Departments of 1Physiology and Biophysics and 2Medicine, Georgetown University School of Medicine, Washington, District of Columbia 20007
Submitted 24 October 2002 ; accepted in final form 21 June 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
renin-angiotensin system; renal mass ablation; mRNA binding proteins; sexual dimorphism
Circulating GH has been reported to increase plasma ANG II, and vice versa, indicating that the regulatory mechanisms governing these two peptide hormones are interrelated (2, 14, 29, 30). A growing volume of literature has also illustrated that in addition to its classic effects, ANG II has growth-promoting properties. ANG II binds to specific cell surface receptors, activates intracellular signaling pathways associated with cell growth, and induces proliferation and hypertrophy in a variety of cells (7, 20, 21, 28). Also, blocking angiotensin type 1 receptors (AT1Rs) has been shown to attenuate the increase in renal function observed 7 days post-UNX (25). However, it is not known whether AT1Rs play a role in the immediate changes associated with compensatory renal growth.
AT1Rs fall into a class of proteins that can be tightly controlled posttranscriptionally. Posttranscriptional gene regulation provides a mechanism for fine-tuning the expression of genes that are involved in crucial cell functions like growth, differentiation, signaling, and development. We have previously described cytosolic proteins that bind to the 5' leader sequence (5'LS) of AT1R mRNA. Our studies demonstrated that the activity of AT1R 5'LS RNA binding proteins are regulated by sex steroids and that AT1R 5'LS binding proteins play a role in regulating AT1R expression (12).
Taken together, these findings are consistent with the concept that ANG II has growth-promoting abilities in the kidney and, if upregulated by GH, may play a role in the early compensatory renal growth response in the male animal. In the present study, the effect of UNX on AT1R expression and AT1R 5'LS binding protein activity was studied in the glomeruli of adult male and female UNX rats. In addition, the role of GH in regulating glomerular AT1R expression and AT1R5'LS binding protein activity was examined in the male UNX rat.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of GH suppression on compensatory renal growth. Adult male rats were anesthetized with isoflurane (3.0% isoflurane in O2, 0.5 l/min) and equipped with a Silastic catheter in the right jugular vein. Beginning immediately after UNX, animals were intravenously injected twice daily (at 0830 and 1330) with either saline vehicle (0.9% sterile NaCl, 0.1 ml, n = 6) or [N-Ac-tyr1,D-Arg2]GRF-(129)amide (GRF-AN), a synthetic peptide antagonist to GH-releasing factor (GRF; 200 µg/kg twice daily, n = 6; PRGF-80, Bachem, Torrance, CA). The animals were housed in separate cages and allowed to recover with food and water ad libitum. We have previously shown that this dosage regimen is effective in suppressing the pulsatile release of GH in intact rats (16). The catheters were kept patent with 0.1 ml of 250 U/ml sodium heparin after each injection. Body weights and excised (control) kidney weights were obtained at the onset of the experiment before the kidneys were snap-frozen. After 48 h of treatment, animals were weighed and killed. The remnant kidneys were excised, weighed, flash-frozen, and stored at 80°C. Control and remnant kidneys were then assayed for glomerular AT1R expression and AT1R 5'LS binding protein activity.
Effect of the AT1R antagonist losartan on compensatory renal growth. Microosmotic pumps (model 1003D, Alzet, Palo Alto, CA) containing either saline vehicle (0.9% sterile NaCl, 0.1 ml, n = 8) or the AT1R antagonist losartan (10 mg · kg1 · day1, n = 11; Merck Research Laboratories, Rahway, NJ) were inserted subcutaneously on the dorsum of the neck under sterile conditions. The wound was closed, and the animals were allowed to recover. After 24 h postimplantation of the microosmotic pumps, both saline and losartan groups underwent a left nephrectomy. Body weights and excised kidney weights were obtained, and the control kidneys were then immediately frozen in liquid nitrogen and stored at 80°C until assay. After 48 h, the animals were weighed and killed, and the remnant kidneys were excised, weighed, flash-frozen, and stored at 80°C until use. Control and remnant kidneys were then assayed for glomerular AT1R expression, binding protein activity, and total protein content.
