Targeting inflammation: new therapeutic approaches in chronic kidney disease (CKD).

Pharmacological Research 81 (2014) 91–102

Contents lists available at ScienceDirect

Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs

Review

Targeting inflammation: New therapeutic approaches in chronic kidney disease (CKD) Daniela Impellizzeri a , Emanuela Esposito a , James Attley b , Salvatore Cuzzocrea a,c,∗ a b c

Department of Biological and Environmental Sciences, University of Messina, Viale Ferdinando Stagno D’Alcontres, Messina 31-98166, Italy Prismic Pharmaceuticals, Scottsdale, AZ, USA Manchester Biomedical Research Centre, Manchester Royal Infirmary, University of Manchester, United Kingdom

a r t i c l e

i n f o

Article history: Received 30 November 2013 Received in revised form 18 February 2014 Accepted 24 February 2014 Keywords: Inflammation Oxidative stress Chronic kidney disease Bardoxolone methyl PEA Mast cell Chemical compounds studied in this article: Palmitoylethanolamide (PEA) (PubChem CID: 4671) Bardoxolone methyl (PubChem CID: 400769) Captopril (PubChem CID: 44093) Tempol (PubChem CID: 137994) N-acetyl-L-cysteine (NAC) (PubChem CID: 12035) Trolox (PubChem CID: 40634) AST-120 (activated charcoal) (PubChem CID: 297) CDDO-imidazolide (PubChem CID: 9958995) RTA 405 (Bardoxolone methyl analogue) (PubChem CID: 400769) Dihydro-CDDO-trifluoroethyl amide (dh404) (Bardoxolone methyl analogue) (PubChem CID: 400769) Fenofibrate (PubChem CID: 3339) WY 14643 (PubChem CID: 5694) Ciprofibrate (PubChem CID: 2763) Rosiglitazone (PubChem CID: 77999) Pioglitazone (PIO) (PubChem CID: 4829)

a b s t r a c t Chronic inflammation and oxidative stress, features that are closely associated with nuclear factor (NF-␬B) activation, play a key role in the development and progression of chronic kidney disease (CKD). Several animal models and clinical trials have clearly demonstrated the effectiveness of angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) therapy to improve glomerular/tubulointerstitial damage, reduce proteinuria, and decrease CKD progression, but CKD treatment still represents a clinical challenge. Bardoxolone methyl, a first-in-class oral Nrf-2 (nuclear factor erythroid 2-related factor 2) agonist that until recently showed considerable potential for the management of a range of chronic diseases, had been shown to improve kidney function in patients with advanced diabetic nephropathy (DN) with few adverse events in a phase 2 trial, but a large phase 3 study in patients with diabetes and CKD was halted due to emerging toxicity and death in a number of patients. Instead, palmitoylethanolamide (PEA) a member of the fatty acid ethanolamine family, is a novel non-steroidal, kidney friendly anti-inflammatory and anti-fibrotic agent with a well-documented safety profile, that may represent a potential candidate in treating CKD probably by a combination of pharmacological properties, including some activity at the peroxisome proliferator activated receptor alpha (PPAR-␣). The aim of this review is to discuss new therapeutic approaches for the treatment of CKD, with particular reference to the outcome of two therapies, bardoxolone methyl and PEA, to improve our understanding of which pharmacological properties are responsible for the anti-inflammatory effects necessary for the effective treatment of renal disease. © 2014 Elsevier Ltd. All rights reserved.

Abbreviations: ACEI, Angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; CKD, chronic kidney disease; DN, diabetic nephropathy; ESRD, end stage renal disease; PEA, palmitoylethanolamide; PPAR␣, proliferator activated receptor alpha; TNF-␣, tumor necrosis factor-␣; RAAS, renin-angiotensin- aldosterone system; GFR, glomerular filtration rate; AngII, angiotensin II; NF-␬B, nuclear factor; TGF-␤, transforming growth factor-␤; MCP-1, monocyte chemotactic protein-1; iNOS, inducible nitric oxide synthase; Nrf2-Keap1, nuclear factor erythroid 2-related factor 2-Kelch-like ECH-associated protein 1; MC, mast cell. ∗ Corresponding author at: Department of Biological and Environmental Sciences, University of Messina, Viale Ferdinando Stagno D’Alcontres, 31-98166 Messina, Italy. Tel.: +39 090 6765208. E-mail address: [email protected] (S. Cuzzocrea). http://dx.doi.org/10.1016/j.phrs.2014.02.007 1043-6618/© 2014 Elsevier Ltd. All rights reserved.

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Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chronic kidney disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Inflammation as a cause of CKD progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. The role of oxidative stress on CKD progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Therapeutic treatment for CKD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Antioxidant treatment in renal disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Clinical antioxidant therapy in CKD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New therapeutic approaches in CKD: bardoxolone methyl and PEA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Bardoxolone methyl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Nrf2-Keap1 signaling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Experimental studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. PEA: experimental evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Mechanism of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. The role of PPAR-␣ receptors in kidney disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. PEA as a PPAR-␣ agonist in kidney disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9. Clinical evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Oxidative stress is at the heart of the complex pathogenesis of both acute and chronic kidney diseases [1,2]. Acute kidney injury (AKI) affects millions of people and has been associated with increased mortality and hospital length of stay as well as putting the patient at risk for future chronic kidney disease (CKD) [3]. Oxidative stress is known to play an important role in the development of renal diseases such as glomerulonephritis, drug-induced nephrotoxicity, and CKD [4]. CKD is associated with mitochondrial dysfunction that leads to an imbalance between reactive oxygen species (ROS) and the natural anti-oxidants that normally quench these pathological free radicals. Treatment of CKD can slow its progression but the therapies remain limited. Blood pressure control using angiotensin-converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARBs) has the greatest weight of evidence, but still are often inactive. Thus, the need for new approaches to manage the exponentially increasing number of patients with CKD is urgent. One such approach that has been considered is to augment the natural cytoprotective responses of the body using small molecule activators such as bardoxolone methyl. The synthetic triterpenoid bardoxolone methyl (CDDO-methyl ester) and its analogs are the most potent inducers of the Nrf2/Keap1 pathway [5,6]. The structure and activity profile of bardoxolone methyl resembles those of the cyclopentenone prostaglandins, the endogenous activators of Nrf2, which favor the resolution of inflammation [7]. Similarly to cyclopentenone prostaglandins, bardoxolone methyl has anti-inflammatory activity by inhibiting the IKK␤/NF-␬B signaling pathway [8]. Moreover, because the clinical development of bardoxolone methyl was halted due to unforeseen toxicity in phase 3, there is a need to determine if this toxicity is related to the mechanism of its beneficial effect in CKD or to an off-target effect. On the other hand, mast cell infiltration and activation have been well documented in several experimental models of inflammation and are gaining increasing interest as key factors in the onset and progression of renal disease [9]. Palmitoylethanolamide (PEA) is an endogenous fatty acid amide belonging to the family of the N-acylethanolamines. Recently, several studies have demonstrated that PEA is an important analgesic, anti-inflammatory, and neuroprotective mediator, acting at several molecular targets in both central and sensory nervous systems as well as immune cells (i.e. mast cells) [9]. A recent study also demonstrated that ultramicronized PEA attenuated the renal dysfunction

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and injury associated with ischemia reperfusion (IR) of the mouse kidney [9]. Another study in a compression model of spinal cord injury [10] showed that ultramicronized PEA treatment effectively reduces mast cell infiltration and activation, which occurs not only in inflammation, but also in inflammatory hyperalgesia and neuropathic hyperalgesia. Thus, because of its important pharmacological properties and a good safety profile, PEA could represent an alternative approach to delay renal disease progression. In this review, we discuss the possible therapeutic approaches in renal diseases and in particular the effects of bardoxolone methyl and PEA in the pathophysiology of CKD (Fig. 1).

