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1 Evans Department of Medicine, Boston University Medical Center, Boston, MA, USA
2 Department of Surgery, University of Tennessee, Knoxville, TN, USA
* To whom correspondence should be addressed. E-mail: racohen{at}med-med1.bu.edu.
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 using immuno-histochemistry, western blotting, immuno-precipitation, and HPLC-UV. MnSOD activity decreased approximately 50 % in angiotensin II-infused rat kidneys (24 ± 4.6 vs. 11 ± 5.2 units/mg) without a change in protein expression. Immuno-histochemical staining showed 3-nitrotyosine predominantly in distal tubules and collecting duct cells in the angiotensin II-infused rat kidneys. By two-photon microscopy, 3-nitrotyrosine co-localized with MnSOD. Total 3-nitrotyrosine content in kidney homogenates was increased in angiotensin II-infused rat kidney (3.2 ± 1.9 in sham-treated vs. 9.5 ± 2.3 ng/mg tissue by HPLC-UV detection). Using tracer amounts of tyrosine-nitrated recombinant MnSOD, the most sensitive technique to detect tyrosine nitration of MnSOD was by 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 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.
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