The Protective Effects Of Pachymic Acid in Acute Kidney Injury
Mar 25, 2022
Contact: joanna.jia@wecistanche.com / WhatsApp: 008618081934791
GUI-PING JIANG,YUE-JUANLIAO, L-LI HUANG,XU-JIAZENG & XIAO-HUI LIAO
Abstract
Acute kidney injury (AKI) is a common clinical disease. Ferroptosis, a new type of regulatory cell death, serves an important regulatory role in AKI(Acute kidney injury). Pachydermic acid (PA), a lanostane-type triterpenoid from Poria cocos, has been reported to be protective against AKI(Acute kidney injury). However, the protective mechanism of PA(Pachymic acid) in AKI(Acute kidney injury) is not yet fully understood. The present study aimed to investigate the effect and molecular mechanism of PA(Pachymic acid) on ferroptosis in renal ischemia-reperfusion injury in vio. A total of 30 mice were intra-peritoneally injected with 5,10 and 20 mg/kg PA(Pachymic acid) for 3 days. A bilateral renal pedicle clip was used for 40 min to induce renal ischemia-reperfusion injury and establish the model. The results demonstrated that treatment with PA(Pachymic acid) decreased serum creatinine and blood urea nitrogen, and ameliorated renal pathological damage. Transmission electron microscopy revealed no characteristic changes in ferroptosis in the mitochondria of the renal tissue in the high-dose PA(Pachymic acid) group and only mild edema. Furthermore, treatment with PA(Pachymic acid) increased glutathione expression and decreased the expression levels of malondialdehyde and cyclooxygenase 2. Treatment with PA(Pachymic acid) enhanced the protein and mRNA expression levels of the ferroptosis related proteins, glutathione peroxidase 4 (GPX4), solute carrier family 7(cationic amino acid transporter,y+system) member 11(SLC7All), and heme oxygenase 1(HO-1)in the kidney, and increased the expression levels of nuclear factor erythroid-derived 2 like 2 (NRF2) signaling pathway members. Taken together, the results of the present study suggest that PA(Pachymic acid) has a protective effect on ischemia-reperfusion induced acute kidney injury in mice, which may be associated with the inhibition of ferroptosis in the kidneys through direct or indirect activation of NRF2, and upregulation of the expression of the downstream ferroptosis related proteins, GPX4, SLCTA11, and HO-1.
dr vita opc fda approved for kidney
CLICK HERE TO PART Ⅰ
Discussion
Previous studies have demonstrated that PA(Pachymic acid) ameliorates renal injury in AKI(Acute kidney injury) and chronic kidney disease via anti-inflammatory and antioxidative stress(21,22). However, whether PA(Pachymic acid) inhibits ferroptosis has not yet been investigated. To the best of our knowledge, the present study was the first to investigate the effect of PA(Pachymic acid) on ferroptosis in IRI-AKI(Acute kidney injury).In the present study, bilateral renal pedicle clamping in mice for 40 min followed by reperfusion for 24 h was used to successfully establish IRI and induce AKI(Acute kidney injury). The results demonstrated that Scr and BUN levels were significantly elevated in the model group, and renal tubules were significantly damaged, based on H&E staining and the Paller score. Thus, the IRI-AKI(Acute kidney injury) model was constructed. Notably, administration of PA(Pachymic acid) significantly decreased the IRI enhanced Scr and BUN serum levels, and alleviated kidney injury in a dose-dependent manner.
