Immunohistochemical Analysis of TGF-β1 Expression in Rat Kidneys Using a Model of Hydrogen Sulfide and Cyclosporine A Exposure

Pathological aging of the kidneys is characterized by cellular senescence, inflammation, and fibrosis. The main regulator in this process is TGF-β1, which is secreted as part of the senescence-associated secretory phenotype (SASP). We have modeled accelerated aging using H2S and CsA, which induce oxidative stress.

The aim of the study was to evaluate the effect of H₂S and CsA, both individually and in combination, on the expression of TGFβ1 and the development of senescent changes in the kidneys of aged laboratory rats.

Materials and methods S . The study was conducted on 44 male white rats older than 18 months in 4 groups: control, H2 (0.26 mmol/day, 10 days), CsA (20 mg/kg/day, 25 days), and H2S+CsA. The kidney paraffin sections were analyzed morphologically and immunohistochemically (QuPath TGFβ1positive cell percentage). Statistical analysis: Kruskal–Wallis test. Results . The highest percentage of TGF-β1positive cells was found in the combined exposure group: up to 7.86 [7,64; 8,03] % in the tubules and up to 7.77 [7,61; 7,95] % in the interstitium. In the isolated exposure to H2S and CsA, the percentage of TGF-β1-positive cells was less: 4.97 [4,68; 5,66] % и 5.71 [5,47; 5,81] % in the tubules, respectively. Conclusion . The combined effect of H₂S and CsA synergistically increases the proportion of TGF-β1-positive cells in the tubules and interstitium of the kidneys, promoting senescence and fibrotic remodeling. TGF-β1 acts as a key marker and driver of pathological kidney aging.

1. Buyan MI, Andrianova NV, Plotnikov EYu. Age-related changes and loss of renal tissue resistance to damage: the role of reducing the number of kidney progenitor cells during aging. Problems of geroscience . 2023;3:127133 (In Russ.). DOI: http://doi.org/10.37586/2949-4745-3-2023-127-133.

2. Kaplunova OA. Age features of human renal arterial vessels. Medical Herald of the South of Russia . 2019;4:5158 (In Russ.). DOI: http://doi.org/10.21886/2219-8075-2019-10-4-51-58.

3. Anikonova LI, Vorobyeva OA, Bakulina NV. ATTR amyloidosis a systemic disease involving the kidneys. Nephrology and Dialysis . 2022;24(3):441–456 (In Russ.). DOI: http://doi.org/10.28996/2618-9801-2022-3-441-456.

4. Sentyabreva AV, Tsvetkov IS, Makarova OV, Kosyreva AM. Morphological characteristics of internal organs of sexually mature and old male Wistar rats with long-term consumption of aluminum chloride. Morphological Bulletin . 2025;33(2):id–950 (In Russ.). EDN: https://elibrary.ru/SXGGKD.

5. Shkuratova IA, Drozdova LI, Petrova IM Khatsko SL, Bytov M. V. Sokolova OV. Age-related aspects of structural and functional degeneration of chicken organ tissues under industrial conditions. Agricultural Scientific Journal . 2025;2:108–116 (In Russ.). DOI: http://doi.org/10.28983/asj.y2025i2pp108-116.

6. Herranz N, Gil J. Mechanisms and functions of cellular senescence. Journal of Clinical Investigation . 2018;128(4):1238–1246. DOI: http://doi.org/10.1172/JCI95148.

7. Bednov IA, Shur VYu, Trizno EV, Davydova DR, Trizno MN, Rstakyan AR. Pathophysiological mechanisms of agerelated vascular changes and potential correction pathways. International journal of medicine and psychology . 2023;6(2):54–67. EDN: https://elibrary.ru/CCUKZM.

8. Buret AG, Allain T, Motta JP, Wallace JL. Effects of Hydrogen Sulfide on the Microbiome: From Toxicity to Therapy. Antioxid Redox Signal . 2022;36(4–6):211–219. DOI: http://doi.org/10.1089/ars.2021.0004.

9. Lu QB, Ding Y, Fu X, Sun HJ, Zhang JR. Hydrogen sulfide in health and diseases: cross talk with noncoding RNAs. American Journal of Physiology Cell Physiology . 2023;324(4):C856–C877. DOI: http://doi.org/10.1152/ajpcell.00507.2022.

10. Kimura H. Hydrogen sulfide (H2S) and polysulfide (H2Sn) signaling: The first 25 years. Biomolecules . 2021;11(6):896. DOI: http://doi.org/10.3390/biom11060896.

11. Muñoz-Espín D, Cañamero M, Maraver A, Gómez-López G, Contreras J, Murillo-Cuesta S, et al. Programmed cell senescence during mammalian embryonic development. Cell . 2013;155(5):1104–1118. DOI: http://doi.org/10.1016/j.cell.2013.10.019.

12. Evangelou K, Gorgoulis VG. Sudan Black B, The specific histochemical stain for lipofuscin: A novel method to detect senescent cells. Methods in Molecular Biology . 2017;1534:111–119. DOI: http://doi.org/10.1007/9781-4939-6670-7_10.

