Neonatal Angiotensin-Converting Enzyme Inhibition in the Rat Induces Persistent Abnormalities in Renal Function and Histology
Recently, we reported that neonatal blockade of the renin-angiotensin system in the rat produces irreversible abnormalities in renal histology associated with increased diuresis. In the present study, we assessed the long-term consequences of neonatal angiotensin-converting enzyme inhibition on renal function. Rats were injected with 10 mg·kg−1·d−1 enalapril or vehicle from day 3 to day 24 after birth. Urine concentrating ability, renal function, and renal histology were assessed in 16-week-old rats. There was a twofold increase in diuresis and water intake in enalapril-treated rats throughout the study course. Urine osmolality after 24 hours of water deprivation was 1008±108 and 2549±48 mOsm·kg−1 (P<.05) in enalapril- and vehicle-treated rats, respectively. Glomerular filtration rate (0.54±0.03 versus 0.75±0.06 mL·min−1·100 g body wt−1, P<.05) and effective renal plasma flow (1.76±0.09 versus 2.19±0.14 mL·min−1·100 g body wt−1, P<.05) were reduced in neonatally enalapril-treated versus control rats. Absolute and fractional urinary sodium excretion values were elevated (P<.05) in enalapril-treated rats. Semiquantitative assessment of renal histology demonstrated statistically significant degrees of papillary atrophy, interstitial fibrosis and inflammation, tubular atrophy and dilatation, and focal glomerulosclerosis in neonatally enalapril-treated rats. In conclusion, neonatal angiotensin-converting enzyme inhibition in the rat produces irreversible alterations in renal function and morphology, demonstrating the importance of an intact renin-angiotensin system neonatally for normal renal development.
A number of studies have demonstrated that treatment of the spontaneously hypertensive rat with ACE inhibitors initiated after weaning during the developmental phase of hypertension produces a persistent reduction in BP that is evident long after therapy is stopped.1 2 3 However, these interventions did not completely normalize BP. Accordingly, we performed a study in which we attempted to fully normalize long-term BP by administering ACE inhibitors from an even earlier age.4 Unexpectedly, we found that the spontaneously hypertensive rat, as well as its normotensive counterpart the Wistar-Kyoto rat, treated with an ACE inhibitor from 3 to 24 days after birth developed a persistent increase in diuresis and water consumption after weaning. Furthermore, these rats showed pronounced changes in renal histology.
A subsequent study5 in different rat strains revealed that these abnormalities in fluid handling and renal histology, mainly characterized by papillary atrophy and chronic tubulointerstitial inflammation, developed after neonatal blockade of the RAS with different ACE inhibitors or the AT1 receptor antagonist losartan. In addition, this effect was dose dependent. These findings indicated that activation of AT1 receptors during the first postnatal weeks is crucial in the normal development of the rat kidney, results that recently have been confirmed by others.6
Although the RAS has been well characterized as a regulator of circulatory homeostasis and long-term BP control in adult animals,7 its role during the fetal and perinatal periods is less clear. However, an important role for the RAS during development has been suggested as the system is activated during late fetal and early postnatal life.8 It is now well recognized that all components of the RAS are expressed in the fetal kidney and developmentally regulated in a tissue-specific manner.9 The renin,10 11 12 13 ACE,12 13 14 15 and AT1 receptor16 genes show increased expression in the kidney during neonatal maturation compared with levels in the adult rat kidney. In addition, kidney Ang II levels are high immediately after birth and decline with aging.13 These results indicate that intrarenally formed Ang II may be important in the regulation of renal hemodynamics, tubular function or growth, and differentiation in the developing mammalian kidney.
It remains to be elucidated whether, and to what extent, a lack of neonatal Ang II stimulation in the kidney and its associated abnormalities in kidney histology impair long-term renal function. Therefore, we assessed renal function in adult rats subjected to enalapril treatment from 3 to 24 days of age.