2. Chronic kidney disease 2.1. Inflammation as a cause of CKD progression CKD is characterized by a progressive loss of renal function, chronic inflammation, oxidative stress, vascular remodeling, and glomerular and tubulointerstitial scarring. Diabetic nephropathy (DN) is the leading cause of CKD and end-stage renal disease (ESRD) [11]. The incidence of CKD is increasing in both developed and developing nations. Increased circulating levels of inflammatory markers, such as C-reactive protein (CRP) and the pro-inflammatory cytokines interleukin (IL)-6, and tumor necrosis factor-␣ (TNF-␣), have been described in CKD patients [12]. The prevalence of inflammation varies from 30 to 75% depending on multiple factors, such as residual renal function, geographic and genetic differences and dialysis therapy [12]. Moreover, dialytic therapy or kidney transplantation may even induce neurological complications. Dialysis can directly or indirectly be associated with dialysis dementia, disequilibrium syndrome, aggravation of atherosclerosis, cerebrovascular accidents due to ultrafiltration-related arterial hypotension, hypertensive encephalopathy, Wernicke’s encephalopathy, hemorrhagic stroke, subdural hematoma, osmotic myelinolysis, opportunistic infections, intracranial hypertension and mononeuropathy [13]. Among these factors, oxidative stress has attracted a great deal of interest from researchers. Oxidative stress appears to increase in the serum of CKD patients because of increased oxidant activity as well as a reduced anti-oxidant defense system, which is accompanied by kidney dysfunction and/or severe cardiorenal syndrome [14].


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stress in CKD patients remains to be determined, however, because population-based clinical studies have not been conducted [22]. 2.3. Therapeutic treatment for CKD

Fig. 1. Pathogenesis of CKD. ROS are strongly implicated in the pathological signaling leading to CKD such as renin-angiotensin system (RAS) activation. AngII can activate the NF-␬B pathway that is involved in the control of the transcription of a variety of cellular genes that regulate the inflammatory response by the production of cytokines, chemokines, cell adhesion molecules, and MC activation. In addition, AngII may also cause renal fibrosis by production of TGF-␤ and the induction of extracellular matrix proteins (MMPs) such as type I procollagen, fibronectin, and collagen type IV.

2.2. The role of oxidative stress on CKD progression The structural characteristics of CKD include increased tubular atrophy, interstitial fibrosis, glomerulosclerosis, renal vasculopathy and reduced renal regenerative capability. These characteristics may be caused, at least in part, by the gradual loss of renal energy through development of mitochondrial dysfunction and resultant, increasing oxidative stress [15]. The role of oxidative stress has attracted increasing attention in the field of CKD, cardiorenal syndrome and their preventive strategies [16]. Oxidative stress and inflammation are common features found in patients with CKD [16,17]. Several lines of evidence suggest that CKD is a pro-oxidant state. Specifically (i) oxidation markers of lipid, protein and DNA are increased in the serum of CKD patients; (ii) oxidative markers, such as hypochlorous acid (HOCl)-modified lipoproteins and advanced glycation end products (AGEs), are accumulated in atherosclerotic lesions of CKD patients [18,19]; (iii) there are numerous defects in the anti-oxidant defence mechanisms, resulting in a decreased elimination and clearance of ROS [16,20]. Increases in the circulating levels of oxidative markers have been documented in patients with CKD [14,21]. Perturbations, therefore, in cellular oxidant handling influence downstream cellular signaling and, in the kidney, promote renal cell apoptosis and senescence, decreased regenerative ability of cells, and fibrosis. These factors have a stochastic deleterious effect on kidney function. The majority of studies investigating anti-oxidant treatments in CKD patients show a reduction in oxidative stress and many show improved renal function [16]. Dialysis treatment appears to be ineffective in correcting this oxidative stress. The overall prevalence of oxidative

CKD is a serious public health problem, which carries a high morbidity and mortality; its treatment still represents a clinical challenge. The renin-angiotensin-aldosterone system (RAAS) is a major pathway involved in the pathogenesis and progression of DN [23] and RAAS blockade is an effective therapeutic strategy to reduce proteinuria and slow progression of diabetic and nondiabetic CKD. An ACE inhibitor, (captopril), was the first drug treatment shown to be effective in slowing the progression of DN in 1993 by Lewis et al. [24]. ACEI and ARBs are standard drugs for treating primary hypertension, however, they are each especially effective in slowing the progressive decay of glomerular filtration rate (GFR) in CKD [24,25]. There are two widely accepted mechanisms by which ACEI and ARBs are understood to be beneficial agents in CKD: (i) hemodynamic/antihypertensive actions and (ii) anti-inflammatory/anti-fibrotic actions. The reduction of angiotensin II (AngII) levels (and subsequent reduction in aldosterone levels) is central to both of these pathways. AngII activates nuclear factor (NF-␬B), upregulates adhesion molecules, and may directly stimulate proliferation of lymphocytes [26]. The net result of these actions is to create a local inflammatory environment in areas where AngII is in high concentration, namely the kidney. AngII may also foster fibrosis via interactions with transforming growth factor-␤ (TGF-␤) and the induction of extracellular matrix proteins such as type I procollagen, fibronectin, and collagen type IV [27]. In addition, animal models have implicated aldosterone to be directly involved with mechanisms of endothelial dysfunction, inflammation, and fibrosis [28]. As ACE inhibitors and ARBs each slow progression individually, the question has arisen as to whether their combination would provide additional advantage [29]. Studies are ongoing to assess the value of interrupting the pathway simultaneously at multiple sites, and it has only very recently been reported that not only is the combination ineffective in decreasing cardiovascular and renal morbidity, but also may have an increased safety risk [30,31] (see further below). Glycemic control also reduces the progression of renal disease as judged by the mitigation of increasing albuminuria in both type 1 and type 2 diabetes [32]. Several metabolic disturbances of CKD also may prove to be useful therapeutic targets but have been insufficiently tested. These include acidosis, hyperphosphatemia, and vitamin D deficiency. In patients with dyslipidemia, statin therapy is also appropriate to reduce the risk of cardiovascular disease. Moreover, drugs aimed at other potentially damaging systems and processes, including endothelin, fibrosis, oxidation, and advanced glycation end products, are at various stages of development [33]. 2.4. Antioxidant treatment in renal disease Epidemiological studies have demonstrated association between inflammatory and oxidative stress markers with cardiovascular and renal outcomes in CKD [34]. Experimental data in animal models suggest beneficial effects of antioxidant agents, but results in human studies are limited and controversial. In early experimental diabetes mellitus in hypertensive rats, the administration of tempol, an antioxidant superoxide dismutase (SOD) mimetic, corrected the oxidative imbalance and improved oxidative stress-induced renal injury, decreasing albuminuria and fibrosis [35]. Similar protection was afforded by the antioxidants N-acetyl-L-cysteine (thiol) and kallistatin in Dahl salt-sensitive rats [36]. In addition, N-acetyl-L-cysteine (NAC) pretreatment reduced endothelial dysfunction caused by uremic toxins by reducing ROS-dependent expression of NF-␬B [37]. NAC reduced

