Nitrolipids in Kidney Physiology and Disease

Abstract

The kidneys are vital organs responsible for maintaining body fluid homeostasis within proper physiologic ranges. Kidney disease is an epidemic clinical problem causing significant morbidity and mortality, and current treatments are limited to renin-angiotensin system blockade or renal replacement therapy for the majority of affected individuals. There is a critical, unmet need for novel pharmacological agents to improve the outcome of patients with kidney disease. Nitro-oleic acid (NO₂-OA) is an endogenously generated electrophilic compound with the capacity to modify thiols in proteins, altering their function. The most important targets appear to be the Keap1/Nrf2 and NF-κB pathways, which have widespread effects on antioxidant, detoxifying, and inflammatory responses in cells and tissues. Through these and potentially additional protective actions, NO₂-OA may be capable of preserving or enhancing kidney function in acute and chronic kidney diseases.

Introduction

The kidneys maintain body fluid composition within narrow physiologic ranges, preserving appropriate osmolarity, ionic composition, pH, and volume while excreting metabolic and environmental wastes and toxins. This complex task is achieved by the approximately one million nephrons in each kidney. Each nephron is comprised of a glomerulus performing plasma filtration, and a complex and convoluted renal tubule that refines the composition of what will ultimately become the urine. If these structures become damaged, the kidney is unable to function appropriately.

Nitrated fatty acids (NO₂-FA) are a family of endogenously-produced bioactive electrophiles formed by the nitration of dietary lipids, including linoleic and oleic acids. Nitro-oleic acid (NO₂-OA) is a promising therapeutic NO₂-FA which post-translationally modifies protein thiols through reversible Michael addition, exerting potent effects in diverse disease models.^1–6 Fatty acids in plasma bind to proteins such as albumin and fatty-acid binding proteins through hydrophobic and van der Waals interactions, and as such are not filtered at the glomerulus and are excreted only in minute quantities in the urine. By contrast, electrophilic NO₂-FA are capable of forming reversible covalent Michael adducts with low-molecular weight thiols such as glutathione (GSH), yielding compounds that are significantly more hydrophilic than the parent fatty acid. Following a single oral dose of [¹⁴C] 10-NO₂-OA administered to rats, 34.9% of recovered radioactivity was observed in the urine, while 47.9% was present in feces⁷. Both N-acetylcysteine adducts as well as β–oxidation and ω-carboxylation products were detected in the urine, suggesting that GSH adducts are formed and undergo further metabolism. Electrophilic NO₂-FA N-acetylcysteine adducts, cysteine adducts and β–oxidation/ ω-carboxylation metabolites are also detected in the urine of healthy human volunteers⁸ and humans receiving an IV formulation of NO₂-OA⁹. It is not definitively known whether urinary NO₂-OA, its conjugates, and its metabolites are derived primarily from filtration or tubular secretion. However, other oxidized lipid mediators such as leukotrienes and prostaglandins are secreted into the tubular lumen as hydrophilic conjugates following Phase II metabolism^10,11. These results suggest that NO₂-FA and NO₂-FA adducts may reach target cells in the kidney either from the apical or basolateral sides. It has been postulated that the reversibility of the Michael adduct through thiol β-elimination may establish new equilibria and form bioactive NO₂-OA in the glomerular filtrate and urine, leading to cytoprotective signaling activities in the renal tubules and collecting system, as well as distally in the bladder and urinary tract.

Spectrum and Mechanisms of Kidney Disease

Chronic kidney diseases (CKD) are a result of longstanding conditions such as diabetes or hypertension and generally represent permanent and irreversible injury. On the other hand, acute kidney injury (AKI) is usually incited by a specific event such as ischemia, sepsis, or toxin exposures. The term acute tubular necrosis (ATN) has often been used interchangeably for AKI, since renal tubules are usually the focus of injury. While some AKI is reversible and resolves with tubular regeneration, AKI can also progress to permanent CKD if the injury is severe¹². Reduction-oxidation (redox) imbalance and inflammation play a role in pathogenesis of both AKI and CKD, and may be ameliorated by nitrated fatty acids via the mechanisms discussed below.

