(Hypertension. 1997;29:216.)
© 1997 American Heart Association, Inc.
Arthur C. Corcoran Memorial Lecture |
Aberrant Renal Vascular Morphology and Renin Expression in Mutant Mice Lacking Angiotensin-Converting Enzyme
From the Division of Pediatric Nephrology, Department of Pediatrics, University of Virginia Health Sciences Center, Charlottesville, and Departments of Pathology (J.H.K., O.S.) and Medicine (J.H.K.), University of North Carolina, Chapel Hill.
Correspondence to R. Ariel Gomez, MD, Chief, Pediatric Nephrology Division, Department of Pediatrics, University of Virginia Health Sciences Center, 300 Lane Rd, MR4 Bldg, Room 2001, Charlottesville, VA 22908
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Abstract |
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To determine whether angiotensin-converting enzyme plays a role in the development and maintenance of normal renal architecture, the renal morphology of 10- to 12-month-old female mice homozygous for a disruption of the converting enzyme gene was compared with that of age-matched wild-type mice. Tubular obstruction, dilatation, and atrophy were present in all kidneys from the homozygous mutant mice but absent in wild types; two kidneys from 4 mutant mice but none from the wild types were hydronephrotic. The entire arterial vascular tree, microdissected from mice with no converting enzyme, was grossly distorted in comparison to the vasculature of wild-type mice; all intrarenal arterial vessels were widened and thickened, including the terminal (afferent) arterioles. In wild-type mice kidneys, renin-positive cells were detected exclusively in a juxtaglomerular localization. In contrast, abnormal distribution of renin immunostaining was observed in mice without converting enzyme; scattered renin-positive cells were seen along the arterial vessels, often in a perivascular localization, and interstitial reninpositive cells surrounded glomeruli. Kidney renin mRNA was increased more than 32-fold in the mutant mice compared with wild types. Northern blot analysis revealed that this increase included the accumulation of large amounts of smaller renin RNA transcripts. In summary, mice lacking the converting enzyme exhibit abnormal renal vessels and tubules. Renin synthesis is increased, accompanied by the presence of small renin mRNA species, and renin is present mainly in interstitial and perivascular cells. We conclude that angiotensin-converting enzyme is necessary to preserve normal kidney architecture and the normal pattern of renin expression.
Key Words: vessels • arterioles • kidney • knockout mice • renovascular
Abbreviations: Ang = angiotensin • AT1 = angiotensin II type 1 receptor • PCR = polymerase chain reaction • RAS = renin-angiotensin system
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Introduction |
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The activation of the RAS in the developing kidney1–3 has led to the hypothesis that its active component, Ang II, may play an important role in renal development.4 Intervention studies in which pharmacological blockers of the RAS were used to treat newborn rats have confirmed this notion.5,6 Both ACE inhibitors and AT1 blockers produce similar renal abnormalities when given to newborn animals.5,6 The most prominent macroscopic renal pathology in these animals is atrophy of the renal medulla; microscopically, the renal tubules are atrophic and dilated, with tubulointerstitial fibrosis.5,6 A similar pathology has been described in some human neonates after treatment of their mothers with ACE inhibitors in late pregnancy.7
While the results of pharmacological interventions could be due to nonspecific effects of the drugs,8 recent gene-targeting studies in mice have underlined the essential role of the RAS for renal development. Mice lacking functional copies of either the angiotensinogen9,10 or the ACE gene (Ace)11,12 develop a renal pathology similar to that of rats treated with RAS blockers neonatally. Vascular changes were observed in both the mutant mice and in the rats treated with AT1 antagonist neonatally. Thus, in the rats, distorted, widened cortical arterioles were described,6 while in the mutant mice, hypercellular vascular walls were described.9–12
The aim of the present study was to examine the vascular pathology of Ace-deficient mice in more detail. ACE inhibition is a strong stimulus for the recruitment of cells of the smooth muscle layer to renin-producing cells.13,14 Therefore, we hypothesized that metaplasia of smooth muscle cells to renin-producing cells might account for the hypercellular vascular walls in Ace mutant mice. The present study, in which we examined renin RNA expression and the distribution of immunoreactive renin in tissue sections and dissected arterial vessels of Ace knockout mice, does not support the hypothesis.