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kidney MDA levels in a mouse model of diabetic nephropathy [38]. In a model of ischemia reperfusion and cyclosporin toxicity after unilateral nephrectomy, the inhibition of the mitochondrial enzymes monoamine oxidases with pargyline (either immediately before or after ischemia reperfusion) improved renal function and renal inflammation 28 days following surgery [39]. Pargyline administrated before ischemia reperfusion significantly reduced apoptosis, necrosis, and fibrosis. This effect was associated to decreased expression of TGF-ˇ1, collagen types I, III, and IV and to the normalization of SOD1, catalase, and inflammatory gene expression. A previous study also demonstrated that tempol reduces renal disfunction in an in vivo rat model of renal ischemia/reperfusion injury [40]. In addition, the majority of in vivo studies using Trolox, an analog of ␣-tocopherol, have also reported beneficial effects in acute cases of renal injury such as ischemia reperfusion, due to rapid solubility and potency [41]. Ishikawa and colleagues [42] demonstrated a decrease in kidney O2 − levels and improved renal function in hemi-nephrectomized rats on a Coenzyme Q10 (CoQ10 ) supplemented diet. Moreover, administration of MitoQ a mitochondrial-targeted derivative of endogenous CoQ10, in a rat model of diabetic nephropathy decreased mesangial expansion and tubulointerstitial fibrosis, thereby improving renal function [43]. In a model of chronic renal failure [44], AST-120, an oral carbonic adsorbent, improved oxidative stress in endothelial cells, measured as the oxidized/unoxidized albumin ratio. This effect was caused by a reduction in the blood level of indoxyl sulfate, a uremic toxin that induces ROS. In addition, allopurinol and its metabolite, oxypurinol, are xanthine oxidoreductase inhibitors that lower serum uric acid levels. Allopurinol treatment of diabetic mice attenuated hyperuricaemia, albuminuria, and tubulo-interstitial injury [45]. In another model of the remnant (CRF) kidney [46], the administration of omega-3 fatty acids (O3FA), effective in mitigating atherosclerosis, significantly lowered several components of oxidative stress and markers of inflammatory and fibrotic response. Furthermore, O-3FA attenuated tubulointerstitial fibrosis and inflammation in the remnant kidney. In anti-Thy1 glomerulonephritis, treatment with parthenolide, an anti-inflammatory agent related to the triterpenoid family, diminished renal inflammation via NF-␬B inhibition, decreased monocyte chemotactic protein-1 (MCP-1) and inducible nitric oxide synthase (iNOS), and improved proteinuria, tubular and glomerular damage [47]. 2.5. Clinical antioxidant therapy in CKD Several clinical studies have been performed to examine the efficacy of antioxidant interventions on oxidative stress markers in patients with CKD [14,48,49]. Unfortunately, there are only a few randomized controlled clinical trials that studied the impact of antioxidant interventions in patients with CKD. SPACE (secondary prevention with anti-oxidants of cardiovascular disease in end-stage renal disease) was a clinical trial involving 196 patients with ESRD who were randomized to receive either 800 IU of ␣tocopherol (vitamin E) per day or matching placebo [50]. During a median follow-up period of 519 days, a statistically significant reduction in the primary composite outcome, consisting of myocardial infarction, ischemic stroke, peripheral vascular disease and unstable angina, was found in patients receiving the vitamin E supplementation. In the second randomized controlled trial, Nacetylcysteine at an oral dose of 600 mg was given twice daily over a period of 15 months to patients receiving haemodialysis for ESRD [51]. This regimen was found to significantly suppress primary outcomes of cardiac events including fatal and nonfatal myocardial infarction, cardiovascular disease (CVD)-related death, need for coronary angioplasty or coronary bypass surgery, ischemic stroke, peripheral vascular disease with amputation, or

need for angioplasty. However, no beneficial effects of vitamin E or N-acetylcysteine administration were observed on all-cause mortality, suggesting that additional strategies are required to improve overall survival in dialysis patients [51]. Several studies in human CKD patients have also shown the benefit of treatment with allopurinol. For example, Kao et al. reported that allopurinol ameliorated endothelial dysfunction and prevented increased left ventricular mass in patients with CKD [52] and Siu et al. reported that allopurinol slowed progression of CKD through its ability to lower serum uric acid levels [53]. Subgroup analysis of some lipid-lowering trials, which included CKD patients, suggested that statin treatments might also reduce serum inflammatory and oxidative markers [54]. Additional studies addressing the clinical impact of a novel class of antioxidants, including endogenous antioxidants such as heme oxygenase (HO-1), for reducing oxidative stress are needed in CKD patients [55].

