Renal proliferation and apoptosis against ascorbic acid administration in a model of acute radiation nephropathy
- Authors: Demyashkin G.A.1,2, Uruskhanova Z.E.1, Koryakin S.N.3, Parshenkov M.A.4, Dubovaya T.K.5, Rodionova G.M.4, Shchekin V.I.6, Ivchenko Y.V.6, Ionova O.V.6
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Affiliations:
- I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia
- National Medical Research Center of Radiology, Moscow, Russia.
- National Medical Research Center of Radiology, Moscow, Russia
- The Institute of Pharmacy of the Sechenov University
- N.I. Pirogov Russian National Research Medical University, Moscow, Russia
- The Institute of Clinical Medicine of Sechenov University
- Section: Original Study Articles
- Submitted: 25.03.2024
- Accepted: 25.06.2024
- Published: 29.07.2024
- URL: https://j-morphology.com/1026-3543/article/view/629410
- DOI: https://doi.org/10.17816/morph.629410
- ID: 629410
Cite item
Abstract
BACKGROUND: Radiation exposure, an integral part of the treatment of malignant neoplasms, carries a risk of radiation nephropathy due to the high radiosensitivity of the kidneys. The study of renal tissue proliferation and apoptosis is one of the keys to understanding the mechanisms of radiation damage and developing treatment strategies.
AIM: Evaluation of intrarenal regulation, proliferation, and apoptosis during pre-radiation administration of ascorbic acid.
METHODS: Wistar rats (n=90) were divided into groups: I - control (n=15); II - irradiation, 2 Gy dose (n=15); III - irradiation, 8 Gy dose (n=15); IV - irradiation, 2 Gy dose + ascorbic acid (intraperitoneal injection; dose 50 mg/kg) (n=15); V - irradiation, 8 Gy dose + ascorbic acid (intraperitoneal injection; dose 50 mg/kg) (n=15); VI - ascorbic acid (intraperitoneal injection; dose 50 mg/kg) (n=15). Kidney slides were stained with hematoxylin and eosin. In addition, immunohistochemical evaluation of the expression level of Ki-67- and Cas-3-positive cells was performed.
RESULTS: The histological study showed that pre-radiation administration of ascorbic acid (intraperitoneal injection; dose 50 mg/kg) in the model of acute radiation nephropathy induced by local irradiation with electrons at doses 2 Gy and 8 Gy contributed to statistical reduction of pathomorphologic changes. According to the results of immunohistochemical evaluation of proliferation and apoptosis - distribution of Ki-67- and Cas-3-positive cells in the tubules, epitheliocytes of proximal and distal tubules of nephrons in mono-irradiation groups revealed activation of the terminal stage of cell death, which correlated with the dose of electron irradiation. At the same time, in the experimental groups with pre-irradiation administration of ascorbic acid a statistically significant decrease in the intensity of apoptosis was recorded.
CONCLUSION: Pre-radiation administration of ascorbic acid statistically reduces the strength of radiation-induced kidney damage, as well as the effect of electron irradiation on the life cycle of tubular cells, epitheliocytes of nephron tubules, while increasing the effectiveness of antioxidant defense.
Full Text
At pre-radiation administration of ascorbic acid in groups IV and V, a decrease in the degree of pathomorphologic changes was observed (Fig. 1).
The magnitude of the lesions of the tubules (thrombotic microangiopathy, collapse), nephron tubules and interstitial component are presented in Table 1.
Immunohistochemical study. In the context of studying the mechanisms of life cycle regulation, the key factor of which is DNA synthesis, we evaluated proliferative activity - Ki-67, in the form of specific nuclear staining in endothelial cells of tubules, podocytes and nephrocytes, as well as epitheliocytes of proximal and distal nephrons (Fig. 2 A, B).
In kidney micropreparations of groups II and III we found differences in Ki-67 expression levels compared to the control group: insignificant decreases in positive cells, predominantly in the tubules (p<0.05) (Fig. 2 A, B).