Redox imbalance is the overabundance of oxidizing species due to excessive production or insufficient scavenging by antioxidant mechanisms and plays a major role in kidney disease pathology¹³. Reactive species cause persistent cell and tissue damage through off-target oxidation of proteins, lipids, and nucleic acids, and can activate multiple pro-fibrotic and pro-inflammatory signaling cascades, including Nuclear Factor-κB (NF-κB), Transforming Growth Factor-β (TGF-β), and p38 Mitogen Activated Protein Kinase (MAPK)^14–17. Oxidant generation has been attributed to increased abundance of NADPH oxidase (Nox) enzymes as well as to mitochondrial dysfunction^13,18.

Inflammation is another unifying mechanism in the initiation and progression of kidney disease^16–18. A variety of inflammatory cells infiltrate the kidney in AKI and CKD^19–21. Inflammation correlates with loss of renal function in animals and humans, and interventions that limit inflammation are protective^22–24. Inflammatory cells contribute to tissue scarring/fibrosis via production of transforming growth factor-β (TGF-β) and other growth factors, which stimulate myofibroblast differentiation and deposition of extracellular matrix²⁵.

NO₂-OA reduces redox imbalance and inflammation through targeting of the Kelch-like ECH-associated protein 1 (Keap1) / Nuclear factor erythroid 2-related factor 2 (Nrf2) and Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways, respectively. Nrf2 is a transcription factor regulating expression of over 250 genes under the control of the antioxidant response element (ARE) motif. These targets have roles in phase II conjugation reactions, reduction reactions, multidrug resistance, catabolism of oxidants, and biosynthesis of reductants and small-molecule antioxidants such as glutathione^26,27. Redox- and electrophile-induced modification of critical cysteine thiolates in the Nrf2 repressor Keap1 leads to increased Nrf2 activity to protect the cell from these stressors. Encouragingly, one recent meta-analysis of genome-wide association studies revealed that alterations in Nrf2 signaling is a central hub connecting the metabolic and inflammatory pathways implicated in CKD²⁸. A variety of available pharmacological activators of Nrf2, including NO₂-OA, are electrophiles that inactivate the Keap1 repressor²⁹ and initiate gene expression programs downstream of Nrf2 which prime tissues to respond to insult. It is worth noting that the responses to Nrf2 activation are specific with regards to cell and tissue type, and overlapping but non-identical responses have been observed with different pharmacologic and genetic models of Nrf2 activation^30–32.

NO₂-OA also has anti-inflammatory properties via electrophilic reaction with key cysteine residues in both the NF-κB p65 subunit and a related protein, inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ)^33–36. Indeed, LPS-induced pro-inflammatory cytokine secretion and leukocyte recruitment were diminished by NO₂-OA^4,33,37,38. These anti-inflammatory effects of NO₂-OA could occur through direct NF-κB inhibition, but there is evidence for indirect effects since maintenance of a reducing intracellular environment by Nrf2 would dampen NF-κB activation^39–41. Crosstalk between Nrf2 and NF-kB also occurs, since the attenuation of NF-κB enhances Nrf2 activity through reduced expression of Keap1⁴². Further, NO₂-OA suppresses the signal transducer and activator of transcription (STAT) signaling pathway, which is proinflammatory³⁴.

NO₂-OA has potential renoprotective effects through engagement of other pathways. Heat shock proteins (HSPs) are protective chaperones upregulated at times of cellular stress, and are upregulated by NO₂-OA^43,44. Similarly, NO₂-FA including NO₂-OA and nitro-conjugated linoleic acid (NO₂-cLA) enhance expression of heme oxygenase 1 (HO-1), effects that have been well-documented in vascular endothelium^45–47. HO-1 prevents heme toxicity through its degradation, while simultaneously generating protective biliverdin/bilirubin, carbon monoxide, and upregulating ferritin⁴⁸. These properties are believed to confer protective effects in kidney disease⁴⁹. Yet another mechanism of cellular protection is through peroxisome proliferator-activated receptors (PPARs), with PPAR-γ being upregulated by NO₂-OA^50,51. Additional pleiotropic actions are suggested by other chapters in this volume. Taken together, these studies support the primary concept that NO₂-OA may be therapeutic for renal disease through multiple additive or synergistic pathways. The existing experimental trials testing NO₂-OA in kidney-related diseases are summarized in Table 1 and described in detail below.

Table 1. Summary of literature describing renal effects of NO₂-OA.

Relevant information from each publication including species/strain, injury model, drug and dosing, as well as a summary of findings, are included.

