Glycyrrhizic Acid Protects Glomerular Podocytes Induced by High Glucose by Modulating SNARK/AMPK Signaling Pathway

Abstract

Objective

Diabetic nephropathy is one of the most important microvascular complications of diabetes, which mainly refers to glomerular capillary sclerosis. Podocytes are an important part of glomerular capillaries. Previous clinical and basic studies have shown that fibrosis is the main factor of diabetic nephropathy. This study aimed to assess the protective mechanism of glycyrrhizic acid (GA) on glomerular podocytes induced by high glucose as we hypothesized that GA may have antifibrotic and anti-inflammatory effects on podocytes through regulation of the adenosine 5′-monophosphate-activated protein kinase (AMPK)/sucrose nonfermenting AMPK-related kinase (SNARK) signaling pathway.

Methods

SNARK siRNA was used to transfect podocytes. Real-time quantitative polymerase chain reaction and immunofluorescence staining assays were used for molecular and pathological analysis. The expression levels of key pathway proteins (including TGF-β1, α-SMA, SITR1, AMPKα, LKB1, PGC-1α, NF-κB, IL-6, and TNF-α) were verified by Western blotting. The expression of inflammatory factors in podocytes was detected by ELISA.

Results

We demonstrated that GA decreased the expression of podocyte fibrosis signaling pathway-related factors by upregulating the AMPK pathway and its related factors. However, after transfection of podocytes with SNARK siRNA, there was an increased expression of fibrosis-related factors and inflammation-related factors.

Conclusion

GA can protect podocytes and alleviate fibrosis and inflammation induced by high glucose, which is related to the AMPK signaling pathway. Meanwhile, knockdown of SNARK protein can inhibit the AMPK signaling pathway, aggravate fibrosis, and increase inflammation.

Keywords

podocyte / glomerular fibrosis / glycyrrhizic acid / diabetic nephropathy / AMPK / SNARK

References

[1] TsoutsoukiJ, WunnaW, ChowdhuryA, et al.. Advances in the management of diabetes: therapies for type 2 diabetes. Postgrad Med J, 2020, 96(1140): 610-618

[2] LibiantoR, DavisTM, EkinciEI, et al.. Advances in type 2 diabetes therapy: a focus on cardiovascular and renal outcomes. Med J Aust, 2020, 212(3): 133-139

[3] HorikoshiS, FukudaN, TsunemiA, et al.. Contribution of TGF-beta1 and Effects of Gene Silencer Pyrrole-Imidazole Polyamides Targeting TGF-beta1 in Diabetic Nephropathy. Molecules, 2020, 25(4): 950

[4] ChungJY, ChanMK, LiJS, et al.. TGF-beta Signaling: From Tissue Fibrosis to Tumor Microenvironment. Int J Mol Sci, 2021, 22(14): 7575

[5] WangY, JiaL, HuZ, et al.. AMP-activated protein kinase/myocardin-related transcription factor-A signaling regulates fibroblast activation and renal fibrosis. Kidney Int, 2018, 93(1): 81-94

[6] CoughlanKA, ValentineRJ, RudermanNB, et al.. AMPK activation: a therapeutic target for type 2 diabetes?. Diabetes Metab Syndr Obes, 2014, 7: 241-253

[7] LiNS, ZouJR, LinH, et al.. LKB1/AMPK inhibits TGF-beta1 production and the TGF-beta signaling pathway in breast cancer cells. Tumour Biol, 2016, 37(6): 8249-8258

[8] LefebvreDL, BaiY, ShahmolkyN, et al.. Identification and characterization of a novel sucrose-non-fermenting protein kinase/AMP-activated protein kinase-related protein kinase, SNARK. Biochem J, 2001, 355(2): 297-305

[9] GotoK, KatoN, ChungRT, et al.. Anti-hepatocellular carcinoma properties of the anti-alcoholism drug disulfiram discovered to enzymatically inhibit the AMPK-related kinase SNARK in vitro. Oncotarget, 2016, 7(46): 74987-74999

[10] CourchetJ, LewisTL, LeeS, et al.. Terminal axon branching is regulated by the LKB1-NUAK1 kinase pathway via presynaptic mitochondrial capture. Cell, 2013, 153(7): 1510-1525

