Lack of angiotensin II–facilitated erythropoiesis causes anemia in angiotensin-converting enzyme–deficient mice (original) (raw)
Anemia. Evaluation of ACE.1 and ACE.2 mice demonstrated a persistent anemia (Figure 1). Both wild-type mice and mice heterozygous for the ACE.1 or ACE.2 mutation had hematocrits that averaged 47–50%. In contrast, the average hematocrits of ACE.1 and ACE.2 knockout mice were 39% and 38% respectively. This reduction was both consistently observed and highly significant for both knockout genotypes as compared with control mice (P < 0.0001). Hemoglobin levels were also reduced in both knockout genotypes (Table 1). There were no significant differences in MCV, MCH, and MCHC, indicating a normocytic, normochromic anemia. None of the genotypes showed a gross abnormality of platelets or white cell number.
Hematocrit. Tail vein blood was collected into microcapillary tubes and the hematocrit was determined for ACE.1 and ACE.2 knockout (KO), wild-type (WT), and heterozygous (HZ) mice. The number of mice in each group was as follows: ACE.1 KO, 12; ACE.1 WT, 13; ACE.1 HZ, 11; ACE.2 KO, 24; ACE.2 WT, 15; ACE.2 HZ, 14. The reduction in hematocrit between knockout and wild-type mice was highly significant, with P < 0.0001 for both knockout genotypes as compared with control mice. No significant difference was observed between wild-type and heterozygous mice. Data are presented as mean ± SE. RBC, red blood cell.
Blood and serum chemistries of ACE.1 and ACE.2 mice
ACE.1 mice were used to study iron metabolism by evaluating serum iron, total iron binding capacity, and serum iron saturation. As presented in Table 1, the values for the ACE.1 knockout mice were no different from those measured in littermate wild-type or heterozygous animals. Stainable bone marrow iron was readily apparent in both ACE.1 and ACE.2 knockout mice (data not shown). These data argue against iron deficiency as the cause of anemia. At necropsy, the histologic appearance of the bone marrow in knockout animals was not different from the bone marrow of control mice. Specifically, the cellularity of bone marrow from knockout mice was greater than 95% with a myeloid-to-erythroid ratio of 4:1. Megakaryocytes were relatively numerous and diffusely spaced. Normal maturation was observed in myeloid, erythroid, and megakaryocytic lineages.
We also investigated hemolysis as a factor contributing to the anemia. This was evaluated in ACE.1 and ACE.2 mice by studying serum indirect bilirubin, lactate dehydrogenase, and reticulocyte levels (Table 1). As with the evaluation of iron, we found no significant differences in these indices between knockout mice and both littermate wild-type and heterozygous animals. While serum haptoglobin is useful in assessing hemolysis in humans, this measurement is not easily applied to mice, since even wild-type mice have undetectable serum haptoglobin levels. Thus, our data suggest that hemolysis does not play a major role in the anemia observed in these animals.
Erythropoietin. In the evaluation of human anemia due to inhibition of the renin-angiotensin system, erythropoietin levels have been controversial; some groups have reported lowered levels, while others find no significant change (4). To measure plasma erythropoietin in ACE.1 and ACE.2 mice, animals were exsanguinated by cardiac puncture and erythropoietin was measured by radioimmunoassay (Figure 2). These data show that ACE.1 and ACE.2 knockout mice have significantly higher plasma erythropoietin levels than wild-type or heterozygous littermate controls. For example, ACE.1 knockout mice averaged 2.72 ± 0.29 mU/ml, while wild-type mice averaged 1.56 ± 0.16 mU/ml (P < 0.01). A similar comparison of ACE.2 knockout mice (2.46 ± 0.42 mU/ml) with wild-type (1.59 ± 0.24 mU/ml) was also significant (P < 0.05). While the ACE.1 knockout animals tended to have higher serum erythropoietin levels than the ACE.2 knockout animals, this difference did not achieve statistical significance. Thus, despite a relatively elevated plasma erythropoietin, ACE.1 and ACE.2 knockout mice are anemic.
