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C O M P L E T E T I T L E : Oral delivery of nanoparticle urolithin A normalizes cellular stress and improves survival in mouse model of cisplatin-induced AKI
F U L L T E X T S O U R C E : Physiology
Abstract
The popular anticancer drug cisplatin causes many adverse side effects, the most serious of which is acute kidney injury (AKI). Emerging evidence from laboratory and clinical studies suggests that the AKI pathogenesis involves oxidative stress pathways; therefore, regulating such pathways may offer protection. Urolithin A (UA), a gut metabolite of the dietary tannin ellagic acid, possesses antioxidant properties and has shown promise in mouse models of AKI. However, therapeutic potential of UA is constrained by poor bioavailability. We aimed to improve oral bioavailability of UA by formulating it into biodegradable nanoparticles that use a surface-conjugated ligand targeting the gut-expressed transferrin receptor. Nanoparticle encapsulation of UA led to a sevenfold enhancement in oral bioavailability compared with native UA. Treatment with nanoparticle UA also significantly attenuated the histopathological hallmarks of cisplatin-induced AKI and reduced mortality by 63% in the mouse model. Expression analyses indicated that nanoparticle UA therapy coincided with oxidative stress mitigation and downregulation of nuclear factor erythroid 2-related factor 2- and P53-inducible genes. Additionally, normalization of miRNA (miR-192-5p and miR-140-5p) implicated in AKI, poly(ADP-ribose) polymerase 1 levels, antiapoptotic signaling, intracellular NAD+, and mitochondrial oxidative phosphorylation were observed in the treatment group. Our findings suggest that nanoparticles greatly increase the oral bioavailability of UA, leading to improved survival rates in AKI mice, in part by reducing renal oxidative and apoptotic stress.
INTRODUCTION
Acute kidney injury (AKI) is a life-threatening condition that can arise from a myriad of factors, such as autoimmunity, sepsis, malignancies, ischemic/hypovolemic trauma, and nephrotoxin exposure (2, 5, 11, 35). Among these, AKI resulting from cisplatin (CIS) is prevalent and affects 20–30% of patients receiving this commonly prescribed antineoplastic drug (31, 36). The pathophysiological manifestations of CIS-induced nephrotoxicity are complex, but multiple stress events stemming from oxidative damage, inflammation, and apoptosis/necaenorhabditisis are believed to play a role in promulgating the destruction of the renal microarchitecture (23, 24, 36, 39). The attenuation of cellular and systemic stress may therefore hold the key to treating CIS-induced AKI, but a clinically translatable therapy for any form of AKI remains elusive.
Urolithin A (UA) is the gut-microbial metabolite of ellagic acid, a polyphenol found in nuts, berries, and pomegranates (7). Both compounds have garnered scientific interest because of their antioxidant and anti-inflammatory properties (6, 20, 26). Our laboratory has previously reported on the superior benefits of UA over its precursor ellagic acid in the reduction of plasma cytokines and inhibition of intracellular inflammatory and apoptotic signaling after a sublethal nephrotoxic dose of CIS in the rat model (21). Although these results are promising, neither UA nor ellagic acid is well absorbed by the gastrointestinal tract. The poor oral bioavailability of UA and the resulting need for frequent and high doses are serious limitations for therapeutic use.
The implementation of biocompatible and biodegradable aliphatic polyester nanoparticles is a promising approach to circumvent the oral bioavailability problem (13, 15, 41). More than just passive vehicles for drug delivery, nanoparticles that are conjugated with ligands actively target cognate receptors on the cell surface to promote receptor-mediated endocytosis (16, 25, 42). One such example of active targeting uses the transferrin receptor (TfR1), which is expressed in the villous epithelium, duodenal crypts, and Peyer’s patches of the small intestine (4, 47, 58). We found that gambogic acid (GA), a noncompetitive ligand of TfR1, can be conjugated to traditional nanoparticles such as polylactic-coglycolic acid (PLGA) to facilitate their trafficking through the gastrointestinal barrier via TfR1 interaction (46). This discovery led to the development of next-generation precision polymer nanosystems (P2Ns) containing additional free carboxyl moieties for GA ligand conjugation (19). With increased GA density per particle, P2Ns-GA is internalized more readily than their PLGA-GA counterparts in both human cell lines and rodent models (19, 46).
