Renoprotective effect of Limonium duriusculum (de Girard) Kuntze via modulation of oxidative stress/ UPR markers and inflammation during cyclosporine-induced nephrotoxicity in rats

Document Type : Original Article


1 Laboratoire de Biologie et Environnement, Faculté des Sciences de la Nature et de la Vie, Université Frères Mentouri Constantine 1, Route Aïn El Bey, 25000, Constantine, Algérie

2 Unité de Recherche, Valorisation des Ressources Naturelles, Molécules Bioactives et Analyses Physicochimiques et Biologiques (VARENBIOMOL), Université Frères Mentouri, Constantine 1, Route Aïn El Bey, 25 000 Constantine, Algérie

3 Laboratory of Molecular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland

4 Department of Nephrology and Hypertension, IIS-Fundacion Jimenez Diaz UAM, Madrid, Spain



Objective(s): The present study aimed to explore the mechanisms underlying the potency of the renoprotective effect of the EtOAc fraction of Limonium duriusculum (EALD) (Plumbaginaceae) against cyclosporine A (CsA), in comparison to vitamin E (Vit. E).
Materials and Methods: In the in-vivo experiment, a model of CsA-induced nephrotoxicity was established by dosing male Wistar rats with 25 mg/kg, for 14 days. The protective effect of EALD was investigated through pretreatment of rats with a dose of 200 mg/kg for 14 days, compared to the oral administration of Vit. E at 100 mg/kg. Renal function and markers of oxidative stress were then assessed. Furthermore, a complementary in-vitro study was carried out to evaluate CsA-induced endoplasmic reticulum stress (ERS) and inflammation on cell culture (3T3 cells and MCT cells) using western blot and quantitative RT-PCR..
Results: Pretreatment of rats with EALD significantly attenuated the elevated levels of renal dysfunction markers (BUN, creatinine) and suppressed malondialdehyde (MDA) levels; It also significantly regulated the changes in superoxide dismutase (SOD), reduced glutathione (GSH), glutathione peroxydase (GPx), and glutathione S-transferase (GST) levels as compared to Vit. E, demonstrating a more effective recovery in renal tissues.  Treatment of cells with CsA was linked to the expression of ERS and inflammatory markers activating transcription factor (ATF4), inositol-requiring enzyme 1α (IRE1α), binding immunoglobulin protein (BiP), and monocyte chemoattractant protein-1 (MCP1). In contrast, pretreatment of cells with EALD resulted in a significant decrease in both ERS and inflammatory markers.
Conclusion: These findings indicate the renoprotective potential of L. duriusculum, as it demonstrated the ability to ameliorate CsA-induced renal dysfunction through its distinctive antioxidant properties.


