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Expression of Renin and Its mRNA in the Adult Rat Kidney With Chronic Ureteral Obstruction
Expression of Renin and Its mRNA in the Adult Rat Kidney With Chronic Ureteral Obstruction Samir Sayem EI-Dahr, MD, R. Ariel Gomez, MD, Gopal Khare, Michael J. Peach, PhD, Robert M. Carey, MD, and Robert L. Chevalier, MD • Angiotensin II has been implicated in mediating renal vasoconstriction resulting from chronic unilateral ureteral obstruction (UUO) in both mature and developing animals. We have previously shown that chronic neonatal UUO results in increased distribution of renin and its mRNA in the obstructed kidney, as well as of immunoreactive renin in the intact opposite kidney. The present study was designed to evaluate the effects of 24 hours versus 4 weeks of UUO on the distribution of renin mRNA and its protein in the adult rat kidney. Renin was detected by immunocytochemistry using a polyclonal anti-rat renin antibody. Renin mRNA was localized by in situ hybridization to an oligonucleotide complementary to renin mRNA. UUO of 24 hours' or 4 weeks' duration did not alter the distribution of renin and its mRNA in the obstructed kidneys as compared with sham-operated kidneys, although kidneys obstructed for 4 weeks had a significant increase in the percent of renin-containing juxtaglomerular apparatuses (JCA) when compared with the intact opposite kidneys (P < 0.05). Compensatory hypertrophy was not present in the intact opposite kidneys after 24 hours of UUO and distribution of renin gene expression was not altered at that time. However, 4 weeks following contralateral UUO, the intact kidneys were hypertrophied and showed a decrease in renin gene expression relative to the obstructed and sham-operated kidneys. We conclude that unlike UUO during early development, chronic UUO in the mature animal does not activate renin gene expression nor alter renin distribution in the obstructed kidneys. Renin gene expression is suppressed in the hypertrophied kidney with prolonged contralateral UUO. The response of intrarenal renin to chronic UUO appears to be 8gedependent, with the mature animal having an attenuated response compared with that of the developing animal. © 1990 by the National Kidney Foundation, Inc. INDEX WORDS: Ureteral obstruction, chronic; Sprague-Dawley rat; renin; immunocytochemistry; mRNA; in situ hybridization.
I
NCREASED ACTIVITY of the renin-angiotensin system (RAS) has been shown to play a role in renal vasoconstriction resulting from chronic unilateral ureteral obstruction (UUO) in both mature and developing animals; angiotensinconverting enzyme (ACE) inhibitors significantly improve the hemodynamics of chronically obstructed kidneys I 5 and single nephrons. 6 However, it is not clear whether the intrarenal RAS is activated in the ipsilateral kidney with chronic UUO. In one study, renin depletion did not attenuate the increased renal vascular resistance resulting from 24 hours of UUO in the adult rat. 7 Furthermore, the distribution of immunoreactive renin was not altered in chronically hydronephrotic adult mouse kidneys.8 Equally important is the response of intrarenal renin in the intact hypertrophied kidney with contralateral UUO in which renal blood flow is typically increased in response to chronic UUO.I-3.9 Recent molecular studies have demonstrated that the expression of intrarenal renin and its mRNA is developmentally regulated. Unlike the newborn kidney, where renin synthesis takes place along most of the length of afferent arteriole, in the adult
kidney renin is synthetized and stored in the juxtaglomerular cells only. \0-\2 Using immunocytochemical and in situ hybridization techniques, we have shown that l-month-old rats subjected to UUO at day 2 of life manifest a significant increase in renin content and increased distribution of microvascular renin and its mRNA in the obstructed kidneys as compared with sham-operated controls. \3 Of note, the distribution of immunoreactive renin was also found to be increased in the intact opposite kidney. \3 The effects of chronic UUO on the intrarenal distribution of renin gene expression in the adult rat are unknown. Due to the From the Departments of Pediatrics, Pharmacology, and Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, I'lt. Supported by National Institute of Health Grant No. DK 40558 (R.L.C.). R.A.G. is supported by NIH Grant No. HL 41889. Dr S. S. El-Dahr is a research fellow of the American Heart Association, Virginia Affiliate. Address reprint requests to Robert L. Chevalier, MD, University of Virginia, Health Sciences Center, Department of Pediatrics, MR4/2034, Charlottesville, I'lt 22908. © 1990 by the National Kidney Foundation, Inc. 0272-6386/90/1506-0009$3.00/0
American Journal of Kidney Diseases, Vol XV, No 6 (June), 1990: pp 575-582
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EL-DAHR ET AL
inherent differences in the activity of the intrarenal RAS between newborn and adult animals, the response of intrarenal renin to UUO in the adult should be less than that observed in the developing animal. '3 The present study was performed in adult rats and was designed to compare the effects of 24 hours versus 4 weeks of UUO on the distribution of renin mRNA and its protein along the microvasculature of the obstructed kidneys, as well as of the intact opposite kidneys . MATERIALS AND METHODS Ten adult Sprague-Dawley rats (200 to 250 g, approximately 10 weeks of age) were subjected to either sham-operation or UUO and were studied 24 hours later (n = 5 in each group). Additional rats were subjected to either sham-operation or UUO and were studied 4 weeks later (n = 6 in each group). Following anesthesia with intraperitoneal pentobarbital sodium (50 mg/kg) (Abbott Laboratories , Chicago, IL), a small incision was made in the left lower quadrant of the abdomen and Ihe distal ureler was ligaled (VVO) or left undisturbed (sham). 13 The abdominal wall was then closed in two layers and the animal was allowed to recover from anesthesia. Subsequently, the animals were housed in separate cages until the time of the study, and they were fed regular rat chow (Purina 5012, Ralston-Purina, St Louis, MO) and were allowed free access to drinking water. At the time of the study, after an overnight fast but free access to water, the animals were anesthetized as described above and mean arterial pressure (MAP) was continuously recorded via a right carotid catheter by means of a Statham 23-10 pressure transducer (Englewood, CO) coupled to a Hewlett-Packard 7754 B recorder (Gould Inc, Medical Products Division, Oxhard, CAl. After a IO-rninute equilibration period, I mL of blood was withdrawn from the carotid catheter and collected in chilled EDTA-containing tubes. Blood samples were immediately centrifuged at 4°C for IS minutes and plasma kept at - 70°C for measurement of plasma renin activity according to the method of Sealy and Laragh. 14 The left ureteral diameter was measured with calipers in all rats midway between the renal pelvis and the bladder. The rats were then killed with saturated potassium chloride solution injected through the carotid catheter. The kidneys were removed, decapsulated, weighed, and processed for immunocytochemistry and in silu hybridization histochemistry as described below.