Glomerular isolation. Frozen kidneys were thawed in sterile ice-cold PBS, pH 7.4, and minced with a sterile razor blade. The glomeruli were isolated by using a modification of the method of Wilkes (26). The cortex was minced to a pastelike consistency and resuspended in PBS buffer. Each kidney was buttered
through a 90-µm bronze sieve (USA Standard Testing Sieve, West Chester, PA) and poured through successive sieves (180-, 150-, 75-µm pore size). The material retained on the 75-µm sieve was harvested in 50-ml conical centrifuge tubes and centrifuged twice with fresh PBS at 120 g for 90 s. The resulting glomerular suspension was examined by light microscopy, and only samples with >95% glomerular purity were used. The glomerular suspension protein concentration was determined using the Bradford method (Bio-Rad Protein Assay), and the suspension was frozen in liquid nitrogen.
Radioligand binding assay. To measure AT1R density (Bmax) and receptor affinity (Kd), radioligand binding assays were performed in isolated glomeruli. For saturation experiments, 0.054.0 nM 125I-[Sar1,Ile8]ANG II was incubated with 2.5 µg of glomeruli for 3 h at room temperature in binding buffer (10 mM Na2HPO4, 120 mM NaCl, 5 mM EGTA, 0.01% BSA) in the presence of 5 µM PD-123319 (AT2R antagonist). Nonspecific binding was determined by the amount of tracer bound in the presence of 200 nM unlabeled ANG II. Incubation was immediately followed by filtration through glass-fiber filters (Whatman GF/C, Hillsboro, OR) on a Brandel cell harvester, and the filters were then counted in a gamma spectrophotometer (Cobra II, Packard). All radioligand receptor binding assays were performed in triplicate in a final volume of 300 µl. Specific binding was defined as the total binding minus nonspecific binding. To determine Bmax and Kd, Scatchard plots were generated from the saturation curves using the Prism software program.
RNA EMSA to assess RNA binding protein activity. Isolated glomeruli were thawed on the day of use and homogenized in 1 ml of a 25 mM Tris buffer, pH 7.4, containing 0.1 mM EDTA, 40 mM KCl, 1% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 0.2 U/ml aprotinin, and 10 mg/ml antipain with a glass homogenizer (model SL1200 StedFast Stirrer, Fisher Scientific). The homogenate was centrifuged at 300 g for 10 min at 4°C. The supernatant was recovered and then centrifuged at 20,000 g for 20 min at 4°C. The resulting supernatant was layered on top of a 30% sucrose cushion containing 10 mM Tris, pH 7.6, 1mMC [PDB] 2H3O2K, 1.5 mM Mg(C2H3O2)2, and 2 mM dithiothreitol. The samples were centrifuged at 230,000 g for 3 h at 4°C. The supernatant (hereafter referred to as the glomerular cytosolic extract) was collected and stored at 70°C. The protein concentration of the glomerular cytosolic extract was measured using the Bradford method with BSA as a standard (Bio-Rad Protein Assay).
The 5'LS (271 nt) of rat AT1aR cDNA was subcloned into a pCR3 vector (Invitrogen) by PCR. The plasmid was linearized using the restriction enzyme Xho1. The radiolabeled 5'LS RNA of rAT1a was prepared using T7 RNA polymerase and -[32P]GTP according to Promega Biotech's protocol for in vitro synthesis of high-specific activity, single-stranded RNA probes. After electrophoresis in an 8 M urea-5% polyacrylamide gel, the RNA probe was eluted from the gel in an elution buffer containing 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, and 0.1% SDS for 12 h at 60°C. The radiolabeled cRNA was precipitated with 7.5 M ammonium acetate and ethanol and resuspended in 150 µlof diethyl pyrocarbonate-H2O.
The RNA EMSA was performed by incubating 2030 µg of glomerular cytosolic extract with 2 µl of 100 mM DTT, 40 U of RNasin inhibitor, 105 cpm of 32P-labeled 5'LS cRNA probe, and 4 µl of 10x binding buffer containing 100 mM HEPES, pH 7.6, 400 mM KCl, 30 mM MgCl2 and 50% glycerol in 30 µl. The solution was incubated at 30°C for 20 min. Four microliters of T1 RNAse (1 U/µl), which degrades single-stranded RNA after G residues, and 4 µl of heparin sulfate (100 mg/ml), which inhibits nonspecific RNA-protein complex formation, was added to the solution and incubated for 15 min. A gel loading buffer (34 µl) of 50% glycerol, 0.25% bromophenol blue, 0.25% xylene cyanole, and 1 mM EDTA was added to the sample, followed by electrophoresis at 200 V on a 4% polyacrylamide gel (59:1 acrylamide:bis-acrylamide) containing 45 mM Tris-borate, pH 8.3, and 1 mM EDTA. After electrophoresis, the gel was transferred to filter paper, dried, exposed to a PhosphorImager screen (Molecular Dynamics), and AT1R 5'LS binding protein activity was quantitated by Image Quant software (IQMac V1.2).