In this study, our goal is to assess the feasibility of using P2Ns-GA-encapsulated UA (P2Ns-GA UA) to treat severe AKI arising from a lethal dosage of CIS. Although there has been an abundance of excellent research on the etiology, prevention, and treatment of CIS nephrotoxicity in rodent models, the majority of focus has been on the early to mid phases of CIS-induced AKI. Considerably fewer studies, however, have monitored progression of the disease and the efficacy of its proposed treatment to their impactful end point, the presence of kidney failure and death. To fully understand the extent of the renal protective potential of P2Ns-GA UA, we conducted a CIS survival study in mice. This study was followed by analysis of intracellular markers to survey if any salient reductions in cytotoxic stress have also been accomplished.
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RESULTS
Nanoparticle encapsulation of UA and bioavailability.
To determine whether P2Ns could improve the oral bioavailability of UA, we encapsulated the drug using the emulsion-diffusion-evaporation method in either unconjugated (P2Ns) or GA-conjugated (P2Ns-GA) formats. Overall, an entrapment efficiency of ~35% was achieved in both nanoparticle formats. Evaluation of particle properties, such as size, shape, polydispersity index, pH, and ζ-potential (Fig. 1, A and B), revealed that UA loading had not altered the particle characteristics significantly. We compared the pharmacokinetics of P2Ns and P2Ns-GA UA against plain UA in the healthy rat model. An oral dose of 50 mg/kg was chosen for plain UA because it was the highest plain UA dosage reported in Guada et al. (21), and this served as the baseline in our bioavailability experiments. The serum peak concentration and area under the curve of P2Ns-UA were moderately increased (2-fold change) over plain UA (Fig. 1, C and D). In contrast, P2Ns-GA UA exhibited significantly better bioavailability, with an approximately sevenfold change in serum peak concentration and area under the curve compared with plain UA (Fig. 1, C and D). Because of the superiority of P2Ns-GA in the oral delivery of UA, we proceeded to test its therapeutic potential in CIS-induced AKI.
Fig. 1
Nanoparticle formulation and bioavailability. A: dynamic light scattering measurement of nanoparticle size and the polydispersity index (PDI). A table showing pH and ζ-potential (means ± SE) is shown. B: scanning electron photomicrograph of precision polymer nanosystems (P2Ns)-gambogic acid (GA) nanoparticles with urolithin A (UA). Scale bar = 1 µm. C: healthy male Sprague-Dawley rats were given a single dose of plain or nanoparticle-formulated UA (50, 25, or 10 mg/kg for each animal) by oral gavage. Plasma UA concentrations were determined by validated LC-MS at various time points 0.5–48 h postdosing. D: summary of the area under the curve (AUC), time to achieve peak concentration (Tmax), and peak concentration (Cmax) of the four groups.
Effect of nanoparticle UA on mouse survival.
For the survival experiments, two groups of male C57BL/6J mice (n = 8 mice/group) were each given a single intraperitoneal dose (20 mg/kg) of CIS known to cause lethal AKI (43). Nanoparticle P2Ns-GA UA was then administered via oral gavage to the treatment group, whereas the CIS control group received no further dosing. Age- and sex-matched C57BL/6J mice without CIS or UA treatment (n = 4) were used as negative controls. Mice were monitored daily and were euthanized immediately when preestablished criteria for morbidity were met or upon study termination (day 19). However, some mice prematurely died before being euthanized and having their organs harvested. The CIS control group began dying by day 8, with 100% dead by day 15. In contrast, treatment with P2Ns-GA UA was found to have a positive effect on survival, delaying the onset of death by 1 day in those that died and reducing overall mortality by 63% (Fig. 2A). Furthermore, mice that survived in the P2Ns-GA UA group appeared healthy at day 19, suggesting that they may have been recovering from CIS-induced injuries. The P2Ns-GA UA group also displayed progressively less reduction in weight loss after CIS injection and had no discernable changes in kidney mass-to-body weight ratio at euthanasia (Fig. 2, B and C). Finally, blood urea nitrogen and serum creatinine levels of euthanized mice (15–19 days) showed that these parameters had returned to normal for P2Ns-GA UA-treated mice, whereas they were still significantly elevated for the CIS control group (Fig. 2D). Kidney tissues from both early (8–9 days) and late (15–19 days) euthanized mice were harvested and used for subsequent analyses.