[11] SunXL, LessardSJ, AnD, et al.. Sucrose nonfermenting AMPK-related kinase (SNARK) regulates exercise-stimulated and ischemia-stimulated glucose transport in the heart. J Cell Biochem, 2019, 120(1): 685-696

[12] LiuB, GanX, ZhaoY, et al.. Inhibition of HMGB1 reduced high glucose-induced BMSCs apoptosis via activation of AMPK and regulation of mitochondrial functions. J Physiol Biochem, 2021, 77(2): 227-235

[13] LiuY. New insights into epithelial-mesenchymal transition in kidney fibrosis. J Am Soc Nephrol, 2010, 21(2): 212-222

[14] TangQ, CaoY, XiongW, et al.. Glycyrrhizic acid exerts protective effects against hypoxia/reoxygenation-induced human coronary artery endothelial cell damage by regulating mitochondria. Exp Ther Med, 2020, 20(1): 335-342

[15] WangYJ, WangZ, ZhaoTQ, et al.. Effect of Glycyrrhizic Acid on High Glucose Induced Podocyte Injury in Mice. J Ningxia Med Univ (Chinese), 2022, 44(03): 267-271

[16] RajabBS, AlbukhariTA, KhanAA, et al.. Antioxidative and Anti-Inflammatory Protective Effects of beta-Caryophyllene against Amikacin-Induced Nephrotoxicity in Rat by Regulating the Nrf2/AMPK/AKT and NF-kappaB/TGF-beta/KIM-1 Molecular Pathways. Oxid Med Cell Longev, 2022, 2022: 4212331

[17] ZhangYL, LiPB, HanX, et al.. Blockage of Fibronectin 1 Ameliorates Myocardial Ischemia/Reperfusion Injury in Association with Activation of AMP-LKB1-AMPK Signaling Pathway. Oxid Med Cell Longev, 2022, 2022: 6196173

[18] UmanathK, LewisJB. Update on Diabetic Nephropathy: Core Curriculum. Am J Kidney Dis, 2018, 71(6): 884-895

[19] BhattDL, SzarekM, PittB, et al.. Sotagliflozin in Patients with Diabetes and Chronic Kidney Disease. N Engl J Med, 2021, 384(2): 129-139

[20] ChenJ, ChenJK, HarrisRC, et al.. EGF receptor deletion in podocytes attenuates diabetic nephropathy. J Am Soc Nephrol, 2015, 26(5): 1115-1125

[21] WuM, YangZ, ZhangC, et al.. Inhibition of NLRP3 inflammasome ameliorates podocyte damage by suppressing lipid accumulation in diabetic nephropathy. Metabolism, 2021, 118: 154748

[22] SongS, QiuD, ShiY, et al.. Thioredoxin-interacting protein deficiency alleviates phenotypic alterations of podocytes via inhibition of mTOR activation in diabetic nephropathy. J Cell Physiol, 2019, 234(9): 16485-16502

[23] GilCL, HookerE, LarriveeB, et al.. Diabetic Kidney Disease, Endothelial Damage, and Podocyte-Endothelial Crosstalk. Kidney Med, 2020, 3(1): 105-115

[24] GuoQ, ZhongW, DuanA, et al.. Protective or deleterious role of Wnt/beta-catenin signaling in diabetic nephropathy: An unresolved issue. Pharmacol Res, 2019, 144: 151-157

[25] ChaudhuriA, GhanimH, AroraP, et al.. Improving the residual risk of renal and cardiovascular outcomes in diabetic kidney disease: A review of pathophysiology, mechanisms, and evidence from recent trials. Diabetes Obes Metab, 2022, 24(3): 365-376

[26] GrahammerF, SchellC, HuberTB, et al.. The podocyte slit diaphragm—from a thin grey line to a complex signalling hub. Nat Rev Nephrol, 2013, 9(10): 587-598

[27] WangD, SantS, FerrellN, et al.. A Biomimetic In Vitro Model of the Kidney Filtration Barrier Using Tissue-Derived Glomerular Basement Membrane. Adv Healthc Mater, 2021, 10(16): e2002275

[28] YoshidaS, WeiX, ZhangG, et al.. Endoplasmic reticulum-associated degradation is required for nephrin maturation and kidney glomerular filtration function. J Clin Invest, 2021, 131(7): e143988

