Main

The renin-angiotensin system plays a central role in the regulation of vascular tone and fluid homeostasis. In addition, ANG II has been shown to stimulate growth in a variety of tissues, including the kidney(1). Compared with that in the adult, the activity of the renal renin-angiotensin system is markedly activated in early development(2). Blocking ANG II by ACE inhibition or ANG AT1 receptor inhibition in fetal life causes hypotension, growth impairment, and abnormal renal development(3, 4). These reports suggest that ANG is necessary for normal renal development.

The cellular mechanisms underlying ANG-dependent stimulation of renal development have not been elucidated. The present study was therefore designed to investigate the role of endogenous ANG II in the regulation of autocrine and paracrine compounds known to mediate cell proliferation, extracellular matrix deposition, and cell-cell and cell-matrix interactions. ANG II has been shown to induce proximal tubular cell hypertrophy through stimulation of TGF-β1(5). Alternatively, the growth response to ANG II may be mediated by conversion of TGF-β1 to its active form(6). In addition, TGF-β1 inhibits tubular cell migration(7) and stimulates deposition of extracellular matrix, although inhibiting its degradation(8). In contrast to TGF-β1, EGF stimulates renal differentiation(9). ANG II potentates the mitogenic action of EGF on proximal tubular cells(10), and inhibition of AT1 receptors decreases the renal expression of EGF in the neonatal rat(11).

Clusterin (sulfated glycoprotein-2) is a glycoprotein produced by mesenchymal cells in renal embryogenesis and by renal tubular cells in response to tubular injury(12). It appears to play a major role in promoting epithelial cell aggregation(13). Inhibition of AT1 receptors increases the renal expression of clusterin in the neonatal rat, suggesting that clusterin expression is normally suppressed by ANG II(11).

The present study was designed to determine whether inhibition of endogenous ANG II production during renal development modulates steady state levels of renal mRNA for TGF-β1, prepro EGF, and clusterin. The postnatal rat was chosen for investigation because only 10% of nephrons are present at birth in this species, with the remainder being formed by 14 d of age(14). Thus, the early postnatal period in the rat is analogous to the midgestational period in the human. In a recent report, all of the components of the renin-angiotensin system have been demonstrated in the human fetal kidney(15).

METHODS

Studies were performed on 28 neonatal Sprague-Dawley rats. Each pregnant rat was put into a separate cage and was allowed free access to regular rat chow and tap water. At birth, neonatal rats were allocated to eight individuals per litter, and the mothers suckled their newborns up to harvest. The experimental protocol was approved by the Animal Care Committee of the University of Virginia.

Beginning on d 1 of life, rats were weighed and gavage-fed a daily dose of enalapril (Sigma Chemical Co., St. Louis, MO) (30 mg/kg of body weight, dissolved in distilled water, n = 16), or vehicle (distilled water, n = 12). This dose of enalapril has been shown to block the pressor response to ANG I in rats(16). Rats were killed at 14 d of age. Kidneys were removed, decapsulated, weighed, and processed for RNA analysis.

Northern blots. Total renal RNA was extracted by a modified method of Chomczynski and Sacchi(17) using guanidium isothiocyanate. A 20-μg RNA sample was electrophoresed on denaturing 1.2% agarose-formaldehyde gel, transferred to a nylon membrane by capillary action, heated, and exposed to UV (254 nm). Northern blot membranes were hybridized, using the method of Church and Gilbert(18), with the use of a rat TGF-β1 cDNA (gift of Su Wen Qian, National Cancer Institute, Bethesda, MD), a mouse EGF cDNA (gift of Graeme I. Bell, Howard Huges Medical Institute, University of Chicago, Chicago, IL), a clusterin cDNA (gift of M. Tenniswood, University of Ottawa, Ottawa, Canada), and a rat renin cDNA (gift of Kevin Lynch, University of Virginia, Charlottesville, VA). cDNAs were labeled with 32P by random priming to specific activities equal to or higher than 1 × 108 cpm/μg. In addition, blots were hybridized with a housekeeping gene, a 780-bp cDNA fragment of human GAPDH (American Type Culture Collection, Rockville, MD), to control for equal loading. Hybridization signals were detected and quantitated by a PhosphorImager system (Molecular Dynamics, Sunnyvale, CA). The ratio of each mRNA to GAPDH mRNA was calculated for each group.

