Vol. 131. No. 6 Printed L~I U..S A.
Renal Growth Hormone Receptor Relationship to Renal Insulin-Like System EDWARD
CHIN,
JIAN ZHOU,
AND
CAROLYN
Gene Expression: Growth Factor
A. BONDY
Developmental Endocrinology Branch, National Institute of Child Health and Human National Institutes of Health, Bethesda, Maryland 20892
Development,
ABSTRACT In order to elucidate potential sites of direct GH action on the kidnev. we used in situ hvbridization to localize GH receutor (GHR) gene expression during the course of development and in the adult rat. In order to illuminate potential interactions between GH and insulinlike growth factor-I (IGF-I) in regulating renal function, we compared the anatomical localization of GHR messenger RNA (mRNA) with that for the IGF-I receptor and for IGF-I in the rat kidney. Low levels of GHR mRNA were present in the kidney from before birth and increased in abundance until postnatal day 40. Hypophysectomy resulted in a decrease and GH treatment resulted in an increase in renal GHR mRNA levels. Renal GHR mRNA was most abundant in the proximal straight tubule, with lesser levels present in the medullary
thick ascending limb (MTAL), and it was not detected in the glomerulus or inner medulla. In contrast, IGF-I receptor mRNA was concentrated in the glomerulus, distal nephron, and collecting system. The only point of convergence for GHR and IGF-I receptor mRNAs was in the MTAL, where IGF-I mRNA was localized. This segregation of GHR and IGF-I receptor gene expression in the kidney suggests that each hormone has distinct spheres of action along the nephron, with GH acting directly on the proximal straight tubule, whereas IGF-I may act on the glomerulus, distal nephron, and collecting duct. GHR expression in the MTAL, which is the site of renal IGF-I synthesis, supports the view that GH has a direct effect on renal IGF-I synthesis. Finally, it appears that in the kidney, as in other GH-sensitive tissues, GH may regulate its receptor levels. (Endocrinology 131: 3061-3066, 1992)
G
renal and glomerular enlargement without evidence of sclerosis (11-13). The recent cloning of the GH receptor (GHR; Ref. 14) has made it possible to begin dissecting the direct and indirect aspectsof GH action in a given tissueby meansof mapping the distinctive sites of GHR, IGF-I, and IGF-I receptor gene expression. Underlying this approach is the view that GHR gene expressionin the absenceof evidence of IGF-I receptor gene expression suggeststhat GH acts directly, i.e. without the mediation of IGF-I, in that site. Conversely, the localization of IGF-I receptor messengerRNA (mRNA) in a site without evidence of local GHR gene expressionwould indicate that effects of GH on this tissuewould likely be mediated by IGF-I. Moreover, the presence of GHR mRNA in cells which also contain IGF-I mRNA indicates that stimulation of IGF-I synthesis is a likely feature of GH action in these particular cells. Finally, coexpression of GHR and IGF-I receptors would support the possibility of combined or complementary GH and IGF-I action, i.e. the dual effector hypothesis(15).
H HAS important effects on a number of aspects of renal function, including renal plasma flow, glomerular filtration rate, renal gluconeogenesis,and tubular phosphate reabsorption (reviewed in Ref. 1). It is unclear, however, to what extent these parameters are affected by GH directly and to what extent by the GH-stimulated production of insulin-like growth factor-I (IGF-I). GH stimulates the synthesis and secretion of hepatic IGF-I, which contributes largely to the circulating IGF-I pool (2). GH also stimulates the local synthesis of IGF-I in specific peripheral tissues, including the kidney (3-5). Given the observation that there is a time lag of several hours between GH administration and the ensuing renal hemodynamic changes, and the fact that administration of IGF-I produces a prompt increase in renal plasma flow and glomerular filtration rate, it has been inferred that GH’s hemodynamic effects are largely mediated by IGF-I (6, 7). Some metabolic effects, however, may be due to GH itself, since GH has been shown to induce phosphate absorption and gluconeogenesisby renal tubules in vitro (8, 9). It is possible, however, that GH acts by stimulating IGF-I production in these assays,and since IGF-I has been shown to have similar in vitro effects (9, lo), the issueremainsunclear. GH and IGF-I also have significant effects on renal size and morphology. Excess GH levels are associated with renal hypertrophy and glomerular sclerosis,whereas excesscirculating IGF-I in the absenceof elevated GH levels results in Received July 14, 1992. Address all correspondence and requests for reprints to: Dr. Edward Chin, National Institutes of Health, Building 10, Room lON262, Bethesda, Maryland 20892.
Materials Experimental
and Methods
animals
Sprague-Dawley rats were obtained from Taconic Farms (Germantown, NY) and used in a protocol approved by the NlCHD Animal Use Committee. Embryonic day 20 (E20) fetuses were removed from timed pregnant dams by hysterotomy, their kidneys removed and frozen over dry ice. Kidneys were also obtained from male rats 5, 16, 40, and approximately 100 days after birth (n = 3 at each age group). Frozen sections were cut at a thickness of 10 pm, thaw-mounted on poly-Llysine-coated slides, and stored at -70 C until use.