In the groups of pre-radiation administration of ascorbic acid, a slight increase in the number (in percent) of Ki-67-positive cells was observed relative to the mono-irradiation groups (ROD 2 Gy and ROD 8 Gy) (p<0.05) (Fig. 2 A, B).
There were no statistical differences in the distribution of Ki-67 positive cells (p<0.05) between the ascorbic acid mono-injection and control groups (Fig. 2 A, B) (Fig. 2 A, B).
Immunohistochemical evaluation of the terminal stage of apoptosis revealed caspase-3-positive cells in the tubules and in the tubules of the nephron, the distribution of which varied between groups. The characteristic feature of these cells is the presence of partially brown cytoplasm and brown-yellow coloration of the nuclei (Fig. 3 A, B).
In groups II (ROD 2 Gy) and III (ROD 8 Gy), we found a significant increase in the number of cells stained with antibodies to caspase-3 relative to the groups, predominantly epitheliocytes, dilated or atrophied tubules of the nephron, with higher statistical significance in the distal nephron compared to the proximal nephron (p<0.05). When caspase-3 immunolabeled cells were distributed in renal tubules, the mean cell counts were 47±6 (ROD 2 Gy) and 72±6 (8 Gy) per 40 tubules (p<0.05) (Figure 3 A, B).
In the groups receiving ascorbic acid before electron irradiation, a decrease in the level of nuclear staining in nephron tubule cells and renal tubules was recorded: the average number of cells was 34±5 (ROD 2 Gy + AK) and 45±3 (ROD 8 Gy + AK) per 40 tubules, which as a percentage was statistically lower than in groups II and III, respectively (p<0.01; p<0.001) (Fig. 3 A, B) (Fig. 3 A, B). `
In group VI (ascorbic acid monotherapy) changes in caspase-3 expression were not statistically significant compared to the control group (p<0.05) (Fig. 3 A, B).
Thus, according to the results of immunohistochemical analysis of proliferation and apoptosis assessment - distribution of Ki-67- and Cas-3-positive cells in tubules, epitheliocytes of proximal and distal tubules of nephrons, revealed activation of terminal stage of cell death. This phenomenon demonstrates a direct dependence on the dose of electron irradiation - ROD 8 Gy. On the contrary, pre-radiation administration of ascorbic acid statistically significantly reduces the intensity of apoptosis.
DISCUSSION
This study is devoted to immunohistochemical investigation of proliferation and apoptosis of vascular tubules, epitheliocytes of proximal and distal tubules of nephrons against the background of ascorbic acid administration in the model of radiation nephropathy induced by exposure to electrons at 2 Gy ROD and 8 Gy ROD.
It is known that radiotherapy is one of the effective methods of treatment of TNF [15, 16]. However, ionizing radiation can cause damage to healthy tissues falling into the irradiation zone [17]. In modern radiobiology, the following types of radiation therapy of the kidney are most often used: X-rays, alpha-rays, photons, electrons, etc. The main objective is to increase its safety and reduce the associated side effects on the surrounding tissues.
Exposure to electrons induces the release of reactive oxygen species (ROS), excessive accumulation of which leads to inhibition of oxidative stress affecting the cell membrane, cell organelles, etc. [18]. [18]. Such damage can alter intracellular signaling cascades by activating genes associated with apoptosis signaling. Also, RFKs can affect DNA, causing mutations of genetic material, reducing cell proliferation and increasing apoptosis [19].
Thanks to recent studies, it is known that radiation-induced cellular aging plays a significant role in the progression of diseases of various organs [20]. In particular, radiation effects on cerebral microvascular endothelial cells demonstrate a link between a single exposure to radiation and the development of neurodegenerative changes in neural tissue [21].
The pathogenetic mechanisms of radiation-induced nephritis are due to the involvement of all components of the kidney, including glomerular, tubular and stromal-vascular components [22].
Ki-67 protein is a marker of cell proliferation and serves as the most important prognostic factor [23].
