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Am J Physiol Regul Integr Comp Physiol 279: R1050-R1060, 2000;
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Vol. 279, Issue 3, R1050-R1060, September 2000

IGF-I treatment attenuates renal abnormalities induced by neonatal ACE inhibition

Annika B. M. Nilsson1, Nicoleta Nitescu1, Yun Chen1, Gregor S. Guron1, Niels Marcussen2, Göran L. Matejka3, and Peter Friberg1,4

1 Department of Physiology, Institute of Physiology and Pharmacology, Göteborg University, Medicinaregatan 11, S-413 90 Göteborg; 3 Department of Internal Medicine and 4 Clinical Physiology, Sahlgrenska University Hospital, S-41345 Göteborg, Sweden; and 2 University Institute of Pathology, Aarhus Kommunehospital, DK-8000 Aarhus, Denmark


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

An intact renin-angiotensin system (RAS) during nephrogenesis is essential for normal renal development. We have shown previously that neonatal inhibition of the RAS, either with ANG II type 1-receptor blockade or angiotensin-converting enzyme (ACE) inhibition, induces irreversible renal abnormalities. The aim of the present study was to investigate whether an interrupted RAS can be compensated for by exogenous administration of another important renal growth-promoting factor, the insulin-like growth factor-I (IGF-I). Rats were treated daily with either the ACE inhibitor enalapril (10 mg/kg), recombinant human IGF-I (3 mg/kg), or the combination enalapril + IGF-I from perinatal day 3 to 13. Urinary concentrating ability, renal function, and renal morphology were assessed at adult age. The gene expression and localization of IGF-I, its receptor, and the growth hormone receptor (GHR) were investigated during ongoing ACE inhibition. The present study demonstrates normalized renal function and histology in enalapril + IGF-I-treated animals. Ongoing ACE inhibition suppressed the medullary IGF-I mRNA expression and altered the local distribution of both IGF-I and GHR. Thus the present study provides evidence for an interaction between the RAS and GH/IGF-I axis in renal development.

renin-angiotensin system; renal development; angiotensin-converting enzyme; insulin-like growth factor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NEONATAL ANGIOTENSIN-CONVERTING enzyme (ACE) inhibition or ANG II type 1 (AT1)-receptor antagonism during the first weeks of life in rats and pigs induces irreversible renal histopathological changes in the long term, mainly characterized by papillary atrophy, tubular dilatation, vascular alterations, chronic interstitial inflammation, and fibrosis (21, 29). These histological abnormalities are associated with an impairment in urinary concentrating ability of renal origin (28), a reduced baseline renal interstitial hydrostatic pressure (50), sodium retention during chronic dietary sodium loading, and a modest renal potassium loss during dietary potassium restriction (27 and A. B. M. Nilsson, P. Friberg, M. A. Adams, unpublished data); defects that may be attributed to the papillary atrophy.

The basis for the development of these renal alterations is formed by the notion that all components of the renin-angiotensin system (RAS) are expressed in the immature rat kidney and show an upregulated gene expression perinatally compared with the adult, leading to high intrarenal ANG II levels (22, 70). Compelling in vitro evidence shows trophic effects of ANG II on different renal cell types (67), findings that support the contention of an involvement of ANG II in renal differentiation and growth during development (31).

Because ANG II is known to promote renal growth, one may surmise that at least part of such an effect may be mediated via the insulin-like growth factor (IGF) system, as IGFs (IGF-I and IGF-II) and the IGF-I receptor (IGF-IR) are all present at various sites in the kidney and are involved in several aspects of renal growth and development (10, 17, 42). Interruption of the RAS during early postnatal development could then possibly attenuate necessary renal development influenced by IGFs. IGF-I has also been implicated in modulating kidney regeneration and repair after renal ischemic events (18, 43). In addition, targeted mutagenesis of the genes encoding for IGF-I and IGF-II, and particularly IGF-IR, showed almost no survival and severe retardation of fetal growth (4, 38). Similar conclusions were drawn by Powell-Braxton et al. (53), who claimed the vital importance of IGF-I for normal embryonic growth in mice. Recent reports from this group showed grossly abnormal kidneys in IGF -/- mice (55).