Twenty-nine male Wistar rats (Möllegaard Breeding Center Ltd, Ejby, Denmark) were used. Pregnant rats were carefully observed near end-gestation for determination of the exact date of birth. Sex was determined in 2-day-old pups, and litters containing only males were transported to our facility where they were randomized into two groups: one group received daily intraperitoneal injections of 10 mg·kg−1 enalapril maleate (Merck, Sharp & Dohme) (n=17), and the other group received saline vehicle (n=12) in equivalent volumes (10 mL·kg−1) from 3 to 24 days of age. No deaths occurred during the study. Pups were weighed daily during the treatment period so that correct dosing could be determined. After treatment was stopped, 11 enalapril- and 11 saline-treated rats were followed weekly in metabolic cages for measurements of water intake and urine production. Water deprivation tests and assessment of renal function in anesthetized rats were performed on all (n=17 and n=12) adult rats at 15 to 16 weeks of age. Rats had free access to normal rat chow (120 mmol·kg−1 NaCl, 80 mmol·kg−1 KCl) and water throughout the study and were kept in rooms with a controlled temperature of 24°C and a 12-hour dark/light cycle. All experiments were approved by the regional ethics committee in Göteborg.
Fluid handling was assessed with the use of metabolic cages at six different time points (ages 7, 9, 11, 13, 15, and 16 weeks) off neonatal intervention. Rats were kept in metabolic cages for 3 days with measurements performed during steady-state conditions on day 3. Water intake and urine volume (milliliters per kilogram per day) were measured, and urinary sodium, potassium, and osmolality were analyzed in collected urine. In addition, water intake was measured weekly in rats kept individually in normal cages.
Urine concentrating ability was assessed by water deprivation in adult rats at 15 to 16 weeks of age, ie, 12 weeks off neonatal treatment. After 3 days in metabolic cages, rats were deprived of water, and osmolality was measured in urine collected between 12 and 24 hours after initiation of water deprivation. Sodium was measured with a flame spectrophotometer (model FLM, Radiometer) and osmolality by the method of freezing point depression (Wide Range Advanced Osmometer model 3MO, Advanced Instruments Inc).
Assessment of Renal Function in Anesthetized Rats
Experiments were performed on 16-week-old rats weighing approximately 370 to 400 g, ie, 13 weeks off neonatal intervention. GFR and ERPF were estimated by clearance of 51Cr-EDTA (100 μCi·mL−1, Amersham Laboratories) and 125I-hippuran (108 μCi·mL−1, Institutt før Energiteknikk), respectively. Rats were initially anesthetized with methohexital (75 mg·kg−1 IP) and tracheotomized with a polyethylene catheter (PE-240). Rats were kept on spontaneous breathing, and body temperature was maintained at 38°C throughout the experiment. The left jugular vein and tail artery were cannulated with PE-50 tubing. The urinary bladder was cannulated through a midline abdominal incision by a PE-160 catheter with a collar inside the bladder to ensure correct positioning. Anesthesia was maintained by an intravenous bolus of chloralose (0.5 mL, 14 mg·kg−1) followed by continuous infusion (35 mg·kg−1·h−1, 6 mL·kg−1·h−1). Chloralose was dissolved in a buffer solution of the following composition (mmol/L): glucose 92, NaHCO3 13, NaCl 62, and Na2B4O7·H2O (borax) 26. After completion of surgery, an intravenous bolus containing 51Cr-EDTA (35 μCi·kg−1) and 125I-hippuran (15 μCi·kg−1) in 0.5 mL saline was administered, followed by a continuous intravenous infusion of 100 μCi 51Cr-EDTA·kg−1·h−1 and 55 μCi 125I-hippuran·kg−1·h−1 in a volume of 9 mL·kg−1·h−1.
