Molecular Medicine

, Volume 21, Issue 1, pp 442–452 | Cite as

d-Amino Acid Substitution of Peptide-Mediated NF-κB Suppression in mdx Mice Preserves Therapeutic Benefit in Skeletal Muscle, but Causes Kidney Toxicity

  • Daniel P. Reay
  • Sheldon I. Bastacky
  • Kathryn E. Wack
  • Donna B. Stolz
  • Paul D. Robbins
  • Paula R. Clemens
Research Article


In Duchenne muscular dystrophy (DMD) patients and the mdx mouse model of DMD, chronic activation of the classical nuclear factor-κB (NF-κB) pathway contributes to the pathogenesis that causes degeneration of muscle fibers, inflammation and fibrosis. Prior studies demonstrate that inhibition of inhibitor of κB kinase (IKK)-mediated NF-κB activation using l-isomer NF-κB essential modulator (NEMO)-binding domain (NBD) peptide-based approaches reduce muscle pathology in the mdx mouse. For our studies, the NBD peptide is synthesized as a fusion peptide with an eight-lysine (8K) protein transduction domain to facilitate intracellular delivery. We hypothesized that the d-isoform peptide could have a greater effect than the naturally occurring l-isoform peptide due to the longer persistence of the d-isoform peptide in vivo. In this study, we compared systemic treatment with low (1 mg/kg) and high (10 mg/kg) doses of l- and d-isomer 8K-wild-type-NBD peptide in mdx mice. Treatment with both l- or d-isoform 8K-wild-type-NBD peptide resulted in decreased activation of NF-κB and improved histology in skeletal muscle of the mdx mouse. However, we observed kidney toxicity (characterized by proteinuria), increased serum creatinine, activation of NF-κB and pathological changes in kidney cortex that were most severe with treatment with the d-isoform of 8K-wild-type-NBD peptide. The observed toxicity was also seen in normal mice.



This work was supported in part by by the Kidney Imaging Core of the Pittsburgh Center for Kidney Research (NIH grant P30-DK079307). In addition, some of the transmission electron microscopy equipment used in this study was funded by grant # 1S10RR019003-01. The work was funded by the Department of Veterans Affairs (VA) Medical Center Merit Review Grant and departmental funds (PRC). We would like to thank Katie Clark and the UPMC Presbyterian Hospital Pathology Department Electron Microscopy Laboratory for their contributions to this manuscript.