The aim of the present study was to examine whether IGF-I, administered exogenously, could attenuate or prevent the renal medullary damage and normalize the impaired ability to concentrate urine that occurs after neonatal ACE inhibition in rats. Thus recombinant human IGF-I (rhIGF-I) was administered concomitantly with the ACE inhibitor enalapril to neonatal rats to explore whether the urinary concentrating ability, renal function, and renal morphological features were affected long term. Because the results from this combined treatment showed a completely reversed renal medullary function, we studied the mRNA expression of IGF-I, IGF-IR, and the growth hormone receptor (GHR) in the renal medulla and cortex during ongoing enalapril treatment. We wanted to investigate whether neonatal ACE inhibition induced an attenuation of the IGF-I gene expression and if so, whether this was correlated with increasing mRNA expression of IGF-IR and GHR. Furthermore, we examined the distribution of IGF-I and GHR in the neonatal kidney by immunohistochemical staining.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

General Procedures

Two-day-old male Wistar pups were purchased (Möllegaard Breeding Centre), divided into four groups, and treated from 3 to 13 days of age twice daily with intraperitoneal injections of 1) isotonic saline (Control, 2 × 10 ml/kg), 2) enalapril maleate (Enalapril, 2 × 5 mg/kg; Merck, Sharp and Dohme, Sollentuna, Sweden), 3) rhIGF-I (IGF-I, 2 × 1.5 mg/kg; Pharmacia-UpJohn, Stockholm, Sweden), or 4) combined enalapril and rhIGF-I treatment (Enalapril + IGF-I). The rats were kept at the local animal department with free access to normal rat chow and tap water, controlled room temperature of 24°C, and a 12:12-h dark-light cycle. All experiments were approved by the regional animal ethics committee in Göteborg.

Urinary Concentrating Ability, Renal Function, And Renal Morphology

Rats were treated twice daily from 3 to 13 days of age with saline (n = 7-11), enalapril (n = 6-9), rhIGF-I (n = 7-11), or enalapril + rhIGF-I (n = 7-11) (see General Procedures). Body weights were followed from 3 days of age until death at 24 wk of age.

Metabolic balance studies. At 7, 10, and 12 wk of age, the rats were put individually in metabolic cages with free access to powdered rat chow (120 mmol/kg NaCl and 80 mmol/kg KCl; Lactamin) and tap water for 48 h, followed by basal water intake (WI), urine production (UV), and urine osmolality (Uosm) measurements for 24 h when food and water were withdrawn. After 24 h of thirst provocation, urine was collected under paraffin oil over 20 h and referred to as maximal urinary concentrating ability (Uosmmax). At 18 wk of age, rats, individually put in metabolic cages, were again deprived of water for 24 h, and urine was collected over the consecutive 20 h. Uosm was determined by freezing-point depression (Wide Range Advanced Osmometer model 3MO; Advanced Instruments, Needham Heights, MA), and WI and UV were determined by weighing, assuming the density to be 1 g/ml.

Renal clearance experiments. At 24 wk of age, rats were anesthetized initially by a single intraperitoneal injection of Mebumal (60 mg/kg; Apoteksbolaget, Umeå, Sweden), tracheotomized for spontaneous breathing (polyethylene tubing, PE-240), and kept at a rectal temperature of 38°C by external heating throughout the experiment. The jugular vein and carotid artery were catheterized (PE-50) for continuous infusions of isotopes (20 µCi 51Cr-EDTA kg/h and 10 µCi 125I-Hippuran kg/h iv) and anesthesia (12 mg · kg-1 · h-1 ia) in a total volume of 7 ml · kg-1 · h-1. The urinary bladder was catheterized through a midline abdominal incision (PE-160) for urine sampling. After an equilibration period of 45 min, urine was collected in preweighed test tubes over two consecutive 20-min clearance periods with midpoint arterial blood sampling (0.3 ml), substituted with isotonic saline to replace volume loss. Mean arterial pressure (MAP) and heart rate were monitored continuously on a Grass polygraph (model 7D; Grass Instruments, Quincy, MA) by means of a pressure transducer (DPT-6100; Peter von Berg Medizintechnik, Kirchseeon/Eglharting, Germany) and sampled via the software PClab v.4.1 (3). The glomerular filtration rate (GFR), effective renal blood flow (ERBF), and fractional excretion of sodium (FENa) and potassium (FEK) were obtained by analyzing the urine and plasma samples for radioactivity by a scintillation counter (Packard, Amana, IA) and sodium and potassium concentrations by flame spectrophotometry (Radiometer, Copenhagen, Denmark). At the end of the acute experiment, kidneys were removed and immersion-fixed in 4% buffered formaldehyde (pH 7.4) until cut and stained for morphological evaluations by means of detailed morphometry.