Fig. 2
Effect of precision polymer nanosystems (P2Ns)-gambogic acid (GA) urolithin A (UA) on mouse survival and other physiological markers after cisplatin (CIS)-induced acute kidney injury (AKI). A: survival plot of male C57BL/6J mice in CIS only (CIS control), P2Ns-GA UA, and negative (Neg) control groups (n = 4–8/group). B: weight loss plot of CIS control and P2Ns-GA UA groups compared with initial animal weight before CIS injection (n = 4/group). C: ratio of kidney mass to body weight at euthanasia (n = 4/group). D: blood urine nitrogen (BUN) and serum creatinine levels at euthanasia (n = 4/group). All data are expressed as means ± SE and were analyzed using an unpaired Student’s t-test between two groups or ANOVA among multiple groups followed by a Tukey’s posttest. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the CIS control group.
Histological evaluation of CIS nephrotoxicity and treatment efficacy.
At the time of euthanasia, the CIS control group had undergone extensive kidney injury as evidenced by histological examination with both H&E and PAS staining. Compared with the negative control and P2Ns-GA UA groups, the CIS control group showed greater interstitial expansion along with increased necrosis and atrophy of the tubules (Fig. 3A). ImageJ analyses of H&E-stained images indicated that the cortical and medullary area occupied by the interstitial space was ~37% in the CIS control group but only 11% and 2% for the P2Ns-GA UA and negative control groups, respectively (Fig. 3C). At ×40 magnification, PAS staining further revealed tubular dilation, apoptotic bodies, prominent PAS-positive renal casts within tubular lumens, and thickening of glomerular basement membranes (Fig. 3B). Cross-sectional glomerular diameter also increased in the CIS control group (Fig. 3D), which suggested compensatory glomerular hypertrophy. In contrast, P2Ns-GA UA treatment significantly negated these morphological abnormalities.
Fig. 3
Histopathological evaluation of kidney sections. A: representative hematoxylin and eosin (H&E)-stained micrographs of each group (n = 4) showing the kidneys at study termination. Original ×4 magnified images (left) and images digitally magnified to the boxed areas (right) are shown side by side. The cisplatin (CIS) control group showed marked tubular injury as evidenced by increased necrosis and atrophy and expansion of interstitial space. Boxed areas indicate magnified renal tubules (scale bars = 200 µm for original images and 50 µm for zoomed images). B: representative periodic acid-Schiff (PAS)-stained micrographs of each group (n = 4) showing kidney injury and apoptosis in the CIS control group at study termination. *Detached cells in dilated tubules (top left) and Tamm-Horsfall protein-containing renal casts (bottom left). Diameters of glomeruli also appeared enlarged in the CIS control group, along with thickening of the basement membrane. The magenta color represents PAS-positive staining. Original magnification: ×40 (scale bars = 50 µm). C: quantification of interstitial expansion (i.e., percent area occupied by interstitial space) observed for each group (n = 4 animals/group). D: quantification of glomerular diameter observed under ×40 PAS staining for each group (n = 30/group). All data are expressed as means ± SE and were analyzed using ANOVA between multiple groups followed by a Tukey’s posttest. ****P < 0.0001 vs. the CIS control group; #P < 0.05 vs. the negative (Neg) control group. P2Ns-GA UA, precision polymer nanosystems-gambogic acid-encapsulated urolithin A.
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