3. New therapeutic approaches in CKD: bardoxolone methyl and PEA 3.1. Bardoxolone methyl Multiple studies have demonstrated associations between measures of inflammation and reduced kidney function, as well as mortality, in patients with CKD [56,57]. Many renal pathogenic stimuli, including angiotensin II, contribute to inflammation by increasing the production of reactive oxygen species and activating the pro-inflammatory NF-␬B signaling pathway [58]. To date, therapies used to slow progression of CKD have focused on controlling blood pressure by targeting specific components of the RAAS system. In recent years, several approaches, including endothelin receptor antagonists (e.g. avosentan), glucosaminoglycans (e.g. sulodexide), advanced glycation end-product (AGE) inhibitors (e.g. pyridorin), erythropoiesis-stimulating agents (e.g. darbepoetin alpha), HMGCoA reductase inhibitors alone or in combination with other cholesterol-lowering agents (e.g. simvastatin + ezetimibe) and RAAS inhibitor combination strategies, ACEI plus ARB or ACEI or ARB plus a direct renin inhibitor (DRI) have produced generally disappointing results [59–61]. In that regard, a recent editorial in NEJM [30] reported that the ongoing telmisartan alone and in combination with ramipril global endpoint trial (ONTARGET), which combined an ACE inhibitor and an ARB, and the aliskiren trial in type 2 diabetes using cardiorenal endpoints (ALTITUDE), which combined an ACE inhibitor or an ARB with the direct renin inhibitor aliskiren, failed to show cardiovascular or renal protection. Fried et al. also reported in the journal “The New England Journal of Medicine, NEJM” [31] the results of the veterans affairs nephropathy in diabetes (VA NEPHRON-D) trial, in which patients with type 2 diabetes who had macroalbuminuria and an estimated glomerular filtration rate of 30.0 to 89.9 ml per minute per 1.73 m2 of body-surface area received an ARB (losartan) with or without an ACE inhibitor (lisinopril); no significant effect on renal outcome was observed. Therefore, three trials not only show that combination therapy with an ACE inhibitor and an ARB does not decrease cardiovascular and renal morbidity, but also suggest that dual therapy carries an increased risk. Indeed, two of the three trials, ALTITUDE and the VA NEPHRON-D trial were stopped early for safety reasons [30]. Thus, the association between inflammation and progressive kidney disease has directed attention toward new pathways for intervention. Nrf2 activators and/or enhancers are a recent and novel class of candidate therapeutic agents for the treatment of chronic and acute kidney injury and diseases, including CKD, ischemia-or chemical-induced acute kidney injury (AKI) as well as DN [22]. A number of pharmaceutical companies,


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Fig. 2. The structure of bardoxolone methyl and its mechanism of action. Fig. 2A shows the chemical structure of bardoxolone methyl, a derivative of the natural product oleanolic acid. Fig. 2B shows its mechanism of action. Specifically, bardoxolone methyl activates Nrf2, a transcription factor responsible for the constitutive and inducible expression of antioxidant response element-regulated genes and is recognized to be a major cellular defense. When Nrf2 is released from its repressive cytosolic protein Keap 1, after it is translocated to the nucleus, Nrf2 activates genes that encode phase II detoxifying enzymes and antioxidant enzymes. In addition to reducing the expression of proinflammatory mediators, including cytokines and adhesion molecules, Nrf2 appears to inhibit NF-␬B activation by regulating anti-inflammatory enzymes mechanism against oxidative stress.

therefore, established research programs to discover and develop potent, selective activators of Nrf-2, the first to reach full clinical development being the triterpenoid, bardoxolone methyl (Fig. 2A). Bardoxolone methyl is a novel synthetic triterpenoid antioxidant inflammation modulator. Antioxidant inflammation modulators potently induce the antioxidant and cytoprotective transcription factor Nrf2, reduce the pro-inflammatory activity of the IKK-␤/NF␬B pathway, increase the production of antioxidant and reductive molecules, and decrease oxidative stress, thereby restoring redox homeostasis in areas of inflammation [62,63]. Nrf2 activation is suppressed in animal models of CKD [64] and data from animals with genetic deletion of Nrf2 suggest that the transcription factor plays an important role in maintaining the function and structure of the kidney [65]. Histological analysis of kidney tissue in Nrf2knockout mice shows impaired antioxidant activity and increased oxidative damage, including enlarged glomeruli, mesangial cell proliferation, thickening of the glomerular basement membrane, and glomerulosclerosis. Kidney function is compromised in these animals, as evidenced by decreased creatinine clearance and shortened lifespan [65,66]. Compelling experimental and early-phase clinical data showed that bardoxolone methyl reduces oxidative stress through Nrf2 activation that supports resolution of inflammation through NF-␬B inhibition and increases estimated (eGFR) in patients with type 2 diabetes mellitus and CKD [67]. The suggestion that this drug may actually improve GFR in patients with diabetic nephropathy is intriguing; bardoxolone methyl treatment generated great interest but raised concerns as well, based on the adverse event profile of the drug [68]. Thus, as the proposed

mechanistic target of bardoxolone methyl, the Nrf2 signaling pathway is nearly omnipresent throughout the human body and the question of whether or not any beneficial effects in the kidney produced by targeting this pathway outweigh the propensity to cause negative effects in other tissues and body systems must remain. Interestingly, another Nrf2-active compound, resveratrol, also shows promise in treating patients with CKD by reducing inflammation and oxidative stress. Although there are no reports of adverse effects related to the use of resveratrol in humans, even at high doses, clinical trials must be developed to explore resveratrol effects in CKD [69]. It is necessary, however, to examine the transcriptional regulatory cofactors associated with Nrf2 in order to understand its role in the pathogenesis of kidney disease and how the system might be affected by pharmacological intervention. 3.2. Nrf2-Keap1 signaling pathway The Nrf2-Keap1 (nuclear factor erythroid 2-related factor 2-Kelch-like ECH-associated protein 1) system is one of the most critical cytoprotective mechanisms acquired in vertebrates over the course of evolution. Nrf2 is a basic-region leucine zipper transcription factor that regulates basal activity and inducible expression of a battery of environmental stress response genes [70]. Nrf2 is normally maintained in the cytoplasm by interaction with the cytosolic repressor protein Keap1, which promotes ubiquitination and proteosomal degradation of Nrf2 [71]. Exposure to both endogenous and exogenous molecules as reactive oxygen species, 15-deoxy-delta12,14-prostaglandin J2, dithiolethiones,

