Intracellular angiotensin II production in diabetic rats is correlated with cardiomyocyte apoptosis, oxidative stress, and cardiac fibrosis

Abstract

Objective: Many of the effects of angiotensin (Ang) II are mediated through specific plasma membrane receptors. However, Ang II also elicits biological effects from the interior of the cell (intracrine), some of which are not inhibited by Ang receptor blockers (ARBs). Recent in vitro studies have identified high glucose as a potent stimulus for the intracellular synthesis of Ang II, the production of which is mainly chymase dependent. In the present study, we determined whether hyperglycemia activates the cardiac intracellular renin-Ang system (RAS) in vivo and whether ARBs, ACE, or renin inhibitors block synthesis and effects of intracellular Ang II (iAng II).

Research design and methods: Diabetes was induced in adult male rats by streptozotocin. Diabetic rats were treated with insulin, candesartan (ARB), benazepril (ACE inhibitor), or aliskiren (renin inhibitor).

Results: One week of diabetes significantly increased iAng II levels in cardiac myocytes, which were not normalized by candesartan, suggesting that Ang II was synthesized intracellularly, not internalized through AT(1) receptor. Increased intracellular levels of Ang II, angiotensinogen, and renin were observed by confocal microscopy. iAng II synthesis was blocked by aliskiren but not by benazepril. Diabetes-induced superoxide production and cardiac fibrosis were partially inhibited by candesartan and benazepril, whereas aliskiren produced complete inhibition. Myocyte apoptosis was partially inhibited by all three agents.

Conclusions: Diabetes activates the cardiac intracellular RAS, which increases oxidative stress and cardiac fibrosis. Renin inhibition has a more pronounced effect than ARBs and ACE inhibitors on these diabetes complications and may be clinically more efficacious.

FIG. 1.

Ang II levels in cardiac myocytes and plasma. Ang II was measured by a competitive ELISA in cardiac myocytes (A) and plasma (B) of control rats (Cont); diabetic rats (STZ); and diabetic rats treated with insulin (Ins), aliskiren (Alsk), candesartan (Cand), or benazepril (Bnz). Values are expressed as means ± SE, n = 6. C–F: Intracellular localization of Ang II (yellow dots, indicated by white arrow), as determined by confocal immunofluorescence microscopy, in heart sections from control rats (C), diabetic rats (D), diabetic rats treated with aliskiren (E), and diabetic rats treated with candesartan (F). Myocyte profiles were identified by costaining with anti-sarcomeric actin (red) and laminin (yellow, peripheral staining). The blue color indicates nuclear staining by DAPI. Magnification ×1,200. G: Quantitative representation of Ang II fluorescence intensity in heart sections (from five images per heart and three hearts per group). Values are expressed as means ± SE, n = 15. *P < 0.05 vs. control, †P < 0.05 vs. diabetic rats without any treatment. (Please see http://dx.doi.org/10.2337/db08-0805 for a high-quality digital representation of this figure.)

FIG. 2.

Representative confocal immunofluorescence images of AGT and renin staining in hearts from control and diabetic rats. Pictures shown are merged images of staining for anti-sarcomeric actin (red), laminin (peripheral red staining in top), AGT (top) or renin (bottom) (green and yellow staining), and nuclei (blue). Magnification ×900. (Please see http://dx.doi.org/10.2337/db08-0805 for a high-quality digital representation of this figure.)

FIG. 3.

Measurement of oxidative stress in heart sections by DHE staining. These are representative images of DHE-stained heart sections from control rats (A); diabetic rats (B); and diabetic rats treated with insulin (C), aliskiren (D), candesartan (E), or benazepril (F). Magnification ×60. G: DHE fluorescence intensity was calculated from five images per heart and three hearts per group. Values are expressed as means ± SE (n = 15). *P < 0.05 vs. control, †P < 0.05 vs. diabetic rats without any treatment. (Please see http://dx.doi.org/10.2337/db08-0805 for a high-quality digital representation of this figure.)

FIG. 4.

Detection of apoptosis in cardiac myocytes by TUNEL assay and cleaved caspase-3 staining. A and B: TUNEL assay on heart sections from control (A) and diabetic (B) rats. D and E: Staining for cleaved caspase-3 in the hearts of control (D) and diabetic (E) rats. C and F: Quantification of TUNEL⁺ (C) and cleaved caspase-3⁺ (F) cells (∼25,000 cells were counted in each case). Values are expressed as means ± SE. *P < 0.05 vs. control, †P < 0.05 vs. diabetic rats without any treatment. (Please see http://dx.doi.org/10.2337/db08-0805 for a high-quality digital representation of this figure.)

FIG. 5.

Detection of cardiac fibrosis by Masson's Trichrome staining in heart sections from control rats (A); diabetic rats (B); and diabetic rats treated with insulin (C), aliskiren (D), candesartan (E), or benazepril (F). Representative images of five sections per heart and three hearts per group were observed with a ×40 objective. (Please see http://dx.doi.org/10.2337/db08-0805 for a high-quality digital representation of this figure.)

FIG. 6.

Schematic representation of the relationship between hyperglycemia, iAng II, and pathological effects. In hyperglycemia, there is an increase in glucose oxidation through the tricarboxylic acid cycle in mitochondria, which results in enhanced generation of reactive oxygen species. Overproduction of superoxide inhibits glyceraldehyde-3-phosphate dehydrogenase activity, resulting in an accumulation of upstream metabolites of the glycolytic pathway. Increased levels of glyceraldehyde-3-phosphate (GAD-3P) cause activation of PKC isoforms through diacylglycerol (DAG) production and synthesis of advanced glycation end products (AGEs). There is increased shuttling of glucose through the hexosamine biosynthesis pathway, resulting in the modification of transcription factors through o-glycosylation. All of these products of hyperglycemia, i.e., oxidative stress, AGEs, PKC, and o-glycosylation of transcription factors, activate expression of RAS components. Cardiac myocytes synthesize and retain Ang II intracellularly in hyperglycemia, whereas cardiac fibroblasts increase both intra- and extracellular Ang II. iAng II could directly increase oxidative stress and cellular apoptosis through unidentified mechanisms and/or could enhance expression of RAS components through a positive feedback mechanism, resulting in enhanced extracellular Ang II levels as well, particularly via cardiac fibroblasts. Extracellular Ang II in turn causes oxidative stress, cardiac myocyte apoptosis, and cardiac fibrosis through the AT₁ receptor. Interrupting this cycle by blocking Ang II synthesis provides protection from hyperglycemia-induced pathological events. ACE inhibitors or ARBs would block only the extracellular synthesis or actions, respectively, of Ang II; whereas a renin inhibitor would block both intra- and extracellular Ang II synthesis, the latter providing an explanation for the more pronounced effects of aliskiren observed in this study.