Renal morphological evaluation. The kidneys were cut in 2-mm slices vertical to their long axis with the use of a device with parallel razor blades (see figure in Ref. 24). The slices were embedded in paraffin, and serial sections of 3 µm were cut and stained with periodic-acid Schiff (PAS), hematoxylin-eosin, and Masson-Trichrome. The sections were evaluated, and it was decided to use the PAS sections for the morphological evaluation. Sections were placed in an Olympus microscope (Vanox AHBT3) that was connected to a computer via a video camera. The computer generated a grid with a preselected number of points.

The zonal definition of cortex, outer and inner stripe of the outer medulla (OSOM and ISOM), and inner medulla (IM) by Kriz and Bankir (37) was used. Every second of the 3-µm thick PAS-stained sections was systematically sampled and viewed by a ×4 objective. The fields of vision were also systematically sampled with the use of a motorized stage with predetermined steps. In this way, ~40 fields were examined per kidney (the total number of points hitting each kidney was 178 ± 10). The number of points typically used in point counting is in the order of 150, which gives a solid unbiased result (25, 40). The volume fraction of each zone, given as per cent, was calculated from the number of points hitting each zone divided by the total number of points hitting the kidney. The absolute volume was then obtained by multiplying the volume fraction of each zone by the kidney weight, assuming that the density of kidney tissue is 1 g/cm3.

The volume fractions of glomerular tufts, distal and proximal tubular cells and lumens, and atrophic cells, vessels, and the interstitium were investigated in the cortex and OSOM. A ×20 objective was used, and the computer generated a grid of 12 points (out of which only 6 were used for the counting of proximal tubules). Each field of vision was systematically sampled, and a total of ~50 fields were examined (per kidney 456 ± 26 points were investigated). The volume fractions of the structures were calculated from the number of points hitting each structure divided by the total number of points hitting the cortex and OSOM.

An investigation of the vessel wall thickness (WT), including media, intima, and the adventitia, was determined in interlobular (outer diameter of 125-175 µm), arcuate (40-60 µm), and cortical radial (15-25 µm) arteries by subtracting the lumen diameter from the outer diameter divided by two. Results are given as WT/outer diameter (%). Between 35 and 50 vessels were evaluated in each kidney.

Cortical and Medullary IGF-I, IGF-IR, and GHR Gene Expression

Male Wistar pups were given daily injections of either enalapril (n = 51) or isotonic saline (n = 51) from age 3 to 13 days. Five pups from each group were killed 24 h after the previous injection on days 4, 5, 7, 9, 11, 13, 15, 20, 30, and 112 (16 wk of age). Both kidneys were excised, weighed, and divided into cortical and medullary regions, snap-frozen in liquid nitrogen and stored at -80°C before preparation of total nucleic acids (TNA). Simultaneously, liver samples were collected, snap-frozen, and stored at -80°C before preparation of total RNA. The specific mRNA expression of IGF-I, IGF-IR, and GHR were analyzed in the renal cortex, medulla, and liver (IGF-I only) by solution hybridization assay.

Preparation of TNA. TNA were prepared according to Durnam and Palmiter (15). Briefly, the tissues were homogenized in an SDS-containing buffer with a polytron (T25; IKA Labortechnik, Staufen, Germany). The samples were digested with proteinase K and then extracted with phenol and chloroform. The TNA were precipitated by adding ethanol, dissolved in a SDS, EDTA, and Tris solution, and measured by means of total DNA content with the use of fluorimetry (DyNA Quant 200; Hoefer Pharmacia Biotech).

Preparation of total RNA. Total RNA from liver tissues was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction with the use of Tri Reagent (Sigma), an improved single-step method reported by Chomczynski and Sacchi (12). RNA concentration was determined by spectrophotometer at 260 nm, and its purity was judged by the 260/280 ratio.

RNA probes. The 170-bases IGF-I RNA probe is complementary to part of exon 2 and 3 of rat IGF-I gene (59). The rat IGF-IR probe consists of 265 bases complementary to part of the 5'-untranslated sequence and to a region encoding the signal peptide and the first 53 amino acids of the IGF-IR alpha -subunit (65). The GHR is a 560-bases fragment of the rat GHR cDNA that encodes the early part of the extracellular domain of the GHR (44). The quality of the IGF-I, IGF-IR, and GHR assays was analyzed on a denaturing polyacrylamide gel (5% acrylamide/8 M urea) by means of the RNase Protection Assay kit from Ambion (Intermedica).