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Methods |
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Animals
The generation of mice deficient in ACE as a consequence of disruption of exon 14 of the Ace gene in strain 129 mouse embryonic stem cells has been described previously.11 Male chimeras derived from the stem cells were mated with C57BL/6J female mice to obtain heterozygous F1 hybrids.11 The F1 heterozygotes were mated to generate F2 offspring.11 For the present study, we used 10- to 12-month-old female mice homozygous for the Ace mutation (Ace -/-) from the F2 generation (C57BL/6jx129/01a).11 The mice were genotyped as described.11 The animals were housed in a room where a constant temperature (22±2°C) and a 12-hour light/dark cycle were maintained. Age-matched female C57BL/6J mice, housed in the same room, were used as wild-type (Ace +/+) controls. All procedures were performed in accordance with the guidelines of the American Physiological Society and had been approved by the University of Virginia Animal Care Committee. Mice were killed by exsanguination under pentobarbital anesthesia (30 mg/kg body weight IP). Kidneys were excised, weighed, snap-frozen, and kept at -80°C for RNA extraction, or fixed and paraffin embedded for histology and immunohistochemistry, or processed for microdissection.
Renin Immunohistochemistry
Immunohistochemical detection of renin was performed as described previously.13,15 Briefly, after deparaffinization, 5- to 7-µm kidney sections were layered with a specific anti-rat-renin polyclonal antibody (dilution 1:2500; kind gift by Dr T. Inagami, Nashville, Tenn). After addition of the secondary antibody (biotin-conjugated anti-rabbit immunoglobulin G made in goat; Vector Lab), the sections were incubated with avidin-biotinylated horseradish peroxidase complex (Vectastain ABC kits; Vector Lab) and exposed to 0.1% diaminobenzidine tetrahydrochloride and 0.02% H2O2 as a source of peroxidase substrate. Each slide was counterstained with hematoxylin. As negative controls, the primary antiserum was replaced by nonimmune rabbit serum. Immunohistochemistry for renin was performed with sections obtained from four kidneys from four Ace -/- mice and four kidneys obtained from four age-matched Ace +/+ mice.
Microvascular Dissection
To obtain an integrated view of the branching pattern of the renal vessels and the distribution of renin within the kidney, the entire renal arterial tree was dissected as described previously for rats.6,16 Briefly, kidneys were digested in 3N HCl at 42°C for 30 minutes and sonicated for 60 seconds. The vasculature was then dissected using a pair of 1-µm tungsten needles (Fine Science Tools Inc) under x10 to x30 magnification (StereoZoom 5; Bausch & Lomb). The arterial tree was mounted on positively charged slides (Superfrost; Fisher), air dried, and stained for renin as described above except that no counterstain was added. Vascular trees were dissected from two Ace -/- kidneys and two Ace +/+ kidneys.
Cloning of a Mouse Renin-1 cDNA
To detect mouse renin mRNA by hybridization, a partial mouse renin-1 probe was cloned from mouse kidney RNA by reverse transcription and PCR. One microgram of mouse kidney RNA was reverse transcribed by Moloney murine leukemia virus reverse transcriptase (GenHunter Corp) at 37°C for 50 minutes, using the primer 5'-GTCAAACTTGGCCAGCATGA-3'. For subsequent PCR, the primer 5'-ATGCCTCTCTGGGCACTCTT-3' was added. The two primers, based on the known nucleotide sequence of the mouse renin-1 gene, yielded a fragment of renin-1 cDNA that corresponds to nucleotides 33 to 583 of the cDNA.17 Two of 20 µL reverse-transcription product was subjected to 30 cycles of PCR at 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute with AmpliTaq polymerase (Perkin-Elmer) in a Perkin-Elmer N801-0150 thermocycler. After agarose gel electrophoresis, the fragment of the expected size (550 base pairs) was excised, purified, and cloned into the pCNTR vector using the General Contractor DNA Cloning System (5 Prime-3 Prime Inc). Sequence analysis confirmed that the cloned partial cDNA was identical to the known mouse renin-1 sequence.17
Northern and Dot Blot Hybridization
Total RNA was extracted using the single-step method of Chomczynski18 with the TRI reagent (Molecular Research Center Inc). Two kidneys from two Ace -/- mice and three kidneys from three Ace +/+ mice were used for RNA extraction. For Northern blot analysis, 10 µg of total RNA was electrophoresed in a 1.2% agarose-formaldehyde gel and transferred to positively charged nylon membranes (Zeta-Probe, Bio-Rad Laboratories) as previously described.1,13 For dot blot quantification of renin RNA, serial dilutions of RNA were transferred to Zeta-Probe membranes with a dot blot manifold (Bio-Rad Laboratories).