Plasma erythropoietin. Anesthetized mice were bled by cardiac puncture and plasma was immediately frozen. Erythropoietin levels were determined by radioimmunoassay. The number of mice in each group was as follows: ACE.1 KO, 10; ACE.1 WT, 9; ACE.1 HZ, 12; ACE.2 KO, 8; ACE.2 WT, 13; ACE.2 HZ, 9. The P value comparing ACE.1 knockout mice with wild-type mice is less than 0.01. A similar comparison of ACE.2 knockout mice with wild-type gave P < 0.05. Thus, the anemia present in ACE.1 and ACE.2 knockout mice was associated with elevated plasma levels of erythropoietin. Data are presented as mean ± SE.
Renal function. We considered renal failure as a possible explanation for anemia in these mice. In order to compare the renal function of the two strains of mice, we evaluated the serum creatinine and creatinine clearance of a large cohort of animals (Figure 3, a and b). These data show a discrepancy between the renal function of the ACE.1 and ACE.2 knockout mice. ACE.1 knockout mice have chemical evidence of renal failure as indicated by an elevation of serum creatinine and a significant reduction of renal creatinine clearance. For instance, the creatinine clearance of ACE.1 knockout mice was 138 ± 15 ml/d, while littermate wild-type mice had a creatinine clearance of 272 ± 46 ml/d (P < 0.05). In contrast, ACE.2 knockout mice had no evidence of renal failure, in that their serum creatinine and creatinine clearance values were not significantly different from wild-type littermate controls. The difference in renal function between ACE.1 and ACE.2 knockout mice is consistent with the known renal histology of the two strains of mice (9). ACE.1 knockout mice have a renal lesion typified by medullary underdevelopment. In contrast, the kidneys of most ACE.2 knockout mice show normal renal medullary development. As anticipated, heterozygous mice of both ACE strains have renal function indistinguishable from that of wild-type mice. Thus, while renal failure may be a potential explanation for anemia in the ACE.1 knockout mice, it seems very unlikely that renal failure is the explanation for the anemia observed in ACE.2 knockout animals.
Renal function. (a) Serum creatinine was measured from venous blood obtained from the tail. The number of mice in each group was as follows: ACE.1 KO, 11; ACE.1 WT, 12; ACE.1 HZ, 14; ACE.2 KO, 30; ACE.2 WT, 50; ACE.2 HZ, 50. (b) The creatinine clearance was measured as described in Methods. The number of mice in each group was as follows: ACE.1 KO, 5; ACE.1 WT, 6; ACE.1 HZ, 6; ACE.2 KO, 30; ACE.2 WT, 21; ACE.2 HZ, 19. All data are presented as mean ± SE. ACE.1 mice have a significant elevation of serum creatinine as compared with wild-type and heterozygous mice (P < 0.01). They also have a significant reduction of creatinine clearance as compared with these same control animals (P < 0.05). In contrast, ACE.2 knockout mice have no evidence of renal failure in that their serum creatinine and creatinine clearance values were not significantly different from wild-type littermate controls. While the creatinine clearance of ACE.1 and ACE.2 heterozygous mice is somewhat elevated above the levels of wild-type mice, these differences do not reach statistical significance. The difference in serum creatinine between ACE.1 wild-type mice and ACE.2 wild-type mice is probably due to the use of different reference laboratories to obtain these data.
Plasma peptide levels. An important difference between ACE.1 and ACE.2 knockout mice is the effect of the mutated ACE gene. ACE.1 knockout mice are null for all ACE activity. In contrast, ACE.2 knockout mice lack tissue-bound ACE but do have some restoration of ACE activity in plasma. Despite this difference, both the blood pressure and the degree of anemia present in these two strains of knockout mice are essentially indistinguishable. To investigate this in detail, we measured the plasma levels of the peptides angiotensin I and angiotensin II by radioimmunoassay (Figure 4, a and b, and Table 1). As an important control for this study, we determined angiotensin I and angiotensin II levels in the plasma of angiotensinogen knockout mice. These animals genetically lack angiotensin I and angiotensin II, allowing us to determine the cross-reactivity of our assay for extraneous peptides. This value was subtracted from the ACE.1 and ACE.2 measurements to obtain the final data. As one would anticipate, both ACE.1 and ACE.2 knockout mice have elevated levels of angiotensin I and much-decreased angiotensin II. In contrast, heterozygous mice have plasma angiotensin II levels that are not significantly different from those of wild-type mice. They achieve this, and a normal blood pressure, through the upregulation of angiotensin I levels (Figure 4b, Table 1). There is a small difference in the plasma levels of angiotensin II between ACE.1 and ACE.2 knockout mice (12.5 ± 2.9 vs. 22.8 ± 4.0 pg/ml, P < 0.05). While this difference is significant, knockout mice of both strains possess only a small fraction of the plasma angiotensin II present in wild-type mice. Interestingly, while angiotensin II levels in ACE.1 knockout mice are reduced by about 90%, they are not zero, probably because of non–ACE-dependent degradation of the elevated levels of angiotensin I present in these animals.