It should be noted that Ki-67 protein is not expressed in the G0 phase, which is characteristic of most atypical cells under hypoxic conditions. Interestingly, it is this phase of the cell cycle that is associated with increased resistance to radiation exposure. Consequently, low expression of Ki-67 in neoplastic cells may indicate not only their hypoxic state but also potential radioresistance, which is important in assessing the efficacy of radiation therapy [24, 25]. In our study, exposure to electrons at 2 Gy and 8 Gy ROD caused a slight decrease in Ki-67 protein level (in percentage) compared to the control group, which may be due to the time factor. However, this hypothesis is the subject of further studies.
Apoptosis is one of the responses to cytotoxic stress and plays a key role in the pathogenesis of renal dysfunction [26]. Apoptosis is regulated by both external (via "death receptors") and internal (mitochondrial) mechanisms. The extrinsic pathway is triggered by specific ligands (Fas ligand and tumor necrosis factor α) [22]. The intrinsic pathway is activated in response to various forms of cellular stress: hypoxia, ischemia, oxidative stress, etc. [27]. [27]. Thus, under the influence of stress, a biochemical cascade leading to apoptosis is triggered. This process is characterized by chromatin condensation and DNA fragmentation. Apoptosis is confirmed by an increase in the number of cells positive for caspase-3, a marker of the terminal stage of apoptosis. The increase in the number of caspase-3-positive cells in kidney structures detected in the present study against the background of a decrease in Ki-67 protein after a single electron irradiation indicates a shift in the balance between cell proliferation and apoptosis towards the latter. The obtained results partially coincide with the data of other authors who used other types of radiation. Cell pool reduction occurs due to modulation of GSK3-, ERK-, and Ras/Raf/MEK-1 signaling pathways and deactivation of Bcl-2 and induction of p53 protein [28, 29].
In a number of studies, it has been demonstrated that ascorbic acid has protective properties, which contributes to the repair of damaged DNA, as well as to the reduction of radiation-induced injury [30-32]. Pre-radiation administration of ascorbic acid resulted in statistically significant improvement in pathomorphologic changes of renal structures compared to mono-irradiated groups (ROD 2 Gy and ROD 8 Gy). Similar results were obtained by other authors when studying the protective properties of natural antioxidants, in particular, Amphora algae extract, which led to a decrease in inflammatory reactions and improved the functional state of the kidneys in rats [33]. However, it should be noted that the researchers used gamma rather than β-particles; also doses different from ours.
Thus, the obtained results reveal the molecular mechanisms of regulation of proliferation and apoptosis of glomerular, tubular and stromal-vascular component cells of kidneys in the model of radiation nephropathy caused by electron irradiation at 2 Gy and 8 Gy ROD. Pre-radiation administration of ascorbic acid, according to morphological and immunohistochemical data, confirms its protective properties.
Table 1: Evaluation of pathomorphologic changes in control and experimental groups (in points)
Data are presented as mean ± standard deviation (M±SD). *Median [minimum-maximum]: 0 (none), 1 (mild), 2 (moderate), 3 (severe). Statistically significant differences compared to the control group are labeled in the table as Irradiation (a) and Irradiation + AA (b); p < 0.05
FIGURES
Figure 1: Kidneys of control and experimental groups. Hematoxylin and eosin staining, magnification. × 200.
Figure 2. Kidneys of control and experimental groups. A - immunohistochemical study with antibodies to Ki-67, mag.× 400. B - histogram. Experimental groups (Figure 2B) are numbered according to the study design. Statistically significant differences are indicated by symbols: * - comparison with control group (p<0.05), ** - comparison of group IV with group II (Single Local Dose 2 Gy + AA and Single Local Dose 2 Gy ) (p<0.01), *** - comparison of group V with group III (Single Local Dose 8 Gy + AA and Single Local Dose 8 Gy) (p<0.001).
Figure 3. Kidneys of control and experimental groups. A - immunohistochemical study with antibodies to caspase-3, mag.× 400. B - histogram. Experimental groups (Figure 3B) are numbered according to the study design. Statistically significant differences are indicated by symbols: * - comparison with control group (p<0.05), ** - comparison of group IV with group II (Single Local Dose 2 Gy + AA and Single Local Dose 2 Gy ) (p<0.01), *** - comparison of group V with group III (Single Local Dose 8 Gy + AA and Single Local Dose 8 Gy) (p<0.001).
