Growth hormone regulation of glomerular AT1 angiotensin receptors in adult unine - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:49 |只看该作者 |倒序浏览 |打印

作者：Ka-Yin K. Mok, Kathryn Sandberg, Joseph M. Sweeny, Wei Zheng, Sunghou Lee,  Susan E. Mulroney作者单位：Departments of Physiology and Biophysics and Medicine, Georgetown University School of Medicine, Washington, District of Columbia 20007


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      【摘要】  W4 d. G# H( P, o
   Sex differences exist in the mechanisms initiating early compensatory renal growth after unilateral nephrectomy (UNX); remnant kidney growth is growth hormone (GH) independent in adult female rats and GH dependent in adult male rats. The present study determined whether sex differences also exist in angiotensin type 1 receptor (AT 1 R) regulation during early remnant kidney (REM) growth after UNX, and if so, whether GH modulates AT 1 R expression after UNX in the male rat. Scatchard analysis of radioligand binding in glomeruli demonstrated that 48 h post-UNX, AT 1 R density (B max ) was significantly decreased by 20% in female REM compared with control kidneys. In contrast, male REM glomerular B max was significantly increased by 28% compared with control kidneys. Furthermore, GH-suppressed male rats displayed attenuated REM growth, which was associated with a 35% decrease in AT 1 R B max. Losartan treatment also decreased REM AT 1 R B max by 55%. The activity of mRNA binding proteins that bind to the 5' leader sequence of the AT 1 R was regulated by UNX and GH treatment in an inverse manner to AT 1 R expression. These findings suggest that in rats 1 ) there are sex differences in the regulation of glomerular AT 1 R expression after UNX; 2 ) the increase in AT 1 R binding sites in the male REM is regulated by GH and mediates early remnant kidney growth; and 3 ) AT 1 R 5' leader sequence mRNA binding proteins play a role in UNX and GH regulation of glomerular AT 1 Rs in both males and females. 8 Q. |: r; O1 S* n# z
      【关键词】 reninangiotensin system renal mass ablation mRNA binding proteins sexual dimorphism
   THE MECHANISMS RESPONSIBLE for compensatory renal growth after uninephrectomy (UNX) include a number of endocrine, biochemical, and molecular changes ( 5, 6, 24 ). In adult male rats, UNX results in the rapid growth of the remaining kidney through hypertrophy, with only a minimal hyperplasic component ( 5, 6, 24 ). We have previously determined that this accelerated growth occurs via a growth hormone (GH)-dependent mechanism ( 8 ), independently of the IGF-I system ( 15, 17 ). In contrast, the early stage of compensatory renal growth in female rats has a significant hyperplastic component, is GH independent, and is associated with a significant increase in renal IGF-I ( 17, 18 ). This mechanism in the female is similar to that observed in the juvenile UNX animal ( 15 - 17 ). Because the increase in GH is dissociated from renal IGF-I in male remnant kidney growth, it has been postulated that GH is acting directly on the kidney, or through another factor. A potential candidate is ANG II.5 r; l7 L& ]1 I# V3 {0 U* U! t
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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 (AT 1 Rs) has been shown to attenuate the increase in renal function observed 7 days post-UNX ( 25 ). However, it is not known whether AT 1 Rs play a role in the immediate changes associated with compensatory renal growth.
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AT 1 Rs 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 AT 1 R mRNA. Our studies demonstrated that the activity of AT 1 R 5'LS RNA binding proteins are regulated by sex steroids and that AT 1 R 5'LS binding proteins play a role in regulating AT 1 R 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 AT 1 R expression and AT 1 R 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 AT 1 R expression and AT 1 R5'LS binding protein activity was examined in the male UNX rat.

