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Klotho preservation by Rhein promotes toll-like receptor 4 proteolysis and attenuates lipopolysaccharide-induced acute kidney injury

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Abstract

Renal anti-aging protein Klotho exhibits impressive properties of anti-inflammation and renal protection, however is suppressed early after renal injury, making Klotho restoration an attractive strategy of treating renal inflammatory disorders. Here, we reported that Klotho is enriched in macrophages and Klotho preservation by Rhein, an anthraquinone derived from medicinal plant rhubarb, attenuates lipopolysaccharide (LPS)-induced acute inflammation essentially via promoting toll-like receptor 4 (TLR4) degradation. LPS-induced pro-inflammatory NF-κB signaling and cytokine expressions coincided with Klotho repression and toll-like receptor 4 (TLR4) elevation in macrophages, renal epithelial cells, and acutely- inflamed kidney. Intriguingly, Rhein treatment effectively corrected the inverted alterations of Klotho and TLR4 and mitigated the TLR4 downstream inflammatory response in a Klotho restoration and TLR4 repression-dependent manner. Klotho inducibly associated with TLR4 after LPS stimulation and suppressed TLR4 protein abundance mainly via a proteolytic process sensitive to the inhibition of Klotho’s putative β-glucuronidase activity. Consistently, Klotho knockdown by RNA interferences largely diminished the anti-inflammatory and renal protective effects of Rhein in a mouse model of acute kidney injury incurred by LPS. Thus, Klotho suppression of TLR4 via deglycosylation negatively controls TLR-associated inflammatory signaling and the endogenous Klotho preservation by Rhein or possibly other natural or synthetic compounds possesses promising potentials in the clinical treatment of renal inflammatory disorders.

Key messages

• Klotho is highly expressed in macrophages and repressed by LPS in vitro and in vivo.

• Klotho inhibits LPS-induced TLR4 accumulation and the downstream signaling.

• Klotho decreases TLR4 via a deglycosylation-associated proteolytic process.

• Rhein effectively prevents acute inflammation-incurred Klotho suppression.

• Rhein reversal of Klotho attenuates LPS-induced acute inflammation and kidney injury.

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References

  1. Zhao Y, Banerjee S, Dey N, LeJeune WS, Sarkar PS, Brobey R, Rosenblatt KP, Tilton RG, Choudhary S (2011) Klotho depletion contributes to increased inflammation in kidney of the db/db mouse model of diabetes via RelA (serine)536 phosphorylation. Diabetes 60:1907–1916

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E et al (1997) Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 390:45–51

    Article  PubMed  CAS  Google Scholar 

  3. Kurosu H, Ogawa Y, Miyoshi M, Yamamoto M, Nandi A, Rosenblatt KP, Baum MG, Schiavi S, Hu MC, Moe OW, Kuro-o M (2006) Regulation of fibroblast growth factor-23 signaling by klotho. J Biol Chem 281:6120–6123

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Urakawa I, Yamazaki Y, Shimada T, Iijima K, Hasegawa H, Okawa K, Fujita T, Fukumoto S, Yamashita T (2006) Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature 444:770–774

    Article  PubMed  CAS  Google Scholar 

  5. Imura A, Tsuji Y, Murata M, Maeda R, Kubota K, Iwano A, Obuse C, Togashi K, Tominaga M, Kita N, Tomiyama KI, Iijima J, Nabeshima Y, Fujioka M, Asato R, Tanaka S, Kojima K, Ito J, Nozaki K, Hashimoto N, Ito T, Nishio T, Uchiyama T, Fujimori T, Nabeshima YI (2007) Alpha-klotho as a regulator of calcium homeostasis. Science 316:1615–1618

    Article  PubMed  CAS  Google Scholar 

  6. Almilaji A, Honisch S, Liu G, Elvira B, Ajay SS, Hosseinzadeh Z, Ahmed M, Munoz C, Sopjani M, Lang F (2014) Regulation of the voltage gated K channel Kv1.3 by recombinant human klotho protein. Kidney Blood Press Res 39:609–622

