(T1130-01-03) Urolithin-A Laden Nanoparticles Upregulate Heme Oxygenase (HO-1) and Enhance Mitochondrial Quality Control in Mouse Model of Cisplatin-Induced Acute Kidney Injury

Purpose: Cisplatin is a widely used chemotherapeutic agent since its approval by the FDA in 1978. It is associated with multiple off-target toxicities, including acute kidney injury (AKI), limiting its clinical use[1]. Urolithin A (UA), a gut metabolite of dietary ellagic acid, exhibits antioxidant properties and has demonstrated potential in AKI mouse models[2]. However, its therapeutic efficacy is hindered by poor oral bioavailability. To address this limitation, we encapsulated UA in biodegradable nanoparticles with surface-conjugated ligands (P2Ns-NAR [UA]) targeting gut-expressed folate receptors improving oral bioavailability of encapsulated drug[3]. Heme oxygenase-1 (HO-1) is an inducible stress response gene that becomes upregulated as a protective measure in various disease conditions affecting several organs[4, 5]. Emerging evidence suggests that HO-1 plays a nephroprotective role by regulating mitochondrial quality control in cisplatin-induced AKI. Here, we investigated the novel protective mechanism of UA-laden nanoparticles via HO-1 against cisplatin-induced oxidative injury in the kidneys by modulating mitochondrial quality control.

Methods: nUA was prepared using oil-in-water emulsification method. In vitro studies were conducted using HK-2 cells to investigate the potential of HO-1 in mitigating cisplatin-induced acute kidney injury (AKI). For in vivo assessment, 10-week-old male C5BL/6J mice were divided into four groups (n=5): one negative control and three test groups. The test groups received cisplatin (C) at 7mg/kg intraperitoneally once a week (mimicking therapeutic regimen, but in healthy rodents); cisplatin (C) alone, C+P2Ns (UA) at 20mg/kg, and C+P2Ns-NAR (UA) at 10mg/kg. Healthy mice served as the control group which received saline. P2Ns (UA) and P2Ns-NAR (UA) were administered three times a week via oral gavage over a four-week study period. Upon completion, the animals were euthanized; urine, plasma, and kidney samples were analyzed. Histological assessments were also conducted to evaluate fibrosis levels in kidney tissues.

Results: P2Ns-NAR (UA) showed a significant reduction in urinary level of creatinine and BUN as compared to the high level observed in cisplatin control. Our treatment further ameliorates the increased level of kidney injury biomarkers observed in the serum and plasma via multiplex assay. We also evaluated the immunoinflammatory markers which were altered in cisplatin-induced AKI mice. A significant increase in pro-inflammatory cytokines such as IL-6, IL-8 and IL-1β were observed, however, our treatment P2Ns-NAR (UA) offered protection by reducing the level of pro-inflammatory cytokines and increasing anti-inflammatory cytokines. Our results further showed a notable upregulation of HO-1 gene expression and protein levels. Additionally, there were increased levels of Pink1, parkin, and LC3b, which are indicative of enhanced mitochondrial quality control mechanisms such as mitophagy. Mitophagy is a selective form of autophagy that removes damaged or dysfunctional mitochondria, thereby maintaining mitochondrial homeostasis. Conversely, levels of Drp1, a protein involved in mitochondrial fission, were decreased, suggesting a reduction in mitochondrial fragmentation. Furthermore, we observed increased levels of Mfn1 and mfn2 in our treatment group, proteins involved in mitochondrial fusion potentially contributing to mitochondrial network stabilization. Kidney specimen stained with Masson's Trichrome also reveals massive fibrosis with accumulation of collagen and extracellular matrix in cisplatin group but showed a significant decrease in our treatment group. Our treatment markedly reduced cisplatin-induced inflammation and oxidative stress in the kidneys of AKI mice. Furthermore, In-vitro assessment revealed increased HO-1 percentage positive cells in our treatment group as opposed cisplatin control.