After a 45-minute equilibration period, two consecutive 30-minute clearance periods were performed. Results are presented as mean values of the two clearance periods. Urine was collected in preweighed vials, and urine volume and radioactivity were determined. Urine density was assumed to be 1.000 g·mL−1 urine. Arterial blood samples (0.5 to 0.8 mL) were obtained at the midpoint of each urine sampling period, and plasma was analyzed for radioactivity. From each blood sample, erythrocytes were resuspended in a 4% albumin/saline solution and transfused back into the rat. Sodium concentrations in plasma and urine were estimated in nine neonatally enalapril- and eight vehicle-treated rats. Mean arterial pressure (MAP) and heart rate were recorded continuously with Statham pressure transducers connected to a Grass polygraph. Filtration fraction (percent) was calculated as (GFR/ERPF)·100. Renal vascular resistance was calculated as MAP/ERPF and expressed in millimeters of mercury per milliliter per minute per 100 g body weight.
After completion of the experiment, kidneys were removed, decapsulated, rapidly weighed, and immersion-fixed. Radioactivity in plasma and urine was measured in a three-channel scintillation counter (model 5019, Packard Co).
Kidneys were immersion-fixed with 4% formaldehyde in phosphate-buffered saline, pH 7.4, embedded in paraffin, and sliced in 3-μm-thick coronal sections. One section from each kidney was stained with hematoxylin and eosin and one with Ladewig's trichrome for examination by light microscopy. Assessments were made by an investigator blind to treatment group. Histological changes were scored semiquantitatively with the use of an arbitrary scale: 0=normal, 1=mild changes, 2=moderate changes, and 3=severe changes. For papillary atrophy scores, a score of 3 corresponded to a complete loss of the papilla, and scores 2 and 1 indicated an atrophy of approximately two thirds and one third of the papillary parenchyma, respectively.
Values are expressed as mean±SE. Body weights and fluid handling data were analyzed with ANOVA for repeated measurements. Cross-sectional data at 15 to 16 weeks of age were analyzed with unpaired two-tailed Student's t test. The Wilcoxon rank sum test was used on renal histopathologic parameters. A value of P<.05 was considered statistically significant.
Body and Kidney Weights
Neonatal enalapril treatment inhibited somatic growth during the treatment interval. An attenuation in body weight gain became evident after 2 weeks of treatment, resulting in an 18% reduction (47±1 versus 58±1 g, P<.05) in body weight on the last day of treatment, 24 days after birth. However, body weights in enalapril-treated rats returned to control levels from 7 weeks of age onward. Body weights in 16-week-old rats subjected to acute assessment of renal function were 380±11 and 390±10 g in enalapril- and vehicle-treated rats, respectively. Absolute (2.27±0.07 versus 2.24±0.10 g, enalapril- versus vehicle-treated rats) and fractional (6.09±0.17 versus 5.82±0.31 g·kg·body wt−1) wet kidney weights did not differ between groups.
Neonatally enalapril-treated rats showed a persistent, approximately twofold increase in diuresis (P<.05) (Fig 1⇓, top) and water intake (P<.05) (Fig 1⇓, bottom) throughout the study. Enalapril-treated rats had a significant increase in urinary sodium excretion when measured repeatedly in metabolic cages (mean of six repeated measurements: 1.55±0.08 versus 1.24±0.05 mmol·d−1, enalapril- versus vehicle-treated rats; P<.05). Urinary potassium excretion did not differ between the groups.
Urine Concentrating Ability
Enalapril-treated rats with free access to water showed a clear reduction in baseline urine osmolality (518±53 versus 1236±114 mOsm·kg−1, P<.05) measured under steady-state conditions in metabolic cages (Fig 2⇓). This difference between groups persisted after 24 hours of water deprivation (1008±108 versus 2549±48 mOsm·kg−1, P<.05) (Fig 2⇓). Both groups were able to respond to water deprivation and did so, in relative terms, to the same extent (increases in urine osmolality, 93±12% and 124±21%, enalapril- and vehicle-treated rats, respectively).
