Hypertension caused by
angiotensin II is characterized by an increase in tissue
oxidant stress as evidenced by increased quantities of reactive
oxygen and
nitrogen species.
Manganese superoxide dismutase (MnSOD) is a key mitochondrial
antioxidant enzyme that is inactivated in conditions of
oxidant stress by reacting with
peroxynitrite to form
3-nitrotyrosine in its active site. The increase in
3-nitrotyrosine content in MnSOD in the kidney of
angiotensin II-infused rats was assessed in this study by immunohistochemistry, Western blotting, immunoprecipitation, and HPLC with UV detection (HPLC-UV). MnSOD activity decreased approximately 50% in
angiotensin II-infused rat kidneys (24 +/- 4.6 vs. 11 +/- 5.2 U/mg) without a change in
protein expression. Immunohistochemical staining showed
3-nitrotyrosine predominantly in distal tubules and collecting duct cells in the
angiotensin II-infused rat kidneys. By two-photon microscopy,
3-nitrotyrosine colocalized with MnSOD. Total
3-nitrotyrosine content in kidney homogenates was increased in
angiotensin II-infused rat kidney [3.2 +/- 1.9 (
sham treated) vs. 9.5 +/- 2.3 ng/mg
protein by HPLC-UV detection]. With tracer amounts of
tyrosine-nitrated recombinant MnSOD, the most sensitive technique to detect
tyrosine nitration of MnSOD was immunoprecipitation from tissue with anti-MnSOD antibody, followed by detection of
3-nitrotyrosine by Western blotting or HPLC. By HPLC,
3-nitrotyrosine content of kidney MnSOD increased 13-fold after
angiotensin II infusion, representing an increase from approximately one-twentieth to one-fifth of the total
3-nitrotyrosine content in
sham-treated and
angiotensin II-infused rat kidney, respectively.
Angiotensin II-induced
hypertension is accompanied by increased
tyrosine nitration of MnSOD, which, because it inactivates the
enzyme, may contribute to increased
oxidant stress in the kidney.