Supplementary material

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  1. 1.
    Hoffman EP, Brown RH, Kunkel LM. (1987) Dystrophin: the protein product of the Duchene muscular dystrophy locus. Biotechnology. 24:457–66.Google Scholar
  2. 2.
    Koenig M, Monaco AP, Kunkel LM. (1988) The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell. 53:219–28.CrossRefGoogle Scholar
  3. 3.
    Bushby K, et al. (2010) Diagnosis and management of Duchenne muscular dystrophy, part 1: diagnosis, and pharmacological and psychosocial management. Lancet Neurol. 9:77–93.CrossRefGoogle Scholar
  4. 4.
    Angelini C, Peterle E. (2012) Old and new therapeutic developments in steroid treatment in Duchenne muscular dystrophy. Acta Myol. 31:9–15.PubMedPubMedCentralGoogle Scholar
  5. 5.
    Biggar WD, Gingras M, Fehlings DL, Harris VA, Steele CA. (2001) Deflazacort treatment of Duchenne muscular dystrophy. J. Pediatr. 138:45–50.CrossRefGoogle Scholar
  6. 6.
    Drachman DB, Toyka KV, Myer E. (1974) Prednisone in Duchenne muscular dystrophy. Lancet. 2:1409–12.CrossRefGoogle Scholar
  7. 7.
    Ervasti JM, Campbell KP. (1991) Membrane organization of the dystrophin-glycoprotein complex. Cell. 66:1121–31.CrossRefGoogle Scholar
  8. 8.
    Ervasti JM, Campbell KP. (1993) A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J. Cell Biol. 122:809–23.CrossRefGoogle Scholar
  9. 9.
    Brenman JE, Chao DS, Xia H, Aldape K, Bredt DS. (1995) Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy. Cell. 82:743–52.CrossRefGoogle Scholar
  10. 10.
    Lai Y, et al. (2009) Dystrophins carrying spectrinlike repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy. J. Clin. Invest. 119:624–35.CrossRefGoogle Scholar
  11. 11.
    Ervasti JM, Campbell KP. (1993) Dystrophin-associated glycoproteins: their possible roles in the pathogenesis of Duchenne muscular dystrophy. Mol. Cell. Biol. Hum Dis Ser. 3:139–66.PubMedGoogle Scholar
  12. 12.
    Acharyya S, et al. (2007) Interplay of IKK/NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy. J. Clin. Invest. 117:889–901.CrossRefGoogle Scholar
  13. 13.
    Kumar A, Boriek AM. (2003) Mechanical stress activates the nuclear factor-kappaB pathway in skeletal muscle fibers: a possible role in Duchenne muscular dystrophy. FASEB J. 17:386–96.CrossRefGoogle Scholar
  14. 14.
    Messina S, et al. (2006) Lipid peroxidation inhibition blunts nuclear factor-kappaB activation, reduces skeletal muscle degeneration, and enhances muscle function in mdx mice. Am. J. Pathol. 168:918–26.CrossRefGoogle Scholar
  15. 15.
    Messina S, et al. (2006) Nuclear factor kappa-B blockade reduces skeletal muscle degeneration and enhances muscle function in Mdx mice. Exp. Neurol. 198:234–41.CrossRefGoogle Scholar
  16. 16.
    Monici MC, Aguennouz M, Mazzeo A, Messina C, Vita G. (2003) Activation of nuclear factor-kappaB in inflammatory myopathies and Duchenne muscular dystrophy. Neurology. 60:993–7.CrossRefGoogle Scholar
  17. 17.
    Reay DP, et al. (2011) Systemic delivery of NEMO binding domain/IKKgamma inhibitory peptide to young mdx mice improves dystrophic skeletal muscle histopathology. Neurobiol. Dis. 43:598–608.CrossRefGoogle Scholar
  18. 18.
    Abdel-Salam E, Abdel-Meguid I, Korraa SS. (2009) Markers of degeneration and regeneration in Duchenne muscular dystrophy. Acta Myol. 28:94–100.PubMedPubMedCentralGoogle Scholar
  19. 19.
    De PL, et al. (2012) Increased muscle expression of interleukin-17 in Duchenne muscular dystrophy. Neurology. 78:1309–14.CrossRefGoogle Scholar
  20. 20.
    Messina S, et al. (2011) Activation of NF-kappaB pathway in Duchenne muscular dystrophy: relation to age. Acta Myol. 30:16–23.PubMedPubMedCentralGoogle Scholar
  21. 21.
    Pahl HL. (1999) Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene. 18:6853–66.CrossRefGoogle Scholar
  22. 22.
    Gilmore TD. (2006) Introduction to NF-kappaB: players, pathways, perspectives. Oncogene. 25:6680–4.CrossRefGoogle Scholar
  23. 23.
    May MJ, Marienfeld RB, Ghosh S. (2002) Characterization of the Ikappa B-kinase NEMO binding domain. J. Biol. Chem. 277:45992–6000.CrossRefGoogle Scholar
  24. 24.
    May MJ, et al. (2000) Selective inhibition of NF-kappaB activation by a peptide that blocks the interaction of NEMO with the IkappaB kinase complex. Science. 289:1550–4.CrossRefGoogle Scholar
  25. 25.