[29] LuQ, HouQ, CaoK, et al.. Complement factor B in high glucose-induced podocyte injury and diabetic kidney disease. JCI Insight, 2021, 6(19): e147716

[30] LinCL, HsuYC, HuangYT, et al.. A KDM6A-KLF10 reinforcing feedback mechanism aggravates diabetic podocyte dysfunction. EMBO Mol Med, 2019, 11(5): e9828

[31] ShengL, ZhuangS. New Insights Into the Role and Mechanism of Partial Epithelial-Mesenchymal Transition in Kidney Fibrosis. Front Physiol, 2020, 11: 569322

[32] LiL, FengY, ZhangJ, et al.. Microtubule associated protein 4 phosphorylation-induced epithelial-to-mesenchymal transition of podocyte leads to proteinuria in diabetic nephropathy. Cell Commun Signal, 2022, 20(1): 115

[33] CherneyDZI, DekkersCCJ, BarbourSJ, et al.. Effects of the SGLT2 inhibitor dapagliflozin on proteinuria in non-diabetic patients with chronic kidney disease (DIAMOND): a randomised, double-blind, crossover trial. Lancet Diabetes Endocrinol, 2020, 8(7): 582-593

[34] PerkovicV, JardineMJ, NealB, et al.. Canagliflozin and Renal Outcomes in Type 2 Diabetes and Nephropathy. N Engl J Med, 2019, 380(24): 2295-2306

[35] Gujarati NA, Leonardo AR, Vasquez JM, et al. Loss of Functional SCO2 Attenuates Oxidative Stress in Diabetic Kidney Disease. Diabetes, 2021,db210316

[36] GalvanDL, LongJ, GreenN, et al.. Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice. J Clin Invest, 2019, 129(7): 2807-2823

[37] SunW, WangY, ZhengY, et al.. The Emerging Role of Sestrin2 in Cell Metabolism, and Cardiovascular and Age-Related Diseases. Aging Dis, 2020, 11(1): 154-163

[38] JuszczakF, CaronN, MathewAV, et al.. Critical Role for AMPK in Metabolic Disease-Induced Chronic Kidney Disease. Int J Mol Sci, 2020, 21(21): 7994

[39] SongS, ShiC, BianY, et al.. Sestrin2 remedies podocyte injury via orchestrating TSP-1/TGF-beta1/Smad3 axis in diabetic kidney disease. Cell Death Dis, 2022, 13(7): 663

[40] WuM, ChenG, LiYP, et al.. TGF-beta and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease. Bone Res, 2016, 4: 16009

[41] ZhangYD, ZhaoSC, ZhuZS, et al.. Cx43- and Smad-Mediated TGF-beta/ BMP Signaling Pathway Promotes Cartilage Differentiation of Bone Marrow Mesenchymal Stem Cells and Inhibits Osteoblast Differentiation. Cell Physiol Biochem, 2017, 42(4): 1277-1293

[42] LiJ, LiN, YanS, et al.. Melatonin attenuates renal fibrosis in diabetic mice by activating the AMPK/PGC1alpha signaling pathway and rescuing mitochondrial function. Mol Med Rep, 2019, 19(2): 1318-1330

[43] KimH, MoonSY, KimJS, et al.. Activation of AMP-activated protein kinase inhibits ER stress and renal fibrosis. Am J Physiol Renal Physiol, 2015, 308(3): F226-F236

[44] GamadN, MalikS, SuchalK, et al.. Metformin alleviates bleomycin-induced pulmonary fibrosis in rats: Pharmacological effects and molecular mechanisms. Biomed Pharmacother, 2018, 97: 1544-1553

[45] WangL, TianY, ShangZ, et al.. Metformin attenuates the epithelial-mesenchymal transition of lens epithelial cells through the AMPK/TGF-beta/Smad2/3 signalling pathway. Exp Eye Res, 2021, 212: 108763

[46] van de VisRAJ, MoustakasA, van der HeideLP, et al.. NUAK1 and NUAK2 Fine-Tune TGF-beta Signaling. Cancers (Basel), 2021, 13(13): 3377

[47] KolliopoulosC, RajaE, RazmaraM, et al.. Transforming growth factor beta (TGFbeta) induces NUAK kinase expression to fine-tune its signaling output. J Biol Chem, 2019, 294(11): 4119-4136