    Article  PubMed  CAS  Google Scholar 

  7. Tsujikawa H, Kurotaki Y, Fujimori T, Fukuda K, Nabeshima Y (2003) Klotho, a gene related to a syndrome resembling human premature aging, functions in a negative regulatory circuit of vitamin D endocrine system. Mol Endocrinol(Baltimore, Md) 17:2393–2403

    Article  CAS  Google Scholar 

  8. Kurosu H, Yamamoto M, Clark JD, Pastor JV, Nandi A, Gurnani P, McGuinness OP, Chikuda H, Yamaguchi M, Kawaguchi H, Shimomura I, Takayama Y, Herz J, Kahn CR, Rosenblatt KP, Kuro-o M (2005) Suppression of aging in mice by the hormone klotho. Science (New York, NY) 309:1829–1833

    Article  CAS  Google Scholar 

  9. Satoh M, Nagasu H, Morita Y, Yamaguchi TP, Kanwar YS, Kashihara N (2012) Klotho protects against mouse renal fibrosis by inhibiting Wnt signaling. Am J Physiol Renal Physiol 303:F1641–F1651

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Doi S, Zou Y, Togao O, Pastor JV, John GB, Wang L, Shiizaki K, Gotschall R, Schiavi S, Yorioka N, Takahashi M, Boothman DA, Kuro-o M (2011) Klotho inhibits transforming growth factor-beta1 (TGF-beta1) signaling and suppresses renal fibrosis and cancer metastasis in mice. J Biol Chem 286:8655–8665

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Imura A, Iwano A, Tohyama O, Tsuji Y, Nozaki K, Hashimoto N, Fujimori T, Nabeshima Y (2004) Secreted klotho protein in sera and CSF: implication for post-translational cleavage in release of klotho protein from cell membrane. FEBS Lett 565:143–147

    Article  PubMed  CAS  Google Scholar 

  12. Chang Q, Hoefs S, van der Kemp AW, Topala CN, Bindels RJ, Hoenderop JG (2005) The beta-glucuronidase klotho hydrolyzes and activates the TRPV5 channel. Science (New York, NY) 310:490–493

    Article  CAS  Google Scholar 

  13. Buendia P, Carracedo J, Soriano S, Madueno JA, Ortiz A, Martin-Malo A, Aljama P, Ramirez R (2015) Klotho prevents NFkappaB translocation and protects endothelial cell from senescence induced by uremia. J Gerontol A Biol Sci Med Sci 70:1198–1209

    Article  PubMed  CAS  Google Scholar 

  14. Maekawa Y, Ishikawa K, Yasuda O, Oguro R, Hanasaki H, Kida I, Takemura Y, Ohishi M, Katsuya T, Rakugi H (2009) Klotho suppresses TNF-alpha-induced expression of adhesion molecules in the endothelium and attenuates NF-kappaB activation. Endocrine 35:341–346

    Article  PubMed  CAS  Google Scholar 

  15. Gao Y, Chen X, Fang L, Liu F, Cai R, Peng C, Qi Y (2014) Rhein exerts pro- and anti-inflammatory actions by targeting IKKβ inhibition in LPS-activated macrophages. Free Radic Biol Med 72:104–112

    Article  PubMed  CAS  Google Scholar 

  16. Theodoros Eleftheriadis GP, Vassilios Liakopoulos, Ioannis Stefanidis, Brian R. Lawson (2012) Toll-like receptors and their role in renal pathologies. Inflamm Allergy Drug Targets 11: 464–477

  17. Oh HJ, Nam BY, Lee MJ, Kim CH, Koo HM, Doh FM, Han JH, Kim EJ, Han JS, Park JT, Yoo TH, Kang SW, Han DS, Han SH (2015) Decreased circulating klotho levels in patients undergoing dialysis and relationship to oxidative stress and inflammation. Perit Dial Int : J Int Soc Perit Dial 35:43–51

