messenger in the signaling transduction, is involved in obese-associated kidney fibrosis [
18] and in diabetic nephropathy [
19]. It has been previously reported that fatty acid modulates mitochondrial ROS production by several mechanisms, including interactions among components of the respiratory chain, there by inhibiting the electron transport [
20]. Nitric oxide (NO) is produced from the conversion of L-arginnine by NO synthase (NOS) and mediated a variety of biology processes such as ROS production. A recent study demonstrated that cholesterol downregulated NOS2 gene level and protein expression in kidneys of FVB/N mice fed with 1% cholesterol diet for 6–8 weeks [
21]. Consistent with this, we showed HFD increased NOS2 mRNA level in 5/6Nx rats with high-fat diet for 12 weeks. These data suggested that cholesterol may mediating ROS production in different stages of chronic kidney diseases. In vitro results shown in Fig.
3D demostrated that Atoravstatin treatment have no effect on mRNA level of NOX4 but decreased it’s protein expression dramatically (Fig.
3A). The underlying mechanism is still unknown, but it may be associated with post translational modifications of NOX4 and NOS2 induced by statins [
22]. Our finding showed that cholesterol also increased ROS production which can be mediated by NAPDH oxidase and mitochondrial damage in the kidney. These data suggests that during dyslipidemia both fatty acid and cholesterol trigger ROS production by mitochondrial and enzymatic pathways, leading to kidney injuries.
Evidence has shown that accumulation of lipid droplets in proximal tubular epithelial cells could be one of the causes to induce ROS overproduction [
23]. Although a protective role of statins by lipid-lowering in lipid-associated tissue injuries has well been known, the potential benefits of statins beyond lipid-lowering are not well established [
24]. Our previous study demonstrated that statins prevented inflammation induced by lipid in the kidney, which was not necessarily associated with its property inhibiting synthesis of cholesterol, but via directly acting on inflammasome. Here our data support a direct role of statins in suppressing ROS production induced by cholesterol by decreasing NOX2/NOX4 protein expression and improving mitochondrial dysfunction.
In cholesterol overload tubular cells, NOX4 is constitutively active producing hydrogen peroxide (H
2O
2) as the prevalent ROS detected, whereas other NOXs (NOX1, NOX2 and NOX5) present in the kidney generate superoxide radical anions as products [
7]. The high expression of NOX4 would transfer electrons to reduce molecular oxygen to form O
2− which are considered as main producers of ROS. NOX4 is the crosslink between NAPDH oxidase and mitochondrial dysfunction and induce ROS production, as NOX4 exists in the outer mitochondrial membrane [
25]. NOX4 upregulation decreased mitochondrial biogenesis and induced mitochondrial electron transport chain (mETC) deficiencies. In the present study, cholesterol markedly induced NOX4 protein expression and mitochondrial dysfunction, which was associated with ROS generation in the collecting duct cells. High-fat diet also induced NOX4 protein and gene expression, which presumably causing ROS in the kidney, leading to tubular injuries. Statins decreased protein/gene expression of NOX4 and improved mitochondrial function in vitro, which is supposed to be protective. Our previous study showed that statins ameliorated cholesterol-induced inflammation by promoting the degradation of NLRP3 inflammasome components in the kidney. Since NLRP3 inflammasome assembly could be triggered by ROS [
14], ROS inhibition by statins is supposed to further attenuate NLRP3 activation. Therefore, the protective role of statins in lipid-induced kidney injuries is likely attributed to both inhibiting NLRP3 inflammasome activation and promoting degradation of NLRP3 components. Although the mechanism by which statins downregulates NOX4 expression was not examined, our data indeed support the conception that statins protect kidney injuries during obesity independent of its property of lowering cholesterol.
In conclusion, statins prevented ROS production induced by cholesterol in the kidney, likely through inhibiting NOX2 and NOX4 protein expression and improving mitochondrial function. Statins may be a therapeutic option in treating cholesterol-associated kidney diseases.
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