Main Subjects

1. Colombo MD, Perego R, Bellia G. Cyclosporine-associated nephrotoxicity. Open J Nephrol 2013; 3:168-180.
2. Bobadilla NA, Gamba G.  New insights into the pathophysiology of cyclosporine nephrotoxicity: A role of aldosterone. Am J Physiol Renal Physiol 2007; 293: F2-F9. 
3. Lai Q, Luo Z, Wu C, Lai S, Wei H, Li T, et al. Attenuation of cyclosporine A induced nephrotoxicity by schisandrin B through suppression of oxidative stress, apoptosis and autophagy. Int Immunopharmacol 2017; 52: 15-23.
4. Pallet N, Bouvier N, Legendre C, Gilleron J, Codogno P, Beaune P, et al.Autophagy protects renal tubular cells against cyclosporine toxicity. Autophagy 2008; 4:783-791.
5. El Bassossy HM, Hassanien MA, Bima A, Ghoneim FM, Elsamanoudy AZ. Renal oxidative stress and inflammatory response in perinatal cyclosporine-A exposed rat progeny and its relation to gender. J Microsc Ultrastruct 2019; 7:44-49.  
6. Liu SH, Yang CC, Chan DC, Wu CT, Chen LP, Huang JW, et al. Chemical chaperon 4-phenylbutyrate protects against the endoplasmic reticulum stress-mediated renal fibrosis in vivo and in vitro. Oncotarget 2016; 7:22116-22127.  
7. Smith JA. Regulation of cytokine production by the unfolded protein response; implications for infection and autoimmunity. Front Immunol 2018; 9: 422-442.
8. Wu D, Huang LF, Chen XC, Huang XR, Li HY, AN N, et al. Research progress on endoplasmic reticulum homeostasis in kidney diseases. Cell Death Dis 2023; 14:473-484.
9. Liu X, Zhang R, Huang L, Zheng Z, Vlassara H, Striker G, et al. Excessive oxidative stress contributes to increased acute ER stress kidney injury in aged mice. Oxid Med Cell Longev 2019; 2019: 2746521-2746535.
10. Wang N, Mao L, Yang L, Zou J, Liu K, Liu M, et al. Resveratrol protects against early polymicrobial sepsis-induced acute kidney injury through inhibiting endoplasmic reticulum stress-activated NF-KB pathway. Oncotarget 2017; 8: 36449-36461.
11. Cid TP, Garcıa JRC, Alvarez FC, De Arriba G. Antioxidant nutrients protect against cyclosporine A nephrotoxicity. Toxicology 2003; 189: 99-111. 
12. Burdmann EA, Andoh TF, Yu L, Bennett WM. Cyclosporine nephrotoxicity. Semin Nephro 2003; 23:465-476. 
13. Rezzani R. Exploring cyclosporine A-side effects and the protective role-played by antioxidants: the morphological and immunohistochemical studies. Histol Histopathol 2006; 21: 301-316.
14. Pereira DM, Valentao P, Correia-da-Silva G, Teixeira N, Andrade PB. Translating endoplasmic reticulum biology into the clinic: A role for ER-targeted natural products? Nat Prod Rep 2015; 32:705-722. 
15. Kunitake H, Koreedab K, Mii M. Morphological and cytological characteristics of protoplast-derived plants of statice (Limonium perezii Hubbard). Scientia Horticulturae 1995; 60 305-312.
16. Quézel P, Santa S. New flora of Algeria and regions southern deserts: Editions of the National Center for Scientific Research; 1962, CNRS, Paris, 1170.
17. Kerkatou M, Redouane-Salah A, León F, Brouard I, Mosset P, Menad A, et al. Secondary metabolites and antioxidant activity of Limonium duriusculum (de Girard) Kuntze extracts. Asian J Chem 2016; 28:2695-2700.
18.  Aniya Y, Miyagi C, Nakandakari A, Kamiya S, Imaizumi N, Ichiba T. Free radical scavenging action of the medicinal herb Limonium wrightii from the Okinawa islands. Phytomedicine 2002; 9:239-244. 
19, Tang XH, Gao J, Chen J, Xu LZ, Tang YH, Zhao XN, et al. Mitochondrial modulation is involved in the hepatoprotection of Limonium sinense extract against liver damage in mice. J Ethnopharmacol 2008; 120:427-431.
20. Yamashiro S, Noguchi K, Matsuzaki T, Miyagi K, Nakasone J, Sakanashi M, et al. Cardioprotective effects of extracts from Psidium guajava L. and Limonium wrightii, Okinawan medicinal plants, against ischemia-reperfusion injury in perfused rat hearts. Pharmacology 2003;67:128-135.
21.  Hamadou M H, Kerkatou M, Zucal C, Bisio A, Provenzani A, Inga A, et al. Limonium duriusculum (de Girard) Kuntze exhibits anti-inflammatory effect Via NF-κ B pathway modulation. Braz Arch Biol Technol 2021; 64:e21200179-e21200193.
22.  Hamadou MH, Kerkatou M, Gatto P, Pancher M, Bisio A, Inga A, et al. Apigenin rich-Limonium duriusculum (de Girard) Kuntze promotes apoptosis in HCT116 cancer cells. Nat Prod Res 2019; 35:2910-2914
23.  Medini F, Megdiche W, Mshvildadze V, Pichette A, Legault J, St-Gelais A, et al. Antiviral-guided fractionation and isolation of phenolic compounds from Limonium densiflorum hydroalcoholic extract. C. R. Chimie 2016; 19: 726-732.