Morphometric and immunohistochemical study. Kidneys from six control and six enalapril-treated rats were fixed in Bouin's solution, embedded in paraffin, sectioned at 4 μm, and stained with periodic acid-Schiff. Paired kidneys from the same rats were fixed in 10% formalin, embedded in paraffin, and sectioned. Using periodic acid-Schiff-stained sections, the relative area of 25 glomeruli from each animal was measured using a digital imaging system (Mocha, Jandel Scientific, San Rafael, CA). Using methylene green-stained formalin-fixed sections, tubular intraluminal areas were measured in 10 non-overlapping fields viewed under 400× magnification.

For each antibody, kidneys from five to six rats were studied for each group. Immunoreactive TGF-β1, EGF, and clusterin were detected with the avidin-biotin immunoperoxidase method (Vectastain, Vector Labs, Burlingame, CA). Antibodies used included a 1:50 dilution of rabbit monoclonal antihuman TGF-β1 antibody (Promega, Madison, WI), 1:400 dilution of rabbit polyclonal anti-rat EGF antibody (Biomedical Technologies, Stoughton, MA), or 1:50 dilution of rabbit monoclonal anti-rat clusterin antibody (Upstate Biotechnology, Lake Placid, NY). As a negative control, the primary antiserum was replaced by nonimmune rabbit serum, and no staining occurred. Sections were counterstained with methylene green and examined in blinded fashion by light microscopy. The mean luminal area of clusterin-stained tubules was compared with that of nonstained tubules.

Statistical analysis. Data are presented as mean ± SEM. Comparisons between groups for each age were performed by one-way ANOVA, the Student-Newman-Keuls test, and t test. Statistical significance was defined as p < 0.05.

RESULTS

Mortality. A death rate of 40% was observed in the enalapril-treated group. There was no mortality in the vehicle-treated group.

Body weight and kidney weight. On d 7, the body weight of pups receiving enalapril was significantly lower than that of the control group (Fig. 1A). This difference persisted to d 14 (Fig. 1A). By d 14, kidney weight was also significantly decreased in the enalapril group (Fig. 1B), but the kidney/body weight ratio was not different between the groups.

Figure 1
figure 1

(A) Body weight of neonatal rats during study. (B) Kidney weight for each group on d 14. (C) Relative glomerular area for each group on d 14. (D) Relative tubular luminal area for each group on d 14. Solid bar, control group; hatched bar, enalapril-treated group; *p < 0.05 vs control.

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Glomerular and tubular luminal area. Compared with the control group, enalapril treatment reduced mean glomerular area by 27% (Fig. 1C). Despite the reduction in glomerular area, there was no glomerular sclerosis or infarction (Fig. 2C). Enalapril treatment doubled mean tubular luminal area (Figs. 1D and 2D).

Figure 2
figure 2

Photomicrographs of representative rat kidneys obtained at the end of the study period. (A) Immunostaining (brown) EGF in control group. EGF is widely distributed in distal tubules. (B) Immunostaining EGF in enalapril-treated group. There is widespread tubular dilatation and markedly reduced EGF immunoreactivity. Glomeruli appear normal. (C) Immunostaining clusterin in control group. There is no identifiable staining. (D) Immunostaining clusterin in enalapril-treated group. Scattered dilated tubules contain immunoreactive clusterin (arrowhead).

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TGF- β 1 immunoreactivity. Immunoreactive TGF-β1 was diffusely distributed along the luminal border of tubular epithelial cells, with no detectable difference in distribution or intensity between groups (not shown).

EGF immunoreactivity. Immunoreactive EGF was localized to the cytoplasm of control distal tubules as shown in Fig. 2A. Enalapril treatment markedly suppressed tubular EGF immunostaining (Fig. 2B).