About the authors
Grigory Alexandrovich Demyashkin
I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia;National Medical Research Center of Radiology, Moscow, Russia.
Author for correspondence.
Email: dr.dga@mail.ru
ORCID iD: 0000-0001-8447-2600
SPIN-code: 5157-0177
Zhanna Eisaevna Uruskhanova
I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia
Email: jey.149@yandex.ru
ORCID iD: 0009-0009-2291-3680
Сo-researcher of First Moscow State University named after I. M. Sechenov. I.M. Sechenov First Moscow State University of the Ministry of Health of Russia (Sechenov University), Moscow, Russia
Sergey Nikolayevich Koryakin
National Medical Research Center of Radiology, Moscow, Russia
Email: korsernic@mail.ru
ORCID iD: 0000-0003-0128-4538
PhD, Head of the Department of Radiation Biophysics «NMRC of Radiology»;
Mikhail Alekseevich Parshenkov
The Institute of Pharmacy of the Sechenov University
Email: misjakj@gmail.com
ORCID iD: 0009-0004-7170-8783
student
Russian Federation, Bolshaya Pirogovskaya str., 2, p.4, Moscow, 119435Tatyana Kleonikovna Dubovaya
N.I. Pirogov Russian National Research Medical University, Moscow, Russia
Email: gusvbr@mail.ru
ORCID iD: 0000-0001-7936-180X
SPIN-code: 4254-6082
Galina Mikhailovna Rodionova
The Institute of Pharmacy of the Sechenov University
Email: rodionovagalinam@mail.ru
ORCID iD: 0000-0002-0536-9590
Vladimir Ivanovich Shchekin
The Institute of Clinical Medicine of Sechenov University
Email: dr.shchekin@mail.ru
ORCID iD: 0000-0003-3763-7454
student
Russian Federation, Bolshaya Pirogovskaya str., 2, p.4, Moscow, 119435Yuliya Valerievna Ivchenko
The Institute of Clinical Medicine of Sechenov University
Email: ivchenko_yu_v@student.sechenov.ru
ORCID iD: 0000-0003-1336-7277
student
Russian Federation, Bolshaya Pirogovskaya str., 2, p.4, Moscow, 119435Olga Vladislavovna Ionova
The Institute of Clinical Medicine of Sechenov University
Email: olgaionova99@mail.ru
ORCID iD: 0009-0007-9137-6597
student
Russian Federation, Bolshaya Pirogovskaya str., 2, p.4, Moscow, 119435References
- R. Pinto et al. In Vivo Studies on Radiofrequency (100 kHz–300 GHz) Electromagnetic Field Exposure and Cancer: A Systematic Review. International Journal of Environmental Research and Public Health. 2023. https://doi.org/10.3390/ijerph20032071
- Wild CP, Espina C, Bauld L, Bonanni B, Brenner H, Brown K, Dillner J, Forman D, Kampman E, Nilbert M, Steindorf K, Storm H, Vineis P, Baumann M, Schüz J. Cancer Prevention Europe. Molecular Oncology. 2019;13(3):528-534. doi: 10.1002/1878-0261.12455
- Wei J, Wang B, Wang H, Meng L, Zhao Q, Li X, et al. Radiation-induced normal tissue damage: oxidative stress and epigenetic mechanisms. Oxidative Medicine and Cellular Longevity. 2019;2019:3010342. doi: 10.1155/2019/3010342
- O'Donoghue J. Relevance of external beam dose-response relationships to kidney toxicity associated with radionuclide therapy. Cancer Biotherapy and Radiopharmaceuticals. 2004;19(3):378-87. doi: 10.1089/1084978041425025
- Dawson, L., Kavanagh, B., Paulino, A., Das, S., Miften, M., Li, X., Pan, C., Haken, R., & Schultheiss, T. Radiation-associated kidney injury. International journal of radiation oncology, biology, physics. 2010;76(3);108-15. https://doi.org/10.1016/j.ijrobp.2009.02.089