Conclusion: Overall, these findings suggest that urolithin-A nanoparticle activates the expression of HO-1 which modulates mitochondrial dynamics and quality control processes, potentially contributing to the amelioration of cisplatin-induced kidney injury. The upregulation of HO-1 and the enhancement of mitochondrial quality control mechanisms, such as mitophagy, may play a crucial role in protecting the kidneys from cisplatin-induced damage.

References: 1. Wang, D. and S.J. Lippard, Cellular processing of platinum anticancer drugs. Nat Rev Drug Discov, 2005. 4(4): p. 307-20.
2. Zou, D., et al., Oral delivery of nanoparticle urolithin A normalizes cellular stress and improves survival in mouse model of cisplatin-induced AKI. Am J Physiol Renal Physiol, 2019. 317(5): p. F1255-f1264.
3. Heyns, I.M., et al., Rationally Designed Naringenin-Conjugated Polyester Nanoparticles Enable Folate Receptor-Mediated Peroral Delivery of Insulin. ACS Appl Mater Interfaces, 2023. 15(39): p. 45651-45657.
4. Hull, T.D., et al., Heme oxygenase-1 regulates mitochondrial quality control in the heart. JCI Insight, 2016. 1(2): p. e85817.
5. Luu Hoang, K.N., J.E. Anstee, and J.N. Arnold, The Diverse Roles of Heme Oxygenase-1 in Tumor Progression. Front Immunol, 2021. 12: p. 658315.

Acknowledgements: This study is supported by NIH-R01DK128597


Figure 1: Effect of orally bioavailable P2Ns-NAR (UA) on Cisplatin-Induced Acute Kidney Injury: A.) Schematic of treatment protocol utilized in this study. Three groups of mice (n=5) received a single weekly dose of cisplatin over four weeks, while two treatment groups, P2Ns (UA) and P2Ns-NAR (UA), were administered by oral gavage, three doses per week for four weeks at doses of 20mg/kg and 10mg/kg body weight, respectively with cisplatin only as positive control and healthy group as negative control. B.) Changes in urinary BUN and creatinine levels in mice with cisplatin-induced acute kidney in mice. C.) Changes in plasma BUN. D.) Changes as observed in mice body weight at 4 weeks. Secretion levels of kidney injury biomarkers: E.) Cystatin, F.) NGAL, and G.) Osteopontin OPN. Relative mRNA expressions of pro-inflammatory cytokines induced by cisplatin in AKI mice: H.) IL-6, I.) IL-1β, and J.) TLR4. Student ‘s t-test/one way ANOVA were used for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.0001 for comparison with cisplatin group). All values are presented as mean ± SEM.


Figure 2: Flow cytometry analysis of percentage heme oxygenase (HO-1) positive cells following cisplatin induced injury in HK-2 cells. A.) Flow cytometry results; B.) Quantified % HO-1 positive cells. Effect of treatments on cisplatin induced inflammation in HK-2 cells. C.) IL-6 and D.) IL-8). Student ‘s t-test/one way ANOVA were used for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.0001 for comparison with cisplatin group). All values are presented as mean ± SEM.


Figure 3: A.) Kidney specimen stained with Masson's Trichrome (a) Healthy control group; revealed no significant amount of fibrous tissue; (b) Cisplatin group revealed massive fibrosis with accumulation of collagen and extracellular matrix components (c and d) our treatment groups revealed retraction of fibrosis as compared to the cisplatin group. (Masson's Trichrome, X50). B.) Our treatment (P2Ns-NAR [UA]) increases protein expression of HO-1 in kidney tissues. Representative western blots analysis of HO-1. C.) Densitometric quantification using Chemidoc MP imaging system and analyzed using image Lab6.1 Biorad (n=3) after normalization with βactin. D.) Our treatment increases relative mRNA expression of HO-1 in kidney tissues. Mitophagy is increased in cisplatin-induced acute kidney injury (E-G). HO-1 expression increases mitochondrial fusion and inhibits fission. Expression of fusion markers H.) mitofusin 1 (Mfn1) and I.) mitofusin2 (Mfn2). Expression of fission marker H.) and dynamic-related protein-1 (DRP1). Student ‘s t-test/one way ANOVA were used for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.0001 for comparison with cisplatin group). All values are presented as mean ± SEM.