Renal Function in Anesthetized Rats
Neonatally enalapril-treated rats showed a 28% reduction in GFR (P<.05) and a 20% reduction in ERPF (P<.05) (Table 1⇓ and Fig 3⇓). Absolute values for GFR were 1.98±0.12 and 2.86±0.16 mL·min−1 (P<.05) and for ERPF were 6.69±0.42 and 8.53±0.54 mL·min−1 (P<.05) in enalapril- and vehicle-treated rats, respectively. In relative terms, the reduction in GFR was more pronounced than the reduction in ERPF in enalapril-treated rats, resulting in a decreased filtration fraction (Table 1⇓). In accordance with data obtained in metabolic cages, neonatally enalapril-treated rats had an almost twofold increase in diuresis (Table 1⇓). This difference was exaggerated further when related to changes in GFR (urine volume/GFR, 2.26±0.37% and 0.85±0.25%, P<.05, in neonatally enalapril- and vehicle-treated rats, respectively).
Mean arterial pressure, heart rate, and renal vascular resistance did not differ between the groups (Table 1⇑). Both absolute and fractional urinary sodium excretion values were elevated in enalapril-treated rats (Table 1⇑).
Kidneys from neonatally enalapril-treated rats showed gross morphological changes that were evident macroscopically. The kidney surface appeared granulated and irregular compared with the smooth surface of control kidneys. Semiquantitative analysis demonstrated statistically significant degrees of papillary atrophy, interstitial chronic inflammation and fibrosis, tubular atrophy and dilatation, and focal glomerulosclerosis in enalapril-treated rats (P<.05; Table 2⇓, Fig 4⇓). In addition, interlobular arteries appeared abnormal, with thickened walls, observations that were made in all enalapril-treated rats (Fig 4⇓). The interstitial inflammatory changes consisted of focal infiltrates of predominantly mononuclear inflammatory cells. The degree of interstitial inflammation and fibrosis did not differ significantly between the cortex and medulla following semiquantification. Atrophic tubules with flattened tubular epithelium and dilatation were present in all enalapril-treated rats and localized predominantly to areas of focal interstitial fibrosis. The papillary atrophy score of 2.1±0.2 (arbitrary units) in enalapril-treated rats corresponded to an approximate atrophy of two thirds of the papillary parenchyma. Glomeruli of enalapril-treated rats showed mild degrees of focal segmental glomerulosclerosis and synechial adhesions to Bowman's capsule. Affected glomeruli were mainly localized to areas of extensive interstitial fibrosis and inflammation.
The major findings in the present study are that neonatal inhibition of the RAS provokes persistent abnormalities in renal function characterized by (1) an impaired urine concentrating ability associated with a marked increase in diuresis and polydipsia and (2) a reduction in GFR and ERPF. In addition, neonatally enalapril-treated rats demonstrated increased urinary sodium excretion. Confirming previous results,5 neonatal ACE inhibition induced irreversible abnormalities in renal morphology, mainly consisting of papillary atrophy, chronic interstitial inflammation, interstitial fibrosis, and tubular atrophy. Taken together, these findings demonstrate that inhibition of the RAS for a brief period of time postnatally induces irreversible abnormalities in renal morphology and function, suggesting an important role for the RAS in normal renal development.
With the use of gene targeting technology, homozygous mice deficient in angiotensinogen17 18 and ACE19 20 developed alterations in renal histology similar to those reported in the present study: tubular dilatation, interstitial inflammation and fibrosis, and thickened vascular walls of interlobular arteries. Niimura et al18 also reported the presence of papillary atrophy in mice deficient in angiotensinogen. Carpenter et al20 described, in accordance with the present study, that the most notable abnormality in ACE mutant mice was a defect in urine concentrating ability associated with an anatomic disorganization of the renal medulla. Thus, gene knockout experiments, in agreement with our findings, prove the fundamental importance of the RAS in renal development. In addition, AT1 receptor antagonism during late gestation21 and neonatally during the first 2 weeks after birth6 in the rat produced renal histological abnormalities resembling those reported here. However, no studies have investigated the long-term consequences of neonatal blockade of the RAS on renal function in relation to the observed abnormalities in kidney morphology.