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triterpenoids, isothiocyanates, can modify reactive cysteines in Keap1 [72], thus rescuing Nrf2 from proteosomal degradation [73] and may also facilitate the nuclear localization of Nrf2 [74]. In the nucleus, Nrf2 heterodimerizes with other transcription factors, leading to the binding of Nrf2 to the cis-regulatory, anti-oxidant response element (ARE) located in the promoter region of Nrf2 target genes, thereby activating their transcription. A series of proteins have been identified that may modulate Nrf2 transcription activation, thereby showing the influence of multiple signaling pathways on Nrf2 responses [75]. Nrf2 regulates the transcription of cytoprotective genes including those encoding antioxidant and phase II-detoxifying enzymes such as catalase, SOD, HO-1, NAD(P)H:quinone oxidoreductase 1, glutathione peroxidase-2 and glutathione S-transferase [70]. Since Nrf2 is ubiquitously expressed, it plays a critical role in protecting many cell types and organ systems from oxidative stress [76]. Nrf2-deficient mice had enhanced susceptibility to pulmonary inflammation [77], acetaminophen hepatotoxicity [78] and neurodegenerative disease [79]. Moreover, ablation of the Nrf2 gene in mice has been shown to cause lupus-like autoimmune nephritis, and worsen diabetesinduced oxidative stress, inflammation and renal injury [65,80]. Nfr2 knockout mice were more susceptible either to ischemia reperfusion injury or to cisplatin-induced nephrotoxicity [81,82], and a protective role of Nrf2 activation has been demonstrated, as in the case of a cholestatic liver injury model where disruption of Keap1 resulted in sustained activation of hepatic Nrf2-regulated detoxifying enzymes and antioxidative stress genes, with attenuation of liver injury [83]. A recent study also explored the role of Nrf2 in AKI by amplifying Nrf2 activation in vivo and in vitro with the synthetic triterpenoid CDDO-imidazolide. Mice treated with CDDO-imidazolide and undergoing experimental bilateral ischemic AKI had improved survival and renal function [84]. Therefore, bardoxolone methyl promotes activation of Nrf2, which is released from the suppressor Keap1 and translocates into the nucleus where it regulates transcription of antioxidant genes containing ARE sequences in their promoter region. These genes are involved in the elimination and/or removal of ROS and inhibition of NF-␬B (Fig. 2B) [22]. In particular, bardoxolone methyl is able to upregulate the renal mRNA expression of critical Nrf2 target genes, NAD(P)H:quinoneoxidoreductase 1 (NQO1) protein expression, and NQO1 and Glutathione reductase (GSR) enzyme activity, as well as total kidney glutathione content. These data may result, at least in part, from reduced protein expression of renal tubular megalin [85]. 3.3. Experimental studies Preclinical studies have shown that bardoxolone methyl ameliorated murine ischemic acute kidney injury and increased the expression of the renal-protective genes Nrf2, PPAR␥, and HO1 [86]. Treatment with the bardoxolone methyl analog RTA 405 attenuated blood pressure increase and endothelial dysfunction in a 5/6 nephrectomy model of pressure overload [87]. In a mouse model of protein overload proteinuria, early administration of RTA 405 limited interstitial inflammation and fibrosis and reduced oxidative stress in the kidney [87]. RTA 405, a triterpenoid analog of bardoxolone methyl, was used as a surrogate molecule since bardoxolone methyl undergoes rodent-specific metabolism to toxic moieties. Further, potential therapeutic mechanisms of bardoxolone in DN were investigated in a type 2 diabetes model, the Zucker diabetic fatty (ZDF) rat. At 3-months of age, rats received treatment with two dosages of the bardoxolone methyl analog RTA 405 alone or in combination with the ACE inhibitor ramipril starting at a phase of overt nephropathy [87]. The results were unexpected. RTA 405 caused adverse changes in the physical status of ZDF rats as early as 1 month after starting treatment.

Acute reductions in food intake and diuresis with decline in body weight, worsening of dyslipidaemia and increase in blood pressure were recorded. Early elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels suggested acute hepatic toxicity. Later, due to the possibility of degradation products having been present in the RTA 405 used in this study, another synthetic triterpenoid derivative of bardeloxone methyl and RTA 405, dihydro-CDDO-trifluoroethyl amide (dh404) [88] was also administered to ZDF rats, but did not show any beneficial effects on renal disease, instead causing a trend toward an increase in proteinuria, glomerulosclerosis, tubular casts and interstitial inflammation. This supports the continuing use of animal models of human disease, where possible, as a critical step for the investigation of new drugs before proceeding with human administration to provide insights into pathogenetic mechanisms as well as unexpected side effects. 3.4. Clinical studies Initially, bardoxolone methyl was evaluated for potential anticancer activity and during phase I trials, was shown, unexpectedly, to improve renal function, assessed in terms of serum creatinine and creatinine clearance, especially in patients who had previously suffered from CKD [89]. These observations suggested that bardoxolone methyl might induce potential renoprotective actions in patients with CKD and type 2 diabetes mellitus. This was initially noted in an exploratory phase 2 open-label trial and then in a larger randomized clinical trial. In the first trial, 20 patients with moderate-severe diabetic CKD were evaluated after 8 weeks of bardoxolone methyl treatment [67]. Notably, there was a significant increase in estimated GFR at 4 weeks using a dosage of 25 mg/day that became more significant after a further 4 weeks treatment during which a dosage of 75 mg/day was used. Serum creatinine and BUN decreased and creatinine clearance increased, without any changes in total excretion or tubular secretion of creatinine. Notably, a slight, but significant albuminuria was observed in patients with CKD in association with bardoxolone methyl administration. Adverse events were generally manageable and mild to moderate in severity with the most common, muscle spasms, affecting 35% of patients. In a phase 2, double-blind, randomized, placebo-controlled trial (the BEAM study), bardoxolone methyl at three dosage levels (25, 75 and 150 mg/day) was associated with improvement in the estimated GFR in patients with advanced CKD and type 2 diabetes at 24 weeks [68]. The improvement persisted at 52 weeks, suggesting that bardoxolone methyl may have promise for the treatment of CKD. Adverse events were generally manageable and mild to moderate in severity. The most frequently reported adverse event in the bardoxolone methyl group was again muscle spasm, which although generally mild, had a dose-related incidence from 42 to 61%. Additionally, 71% of all bardoxolone methyl treated patients had transient ALT elevations between 2 and 4 weeks after the start of therapy and 11% had elevations that were three times the upper limit of normal although they did not persist. Problematically, an editorial in the American Journal of Nephrology (Tayek and Kalantar-Zadeh) [85] pointed out that the calculation of eGFR in the BEAM study was based on the level of serum creatinine, which is influenced by muscle mass and probably dietary meat intake. A dramatic weight loss differential of 5–10 kg was observed in patients who received bardoxolone methyl compared with placebo. The bardoxolone weight loss was 7.7–10.1 kg compared to 2.4 kg in the placebo group. It is possible, therefore, that the efficacy attributed to bardoxolone methyl in improving kidney function in the BEAM study was not produced by an anti-inflammatory effect on the kidney. Following the reported successful outcome of the BEAM study, a multinational, double-blind, placebo-controlled phase 3 outcomes