    Article  CAS  Google Scholar 

  18. Zhou X, Chen K, Lei H, Sun Z (2015) Klotho gene deficiency causes salt-sensitive hypertension via monocyte chemotactic protein-1/CC chemokine receptor 2-mediated inflammation. J Am Soc Nephrol : JASN 26:121–132

    Article  PubMed  CAS  Google Scholar 

  19. Wang Y, Sun Z (2009) Klotho gene delivery prevents the progression of spontaneous hypertension and renal damage. Hypertension 54:810–817

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Zhang Q, Liu L, Lin W, Yin S, Duan A, Liu Z, Cao W (2017) Rhein reverses klotho repression via promoter demethylation and protects against kidney and bone injuries in mice with chronic kidney disease. Kidney Int 91:144–156

    Article  PubMed  CAS  Google Scholar 

  21. Zhang Q, Yin S, Liu L, Liu Z, Cao W (2016) Rhein reversal of DNA hypermethylation-associated klotho suppression ameliorates renal fibrosis in mice. Sci Rep 6:34597

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Chen X, Peng S, Zeng H, Fu A, Zhu Q (2015) Toll-like receptor 4 is involved in a protective effect of rhein on immunoglobulin a nephropathy. Indian J Pharmacol 47:27–33

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Wu C, Cao H, Zhou H, Sun L, Xue J, Li J, Bian Y, Sun R, Dong S, Liu P et al (2015) Research progress on the antitumor effects of Rhein: literature review. Anti Cancer Agents Med Chem

  24. Zeng CC, Liu X, Chen GR, Wu QJ, Liu WW, Luo HY, Cheng JG (2014) The molecular mechanism of rhein in diabetic nephropathy. Evidence-based complementary and alternative medicine. eCAM 2014:487097

    PubMed  Google Scholar 

  25. Zhang K, Jiao XF, Li JX, Wang XW (2015) Rhein inhibits lipopolysaccharide-induced intestinal injury during sepsis by blocking the toll-like receptor 4 nuclear factor-kappaB pathway. Mol Med Rep 12:4415–4421

    Article  PubMed  CAS  Google Scholar 

  26. Yu C, Qi D, Sun JF, Li P, Fan HY (2015) Rhein prevents endotoxin-induced acute kidney injury by inhibiting NF-kappaB activities. Sci Rep 5:11822

    Article  PubMed  PubMed Central  Google Scholar 

  27. Murad S (2014) Toll-like receptor 4 in inflammation and angiogenesis: a double-edged sword. Front Immunol 5:313

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Wang Y, Chen T, Han C, He D, Liu H, An H, Cai Z, Cao X (2007) Lysosome-associated small Rab GTPase Rab7b negatively regulates TLR4 signaling in macrophages by promoting lysosomal degradation of TLR4. Blood 110:962–971

    Article  PubMed  CAS  Google Scholar 

  29. Liaunardy-Jopeace A, Gay NJ (2014) Molecular and cellular regulation of toll-like receptor-4 activity induced by lipopolysaccharide ligands. Front Immunol 5:473

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. da Silva CJ, Ulevitch RJ (2002) MD-2 and TLR4 N-linked glycosylations are important for a functional lipopolysaccharide receptor. J Biol Chem 277:1845–1854

    Article  CAS  Google Scholar 

  31. Cao W, Bao C, Padalko E, Lowenstein CJ (2008) Acetylation of mitogen-activated protein kinase phosphatase-1 inhibits toll-like receptor signaling. J Exp Med 205:1491–1503

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Yin S, Zhang Q, Yang J, Lin W, Li Y, Chen F, Cao W (2017) TGFβ-incurred epigenetic aberrations of miRNA and DNA methyltransferase suppress klotho and potentiate renal fibrosis. Biochim Biophys Acta (BBA) - Mol Cell Res 1864:1207–1216

    Article  CAS  Google Scholar 

  33. Qin T, Du R, Huang F, Yin S, Yang J, Qin S, Cao W (2016) Sinomenine activation of Nrf2 signaling prevents hyperactive inflammation and kidney injury in a mouse model of obstructive nephropathy. Free Radic Biol Med 92:90–99

