Manipulation of components of the renin angiotensin system in renal proximal tubules fails to alter atherosclerosis in hypercholesterolemic mice

Abstract

Background and objective: Whole body manipulation of the renin-angiotensin system (RAS) consistently exerts profound effects on experimental atherosclerosis development. A deficit in the literature has been a lack of attention to the effects of sex. Also, based on data with gene-deleted mice, the site of RAS activity that influences lesion formation is at an unknown distant location. Since angiotensin (AngII) concentrations are high in kidney and the major components of the RAS are present in renal proximal tubule cells (PTCs), this study evaluated the role of the RAS in PTCs in atherosclerosis development.

Methods and results: Mice with an LDL receptor -/- background were fed Western diet to induce hypercholesterolemia and atherosclerosis. We first demonstrated the role of AT1 receptor antagonism on atherosclerosis in both sexes. Losartan, an AngII type 1 (AT1) receptor blocker, had greater blood pressure-lowering effects in females than males, but equivalent effects between sexes in reducing atherosclerotic lesion size. To determine the roles of renal AT1a receptor and angiotensin-converting enzyme (ACE), either component was deleted in PTCs after weaning using a tamoxifen-inducible Cre expressed under the control of an Ndrg1 promoter. Despite profound deletion of AT1a receptor or ACE in PTCs, the absence of either protein did not influence development of atherosclerosis in either sex. Conversely, mice expressing human angiotensinogen and renin in PTCs or expressing human angiotensinogen in liver but human renin in PTCs did not change atherosclerotic lesion size in male mice.

Conclusion: Whole-body AT1R inhibition reduced atherosclerosis equivalently in both male and female mice; however, PTC-specific manipulation of the RAS components had no effects on hypercholesterolemia-induced atherosclerosis.

Keywords: AT1 receptor; angiotensin; angiotensin-converting enzyme; angiotensinogen; atherosclerosis; kidney; proximal tubules; renin.

Figure 1

Losartan reduced systolic blood pressure and attenuated atherosclerosis in both male and female LDL receptor −/− mice. Both male and female LDL receptor −/− mice were fed Western diet and infused with either vehicle or losartan (12.5 mg/kg/day) for 12 weeks. N = 9–10/group. (A) Plasma renin concentrations (PRC), (B) systolic blood pressure (SBP), (C) plasma total cholesterol concentrations (TC), and (D) percent atherosclerotic lesion area. Data of PRC was log-transformed to meet normal distribution assumptions for statistical analysis. Two-way ANOVA was used to evaluate the interaction between sex and treatment. P-value was adjusted using the Bonferroni method in the post hoc test that examined the effect of losartan on each parameter in both sexes. P (sex × treatment) = 0.008 for (A) and 0.001 for (B), respectively.

Figure 2

Validation of Ndrg1-Cre activation and distribution. Ndrg1-CreERT2^0/0 and Ndrg1-CreERT2^+/0 mice were injected intraperitoneally with tamoxifen 150 mg/kg/day for 5 consecutive days. Kidneys were harvested two weeks after the completion of tamoxifen injection. (A) PCR for detecting Cre recombinase in tail DNA, (B) X-gal staining of mouse kidneys, and (C) membrane-localized tdTomato and EGFP proteins detected by confocal microscopy in kidney sections.

Figure 3

AT1aR deletion in PTCs had no effect on atherosclerosis in hypercholesterolemic mice. (A) Experimental protocol, (B) qPCR of renal Agtr1a, (C) RNAscope of renal Agtr1a, (D) plasma renin concentrations (PRC), (E) systolic blood pressure (SBP), (F) plasma total cholesterol concentrations (TC), and (G) percent atherosclerotic lesion area. N = 11–13/group. Two-way ANOVA was used to evaluate the interaction between sex and treatment (B,D–G). P-value was adjusted using the Bonferroni method in the post hoc test that examined the effect of AT1aR deletion in PTCs. G, glomerulus; PTC, proximal tubule cells.

Figure 4

ACE deletion in PTCs had no effect on atherosclerosis in hypercholesterolemic mice. (A) Experimental protocol, (B) qPCR of renal Ace, (C) immunostaining of ACE in kidney, (D) systolic blood pressure (SBP), (E) plasma total cholesterol concentrations (TC), and (F) percent atherosclerotic lesion area. N = 7–13/group. Two-way ANOVA was used to evaluate the interaction between sex and genotypes (B,D–F). White-corrected covariance matrix was incorporated into the two-way ANOVA for analyzing data with heteroscedasticity (B,E). G, glomerulus; PTC, proximal tubule cells; V, vessel.

Figure 5

Human AGT and renin in PTCs had no effect on atherosclerosis in hypercholesterolemic mice. (A) Experimental protocol, (B) qPCR of renal human AGT (hAGT), (C) qPCR of renal human renin (hREN), (D) immunostaining of human AGT (hAGT) in kidney, (E) plasma renin concentrations (PRC), (F) systolic blood pressure (SBP), (G) plasma total cholesterol concentrations (TC), and (H) percent atherosclerotic lesion area. N = 3–6/group. Mann–Whitney U test was performed to determine the difference in the abundance of renal hAGT (B) or hREN (C) mRNA between two groups. Kruskal–Wallis one-way ANOVA on Ranks test was performed to determine the difference in each parameter among four groups (E–H). ND, not detectable; G, glomerulus.

Figure 6

Human AGT in liver and human renin in PTCs had no effect on atherosclerosis in hypercholesterolemic mice. (A) Experimental protocol, (B) qPCR of renal human AGT (hAGT), (C) ELISA of plasma human AGT (hAGT), (D) immunostaining of human AGT (hAGT) in kidney, (E) qPCR of renal human renin (hREN), (F) RNAscope of human renin (hREN) in kidney, (G) plasma renin concentrations (PRC), (H) systolic blood pressure (SBP), (I) plasma total cholesterol concentrations (TC), and (J) percent atherosclerotic lesion area. N = 7–9/group. Mann–Whitney U test was performed to compare renal abundance of hREN mRNA between two Kap-hREN transgenic mouse groups (E) Kruskal–Wallis one-way ANOVA on Ranks test (G–I) or one-way ANOVA test (J) was performed to compare each parameter among the three groups. ND, not detectable; G, glomerulus; PTC, proximal tubule cells.