study (BEACON) was started in June 2011, to evaluate bardoxolone methyl’s impact on progression to ESRD or cardiovascular death in patients with stage 4 CKD and type 2 diabetes. In this study, 2185 patients with type 2 diabetes and stage 4 chronic kidney disease were enrolled and were randomized to receive either 20 mg/day of bardoxolone methyl or placebo. The study, however, was halted prematurely upon the recommendation of the Independent Data Monitoring Committee due to concerns about an excess of serious adverse events and mortality in the bardoxolone methyl arm. Due to the early termination, median follow-up amounted to only 9 months, and during that time the same proportion of patients in each group (6%) hit the primary composite endpoint. In the bardoxolone methyl group, ESRD developed in 43 patients, and 27 patients died from cardiovascular causes; in the placebo group, ESRD developed in 51 patients, and 19 patients died from cardiovascular causes. Overall, however, a total of 96 patients in the bardoxolone methyl group were hospitalized for or died from heart failure, as compared with 55 in the placebo group. Estimated GFR, blood pressure, and the urinary albumin-tocreatinine ratio increased significantly and body weight decreased significantly in the bardoxolone methyl group, as compared with the placebo group [90], an effect that was also seen in the earlier BEAM study [54]. The reasons for these adverse events are unclear. Chertow and colleagues speculated that fluid retention, increased afterload, and higher heart rate contributed to heart failure in patients in the bardoxolone methyl group [90]. In addition, direct toxic effects are possible [90]. Unfortunately, in the mentioned trials with bardoxolone no information is provided about the precise genes and enzyme activities that were upregulated, thus the answer to the question “why” still remains elusive. There has been no information provided about the future development status of the compound. In the absence of such information, it must be assumed that it will not be progressed. Unfortunately, as pointed out in an accompanying editorial to the bardoxolone study report in the NEJM [91] the failure rate of new drug therapies in clinical trials is extraordinarily high, exceeding 90% overall; even in phase 3 trials, it is still approximately 50%. In addition to bardoxolone methyl, a series of other new therapies for CKD have foundered over the course of drug development highlighting a need for improvement in the development of drugs for this condition. Recently, Reisman et al. [85] as part of the long-term toxicology program of the compound investigated whether the bardoxolone methyl-induced albuminuria may result from the downregulation of megalin, a protein involved in the tubular reabsorption of albumin and lipid-bound proteins. Administration of bardoxolone methyl to cynomolgus monkeys at 30 mg/kg/day for 28 days significantly decreased the protein expression of megalin in renal tubules, which inversely correlated with the urine albumin-to-creatinine ratio. Daily oral administration of bardoxolone methyl at doses up to 300 mg/kg/day to monkeys for 1 year, however, did not lead to any adverse effects on renal histopathological findings, but reduced serum creatinine and BUN, as observed in patients with CKD. It should be noted, however, that in the 28-day study, the bardoxolone methyl-treated monkeys showed a minimal but significant loss in body weight compared to controls at 28 days and in the 12month study all bardoxolone methyl-treated groups gained less weight than controls over the course of the study. This mirrors the loss in body weight seen in the BEAM and BEACON studies (mentioned above) in bardoxolone methyl-treated patients with CKD and type 2 diabetes [68]. These findings suggest that the increase in albuminuria that accompanies bardoxolone methyl administration may result, at least in part, from reduced expression of megalin, which seems to occur without major adverse effects, apart from some effect on body weight and with strong induction of Nrf2 target genes [85]. Although it remains mechanistically interesting, the adverse safety profile of bardoxolone methyl renders it unsuitable

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for therapeutic use. The search for new therapies, therefore, must continue. 3.5. PEA: experimental evidence PEA is a saturated fatty acid derivative in which the carboxylate function is amidated by the primary amine of ethanolamine [92]. PEA is a naturally occurring N-acylethanolamine (NAE) endorsed with pleiotropic effects, collectively considered to play protective and homeodynamic roles in the animal [93] and vegetable kingdoms [94]. Like other NAEs, PEA acts locally, and its tissue levels are tightly regulated through a balance between anabolic and catabolic pathways, respectively catalyzed by the biosynthetic enzyme NAPE-selective phospholipase D and the degradative N-acylethanolamine-hydrolyzing acid amidase (Fig. 3) [92]. PEA, besides penetrating the cells by passive transfer, due to its high lipophilicity, is taken up by cells through a facilitated transport system that is apparently similar in neuronal and immune cells (i.e., mast cells MCs) and pharmacologically distinct from that of the PEA analog anandamide [95]. PEA was first discovered in the late 1950s, when it was shown that the anti-allergic and anti-inflammatory activity exerted by dietary supplementation with egg yolk, peanut oil or soybean lecithin [96] was due to a specific lipid fraction corresponding to PEA [97]. Anti-inflammatory and protective activities of PEA were confirmed in several models of inflammation, i.e. carrageenan-induced paw edema, adjuvant-induced arthritis, tuberculin hypersensitivity and ischemia reperfusion injury [9,92,98]. Our studies also demonstrated that ultramicronized PEA treatment protected against MPTP-induced neurotoxicity in a mouse model of Parkinson’s disease and protected the neurovascular unit and reduced secondary injury after traumatic brain injury in mice [99,100]. Repeated ultramicronized PEA administration significantly reduced the degree of spinal cord inflammation and tissue injury, neutrophil infiltration, nitrotyrosine formation, pro-inflammatory cytokine expression, NF-␬B activation, and apoptosis. Moreover, ultramicronized PEA treatment significantly ameliorated the recovery of motor function [101]. In a more recent paper by Esposito et al. [10] significantly less mast cell (MC) density and degranulation were also observed after experimental SCI in the spinal cord tissues collected from mice that had been treated with ultramicronized PEA. In addition, a veterinary dermatological trial in 2001 reported that one-month treatment with PEA resulted in decreased pruritus, erythema and alopecia in cats affected with hypersensitivity skin disorders, i.e., eosiniphilic plaques and eosinophilic granuloma [102]. Moreover, a recently published paper showed that a single oral dose of PEA (10 mg/kg) significantly reduced the wheal and flare reaction in dogs with skin hypersensitivity [103]. Oxidative stress has been implicated as one of the major factors influencing the progression of renal injury [104] also contributing to the induction of pro-inflammatory and pro-fibrotic pathways [105,106]. ROS and RNS are strong inducers of MC activation and thus their subsequent release of primary and secondary pro-inflammatory and pro-fibrotic cytokines and growth factors. Compelling experimental evidence supports the hypothesis that renal infiltration by MCs plays a relevant role in the progression of renal disease from inflammation to fibrosis. In that regard, MCs represent important cellular targets of PEA intervention. A previous study clearly demonstrated that PEA significantly reduced MCs infiltration in kidney tissues in a mouse model of renal ischemia-reperfusion injury IRI (as will be discussed in another section) [9]. A recent work also demonstrated the potential beneficial effects of long-term treatment with PEA on conditions associated with kidney disease, such as hypertension. In the spontaneously hypertensive rat (SHR), PEA administered daily by subcutaneous injection at 30 mg/kg/day for 35 days was able to lower systolic blood pressure and to prevent renal damage through a reduction of

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Fig. 3. The biosynthesis of PEA. N-Acylated ethanolamines (NAE) are naturally occurring lipids that have different bioactivities. PEA is a naturally occurring NAE with protective effects. It is produced “on demand”, it is not stored in vescicles. Phosphatidylethanolamine, a cell membrane phospholipid characteristic of nervous tissue, forms N-acylphosphatidylethanolamines (NAPEs) when cells are subjected to potentially harmful stimuli. After being cleaved by phospholipases (NAPE-PLD), NAPEs can be transformed into NAE. PEA is metabolized by cellular enzymes, fatty acid amide hydrolase (FAAH) or N-acylethanolamine-hydrolyzing acid amidase (NAAA), the latter of which has more specificity toward PEA over other fatty acid amides.