For hybridization, the renin cDNA was radiolabeled with 
[32]P-deoxy-CTP (DuPont NEN) by PCR labeling. Fifteen to 20 ng of the insert of the cloned renin-1 was amplified using the same primers and cycling parameters as described above. The final concentrations in the PCR reaction were 1 µmol/L for both primers, 0.83 µmol/L [32]P-deoxy-CTP (3000 Ci/mmol), 1.88 µmol/L deoxy-ATP, -GTP and -TTP, 1.5 µmol/L MgCl2, 50 mmol/L KCl, and 10 mmol/L TRIS-Cl pH 8.4. Northern and dot blot membranes were hybridized by a modification of the method of Church and Gilbert,19 as previously described.1,13 The hybridization solution contained 0.25 mol/L sodium phosphate, pH 7.2, 7% sodium dodecyl sulfate, 1 mmol/L EDTA, 0.1% bovine serum albumin, and 10 mg/L salmon sperm DNA. The posthybridization washes were performed at decreasing concentrations of sodium phosphate (final concentration, 20 mmol/L) and sodium dodecyl sulfate (final concentration, 1%; all chemicals from Sigma Chemical Co). Both the hybridization and the posthybridization washes were performed at 65°C. For autoradiography, membranes were exposed to Kodak XAR films or to a phosphoimager screen (Molecular Dynamics Inc). Signals were quantified after scanning the phosphoimager screen using the Image-Quant software (Molecular Dynamics).
Comparisons between groups were made by t test for independent samples. Results are presented as mean±SEM.
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Results |
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The Ace -/- mice at the time of sacrifice were of normal appearance except that their body weights were lower than the weights of the age-matched Ace +/+ controls (28.2±1.5 versus 36.7±2.7 g, n=4 each, P<.01). The kidneys, however, were abnormal, and two kidneys from two different Ace -/- mice but none from the wild-type mice showed overt hydronephrosis. Histological sections demonstrated that in these two hydronephrotic kidneys, only a small rim of renal tissue surrounded a grossly distended renal pelvis (not shown). If these two hydronephrotic kidneys were excluded from the analysis, the Ace -/- kidneys were smaller than Ace +/+ kidneys (173.2±3.3 versus 232.4±5.1 mg, P<.001) but the ratios of kidney weight to body weight did not differ significantly in the two genotypes (Ace -/-, 6.23±0.14 mg/g; Ace +/+, 6.41±0.35 mg/g; P>.2).
Histological examination of tissue sections (Fig 1) showed marked atrophy of the renal papilla as described previously by others.12 Obstruction, dilatation, and atrophy of cortical and medullary tubules was observed in the Ace -/- mice but not in the wild-type mice. Dilated Bowman’s spaces were noted in Ace -/-, and there was interstitial fibrosis (Fig 1B).
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Arterial vessels were prominent in the tissue sections from the Ace -/- mice; the vessels were widened and exhibited thickened, hypercellular vascular walls (Fig 1C). Glomeruli were often seen close to the arterial vessels that had a larger diameter than normal terminal arterioles (Fig 1B and 1C), and this frequent pairing of abnormal vessels with glomeruli suggested that the widened, hypercellular vessels were afferent arterioles or interlobular arteries. Microvascular dissection also demonstrated abnormal arterial vessels in the Ace -/- mice (Fig 2). Contrary to the predictable progressive branching and narrowing of the renal vasculature of the wild-type mice, the vascular tree of Ace -/- was grossly distorted (Fig 2). The terminal (afferent) arterioles were fewer, thicker, and shorter (Fig 2).
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Renin immunohistochemistry demonstrated strong (normal) staining in the juxtaglomerular region in wild-type mice with a complete absence of staining in the tubules (Fig 1A). In contrast to the wild type, the Ace -/- mice showed cells staining strongly positive for renin in the outer layers of arterial vessels, and in periglomerular and interstitial locations (Fig 1B through 1D). Cells in the smooth muscle layer of the hypercellular vessels did not stain for renin (Fig 1C). Renin-positive cells were frequently present surrounding sclerotic or obsolescent glomeruli or vessels (Fig 1D). Microvascular dissection showed patches of renin staining scattered throughout the vascular tree (Fig 2). The tubules of the Ace -/- mice showed generalized renin staining of modest intensity (Fig 1B through 1D).
Renin mRNA was elevated more than 32-fold in Ace -/- kidneys, as measured by dot blot (Fig 3). The highest dilution of RNA tested in the Ace -/- mice still yielded a higher signal than the lowest dilution tested in the Ace +/+ mice (Fig 3). Northern blotting revealed that in RNA from Ace -/- mice, the renin-1 probe hybridized not only to a band of expected size but also to smaller transcripts, down to 
200 nucleotides (Fig 4). In RNA from wild-type mice, hybridization was mainly to a single transcript of the expected size (Fig 4). This phenomenon was specific for renin RNA in the Ace -/- mice, as smaller species were not observed in RNA from Ace -/- mice hybridized with GAPDH or transforming growth factor-ß cDNAs (Fig 4). These control hybridizations and ethidium bromide staining of the membrane (not shown) excluded the possibility that a general degradation of RNA was present in the samples from the Ace -/- kidneys. The finding of smaller transcripts hybridizing with the renin probe was repeated on a second Northern blot (not shown).