METHODS- ]. p8 E  t' o8 {& T4 q
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UNX. Under sterile conditions approved by the Georgetown University Animal Care and Use Committee, adult (12-15 wk of age) male and female Wistar rats were anesthetized with isoflurane (3.0% isoflurane in O 2, 0.5 l/min) and underwent sham (kidney gently manipulated and left in place) or left renal nephrectomy. The left kidney was exposed by a flank incision, the renal artery and vein were tied off, and the kidney was removed and weighed. The adrenal gland was not removed. The kidney was bisected, and medullary tissue was removed with sterile scissors. Both halves of the renal cortex were immediately snap-frozen in liquid nitrogen and stored at -80°C. One-half of the cortex was used to determine AT 1 R 5'LS binding protein activity by RNA gel EMSA. The other half of the cortex was used to determine AT 1 R binding. The flank incision was sutured, and the animals were allowed to recover with food and water ad libitum. After 18, 24, or 48 h ( n = 5-18/group), the right kidney (i.e., remnant kidney) was removed and processed using the same procedures as the left kidney (control kidney). Kidney growth was based on wet weight because we have previously found that the increase in kidney dry weight induced by UNX is proportional to wet weight and total protein ( 27 ).
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Effect of GH suppression on compensatory renal growth. Adult male rats were anesthetized with isoflurane (3.0% isoflurane in O 2, 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-tyr 1, D -Arg 2 ]GRF-(1-29)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 AT 1 R expression and AT 1 R 5'LS binding protein activity.

Effect of the AT 1 R 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 AT 1 R antagonist losartan (10 mg · kg -1 · day -1, 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 AT 1 R 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 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.  f! }# \4 W9 s) l+ J8 t3 Q* c

Radioligand binding assay. To measure AT 1 R density (B max ) and receptor affinity ( K d ), radioligand binding assays were performed in isolated glomeruli. For saturation experiments, 0.05-4.0 nM 125 I-[Sar 1,Ile 8 ]ANG II was incubated with 2.5 µg of glomeruli for 3 h at room temperature in binding buffer (10 mM Na 2 HPO 4, 120 mM NaCl, 5 mM EGTA, 0.01% BSA) in the presence of 5 µM PD-123319 (AT 2 R 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 B max and K d, Scatchard plots were generated from the saturation curves using the Prism software program.& n; Z  B$ a3 J6 x. V: Z
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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  2 H 3 O 2 K, 1.5 mM Mg(C 2 H 3 O 2 ) 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).
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The 5'LS (271 nt) of rat AT 1a R cDNA was subcloned into a pCR3 vector (Invitrogen) by PCR. The plasmid was linearized using the restriction enzyme Xho 1. The radiolabeled 5'LS RNA of rAT 1a was prepared using T7 RNA polymerase and -[ 32 P]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 1-2 h at 60°C. The radiolabeled cRNA was precipitated with 7.5 M ammonium acetate and ethanol and resuspended in 150 µlof diethyl pyrocarbonate-H 2 O.& n7 B7 Z, j9 ?. u3 U7 A

The RNA EMSA was performed by incubating 20-30 µg of glomerular cytosolic extract with 2 µl of 100 mM DTT, 40 U of RNasin inhibitor, 10 5 cpm of 32 P-labeled 5'LS cRNA probe, and 4 µl of 10 x binding buffer containing 100 mM HEPES, pH 7.6, 400 mM KCl, 30 mM MgCl 2 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 (3-4 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 AT 1 R 5'LS binding protein activity was quantitated by Image Quant software (IQMac V1.2).# z. `- b4 K: R' `4 ^; {
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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 P6 Z/ N1 U; m- z; W

