ASN's Mission

To create a world without kidney diseases, the ASN Alliance for Kidney Health elevates care by educating and informing, driving breakthroughs and innovation, and advocating for policies that create transformative changes in kidney medicine throughout the world.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on X

Kidney Week

Abstract: SA-PO265

Hydralazine Adduct Formation on Myeloperoxidase Contributes to Development of Drug-Associated ANCA Vasculitis

Session Information

Category: Pharmacology (PharmacoKinetics, -Dynamics, -Genomics)

  • 2000 Pharmacology (PharmacoKinetics, -Dynamics, -Genomics)

Authors

  • Xi, Gang, The University of North Carolina at Chapel Hill Kidney Center, Chapel Hill, North Carolina, United States
  • McInnis, Elizabeth A., The University of North Carolina at Chapel Hill Kidney Center, Chapel Hill, North Carolina, United States
  • Lardinois, Olivier, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States
  • Poulton, John S., The University of North Carolina at Chapel Hill Kidney Center, Chapel Hill, North Carolina, United States
  • Chen, Dhruti P., The University of North Carolina at Chapel Hill Kidney Center, Chapel Hill, North Carolina, United States
  • Zeitler, Evan, The University of North Carolina at Chapel Hill Kidney Center, Chapel Hill, North Carolina, United States
  • Derebail, Vimal K., The University of North Carolina at Chapel Hill Kidney Center, Chapel Hill, North Carolina, United States
  • Falk, Ronald, The University of North Carolina at Chapel Hill Kidney Center, Chapel Hill, North Carolina, United States
Background

Hydralazine exposure, an anti-hypertensive agent and a carbonyl scavenger, is associated with the development of ANCA vasculitis. We propose a pathogenic mechanism for hydralazine associated ANCA vasculitis hinging on formation of hydralazine adducts on myeloperoxidase (MPO), the primary immunogen for MPO ANCA.

Methods

In vitro hydralazine studies were performed using horse metmyoglobin (Mb) and human MPO. Hydralazine labeled Mb was digested by trypsin and peptides were analyzed with reverse phase HPLC and electrospray mass spectrometry. In addition, hydralazine labeled MPO was separated using PAGE gels and the hydralazine adduct was detected using an anti-hydralazine antibody. Commercially anti-MPO antibodies recognizing different portions of MPO were used to investigate the conformational change of the MPO heavy chain after hydralazine adducts formation. In addition, an immunoprecipitation assay was performed to detect hydralazine adducts on circulating MPO from patients. Furthermore, anti-MPO IgM and IgG antibodies were measured using ELISAs. Purified IgM and IgG from patients and healthy controls’ plasma were investigated their ability to recognize control MPO or hydralazine labeled MPO.

Results

In vitro studies showed that carbonyl groups formation on primary amines of a protein in the presence of aldehydic products, such as acrolein, is required for hydralazine to bind a protein. Mass spectrometry data showed that hydralazine bound to a carbonyl group of Mb. Under similar conditions, hydralazine adducts were formed on MPO. Importantly, the hydralazine adducts on MPO could be detected in plasma from hydralazine associated ANCA patients but not from patients with non-hydralazine associated ANCA or healthy subjects. Using commercially anti-MPO antibodies, we demonstrated that hydralazine adducts formation on MPO resulted in conformation changes. In addition, purified IgG and IgM autoantibodies from hydralazine associated ANCA patients were reactive against hydralazine labeled MPO.

Conclusion

Under appropriate reactive conditions, hydralazine adducts were able to be formed on MPO in some subjects who were exposed to hydralazine. Hydralazine adducts induced MPO conformational changes which may facilitate autoantibody development, leading to hydralazine associated ANCA vasculitis.

Funding

  • NIDDK Support