Renin Immunocytochemistry Immunocytochemical localization of renin was performed using the ABC immunoperoxidase technique as described previously. 10 13 The primary antibody is a specific polyclonal antiral renin antibody (diluted 112,500, gift of Dr T. Inagami, Department of Biochemistry, Vanderbilt University, Nashville, TN). As negative controls, the primary antiserum was replaced by nonimmune rabbit serum, and no staining occurred. An average of two to three sections per kidney (7 -I'm thick) were examined by light microscopy. The number of reninstained and the total number of juxtaglomerular apparatuses (1GA) were counted in each section. With the aid of an ocular
micrometer, the total length of the afferent arteriolar segments (AA) and the length of renin immunostaining along the AA were measured. All measurements were performed on coded slides such that the investigators were not aware of the treatment groups. Since the total number of JGA varied among sections, the foll owing immunohistochemical ratios were determined and compared among groups as previously described 10 \3: %JGA = (the number of stained JGA/total number of JGA) x 100; %LAA = (length of stained AA/total length of AA) x 100. The ratios thus obtained from each slide were pooled and averaged for each kidney. Only JGA with a vas afferens entering the glomerulus were used for computation. 10· \3
Hybridization Histochemistry Kidneys from four rats in each group were processed for in situ hybridization as previously described." ·I3·" Briefly, after fixation in 4 % paraformaldehyde in 0.12 mollL sodiumphosphate (pH 7.4 for I hour) and overnight cryprotection in 30% sucrose in 0 . 12 mollL sodium phosphate (pH 7.4), kidney sections (IO-I'm) were cut in a cryostat (Histostat Microtome, American Optical, Buffalo, NY) and mounted immediately on acid-cleaned gelatin-coated slides. Subsequently, 200 I'L of prehybridization buffer" ·13 was placed on each section and the slides were incubated at 37 °C for I hour. The sections were then hybridized at 37 °C for 24 hours to an oligonucleotide complementary to nucleotides 313-340 of rat renin mRNA.II.13 The oligonucleotide was 3 '-end-labeled with a-thio-[35S]-dATP to a total specific activity of 2 x 107 cpm/pmol. Equal counts of HS-labeled sense oligonucleotide were added to some kidney sections as a control for the specificity of the hybridization signals. In addition, a 200-fold molar excess of unlabeled oligonucleotide was added to some sections as a background control. Following hybridization, the kidney sections were washed in decreasing concentrations of SSPE (I x SSPE is 0.15 mollL NaCI, 0 .01 mol/L sodium phosphate, 0 .001 mollL EDTA, pH 7.4) as described previouslyll .13 with the last wash as the high stringency wash in 0.1 x SSPE with 0 .014 mollL 2-mercaptoethanol and I % sodium thiosulfate (20 minutes, 53°C). Following washes, the sections were postfixed in 0.05 mollL TRIS-saline-azide followed by 4 % paraformaldehyde in 0.12 mol/L sodium phosphate, pH 7.4. After dehydration, the sections were submitted to contact and emulsion autoradiography (NTB2, Kodak, Rochester, NY). Developing time was 14 days in all groups. An average of two to three sections per kidney was used for computations. In each section, the number of hybridization signals (grains) was counted in each juxtaglomerular area by computer digitization (Gould IP 8500, courtesy of Biomedical Engineering Department, University of Virginia) . The criteria used for identifying JGAs were the same as those described in the immunocytochemistry method section (see above). All measurements were performed on coded slides such that the investigator was unaware of the treatment groups. The numbers thus obtained were divided into class intervals and plotted in a frequency distribution histogram as a function of the percent of JGAs containing the number of grains in the corresponding class intervals. In addition, the percent of JGAs containing renin mRNA was determined by dividing the number of JGA containing the hybridization signals by the total number of JGA.
RENIN AND ITS MRNA IN URETERAL OBSTRUCTION
Statistical Analysis Comparisons among groups were performed by one-way analysis of variance and between the left and right kidneys in each group by paired t test. Comparisons between frequency distributions were performed using the Kruskall-Wallis test. Significance was defined as P < 0.05. Results are expressed as means ± SEM.
RESULTS
Effect of uua on Somatic and Renal Growth
There was no change in body weight in rats following 24 hours of UUO or sham operation. However, rats subjected to 4 weeks of UUO gained significantly less weight than their sham-operated counterparts. Kidneys subjected to 24-hour UUO were heavier than the intact opposite and sham-operated kidneys. Following 4-week UUO, the obstructed kidneys were significantly smaller than the intact opposite kidneys, and by that time, the intact opposite kidneys had increased their weights by more than 35 % over those of sham kidneys. A significant correlation was found between left kidney weights and body weights of the 24-hour UUO group (r = 0.9; P = 0.03). No other correlations were found between body weights and either left or right kidney weights in the remaining treatment groups. The left ureteral diameter was significantly dilated in both UUO groups, and greater in 4-week than in 24-hour UUO rats (Table 1). Effect of uua on Plasma Renin Activity and Blood Pressure
Twenty-four-hour sham-operated and UUO rats had a higher plasma renin activity (24-hour sham [n = 4], 12.5 ± 2.2; 24-hour UUO [n = 5], 14.5
Table 1. Group
24-Hour sham (n = 5) 24-Hour UUO (n = 5) 4-Week sham (n = 6) 4-Week UUO (n = 6)
577
± 3.5 ng/mLlh) than 4-week sham-operated and UUO animals (4-week sham [n = 6], 6.7 ± 1.0; 4-week UUO [n = 6], 4.8 ± 1.4 ng/mLlh), although statistical significance was achieved only between 24-hour UUO and 4-week UUO groups (P < 0.05). Neither 24-hour nor 4-week UUO had an appreciable effect on MAP as compared with that of sham-operated animals (Table 1).
Effect of 24-hour uua on the Distribution of Renin and Its mRNA
Kidney sections that were hybridized in the presence of excess unlabeled probe showed no hybridization signals. Background labeling was minimal in all sections examined (average of less than 20 grains per high power field). The hybridization signals were limited to the arterial tree. Tubular structures or peri tubular capillaries showed no specific hybridization signals. The kidney sections that were hybridized with the labeled sense oligonucleotide also showed no specific hybridization signals. Chronic UUO of 24 hours' duration did not affect the distribution of renin or its mRNA in either the obstructed or the intact opposite kidneys as compared with sham-operated kidneys. In both kidneys of 24-hour UUO, as well as of 24-hour sham groups, immunoreactive renin and renin mRNA in situ hybridization signals were confined to the vascular pole of the glomerulus and were not observed upstream along the AA (Figs lA and 2A). In addition, %JGA and %LAA with immunoreactive renin and %JGA with renin mRNA were not different among the kidneys of these groups (Table 2).