Reference	Species	Model	Drug	Dose	Effect of Drug
1	Mouse (B6129SF2/J)	AKI - Bilateral I/R (30min)	NO2-OA	500µg/kg starting 50min after ischemia and every 6 hours for 24 hours.	↓ histologic injury score, plasma Cre/BUN, and tissue injury markers (MPO, TNF- α, ICAM-1, IL-1β, NOX,)
52	Human (tubular epithelial cell)	AKI – Oxygen/Glucose Deprivation	NO2-OA	1.25µM for 45min prior to deprivation	↓ Bax activation and inhibition of apoptotic cascade
53	Human (tubular epithelial cell)	AKI – Oxygen/Glucose Deprivation	NO2-OA	1.25µM for 45min prior to deprivation	↓ NOX2/4, ↑ Nrf2 targets
54	Mouse (C57BL/6J)	AKI – Sepsis (10mg/kg LPS I.P.)	NO2-OA	200µg/kg*d (osmotic minipump) starting 48hr prior to LPS exposure	↓ Inflammation, cardiac, hepaBc, renal Bssue injury (↓ Renal MCP-1, ICAM-1, VCAM-1, iNOS, COX-2, PGE2 and ↓ Plasma Cre/BUN)
59	Mouse (C57BL/6)	AKI – Cisplatin (20mg/kg I.P.)	NO2-OA	2mg/kg*d (given for 48hr as pre-treatment only via osmotic minipump)	↓ Plasma Cre/BUN; ↓ histologic injury score
					Caveat: NO2-OA may have indirect effects on cisplatin metabolism leading to lower exposure
74	Mouse (db/db)	CKD - Diabetic nephropathy	NO2-OA + Losartan	5mg/kg*d (NO2-OA, osmotic minipump) and 10mg/kg*d (losartan, diet) for 2 weeks	↓ markers of oxidaBve stress (TBARS, NOX2/4) and inflammation (TNF-α, COX-2, urine PGE2), ↓ glomerulosclerosis and fibrosis
3	Mouse (C57BL/6J)	CKD – Ang II hypertension (500ng/kg*min osmotic minipump)	NO2-OA GW9622	5mg/kg*d (osmotic minipump) or 10mg/kg (jugular vein infusion); 10mg/kg (jugular vein infusion)	↓ systolic and diastolic arterial pressure Effects were PPAR-γ independent
	Rat (2nd order mesenteric artery)	Ang II vasoconstriction dose response	NO2-OA	2.5–5µM	↓ mesenteric artery contraction by Ang II
78	Mouse (C57BL/6) sEH C521S mut and WT	CKD – Ang II hypertension (1mg/kg*d osmotic minipump)	NO2-OA	5mg/kg*d (osmotic minipump) starting 3 days after Ang II exposure	↓Mean arterial pressure, cardiac hypertrophy; inhibition of sEH; ↑ vasorelaxation; effects were negated in sEH “redox dead“ mutant mice
			Conjugated linoleic acid + sodium nitrite (to generate nitro fatty acids)	10mg/kg*d cLA + 20mg/kg*d NaNO2 5 days oral gavage	Inhibition of sEH Generation of NO2-FA (Documented with same dietary protocol in independent manuscript94)
79	Mouse (BALB/c)	AKI – Adriamycin (10mg/kg I.P.)	NO2-OA	5mg/kg*d (osmotic minipump) starting 2 days prior to adriamycin exposure	↓ glomerulosclerosis, podocyte loss, tubulointersBBal fibrosis, oxidative stress
					Caveat: NO2-OA may have indirect effects on adriamycin metabolism leading to lower exposure

Abbreviations: Angiotensin II (Ang II), Bcl-associated X protein (Bax), blood urea nitrogen (BUN), Creatinine (Cre), Cyclooxygenase 2 (COX-2), PPAR-γ inhibitor (GW9622), inducible Nitric Oxide Synthase (iNOS), Intercellular Adhesion Molecule 1 (ICAM-1), Interleukin 1 Beta (IL-1β), intraperitoneal (I.P.), Lipopolysaccharide (LPS), Monocyte Chemoattractant Protein 1 (MCP-1), Myeloperoxidase (MPO), NAD(P)H Oxidase (NOX), nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2), Prostaglandin E2 (PGE₂), soluble Epoxide Hydrolase (sEH), Tissue Necrosis Factor alpha (TNF-α), Vascular Cell Adhesion Molecule 1 (VCAM-1), Thiobarbituric acid reactive substances (TBARS).

