Intrarenal angiotensin-converting enzyme induces hypertension in response to angiotensin I infusion

Abstract

The contribution of the intrarenal renin-angiotensin system to the development of hypertension is incompletely understood. Here, we used targeted homologous recombination to generate mice that express angiotensin-converting enzyme (ACE) in the kidney tubules but not in other tissues. Mice homozygous for this genetic modification (ACE 9/9 mice) had low BP levels, impaired ability to concentrate urine, and variable medullary thinning. In accord with the ACE distribution, these mice also had reduced circulating angiotensin II and high plasma renin concentration but maintained normal kidney angiotensin II levels. In response to chronic angiotensin I infusions, ACE 9/9 mice displayed increased kidney angiotensin II, enhanced rate of urinary angiotensin II excretion, and development of hypertension. These findings suggest that intrarenal ACE-derived angiotensin II formation, even in the absence of systemic ACE, increases kidney angiotensin II levels and promotes the development of hypertension.

Figure 1.

Generation of ACE 9/9 mice. (A) Structure of the ACE wild-type allele. Arrows indicate somatic and testis promoters. Boxes indicate exon 1 through exon 25 of somatic ACE. (B) Homologous recombination construct. With use of a BssHII restriction site on the ACE gene, a neomycin resistance (Neo^R) cassette, and kidney-specific (Ksp) cadherin promoter upstream of a human β-globin minimal promoter (Ksp cadherin/Bg) were inserted sequentially. (C) ACE. 9 modified allele. As a result of homologous recombination, the structural portion of the ACE gene is now under the control of the Ksp cadherin/Bg promoter. The effects of the somatic ACE promoter are minimized because of the presence of the Neo^R cassette as any transcripts originated by this promoter would terminate with the neomycin construct.

Figure 2.

ACE expression is restricted to the kidneys in ACE 9/9 mice. (A) ACE activity was determined by REA in plasma and tissue homogenates and expressed as activity units per μg of protein or μl of plasma. (B) Western blot analysis of ACE expression. Whole tissue homogenates were probed against ACE and β-actin to confirm the results from the ACE activity analysis. (C) Immunohistochemical analysis of ACE expression in the kidneys. ACE positive structures were identified using an antibody against the carboxyl terminus of ACE. They are shown as brown areas in the pictures and are also identified by the black arrows. Images are representative sections of proximal tubules (top), blood vessels (middle), and the medulla (bottom) of WT (left) and ACE 9/9 mice (right). **P < 0.01 versus WT mice by unpaired t test analysis, n = 4 to 7.

Figure 3.

ACE 9/9 mice are unable to concentrate urine. For these studies mice were housed individually in metabolic cages to collect samples and the results are expressed as daily averages. Urine osmolality was determined by vapor-based osmometry before and after 24 hours of water deprivation. **P < 0.01 versus WT mice by unpaired t test analysis, n = 8 to 9.

Figure 4.

Changes in systolic BP (SBP, A) and body weight (B) in response to chronic angiotensin I infusions. Systolic BP was determined by tail-cuff plethysmography. *P < 0.05 versus respective genotype and time-matched controls by two-way ANOVA (SBP) and changes within the same group before and after treatment by repeated measures design (body weight), n = 11 to 15.

Figure 5.

Kidney Ang I and Ang II increase during chronic Ang I infusions in ACE 9/9 mice. Ang I and Ang II content were measured by radioimmunoanalysis in total kidney homogenates obtained from ACE 9/9 or WT mice after 3 weeks of infusions. The results are expressed as per gram of kidney weight (KW). *P < 0.05 versus respective controls by unpaired t test analysis, n = 5 to 8 for Ang I analysis and 11 to 15 for Ang II analysis.

Figure 6.

Urinary Ang II excretion increases during chronic Ang I infusions in ACE 9/9 mice. Urine samples were collected by housing the mice individually in metabolic cages for 72 hours, during which the mice had free access to food and water (results are expressed as daily average). Ang II concentration was measured by radioimmunoanalysis. Systolic BP was determined by tail-cuff plethysmography. *P < 0.05 versus respective genotype and time-matched controls by two-way ANOVA, n = 7 to 13. The Pearson's test was used to determine a correlation in (B).

Figure 7.

Changes in kidney ACE (left) and AT1R (right) mRNA and protein expression in response to chronic angiotensin I infusions. ACE and AT1aR mRNA expression were determined by quantitative real time-PCR. Protein expression was determined by Western blot. In both cases, whole kidney homogenates were used and the results normalized against β-actin expression. *P < 0.05 versus respective controls by unpaired t test analysis, n = 11 to 15.