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Discussion |
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We have studied the renal morphology and renin expression in mice lacking a normally functioning Ace gene. Our results indicate that ACE is essential for the preservation of normal renal morphology and a normal pattern of renin expression. Mice lacking a functional Ace gene have grossly distorted arterial vessels with hypercellular lesions of the medial layer. These vascular abnormalities are not, however, due to the recruitment of vascular smooth muscle cells for renin synthesis because the cells within the smooth muscle layer do not stain for renin. Rather, the renin-positive cells surround blood vessels and glomeruli in the Ace -/- mice. Total renin mRNA is vastly increased in the Ace -/- mouse kidneys, and small RNA transcripts hybridizing to the renin cDNA are clearly present in large amounts.
Renal vascular and tubular lesions in animals treated neonatally with ACE inhibitors have been previously described.5,6 The results of the current study and of other reports from studies targeting the Ace gene11,12 establish that the renal abnormalities induced by ACE inhibitors are readily understandable as being due to the lack of ACE activity and not to some unrelated toxicity of these drugs.8
The renal arterial vessels of the Ace -/- mice exhibit a striking pathology: the cortical arterioles are widened and thickened, and the vascular walls are hypercellular. We note that glomeruli are frequently paired with arterial vessels that have diameters equal to or greater than the glomeruli themselves. Our renin-staining experiments exclude the possibility that metaplasia of smooth muscle cells to renin-producing cells plays a role in these hypercellular lesions, although it is strikingly apparent that the hyperplastic vessels are usually surrounded by cells that stain strongly for renin. It is tempting to speculate that the renin-producing cells are producing some factor(s) that can stimulate smooth muscle cell growth even in the absence of ACE. Further experiments will be required to address this possibility.
The distribution of immunoreactive renin was highly abnormal in the Ace -/- mice. Wild-type mice exhibited renin staining only in the juxtaglomerular area. In Ace -/- mice, renin-positive cells were observed in the outer layers of vessels throughout the vascular tree (as indicated above) and in the interstitium surrounding glomeruli. ACE inhibition is a strong stimulus for the recruitment of cells to produce renin,13,14 so we were not surprised to find renin synthesis outside the juxtaglomerular area in mice lacking a functional Ace gene. However, our observation that the renin-producing cells in the Ace -/- mouse kidneys were perivascular was surprising. Renin immunoreactivity in periglomerular and perivascular cells has occasionally been described in kidneys of patients with end-stage renal disease,20,21 and "nests" of renin-positive cells have been described in human kidneys with segmental renal hypoplasia.21,22 However, we are not aware of reports describing the extensive interstitial and perivascular distribution of renin-producing cells that was seen in the Ace -/- mice in the present study. Unlike the wild-type mice, the Ace -/- mice also showed generalized renin staining in the renal tubules, albeit in lesser amounts than in the perivascular cells. This tubular staining for renin could be due to uptake of plasma renin, which is presumably elevated in the Ace -/- mice, or to an increased synthesis of renin by tubular cells.23,24
We expected an increase in renin RNA in the Ace -/- mouse kidneys, because ACE inhibition also stimulates renin synthesis.13 However, the presence of considerable amounts of smaller transcripts hybridizing with the renin cDNA was not expected and to the best of our knowledge has not been described previously. We excluded the possibility that general degradation of kidney mRNA was responsible for these irregular transcripts. Others have described that the stimulation of renin synthesis by cyclic AMP involves an increase in the stability of renin mRNA.25,26 Thus, one could speculate that lifelong stimulation of renin in the Ace -/- mice might reveal a pathway in the breakdown of renin mRNA that is not readily apparent under normal circumstances. Other experiments are needed to address this question.
