Immunohistochemical expression of intrarenal renin angiotensin system components in response to tempol in rats fed a high salt diet

Abstract

Aim: To determine the effect of tempol in normal rats fed high salt on arterial pressure and the balance between antagonist components of the renal renin-angiotensin system.

Methods: Sprague-Dawley rats were fed with 8% NaCl high-salt (HS) or 0.4% NaCl (normal-salt, NS) diet for 3 wk, with or without tempol (T) (1 mmol/L, administered in drinking water). Mean arterial pressure (MAP), glomerular filtration rate (GFR), and urinary sodium excretion (UVNa) were measured. We evaluated angiotensin II (Ang II), angiotensin 1-7 (Ang 1-7), angiotensin converting enzyme 2 (ACE2), mas receptor (MasR), angiotensin type 1 receptor (AT1R) and angiotensin type 2 receptor (AT2R) in renal tissues by immunohistochemistry.

Results: The intake of high sodium produced a slight but significant increase in MAP and differentially regulated components of the renal renin-angiotensin system (RAS). This included an increase in Ang II and AT1R, and decrease in ACE-2 staining intensity using immunohistochemistry. Antioxidant supplementation with tempol increased natriuresis and GFR, prevented changes in blood pressure and reversed the imbalance of renal RAS components. This includes a decrease in Ang II and AT1R, as increase in AT2, ACE2, Ang (1-7) and MasR staining intensity using immunohistochemistry. In addition, the natriuretic effects of tempol were observed in NS-T group, which showed an increased staining intensity of AT2, ACE2, Ang (1-7) and MasR.

Conclusion: These findings suggest that a high salt diet leads to changes in the homeostasis and balance between opposing components of the renal RAS in hypertension to favour an increase in Ang II. Chronic antioxidant supplementation can modulate the balance between the natriuretic and antinatriuretic components of the renal RAS.

Keywords: Angiotensin 1-7; Angiotensin II; High sodium diet; Kidney; Tempol.

Figure 1

The graphs show the quantitative evaluation of Angiotensin II immunostaining in renal tissue. Data are expressed as mean ± SEM; ^aP < 0.05 vs the respective NS group, ^cP < 0.05 vs the respective group without tempol. The photomicrographs represent Ang II immunostaining in NS, HS, NS-T and HS-T groups. Original magnification: 400 ×. IOD: Integrated optic density; NS: Normal salt diet group; HS: High salt diet group; NS-T: Normal salt diet plus tempol group; HS-T: High salt diet plus tempol group.

Figure 2

The graphs show the quantitative evaluation of angiotensin converting enzyme 2 immunostaining in renal tissue. Data are expressed as mean ± SEM; ^aP < 0.05 vs the respective NS group, ^cP < 0.05 vs the respective group without tempol. The photomicrographs represent ACE2 immunostaining in NS, HS, NS-T and HS-T groups. Original magnification: 400 ×. IOD: Integrated optic density; NS: Normal salt diet group; HS: High salt diet group; NS-T: Normal salt diet plus tempol group; HS-T: High salt diet plus tempol group; ACE2: Angiotensin converting enzyme 2.

Figure 3

The graphs show the quantitative evaluation of Angiotensin 1-7 immunostaining in renal tissue. Data are expressed as mean ± SEM; ^aP < 0.05 vs the respective NS group, ^cP < 0.05 vs the respective group without tempol. The photomicrographs represent Ang 1-7 immunostaining in NS, HS, NS-T and HS-T groups. Original magnification: 400 ×. IOD: Integrated optic density; NS: Normal salt diet group; HS: High salt diet group; NS-T: Normal salt diet plus tempol group; HS-T: High salt diet plus tempol group.

Figure 4

The graphs show the quantitative evaluation of Mas receptor immunostaining in renal tissue. Data are expressed as mean ± SEM; ^aP < 0.05 vs the respective NS group, ^cP < 0.05 vs the respective group without tempol. The photomicrographs represent MasR immunostaining in NS, HS, NS-T and HS-T groups. Original magnification: 400 ×. IOD: Integrated optic density; NS: Normal salt diet group; HS: High salt diet group; NS-T: Normal salt diet plus tempol group; HS-T: High salt diet plus tempol group; MasR: Mas receptor.

Figure 5

The graphs show the quantitative evaluation of angiotensin type 1 receptor immunostaining in renal tissue. Data are expressed as mean ± SEM; ^aP < 0.05 vs the respective NS group, ^cP < 0.05 vs the respective group without tempol. The photomicrographs represent AT1R immunostaining in NS, HS, NS-T and HS-T groups. Original magnification: 400 ×. IOD: Integrated optic density; NS: Normal salt diet group; HS: High salt diet group; NS-T: Normal salt diet plus tempol group; HS-T: High salt diet plus tempol group; AT1R: Angiotensin type 1 receptor.

Figure 6

The graphs show the quantitative evaluation of angiotensin type 2 receptor immunostaining in renal tissue. Data are expressed as mean ± SEM; ^aP < 0.05 vs the respective group without tempol. The photomicrographs represent AT2R immunostaining in NS, HS, NS-T and HS-T groups. Original magnification: 400 ×. IOD: Integrated optic density; NS: Normal salt diet group; HS: High salt diet group; NS-T: Normal salt diet plus tempol group; HS-T: High salt diet plus tempol group; AT2R: Angiotensin type 2 receptor.