Clusterin immunoreactivity. Immunoreactive clusterin was not detectable in control kidneys (Fig. 2C), but was localized to scattered dilated tubules in kidneys from enalapril-treated rats (Fig. 2D). The median luminal area of clusterin-staining tubules was 4-fold greater than that of nonstaining tubules (p < 0.05, ANOVA on rank sum).

TGF-β1 mRNA expression. Treatment for 14 d with enalapril reduced steady state renal TGF-β1 mRNA expression by nearly 50% (Figs. 3 and 4A).

Figure 3
figure 3

Representative Northern blot for TGF-β1, prepro EGF, clusterin, and renin (upper row). Shown also (lower row) is GAPDH for each lane above. C, control; E, enalapril-treated group.

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Figure 4
figure 4

(A) Ratio of renal TGF-β1 to GAPDH mRNA for each group. (B) Ratio of renal prepro EGF to GAPDH mRNA. (C) Ratio of renal clusterin to GAPDH mRNA. (D) Ratio of renal renin to GAPDH mRNA. Solid bar, control group; hatched bar, enalapril-treated group; *p < 0.05 vs control.

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EGF mRNA expression. Enalapril treatment decreased renal EGF mRNA expression by 81% (Figs. 3 and 4B).

Clusterin mRNA expression. In contrast, enalapril treatment increased renal clusterin mRNA expression over 3-fold that of the control group (Figs. 3 and 4C).

Renin mRNA expression. Enalapril nearly doubled renal renin mRNA expression (Figs. 3 and 4D).

DISCUSSION

The present study demonstrates that ACE inhibition by enalapril impairs somatic and renal growth in the neonatal rat. Moreover, renal development is impaired as evidenced by reduced glomerular volume and tubular dilatation. The increase in renal renin expression is the expected consequence of recruitment of renin-secreting cells resulting from ANG II inhibition(16). The results are consistent with reports of intrauterine growth retardation, renal tubular dysplasia, and neonatal death resulting from the maternal use of ACE inhibitors during pregnancy in humans, primates, sheep, and rabbits(3, 19–21). The long-term consequences of ACE inhibition or ANG II antagonism in the neonatal rat include papillary atrophy and impaired urine concentrating ability in adulthood(22). Mice homozygous for a null mutation in the angiotensinogen gene show delayed glomerular maturation, abnormal renal vascular development, and renal papillary hypoplasia(23). It should be noted that, in these mice, the abnormalities present in the kidneys are not present in the heart, liver, or major vessels. Although the effects of enalapril in neonatal rats demonstrated in the present study may not be limited to the kidney, the studies of angiotensinogen-deficient mice suggest that this organ is the most severely affected by ACE inhibition. Interference with normal renal maturation by ACE inhibitors has been reported even for amphibians: tadpoles exposed to water containing enalapril develop dysplastic changes in the mesonephros(4).

Administration of losartan (an AT1 receptor antagonist) to neonatal rats also impairs somatic and renal growth, by interfering with renal vascular and glomerular development(4). This effect was not obtained with administration of an AT2 receptor inhibitor, suggesting that the primary effect of ACE inhibitors on renal growth is mediated by AT1 receptors(4). Although the fetal consequences of maternal ACE inhibition have been ascribed to fetal hypotension and renal ischemia(3), administration of hydralazine to neonatal rat pups does not impair renal growth or result in renal dysgenesis(4, 24). Enalapril (but not hydralazine) causes growth retardation in maturing glomeruli in the rat(25). This suggests that the changes observed in the present study are the result of inhibition of ANG II formation rather than being a response to hypotension. We have also recently reported that administration of losartan to neonatal rats impairs growth of the normal kidney, but does not further decrease growth of the kidney subjected to chronic ureteral obstruction(11). ANG II induces cellular hypertrophy in cultured proximal tubular cells(10), whereas administration of exogenous ANG II to neonatal rats stimulates renal growth and glomerular maturation(26). These results suggest a dominant role for ANG in the overall regulation of renal growth during the period of nephrogenesis.

