Perturbed medullary tubulogenesis in neonatal rat exposed to reninangiotensin system inhibition
Daina Lasaitiene1,
Yun Chen1,
Gregor Guron1,
Niels Marcussen4,
Andrzej Tarkowski3,
Esbjorn Telemo3 and
Peter Friberg1,2
1Department of Physiology, Institute of Physiology and Pharmacology, 2Department of Clinical Physiology, 3Department of Rheumatology and Inflammation Research, University of Gothenburg, Sweden and 4Department of Pathology, University of Aarhus, Denmark
Correspondence and offprint requests to: Daina Lasaitiene, MD, Department of Physiology, Institute of Physiology and Pharmacology, University of Gothenburg, Box 432, S-405 30, Gothenburg, Sweden. Email: daina.lasaitiene{at}fysiologi.gu.se
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Abstract
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Background. Pharmacological interruption of the angiotensin II type-1 receptor (AT1) signalling during nephrogenesis in rats induces irreversible abnormalities in kidney morphology, comprising papillary atrophy and tubulointerstitial damage, which are characterized by tubular dilatation/atrophy and interstitial inflammation/fibrosis. This study determined the time course for development of tubular structural and inflammatory changes and possible cytokine production in the renal medulla of newborn rats exposed to angiotensin-converting enzyme (ACE) inhibition. Additionally, medullary expression of E-cadherin, a marker for tubular formation, was investigated in ACE-inhibited rats.
Methods. Newborn rats were exposed (postnatal days 012) to ACE inhibitor enalapril and killed at days 1, 2, 4, 9 and 13. One kidney was used for morphological evaluation and the other for immunohistochemistry, using antibodies directed against monocytes/macrophages, T cells and E-cadherin on frozen sections. In a separate experiment, rats were treated for 9 days and had their kidneys processed for western immunoblot and immunohistochemistry, where antibodies directed against monocyte chemoattractant protein-1 (MCP-1) and tumour necrosis factor-
(TNF-
) were used on paraffin sections.
Results. In renal medulla from enalapril-treated rats, volume fractions of tubular lumens and interstitium were increased from postnatal days 2 and 4, respectively, while that of tubular cells was decreased from 4 days of age. Concomitant loss and/or reduction in E-cadherin expression (from day 2) was observed in dilated medullary tubules of enalapril-treated rats. Furthermore, in the medulla of enalapril-treated rats, the increased number of ED2+ (resident macrophages) cells, followed by the increase in ED1+ (monocytes/macrophages) and CD4+ T cells, was observed at days 9 and 13, respectively. This was accompanied by increased medullary expression of TNF-
at day 9.
Conclusions. Neonatal ACE inhibition perturbs medullary tubulogenesis, as indicated by tubular dilatation and a lack of E-cadherin expression in these tubules. Macrophage/monocyte-mediated immune response is a secondary event, coincidentally associated with the up-regulation of TNF-
.
Keywords: angiotensin-converting enzyme inhibition; E-cadherin; inflammation; reninangiotensin system; renal development; tubulogenesis
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Introduction
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We and others have demonstrated an abnormal renal development in the rat following neonatal interruption of the reninangiotensin system (RAS) [1]. Given the fact that abnormal renal development is linked to subsequent susceptibility to cardiovascular disease in adulthood [2], with the RAS being a candidate in perinatal programming [3], it is of interest to study RAS-mediated renal development. Human kidney development is complete in utero by week 36 [4], whereas in the rat nephrogenesis continues into the second postnatal week [5]. Since rats are born with immature kidneys, with the first two postnatal weeks corresponding to the second and third trimester in humans, the neonatal rat model appears useful for studying the mechanisms of kidney development in the human fetus.
Neonatal angiotensin-converting enzyme (ACE) inhibition or angiotensin II type-1 (AT1) receptor antagonism, but not AT2 receptor blockade, produces irreversible abnormalities in kidney morphology and function, indicating that an intact signalling through AT1 receptors is a prerequisite for normal renal development [1]. The functional consequences of neonatal ACE inhibition have been investigated extensively and the main defect is a marked impairment in urine concentrating ability that is linked to renal papillary atrophy and persistent tubulointerstitial damage [1]. Tubulointerstitial damage comprises tubular dilatation/atrophy and interstitial inflammation/fibrosis [1]. The interstitial inflammatory changes consist of focal, predominantly mononuclear cell infiltrates, which are present in all kidney zones of adult rats long after cessation of neonatal RAS inhibition [1]. Although it has been shown that a lack of AT1 receptor stimulation in the developing kidney inevitably leads to persistent tubulointerstitial abnormalities long-term, little is known about when and why these changes develop.