Solution hybridization assay. The amount of specific mRNA was determined with the use of 35S-UTP-labeled RNA probes (15). The probes were prepared according to the method of Melton et al. (47) and were hybridized to TNA samples at 70°C for at least 16 h. Hybridization was performed in 20 µl of 0.6 mol/l NaCl, 20 mmol/l Tris · HCl (pH 7.5), 4 mmol/l EDTA, 0.1% (wt/vol) SDS, 0.75 mmol/l dithiothreitol, 25% (vol/vol) formamide; 10,000-20,000 cpm of 35S-UTP-labeled probe was used per incubation. The samples were exposed to RNases, and the hybrids were precipitated with 100 µl TCA (6 mol/l), collected on glass microfiber filters, and analyzed in a liquid scintillation counter (1900 TR; Packard Instrument). A standard curve constructed from synthetic RNA standard complementary to the probe was included in each assay, and the radioactivity of the TNA samples, analyzed in duplicate, was compared with the standard curve. Tubes including hybridization buffer and probe served only as blanks.

Renal IGF-I and GHR Localization

Rats were treated twice daily from 3 to 13 days of age (Enalapril n = 3; saline n = 3) when killed; both kidneys were excised, sliced in five transversal sections, fixed in 4% buffered formaldehyde (pH 7.4), dehydrated, and embedded in paraffin. Five-micrometer sections were cut and boiled 2 × 10 min in a citrate buffer (0.01 M Na citrate, pH 6.0) in a microwave oven. The sections were rinsed and blocked with 10% fat-free milk (FFM) diluted in Tris-buffered saline (TBS); thereafter they were incubated with either anti-IGF-I (K1792, polyclonal antibody/rabbit 1:1,000) or anti-GHR (MAb 263, monoclonal antibody/rat) at 4°C overnight. Control sections were incubated with nonimmune rabbit sera with the primary antibodies omitted or with an unrelated mouse antibody at the same concentration as MAb 263. The sections were rinsed and incubated with HRP-labeled F(ab)2-fragments diluted in TBS/5% FFM for 60 min. The sections were stained with a standard DAB staining (3,3'-diaminobenzidine tetrahydrochloride and 0.01% H2O2 in TBS). The sections were then dehydrated and mounted. One section from each animal was stained with hematoxylin-eosin for morphological evaluation. The specificity of the antibodies were tested in a preabsorption test with the respective relating proteins.

Calculations

Standard equations for clearance calculations were used to obtain GFR (by means of 51Cr-EDTA clearance) and ERBF (by means of 125I-Hippuran clearance). Filtration fraction was calculated as GFR/ERBF, and FENa and FEK were obtained from respective clearances corrected by GFR. Linear regression and the correlation coefficient for Uosmmax as a function of the volume fraction of IM zone were evaluated in the total material, n = 34 (Microsoft Excel v7.0).

Statistical Analysis

All values are expressed as means ± SD. One-way ANOVA for repeated measurements and Dunnett's multiple comparison test for treatment effects vs. control were used (Microsoft Excel v7.0, Astute v1.7, Module 1 v1.1). Univariate and multivariate repeated-measures analyses were used for the evaluation of the time course of gene expression between the groups (SYSTAT). A P value of <0.05 was considered statistically significant. The differences between group averages are presented as confidence intervals (CI) of a 95% significance level.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Neonatal growth. The somatic growth of enalapril-treated rats was attenuated from 13 days of age compared with controls (P < 0.05, Fig. 1). However, from 3 wk of age, enalapril-treated rats gained proportionally more than controls, and by adult age, body weights were similar (Fig. 1, inset); only enalapril + IGF-I-treated rats remained smaller (CI 26-144 g vs. controls). Sole IGF-I treatment did not affect the neonatal growth development per se. However, IGF-I treatment attenuated the reduced weight gain induced by sole enalapril treatment (Fig. 1, P < 0.05 vs. enalapril), still demonstrating reduced body weights in the combined treatment group vs. controls (P < 0.05).


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  		Fig. 1.   Somatic growth in neonatal rats treated twice daily from 3 to 13 days of age with isotonic saline (control, 10 ml/kg, n = 9), enalapril (5 mg · kg-1 · 24 h-1, n = 6), insulin-like growth factor-I (IGF-I, 1.5 mg · kg-1 · 24 h-1, n = 9), or the combination of enalapril + IGF-I (5 + 1.5 mg · kg-1 · 24 h-1, n = 10). Enalapril-treated rats showed a significant attenuation of growth from 13 days of age (* P < 0.05 vs. controls), however, by adult age, body weights were similar to controls (inset). Combined IGF-I treatment attenuated the transient growth retardation induced by neonatal angiotensin-converting enzyme (ACE) inhibition ($ P < 0.05 vs. enalapril; * P < 0.05 vs. control). Values are means ± SD.