GPX4 serves an important role in ferroptosis (30). Small molecules, such as elastin, RSL-3, and RSL-5(13), through different signaling pathways, directly or indirectly inhibit GPX4 activity, thus decreasing cell antioxidant capacity, causing reactive oxygen species overload and membrane lipid peroxidation reactions, and ultimately damaging cell membrane integrity and causing cell ferroptosis (13). In GPX4 knockout mice, GPX4 deficiency leads to spontaneous acute renal failure and an increased rate of early mortality, whereas ferroptosis of renal tubular epithelial cells is the main cause of renal failure in GPX4 knockout mice (30). Thus, increasing GPX4 activity effectively inhibits ferroptosis. The synthesis of GSH, a cofactor necessary for GPX4 function, directly increases GPX4 activity. GSH synthesis is influenced by cystine. The transport of cystine from the extracellular to the intracellular space is largely dependent on the cystine/glutamate reverse transporter (system xc-)on the cell membrane. This transporter is a heterodimer composed of two subunits, SLC7Al1(xCT) and SLC3A2. It mainly transports glutamate to the extracellular space and mediates cysteine entry into the cell (28). Inhibiting the activity of the system xc-affects the synthesis of GSH by inhibiting the absorption of cystine, thereby leading to inactivity of GPX4, a decrease in cell antioxidant capacity, accumulation of lipid reactive oxygen species, and ultimately oxidative damage and ferroptosis (31). Yang et al (32)confirmed that prostaglandin-endoperoxide synthase 2(PTGS2), encoding COX-2, is a suitable marker for the lipid peroxidation that occurs during GPX4-regulated ferroptosis. Thus, PTGS2 upregulation has been suggested to be a downstream marker of ferroptosis. Enhancing system xc-, promoting the absorption of cystine, increasing the production of GSH, and increasing the activity of GPX4 may inhibit ferroptosis and decrease renal injury (32,33). The results of the present study demonstrated that the system xc-activity decreased, GSH synthesis decreased, GPX4 activity decreased, MDA and Cox-2 expression increased, and ferroptosis occurred in IRI-AKI(Acute kidney injury) mice. In addition, treatment with PA(Pachymic acid) significantly increased the expression levels of GSH, xCT, and GPX4, and decreased the expression levels of MDA and Cox‑2, thus suggesting that treatment with moderate and high doses of PA(Pachymic acid) enhances system xc‑ activity, promotes the absorption of cystine, increases GSH production, increases GPX4 activity and inhibits ferroptosis.
NRF2, a stress‑inducible transcription factor, has emerged as a key regulator of both lipid peroxidation and ferroptosis (23). Notably, a number of the proteins and enzymes (such as GPX4, xCT, and the glutamate‑cysteine ligase catalytic and modifier subunits) are responsible for preventing lipid peroxidation, and thus initiation of ferroptosis, are NRF2 target genes (23). NRF2 serves a key role in regulating iron/heme metabolism (34). Following activation of NRF2, NRF2 upregulates iron metabolism-related proteins involved in ferroptosis, such as ferritin (Ferritin light chain and Ferritin heavy polypeptide 1), the intracellular iron storage proteins (35), iron chelatase, and HO‑1 (36). Adedoyin et al (37) reported that HO‑1 in proximal renal tubular epithelial cells can resist ferroptosis induced by Erastin in AKI(Acute kidney injury), thus, decreasing AKI(Acute kidney injury). In addition, NRF2 regulates proteases involved in GSH synthesis and metabolism, such as glutamine‑cysteine ligase catalytic and regulatory subunits, glutathione synthase, and a subunit of the cystine/glutamate transporter xCT (38‑40). GPX4 is a target regulatory gene of NRF2 (41). Li et al (42) demonstrated that pretreatment with Roxadustat (FG‑4592) attenuates folic acid‑induced kidney injury through anti‑ferroptosis via the Akt/GSK‑3β/NRF2 pathway. The results of the present study demonstrated that administration of moderate and high doses of PA(Pachymic acid) directly or indirectly promoted the activation of the NRF2 signaling pathway, upregulated the expression levels of the downstream ferroptosis regulated proteins, GPX4, xCT, and HO‑1, increased GSH levels in renal tissue, and decreased the levels of the ferroptosis‑associated lipid peroxidation protein, COX‑2 and the lipid oxidation indicator, MDA.