We focused our study on the tubulointerstitium within the renal medulla, since atrophy of the inner medulla, accompanied by urine concentrating disability, is the predominant structuralfunctional defect in rats subjected to neonatal ACE inhibition [1]. The aims of this study were: (i) to determine the time course for development of tubular structural changes and to quantify them in rats treated neonatally with ACE inhibition; (ii) to explore whether the infiltration of inflammatory cells precedes or is secondary to tubular structural abnormalities; (iii) to study the expression of inflammatory cytokinesmonocyte chemoattractant protein-1 (MCP-1) and tumour necrosis factor-
(TNF-
)given their established role in the initiation and/or progression of renal tubulointerstitial damage in adult kidneys [6,7].
In the present study, medullary tubular dilatation in ACE-inhibited rats was the earliest finding, evident already 48 h after instillation of the ACE inhibitor treatment. This finding suggests that neonatal ACE inhibition interferes primarily with tubular development. In order to gain further insight into the mechanisms behind the perturbed tubular epithelial development in ACE-inhibited rats, we investigated, as an additional aim, the expression and distribution of E-cadherin, given the fact that E-cadherin is a specific marker for tubulogenesis [8] and a key molecule for establishment of epithelial cellcell junctional complexes [9].
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Subjects and methods
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General procedures
Time-mated, female Wistar rats (B&K Universal AB, Sollentuna, Sweden) were transported to our facility on the 14th day of pregnancy. They were observed carefully for determination of the day of delivery. Both male and female pups were included in the study. Weight-matched pups were divided into two groups receiving daily i.p. injections of either enalapril maleate (10 mg/kg; Sigma-Aldrich Sweden AB) or isotonic saline vehicle in equivalent volumes of 10 ml/kg from day 0 (within 12 h after birth) to day 12. We have performed previously a detailed time course study of the window during the neonatal phase when renal abnormalities are likely to occur, in terms of both structure and function [10]. That study demonstrated conclusively that the vulnerable age interval for induction of renal abnormalities was restricted to the first 13 days after birth. Based on that knowledge we have now modified the treatment regimen in such a way that we interfere with postnatal kidney development as early as possible, i.e. from days 0 to 12. Rats had free access to standard rat chow and tap water and were kept in rooms with a controlled temperature of 24°C and a 12-h dark (6 pm to 6 am)/light cycle, throughout the study. The regional ethics committee in Gothenburg approved all experiments.
Protocol
Eight pups from each group were killed by decapitation on postnatal days 1, 2, 4, 9 and 13 and both kidneys and spleens were rapidly removed. The right kidney from each animal was weighed and used for morphometrical/stereologic analysis and the left kidney was prepared for immunohistochemistry. Right kidneys were fixed in 4% paraformaldehyde and cut in 2-mm slices vertical to their long axis with the use of a device with parallel razor blades [11]. The slices were embedded in paraffin, and serial sections of 3 µm were cut and stained with haematoxylineosin or Massons trichrome. The left kidney and spleen specimens were embedded in Tissue-Tek O.C.T. compound and snap-frozen in isopentane, pre-chilled by liquid nitrogen and kept at -80°C until cryosectioned. Five-micrometre-thick sections were cross-cut to permit inspection of both medulla and cortex and mounted on superfrost plus glass slides and kept at -20°C until used.
In a separate experiment, ratseight pups in each groupwere treated for 9 days before death. One kidney was dissected into medulla and cortex, snap-frozen in liquid nitrogen and used for the detection of MCP-1 and TNF-
by western immunoblot technique. The other kidney was embedded in paraffin and used for immunohistochemistry.
Morphometrical analysis
Haematoxylineosin sections were used for the morphometrical analysis. Using unbiased stereologic methods, the following estimations were made by the investigator blinded to treatment groups: (i) volumes of the two different zones in the kidney, i.e. cortex together with outer stripe of the outer medulla (cortex + OSOM), and inner stripe of the outer medulla together with inner medulla (ISOM + IM), where the zonal definition determined by Kriz and Bankir [12] was used. A x4 objective was used and each field of vision included a grid with 36 points. The total number of points hitting each kidney averaged 139 ± 16. The volume of cortex + OSOM and ISOM + IM was estimated because of the difficulty to distinguish the boundary between cortex and outer medulla under low magnification; (ii) volumes of tubular epithelium, tubular lumens and interstitium/vessels in the medulla (OSOM + ISOM + IM). A x40 objective was used and each field of vision included a grid with 16 points. The total number of points hitting the medulla averaged 320 ± 110.