Plasma angiotensin peptide levels. Anesthetized mice were bled by cardiac puncture and plasma was immediately frozen. Plasma angiotensin I and angiotensin II peptide levels were determined by radioimmunoassay. The number of mice in each group was as follows: ACE.1 KO, 12; ACE.1 WT, 10; ACE.1 HZ, 11; ACE.2 KO, 11; ACE.2 WT, 13; ACE.2 HZ, 10. (a) Both ACE.1 and ACE.2 knockout mice have a marked reduction of plasma angiotensin II, with P < 0.0001 compared with either wild-type or heterozygous mice. (b) Both ACE.1 and ACE.2 knockout mice have a marked reduction of the angiotensin II / angiotensin I ratio. This is due to a reduction of plasma angiotensin II and an elevation of plasma angiotensin I levels (see Table 1). While ACE.1 and ACE.2 heterozygous mice have normal levels of plasma angiotensin II (a), the elevation of angiotensin I present in these mice resulted in a significant reduction of the angiotensin II / angiotensin I ratio as compared with wild-type mice (P < 0.01). Data are presented as mean ± SE.
Acetyl-SDKP is a peptide that is normally degraded by the amino-terminal catalytic region of ACE. While not extensively studied, this peptide has been implicated as a bone marrow stem cell suppressor (16–18). Using a radioimmunoassay, we studied plasma acetyl-SDKP levels in the ACE.1 and ACE.2 mice (Figure 5). Both the ACE.1 and ACE.2 knockout mice showed a very significant elevation of plasma acetyl-SDKP as compared with wild-type or heterozygous mice (P < 0.0001). In addition, there was a significant difference between the plasma levels of the ACE.1 knockout mice and those of the ACE.2 knockout mice (3.5 ± 0.4 nM vs. 2.2 ± 0.2 nM, P < 0.01). One might anticipate such a difference in acetyl-SDKP peptide levels between the ACE.1 and ACE.2 knockout mice, since previous analysis has shown that the plasma activity of ACE.2 knockout mice, as measured with the amino-terminal–specific substrate acetyl-SDKP, is almost 90% that of plasma from wild-type mice (9). Thus, ACE.1 and ACE.2 knockout mice have an equivalent degree of anemia, despite the disparity of plasma acetyl-SDKP levels. While one may hypothesize that acetyl-SDKP is not the primary cause of anemia in these mice, formal studies, such as investigations of the kinetics of SDKP inhibition, need to be performed to fully evaluate this question.
Acetyl-SDKP. Venous blood was obtained from the tail and plasma was immediately frozen. Acetyl-SDKP peptide levels were measured by radioimmunoassay. The number of mice in each group was as follows: ACE.1 KO, 7; ACE.1 WT, 6; ACE.1 HZ, 6; ACE.2 KO, 18; ACE.2 WT, 9; ACE.2 HZ, 10. Both ACE.1 and ACE.2 knockout mice have an elevation of plasma acetyl-SDKP as compared with wild-type or heterozygous mice. The highest levels of peptide were present in the ACE.1 knockout mice, animals completely null for ACE activity. Data are presented as the means ± SE.