Characteristics of Rats
Body Weight (g)
Left Kidney Weight (g)
Right Kidney Weight (g)
270 ± 4.9
1.10 ± 0.03
1.10 ± 0.01
0.9 ± 0.02
131 ± 4.2
267 ± 5.1
1.48 ± 0.10*t
1.11 ± 0.01
2.1 ± 0.06t
140 ± 3.9
340 ± 6.0§
1.44 ± 0.07
1.40 ± 0.04
1.0 ± 0.03
122 ± 5.9
319 ± 5.6t:j:
1.17 ± 0.11*t:j:
1.90 ± 0.2t:j:
3.5 ± 0.05*:j:
128 ± 14
left Ureteral Diameter (mm)
MAP (mm Hg)
NOTE. Values are means ± SEM. Nine rats were used for measurement of MAP in the 24-hour UUO group. *P < 0.05 v right kidney; tP < 0.05 v corresponding sham group; :j:P < 0.01 v 24-hour UUO: §P < 0.01 v 24-hour sham.
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EL-DAHR ET AL
Fig 1. Renin localization by immunocytochemistry. Section from an obstructed kidney after (A) 24 hours or (8) 4 weeks of UUO. As in sham-operated kidneys, immunoreactive renin (arrow) is confined to the vascular pole of the glomerulus only. (Original magnification x 200.)
Effect of 4-Week UUO on the Distribution of Renin and Its mRNA Following 4 weeks of UUO, the ipsilateral kidney manifested no detectable changes in the localization of either renin or its mRNA when compared with sham kidneys and both were confined to a juxtaglomerular location (Figs IB and 2B). However, %JGA with immunoreactive renin was significantly lower in the intact opposite kidneys than in the obstructed kidneys (Table 2). In addition, %JGA with detectable renin mRNA was signifi-
cantly lower in these hypertrophied kidneys than in either obstructed or sham kidneys (Table 2). Comparing 24 hours versus 4 weeks of UUO showed no significant differences in the distribution of renin or renin mRNA in the obstructed kidneys. The only difference was seen in the intact opposite kidneys, where %JGA with renin mRNA was lower in the intact opposite kidneys of 4-week UUO than in those of 24-hour UUO (Table 2). The distribution of grain counts per JGA was also similar in the kidneys of all treatment groups (Fig 3).
579
RENIN AND ITS MRNA IN URETERAL OBSTRUCTION
Fig 2. In situ hybridization. Section from an obstructed kidney after (A) 24 hours or (8) 4 weeks of UUO. Similar to the localization of immunoreactive renin, the hybridization signals representing renin mRNA (grains) are confined to the juxtaglomerular portion of the AA. (Original magnification x 200.)
Table 2.
Distribution of Renin and Renin mRNA
%JGA With Immunoreactive Renin Group
24-Hour sham 24-Hour UUO 4-Week sham 4-Week UUO
LK
36.6 36.2 39.8 46.3
± ± ± ±
%LAA With Immunoreactive Renin
RK
2.7 3.4 3.8 3.9'
36.4 34.6 35.4 28.7
± ± ± ±
LK
3.6 3.8 5.7 7.7
29.6 27.6 24.8 29.2
± ± ± ±
%JGA With Renin mRNA LK
RK
2.1 4.0 3.0 1.9
NOTE. Values are means ± SEM. Abbreviations: LK, left kidney; RK, right kidney. 'P < 0.05 v right kidney; tP < 0.05 v right kidneys of other groups.
30.6 30.0 20.9 23.7
± ± ± ±
4.4 4.2 1.0 5.3
44 44 40 42
± ± ± ±
3.0 2.0 3.0 1.0'
RK
41 41 38 27
± ± ± ±
2.0 4.0 3.0 2.0t
580
EL-OAHR ET AL
50.---------------------------------------~
0-04-WK sham 6-64-WK UUOLK A - A 4-WK UUORK e-e24-HR sham D-D24-HR UUOLK .-.24-HR UUORK
40 en
«
C>
-, lL.
o
30
I-
Z
w u
20
0:: W 0...
10
~!
O~----~----~----~-===~~~~~ o 200 400 600 800 1000 NUMBER OF GRAINS PER JGA Fig 3. Frequency distribution histogram of the number of in situ hybridization signals (grains) per JGA. Kruskall-Wallis test showed no significant differences among the different treatment groups in the number of grains per JGA.
There was no correlation between %JGA or %LAA and plasma renin activity in any of the treatment groups. DISCUSSION
The present study demonstrates that the overall distribution of renin gene-expressing cells is not altered in the mature kidney subjected to chronic UUO of 24 hours' or 4 weeks' duration. We have recently provided evidence for activation of renin gene expression in chronically obstructed kidneys of I-month-old rats subjected to UUO at day 2 of life, 13 wherein UUO during early development was associated with persistence of the neonatal pattern of renin distribution along the renal microvasculature. This finding prompted us to conduct the present study to examine the response of intrarenal renin to chronic UUO in the mature animal. We found that in contrast to our previous findings in newborn rats, adult rats with chronic UUO of 24 hours' and 4 weeks' duration do not manifest an increase in the distribution of immunoreactive renin or renin mRNA in the ob-
structed kidneys. Moreover, UUO caused no shift in the intensity of renin mRNA signals per JGA (Fig 3). Thus, it is unlikely that UUO in the adult resulted in an increased renin gene expression in some glomeruli and not others. However, in the neonate, 4 % of JGAs contained a greater number of renin mRNA signaIs.13 The lack of effect of chronic UUO on the distribution of immunoreactive renin has also been demonstrated in the hydronephrotic adult mouse kidney.8 It is unlikely that activation of renin gene expression took place earlier than 24 hours postobstruction or between 24 hours and 4 weeks with return to baseline gene expression at the times of study. The enhanced activity of the intrarenal RAS during early development compared to adulthood lO - 12 presumably contributed to differences between the two age groups with regard to their intrarenal response to UUO. The ability of the ACE inhibitors, captopril and enalapril, to improve significantly the hemodynamics of chronically obstructed kidneys in both adult and young animals contrasts with our renin immunocytochemical and in situ hybridization
RENIN AND ITS MRNA IN URETERAL OBSTRUCTION
581
findings. Since ACE inhibitors also inhibit kininase II, the enzyme that inactivates the vasodilator bradykinin, 16 their salutary effects in chronic UUO may be mediated by vasodilatory kinins rather than by suppressing intrarenal angiotensin II levels. However, the effects of captopril on the hemodynamics of kidneys subjected to chronic UUO have been shown to occur independently of the kinin system. 