The present study reveals that ACE inhibition significantly reduced the expression of TGF-β1 in the maturing kidney. Because the renin-angiotensin system is activated in early development, this is consistent with the increased expression of TGF-β1 by renal proximal tubular cells undergoing hypertrophy after exposure to ANG(5). TGF-β1 enhances the extent of gap junction-mediated intercellular communication between normal rat kidney cells(27). TGF-β1 also regulates the glomerular and renal interstitial production of numerous extracellular matrix molecules, as well as the integrins that are the receptors for these molecules(28). In this regard, enalapril reduces renal TGF-β1 expression and interstitial fibrosis which are stimulated by ureteral obstruction(29). We found that whereas losartan reduced the augmented TGF-β1 expression by the obstructed neonatal rat kidney, there was no effect on TGF-β1 expression by the normal kidney(11). The reasons for these differences may relate to incomplete inhibition of the AT1 receptor by losartan (although a large dose was used), or decreased degradation of bradykinin resulting from ACE inhibition in the present study. The latter is unlikely, because TGF-β1 expression by cultured mesangial cells is unaffected by bradykinin, which stimulates (rather than inhibiting) mesangial proliferation(30). An alternative explanation is the regulation of TGF-β1 expression by ANG receptors other than the AT1 receptor. Thus, whereas losartan increases ANG II levels, ACE inhibition by enalapril reduces ANG II levels. Renal TGF-β1 is localized to cortical distal tubular cells, and expression decreases during early postnatal development in the rat(31). It is likely that TGF-β1 plays a role in controlling interactions between epithelia and surrounding mesenchyme in the developing kidney(32), and ACE inhibition may impair renal growth in part by this mechanism.

Inhibition of renal EGF expression by enalapril in the neonatal rat as found in the present study is similar to the response to losartan(11). EGF is a potent growth promoter for renal proximal tubular cells(33) and regulates tubulogenesis(9, 34). ANG II and EGF are synergistic in stimulating cellular proliferation, by complementary actions at sequential cell cycle loci(35). Renal EGF expression increases progressively in the maturing rat and mouse, after first appearing in distal tubules several days after birth(36, 37). In addition, renal EGF receptors are induced and activated in late gestation in the rat, which suggests their participation in renal growth and development(38). Expression of EGF is also reduced in polycystic kidney disease of mice(39), and administration of exogenous EGF reduces tubular dilatation and improves renal function in this model(40). Moreover, chronic administration of ANG II in the rat increases aortic expression of EGF receptor mRNA(41). Inhibition of ANG II formation by enalapril in the present study may therefore also contribute to impaired renal growth and development by interfering with production of EGF and its receptor.

Clusterin is a glycoprotein expressed by a variety of tissues, that promotes aggregation and adhesion of renal epithelial cells(13). Clusterin expression is increased in the developing glomeruli during epithelial polarization, and may play a significant role in this process(42). In addition, renal clusterin expression is activated in response to injury, including ureteral obstruction, renal ablation, and renal ischemia(12). In the present study, enalapril markedly increased renal clusterin expression in the neonatal kidney, a response similar to that resulting from losartan administration(11). Clusterin was localized to dilated tubules, a pattern similar to that resulting from ureteral obstruction and cystic kidney disease(43, 44). This suggests that clusterin expression is chronically suppressed by ANG II AT1 receptors. However, in a preliminary study, we demonstrated a marked increase in renal clusterin expression after ANG II administration in 3-d-old rats subjected to ureteral obstruction(26). Because this response can be blocked by an AT2 receptor inhibitor, but not by losartan(45), clusterin expression may be stimulated by AT2 receptors, which are more abundant in early life, and inhibited by AT1 receptors, which predominate in the mature kidney(46).

In summary, chronic inhibition of endogenous ANG II in neonatal rats impaired somatic and renal growth, and increased renal renin expression. This was associated with suppression of TGF-β1 and EGF gene expression. These growth factors have well established effects in the kidney, to promote cellular growth and differentiation. The findings are consistent with the hypothesis that ACE inhibitor “fetopathy”(3) is a consequence of impaired expression of growth factors under the regulation of ANG II. Stimulation of the renal expression of clusterin after ACE inhibition may result from inhibition of AT1 receptors.