- Aratani S, Tagawa M, Nagasaka S, Sakai Y, Shimizu A, Tsuruoka S. Radiation-induced premature cellular senescence involved in glomerular diseases in rats. Scientific Reports. 2018;14;8(1):16812. doi: 10.1038/s41598-018-34893-8
- Scholz M, Kraft-Weyrather W, Ritter S, Kraft G. Cell cycle delays induced by heavy ion irradiation of synchronous mammalian cells. International Journal of Radiation Biology. 1994;66(1):59-75. doi: 10.1080/09553009414550951
- Mavragani IV, Nikitaki Z, Kalospyros SA, Georgakilas AG. Ionizing Radiation and Complex DNA Damage: From Prediction to Detection Challenges and Biological Significance. Cancers (Basel). 2019;11(11):1789. doi: 10.3390/cancers11111789
- Carante MP, Ballarini F. Radiation Damage in Biomolecules and Cells. International Journal of Molecular Sciences. 2020;21(21):8188. doi: 10.3390/ijms21218188
- Sia J, Szmyd R, Hau E, Gee HE. Molecular Mechanisms of Radiation-Induced Cancer Cell Death: A Primer. Frontiers in Cell and Developmental Biology. 2020;8:41. doi: 10.3389/fcell.2020.00041
- Ashrafizadeh M, Farhood B, Eleojo Musa A, Taeb S, Najafi M. Damage-associated molecular patterns in tumor radiotherapy. International Immunopharmacology. 2020;86:106761. doi: 10.1016/j.intimp.2020.10676
- Chen J. The Cell-Cycle Arrest and Apoptotic Functions of p53 in Tumor Initiation and Progression. Cold Spring Harbor Perspectives in Medicine. 2016;6(3):a026104. doi: 10.1101/cshperspect.a026104
- Demyashkin G.A., Koryakin S.N., Stepanova Y.Y., Shchekin V.I., Shegai P.V. Morphological characterization of kidneys in rats after targeted irradiation with electrons at doses of 2, 4 and 6 G. Veterinary Physician. 2021;9-16. doi: 10.33632/1998-698Х.2021-5-9-16
- Demyashkin G.A., Koryakin S.N., Stepanova Y.Y., Shchekin V.I., Shegai P.V. Implication of priceless electronic exposure on historarchitectonics of the robbins of Wistar Rats at Dose of 8 Gr. Veterinary Doctor. 2021;20-26. doi: 10.33632/1998-698Х.2021-4-20-26
- Buglione M, Spiazzi L, Urpis M, Baushi L, Avitabile R, Pasinetti N, Borghetti P, Triggiani L, Pedretti S, Saiani F, Fiume A, Greco D, Ciccarelli S, Polonini A, Moretti R, Magrini SM. Light and shadows of a new technique: is photon total-skin irradiation using helical IMRT feasible, less complex and as toxic as the electrons one? Radiation Oncology. 2018; 29;13(1):158. doi: 10.1186/s13014-018-1100-4
- Lee MJ, Son HJ. Electron beam radiotherapy for Kaposi's sarcoma of the toe and web. Journal of Cancer Research & Therapy. 2020;16(1):161-163. doi: 10.4103/jcrt.JCRT_115_18
- Zanoni M, Cortesi M, Zamagni A, Tesei A. The Role of Mesenchymal Stem Cells in Radiation-Induced Lung Fibrosis. International Journal of Molecular Sciences. 2019;20(16):3876. doi: 10.3390/ijms20163876
- Yumusak N, Sadic M, Yucel G, Atilgan HI, Koca G, Korkmaz M. Apoptosis, and cell proliferation in short-term and long-term effects of radioiodine-131-induced kidney damage: an experimental and immunohistochemical study. Nuclear Medicine Communications 2018;39(2):131-139. doi: 10.1097/MNM.0000000000000788