5 Another possibility is that the adult renal microvasculature is more sensitive to angiotensin II than that of the young animal. However, this is contradicted by the findings of Robillard et al,17 who have shown that although the adrenal and vasopressor responses to an increase in plasma angiotensin II concentration were less in fetuses than in adult ewes, the effect of angiotensin II on renal hemodynamics, glomerular filtration rate, and sodium excretion were similar in the two age groups. It should be noted that the renin antibody used in the present study localizes prorenin, as well as active renin. Thus, although the ratio of active to inactive renin cannot be inferred to from the present study, it is possible that the proportion of active renin was enhanced by UUO. Future development of specific antibodies directed against different parts of the renin molecule in the rat would be of a great value in testing this hypothesis. To determine whether UUO results in greater nephron heterogeneity with regard to renin production, renin secretion and gene expression must be quantitated directly in isolated JGAs from obstructed and intact nephrons. The results of the present study indicate that the distribution of renin gene expression is diminished in the intact kidneys with 4 weeks of contralateral UUO. This finding is consistent with the increased renal blood flow typically observed in the hypertropied kidneys .I.3.9 The distribution of immunoreactive renin in the intact opposite kidney of adult animals differed markedly from that previously observed in neonatal animals. In the newborn rat, renin immunodistribution was increased in both the obstructed and intact opposite kidneys,13.18 whereas in the adult rat the distribution of immunoreactive renin was lower in the intact opposite kidney than in the obstructed kidney. These findings indicate that there are age-related differences in the response of intrarenal renin to chronic UUO in both the obstructed and intact opposite kidneys. The apparent difference between %JGA with immunoreactive renin and %JGA with
renin mRNA in the nonobstructed kidneys at 4 weeks could be related to the difference in the sensitivity by which immunocytochemistry and in situ hybridization detect renin and its mRNA, respectively. The correlation between the sensitivities of the two methods has not been established. Another important finding of this study was the lack of correlation between intrarenal renin immunoreactivity and plasma renin activity in all the treatment groups. However, this finding is not surprising, considering a number of recent studies that demonstrate the independence of tissue RAS from the circulating system.12.1920 Similar dissociation between tissue and peripheral renin was found in our study of neonatal rats with chronic UU013; the obstructed kidneys had a significant increase in the proportion of renin-containing and renin-synthetizing JGAs and in active renin content compared with sham-operated kidneys, while plasma renin activity was not different from that of sham-operated rats. 13 In the present study, plasma renin activity levels were significantly higher in 24-hour UUO than 4-week UUO groups. However, 24-hour sham animals also had elevated plasma renin activity values that were not statistically significant from 24-hour UUO rats. It is likely that as a result of surgery, food and water intake were decreased during the ensuing 24 hours. Resulting intravascular volume contraction would therefore lead to increased plasma renin activity in both of the 24-hour groups. After 24 hours of UUO, the obstructed kidneys were heavier than either intact opposite or control kidneys. This is likely to be due to increased water content, rather than true hypertrophy. 21 Unlike the intact kidneys following 4 weeks of contralateral UUO, which underwent a significant compensatory hypertrophy, an increase in mass was not detectable in kidneys with contralateral UUO of 24 hours' duration. This is consistent with previous findings in adult Sprague-Dawley rats showing a significant compensatory increase in weight only 7 days after contralateral UUO.21 It is important to note that in contrast to our previous findings in the newborn rat with chronic UUO, in which the obstructed kidneys suffered a marked growth arrest, 1318 the present study showed only a slight decrease in the weight of the obstructed kidneys of adult rats. We speculate that the enhanced expression of intrarenal renin in the obstructed kidneys of newborn as opposed to adult animals may con-
582
EL-DAHR ET AL
tribute to a greater ischemic injury in the developing than in mature kidneys. In this regard, the effects of chronic UUO on renal function are more severe in the young than in the old animal. 22 In summary, following 24 hours or 4 weeks of UUO in the adult rat, the obstructed kidney does not show evidence of activation of renin gene expression nor alteration in renin distribution. In contrast, compensatory hypertrophy in the intact kidney after 4 weeks of contralateral UUO is associated with a smaller proportion of JGAs express-
ing the renin gene. In view of our previous findings in the newborn animal with chronic UUO,13.\8 we conclude that the response of intrarenal renin to ureteral obstruction is age-related, with the mature kidney manifesting an attenuated response compared with the developing kidney. ACKNOWLEDGMENT The technical assistance of Vicki J. Vallastro is gratefully acknowledged.
REFERENCES I. Chevalier RL, Jones CE: Contribution of endogenous vasoactive compounds to renal vascular resistance in neonatal chronic partial ureteral obstruction. J Urol 136:532-535, 1986 2. Chevalier RL, Peach MJ: Hemodynamic effects of enalapril on neonatal chronic partial ureteral obstruction. Kidney Int 28:891-898, 1985 3. Chevalier RL, Gomez RA: Response of the renin-angiotensin system to relief of neonatal ureteral obstruction. Am J Physiol 255:FI070-FI077, 1988 4. McDougal WS: Pharmacologic preservation of renal mass and function in obstructive uropathy. J UroI128:418-421, 1982 5. Yarger WE, Schocken DD, Harris RH: Obstructive nephropathy in the rat: Possible roles for the renin-angiotensin system, prostaglandins, and thromboxanes in postobstructive renal function. J Clin Invest 65:400-412, 1980 6. Carmines PK, Tanner GA: Angiotensin in the hemodynamic response to chronic nephron obstruction. Am J PhysioI245:F75-F82, 1983 7. Huguenin M, Ott CE, Romero JC, et al: Influence of renin depletion on renal function after release of 24-hour ureteral obstruction. 1 Lab Clin Med 87:58-64, 1976 8. Buhrle Cp, Hackenthal E, Helmchen U, et al: The hydronephrotic kidney of the mouse as a tool for intravital microscopy and in vitro electrophysiological studies of renin-containing cells. Lab Invest 54:462-471,1986 9. Siegel NJ, Upadhaya K, Kashgarian M: Inhibition of indomethacin of adaptive changes in the contralateral kidney after release of unilateral ureteral occlusion. Kidney Int 20:691694, 1981 10. Gomez RA, Cheavalier RL, Sturgill BC, et al: Maturation of the intrarenal renin distribution in Wi star-Kyoto rats. J Hypertens 4:S31-S33, 1986 (suppl 5) 11. Gomez RA, Lynch KR, Chevalier RL, et a1: Distribu-