RESULTS9 b' d( \' _! t# m# n

Effect of UNX on glomerular AT 1 R expression in control and remnant kidneys 18, 24, and 48 h post-UNX in male rats. To determine glomerular AT 1 R expression in control and remnant kidneys, adult male rats underwent UNX, and remnant kidneys were harvested after 18, 24, or 48 h. Compared with control kidneys, remnant kidney weights were significantly increased at 18, 24, and 48 h post-UNX ( Fig. 1 A ). In sham animals, the weights of the right and left kidney were not statistically different and were similar to the weight of the left kidney removed at the time of UNX (data not shown). The accelerated remnant kidney growth at each of these time points post-UNX was associated with an increase in remnant kidney glomerular AT 1 R expression compared with control kidneys ( Fig. 1 B ). Scatchard analysis of AT 1 R binding revealed that B max in the remnant kidney was significantly elevated by 24 h and remained elevated 48 h post-UNX compared with the control kidneys. No significant difference was found between control and remnant kidney glomerular AT 1 RB max at 18 h post-UNX. Furthermore, no significant differences were found between the K d values in the remnant (0.19 ± 0.05 nm) and control (0.23 ± 0.06 nm) kidneys after 24 (data not shown) and 48 h ( Fig. 1 C ). To identify whether the increase in AT 1 R B max was associated with changes in glomerular AT 1 R 5'LS binding protein activity, gel-shift assays were performed on cytosolic extracts from control and remnant male kidneys. Figure 2 A shows representative control and remnant kidney extracts from three animals: the RNA protein complex band indicates the level of binding protein activity (30 µg extract/lane). In each animal, remnant kidney extract AT 1 R 5'LS binding protein activity was significantly reduced compared with control (from same animal). Figure 2 B ( left ) illustrates this significant reduction in AT 1 R 5'LS binding protein activity ( n = 6) compared with the significant increase in AT 1 RB max observed in the same remnant kidneys ( right ).

Fig. 1. Effect of unilateral nephrectomy (UNX) on remnant kidney weight and glomerular angiotensin type 1 receptor (AT 1 R) expression in the adult male rat. A : time course of changes in remnant kidney growth 18 ( n = 7), 24 ( n = 11), and 48 h ( n = 12) after UNX. B : time course of changes in AT 1 R density (B max ) in remnant kidneys 18 ( n = 5), 24 ( n = 18), and 48 h ( n = 18) after UNX. C : representative saturation analysis of 125 I-[Sar 1,Ile 8 ]ANG II binding in glomeruli from control and remnant kidneys 48 h post-UNX. Each point represents triplicate determinations. prot, Protein. Inset : Scatchard analysis of radioligand binding data. * P  Q( G% }4 C9 z9 r: {

Fig. 2. Effect of UNX on glomerular AT 1 R expression and AT 1 R 5' leader sequence (5'LS) binding protein (BP) activity in adult male rats. A : RNA protein complex (RPC) formation (AT 1 R 5'LS BP activity) of control (C) and remnant (R) kidneys is shown for 3 representative rats 48 h post-UNX. RPC represents the binding of 30 µg of glomerular cytosolic extracts from control and remnant kidneys that were incubated with 10 5 cpm of a 32 P-labeled 5'LS AT 1 R cRNA probe. The migration of the radiolabeled 5'LS AT 1 cRNA probe in the absence of glomerular cytosolic extract is shown in lane P. B : effect of UNX on AT 1 R B max and AT 1 R 5'LS BP activity in control (Cont; n = 6) and remnant (Rem; n = 6) kidneys 48 h post-UNX. * P1 B6 @4 S( v9 t$ }- k( q. b0 P1 m

Effect of a GRF antagonist on AT 1 R binding and AT 1 R 5 ' LS binding protein activity in control and remnant kidneys 48 h post-UNX in male rats. To determine whether GH regulates AT 1 R expression during the early stages of compensatory renal growth, GH was suppressed in animals using the GRF antagonist GRF-AN before UNX. AT 1 R 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 in glomerular AT 1 R expression ( Fig. 3 A, right ) compared with control kidneys. This was associated with a significant (-58%) reduction in remnant kidney weight compared with control kidneys ( Fig. 3 B ), which was consistent with our previous findings ( 8 ). To investigate the effect of GH on AT 1 R 5'LS binding protein activity during compensatory renal growth, RNA EMSA studies were performed on glomerular extracts from the same kidneys in which AT 1 R 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 AT 1 R 5'LS binding protein activity (38.5 ± 5.4%, P Fig. 3 A, left ) compared with control kidneys.* Q1 \" O1 j+ v& m- u- D