- Sheikh BY, Sarker MMR, Kamarudin MNA, Mohan G. Antiproliferative and apoptosis inducing effects of citral via p53 and ROS-induced mitochondrial-mediated apoptosis in human colorectal HCT116 and HT29 cell lines. Biomed Pharmacother. 2017;96:834-846. doi: 10.1016/j.biopha.2017.10.038
- Aratani S, Tagawa M, Nagasaka S, Sakai Y, Shimizu A, Tsuruoka S. Radiation-induced premature cellular senescence involved in glomerular diseases in rats. Scientific Reports. 2018;8(1):16812. doi: 10.1038/s41598-018-34893-8
- McRobb LS, McKay MJ, Gamble JR, Grace M, Moutrie V, Santos ED, Lee VS, Zhao Z, Molloy MP, Stoodley MA. Ionizing radiation reduces ADAM10 expression in brain microvascular endothelial cells undergoing stress-induced senescence. Aging (Albany NY). 2017;9(4):1248-1268. doi: 10.18632/aging.101225
- Ahmad A, Mitrofanova A, Bielawski J, et al. Sphingomyelinase-like phosphodiesterase 3b mediates radiation-induced damage of renal podocytes. FASEB J. 2017;31(2):771-780. doi: 10.1096/fj.201600618R
- Freudlsperger C, Freier K, Hoffmann J, Engel M. Ki-67 expression predicts radiosensitivity in oral squamous cell carcinoma. International Journal of Oral & Maxillofacial Surgery. 2012;41(8):965-9. doi: 10.1016/j.ijom.2012.04.014
- Fu DR, Kato D, Endo Y, Kadosawa T. Apoptosis and Ki-67 as predictive factors for response to radiation therapy in feline nasal lymphomas. Journal of Veterinary Medical Science. 2016;78(7):1161-6. doi: 10.1292/jvms.15-0693
- Couture C, Raybaud-Diogène H, Têtu B, Bairati I, Murry D, Allard J, Fortin A. p53 and Ki-67 as markers of radioresistance in head and neck carcinoma. Cancer. 2002;94(3):713-22. doi: 10.1002/cncr.10232
- Kim DH, Park JS, Choi HI, Kim CS, Bae EH, Ma SK, Kim SW. The critical role of FXR is associated with the regulation of autophagy and apoptosis in the progression of AKI to CKD. Cell Death and Disease. 2021;12(4):320. doi: 10.1038/s41419-021-03620-z
- Li G, Wang S, Fan Z. Oxidative Stress in Intestinal Ischemia-Reperfusion. Front Med (Lausanne). 2022 Jan 14;8:750731. doi: 10.3389/fmed.2021.750731
- Wang Q, Zhou Y, Wang X, Evers BM. Glycogen synthase kinase-3 is a negative regulator of extracellular signal-regulated kinase. Oncogene. 2006;25(1):43-50. doi: 10.1038/sj.onc.1209004
- Morel C, Carlson SM, White FM, Davis RJ. Mcl-1 integrates the opposing actions of signaling pathways that mediate survival and apoptosis. Molecular and Cellular Biology. 2009;29(14):3845-3852. doi: 10.1128/MCB.00279-09
- J., Allum AJ, Mussallem JT, Froning CE, Haskins AH, Buckner MA, Miller CD, Kato TA. Ascorbic Acid 2-Glucoside Pretreatment Protects Cells from Ionizing Radiation, UVC, and Short Wavelength of UVB. Genes (Basel). 2020;11(3):238. doi: 10.3390/genes11030238
- Petruk G, Del Giudice R, Rigano MM, Monti DM. Antioxidants from Plants Protect against Skin Photoaging. Oxidative Medicine and Cellular Longevity. 2018;2018:1454936. doi: 10.1155/2018/1454936
- El-Sonbaty SM, Moawed FSM, Elbakry MMM. Amphora algae with low-level ionizing radiation exposure ameliorate D-galactosamine-induced inflammatory impairment in rat kidney. Environmental Toxicology. 2021;36(4):451-459. doi: 10.1002/tox.23050
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