tion of renin mRNA and its protein in the developing rat kidney. Am J Physiol 257:F850-F858, 1989 12. Gomez RA, Lynch KR, Chevalier RL, et al: Renin and angiotensinogen gene expression in maturing rat kidney. Am J Physiol 254:F582-F587, 1988 13. EI-Dahr SS, Gomez RA, Gray SS, et al: In situ localization of renin and its mRNA in neonatal ureteral obstruction. Am J Physiol (in press) 14. Sealy JE, Laragh JH: How to do plasma renin assay. Cardiovascular Med 2: 1079-1086, 1977 15. Stornetta RL, Hawelu-Johnson CL, Guyenet P, et al: Astrocytes synthesize angiotensinogen in brain. Science 242: 1444-1446, 1988 16. Williams GH, Hollenberg NK: Accentuated vascular and endocrine response to SQ 20881 in hypertension. N Engl J Med 297: 184-188, 1977 17. Robillard JE, Gomez RA, VanOrden D, et al: Comparison of the adrenal and renal responses to angiotensin II in fetal lambs and adult sheep. Circ Res 50:140-147, 1982 18. EI-Dahr SS, Chevalier RL, Gomez RA, et al: Renal nerves modulate renin gene expression in the developing kidney with ureteral obstruction. Kidney Int 37:354, 1990 (abstr) 19. Nakamura N, Soubrier F, Menard 1, et al: Nonproportional changes in plasma renin concentration, renal renin content and rat renin messenger RNA. Hypertension 7:855-859, 1985 20. Peters-Haefeli L: Renal cortical renin activity and renin secretion at rest and in response to hemorrhage. Am J Physiol 221:1331-1338,1971 21. Zelman SJ, Zenser TV, Davis BB: Renal growth in response to unilateral ureteral obstruction. Kidney Int 23:594598, 1983 22. Taki M, Goldsmith DI, Spitzer A: Impact of age on effects of ureteral obstruction on renal function. Kidney Int 24:602-609, 1983
I
NCREASED ACTIVITY of the renin-angiotensin system (RAS) has been shown to play a role in renal vasoconstriction resulting from chronic unilateral ureteral obstruction (UUO) in both mature and developing animals; angiotensinconverting enzyme (ACE) inhibitors significantly improve the hemodynamics of chronically obstructed kidneys I 5 and single nephrons. 6 However, it is not clear whether the intrarenal RAS is activated in the ipsilateral kidney with chronic UUO. In one study, renin depletion did not attenuate the increased renal vascular resistance resulting from 24 hours of UUO in the adult rat. 7 Furthermore, the distribution of immunoreactive renin was not altered in chronically hydronephrotic adult mouse kidneys.8 Equally important is the response of intrarenal renin in the intact hypertrophied kidney with contralateral UUO in which renal blood flow is typically increased in response to chronic UUO.I-3.9 Recent molecular studies have demonstrated that the expression of intrarenal renin and its mRNA is developmentally regulated. Unlike the newborn kidney, where renin synthesis takes place along most of the length of afferent arteriole, in the adult
kidney renin is synthetized and stored in the juxtaglomerular cells only. \0-\2 Using immunocytochemical and in situ hybridization techniques, we have shown that l-month-old rats subjected to UUO at day 2 of life manifest a significant increase in renin content and increased distribution of microvascular renin and its mRNA in the obstructed kidneys as compared with sham-operated controls. \3 Of note, the distribution of immunoreactive renin was also found to be increased in the intact opposite kidney. \3 The effects of chronic UUO on the intrarenal distribution of renin gene expression in the adult rat are unknown. Due to the From the Departments of Pediatrics, Pharmacology, and Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, I'lt. Supported by National Institute of Health Grant No. DK 40558 (R.L.C.). R.A.G. is supported by NIH Grant No. HL 41889. Dr S. S. El-Dahr is a research fellow of the American Heart Association, Virginia Affiliate. Address reprint requests to Robert L. Chevalier, MD, University of Virginia, Health Sciences Center, Department of Pediatrics, MR4/2034, Charlottesville, I'lt 22908. © 1990 by the National Kidney Foundation, Inc. 0272-6386/90/1506-0009$3.00/0
American Journal of Kidney Diseases, Vol XV, No 6 (June), 1990: pp 575-582
575
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EL-DAHR ET AL
inherent differences in the activity of the intrarenal RAS between newborn and adult animals, the response of intrarenal renin to UUO in the adult should be less than that observed in the developing animal. '3 The present study was performed in adult rats and was designed to compare the effects of 24 hours versus 4 weeks of UUO on the distribution of renin mRNA and its protein along the microvasculature of the obstructed kidneys, as well as of the intact opposite kidneys . MATERIALS AND METHODS Ten adult Sprague-Dawley rats (200 to 250 g, approximately 10 weeks of age) were subjected to either sham-operation or UUO and were studied 24 hours later (n = 5 in each group). Additional rats were subjected to either sham-operation or UUO and were studied 4 weeks later (n = 6 in each group). Following anesthesia with intraperitoneal pentobarbital sodium (50 mg/kg) (Abbott Laboratories , Chicago, IL), a small incision was made in the left lower quadrant of the abdomen and Ihe distal ureler was ligaled (VVO) or left undisturbed (sham). 13 The abdominal wall was then closed in two layers and the animal was allowed to recover from anesthesia. Subsequently, the animals were housed in separate cages until the time of the study, and they were fed regular rat chow (Purina 5012, Ralston-Purina, St Louis, MO) and were allowed free access to drinking water. At the time of the study, after an overnight fast but free access to water, the animals were anesthetized as described above and mean arterial pressure (MAP) was continuously recorded via a right carotid catheter by means of a Statham 23-10 pressure transducer (Englewood, CO) coupled to a Hewlett-Packard 7754 B recorder (Gould Inc, Medical Products Division, Oxhard, CAl. After a IO-rninute equilibration period, I mL of blood was withdrawn from the carotid catheter and collected in chilled EDTA-containing tubes. Blood samples were immediately centrifuged at 4°C for IS minutes and plasma kept at - 70°C for measurement of plasma renin activity according to the method of Sealy and Laragh. 14 The left ureteral diameter was measured with calipers in all rats midway between the renal pelvis and the bladder. The rats were then killed with saturated potassium chloride solution injected through the carotid catheter. The kidneys were removed, decapsulated, weighed, and processed for immunocytochemistry and in silu hybridization histochemistry as described below.