3061
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RENAL
3062
GHR
GENE
Endo * 1992 Voll31. No 6
EXPRESSION
FIG. 1. Ontogeny
of renal GHR gene expression as shown by in situ hybridization. Hematoxylin and eosin (HE)-stained sections are shown on the left side of the figure, and film autoradiographs of the sections hybridized to the GHR cRNA probe on the right. All sections were hybridized in the same experiment and exposed for the same time. Ad, Adrenal; IM, inner medulla; Cx, cortex; arrowheads, medullary ray. Bar = 1.6 mm.
Hypophysectomy
and GH treatment
Female Sprague-Dawley rats, hypophysectomized (Hx) or shamoperated at 20 days of age, were obtained from Taconic Farms. Hx rats demonstrating a weight gain 10% or less that gained by sham-operated rats for the first 5 postoperative days were judged to have undergone successful pituitary ablation and were apportioned into groups receiving diluent or GH beginning on the sixth day after surgery. Hx rats received 150 rg GH (NIDDK-Rat GH-B-12; AFP-10478C obtained from the National Hormone and Pituitary Program, Baltimore, MD) ip, or an equal volume of diluent twice a day. Rats were given water containing 5% sucrose and rat chow ad libitum. Rats were killed in groups of three (1 sham + diluent, 1 Hx + diluent, 1 Hx + GH) after 1, 2, and 3 days of treatment, at 7, 8, and 9 days post surgery, respectively.
extracellular region, the transmembrane domain, and a portion of the intracellular region (16). The rat IGF-I probe contained 376 bases complementary to part of the IGF-I coding region as well as 3’-untranslated region sequences (17). The rat IGF-I receptor probe contained 265 bases complementary to 15 bases of 5’-untranslated region and to the region encoding the signal peptide and the first 53 amino acids of the a-subunit of the rat type-1 IGF receptor (18). A control sense probe was made by transcribing this receptor clone insert in the reverse direction. The details of the in situ hybridization protocol have been published previously (5). Controls in the form of parallel tissue sections hybridized to a sense probe were hybridized, washed, and exposed in the same experiments. The background or nonspecific signal from these sections was minimal.
Densitometry In situ hybridization
and complementary
The rat GHR probe was a 900-base the region encoding the signal peptide
RNA (cRNA) probes
BglII fragment
complementary to and the coding sequence for the
Results were quantified ously described (19). Two were selected for analysis,
by computer-assisted densitometry as previsections from the left kidney of each animal and all sections were anatomically matched
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RENAL
GHR GENE EXPRESSION
3063
to include papilla, outer medulla, and cortex. All sections from sham, Hx, and GH-treated animals were prepared, hybridized, washed, exposed, and analyzed together. Density readings from the outer stripe of the outer medulla (OS/OM) were taken, and an average value was determined.
Results
FIG. 2. Paired bright and darkfield photomicrographs showing the cellular localization of GHR mRNA in the renal cortex. In the emulsion-coated sections the mRNA hybridization signal appears as black grains in the brightfield and as white grains in the darkfield illumination. GHR mRNA is abundant in the tubular epithelial cells of the PSTs (hollow arrows). GHR mRNA is not detected in the glomerulus (G, arrowheads) or in the proximal convoluted tubules (solid arrows). G, Glomerulus. Bar = 50 ym.
GHR mRNA is detected in the rat kidney as early as embryonic day 20 (E20) and gradually increasesin abundance until postnatal day 40 when stable adult levels are attained (Fig. 1). These changes were seen in all animals from each time period. From E20 GHR mRNA is concentrated in the outer stripe of the outer medulla (OS/OM) and in the medullary rays which penetrate the cortex. By postnatal day 16, GHR mRNA is also detected in the inner stripe of the outer medulla (IS/OM, Fig. 1). GHR mRNA is localized in the straight portion of the proximal tubule (PST), a part of the nephron which is concentrated in medullary rays and OS/OM (Fig. 2). PSTs are identified not only by their distinctive localization in medullary rays and OS/OM but also by the presence of a brush border and linear appearance (Fig. 2). Hybridization is also detected in some proximal tubule segmentslocated in the renal cortex (i.e. not in the OS/OM or medullary rays; Fig. 2) but was not above background levels in glomeruli. Becausethe developmental time course demonstrated by renal GHR gene expression was parallel to the ontogeny of GH-stimulated growth in the rat, we examined the effect of hypophysectomy (Hx) and GH replacement treatment on renal GHR gene expression. Figure 3 shows the results of hypophysectomy in the absenceand presence of GH treatment. Renal GHR mRNA levels were reduced after hypophysectomy and were normalized by GH treatment. The effect of GH on renal GHR mRNA levels was maximal after just 1 day of GH treatment and was maintained after 2 and 3 days of treatment. GHR mRNA levels as measured by computer-assisteddensitometry were similar after 1, 2, and 3 days of GH treatment, so these values were pooled. Mean values for each group, expressedin arbitrary densitometric units were: sham, 155.38 + 8.57 (n = 4); Hx, 116.77 + 1.57 (n = 4); and GH-treated, 137.55 + 4.03 (n = 3). The value for the Hx group was significantly lower than either the sham or GH-treated groups. (P c 0.005 by one-way analysis of variance). We have previously shown that IGF-I mRNA is localized in medullary thick ascendinglimbs (MTAL) and IGF-I receptor mRNA in glomeruli and distal nephron, including MTALs and collecting ducts (5). In order to directly compare anatomical patterns of GHR, IGF-I, and IGF-I receptor gene expression, sequential sections from a single kidney were hybridized to cRNA probes for these different mRNAs, and the results are shown in Fig. 4. The GHR-hybridized sections were deliberately over-exposed (with respect to the proximal tubule signal) in order to bring up the pattern in the inner stripe. GHR, IGF-I receptor, and IGF-I mRNAs are colocalized in TALs occupying the IS/OM. GHR and IGF-I receptor gene expression are both present in the OS/OM and medullary ray but arise from different nephron segments.The IGF-I receptor hybridization signal in the medullary rays arisesfrom distal nephron segmentsand collecting ducts, not
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RENAL
3064
GHR GENE EXPRESSION
Endo. Voll31.
