Molecular and Cellular Biochemistry

, Volume 382, Issue 1–2, pp 185–191

NOX1 abet mesangial fibrogenesis via iNOS induction in diabetes



Both NADPH oxidase (NOX) and inducible nitric oxide synthase (iNOS) are the main sources of reactive oxygen species in kidney. However, their interactions in oxidative stress and contributions to kidney fibrosis during diabetic nephropathy have not been studied. Human mesangial cells were treated with normal glucose (5.6 mmol/L), high glucose (30 mmol/L) in the presence or absence of AGE (200 mg/L). Protein expressions of NOX1, NOX2, NOX4, and iNOS were examined by immunoblotting. NOX was genetically silenced with specific RNAi to study the interactions between NOX and iNOS in diabetic milieu. Superoxide (O·−) and peroxynitrite (ONOO·−) productions were assessed by dihydroethidium and hydroxyphenyl fluorescein, respectively. Fibrotic factors were determined by biochemistry assay. Superoxide, peroxynitrite, TGF-β, and fibronectin productions as well as the protein expressions of NOX1, NOX2, NOX4, and iNOS were increased in the diabetic milieu (high glucose 30 mmol/L plus AGE 200 mg/L). However, abolishment of iNOS induction with 1400W or iNOS RNAi would restore peroxynitrite, TGF-β, and fibronectin productions completely to basal level and attenuate superoxide production. Moreover, NOX1 inhibition not only prevented iNOS induction but also abrogated changes consequent to iNOS induction such as mesangial fibrogenesis.


Inducible nitric oxide synthase (iNOS) Human glomerular mesangial cells (HMCs) Advanced glycosylation end products (AGEs) Oxidative stress (OS) Transform growth factor-β (TGF-β) Fibronectin 