Every 2-mm-thick slice, containing medulla, was investigated, meaning that approximately two sections at early time points (days 1 to 4) and three to five sections at postnatal days 9 and 13 were examined from each kidney. Sections were placed in an Olympus microscope that was connected to a video camera and a computer. The computer generated a point set on the screen, and the video recorded the microscopic field. In systemic order with random start using a stage motor, sections were investigated by point counting, and the number of points hitting each structure was estimated and related to the total number of hits [11]. From estimated volume fraction, absolute volume of ISOM + IM zone was calculated assuming that the specific gravity of kidneys was 1 g/cm3.
Semi-quantitative assessment
Sections stained with haematoxylineosin and Massons trichrome were used for the semi-quantitative assessment of the fibrotic changes. Assessments were made by an investigator blind to treatment group. Fibrotic changes were scored semi-quantitatively with the use of arbitrary scale: 0 = normal, 1 = mild changes [1].
Immunohistochemistry
Immunostaining for monocytes/macrophages (ED1+ cells, ED2+ cells), CD4+ T cells and E-cadherin was performed on cryosections. Kidney and spleen sections were fixed in cold acetone (for 30 s in 50% and for 5 min in 100%) and air-dried, then washed in phosphate-buffered saline (PBS) three times. Sections were incubated with 40 µl of primary antibodies (Table 1) for 1 h in a humid atmosphere at room temperature. After washing three times in PBS all sections were depleted of endogenous peroxidase activity by treatment with 0.3% H2O2 for 5 min. After additional washing in PBS, sections were incubated with 40 µl (1:100) of secondary antibody (horseradish peroxidase (HRP) linked F(ab)2 fragment of sheep anti-mouse IgG (Amersham Pharmacia Biotech AB, Uppsala, Sweden) or HRP-linked goat anti-rabbit IgG (Santa Cruz Biotechnology, CA, USA) for 30 min, followed by three additional rinses and washes with PBS. Binding of peroxidase-labelled secondary antibody was detected by incubation for 810 min with substrate AEC (3-amino-9-ethyl-carbazole; Sigma Chemical, St Louis, MO, USA), containing 8 µl 30% H2O2. After rinsing in distilled water sections were counterstained with Mayers haematoxylin and mounted in aqueous medium. Positive and negative control incubations were performed at the same time to test the specificity of antibodies. Spleen specimens were used as positive control. Spleen and kidney specimens, incubated with 1% bovine serum albumin (BSA) to replace the specific primary antibody, served as negative control.
Immunostaining for MCP-1 and TNF-
was performed on paraffin sections. Before immunostaining, sections were heated at 60°C for 30 min, deparaffinized and boiled in citric acid buffer (0.01 M, pH 6.0) for 30 min. Non-specific binding was blocked by incubation with donkey serum (15 min). Following overnight incubation (at 4°C) with anti-MCP-1 or anti-TNF-
antibodies, sections were depleted of endogenous peroxidase activity and incubated with secondary antibody [HRP-linked donkey anti-goat IgG (Santa Cruz, 1:100) for 30 min. Immunoreactivity was visualized using DAB (3,3'-diaminobenzidine; DAKO, CA, USA).
All antibodies used for immunohistochemistry were diluted in PBS containing 1% of BSA.