Statistics. Data were expressed as means or means ± SE of percent changes from the different groups. Comparisons of control and remnant kidneys were performed using paired Student's t-tests. Comparisons between different groups used unpaired Student's t-tests. Differences were designated significant at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Effect of a GRF antagonist on AT1R binding and AT1R 5'LS binding protein activity in control and remnant kidneys 48 h post-UNX in male rats. To determine whether GH regulates AT1R expression during the early stages of compensatory renal growth, GH was suppressed in animals using the GRF antagonist GRF-AN before UNX. AT1R radioligand analysis was performed in isolated glomeruli from control and male remnant kidneys harvested 48 h post-UNX in the presence and absence of GRF-AN. In contrast to GH-replete animals, remnant kidneys from animals treated with GRF-AN displayed a significant decrease (35.1 ± 6.9%, P < 0.05) in glomerular AT1R expression (Fig. 3A, right) compared with control kidneys. This was associated with a significant (58%) reduction in remnant kidney weight compared with control kidneys (Fig. 3B), which was consistent with our previous findings (8). To investigate the effect of GH on AT1R 5'LS binding protein activity during compensatory renal growth, RNA EMSA studies were performed on glomerular extracts from the same kidneys in which AT1R binding was determined 48 h post-UNX. In contrast to GH-replete UNX animals, remnant kidneys treated with GRF-AN displayed a significant increase in AT1R 5'LS binding protein activity (38.5 ± 5.4%, P < 0.05) (Fig. 3A, left) compared with control kidneys.
|
Effect of AT1R blockade on remnant kidney growth 48 h post-UNX in male rats. To determine whether renal AT1Rs mediate the growth-promoting effects of GH on the remnant kidney, miniosmotic pumps containing saline or the AT1R antagonist losartan (10 mg · kg1 · day1, Merck Research Laboratories) were implanted in adult male rats, and remnant kidney growth and glomerular AT1R Bmax were examined 48 post-UNX. Glomerular AT1R expression was significantly reduced in male remnant kidneys from losartantreated animals compared with their controls (Bmax: control 1,043 ± 67 vs. losartan 787 ± 47 fmol/mg protein, P < 0.05; Fig. 4A). This inhibition of AT1R resulted in a significant decrease in remnant kidney weight (55.3 ± 10.1%, P < 0.01), kidney weight-to-body weight ratios (17.8 ± 2.0%, P < 0.05), and total protein levels (30.5 ± 10.8%, P < 0.05) compared with saline remnant kidneys (Fig. 4, BD).
|
Effect of UNX on glomerular AT1R expression in control and remnant kidneys 18, 24, and 48 h post-UNX in female rats. To further investigate the sex differences in the mechanisms initiating compensatory renal growth, we examined glomerular AT1R expression in control and remnant kidneys from adult female rats 48 h post-UNX. Female remnant kidney growth was consistent with previous studies (18). Furthermore, in sham UNX animals, the weights of the left and right kidney were identical and similar to the weight of the left kidney removed at the time of UNX. In contrast to the adult male remnant kidney, the accelerated remnant kidney growth in female adult rats was associated with a significant decrease in glomerular AT1R expression compared with control kidneys. Scatchard analysis of glomerular AT1R binding revealed a 24% decrease in Bmax in the remnant kidney, without changes in ligand affinity, at 48 h post-UNX compared with the control kidneys (data not shown). In these same female animals, a small but significant increase in AT1R 5'LS binding protein activity (9.4 ± 4.1%, P < 0.05) was observed in the remnant kidney compared with control (Fig. 5).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The observed increase in glomerular AT1R expression in male remnant kidneys was dependent on circulating GH because in the GRF-AN-treated animals, UNX caused a decrease in glomerular AT1R binding sites and blocked the rapid remnant kidney growth. These data suggest that circulating GH mediates in vivo remnant kidney growth by modulating AT1R binding sites. The concept that GH interacts with the renin-angiotensin system is new (9) and has been substantiated only within the last decade (2, 10, 14, 30). However, the mechanisms by which GH and ANG II regulate each other's secretion are not clear (3, 14). The present findings showing that circulating GH regulates glomerular AT1R expression in adult male rats expand on previous studies by Wyse and colleagues, who found that GH regulates AT1Rs in vitro in astrocytes (29) and in vivo in the adrenal, liver, and kidney (30). The mechanism of GH-induced upregulation of AT1Rs could have implications for normal growth and fluid and electrolyte homeostasis, as well as for pathophysiological conditions, such as acromegaly, diabetic nephropathy, and hypertension. Indeed, the specific increase in glomerular AT1R expression is linked to the glomerular hypertrophy that occurs in male remnant kidneys 2 mo post-UNX (Mok K-Y, Mulroney SE, and Sandberg K, unpublished observations). While the present study reported on glomerular AT1R binding and binding protein activity, we hypothesize that the AT1R mechanism acts in all the hypertrophied renal tissue, because losartan treatment blocked compensatory renal growth (Fig. 4). Future studies will look at other areas of the kidney to confirm this notion. It is not known what contribution intrarenal ANG II or other ATR populations have in the compensatory renal growth response. However, the findings that the AT1R blocker losartan significantly attenuates male remnant kidney growth strongly suggest that the AT1R is a key factor in the response. Future studies will examine the GH/AT1R mechanism in the kidney as well as in other AT1R-rich tissues (i.e., adrenal, heart, and vasculature).