24. Tariq M, Morais C, Sobki S, Al Sulaiman M, Al Khader A. N-acetylcysteine attenuates cyclosporin-induced nephrotoxicity in rats. Nephrol Dial Transplant 1999; 14: 923-929
25. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275.
26. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95:58-95.
27. Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 1974; 47: 469-474.
28. Sedlak J, Hanus L. Changes of glutathione and protein bound SH-groups concentration in rat adrenals under acute and repeated stress. Endocrinol Exp1982; 16: 103-109.
29. Habig WH, Pabst MJ, Jakoby WB. Glutathione-S-transferases: The first enzymatic step in mercapturic acid formation. J Biol Chem1974; 249: 7130-7139.
30. Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: Biochemical role as a component of glutathione peroxidase. Science 1973; 179: 588-590. 
31. Wissmann C, Frey FJ, Ferrari P, Uehlinger DE. Cyclosporine induced nephrotoxicity in renal transplant recipients: the role of the transplanted kidney. J Am Soc Nephrol 1996; 7: 2677-2681.
32. Anjaneyulu M, Tirkey N, Chopra K. Attenuation of cyclosporine-induced renal dysfunction by catechin: Possible antioxidant mechanism. Renal Fail 2003; 25: 691-707.
33. El‐Bassossy H M, Eid BG. Cyclosporine A exhibits gender‐specific nephrotoxicity in rats: Effect on renal tissue inflammation. Biochem Biophys Res Commun 2018; 495:468-472. 
34. Raza Z, Naureen Z. Melatonin ameliorates the drug induced nephrotoxicity: Molecular insights. Nefrologia 2020; 40: 12-25. 
35. Greene EL, Paller MS. Oxygen free radicals in acute renal failure. Miner Electrolyte Metab 1991; 17: 124-132.
36. Hussein SA, Ragab OA, El-Eshmawy MA. Protective effect of green tea extract on cyclosporine A induced Nephrotoxicity in rats. Benha Vet Med J 2013; 25:205-217.
37. Malhotra JD, Kaufman RJ. Endoplasmic reticulum stress and oxidative stress: a vicious cycle and double edged sword? Antioxid Redox Signal 2007; 9:2277-2293.
38. Aoyama K, Nakaki T. Glutathione in cellular redox homeostasis: association with the excitatory amino acid carrier 1 (EAAC1). Molecules 2015; 20:8742-8758.
39. Li C, Shi L, Chen D, Ren A, Gao T, Zhao M. Functional analysis of the role of glutathione peroxidase (GPx) in the ROS signaling pathway, hyphal branching and the regulation of ganoderic acid biosynthesis in Ganodermalucidum. Fungal Genet Biol 2015; 82:168-180.
40. Strange RC, Spiteri MA, Ramachandran S, Fryer AA. Glutathione-S-transferase family of enzymes. Mutat Res 2001; 482:21-26.
41. Amrani A, Boubekri N, Benaissa O, Zama D, Benayache F, Benayache S. Protective role of Limonium bonduelli extract against non-enzymatic peroxidation in brain and testes induced by iron in vitro. Int J Phytomed 2017; 9:72-78.
42. Tang XH, Chen J, Yang XL, Yan LF, Gao J. Preservation on calcium homeostasis is involved in mitochondrial protection of Limonium sinense against liver damage in mice. Pharmacogn Mag 2010; 6:191-197.
43. Wang C, Salahudeen AK. Lipid peroxidation accompanies cyclosporine nephrotoxicity: Effects of vitamin E. Kidney Int 1995; 47: 927-934.
44. Medini F, Bourgou S, Lalancette KJ, Snoussi M, Mkadmini K, Coté I, et al. Phytochemical analysis, antioxidant, anti-inflammatory, and anticancer activities of the halophyte Limonium densiflorum extracts on human cell lines and murine macrophages. S Afr J Bot 2015; 99:158-164.
45. Cao SS, Kaufman RJ. Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease. Antioxid Redox Signal 2014; 21:396-413.
46. Tu BP, Weissman JS. Oxidative protein folding in eukaryotes: mechanisms and consequences. J Cell Biol 2004; 164: 341-346.
47. Lenna S, Han R, Trojanowska M. ER stress and endothelial dysfunction. IUBMB Life 2014; 66:530-537. 
48. Yuan Y, Xu X, Zhao C, Zhao M, Wang H, Zhang B, et al. The roles of oxidative stress, endoplasmic reticulum stress and autophagy in aldosterone/mineralocorticoid receptor-induced podocyte injury. Lab Invest 2015; 95:1374-1386.
49. Lee ES, Kim HM, Kang JS, Lee EY, Yadav D, Kwon MH, et al. Oleanolic acid and N-acetylcysteine ameliorate diabetic nephropathy through reduction of oxidative stress and endoplasmic reticulum stress in a type 2 diabetic rat model. Nephrol Dial Transplant 2016; 31:391-400.