Urinary concentrating ability. Conscious neonatally enalapril-treated rats showed an increased baseline UV and decreased Uosm already from 7 wk of age (data not shown). WI was similarly increased, being 253 ± 20 ml · kg-1 · day-1 vs. control 167 ± 40 ml · kg-1 · day-1 (CI 33-139 ml · kg-1 · day-1). The combined enalapril + IGF-I treatment group demonstrated a preserved urinary concentrating ability with normal WI (184 ± 36 ml · kg-1 · day-1 at 7 wk of age; not significant vs. controls) as did the IGF-I-treated rats (WI 145 ± 19 ml · kg-1 · day-1). The group differences in urinary concentrating capacity persisted during aging and were further manifested by thirst provocation (Fig. 2, Uosmmax and UV at 18 wk of age). Moreover, sole IGF-I treatment resulted in an increased urinary concentrating ability compared with controls (CI 149-1,450 mosmol/kgH20).


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  		Fig. 2.   Urine production and osmolality in conscious 18-wk-old controls (n = 9), neonatally enalapril (n = 6)-, IGF-I (n = 9)-, and enalapril + IGF-I (n = 10)-treated rats. The decreased urinary concentrating ability in neonatally enalapril-treated rats (* P < 0.05 vs. controls) was normalized completely by combined IGF-I treatment ($ P < 0.05 vs. enalapril). Moreover, sole IGF-I treatment caused an increased capacity to concentrate urine (* P < 0.05 vs. controls). Values are means ± SD.

Renal function and hemodynamics. Renal function variables at baseline in 24-wk-old anesthetized rats revealed no significant differences among the treatment groups. MAP was also similar, averaging 127 ± 21 mmHg. GFR averaged 0.78 ± 0.20 ml · min-1 · g kidney wt-1, and the clearance of hippuran 2.31 ± 0.66 ml · min-1 · g kidney wt-1. FENa and FEK averaged 0.22 ± 0.20 and 44.0 ± 33.7%, respectively. Kidney weight/body weight ratios were not different between the groups.

Renal histology. Detailed stereological evaluation of the renal morphology demonstrated a maintained renal integrity in the enalapril + IGF-I treatment group (Table 1, Fig. 3), not being statistically different from saline controls in any of the structures investigated in the cortex and OSOM. The absolute volume of the OSOM was, however, increased vs. controls (CI 15-55 mm3/g body wt), and the interlobular vessel WT was increased (Table 1) similar to that in the enalapril-treated rats. IGF-I treatment did not only affect the interlobular vessel WT. This structural hypertrophy or hyperplasia was not seen for the arcuate or the cortical radial vessels (Table 1). Corroborating our previous results, enalapril-treated rats showed gross renal abnormalities, mainly characterized by a decreased volume fraction of the IM zone, which includes the papilla (CI -2.8-14.6 mm3/g body wt), and an increased OSOM zone (CI 15-64 mm3/g body wt) compared with controls. Furthermore, the degree of dilated tubules in the cortex and OSOM, as described by the volume fraction of tubular lumen of both proximal and distal tubules, was increased in enalapril-treated rats (CI 1-7% vs. controls), whereas the volume fraction of proximal tubules in these rats was reduced (CI 3-15%) and the amount of distal tubules was similar to controls. The volume fraction of the interstitium was also increased (CI 1-8% vs. controls), whereas the volume fraction of both glomeruli and vessels was similar in all treatment groups. Neonatally IGF-I-treated rats did not differ from controls in any of the investigated morphological variables, with the exception of an increased IM absolute volume (CI 2-18 mm3/g body wt). The increased size of the papilla corresponded partially to the increased urinary concentrating ability in these rats, being 3,882 ± 502 vs. 3,082 ± 292 mosmol/kgH20 (Fig. 2). There was a positive correlation between maximal Uosm and IM for all treatment groups taken together as shown in Fig. 4.

                              
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  		Table 1.   Renal histological variables in 24-wk-old neonatally treated rats



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  		Fig. 3.   Renal histopathological sections at 24 wk of age A: control, B: papillary atrophy and deranged structures by neonatal enalapril treatment, C: increased inner medullary absolute volume by sole IGF-I administration, and D: the normalized renal architecture by the combination enalapril + IGF-I. A: zonal definition is shown in the control section. Hematoxylin-eosin staining at ×4 magnification. OSOM, outer stripe of the outer medulla; ISOM, inner stripe of the outer medulla; IM, inner medulla.