    Article  PubMed  CAS  Google Scholar 

  34. Yang J, Yin S, Bi F, Liu L, Qin T, Wang H, Cao W (2016) TIMAP repression by TGFβ and HDAC3-associated Smad signaling regulates macrophage M2 phenotypic phagocytosis. J Mol Med 95:273–285

    Article  PubMed  CAS  Google Scholar 

  35. Azuma M, Koyama D, Kikuchi J, Yoshizawa H, Thasinas D, Shiizaki K, Kuro-o M, Furukawa Y, Kusano E (2012) Promoter methylation confers kidney-specific expression of the klotho gene. FASEB J : Off Publ Fed Am Soc Exp Biol 26:4264–4274

    Article  CAS  Google Scholar 

  36. Bacchetta J, Sea JL, Chun RF, Lisse TS, Wesseling-Perry K, Gales B, Adams JS, Salusky IB, Hewison M (2013) Fibroblast growth factor 23 inhibits extrarenal synthesis of 1,25-dihydroxyvitamin D in human monocytes. J Bone Miner Res 28:46–55

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Han X, Li L, Yang J, King G, Xiao Z, Quarles LD, Ellmeier W (2016) Counter-regulatory paracrine actions of FGF-23 and 1,25(OH)2D in macrophages. FEBS Lett 590:53–67

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Cha SK, Hu MC, Kurosu H, Kuro-o M, Moe O, Huang CL (2009) Regulation of renal outer medullary potassium channel and renal K(+) excretion by klotho. Mol Pharmacol 76:38–46

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Warsi J, Abousaab A, Lang F (2015) Up-regulation of excitatory amino acid transporters EAAT1 and EAAT2 by ss-klotho. Neurosignals 23:59–70

    Article  PubMed  Google Scholar 

  40. Abousaab A, Warsi J, Salker MS, Lang F (2016) β-klotho as a negative regulator of the peptide transporters PEPT1 and PEPT2. Cell Physiol Biochem 40:874–882

    Article  PubMed  CAS  Google Scholar 

  41. Cha SK, Ortega B, Kurosu H, Rosenblatt KP, Kuro OM, Huang CL (2008) Removal of sialic acid involving klotho causes cell-surface retention of TRPV5 channel via binding to galectin-1. Proc Natl Acad Sci U S A 105:9805–9810

    Article  PubMed  PubMed Central  Google Scholar 

  42. Moreno JA, Izquierdo MC, Sanchez-Nino MD, Suarez-Alvarez B, Lopez-Larrea C, Jakubowski A, Blanco J, Ramirez R, Selgas R, Ruiz-Ortega M, Egido J, Ortiz A, Sanz AB (2011) The inflammatory cytokines TWEAK and TNFalpha reduce renal klotho expression through NFkappaB. J Am Soc Nephrol: JASN 22:1315–1325

    Article  PubMed  CAS  Google Scholar 

  43. Zhou L, Li Y, Zhou D, Tan RJ, Liu Y (2013) Loss of klotho contributes to kidney injury by derepression of Wnt/beta-catenin signaling. J Am Soc Nephrol: JASN 24:771–785

    Article  PubMed  CAS  Google Scholar 

  44. Zhao Y, Zhao MM, Cai Y, Zheng MF, Sun WL, Zhang SY, Kong W, Gu J, Wang X, Xu MJ (2015) Mammalian target of rapamycin signaling inhibition ameliorates vascular calcification via klotho upregulation. Kidney Int 88:711–721

    Article  PubMed  CAS  Google Scholar 

  45. Hsu SC, Huang SM, Chen A, Sun CY, Lin SH, Chen JS, Liu ST, Hsu YJ (2014) Resveratrol increases anti-aging klotho gene expression via the activating transcription factor 3/c-Jun complex-mediated signaling pathway. Int J Biochem Cell Biol 53:361–371