Gross changes of the cortical and medullary tubules were present in the Ace -/- kidneys, including tubular dilatation, atrophy, and obstruction. Our observations agree with previous reports on the tubular pathology in ACE-deficient mice11,12 and in rats treated neonatally with ACE inhibitor.5 These prior studies also demonstrated that the animals cannot properly concentrate their urine5,12 and that the Ace mutant mice develop renal failure.12 Given the close interrelationship of growing renal vessels and tubules,27 the tubular pathology in the Ace -/- mice could be a consequence of the distorted vascular growth. Alternatively, both abnormalities could be the consequence of absence of Ang II, which is known to be a growth factor for renal vascular28 as well as tubular cells.29,30
Lack of intrarenal Ang II is a likely cause for the abnormal renal morphology in Ace -/- mice, because plasma ACE activity is not detectable in these animals.11 However, we did not measure Ang II in plasma or renal tissue. Enzymes other than ACE might still generate Ang II, particularly in newborn animals.31 Therefore, we cannot exclude the possibility of other mechanisms contributing to the renal pathology of Ace -/- mice. Accumulation of kinins occurs during ACE inhibition,32 and kinins regulate renal papillary blood flow via nitric oxide generation.33 Thus, elevated kinin levels may also play a role in our ACE-deficient mutant mice. In addition, other Ang peptides, especially Ang I and Ang-(1–7), increase after ACE inhibition.32 Since Ang-(1–7) affects the growth of vascular smooth muscle cells,34 the peptide may also be involved in the pathogenesis of the renal damage in ACE-deficient mice. Furthermore, Ang-(1–7) could potentiate the effects of bradykinin.35 Finally, one could speculate that prorenin, which is presumably highly elevated in Ace -/- mice, could affect vascular growth. High prorenin levels have been associated with the proliferation of retinal vessels in diabetic patients.36,37 However, the similarity of the renal lesions induced by disrupting the Ace gene to those seen after disruption of the angiotensinogen gene9,10 suggests that lack of Ang II is the most important factor in the pathogenesis of the renal pathology.
It is of interest that targeting of each of the known Ang II receptors (AT1A, AT1B, and AT2) did not produce renal lesions comparable to those described here,38–40 presumably because only one of the receptor genes was disrupted. Simultaneous inactivation of more than one receptor gene, eg, both AT1 genes, may be necessary to address this issue. However, pharmacological interventions clearly support the notion that lack of stimulation of the AT1 receptor is sufficient to account for the renal pathology induced by ACE inhibition.5,6
In summary, our data show that ACE is essential for the preservation of normal renal vascular and tubular morphology and for a normal distribution of renin expression. Further research will be necessary to uncover the mechanisms responsible for the distorted vascular growth, the ectopic expression of renin in interstitial and perivascular cells, and the extensive presence of smaller renin RNA transcripts in Ace -/- mice. The renal pathology in mice lacking a functioning Ace gene underlines the necessary role of the RAS in the preservation and/or development of renal vessels and tubules.
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Acknowledgments |
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This study was supported by the following NIH grants: The Center of Excellence in Pediatric Nephrology and Urology (DK-44756), the O’Brien Center for Kidney and Urology Research (DK-45179), the Child Health Research Center (HD-22910 to Dr Gomez), NIH grant HL-03470 to Dr Krege and grants GM-20069 and HL-49277 to Dr Smithies. We are grateful to the W.M. Keck Foundation for a grant to the University of North Carolina to support work with animal models. Dr Hilgers was the recipient of a habilitation scholarship (Hi 510/5-1 and 3) from the Deutsche Forschungsgemeinschaft, Bonn, Germany. Dr Krege was a Howard Hughes Medical Institute Physician Research Fellow.
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P. B. Freeburg and D. R. Abrahamson Dissecting the JGA: new functions for JG cells? Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2004; 286(3): R449 - R450. [Full Text] [PDF]
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E. S. Pentz, M. A. Moyano, B. A. Thornhill, M. L. S. Sequeira Lopez, and R. A. Gomez Ablation of renin-expressing juxtaglomerular cells results in a distinct kidney phenotype Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2004; 286(3): R474 - R483. [Abstract] [Full Text] [PDF]
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B. C. Cholewa and D. L. Mattson Role of the renin-angiotensin system during alterations of sodium intake in conscious mice Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2001; 281(3): R987 - R993. [Abstract] [Full Text] [PDF]
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A. C. Marsh, K. J. Gibson, J. Wu, P. C. Owens, J. A. Owens, and E. R. Lumbers Chronic effect of insulin-like growth factor I on renin synthesis, secretion, and renal function in fetal sheep Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2001; 281(1): R318 - R326. [Abstract] [Full Text] [PDF]
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V. F. Norwood, M. Garmey, J. Wolford, R. M. Carey, and R. A. Gomez Novel expression and regulation of the renin-angiotensin system in metanephric organ culture Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2000; 279(2): R522 - R530. [Abstract] [Full Text] [PDF]
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L. L. Woods and R. Rasch Perinatal ANG II programs adult blood pressure, glomerular number, and renal function in rats Am J Physiol Regulatory Integrative Comp Physiol, November 1, 1998; 275(5): R1593 - R1599. [Abstract] [Full Text] [PDF]
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