Red cell mass. A critical question concerning the anemia present in ACE knockout mice is the effect of vasodilation in these animals. Angiotensin II is a potent vasoconstrictor, and the marked reduction of this peptide in the ACE knockout mice should promote vasodilation. Hypothetically, this might lead to volume expansion and a dilutional anemia. We used 51Cr labeling of red blood cells to determine blood volume, red blood cell volume, and plasma volume in ACE.2 knockout mice (Figure 6). ACE.2 knockout mice have a total blood volume comparable with that of wild-type mice (58 μl/g vs. 56 μl/g). This value is similar to the total blood volumes of several strains of mice previously reported in the literature (14, 19). However, ACE.2 knockout mice have a 25% reduction of red cell mass as compared with wild-type mice (22.7 μl/g ± 1.1 μl/g vs. 30.3 μl/g ± 1.7 μl/g, P < 0.01). These data show that the anemia observed in ACE.2 knockout mice is real and not due to excessive volume expansion. Thus, our data indicate that ACE.2 knockout mice have roughly an equivalent reduction of both hematocrit (Figure 1) and red cell mass.
Red cell mass. Whole blood was obtained by cardiac puncture from donor ACE.2 heterozygous mice. After labeling with 51Cr, an aliquot was infused via a carotid artery catheter. After allowing for equilibration, blood was obtained by cardiac puncture and used to determine total blood volume and total red cell volume. An implied plasma volume was then calculated. All data were normalized for the weight of the animal. The number of mice in each group was as follows: ACE.2 KO, 7; ACE.2 WT, 7. While the blood volume of ACE.2 knockout mice is equivalent to that of wild-type mice, the data show that the ACE.2 knockout mice have roughly a 25% reduction of red cell mass (P < 0.01). Data are presented as mean ± SE.
Response of anemia to angiotensin II. In evaluating the anemia in the ACE.2 knockout mice, we questioned whether the anemia was due to the lack of angiotensin II production or to an effect of some other peptide such as acetyl-SDKP accumulation. This can be experimentally investigated by infusing angiotensin II into ACE.2 knockout mice and examining the effect on hematocrit. If the anemia is the result of a nonangiotensin peptide such as acetyl-SDKP, then angiotensin II peptide infusion should have a minimal effect. An important preliminary study was to determine the dose of angiotensin II sufficient to raise the blood pressure of ACE.2 knockout animals to roughly wild-type levels. ACE.2 knockout mice are far more sensitive to exogenous angiotensin II than wild-type mice. As an example, the constant infusion of 1.1 mg/kg/d, a dose of angiotensin II often used to raise the blood pressure of rodents by roughly 40 mmHg, caused extreme hypertension and death in ACE.2 knockout mice. Finally, we determined that the infusion by osmotic minipump of 0.3 mg/kg/d of angiotensin II for 2 weeks was well tolerated and raised the systolic blood pressure of ACE.2 knockout mice from 73 mmHg to 131 mmHg (Figure 7A). This small dose of angiotensin II had a minimal effect on ACE.2 wild-type mice, raising systolic blood pressure from 106 mmHg to 114 mmHg.
Angiotensin II infusion increases hematocrit. A cohort of wild-type and ACE.2 knockout mice were evaluated for systolic blood pressure and hematocrit. Animals were then implanted with osmotic minipumps delivering either angiotensin II (+ Ang) or vehicle (Control). After 2 weeks, blood pressure and hematocrit were reassessed. (a) The systolic blood pressure of the mice before and after angiotensin II infusion. The number of mice in each group was as follows: ACE.2 WT, 6; ACE.2 KO + Ang, 6; ACE.2 KO control, 4. Infusion of small amounts of angiotensin II raised the blood pressure of ACE.2 knockout mice to levels comparable with those of wild-type mice. (b) The hematocrits of the mice described in a were studied before and after angiotensin II infusion. ACE.2 knockout mice treated with angiotensin II showed a significant increase of hematocrit (P < 0.001) to levels near those of wild-type mice.
A cohort of ACE.2 knockout mice was treated with either 0.3 mg/kg/d angiotensin II or saline for 2 weeks (Figure 7). Systolic blood pressure and hematocrit were determined immediately before and at the end of the infusion period. A group of wild-type ACE.2 mice were treated in a similar fashion. This study showed a marked increase of hematocrit in ACE.2 knockout mice receiving angiotensin II from the preinfusion level of 41.2 ± 1.0% to the postinfusion value of 48.3 ± 0.8% (P < 0.001). Thus, treatment of ACE.2 knockout mice with a low dose of angiotensin II raised the blood pressure and corrected the hematocrit to near wild-type levels. This study suggests that the anemia in ACE.2 knockout mice is directly related to the lack of angiotensin II generation in these animals.