Of additional importance are the findings that GH may regulate glomerular AT1R 5'LS binding protein activity in male animals. UNX reduced glomerular AT1R 5'LS binding protein activity in the remnant compared with control kidneys while increasing activity in GRF-AN-treated animals. The studies show that GH significantly elevated glomerular AT1R 5'LS binding protein activity in remnant kidneys 48 h post-UNX and at the same time reduced glomerular AT1R expression compared with control kidneys, supporting our previous studies that AT1R 5'LS binding proteins regulate AT1R expression. Taken together, these data suggest that GH regulates the AT1R through AT1R 5'LS RNA binding proteins in adult male rats during compensatory renal growth. Conversely, in adult female remnant kidneys, glomerular 5'LS binding protein activity was slightly increased, and this was associated with a decrease in glomerular AT1 R expression 48 h post-UNX compared with control kidneys. The finding that the effects of GH on glomerular AT1R5'LS binding protein activity and AT1R expression were far greater in male compared with female animals is consistent with our previous observations of sex differences in the mechanisms governing initial compensatory renal growth. It will be interesting in future studies to determine whether sex differences also exist in GH regulation of 5'LS binding proteins and AT1Rs under normal physiological conditions.
The finding that losartan inhibited GH-dependent remnant kidney growth extends previous studies demonstrating that angiotensin-converting enzyme inhibition attenuated both the increase in renal function (4, 25) and the progression in renal damage induced by UNX (1a, 11). In contrast, Valentin et al. (23) reported that the renal hypertrophy induced by UNX occurred without changes in glomerular AT1R density and in the presence of the angiotensin-converting enzyme inhibitor captopril. The basis for this apparent difference in results is not clear but may be related to the time when glomerular AT1R density was measured. We measured AT1R expression at 0 and 48 h post-UNX, whereas Valentin et al. measured AT1R expression on days 0 and 7. Thus the change in AT1R Bmax at 48 h could have gone undetected by Valentin et al.
Baylis and Wilson (1) first reported that there are sex differences in the development of renal pathology after renal ablation and that testosterone drives the progression of renal damage. We recently expanded on this earlier work by reporting that significant glomerular hypertrophy and tubular damage occurred in remnant kidneys from male, but not female, rats only 2 mo post-UNX (19). Furthermore, gonadal hormone replacement studies indicated that the renal damage was testosterone dependent (19). Additional studies determined that blockade of AT1Rs by antagonist treatment as well as suppression of GH decreased renal growth glomerular hypertrophy and proteinuria 2 mo post-UNX, suggesting that GH (indirectly through AT1Rs) and AT1Rs mediate glomerular hypertrophy and the initiation of renal damage in the UNX male (13). The data presented in this paper suggest that the observed sexual dimorphism in glomerular hypertrophy during compensatory renal growth is a result of the GH-induced upregulation of AT1Rs in the male, but not in the female, UNX rat.
In summary, this study shows that GH upregulates glomerular AT1R expression in the early phase of compensatory renal growth in the adult male, but not in female, rat and that male remnant kidney growth depends on this increase in AT1R expression. These data confirm and extend our previous findings of sexual dimorphism in early compensatory renal growth mechanisms. This study also shows that changes in 5'LS binding proteins are inversely related to changes in AT1 R expression, suggesting that AT1R 5'LS binding proteins play a role as a contributory mechanism in the GH-induced upregulation of AT1Rs during compensatory renal growth in the male rat.
![]() |
DISCLOSURES |
---|
![]() |
FOOTNOTES |
---|
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. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|