13. Papageorgis P. Complex interplay between aging and cancer: Role of TGF-signaling. Critical Reviews in Oncogenesis . 2017;22:313–321. DOI: http://doi.org/10.1615/CritRevOncog.2017025134.

14. Wu J, Niu J, Li X, Wang X, Guo Z, Zhang F. TGF-1 induces senescence of bone marrow mesenchymal stem cells via increase of mitochondrial ROS production. BMC Developmental Biology . 2014;14:21. DOI: http://doi.org/10.1186/1471-213X-14-21.

15. Li J, Zou Y, Kantapan J, Su H, Wang L, Dechsupa N. TGF-β/Smad signaling in chronic kidney disease: exploring post-translational regulatory perspectives (Review). Molecular Medicine Reports . 2024;30(2):143. DOI: http://doi.org/10.3892/mmr.2024.13267.

16. Thomas HY, Ford Versypt AN. A mathematical model of glomerular fibrosis in diabetic kidney disease to predict therapeutic efficacy. Frontiers in Pharmacology . 2024;15:1481768. DOI: http://doi.org/10.3389/fphar.2024.1481768.

17. Sun H, Ke C, Zhang L, Tian C, Zhang Z, Wu S. Long non-coding RNA (LncRNA)-ATB promotes inflammation, cell apoptosis and senescence in transforming growth factor-β1 (TGF-β1) induced human kidney 2 (HK-2) cells via TGFβ/SMAD2/3signaling pathway. Medical Science Monitor . 2020;26:e922029. DOI: http://doi.org/10.12659/MSM.922029.

18. Trizno EV. Prosensent effect of hydrogen sulfide in rat kidneys university proceedings. Volga region. Medical sciences . 2023;4(68):221–230. DOI: http://doi.org/10.21685/2072-3032-2023-4-22.

19. Golovackaya LI, Trizno MN, Golubkina EV, Trizno NN, Dyukareva OS. Study of the mechanism of competitive inhibitory action of methionine against the toxic effect of hydrogen sulfide in vivo based on molecular sistems modelling. Atherosclerosis and dyslipidemias . 2022;3(48):14–20. DOI: http://doi.org/10.34687/22198202.jad.2022.03.0002.

20. Trizno EV, Mazhitova MV, Trizno MN, Davydova DR, Rstakyan AR, Hachatryan GS. Modification of the immune system with hydrogen sulfide and cyclosporine A. International journal of medicine and psychology . 2024;7(3):6–15. DOI: http://doi.org/10.58224/2658-3313-2024-7-3-6-15.

21. Trizno EV, Bednov IA, Trizno MN, Davydova DR, Rstakyan AR. Immunity and hemostasis system indicators in age groups of laboratory rats during the use of cyclosporine A. Immunopathology, Allergology, Infectology . 2023;4:60–65. DOI: http://doi.org/10.14427/jipai.2023.4.60.

22. Alqahtani S, Alqahtani T, Venkatesan K, Sivadasan D, Ahmed R, Sirag N., et al. SASP modulation for cellular rejuvenation and tissue homeostasis: Therapeutic strategies and molecular insights. Cells . 2025;14(8):608. DOI: http://doi.org/10.3390/cells14080608.

23. Kim HS, Choi S-I, Jeung E-B, Yoo Y-M. Cyclosporine A induces apoptotic and autophagic cell death in rat pituitary GH3 cells. PLOS One . 2014;9(10):e108981. DOI: https://doi.org/10.1371/journal.pone.0108981.

24. Liu QF, Ye JM, Yu LX, Dong XH, Feng JH, Xiong Y et al. Klotho mitigates cyclosporine A (CsA)-induced epithelial–mesenchymal transition (EMT) and renal fibrosis in rats. International Urology and Nephrology . 2017;49:345–352. DOI: https://doi.org/10.1007/s11255-016-1439-0.

25. Wu Q, Wang X, Nepovimova E, Wang Y, Yang H, Kuca K. Mechanism of cyclosporine A nephrotoxicity: Oxidative stress, autophagy, and signalings. Food and Chemical Toxicology . 2018;118:889–907. DOI: http://doi.org/10.1016/j.fct.2018.06.054.

26. Chen HJ, Qian L, Li K, Qin YZ, Zhou JJ, Ji XY, et al. Hydrogen sulfide-induced post-translational modification as a potential drug. Genes and Diseases . 2022;10(5):1870–1882. DOI: http://doi.org/10.1016/j.gendis.2022.03.022.

27. Ueda S, Tominaga T, Ochi A, Sakurai A, Nishimura K, Shibata E, et al. TGF-β1 is involved in senescence-related pathways in glomerular endothelial cells via p16 translocation and p21 induction. Scientific Reports . 2021;11:21643. DOI: http://doi.org/10.1038/s41598-021-01150-4.

28. Trizno MN, Kurtusunov BT, Trizno EV, Mazhitova MV. Detection of visceral organ cells hyporesponsive to lipofuscinogenesis. Modern Problems of Science and Education . 2024;(3):48. DOI: http://doi.org/10.17513/spno.33499.