50. El Karoui K, Viau A, Dellis O, Bagattin A, Nguyen C, Baron W, et al. Endoplasmic reticulum stress drives proteinuria-induced kidney lesions via Lipocalin 2. Nat Commun 2016; 7:10330-10342.
51. González-Guerrero C, Cannata-Ortiz P, Guerri C, Egido J, Ortiz A, Ramos AM. TLR4-mediated inflammation is a key pathogenic event leading to kidney damage and fibrosis in cyclosporine nephrotoxicity. Arch Toxicol 2017; 91:1925-1939.
52. Kolattukudy PE, Niu J. Inflammation, Er stress, autophagy and MCP-1/CCR2 Ppathway. Circ Res 2012; 110: 174-189.
53. Hetz C, Papa FR. The unfolded protein response and cell fate control. Molecular Cell 2018; 69:169-181.
54. Pakos-Zebrucka K, Koryga I, Mnich K., Ljujic M, Samali A, Gorman AM. The integrated stress response. EMBO Rep 2016; 17:1374-1395.
55. Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, et al. An integrated stress response regulates aminoacid metabolism and resistance to oxidative stress. Mol Cell 2003; 11: 619-633.
56. Wang C, Li H, Meng Q, Du Y, Xiao F, Zhang Q, et al. ATF4 deficiency protects hepatocytes from oxidative stress via inhibiting CYP2E1 expression. J Cell Mol Med 2014; 18:80-90.
57. Komoike Y, Inamura H, Matsuoka M. Effects of salubrinal on cadmium-induced apoptosis in HK-2 human renal proximal tubular cells. Arch Toxicol 2012; 86:37-44.
58. Ehren JL, Maher P. Concurrent regulation of the transcription factors Nrf2 and ATF4 mediates the enhancement of glutathione levels by the flavonoid fisetin. Biochem Pharmacol 2013; 85:1816-1826.
59. Reyes-Fermín LM, Aparicio-Trejo OE, Avila-Rojas SH, Gómez-Sierra T, Martínez-Klimova E, Pedraza-Chaverri J. Natural antioxidants’ effects on endoplasmic reticulum stress-related diseases. Food Chem Toxicol 2020; 138: 111229.
60. Grootjans J, Kaser A, KaufmanRJ, Richard S, Blumberg RS. The unfolded protein response in immunity and inflammation. Nat Rev Immunol 2016;16:469-484.
61. Chaudhari N, Talwar P, Parimisetty A, d’Hellencourt CL, Ravanan P. Endoplasmic reticulum stress, inflammation, and oxidative stress. Front Cell Neurosci 2014; 8:213.
62. Junjappa RP, Patil P, Bhattarai KR, Kim HR, Chae HJ. IRE1α Implications in endoplasmic reticulum stress-mediated development and pathogenesis of autoimmune diseases.  Front Immunol 2018; 9:1289.
63.  Gendrisch F, Völkel L, Fluck M, Apostolova P, Zeiser R, Jakob T, et al. IRE1 and PERK signaling regulates inflammatory responses in a murine model of contact hypersensitivity. Allergy 2022; 77: 966-678.
64. Zha X, Yue Y, Dong N, Xiong S. Endoplasmic reticulum stress aggravates viral myocarditis by raising inflammation through the IRE1-associated NF-κB pathway. Can J Cardiol 2015; 31:1032-1040.
65. Fougeray S, Bouvier N, Beaune P, Legendre C, Anglicheau D, Thervet E, et al. Metabolic stress promotes renal tubular inflammation by triggering the unfoldedprotein response. Cell Death Differ 2011;  2: e143-152.
66. Gargalovic PS, Gharavi NM, Clark MJ, Pagnon J, Yang WP, He A, et al. The unfolded protein response is an important regulator of inflammatory genes in endothelial cells. Arterioscler Thromb Vasc Biol 2006; 26:2490-2496.
67. Huang H, Jing J, Wang JJ, Sheibani N, Zhang SX. ATF4 is a novel regulator of MCP-1 in microvascular endothelial cells. J Inflamm 2015; 12:31-42.
68. Singh S, Anshita D, Ravichandiran V. MCP-1: Function, regulation, and involvement in disease. Int Immunopharmacol 2021; 101:107598.
69. Mohammed-Ali Z, Lu C, Marway MK, Carlisle RE, Ask K, Lukic D, et al. Endoplasmic reticulum stress inhibition attenuates hypertensive chronic kidney disease through reduction in proteinuria. Sci Rep 2017; 7:41572-41582.
70. Guo R, Liu W, Liu B, Zhang B, Li W, Xu Y. SIRT1 suppresses cardiomyocyte apoptosis in diabetic cardiomyopathy: An insight into endoplasmic reticulum stress response mechanism. Int J Cardiol 2015; 191:36-45.
71. Li S, Ye J, Deng Z, Yu L, Gu X, Liu Q. Ginsenoside-Rg1 inhibits endoplasmic reticulum stress-induced apoptosis after unilateral ureteral obstruction in rats. Ren Fail 2015; 37:890-895.
72.  Jeon BJ, Yang HM, Lyu YS, Pae HO, Ju SM, Jeon BH. Apigenin inhibits indoxyl sulfate-induced endoplasmic reticulum stress and anti-proliferative pathways, CHOP and IL-6/p21, in human renal proximal tubular cells. Eur Rev Med Pharmacol Sci 2015; 19:2303-2310.
73. Wu Q, Li W, Ahao J, Sun W, Yang Q, Chen C, et al. Apigenin ameliorates doxorubicin-induced renal injury via inhibition of oxidative stress and inflammation. Biomed Pharmacother 2021; 137:111308.