Acute kidney injury

NO₂-OA may be useful in treating AKI/ATN. The most common experimental model for AKI is ischemia/reperfusion (I/R) injury. In this model, mice were treated with NO₂-OA starting 50 minutes after the injury. Compared to vehicle-treated counterparts, these mice had improved blood urea nitrogen (BUN) and serum creatinine, which are circulating markers of renal function. Nox subunit expression and proinflammatory cytokines MCP-1, IL-1β, and TNF-α were all reduced in the NO₂-OA treated cohort, suggesting dual mechanisms of protection involving redox imbalance and inflammation¹. Independently, NO₂-OA protected human tubular epithelial cells in vitro from an I/R-like oxygen and glucose deprivation/restoration insult, and this effect was also related to suppression of NADPH oxidases^52,53.

NO₂-OA has similar protective effects in AKI caused by sepsis or nephrotoxins. Septic shock causes AKI through cytokine release, hypoperfusion, and ischemia, and can be induced experimentally through injection of lipopolysaccharide (LPS) endotoxin^54–57. Continuous infusion of NO₂-OA improved both creatinine and BUN while reducing TNF-α and various chemokines and adhesion molecules. NO₂-OA also improved the hypotension related to LPS-induced cardiovascular dysfunction, which would reduce renal ischemia⁵⁴. In an AKI model induced by the nephrotoxin cisplatin, NO₂-OA was again protective, although interpretation must take into account the enhanced detoxification of cisplatin-induced free radicals⁵⁸ that would be expected with activation of antioxidant responses by NO₂-OA⁵⁹.

At least part of the protection afforded by NO₂-OA is likely mediated by Nrf2 activation. The electrophilic triterpenoid and Nrf2 activator, CDDO-imidazolide (CDDO-Im), reduces AKI injury, and was associated with downregulation of inflammatory cytokines and upregulation of protective antioxidant genes⁶⁰. Hypomorphic Keap1 mice with constitutively active Nrf2 also fared better than wild-type and Nrf2 knockout mice in I/R injury. The Keap1 hypomorphs were additionally protected from the development of late-stage fibrosis (AKI-to-CKD progression), and this was associated with higher glutathione levels which reduce redox imbalance⁶¹. Pharmacologic activation of Nrf2 by CDDO-Im given in the first five days after I/R injury also protected against fibrosis and CKD development⁶². This last point is important, since it implies that therapy can be initiated after AKI to protect against long-term sequelae. The protective effect of genetic activation of Nrf2 has been corroborated independently in our laboratory, with Keap1 hypomorphic mice demonstrating a reduction in renal inflammatory markers (TNF-α, RANTES, IL-6, and MCP-1) one day after I/R injury. While there was not a corresponding early improvement in renal function or histologic injury, there was reduction in fibrosis at 10 days, suggesting protection against AKI-to-CKD progression⁶¹.

Other effects may also contribute to protection from AKI. Several studies indicate protection from injury with upregulation of HSPs^63–65 and PPAR-γ^66–68. In addition, induction of HO-1 would be expected to reduce injury^69–71. While these protective mechanisms have not yet been definitively linked to NO₂-OA in the kidney, it is tempting to speculate that NO₂-OA induces protection in AKI via these pathways.

Chronic Kidney Disease

NO₂-OA has been tested as a treatment for diabetic nephropathy, which is the leading cause of renal failure in the developed world^72,73. In murine diabetes caused by leptin receptor mutation, NO₂-OA reduced glomerular injury and the expression of profibrotic TGF-β and extracellular matrix proteins collagen III, fibronectin, and α-smooth muscle actin (α-SMA). Synergism between losartan and NO₂-OA was observed on evaluation of inflammatory markers TNF-α, cyclooxygenase 2, and urinary prostaglandin E2 (PGE2) as well as the oxidative stress marker, thiobarbituric acid-reactive substances (TBARS), and renal NAD(P)H oxidase components⁷⁴. NO₂-OA also downregulates the chemoattractant MCP-1 in LPS-activated macrophages,^33,34 which has been strongly implicated in development of diabetic nephropathy⁷⁵.

Nrf2 activity may be responsible for these protective effects, as Nrf2 demonstrated beneficial effects in streptozotocin (STZ) models of diabetic nephropathy. Nrf2 null mice suffered more severe CKD than their wild-type counterparts after STZ⁷⁶. Pharmacologic activation of Nrf2 using the natural electrophiles sulforaphane or cinnamic aldehyde mitigated oxidative damage and suppressed pro-fibrotic cytokines, and this effect was dependent on the presence of Nrf2⁷⁷. These results suggest that endogenous lipid electrophiles such as NO₂-OA may reduce diabetic kidney injury via enhancement of Nrf2 activity.