In the present study, adult neonatally enalapril-treated rats showed a decreased urine concentrating ability as a consequence of severe histopathologic changes in the renal inner medulla, characterized by a pronounced papillary atrophy. Evidently, this would impair the efficiency of the countercurrent multiplication system and reduce urine concentrating ability. In agreement with a defect in urine concentrating ability of renal origin, enalapril-treated rats responded to water deprivation with a twofold increase in urine osmolality, demonstrating intact endogenous arginine vasopressin release. In addition, we have shown that neonatally enalapril-treated rats are unable to improve their urine concentrating ability after administration of desamino-vasopressin (G.G., unpublished data, 1996). Considering the marked papillary atrophy, involving approximately two thirds of papillary parenchyma, we speculate that the increase in renal sodium excretion in anesthetized enalapril-treated rats was due to a reduced sodium reabsorption in the medullary collecting duct, where urinary sodium excretion is finally adjusted.22 In agreement with data obtained in anesthetized rats, urinary sodium excretion was significantly elevated in enalapril-treated rats when measured repeatedly in metabolic cages. However, in these experiments, daily sodium intake was not measured; therefore, net sodium balance could not be estimated. The contribution of different nephron segments in the altered renal sodium handling of neonatally enalapril-treated rats needs to be further investigated.
During neonatal enalapril administration, rats developed a transient reduction in body weight, which returned to control levels at the age of 7 weeks. This finding is in accordance with previous studies in which blockade of the RAS was induced in the late gestation fetal,21 neonatal,5 6 23 or weanling24 rat. The attenuated gain in body weight may be due to an altered sodium balance during a phase of rapid somatic growth.
It is possible that a reduction in systemic BP and renal hypoperfusion during neonatal enalapril treatment could compromise medullary blood flow, leading to severe hypoxia and papillary atrophy. However, neonatal administration of the vasodilators hydralazine6 or nifedipine (G.G., unpublished data, 1996) has not been associated with abnormalities in kidney histology or function. Furthermore, systemic BP in weanling, losartan-treated rats remained within the range for autoregulation of renal blood flow.24 Still, medullary hypoperfusion and ischemia during neonatal enalapril administration cannot be ruled out as a cause for papillary atrophy in the present study. Interestingly, Niimura et al18 suggested that Ang II may be involved in the maturational growth of the renal papilla. Platelet-derived growth factor-A mRNA was highly expressed in the papilla of control animals and absent in angiotensinogen-deficient transgenic mice developing papillary atrophy.
In the present study, renal interlobular arteries of adult rats treated neonatally with enalapril appeared to have thickened walls, resembling vascular lesions associated with long-standing hypertension, although BP levels were normal. Similar vascular changes, characterized by medial hyperplasia in interlobular arteries and afferent arterioles, have been reported in rats6 21 and mice17 18 19 20 with an inhibited RAS during development. Some investigators concluded that the medial hyperplasia was partially due to a recruitment of renin-producing cells along the renal vasculature as a consequence of an absent feedback inhibition on renin production.6 18 21 However, Niimura et al18 also showed a pronounced proliferation of renin-negative cells in thickened walls of interlobular arteries in angiotensinogen-deficient mice. Thus, the medial hyperplasia in interlobular arteries, developing after blockade of the RAS in immature animals, is not entirely attributed to a recruitment of renin-producing cells and needs further exploration.