Western immunoblot
Dissected renal medullae were homogenized in buffer containing 250 mM sucrose, 10 mM HEPESTris (pH 6.95) and protease inhibitors (CompleteTM mini, Roche). Protein concentration was measured using a Bio-Rad Protein Assay kit. Aliquots of 40 µg proteins were solubilized in Laemmli sample buffer and separated, under reducing conditions, by electrophoresis on 415% TrisHCl gradient gel (Bio-Rad, CA, USA). Proteins were transferred to a PVDF-membrane (Amersham). After blocking in 5% non-fat milk with phosphate-buffered saline plus 0.1% Tween 20 (PBS-T), the membrane was incubated with primary anti-TNF-
(1:300) or anti-MCP-1 (1:100) antibodies diluted with 1% non-fat dry milk in PBS-T for 1 h. The proteins were detected using HRP-linked donkey anti-goat IgG (1:1000, Santa Cruz, 30 min incubation) and ECL detection system (Amersham). The specificity of anti-TNF-
(sc-1350) and anti-MCP-1 (sc-1785) antibodies was tested by their pre-absorption with an excess of specific blocking peptide (sc-1350 P and sc-1785 P; Santa Cruz). Bands were visualized using a Fuji LAS-1000 cooled CCD camera/Dark Box, employing the Image Reader LAS-1000 v 1.1 software, and the density of the bands was analysed with the help of computer program Image Gauge software v 3.45. ß-Actin was used as internal control and the level of TNF-
was expressed as the ratio of TNF-
/ß-actin.
Quantification of kidney infiltrating and residing immune cells
Immunostained sections were placed in an Olympus microscope that was connected to a computer via a video camera. One 5-µm-thick immunostained cross-section from each kidney was viewed by a x40 objective and 3545 fields of vision in cortex, and 3650 fields of vision in medulla were sampled systematically with the use of a motorized stage with predetermined steps. The computer generated a 41 946 µm2 counting frame. All immunostained cells, which fell inside the frame or hit the upper horizontal edge and/or right vertical edge were counted, whereas cells falling outside the frame or hitting the lower horizontal and/or left vertical edge were excluded.
Statistical analysis
Values are expressed as means ± SD. The data were analysed by ANOVA followed by Fishers post-hoc least significant difference test, using the computer software SYSTAT (version 5.02, Evanston, IL). Unpaired two-tailed Students t-test was used when appropriate. P < 0.05 was considered statistically significant.
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Results
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Body and kidney weights
Neonatal enalapril treatment reduced body weight gain and increased kidney weights. Thus, the kidney to body weight ratio was increased in enalapril-treated rats (Table 2).
Kidney histology and expression of E-cadherin
Enalapril treatment reduced the absolute volume of the ISOM + IM zone from postnatal day 9 [26.9 ± 6.6 (enalapril-treated) vs 32.6 ± 7.5 (controls), values are means ± SD (in mm3), P < 0.05] and onwards. In the medulla of enalapril-treated rats, a significant increase in the volume fraction of tubular lumens (Figure 1A), indicating tubular dilatation, was found from postnatal day 2 (Figure 2AD). The volume fraction of interstitium increased (Figure 1B), whereas that of tubular cells decreased from 4 days of age (Figure 1C). In the medulla of enalapril-treated rats, interstitial fibrotic changes were detectable from postnatal day 9 (Table 3).

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Fig. 1. (AC) Volume fraction (Vv %) of tubular lumens (A), interstitium (B) and tubular cells (C) in the rat medulla during neonatal treatmentfrom birth to 12 days of agewith saline vehicle (open bars) or enalapril (10 mg * kg-1 * day-1, filled bars). *P < 0.05 vs saline vehicle. Values are means ± SD (n = 8 per group and time point).
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Fig. 2. (AD) Kidney sections from 2-day-old rats treated with saline vehicle (left column; A and C) or enalapril (right column; B and D) for 48 h. Enalapril-treated rat shows dilatation of medullary collecting tubules (depicted by arrows). Magnification x10 in (A) and (B); x40 in (C) and (D) (haematoxylineosin stain).
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Lateral E-cadherin expression in the epithelial cells of developing medullary collecting tubules was observed in saline-treated rats throughout nephrogenesis, i.e. days 2 to 13 (Figure 3A, C, E and G). Enalapril-treated rats demonstrated lack of/or dramatically reduced epithelial expression of E-cadherin in dilated medullary collecting tubules, the phenomenon, which was observed from day 2 and onwards (Figure 3B, D, F and H).

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Fig. 3. (A, C, E and G) Medullary expression of the E-cadherin protein in 2-day-old (A), 4-day-old (C), 9-day-old (E) and 13-day-old (G) rat kidneys treated with saline vehicle from the day 0 until the day of death. Control kidneys demonstrate lateral E-cadherin expression in the epithelial cells of collecting tubules. Magnification x40. (B, D, F and H) Medullary expression of the E-cadherin protein in 2-day-old (B), 4-day-old (D), 9-day-old (F) and 13-day-old (H) rat kidneys treated with enalapril (10 mg * kg-1 * day-1) from the day 0 until the day of death. Enalapril-treated kidneys demonstrate reduced and/or lost E-cadherin expression in dilated tubules. Arrows depict dilated tubules. Magnification x40.