Fig. 3. Effect of the growth hormone (GH) antagonist [ N -Ac-tyr 1, D -Arg 2 ]GRF-(1-29)amide (GRF-AN) on UNX-induced changes in AT 1 R expression, AT 1 R 5'LS BP activity, and kidney wt (KW) in adult male rats. A : effect of GRF-AN on the UNX-induced changes in AT 1 R expression and AT 1 R 5'LS BP activity 48 h post-UNX in Cont ( n = 6) and Rem ( n = 6) kidneys. * P B : effect of the change in KW (remnant KW-control KW) in control (saline) and GH-suppressed (GRF-AN) adult male rats after UNX ( n = 6/group). * P
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Effect of AT 1 R blockade on remnant kidney growth 48 h post-UNX in male rats. To determine whether renal AT 1 Rs mediate the growth-promoting effects of GH on the remnant kidney, miniosmotic pumps containing saline or the AT 1 R antagonist losartan (10 mg · kg -1 · day -1, Merck Research Laboratories) were implanted in adult male rats, and remnant kidney growth and glomerular AT 1 R B max were examined 48 post-UNX. Glomerular AT 1 R expression was significantly reduced in male remnant kidneys from losartantreated animals compared with their controls (B max : control 1,043 ± 67 vs. losartan 787 ± 47 fmol/mg protein, P Fig. 4 A ). This inhibition of AT 1 R resulted in a significant decrease in remnant kidney weight (-55.3 ± 10.1%, P ± 2.0%, P ± 10.8%, P ( Fig. 4, B - D ).
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Fig. 4. Effect of the AT 1 R antagonist losartan on UNX-induced changes in AT 1 R expression and remnant kidney growth 48 h post-UNX in adult male rats. A : changes in AT 1 R B max in losartan ( n = 7)-treated animals 48 h post-UNX in control and remnant kidneys. B : remnant kidney growth in saline ( n = 8)- and losartan ( n = 11)-treated animals. C : ratio of remnant KW to body weight (KW/BW) in saline ( n = 8)- and losartan ( n = 11)-treated animals. D : remnant kidney total protein levels in saline- and losartan-treated rats ( n = 4/group). ** P *P' C: a$ e/ s9 e; ?9 A- J
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Effect of UNX on glomerular AT 1 R 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 AT 1 R 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 AT 1 R expression compared with control kidneys. Scatchard analysis of glomerular AT 1 R binding revealed a 24% decrease in B max 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 AT 1 R 5'LS binding protein activity (9.4 ± 4.1%, P compared with control ( Fig. 5 ).; p) O3 y4 R/ s8 Y% m
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Fig. 5. Effect of UNX on glomerular AT 1 R expression and AT 1 R5'LS BP activity in the adult female rat. Effect of UNX on changes in AT 1 R B max and AT 1 R 5'LS BP activity in Cont ( n = 6) and Rem ( n = 6) kidneys 48 h post-UNX. * P& T- I; m9 Q3 h& @3 `( r
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DISCUSSION
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This study demonstrated that UNX stimulates a rapid increase in glomerular AT 1 R expression in adult male, but not female, remnant kidneys (by 24 h), which is sustained 48 h post-UNX. The fact that the time course of glomerular AT 1 R upregulation correlates with the initial rapid increase in remnant kidney growth and that an AT 1 R antagonist prevents both the UNX-induced AT 1 R upregulation and increase in renal growth suggests that AT 1 Rs play a role in compensatory renal growth. UNX upregulates glomerular AT 1 R expression in adult male rats by increasing the number of receptor binding sites rather than by increasing receptor ligand affinity. Upregulation of AT 1 R binding sites is likely due to transcriptional and/or posttranscriptional mechanisms that increase receptor synthesis and/or decrease receptor metabolism, whereas post-translational mechanisms are more likely to alter ligand affinity and receptor signaling. Thus it will be interesting in future experiments to examine rates of AT 1 R mRNA turnover and translational efficiency in this model of compensatory renal growth.' ]! H, m3 m3 V6 l