[48] FengX, ChenX, ZaeemM, et al.. Sesamol Attenuates Neuroinflammation by Regulating the AMPK/SIRT1/NF-kappaB Signaling Pathway after Spinal Cord Injury in Mice. Oxid Med Cell Longev, 2022, 2022: 8010670

[49] XuT, WangS, LiX, et al.. Lithium chloride represses abdominal aortic aneurysm via regulating GSK3beta/SIRT1/NF-kappaB signaling pathway. Free Radic Biol Med, 2021, 166: 1-10

[50] GaoC, FeiX, WangM, et al.. Cardamomin protects from diabetes-induced kidney damage through modulating PI3K/AKT and JAK/STAT signaling pathways in rats. Int Immunopharmacol, 2022, 107: 108610

[51] SunHJ, XiongSP, CaoX, et al.. Polysulfide-mediated sulfhydration of SIRT1 prevents diabetic nephropathy by suppressing phosphorylation and acetylation of p65 NF-kappaB and STAT3. Redox Biol, 2021, 38: 101813

[52] KimuraY, YanagidaT, OndaA, et al.. Soluble Uric Acid Promotes Atherosclerosis via AMPK (AMP-Activated Protein Kinase)-Mediated Inflammation. Arterioscler Thromb Vasc Biol, 2020, 40(3): 570-582

[53] KatoK, TokudaH, Matsushima-NishiwakiR, et al.. AMPK limits IL-1-stimulated IL-6 synthesis in osteoblasts: involvement of IkappaB/NF-kappaB pathway. Cell Signal, 2012, 24(8): 1706-1712

[54] OhH, ParkSH, KangMK, et al.. Asaronic Acid Attenuates Macrophage Activation toward M1 Phenotype through Inhibition of NF-kappaB Pathway and JAK-STAT Signaling in Glucose-Loaded Murine Macrophages. J Agric Food Chem, 2019, 67(36): 10069-10078

[55] CaoXJ, WuR, QianHY, et al.. Metformin attenuates diabetic neuropathic pain via AMPK/NF-kappaB signaling pathway in dorsal root ganglion of diabetic rats. Brain Res, 2021, 1772: 147663

[56] LiF, ChenY, LiY, et al.. Geniposide alleviates diabetic nephropathy of mice through AMPK/SIRT1/NF-kappaB pathway. Eur J Pharmacol, 2020, 886: 173449

[57] ManciniSJ, WhiteAD, BijlandS, et al.. Activation of AMP-activated protein kinase rapidly suppresses multiple pro-inflammatory pathways in adipocytes including IL-1 receptor-associated kinase-4 phosphorylation. Mol Cell Endocrinol, 2017, 440: 44-56

[58] HuangW, ShangWL, WangHD, et al.. Sirt1 overexpression protects murine osteoblasts against TNF-alpha-induced injury in vitro by suppressing the NF-kappaB signaling pathway. Acta Pharmacol Sin, 2012, 33(5): 668-674

[59] LeeIY, LimJM, ChoH, et al.. MST1 Negatively Regulates TNFalpha-Induced NF-kappaB Signaling through Modulating LUBAC Activity. Mol Cell, 2019, 73(6): 1138-1149.e6

[60] LiHN, YangQQ, WangWT, et al.. Red nucleus IL-33 facilitates the early development of mononeuropathic pain in male rats by inducing TNF-alpha through activating ERK, p38 MAPK, and JAK2/STAT3. J Neuroinflammation, 2021, 18(1): 150

[61] FadaeiR, BagheriN, HeidarianE, et al.. Serum levels of IL-32 in patients with type 2 diabetes mellitus and its relationship with TNF-alpha and IL-6. Cytokine, 2020, 125: 154832

[62] PangR, GuD. Triptolide Improves Renal Injury in Diabetic Nephropathy Rats through TGF-beta1/Smads Signal Pathway. Endocr Metab Immune Disord Drug Targets, 2021, 21(10): 1905-1911

[63] Zitman-GalT, EinbinderY, OhanaM, et al.. Effect of liraglutide on the Janus kinase/signal transducer and transcription activator (JAK/STAT) pathway in diabetic kidney disease in db/db mice and in cultured endothelial cells. J Diabetes, 2019, 11(8): 656-664

[64] JohnsonDE, O’KeefeRA, GrandisJR, et al.. Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat Rev Clin Oncol, 2018, 15(4): 234-248