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Infiltrating and residing immune cells in the kidney and medullary expression of cytokines
The number of resident macrophages (ED2+ cells) in the medulla of saline-treated rats did not change over the time (Figure 4A). Resident macrophages (ED2+ cells) were more abundant in the medulla of enalapril-treated rats vs controls at days 9 and 13 (Figure 4A).

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Fig. 4. (AC) Quantification of ED2+ cells (resident macrophages) (A), ED1+ cells (monocytes/macrophages) (B) and CD4+ T cells (C) in the rat medulla during neonatal treatmentfrom birth to 12 days of agewith saline vehicle (open bars) or enalapril (10 mg * kg-1 * day-1, filled bars). *P < 0.05 vs saline vehicle. Values are means ± SD (n = 8 per group and time point).
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Monocytes/macrophages (ED1+ cells) and CD4+ T cells in the medulla of saline-treated rats increased (P < 0.05, indicating a statistically significant difference over time) gradually starting from day 4 (Figure 4B and C). Surprisingly, the number of monocytes/macrophages (ED1+ cells) in the medulla (Figure 4B) of enalapril-treated rats was decreased at day 4. An increased number of monocytes/macrophages (ED1+ cells) and CD4+ T cells in the medulla (Figure 4B and C) of enalapril-treated rats vs controls was found only at day 13.
Using immunohistochemistry, both saline and enalapril-treated rats demonstrated medullary tubular expression of TNF-
(Figure 5A and B). Western immunoblot showed a band of
98 kDa, which disappeared after antibodys pre-absorption with blocking peptide, confirming the specificity of anti-TNF-
antibody (Figure 5C). Density analysis of the band revealed a statistically significant increase in medullary expression of TNF-
protein in enalapril-subjected kidneys at day 9 (Figure 5D).
Using immunohistochemistry, MCP-1 expression was low and staining was confined mostly to the vasculature. Western immunoblot analysis revealed levels of MCP-1 expression below detection limit (data not shown).
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Discussion
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The main finding of the present study was medullary tubular dilatation, evident already 48 h after instillation of the ACE inhibitor in the newborn rat, while tubulointerstitial inflammation and fibrosis manifested not until the second postnatal week. This late inflammatory response was characterized by TNF-
over-expression and increased numbers of ED2+, ED1+ and CD4+ T cells, together with interstitial fibrotic changes. In addition, enalapril treatment caused a reduced expression of E-cadherin in dilated medullary collecting tubules, suggesting that suppressed E-cadherin levels could be involved in the development of tubular dilatation. Taken together, the present study demonstrates that the RAS is important in mediating medullary tubulogenesis, and that tubular dilatation is an early event followed by a secondary inflammatory response.
It is well established that interruption of the RAS during nephrogenesis results in papillary atrophy, tubular dilatation and atrophy, chronic interstitial inflammation and fibrosis and renal vascular changes in adulthood [1]. The data have been confirmed by different investigators using similar pharmacological treatments [5,13] or genetically modified mice [14]. The present study extends our knowledge as to the temporal development of the medullary tubulointerstitial damage within the neonatal period. The present results showed that in the medulla of enalapril-treated rats, significant tubular dilatation was evident already 48 h after treatment instillation, followed by interstitial expansion and decreased tubular cell numbers from postnatal day 4. These findings demonstrate that neonatal ACE inhibition induces abnormalities rapidly in developing medullary tubules. The process of tubule formation in the developing kidney requires epithelial cells to undergo shape change and migrate to a specific location, establish complex cellmatrix and intercellular interactions and develop an apical-basolateral polarity [15]. One of the most important types of intercellular interactions required for the establishment of epithelial tissue is that mediated by the members of the classic cadherin family, with E-cadherin being the most studied member [16]. Constitutively expressed in collecting ducts [17], E-cadherin has a fundamental role in the formation of polarized epithelial cells [18], thus, ensuring epithelial physiological function in absorption/secretion and as a barrier [19]. Moreover, the potential role for E-cadherin in tubulogenesis was suggested since E-cadherin appears, subsequent to induction, at lateral surfaces of the differentiated polarized tubular epithelium [20]. Thus, the suppression of E-cadherin in dilated medullary tubules of ACE-inhibited kidneys, as evident from this study, points to perturbed medullary tubulogenesis. Collectively, the RAS is important in mediating normal postnatal medullary tubulogenesis. Moreover, our findings suggest a role for E-cadherin in this process although additional studies are clearly needed to elucidate the interaction between the RAS and E-cadherin during tubulogenesis.