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  		Fig. 4.   The urinary concentrating capacity (Uosmmax) in conscious rats during 24-40 h of thirst provocation correlated to the absolute volume of renal inner medulla (r = 0.591, P = 0.0004) in rats treated neonatally with isotonic saline (n = 9), enalapril (n = 6), IGF-I (n = 9), or enalapril + IGF-I (n = 10). gbw, Grams body wt.

IGF-I, IGF-IR, and GHR gene expression. The IGF-I mRNA expression in the renal cortex peaked at 7 days of age, whereas the medulla showed the highest expression at 30 days of age (Fig. 5). Comparison of the area under the curves in Fig. 5 demonstrated a threefold medullary IGF-I mRNA expression vs. the cortical expression, whereas the IGF-IR gene expression in the cortex was twofold the expression in the renal medulla (Fig. 5). Enalapril treatment significantly suppressed the medullary IGF-I mRNA expression (P < 0.05), evident already 24 h after the first injection. In contrast, the IGF-I gene expression in the cortical tissues was not affected by neonatal ACE inhibition. Neonatal enalapril treatment did not alter the IGF-IR gene expression in the medulla, whereas the renal cortex showed an upregulated expression from 9 to 30 days of age (P < 0.05), i.e., during the last 5 days of enalapril injections and 2 wk after cessation of treatment. The gene expression of GHR was not affected by neonatal ACE inhibition in neither the cortex nor the medulla, demonstrating a threefold cortical expression compared with the renal medulla, both increasing the mRNA levels with age (Fig. 5). The hepatic mRNA expression of IGF-I was about 5- and 12-fold the expression in the renal medulla and cortex, respectively, and was not affected by the neonatal enalapril treatment at any time point (data not shown). The quality of the rat GHR, IGF-IR, and IGF-I probes was demonstrated by RNase protection assay (Fig. 6, respectively).


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  		Fig. 5.   Time course gene expression of IGF-I (top), its receptor (IGF-IR, middle), and the growth hormone receptor (GHR, bottom) in the rat renal cortex (left) and medulla (right) during and after neonatal treatment (from 3-13 days of age) with saline (10 ml · kg-1 · injection-1, open bars) or enalapril (10 mg · kg-1 · day-1, filled bars). P < 0.05 indicates a statistically significant difference over the investigated period (4-112 days of age), and n.s. indicates no difference of significance. Values are means ± SD (n = 5 per group and time point) and expressed as amol/µg DNA.



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  		Fig. 6.   RNase protection assay of total RNA hybridized with 35S-UTP-labeled riboprobes (GHR: lanes 2-5; IGF-IR: lanes 6-9; IGF-I: lanes 10-14). Lanes 3, 6, and 14: undigested probes. Lanes 2, 7, and 13: digested probes. Lanes 5, 9, and 10: probes hybridized with 30 µg of total RNA from renal medulla. Lanes 4, 8, and 11: probes hybridized with 30 µg of total RNA from renal cortex. Lane 12: probe hybridized with 20 µg of total RNA from liver. Lanes 1 and 15: DNA marker.

Local distribution of IGF-I and GHR. Immunohistochemical results in control sections showed either faint background staining (rabbit polyclonal antibody) or no staining at all (unrelated mouse monoclonal antibody). In the control kidneys, IGF-I immunoreactivity (IGF-I-IR) was located to cells of the collecting duct and the thin loop of Henle, in concordance with previous studies (42). GHR appeared to be weakly expressed in the medulla of control animals and located to cells of the collecting duct. In enalapril-treated rats, the medullary morphology was altered with interstitial hypercellularity compared with controls. Both IGF-I-IR and GHR immunoreactivity appeared locally more dense in the interstitial compartment compared with controls (Fig. 7, respectively).


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  		Fig. 7.   Medullary expression of the IGF-I and GHR proteins at 13 days of age and after 10 days of daily intraperitoneal injections of the corresponding vehicle saline (A) or enalapril (10 mg · kg-1 · day-1; B). The control section demonstrates IGF-I bound to the tubular membranes only, whereas the ACE-inhibited kidney shows IGF-I staining also in the interstitium (B, top right). Similarly, the location of GHR is distributed both in the interstitium and the tubular membranes of the neonatally ACE-inhibited medulla (B, bottom left). The hypercellularity in enalapril-treated kidneys is shown in the hematoxylin-eosin staining (B, top left). Inserted bars denote 100 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study shows a beneficial and protective effect of exogenously administrated IGF-I during combined neonatal ACE inhibition by reversing the marked renal structural abnormalities and dysfunctions induced by sole neonatal ACE inhibition. The present study further demonstrates a suppressed mRNA expression of IGF-I and an altered distribution of the IGF-I protein in the renal IM during ongoing ACE inhibition. Thus the present results provide strong support for an interaction between RAS and the growth hormone (GH)/IGF axis in normal renal development.