glomerulosclerosis, and tubulointerstitial fibrosis, modulating key enzymes in ROS/RNS synthesis and/or scavenging and angiotensin II (AT) receptors homeostasis [107]. The effect, however, of orally administered PEA, on blood pressure in humans has not yet been reported. 3.6. Mechanism of action PEA was originally considered to be an endocannabinoid (eCB), since at first it was suggested to be a cannabinoid receptor 2 (CB2) agonist [108]. Currently, several mechanisms have been proposed to explain the anti-inflammatory and anti-hyperalgesic effects of PEA, including: (i) the activation of a cell surface receptor (i.e. CB2like or, alternatively, the orphan GPR55 receptor) or otherwise a nuclear receptor of the peroxisome proliferator-activated receptor (PPAR) family [109,110]; (ii) the down-modulation of mast cell hyperactivity (autocoid local inflammation antagonism mechanism) [111]; (iii) an action as “entourage” compound, i.e. the augmentation of eCB activities at their receptors and/or the inhibition of eCB degradation [112]. Although its presence in mammalian tissues has been known since the 1960s, PEA has emerged only recently among other bioactive N-acylethanolamines as an important endogenous lipid modulator, which, because of its chemical stability, can be also administered exogenously as the active principle of current anti-inflammatory and analgesic preparations [93]. Chronic inflammation and oxidative stress, features that are closely associated with NF-␬B activation play a key role in the development and progression of CKD and its related disorders. In that regard, during kidney inflammation, PEA (identified in numerous articles as an endogenous PPAR-␣ agonist) can modulate the PPAR-␣ pathway that is able to attenuate NF␬B-induced inflammatory factors (IL-1, or TNF˛), inhibit infiltration and activation of MC, reduce masengial matrix proliferation induced by reactive oxidative stress (ROS) which then resulted in albuminuria [9,113]. Thus, by reducing low

grade chronic inflammation and by inhibiting oxidative stress, PEA may reduce risk factors associated with the progression of CKD and have the potential to offer significant benefit to patients with CKD at early and, quite possibly, even at late stages of the disease. Moreover, more studies are required to define the exact mechanism of PEA in the kidney inflammation and delineate additional pathways by which PEA suppresses NF-␬B activation (Fig. 4) [9]. 3.7. The role of PPAR-˛ receptors in kidney disease PPARs, members of the superfamily of ligand regulated transcription factors, are expressed in the cardiovascular system and control diverse vascular functions by mediating appropriate changes of gene expression through binding to specific peroxisome proliferator response elements (PPREs). Prior studies in animal models had described the beneficial roles for PPARs in reducing renal injury and dysfunction [114]. In particular, PPAR-␣, which is highly expressed in kidney, liver, and heart, has been shown to be involved in the control of blood pressure, and hypertensiverelated complications, such as stroke and renal damage [115,116]. The protective role for PPAR-␣ activators identified in cardiovascular diseases has been ascribed to various effects. Beyond their effect on vascular function, PPAR-␣ agonists (fenofibrates) exert a renoprotective effect through anti-inflammatory [117] and anti-oxidant properties via the downregulation of inflammatory cytokines and reduction of oxidative stress [118]. Moreover, the renoprotective effect of exogenous PPAR-␣ agonists (WY 14643 and ciprofibrate) via induced megalin expression has been demonstrated in porcine LLC-PKI (epithelial cell line derived from kidney proximal tubule) and in BALB/c mice made albuminuric by bovine serum albumin (BSA) administration [119]. Additionally, however, in this study the PPAR␥ agonist rosiglitazone was also shown to counteract the reduction in megalin produced by BSA, so multiple systems may be involved. Previous reports also demonstrated


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Fig. 4. Therapeutic targets of PEA in renal diseases. PEA has been shown to be effective in several experimental models of inflammation. PEA is considered an endogenous activator of PPAR-␣, interacting with this receptor to inhibit inflammatory pathways such as NF-␬B and consequently, oxidative stress. In addition, the efficacy of PEA in controlling mast cell (MC) behavior, which likely accounts for its many anti-inflammatory, anti-angiogenic and analgesic effects, is well recognized (ALIA mechanism). MCs are key players in orchestrating several disorders including both acute and chronic inflammatory processes. Thus, the effects of PEA in the control of mast cell degranulation could be an important therapeutic strategy to modulate the pathological scenario associated with renal diseases.

that pioglitazone (PIO), another PPAR␥ agonist, decreased cell proliferation, interstitial fibrosis, and inflammation, and ameliorated PKD progression in PCK rats by inhibition of renal Raf/MEK/ERK and AKT/mTOR/S6 activity in diseased renal cells [120,121]. In addition, clinical evidence suggests a beneficial effect of fibrate treatment in patients with type-2 diabetes [122] and data from FIELD (fenofibrate intervention for event lowering in diabetes) study also indicate promising effects with fenofibrate in preventing progression of diabetes-related microvascular complications [123]. In db/db type-2 diabetic mice, treatment with fenofibrate markedly lowers urinary albumin excretion and improves glomerular mesangial expansion [124]. Although microalbuminuria may rather be a marker for cardiovascular disease [125], its applications as a reversible marker of kidney and vascular damage were reported [126]. Therefore, both clinical observations and rodent experiments suggest that PPAR-˛ activation may play a beneficial role in diabetes induced nephropathy. Although the effects of PPAR-␣ have not been fully investigated, they are shown to be protective in chronic kidney diseases. 3.8. PEA as a PPAR-˛ agonist in kidney disease A recent study clearly demonstrated that PEA significantly attenuated the degree of renal dysfunction, injury, and inflammation caused by ischemia-reperfusion injury and these positive effects were at least in part dependent on the PPAR-˛ pathway [9]. Specifically, it was shown for the first time that in this model of experimental IRthat treatment with PEA ameliorated morphological features of IR (including brush border loss, nuclear condensation, cytoplasmatic swelling, and consistent loss of significant numbers of nuclei from tubular profiles) as verified by macroscopic and histological findings. In particular, treatment with PEA also significantly reduced the lipid peroxidation and nitrotyrosine expression in the kidney possibly in part related to the reduced neutrophil recruitment [9]. PPAR-␣ agonists reduce proteinuria and have been linked to the regulation of the renin angiotensin-system, with a reduction of AT1 and the induction of AT2 receptor expression in kidney. It has been further shown that PPAR-␣ activation using docosahexaenoic acid (DHA) suppressed oxidative stress through the inhibition of Ang II-induced activation of NADPH oxidase and an increase in scavenging enzymes and improvement in endothelial function [127]. In that regard, a recent study demonstrated that PEA treatment