Renin Immunocytochemistry Immunocytochemical localization of renin was performed using the ABC immunoperoxidase technique as described previously. 10 13 The primary antibody is a specific polyclonal antiral renin antibody (diluted 112,500, gift of Dr T. Inagami, Department of Biochemistry, Vanderbilt University, Nashville, TN). As negative controls, the primary antiserum was replaced by nonimmune rabbit serum, and no staining occurred. An average of two to three sections per kidney (7 -I'm thick) were examined by light microscopy. The number of reninstained and the total number of juxtaglomerular apparatuses (1GA) were counted in each section. With the aid of an ocular
micrometer, the total length of the afferent arteriolar segments (AA) and the length of renin immunostaining along the AA were measured. All measurements were performed on coded slides such that the investigators were not aware of the treatment groups. Since the total number of JGA varied among sections, the foll owing immunohistochemical ratios were determined and compared among groups as previously described 10 \3: %JGA = (the number of stained JGA/total number of JGA) x 100; %LAA = (length of stained AA/total length of AA) x 100. The ratios thus obtained from each slide were pooled and averaged for each kidney. Only JGA with a vas afferens entering the glomerulus were used for computation. 10· \3
Hybridization Histochemistry Kidneys from four rats in each group were processed for in situ hybridization as previously described." ·I3·" Briefly, after fixation in 4 % paraformaldehyde in 0.12 mollL sodiumphosphate (pH 7.4 for I hour) and overnight cryprotection in 30% sucrose in 0 . 12 mollL sodium phosphate (pH 7.4), kidney sections (IO-I'm) were cut in a cryostat (Histostat Microtome, American Optical, Buffalo, NY) and mounted immediately on acid-cleaned gelatin-coated slides. Subsequently, 200 I'L of prehybridization buffer" ·13 was placed on each section and the slides were incubated at 37 °C for I hour. The sections were then hybridized at 37 °C for 24 hours to an oligonucleotide complementary to nucleotides 313-340 of rat renin mRNA.II.13 The oligonucleotide was 3 '-end-labeled with a-thio-[35S]-dATP to a total specific activity of 2 x 107 cpm/pmol. Equal counts of HS-labeled sense oligonucleotide were added to some kidney sections as a control for the specificity of the hybridization signals. In addition, a 200-fold molar excess of unlabeled oligonucleotide was added to some sections as a background control. Following hybridization, the kidney sections were washed in decreasing concentrations of SSPE (I x SSPE is 0.15 mollL NaCI, 0 .01 mol/L sodium phosphate, 0 .001 mollL EDTA, pH 7.4) as described previouslyll .13 with the last wash as the high stringency wash in 0.1 x SSPE with 0 .014 mollL 2-mercaptoethanol and I % sodium thiosulfate (20 minutes, 53°C). Following washes, the sections were postfixed in 0.05 mollL TRIS-saline-azide followed by 4 % paraformaldehyde in 0.12 mol/L sodium phosphate, pH 7.4. After dehydration, the sections were submitted to contact and emulsion autoradiography (NTB2, Kodak, Rochester, NY). Developing time was 14 days in all groups. An average of two to three sections per kidney was used for computations. In each section, the number of hybridization signals (grains) was counted in each juxtaglomerular area by computer digitization (Gould IP 8500, courtesy of Biomedical Engineering Department, University of Virginia) . The criteria used for identifying JGAs were the same as those described in the immunocytochemistry method section (see above). All measurements were performed on coded slides such that the investigator was unaware of the treatment groups. The numbers thus obtained were divided into class intervals and plotted in a frequency distribution histogram as a function of the percent of JGAs containing the number of grains in the corresponding class intervals. In addition, the percent of JGAs containing renin mRNA was determined by dividing the number of JGA containing the hybridization signals by the total number of JGA.
RENIN AND ITS MRNA IN URETERAL OBSTRUCTION
Statistical Analysis Comparisons among groups were performed by one-way analysis of variance and between the left and right kidneys in each group by paired t test. Comparisons between frequency distributions were performed using the Kruskall-Wallis test. Significance was defined as P < 0.05. Results are expressed as means ± SEM.
RESULTS
Effect of uua on Somatic and Renal Growth
There was no change in body weight in rats following 24 hours of UUO or sham operation. However, rats subjected to 4 weeks of UUO gained significantly less weight than their sham-operated counterparts. Kidneys subjected to 24-hour UUO were heavier than the intact opposite and sham-operated kidneys. Following 4-week UUO, the obstructed kidneys were significantly smaller than the intact opposite kidneys, and by that time, the intact opposite kidneys had increased their weights by more than 35 % over those of sham kidneys. A significant correlation was found between left kidney weights and body weights of the 24-hour UUO group (r = 0.9; P = 0.03). No other correlations were found between body weights and either left or right kidney weights in the remaining treatment groups. The left ureteral diameter was significantly dilated in both UUO groups, and greater in 4-week than in 24-hour UUO rats (Table 1). Effect of uua on Plasma Renin Activity and Blood Pressure
Twenty-four-hour sham-operated and UUO rats had a higher plasma renin activity (24-hour sham [n = 4], 12.5 ± 2.2; 24-hour UUO [n = 5], 14.5
Table 1. Group
24-Hour sham (n = 5) 24-Hour UUO (n = 5) 4-Week sham (n = 6) 4-Week UUO (n = 6)
577
± 3.5 ng/mLlh) than 4-week sham-operated and UUO animals (4-week sham [n = 6], 6.7 ± 1.0; 4-week UUO [n = 6], 4.8 ± 1.4 ng/mLlh), although statistical significance was achieved only between 24-hour UUO and 4-week UUO groups (P < 0.05). Neither 24-hour nor 4-week UUO had an appreciable effect on MAP as compared with that of sham-operated animals (Table 1).
Effect of 24-hour uua on the Distribution of Renin and Its mRNA
Kidney sections that were hybridized in the presence of excess unlabeled probe showed no hybridization signals. Background labeling was minimal in all sections examined (average of less than 20 grains per high power field). The hybridization signals were limited to the arterial tree. Tubular structures or peri tubular capillaries showed no specific hybridization signals. The kidney sections that were hybridized with the labeled sense oligonucleotide also showed no specific hybridization signals. Chronic UUO of 24 hours' duration did not affect the distribution of renin or its mRNA in either the obstructed or the intact opposite kidneys as compared with sham-operated kidneys. In both kidneys of 24-hour UUO, as well as of 24-hour sham groups, immunoreactive renin and renin mRNA in situ hybridization signals were confined to the vascular pole of the glomerulus and were not observed upstream along the AA (Figs lA and 2A). In addition, %JGA and %LAA with immunoreactive renin and %JGA with renin mRNA were not different among the kidneys of these groups (Table 2).
Characteristics of Rats
Body Weight (g)
Left Kidney Weight (g)
Right Kidney Weight (g)
270 ± 4.9
1.10 ± 0.03
1.10 ± 0.01
0.9 ± 0.02
131 ± 4.2
267 ± 5.1
1.48 ± 0.10*t
1.11 ± 0.01
2.1 ± 0.06t
140 ± 3.9
340 ± 6.0§
1.44 ± 0.07
1.40 ± 0.04
1.0 ± 0.03
122 ± 5.9
319 ± 5.6t:j:
1.17 ± 0.11*t:j:
1.90 ± 0.2t:j:
3.5 ± 0.05*:j:
128 ± 14
left Ureteral Diameter (mm)
MAP (mm Hg)
NOTE. Values are means ± SEM. Nine rats were used for measurement of MAP in the 24-hour UUO group. *P < 0.05 v right kidney; tP < 0.05 v corresponding sham group; :j:P < 0.01 v 24-hour UUO: §P < 0.01 v 24-hour sham.
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EL-DAHR ET AL
Fig 1. Renin localization by immunocytochemistry. Section from an obstructed kidney after (A) 24 hours or (8) 4 weeks of UUO. As in sham-operated kidneys, immunoreactive renin (arrow) is confined to the vascular pole of the glomerulus only. (Original magnification x 200.)