Taken together, the results of the present study suggest that PA(Pachymic acid) may improve renal function and decrease renal injury in IRI‑AKI(Acute kidney injury) mice. This protective effect may be associated with the inhibition of ferroptosis in the kidneys through direct or indirect activation of NRF2, and upregulation of the downstream ferroptosis‑related proteins, GPX4, xCT, and HO‑1 (Fig. 6). However, the present study is not without limitations. The effect of PA(Pachymic acid) on ferroptosis has not been extensively studied. Thus, prospective studies should use elastin, a selective inhibitor of system xc‑, to inhibit the activity of xCT in mice, and subsequently verify whether inhibition of xCT induces ferroptosis in mouse kidney cells, thus leading to AKI(Acute kidney injury) and whether PA(Pachymic acid) inhibits ferroptosis mainly through the xCT‑GPX4 pathway. Further mechanistic studies must be performed in vitro. In addition, PA should be used to treat renal tubular epithelial cells to verify the effect of PA(Pachymic acid) on ferroptosis in vitro.
Figure 6. Schematic diagram of proposed molecular mechanisms for PA(Pachymic acid) regulating ferroptosis in IRI‑AKI(Acute kidney injury). PA(Pachymic acid) can improve renal function and decrease renal injury in IRI‑AKI(Acute kidney injury) mice by activating the NRF2 signaling pathway, GSH, and downstream ferroptosis‑related proteins (GPX4, xCT, and HO‑1) to inhibit ferroptosis in the kidney. PA, pachydermic acid; IRI, ischemia-reperfusion injury; AKI(Acute kidney injury); NRF2, nuclear factor erythroid derived 2 like 2; GSH, glutathione; GPX4, glutathione peroxidase 4; HO‑1, heme oxygenase 1; xCT, glutamate transporter system; Glu, glutamic acid; Cys, cysteine.
References
1. Hoste EAJ, Kellum JA, Selby NM, Zarbock A, Palevsky PM, Bagshaw SM, Goldstein SL, Cerda J, and Chawla LS: Global epidemiology and outcomes of acute kidney injury. Nat Rev Nephrol 14: 607-625.2018.
2. Kaddourah A.Basu RK.Bagshaw SM and Goldstein SL: AWARE Investigators: Epidemiology of acute kidney injury in critically ill children and young adults. N Engl J Med 376: 11-20.2017.
3. Chertow GM, Burdick, Honour M, Bonventre JV, and Bates DW: Acute kidney injury, mortality, length of stay, and costs in hospitalized patients. J Am Soc Nephrol 16:3365-3370,2005.
4. Susantitaphong P, Cruz DN, Cerda J, Abulfaraj M, Alqahtani F Koulouridis I and Jaber BL; Acute Kidney Injury Advisory Group of the American Society of Nephrology: World incidence of AKI(Acute kidney injury): A meta-analysis. Clin JAm Soc Nephrol 8:1482-1493, 2013.
5. Ronco C, Bellomo R, and Kellum JA: Acute kidney injury. Lancet 394:1949-1964,2019.
6. James MT, Bhatt M, Pannu Nand Tonelli M: Long-term outcomes of acute kidney injury and strategies for improved care. Nat Rev Nephrol 16: 193-205,2020.
7. Han SJ and Lee HT: Mechanisms and therapeutic targets of ischemic acute kidney injury. Kidney Res Clin Pract 38: 427-440, 2019.
8. Priante G, GianeselloL, Ceol M, Del Prete D, and England F: Cell death in the kidney.Int J Mol Sci 20:3598,2019.
9. Martin-Sanchez D,Poveda J,Fontecha-Barriuso M, Ruiz-Andres O, Sanchez-Nino MD, Ruiz-Ortega M, Ortiz A and Sanz AB:Targeting of regulated necrosis in kidney disease. Nefrologia 38: 125-135.2018.
10. Kers J.Leemans JCand Linkermann A: An overview of pathways of regulated necrosis in acute kidney injury. Semin Nephrol 36:139-152,2016.