in SHR rats was able to down-regulate kidney AT1 expression and to enhance AT2 [107], suggesting a beneficial effect of PEA not only in reducing AT1-induced detrimental effects, such as upregulation or increase in NADPH oxidase activity, but also through a contribution of AT2 to its renoprotective effect [107]. Thus, concomitant with a decrease in the generation of oxygen and nitrogen molecules, PEA treatment also increased the protective antioxidant defence, specifically Cu/Zn SOD, protecting against kidney damage [107]. Further, Konishi et al. [128] also investigated the significance of chymase-positive MCs in the pathophysiology of renal damage. MC-derived chymase is one of the serine proteases present in the secretory granules of MCs and is an angiotensin II (ATII) forming enzyme, similar to ACE. The modification of renal AT1/AT2 by PEA treatment was also accompanied by a reduction in ACE expression [107], this enzyme being most important for AngII formation in rats, but less important in humans, who have alternative pathways of AngII synthesis. These data support the premise that PEA can modulate the RAAS system, not only by shifting the AT1/AT2 balance toward a more normotensive status, but also by reducing the expression of ACE, most likely associated with a decrease of AngII level [107]. Thus, PEA treatment can reduce progression of renal damage, attenuate up-regulation of the pro-oxidant/pro-inflammatory systems in the kidney and perhaps produce a modulation of the blood pressure elevation often associated with renal disease. These observations point to its potential role in the hemodynamic and non-hemodynamic disorders that contribute to progression of renal disease, expanding its possible therapeutic use in hypertension and kidney diseases. 3.9. Clinical evidence The effect of PEA has been explored in many clinical studies of inflammatory and pain syndromes in both human and in companion animals. Positive results of such PEA treatment were reported in over 20 clinical studies with more than 2000 patients having been successfully treated without any adverse events of note being reported [129]. The first clinical evidence appeared during the 1970s, which demonstrated the ability of PEA to control some symptoms of respiratory tract infections [130]. Following this pioneer work, and on the basis of the ability of PEA to control MC activation, a variety of clinical trials were initiated. Several studies demonstrated that PEA was able to control pain syndrome, particularly in MC sustained pain. A recent paper showed that

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administration of PEA and polydatin (a resveratrol analog that can be isolated from various plant sources, including Giant knotweed rhizome, the dried rhizome and root of Polygonum cuspidatum, a common medicinal plant in China) appeared to be very useful in controlling chronic pelvic pain associated with endometriosis [131]. In concurrence with this report, PEA was found effective in patients with chemotherapy-induced painful neuropathy [132]. Moreover, a recent pilot study was carried out to assess the efficacy and safety of twice daily application of a topical emulsion containing 2% adelmidrol, an analog of PEA, on 20 pediatric patients suffering from mild atopic dermatitis [133]. Complete resolution of symptoms was seen in 80% of patients, postulated to be due to the inhibition of NGF release from cutaneous MCs. Thus, the beneficial effects of PEA have been demonstrated in a number of clinical trials in various inflammatory and pain syndromes and it has an extremely well-documented safety profile [129]. There is, however, limited clinical evidence about its effects in renal diseases. Further clinical research with PEA is needed to investigate its clinical utility in patients with CKD.

4. Conclusions Renal disease may arise due to a number of reasons. In most cases, it is related to a history of diabetes mellitus or hypertension, but glomerulonephritis and autoimmune diseases can also lead to CKD. Given an early diagnosis, the progression of CKD can be slowed down by lowering the blood pressure with ACE inhibitors or ARBs, but alternative therapy that can stop the renal impairment is still unavailable [134]. Because inflammation and oxidative stress are implicated in the pathogenesis of CKD and other complications, compounds capable of attenuating these processes or conditions should attract particular interest for evaluation in treating CKD. The beneficial effects of bardoxolone methyl on DN observed in initial clinical studies have been negated by events from the later BEACON study, given emergent adverse reactions and deaths that have only very recently been described and quantified [56]. There had been hope that bardoxolone methyl offered a novel mechanistic approach in treating CKD but this is now in doubt. It is apparent, therefore, that much more research is necessary to get a better understanding of the new modes of action of kidneyprotective anti-inflammatory molecules [134]. In the meantime, PEA may represent a new important therapeutic approach worthy of further investigation for renal disease. Given its demonstrated lack of side effects, PEA could be a very good candidate for treating inflammation in chronic nephropathy. Actually, this lack of side effects during PEA administration has been repeatedly reported in published clinical studies [129]. Furthermore and most importantly, the anti-inflammatory effect of PEA is not accompanied by tolerance following repeated administration of high doses, enhancing the potential therapeutic utility of the compound [135]. In conclusion, data from our and other laboratories in several experimental models suggest that PEA can reduce the development of renal inflammation and tissue injury, indicating that it may have therapeutic utility in kidney diseases and/or associated conditions. Although considerable effort and attention have been directed to studying the efficacy of PEA in vitro and in vivo models, much work remains to be done to fully elucidate its exact mechanism of action.

Conflict of interest James Attley, one of the authors of this review article, serves as a consultant and advisor to Prismic Pharmaceuticals, and has an equity-based interest in the company.

Acknowledgments The authors would like to thank Giovanni Leotta for his excellent technical assistance during this study and Miss Valentina Malvagni for editorial assistance with the manuscript. References [1] Nangaku M. Chronic hypoxia and tubulointerstitial injury: a final common pathway to end-stage renal failure. J Am Soc Nephrol 2006;17:17–25. [2] Aksu U, Demirci C, Ince C. The pathogenesis of acute kidney injury and the toxic triangle of oxygen, reactive oxygen species and nitric oxide. Contrib Nephrol 2011;174:119–28. [3] Singbartl K, Kellum JA. AKI in the ICU: definition, epidemiology, risk stratification, and outcomes. Kidney Int 2012;81:819–25. [4] Okamura DM, Himmelfarb J. Tipping the redox balance of oxidative stress in fibrogenic pathways in chronic kidney disease. Pediatr Nephrol 2009;24:2309–19. [5] Yates MS, Tauchi M, Katsuoka F, Flanders KC, Liby KT, Honda T, et al. 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The iron cycle in chronic kidney disease (CKD): from genetics and experimental models to CKD patients.

Inflammation, Endothelial Dysfunction and Increased Left Ventricular Mass in Chronic Kidney Disease (CKD) Patients: A Longitudinal Study.

Epigenetic regulation in the acute kidney injury (AKI) to chronic kidney disease transition (CKD).

International Network of Chronic Kidney Disease cohort studies (iNET-CKD): a global network of chronic kidney disease cohorts.

Targeting inflammation as a therapeutic strategy for drug-resistant epilepsies: an update of new immune-modulating approaches.

Asthma. New therapeutic approaches.

New Markers of Inflammation and Tubular Damage in Children with Chronic Kidney Disease.

Risk factors for CKD progression in Japanese patients: findings from the Chronic Kidney Disease Japan Cohort (CKD-JAC) study.

2. Inflammation, Cytokines and Chemokines in Chronic Kidney Disease.

Nutritional strategies to reduce inflammation in chronic kidney disease patients.

Inflammation and nutrition in children with chronic kidney disease.

Targeting inflammation in diabetes: Newer therapeutic options.

Vitamin D and chronic kidney disease-mineral bone disease (CKD-MBD).

Therapeutic Approaches Targeting MYC-Driven Prostate Cancer.