Effect of 4-Week UUO on the Distribution of Renin and Its mRNA Following 4 weeks of UUO, the ipsilateral kidney manifested no detectable changes in the localization of either renin or its mRNA when compared with sham kidneys and both were confined to a juxtaglomerular location (Figs IB and 2B). However, %JGA with immunoreactive renin was significantly lower in the intact opposite kidneys than in the obstructed kidneys (Table 2). In addition, %JGA with detectable renin mRNA was signifi-
cantly lower in these hypertrophied kidneys than in either obstructed or sham kidneys (Table 2). Comparing 24 hours versus 4 weeks of UUO showed no significant differences in the distribution of renin or renin mRNA in the obstructed kidneys. The only difference was seen in the intact opposite kidneys, where %JGA with renin mRNA was lower in the intact opposite kidneys of 4-week UUO than in those of 24-hour UUO (Table 2). The distribution of grain counts per JGA was also similar in the kidneys of all treatment groups (Fig 3).
579
RENIN AND ITS MRNA IN URETERAL OBSTRUCTION
Fig 2. In situ hybridization. Section from an obstructed kidney after (A) 24 hours or (8) 4 weeks of UUO. Similar to the localization of immunoreactive renin, the hybridization signals representing renin mRNA (grains) are confined to the juxtaglomerular portion of the AA. (Original magnification x 200.)
Table 2.
Distribution of Renin and Renin mRNA
%JGA With Immunoreactive Renin Group
24-Hour sham 24-Hour UUO 4-Week sham 4-Week UUO
LK
36.6 36.2 39.8 46.3
± ± ± ±
%LAA With Immunoreactive Renin
RK
2.7 3.4 3.8 3.9'
36.4 34.6 35.4 28.7
± ± ± ±
LK
3.6 3.8 5.7 7.7
29.6 27.6 24.8 29.2
± ± ± ±
%JGA With Renin mRNA LK
RK
2.1 4.0 3.0 1.9
NOTE. Values are means ± SEM. Abbreviations: LK, left kidney; RK, right kidney. 'P < 0.05 v right kidney; tP < 0.05 v right kidneys of other groups.
30.6 30.0 20.9 23.7
± ± ± ±
4.4 4.2 1.0 5.3
44 44 40 42
± ± ± ±
3.0 2.0 3.0 1.0'
RK
41 41 38 27
± ± ± ±
2.0 4.0 3.0 2.0t
580
EL-OAHR ET AL
50.---------------------------------------~
0-04-WK sham 6-64-WK UUOLK A - A 4-WK UUORK e-e24-HR sham D-D24-HR UUOLK .-.24-HR UUORK
40 en
«
C>
-, lL.
o
30
I-
Z
w u
20
0:: W 0...
10
~!
O~----~----~----~-===~~~~~ o 200 400 600 800 1000 NUMBER OF GRAINS PER JGA Fig 3. Frequency distribution histogram of the number of in situ hybridization signals (grains) per JGA. Kruskall-Wallis test showed no significant differences among the different treatment groups in the number of grains per JGA.
There was no correlation between %JGA or %LAA and plasma renin activity in any of the treatment groups. DISCUSSION
The present study demonstrates that the overall distribution of renin gene-expressing cells is not altered in the mature kidney subjected to chronic UUO of 24 hours' or 4 weeks' duration. We have recently provided evidence for activation of renin gene expression in chronically obstructed kidneys of I-month-old rats subjected to UUO at day 2 of life, 13 wherein UUO during early development was associated with persistence of the neonatal pattern of renin distribution along the renal microvasculature. This finding prompted us to conduct the present study to examine the response of intrarenal renin to chronic UUO in the mature animal. We found that in contrast to our previous findings in newborn rats, adult rats with chronic UUO of 24 hours' and 4 weeks' duration do not manifest an increase in the distribution of immunoreactive renin or renin mRNA in the ob-
structed kidneys. Moreover, UUO caused no shift in the intensity of renin mRNA signals per JGA (Fig 3). Thus, it is unlikely that UUO in the adult resulted in an increased renin gene expression in some glomeruli and not others. However, in the neonate, 4 % of JGAs contained a greater number of renin mRNA signaIs.13 The lack of effect of chronic UUO on the distribution of immunoreactive renin has also been demonstrated in the hydronephrotic adult mouse kidney.8 It is unlikely that activation of renin gene expression took place earlier than 24 hours postobstruction or between 24 hours and 4 weeks with return to baseline gene expression at the times of study. The enhanced activity of the intrarenal RAS during early development compared to adulthood lO - 12 presumably contributed to differences between the two age groups with regard to their intrarenal response to UUO. The ability of the ACE inhibitors, captopril and enalapril, to improve significantly the hemodynamics of chronically obstructed kidneys in both adult and young animals contrasts with our renin immunocytochemical and in situ hybridization
RENIN AND ITS MRNA IN URETERAL OBSTRUCTION
581
findings. Since ACE inhibitors also inhibit kininase II, the enzyme that inactivates the vasodilator bradykinin, 16 their salutary effects in chronic UUO may be mediated by vasodilatory kinins rather than by suppressing intrarenal angiotensin II levels. However, the effects of captopril on the hemodynamics of kidneys subjected to chronic UUO have been shown to occur independently of the kinin system. 5 Another possibility is that the adult renal microvasculature is more sensitive to angiotensin II than that of the young animal. However, this is contradicted by the findings of Robillard et al,17 who have shown that although the adrenal and vasopressor responses to an increase in plasma angiotensin II concentration were less in fetuses than in adult ewes, the effect of angiotensin II on renal hemodynamics, glomerular filtration rate, and sodium excretion were similar in the two age groups. It should be noted that the renin antibody used in the present study localizes prorenin, as well as active renin. Thus, although the ratio of active to inactive renin cannot be inferred to from the present study, it is possible that the proportion of active renin was enhanced by UUO. Future development of specific antibodies directed against different parts of the renin molecule in the rat would be of a great value in testing this hypothesis. To determine whether UUO results in greater nephron heterogeneity with regard to renin production, renin secretion and gene expression must be quantitated directly in isolated JGAs from obstructed and intact nephrons. The results of the present study indicate that the distribution of renin gene expression is diminished in the intact kidneys with 4 weeks of contralateral UUO. This finding is consistent with the increased renal blood flow typically observed in the hypertropied kidneys .I.3.9 The distribution of immunoreactive renin in the intact opposite kidney of adult animals differed markedly from that previously observed in neonatal animals. In the newborn rat, renin immunodistribution was increased in both the obstructed and intact opposite kidneys,13.18 whereas in the adult rat the distribution of immunoreactive renin was lower in the intact opposite kidney than in the obstructed kidney. These findings indicate that there are age-related differences in the response of intrarenal renin to chronic UUO in both the obstructed and intact opposite kidneys. The apparent difference between %JGA with immunoreactive renin and %JGA with