One may envisage that the disturbed tubulogenesis in ACE-subjected rats could be prompted by inflammatory changes. However, the present study demonstrated a late inflammatory response, characterized by TNF-
over-expression and increased numbers of ED2+, ED1+ and CD4+ T cells, together with interstitial fibrotic changes. These changes were observed not until the second postnatal week in ACE-inhibited kidneys. In the medulla of enalapril-treated rats, resident macrophage (ED2+ cells) accumulation (from day 9) was followed by monocyte/macrophage (ED1+ cells) and CD4+ T cell recruitment from day 13. Data obtained from this study and from the literature [21] suggest that both the proliferation of resident macrophages (ED2+ cells) and the recruitment of blood-borne inflammatory cells (ED1+, CD4+) contribute to the inflammatory cell accumulation in the interstitium. Although the mechanisms by which tubular distortion produced interstitial inflammation are incompletely understood, our finding of increased expression of inflammatory cytokine TNF-
in the medulla of enalapril-treated rats suggests a role for TNF-
in the establishment of interstitial inflammation in neonatal rats. Indeed, TNF-
, which is produced by tubular epithelial cells and monocytes/macrophages, has been involved in the aetiology of renal tubulointerstitial injury of various origins [7]. In addition to our novel demonstration of TNF-
protein expression by inflamed neonatal rat kidneys, TNF-
has also been detected in healthy developing kidneys, where it could act as a modulator of growth and/or differentiation of various cells [22]. One may discuss the size of detected TNF-
band (
98 kDa), which is somewhat different from those reported in literature [23]. However, the same results were reproduced with the anti-TNF-
antibodies of two different batches, and their specificity was confirmed by neutralization with the blocking peptide. Hence, we believe that we have detected the complex of TNF-
bound to one of its receptors, which are expressed in kidneys [24].
Given the unexpected decrease in the number of monocytes/macrophages (ED1+ cells) in ACE-inhibited kidneys at day 4 after birth, as was found in this study, and the possession of angiotensin-generating system by circulating rat mononuclear cells [25], one could argue that the RAS might be implicated in the postnatal monocyte/macrophage establishment in the rat kidney. Consequently, the inflammatory response following neonatal ACE inhibition would be anticipated somewhat later, as demonstrated in this study. Additionally, the reduced infiltration of monocytes/macrophages at day 4 in ACE-inhibited kidneys could account for early initiation of interstitial fibrosis (already at day 9) given their salutary function in the evolution of renal fibrosis [26].
Moreover, the present results showed that during normal conditions the number of monocytes/macrophages (ED1+ cells) and CD4+ T cells in the medullary interstitium increased concomitantly with the growth of the kidney. The normal kinetics of monocytes/macrophages over the 2 first postnatal weeks in saline-treated rats corroborates findings in mice [27], where the number of macrophages (F 4/80+ cells) increases transiently in the early postnatal period, being maximal at 2 weeks of age. Tissue macrophages have local trophic and/or scavenger functions, thus influencing organogenesis and tissue turnover [27], and, therefore, may be implicated in nephrogenesis, which continues into the second postnatal week in the rat [5].
In summary, our results show that ACE inhibition in the newborn rat perturbs medullary tubulogenesis, microscopically discernible as tubular dilatation and concomitant loss/reduction in E-cadherin expression by dilated tubules already 48 h after treatment instillation. The immune response, characterized by up-regulation of TNF-
, proliferation of resident macrophages and influx of monocytes, becomes evident in the second postnatal week, indicating that the inflammation is a secondary event.
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Acknowledgments
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The technical assistance of Ulla Hovgaard, Ing-Marie Nilsson and Mikhail Dozmorov is gratefully acknowledged. Dr Chen is a recipient of postdoctoral fellowship from Wallenbergs foundation. This study was supported by the Swedish Medical Research Council (Grant 9047). Part of this work was presented at the 18th Scientific Meeting of the International Society of Hypertension (J Hypertens 2000; 18 [Suppl 4]: S85).
Conflict of interest statement. None declared.
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Received for publication: 28.11.02
Accepted in revised form: 11. 7.03