After diabetes, hypertension is the second leading cause of CKD⁷³. To date, NO₂-OA has not been evaluated in hypertension models of CKD. However, NO₂-OA has been found to reduce Angiotensin II-induced hypertension through antagonism of the Angiotensin II type 1 receptor³. In another model of Angiotensin II infusion, 10-NO₂-OA protected against hypertension and improved vasorelaxation through inhibition of soluble epoxide hydrolase. In addition, dietary supplementation with conjugated linoleic acid and sodium nitrite, which increase production of endogenous NO₂-cLA, mimics the antihypertensive effects of NO₂-OA⁷⁸. Studies evaluating renal function in these hypertension models still need to be performed. The potential to simultaneously reduce blood pressure and inflammation suggests that NO₂- OA may extend the scope of therapeutic benefit to CKD patients with either hypertensive or diabetic disease.

Specific glomerular diseases may also be treated by NO₂-OA. In a mouse model of focal segmental glomerulosclerosis (FSGS) induced by adriamycin, NO₂-OA improved renal function as evidenced by reduced proteinuria, plasma creatinine and blood urea nitrogen⁷⁹. NO₂-OA also improved glomerular and tubulointerstitial lesions on histology as well as collagen III, fibronectin, and α-SMA. As in other models of disease, markers of inflammation (TNF-α, IL-1β, and MCP-1) and reactive oxygen species production (Nox subunits) were also down-regulated in the treatment group, suggesting multiple pathways of protection. However, as in the cisplatin AKI studies above, activation of Nrf2-linked Phase II enzymes and oxidoreductases by NO₂-OA may have confounding effects on the renal toxicity of adriamycin. Adriamycin is cytotoxic as a result of two competing mechanisms: DNA intercalation resulting in inhibition of Topoisomerase II by the parent compound, or reduction to a reactive semiquinone which generates damaging reactive oxygen species⁸⁰. While some reports suggest that adriamycin is not a substrate for the Nrf2-regulated oxidoreductase NAD(P)H-quinone oxidoreductase (NQO1)⁸¹, others suggest that NQO1 may bioactivate adriamycin and related quinones to reactive semiquinones^82–84, while some evidence suggests that NQO1 may be important in detoxification of adriamycin⁸⁵. Notably, Nrf2 likely activates additional oxidoreductases in addition to NQO1, with effects on adriamycin metabolism which have not been characterized.

Mice with genetic Nrf2 enhancement have demonstrated protection against nonglomerular CKD in a unilateral ureteral obstruction (UUO) model. Animals with constitutive hypomorphism of the Nrf2 repressor Keap1 displayed reduced renal fibrosis and α-SMA compared to wild-type counterparts after UUO. Nrf2 activity was suppressed by the injury in wild-type mice, but this effect was ameliorated by Keap1 hypomorphism⁶¹. Furthermore, Nrf2 knockout mice were sensitized to UUO, demonstrating greater tubular injury and apoptosis at early stages and greater late-stage fibrosis⁸⁶. These studies indicate that severe injuries can downregulate Nrf2 activity and that loss of this protective pathway is a mechanism for CKD progression.

As in AKI, NO₂-FA are likely to have advantages beyond Nrf2 enhancement. As an endogenously produced molecule, NO₂-FA are less likely to have significant toxicity. This is particularly important since a clinical trial in diabetics using bardoxolone methyl (CDDO-Me), a triterpenoid Nrf2 enhancer, demonstrated an increase in adverse effects leading to early termination of the trial^87,88. Some preclinical studies verified deleterious effects with pharmacologic Nrf2 enhancement⁸⁹. In contrast, NO₂-FA have targets beyond Nrf2, including NF-κB, HSP, PPAR-γ, and HO-1, which have all been implicated in CKD development^49,90–93. These pleiotropic actions uniquely position NO₂-FA to have potent beneficial effects in a variety of pathologies.

Conclusion

Novel therapeutics are critically needed for the treatment of kidney disease. By virtue of its potent ability to improve redox imbalance and reduce inflammation, NO₂-OA may be a promising agent for the treatment of both acute and chronic kidney diseases. Additional investigation is required to determine the safety and efficacy of this agent and how it may be best harnessed to improve the care of patients.