renin mRNA in the nonobstructed kidneys at 4 weeks could be related to the difference in the sensitivity by which immunocytochemistry and in situ hybridization detect renin and its mRNA, respectively. The correlation between the sensitivities of the two methods has not been established. Another important finding of this study was the lack of correlation between intrarenal renin immunoreactivity and plasma renin activity in all the treatment groups. However, this finding is not surprising, considering a number of recent studies that demonstrate the independence of tissue RAS from the circulating system.12.1920 Similar dissociation between tissue and peripheral renin was found in our study of neonatal rats with chronic UU013; the obstructed kidneys had a significant increase in the proportion of renin-containing and renin-synthetizing JGAs and in active renin content compared with sham-operated kidneys, while plasma renin activity was not different from that of sham-operated rats. 13 In the present study, plasma renin activity levels were significantly higher in 24-hour UUO than 4-week UUO groups. However, 24-hour sham animals also had elevated plasma renin activity values that were not statistically significant from 24-hour UUO rats. It is likely that as a result of surgery, food and water intake were decreased during the ensuing 24 hours. Resulting intravascular volume contraction would therefore lead to increased plasma renin activity in both of the 24-hour groups. After 24 hours of UUO, the obstructed kidneys were heavier than either intact opposite or control kidneys. This is likely to be due to increased water content, rather than true hypertrophy. 21 Unlike the intact kidneys following 4 weeks of contralateral UUO, which underwent a significant compensatory hypertrophy, an increase in mass was not detectable in kidneys with contralateral UUO of 24 hours' duration. This is consistent with previous findings in adult Sprague-Dawley rats showing a significant compensatory increase in weight only 7 days after contralateral UUO.21 It is important to note that in contrast to our previous findings in the newborn rat with chronic UUO, in which the obstructed kidneys suffered a marked growth arrest, 1318 the present study showed only a slight decrease in the weight of the obstructed kidneys of adult rats. We speculate that the enhanced expression of intrarenal renin in the obstructed kidneys of newborn as opposed to adult animals may con-
582
EL-DAHR ET AL
tribute to a greater ischemic injury in the developing than in mature kidneys. In this regard, the effects of chronic UUO on renal function are more severe in the young than in the old animal. 22 In summary, following 24 hours or 4 weeks of UUO in the adult rat, the obstructed kidney does not show evidence of activation of renin gene expression nor alteration in renin distribution. In contrast, compensatory hypertrophy in the intact kidney after 4 weeks of contralateral UUO is associated with a smaller proportion of JGAs express-
ing the renin gene. In view of our previous findings in the newborn animal with chronic UUO,13.\8 we conclude that the response of intrarenal renin to ureteral obstruction is age-related, with the mature kidney manifesting an attenuated response compared with the developing kidney. ACKNOWLEDGMENT The technical assistance of Vicki J. Vallastro is gratefully acknowledged.
REFERENCES I. Chevalier RL, Jones CE: Contribution of endogenous vasoactive compounds to renal vascular resistance in neonatal chronic partial ureteral obstruction. J Urol 136:532-535, 1986 2. Chevalier RL, Peach MJ: Hemodynamic effects of enalapril on neonatal chronic partial ureteral obstruction. Kidney Int 28:891-898, 1985 3. Chevalier RL, Gomez RA: Response of the renin-angiotensin system to relief of neonatal ureteral obstruction. Am J Physiol 255:FI070-FI077, 1988 4. McDougal WS: Pharmacologic preservation of renal mass and function in obstructive uropathy. J UroI128:418-421, 1982 5. Yarger WE, Schocken DD, Harris RH: Obstructive nephropathy in the rat: Possible roles for the renin-angiotensin system, prostaglandins, and thromboxanes in postobstructive renal function. J Clin Invest 65:400-412, 1980 6. Carmines PK, Tanner GA: Angiotensin in the hemodynamic response to chronic nephron obstruction. Am J PhysioI245:F75-F82, 1983 7. Huguenin M, Ott CE, Romero JC, et al: Influence of renin depletion on renal function after release of 24-hour ureteral obstruction. 1 Lab Clin Med 87:58-64, 1976 8. Buhrle Cp, Hackenthal E, Helmchen U, et al: The hydronephrotic kidney of the mouse as a tool for intravital microscopy and in vitro electrophysiological studies of renin-containing cells. Lab Invest 54:462-471,1986 9. Siegel NJ, Upadhaya K, Kashgarian M: Inhibition of indomethacin of adaptive changes in the contralateral kidney after release of unilateral ureteral occlusion. Kidney Int 20:691694, 1981 10. Gomez RA, Cheavalier RL, Sturgill BC, et al: Maturation of the intrarenal renin distribution in Wi star-Kyoto rats. J Hypertens 4:S31-S33, 1986 (suppl 5) 11. Gomez RA, Lynch KR, Chevalier RL, et a1: Distribu-
tion of renin mRNA and its protein in the developing rat kidney. Am J Physiol 257:F850-F858, 1989 12. Gomez RA, Lynch KR, Chevalier RL, et al: Renin and angiotensinogen gene expression in maturing rat kidney. Am J Physiol 254:F582-F587, 1988 13. EI-Dahr SS, Gomez RA, Gray SS, et al: In situ localization of renin and its mRNA in neonatal ureteral obstruction. Am J Physiol (in press) 14. Sealy JE, Laragh JH: How to do plasma renin assay. Cardiovascular Med 2: 1079-1086, 1977 15. Stornetta RL, Hawelu-Johnson CL, Guyenet P, et al: Astrocytes synthesize angiotensinogen in brain. Science 242: 1444-1446, 1988 16. Williams GH, Hollenberg NK: Accentuated vascular and endocrine response to SQ 20881 in hypertension. N Engl J Med 297: 184-188, 1977 17. Robillard JE, Gomez RA, VanOrden D, et al: Comparison of the adrenal and renal responses to angiotensin II in fetal lambs and adult sheep. Circ Res 50:140-147, 1982 18. EI-Dahr SS, Chevalier RL, Gomez RA, et al: Renal nerves modulate renin gene expression in the developing kidney with ureteral obstruction. Kidney Int 37:354, 1990 (abstr) 19. Nakamura N, Soubrier F, Menard 1, et al: Nonproportional changes in plasma renin concentration, renal renin content and rat renin messenger RNA. Hypertension 7:855-859, 1985 20. Peters-Haefeli L: Renal cortical renin activity and renin secretion at rest and in response to hemorrhage. Am J Physiol 221:1331-1338,1971 21. Zelman SJ, Zenser TV, Davis BB: Renal growth in response to unilateral ureteral obstruction. Kidney Int 23:594598, 1983 22. Taki M, Goldsmith DI, Spitzer A: Impact of age on effects of ureteral obstruction on renal function. Kidney Int 24:602-609, 1983