The observed increase in glomerular AT 1 R expression in male remnant kidneys was dependent on circulating GH because in the GRF-AN-treated animals, UNX caused a decrease in glomerular AT 1 R binding sites and blocked the rapid remnant kidney growth. These data suggest that circulating GH mediates in vivo remnant kidney growth by modulating AT 1 R 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 AT 1 R expression in adult male rats expand on previous studies by Wyse and colleagues, who found that GH regulates AT 1 Rs in vitro in astrocytes ( 29 ) and in vivo in the adrenal, liver, and kidney ( 30 ). The mechanism of GH-induced upregulation of AT 1 Rs 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 AT 1 R 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 AT 1 R binding and binding protein activity, we hypothesize that the AT 1 R 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 AT 1 R blocker losartan significantly attenuates male remnant kidney growth strongly suggest that the AT 1 R is a key factor in the response. Future studies will examine the GH/AT 1 R mechanism in the kidney as well as in other AT 1 R-rich tissues (i.e., adrenal, heart, and vasculature).; X, I7 Y9 X  e. U/ k2 V7 W! Q$ x
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Of additional importance are the findings that GH may regulate glomerular AT 1 R 5'LS binding protein activity in male animals. UNX reduced glomerular AT 1 R 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 AT 1 R 5'LS binding protein activity in remnant kidneys 48 h post-UNX and at the same time reduced glomerular AT 1 R expression compared with control kidneys, supporting our previous studies that AT 1 R 5'LS binding proteins regulate AT 1 R expression. Taken together, these data suggest that GH regulates the AT 1 R through AT 1 R 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 AT 1 R expression 48 h post-UNX compared with control kidneys. The finding that the effects of GH on glomerular AT 1 R5'LS binding protein activity and AT 1 R 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 AT 1 Rs under normal physiological conditions.0 U1 q6 a" M) L  O
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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 AT 1 R 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 AT 1 R density was measured. We measured AT 1 R expression at 0 and 48 h post-UNX, whereas Valentin et al. measured AT 1 R expression on days 0 and 7. Thus the change in AT 1 R B max at 48 h could have gone undetected by Valentin et al.1 |! j0 ]" K7 [$ [
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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 AT 1 Rs 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 AT 1 Rs) and AT 1 Rs 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 AT 1 Rs in the male, but not in the female, UNX rat.+ w, t* G) m0 }  V4 X8 M
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In summary, this study shows that GH upregulates glomerular AT 1 R 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 AT 1 R 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 AT 1 R expression, suggesting that AT 1 R 5'LS binding proteins play a role as a contributory mechanism in the GH-induced upregulation of AT 1 Rs during compensatory renal growth in the male rat.2 z+ C6 f1 D( p$ a6 K& C

DISCLOSURES

This work was supported by grants from the American Heart Association (Established Investigator, 0040012N), National Kidney Foundation of the National Capital Area, and National Science Foundation (to S. E. Mulroney); and the National Kidney Foundation of the National Capital Area, American Heart Association (Established Investigator, 96-40040), and National Institutes of Health (AG-19291 and R01-HL-57502 to K. Sandberg).+ B  `/ S" i3 k% T
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Degli Uberti EC, Trasforini G, Margutti A, Rossi R, Ambrosio MR, and Pansini R. Stimulation of growth hormone and corticotropin release by angiotensin II in man. Metabolism 39: 1063-1067, 1990.! |5 C8 I, `/ ~  b
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Dworkin LD, Feiner HD, Parker M, and Tolbert E. Effects of nifedipine and enalapril on glomerular structure and function in uninephrectomized SHR. Kidney Int 39: 1112-1117, 1991.
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