11. Pefanis A, Perino FL, Murphy JM, and Cowan PJ: Regulated necrosis in kidney ischemia-reperfusion injury. Kidney Int 96:291-301,2019.
12. Hu Z, Zhang H, Yang SK, Wu X, He D, Cao K, and Zhang W: Emerging role of ferroptosis in acute kidney injury. Oxid Med Cell Longev 2019:8010614,2019.
13. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, et al:Ferroptosis: An iron-dependent form of nonapoptotic cell death. Cell 149: 1060-1072,2012.
14. Li J, Cao F, Yin HL, Huang ZJ, Lin ZT, Mao N, Sun B, and Wang G: Ferroptosis: Past, present, and future.Cell Death Dis 11:88,2020.
15. Linkermann A, Skouta R, Himmerkus N, Mulay SR, Dewitz C, De Zen F,Prokai A,Zuchtriegel G,Krombach F, Welz PS,et al:Synchronized renal tubular cell death involves ferroptosis. Proc Natl Acad Sci USA 111: 16836-16841,2014.
16. Ling H, Zhang Y, Ng KY, and Chew EH: Pachymic acid impairs breast cancer cell invasion by suppressing nuclear factor-kB-dependent matrix metalloproteinase-9 expression. Breast Cancer Res Treat 126: 609-620.2011.
17. Li FF, Yuan Y, Liu Y, Wu QQ, Jiao R, Yang Z, Zhou MQ, and Tang QZ: Pachymic acid protects H9c2 cardiomyocytes from lipopolysaccharide-induced inflammation and apoptosis by inhibiting the extracellular signal-regulated kinase 1/2 and p38 pathways. Mol Med Rep 12: 2807-2813,2015.
18. Li JY, Wu HX, and Yang G: Pachymic acid improves survival and attenuates acute lung injury in septic rats induced by cecal ligation and puncture.Eur Rev Med Pharmacol Sci 21:1904-1910.2017
19. Li TH, Hou CC, Chang CL, and Yang WC: Anti-hyperglycemic properties of crude extract and triterpenes from Poria cocos. Evid Based Complement Alternat Med 2011: 128402,2011.
20. Shah VK, Choi JJ, Han JY, Lee MK, Hong JT, and Oh KW: Pachymic acid enhances pentobarbital-induced sleeping behaviors via GABAA-ergic systems in mice.Biomol Ther(Seoul)22:314-320.2014.
21. Cai ZY, Sheng ZX, and Yao H: Pachymic acid ameliorates sepsis-induced acute kidney injury by suppressing inflammation and activating the Nrf2/HO-1 pathway in rats.Eur Rev Med Pharmacol Sci 21:1924-1931,2017.
22. Chen DQ, Feng YL, Chen L, Liu JR, Wang M, Vaziri ND, and Zhao YY: Poricoic acid A enhances melatonin inhibition of AKI(Acute kidney injury)-to-CKD transition by regulating Gas6/AxINFxB/Nrf2 axis. Free Radic Biol Med 134: 484-497,2019.
23. Dodson M, Castro-Portuguez R, and Zhang DD: NRF2 plays a critical role in mitigating lipid peroxidation and ferroptosis. Redox Biol 23:101107,2019.
24. National Research Council (US)Institute for Laboratory Animal Research: Guide for the Care and Use of Laboratory Animals. Washington(DC): National Academies Press(US);1996.https://www.ncbi.nlm.nih.gov/books/NBK232589/doi: 10.17226/5140.
25. Wei Qand Dong Z: A mouse model of ischemic acute kidney injury: Technical notes and tricks.Am J Physiol Renal Physiol 303:F1487-F1494,.2012.
26. Paller MS, Hoidal JR, and Ferris TF: Oxygen free radicals in ischemic acute renal failure in the rat. J Clin Invest 74:1156-1164, 1984.