  1. 1.
    Lopes AA (2009) End-stage renal disease due to diabetes in racial/ethnic minorities and disadvantaged populations. Ethn Dis 19(1):47Google Scholar
  2. 2.
    Palm F, Nordquist L, Wilcox CS, Hansell P (2011) Oxidative stress and hypoxia in the pathogenesis of diabetic nephropathy. Studies on renal disorders. Humana Press, NY, pp 559–586Google Scholar
  3. 3.
    Trachtman H, Futterweit S, Pine E, Mann J, Valderrama E (2002) Chronic diabetic nephropathy: role of inducible nitric oxide synthase. Pediatr Nephrol 17(1):20–29. doi:10.1007/s004670200004 PubMedCrossRefGoogle Scholar
  4. 4.
    Piao YJ, Seo YH, Hong F, Kim JH, Kim YJ, Kang MH, Kim BS, Jo SA, Jo I, Jue DM, Kang I, Ha J, Kim SS (2005) Nox 2 stimulates muscle differentiation via NF-kappaB/iNOS pathway. Free Radic Biol Med 38(8):989–1001. doi:10.1016/j.freeradbiomed.2004.11.011 PubMedCrossRefGoogle Scholar
  5. 5.
    Fujii J (2011) Introduction to serial reviews: physiological relevance of antioxid/redox genes; learning from genetically modified animals. J Clin Biochem Nutr 49(2):69PubMedCrossRefGoogle Scholar
  6. 6.
    Szabo C (2009) Role of nitrosative stress in the pathogenesis of diabetic vascular dysfunction. Br J Pharmacol 156(5):713–727PubMedCrossRefGoogle Scholar
  7. 7.
    Thallas-Bonke V, Thorpe SR, Coughlan MT, Fukami K, Yap FYT, Sourris KC, Penfold SA, Bach LA, Cooper ME, Forbes JM (2008) Inhibition of NADPH oxidase prevents advanced glycation end product-mediated damage in diabetic nephropathy through a protein kinase C-α-dependent pathway. Diabetes 57(2):460–469PubMedCrossRefGoogle Scholar
  8. 8.
    Huang J, Huang K, Lan T, Xie X, Shen X, Liu P, Huang H (2013) Curcumin ameliorates diabetic nephropathy by inhibiting the activation of the SphK1-S1P signaling pathway. Mol Cell Endocrinol 365(2):231–240. doi:10.1016/j.mce.2012.10.024 PubMedCrossRefGoogle Scholar
  9. 9.
    Xie X, Peng J, Chang X, Huang K, Huang J, Wang S, Shen X, Liu P, Huang H (2013) Activation of RhoA/ROCK regulates NF-kappaB signaling pathway in experimental diabetic nephropathy. Mol Cell Endocrinol 369(1–2):86–97. doi:10.1016/j.mce.2013.01.007 PubMedCrossRefGoogle Scholar
  10. 10.
    Xiao H, Li Y, Qi J, Wang H, Liu K (2009) Peroxynitrite plays a key role in glomerular lesions in diabetic rats. J Nephrol 22(6):800–808PubMedGoogle Scholar
  11. 11.
    Cui W, Li B, Bai Y, Miao X, Chen Q, Sun W, Tan Y, Luo P, Zhang C, Zheng S, Epstein PN, Miao L, Cai L (2013) Potential role for Nrf2 activation in the therapeutic effect of MG132 on diabetic nephropathy in OVE26 diabetic mice. Am J Physiol Endocrinol Metab 304(1):E87–E99. doi:10.1152/ajpendo.00430.2012 PubMedCrossRefGoogle Scholar
  12. 12.
    Star RA (1997) Intrarenal localization of nitric oxide synthase isoforms and soluble guanylyl cyclase. Clin Exp Pharmacol Physiol 24(8):607–610PubMedCrossRefGoogle Scholar
  13. 13.
    Pawluczyk IZA, Harris KPG (2012) Effect of angiotensin type 2 receptor over-expression on the rat mesangial cell fibrotic phenotype: effect of gender. J Renin Angiotensin Aldosterone Syst 13(2):221–231PubMedCrossRefGoogle Scholar
  14. 14.
    Chang PC, Chen TH, Chang CJ, Hou CC, Chan P, Lee HM (2004) Advanced glycosylation end products induce inducible nitric oxide synthase (iNOS) expression via a p38 MAPK-dependent pathway. Kidney Int 65(5):1664–1675. doi:10.1111/j.1523-1755.2004.00602.x PubMedCrossRefGoogle Scholar
  15. 15.
    Sugimoto H, Shikata K, Wada J, Horiuchi S, Makino H (1999) Advanced glycation end products-cytokine-nitric oxide sequence pathway in the development of diabetic nephropathy: aminoguanidine ameliorates the overexpression of tumour necrosis factor-alpha and inducible nitric oxide synthase in diabetic rat glomeruli. Diabetologia 42:878–886PubMedCrossRefGoogle Scholar
  16. 16.
    Wautier JL (2004) Protein glycation: a firm link to endothelial cell dysfunction. Circ Res 95(3):233–238. doi:10.1161/01.res.0000137876.28454.64 PubMedCrossRefGoogle Scholar
  17. 17.
    Candido R (2003) A breaker of advanced glycation end products attenuates diabetes-induced myocardial structural changes. Circ Res 92(7):785–792. doi:10.1161/01.res.0000065620.39919.20 PubMedCrossRefGoogle Scholar
  18. 18.
    Lee HB (2003) Reactive oxygen species-regulated signaling pathways in diabetic nephropathy. J Am Soc Nephrol 14(90003):S241–S245. doi:10.1097/01.asn.0000077410.66390.0f PubMedCrossRefGoogle Scholar
  19. 19.
    Wu F, Tyml K, Wilson JX (2008) iNOS expression requires NADPH oxidase-dependent redox signaling in microvascular endothelial cells. J Cell Physiol 217(1):207–214. doi:10.1002/jcp.21495 PubMedCrossRefGoogle Scholar
  20. 20.
    Fan Q, Liao J, Kobayashi M, Yamashita M, Gu L, Gohda T, Suzuki Y, Wang LN, Horikoshi S, Tomino Y (2004) Candesartan reduced advanced glycation end-products accumulation and diminished nitro-oxidative stress in type 2 diabetic KK/Ta mice. Nephrol Dial Transplant 19(12):3012–3020. doi:10.1093/ndt/gfh499 PubMedCrossRefGoogle Scholar
  21. 21.
    Youn JY, Gao L, Cai H (2012) The p47phox- and NADPH oxidase organiser 1 (NOXO1)-dependent activation of NADPH oxidase 1 (NOX1) mediates endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction in a streptozotocin-induced murine model of diabetes. Diabetologia 55(7):2069–2079. doi:10.1007/s00125-012-2557-6 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Department of EndocrinologyRenmin Hospital of Wuhan UniversityWuhanChina

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