    Peterson JM, et al. (2011) Peptide-based inhibition of NF-kappaB rescues diaphragm muscle contractile dysfunction in a murine model of Duchenne muscular dystrophy. Mol. Med. 17:508–15.CrossRefGoogle Scholar
  26. 26.
    Bessalle R, Kapitkovsky A, Gorea A, Shalit I, Fridkin M. (1990) All-D-magainin: chirality, antimicrobial activity and proteolytic resistance. FEBS Lett. 274:151–5.CrossRefGoogle Scholar
  27. 27.
    Hamamoto K, Kida Y, Zhang Y, Shimizu T, Kuwano K. (2002) Antimicrobial activity and stability to proteolysis of small linear cationic peptides with D-amino acid substitutions. Microbiol. Immunol. 46:741–9.CrossRefGoogle Scholar
  28. 28.
    Hong SY, Oh JE, Lee KH. (1999) Effect of D-amino acid substitution on the stability, the secondary structure, and the activity of membraneactive peptide. Biochem. Pharmacol. 58:1775–80.CrossRefGoogle Scholar
  29. 29.
    Tugyi R, et al. (2005) Partial D-amino acid substitution: Improved enzymatic stability and preserved Ab recognition of a MUC2 epitope peptide. Proc. Natl. Acad. Sci. U. S. A. 102:413–8.CrossRefGoogle Scholar
  30. 30.
    Guttridge DC, Albanese C, Reuther JY, Pestell RG, Baldwin AS Jr. (1999) NF-kappaB controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol. Cell. Biol. 19:5785–99.CrossRefGoogle Scholar
  31. 31.
    De LA, Pierno S, Liantonio A, Conte CD. (2002) Pre-clinical trials in Duchenne dystrophy: what animal models can tell us about potential drug effectiveness. Neuromuscul. Disord. 12 Suppl 1:S142–6.Google Scholar
  32. 32.
    Hindi SM, Sato S, Choi Y, Kumar A. (2014) Distinct roles of TRAF6 at early and late stages of muscle pathology in the mdx model of Duchenne muscular dystrophy. Hum. Mol. Genet. 23:1492–505.CrossRefGoogle Scholar
  33. 33.
    Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. (1999) In vivo protein transduction: delivery of a biologically active protein into the mouse. Science. 285:1569–72.CrossRefGoogle Scholar
  34. 34.
    Pujals S, Sabido E, Tarrago T, Giralt E. (2007) All-D proline-rich cell-penetrating peptides: a preliminary in vivo internalization study. Biochem. Soc. Trans. 35:794–6.CrossRefGoogle Scholar
  35. 35.
    Wang Y, Rangan GK, Tay YC, Wang Y, Harris DC. (1999) Induction of monocyte chemoattractant protein-1 by albumin is mediated by nuclear factor kappaB in proximal tubule cells. J. Am. Soc. Nephrol. 10:1204–13.PubMedGoogle Scholar
  36. 36.
    Zoja C, et al. (1998) Protein overload stimulates RANTES production by proximal tubular cells depending on NF-kappa B activation. Kidney Int. 53:1608–15.CrossRefGoogle Scholar
  37. 37.
    Morigi M, et al. (2002) Protein overload-induced NF-kappaB activation in proximal tubular cells requires H(2)O(2) through a PKC-dependent pathway. J. Am. Soc. Nephrol. 13:1179–89.PubMedGoogle Scholar
  38. 38.
    Tang S, et al. (2003) Albumin stimulates interleukin-8 expression in proximal tubular epithelial cells in vitro and in vivo. J. Clin. Invest. 111:515–27.CrossRefGoogle Scholar
  39. 39.
    Krug AW, Volker K, Dantzler WH, Silbernagl S. (2007) Why is D-serine nephrotoxic and alphaaminoisobutyric acid protective? Am. J. Physiol Renal Physiol. 293: F382–90.CrossRefGoogle Scholar
  40. 40.
    Sanz AB, et al. (2010) NF-kappaB in renal inflammation. J. Am. Soc. Nephrol. 21:1254–62.CrossRefGoogle Scholar
  41. 41.
    Zoja C, Benigni A, Remuzzi G. (1999) Protein overload activates proximal tubular cells to release vasoactive and inflammatory mediators. Exp Nephrol. 7:420–428.CrossRefGoogle Scholar
  42. 42.
    Zoja C, Morigi M, Remuzzi G. (2003) Proteinuria and phenotypic change of proximal tubular cells. J. Am. Soc. Nephrol. 14 Suppl 1:S36–41.CrossRefGoogle Scholar

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Authors and Affiliations

  • Daniel P. Reay
    • 1
    • 2
  • Sheldon I. Bastacky
    • 3
  • Kathryn E. Wack
    • 4
  • Donna B. Stolz
    • 4
    • 5
  • Paul D. Robbins
    • 6
  • Paula R. Clemens
    • 1
    • 2
  1. 1.Department of Veterans Affairs Medical CenterNeurology ServicePittsburghUSA
  2. 2.S520 Biomedical Science Tower, Department of NeurologyUniversity of PittsburghPittsburghUSA
  3. 3.Department of PathologyUniversity of PittsburghPittsburghUSA
  4. 4.Department of Cell BiologyUniversity of PittsburghPittsburghUSA
  5. 5.Center for Biologic ImagingUniversity of PittsburghPittsburghUSA
  6. 6.Department of Metabolism and AgingScripps FloridaJupiterUSA

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