An important role for Ang II in neonatal renal vascular development was recently described in the rat.6 Neonatal administration of losartan from birth to 14 days of age resulted in fewer, thicker, and shorter afferent arterioles and reduced glomerular size and numbers.6 These changes would inevitably reduce glomerular filtration surface area and renal blood flow and could provide an explanation for the reduction in GFR and ERPF demonstrated in the present study. However, no detailed morphometric analysis of glomerular changes was performed in the present study, although semiquantitative analysis revealed focal segmental glomerulosclerosis in neonatally enalapril-treated rats. Consistent with a reduction in GFR, enalapril-treated rats showed focal areas of tubular atrophy and interstitial fibrosis, indicative of a reduced or absent glomerular filtration in these nephrons. A reduction in renal hippuran extraction in enalapril-treated rats could be misinterpreted as a reduction in renal plasma flow. However, measurements of hippuran extraction in a separate group of neonatally enalapril- and vehicle-treated rats did not show any difference between groups (G.G., unpublished data, 1996).
In agreement with findings in the present study, there is now clear evidence for a role of Ang II in neonatal renal growth. AT1 receptor antagonism in the neonatal6 25 and weanling24 rat inhibited renal growth, as indicated by reduced kidney weight, lower kidney DNA content, and decreased proliferating cell number.
In conclusion, neonatal ACE inhibition with enalapril from 3 to 24 days after birth induced a decrease in urine concentrating ability and reduction in GFR and renal blood flow in the adult rat long after cessation of treatment. These rats also developed renal histological abnormalities mainly consisting of papillary atrophy, interstitial inflammation and fibrosis, and tubular atrophy. Thus, normal renal development does strongly depend on an intact RAS during the first 3 weeks after birth.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|AT1||=||angiotensin type 1 (receptor)|
|ERPF||=||effective renal plasma flow|
|GFR||=||glomerular filtration rate|
This study was supported by the Göteborg Medical Society, the Swedish Medical Society, and the Swedish Medical Research Council (9047, 11133). M.A. Adams is funded by a career award in Medicine jointly given by the Health Research Foundation of the Pharmaceutical Manufacturers Association of Canada and the Medical Research Council of Canada. The authors appreciate the skillful technical and photographic assistance of Anna Wickman, Anders Eliasson, Beatrice Sandvik, Thi Hanh Luu, and Ingeborg May. The authors also thank Merck, Sharp & Dohme, Sollentuna, Sweden, for generously providing enalapril.
- Received May 27, 1996.
- Revision received June 17, 1996.
- Revision received July 18, 1996.
Adams MA, Bobik A, Korner PI. Enalapril can prevent vascular amplifier development in spontaneously hypertensive rats. Hypertension. 1990;16:252-260.
Korner PI, Bobik A. Cardiovascular development after enalapril in spontaneously hypertensive and Wistar-Kyoto rats. Hypertension. 1995;25:610-619.
Harrap SB, Van der Merwe WM, Griffin SA, Macpherson F, Lever AF. Brief angiotensin converting enzyme inhibitor treatment in young spontaneously hypertensive rats reduces blood pressure long-term. Hypertension. 1990;16:603-614.
Friberg P, Adams MA, Bobik A, Bohman SO, Gustavsson H, Nilsson H, Petersen J. ACE-inhibition in newborn rats causes kidney damage. J Hypertens. 1990;8(suppl 3):S5. Abstract.
Yosipiv IV, El-Dahr SS. Activation of angiotensin-generating systems in the developing rat kidney. Hypertension. 1996;27:281-286.
Kim H, Krege JH, Kluckman KD, Hagaman JR, Hodgin JB, Best CF, Jennette JC, Coffman TM, Maeda N, Smithies O. Genetic control of blood pressure and the angiotensinogen locus. Proc Natl Acad Sci U S A. 1995;92:2735-2739.
Niimura F, Labosky PA, Kakuchi J, Okubo S, Yoshida H, Oikawa T, Ichiki T, Naftilan AJ, Fogo A, Inagami T, Hogan BLM, Ichikawa I. Gene targeting in mice reveals a requirement for angiotensin in the development and maintenance of kidney morphology and growth factor regulation. J Clin Invest. 1995;96:2947-2954.
Diezi J, Michoud P, Aceves J, Giebisch G. Micropuncture study of electrolyte transport across papillary collecting duct of the rat. Am J Physiol. 1973;224:623-634.