We have reported previously long-term alterations of the renal morphology and function after neonatal RAS blockade (21, 26). These data have been confirmed by others with the use of similar pharmacological treatments (46, 60, 63) or genetically modified models (16, 35-36, 49, 62). The present study demonstrates the pronounced difference between the disturbed histology showing rough cortical surface, disorganized tubules, blurred cortical demarcation, and a more or less complete papillary atrophy after neonatal enalapril treatment compared with the almost completely normalized renal histology by combined IGF-I and enalapril treatment (Fig. 3).

We have hypothesized a potential role for ANG II as a growth-promoting and proliferative factor in renal development, and that neonatal RAS blockade per se inhibits the natural growth and development of the kidney. The necessity of an intact RAS in normal renal development has to date been well established, and more recently, the role for IGF-I in regulating renal development has been confirmed (30, 39, 56). Both RAS and IGF-I demonstrate a developmental pattern, with the highest renal expression in late fetal and early postnatal ages (23, 57). In vitro studies have shown that ANG II is a mitogen for renal vascular (11), mesangial (54), and tubular cells (66) via the AT1 receptor (32). In addition to stimulating cell growth directly, potential mediators of angiotensin growth actions are other growth factors (TGF-beta , PDGF, bFGF, EGF), oncogenes, kinases, changes in intracellular calcium, and mechanical stress (31). IGF-I is produced by cultured metanephroi (56), and blocking its expression prevents growth and development (56, 64). The renal growth-promoting actions of IGF-I are well described (33) and involve direct actions on both metanephroi development (39), glomerular size, and vascularization (13), predominantly mediated via the IGF-IR and regulated by the IGF binding proteins (14, 30). The present study shows proliferative actions of neonatal IGF-I treatment on the renal IM, which increased the urinary concentrating capacity even above control levels in adult animals. Confirming the fundamental role of the papilla in the urinary concentrating mechanism, the present data showed significant correlation between the fractional volume of renal IM and Uosmmax. The interaction between RAS and the GH/IGF-I axis has, however, not been exclusively investigated. Mature settings have demonstrated enhanced ovarian ANG II production and follicular development by IGF-I (69), beneficial effects by GH and IGF-I in chronic ACE inhibitor-treated experimental heart failure (34), an activated RAS by exogenous administration of GH in Lewis dwarf rats (68), whereas GH therapy in children does not affect RAS activity (6). Furthermore, in vitro evidence shows increased expression of IGF-I in tubular cells by ANG II (67).

Intensive research is ongoing as to the intracellular mechanisms mediating the physiological responses to ANG II and IGF-I. The AT1 receptor is a G protein-coupled receptor and mediates its effects by tyrosine phosphorylation and activation of phospholipase C, resulting in increasing levels of D-inositol 1,4,5-tris-phosphate and diacylglycerol, a cascade that increases the intracellular Ca2+ concentrations (5). Stimulation of the IGF-IR increases the tyrosine phosphorylation of several substrates, including insulin receptor substrate 1 (IRS-1) and tyrosine phosphatase (PTP-1D) (1). The IRS-1 is one of the main substrates for the IGF-IR, and it activates several downstream proteins to promote growth and glucose metabolism (2) and to stimulate the induction of, e.g., c-fos (9). Ali et al. (1) have demonstrated an induction and sustained increase in the phosphorylation of IRS-1 and PTP-1D by IGF-I and, moreover, that IRS-1 and PTP-1D were induced by ANG II however only transiently. These findings indicate that there can be convergent intracellular signaling by G protein-coupled and growth factor receptors, e.g., the AT1 and IGF-I receptors.

The present study demonstrates that enalapril directly or indirectly affects the renal expression of IGF-I, IGF-IR, and GHR during ongoing neonatal treatment. The finding that neonatal RAS blockade influences the IGF-I expression, both at the mRNA and protein level in association with the development of an abnormal renal morphology, strongly suggests that ANG II interacts with or signals via the IGF-I/IGF-IR during postnatal renal growth and differentiation. Paracrine secretion of IGF-I is regulated via GHR, and although GHR mRNA levels were unaltered by enalapril treatment, our immunocytochemical data demonstrated a more wide and dense expression of GHR in the medullary interstitium. Discordant findings between mRNA expression patterns and local protein distribution may indicate a differentiated breakdown or translation of the protein, or capture by binding proteins, that in turn feedback altered mRNA expression. Consequently, neonatal RAS blockade resulted in disturbances in at least two parts of the paracrine renal medullary GH/IGF-I system. The more exact molecular link between RAS blockade and alterations in the GH/IGF-I axis remains to be elucidated.