    Article  PubMed  CAS  Google Scholar 

  46. Hsu S-C, Huang S-M, Lin S-H, Ka S-M, Chen A, Shih M-F, Hsu Y-J (2014) Testosterone increases renal anti-agingklothogene expression via the androgen receptor-mediated pathway. Biochem J 464:221–229

    Article  PubMed  CAS  Google Scholar 

  47. Kuwahara N, Sasaki S, Kobara M, Nakata T, Tatsumi T, Irie H, Narumiya H, Hatta T, Takeda K, Matsubara H, Hushiki S (2008) HMG-CoA reductase inhibition improves anti-aging klotho protein expression and arteriosclerosis in rats with chronic inhibition of nitric oxide synthesis. Int J Cardiol 123:84–90

    Article  PubMed  Google Scholar 

  48. Hu MC, Shi M, Zhang J, Pastor J, Nakatani T, Lanske B, Razzaque MS, Rosenblatt KP, Baum MG, Kuro-o M, Moe OW (2010) Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule. FASEB J : Off Publ Fed Am Soc Exp Biol 24:3438–3450

    Article  CAS  Google Scholar 

  49. Ding Y, Kim S, Lee SY, Koo JK, Wang Z, Choi ME (2014) Autophagy regulates TGF-beta expression and suppresses kidney fibrosis induced by unilateral ureteral obstruction. J Am Soc Nephrol: JASN 25:2835–2846

    Article  PubMed  CAS  Google Scholar 

  50. Shi M, Flores B, Gillings N, Bian A, Cho HJ, Yan S, Liu Y, Levine B, Moe OW, Hu MC (2015) Klotho mitigates progression of AKI to CKD through activation of autophagy. J Am Soc Nephrol 27:2331–2345

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Halina Lis NS (1993) Protein glycosylation structural and functional aspects. Eur J Biochem 218:1–27

    Article  Google Scholar 

  52. Rocuts F, Ma Y, Zhang X, Gao W, Yue Y, Vartanian T, Wang H (2010) Carbon monoxide suppresses membrane expression of TLR4 via myeloid differentiation Factor-2 in TC3 cells. J Immunol 185:2134–2139

    Article  PubMed  CAS  Google Scholar 

  53. Ohnishi T, Muroi M, Ki T (2003) MD-2 is necessary for the toll-like receptor 4 protein to undergo glycosylation essential for its translocation to the cell surface. Clin Vaccine Immunol 10:405–410

    Article  CAS  Google Scholar 

  54. Zhou X, Wang X (2014) Klotho: a novel biomarker for cancer. J Cancer Res Clin Oncol 141:961–969

    Article  PubMed  CAS  Google Scholar 

  55. Hu MC, Bian A, Neyra J, Zhan M (2015) Klotho, stem cells, and aging. Clin Interv Aging 1233. https://doi.org/10.2147/cia.s84978

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Acknowledgements

This work was supported by research grants from National Nature Science Foundation of China (81470940 and 81670762).

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  1. Fangfang Bi and Fang Chen contributed equally to this work.

Authors and Affiliations

  1. Nanjing University School of Medicine, Jiangsu Key Lab of Molecular Medicine, 22 Hankou Road, Nanjing, 210093, China

    Fangfang Bi, Fang Chen, Yanning Li, Ai Wei & Wangsen Cao

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  1. Fangfang Bi
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  2. Fang Chen
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Correspondence to Wangsen Cao.

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Use of animal and the experimental procedures were in accordance with the animal use guidelines and approved by the Institutional Animal Care and Use Committee of Nanjing University School of Medicine (Nanjing, China).

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Bi, F., Chen, F., Li, Y. et al. Klotho preservation by Rhein promotes toll-like receptor 4 proteolysis and attenuates lipopolysaccharide-induced acute kidney injury. J Mol Med 96, 915–927 (2018). https://doi.org/10.1007/s00109-018-1644-7

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  • DOI: https://doi.org/10.1007/s00109-018-1644-7

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