Besides the renal morphological and functional benefits of combined IGF-I treatment in the present study, the somatic growth retardation produced by sole ACE inhibition was partly reversed, although not completely normalized. One can speculate that the renal protection by IGF-I in the combined treatment group may account for the benefits in somatic growth, but it may also be attributed to increased somatic growth by IGF-I per se, as previously reported (7, 45). Sole IGF-I treatment in the present study demonstrated, however, similar body growth curves as control rats. Supporting the notion that IGF-I is involved in the induced growth retardation by neonatal ACE inhibition, mice deficient in IGF-I showed a similar onset in body growth attenuation from 15 to 16 days of age (4).

Neonatal RAS blockade may also result in an altered balance of apoptotic and proliferative signaling. We and others (46, 61) have demonstrated increased medullary apoptosis during neonatal ACE inhibition and decreased medullary cell proliferation by means of positive prolife-rating cell nuclear antigen staining. Correspondingly, IGF-I is known to counterbalance programmed cell death (8) and increase cell proliferation (58).

One may envisage that the renal abnormalities produced by neonatal ACE inhibition may be a consequence of decreased oxygen tension in the particularly vulnerable renal IM. Indeed, acute intraperitoneal administration of enalapril, at a dose of 10 mg/kg, produced a persistent 30% reduction in MAP over at least 5 h in the anesthetized 22-day-old rat (28). However, acute administration of an equipotent hypotensive dose of enalaprilat in the anesthetized piglet did not produce any reductions in local renal blood flows or medullary oxygen tension (Nilsson and Friberg, unpublished data), indicating that the ACE inhibitor-induced papillary atrophy is not provoked by renal hemodynamic alterations. Moreover, treatment with the calcium antagonist nifedipine from 3 to 24 days of age did not produce any long-term alterations in renal function and histology, despite pronounced MAP reduction (28) and substantial renal hypoperfusion and medullary hypoxia (Nilsson and Friberg, unpublished data). Collectively, these data lend strong support for the idea of ANG II as having an important role for neonatal renal growth, either directly or via the GH/IGF-I axis.

In conclusion, the present study provides evidence of a protective role for IGF-I in the development of normal renal morphology and function when concomitantly administered during ongoing neonatal ACE inhibition. Plausible explanations for these beneficial effects may include a normalization of the ACE inhibitor-induced disturbance in the GH/IGF-I system with increased apoptotic signaling and/or decreased cell proliferation. Thus exogenous IGF-I can compensate for the absence of AT1-receptor stimulation during renal growth and development. One can speculate that in the physiological situation, AT1-receptor stimulation results in IGF-I-induced cell proliferation and/or inhibition of apoptosis.

Perspectives

Supplement treatment with IGF-I reverses or prevents the induced renal abnormalities by neonatal ACE inhibition, indicating that the postnatal RAS and the GH/IGF-I axis are interacting in renal development. The present study reveals that neonatal ACE inhibition affects the renal GH/IGF-I axis and demonstrates that exogenous administration of IGF-I can compensate for the reduced activation of RAS. These findings may be of clinical importance when ACE inhibitors or AT1-receptor antagonists are used in the newborn. Accordingly, plausible treatment with ACE inhibitors have been discussed in diabetic nephropathy in view of the causal role of the GH/IGF-I axis (19, 20). The interaction between AT1-receptor signaling and the GH/IGF-I axis in nephrogenesis needs, however, further investigation for unravelling of the exact mechanisms.


    ACKNOWLEDGEMENTS

The authors acknowledge Dr. Birgitta Sundelin at Karolinska Hospital in Sweden for histological preparations.


    FOOTNOTES

This study was supported by the Swedish Medical Research Council (no. 9047, 11133) and Astra Hössle AB (Mölndal, Sweden). The authors thank Merck, Sharp and Dohme (Sollentuna, Sweden) for providing enalapril maleate and Pharmacia-UpJohn (Stockholm, Sweden) for providing human recombinant insulin-like growth factor-1.

Address for reprint requests and other correspondence: A. Nilsson, Dept. of Physiology, Institute of Physiology and Pharmacology, Göteborg Univ., Medicinaregatan 11, S-413 90 Göteborg, Sweden (E-mail: annika.nilsson{at}fysiologi.gu.se).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 25 October 1999; accepted in final form 7 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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