1992 No 6
FIG. 3. Effects of hypophysectomy with and without GH treatment on renal GHR mRNA. Rats were Hx or sham-operated at 20 days of age. GH or diluent treatment commenced 5 days afterwards (Hx and Hx/GH, respectively). This figure shows representative film autoradiographs from kidney sections taken from littermates after 3 days of GH or diluent computer-assisted densitometry, after just 1 day of treatment; however, the area demonstrating GHR gene expression was reduced, as was the size of the entire kidney-compared to the sham controls. Bar = 1.26 mm.
from proximal tubules (5). Note that GHR and IGF-I mRNAs are localized in the perinephric fat, and GHR and IGF-I receptor mRNAs are abundant in the adrenal cortex (Fig. 4). The segmental distributions of GHR, IGF-I receptor, and IGF-I mRNAs in the nephron are diagrammed in Fig. 5.
Discussion Previous studies have shown that GHR mRNA is most abundant in the rat liver, but significant expression is also found in other rat tissues,including the kidney (16, 20). Two major transcripts, representing alternatively spliced RNAs (reviewed in Ref. 21) are found in most tissues.A high mol wt transcript (4.5 kilobases)representsthe membrane-bound GH receptor, and a lower weight species (1.2 kilobases) encodes the extracellular portion of this receptor, which is apparently secretedfrom hepatocytes into the circulation and functions as a GH binding protein (22). The liver contains a high percentage of the low mol wt soluble binding protein transcript, whereas in other tissues,including the kidney, the high mol wt receptor transcript is the predominant species (20). The probe used in this study correspondsto the extracellular portion of the GH receptor protein and thus hybridizes to both transcripts. The anatomical pattern of GHR gene expression in the kidney has distinct implications with respect to GH’s physiological effects on renal function. GHR mRNA is most abundant in the straight portion of the proximal tubule, where IGF-I receptor mRNA is not detected, suggestingthat GH directly regulates metabolic functions in this portion of the nephron. The proximal tubule is the site of renal gluco-
neogenesis,and the kidney may produce as much as 50% of the glucose synthesized in the counter-regulatory response (23). Since GH stimulates gluconeogenesisin its capacity as a counter-regulatory hormone, it seemslikely that its direct action on these renal tubules may be an important part of the hypoglycemic response. In support of this view is the observation that GH stimulates gluconeogenesisfrom proximal tubules in vitro (8). The proximal tubule is also a major site of renal phosphate reabsorption, which is stimulated by GH (9). IGF-I has also been reported to stimulate phosphate reabsorption from proximal tubules in vitro (10). This observation could be explained by a low level of IGF-I receptor expression in proximal tubules, perhaps up-regulated by in vitro conditions, or by the contamination of preparations by cortical thick ascendinglimbs. Renal phosphate reabsorption is necessaryto maintain the high serum phosphate levels required during skeletal growth, hence GH’s stimulation of renal phosphate reabsorption is harmonious with its role in promoting somatic growth. It is notable that although both gluconeogenesisand phosphate reabsorption take place in all divisions of the proximal tubule, GHR mRNA is concentrated predominantly in the straight portion. This finding suggeststhat GH may have some additional role in proximal tubule function, which is specific to this latter domain, GHR mRNA is also found in the MTALs, which are concentrated in the IS/OM. Since this is the site of renal IGF-I mRNA localization, it is likely that GH may act here to stimulate renal IGF-I production. In support of this view, hypophysectomy has been shown to reduce and GH treatment to increaserenal IGF-I mRNA content (4, 5). We have
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RENAL
GHR GENE EXPRESSION
3065
CD
IGFR- I
IGF-I
FIG. 5. Schematic
outline of the distributions of GHR, IGF-I receptor IGF-I mRNAs in the rat nephron. PCT, Proximal convolutedtubule;TAL, thick ascending limb of the loop of Henle; DCT, distal convoluted tubule; CD, collecting duct. (IGFR-I),
and
GH treatment. Other studies have also reported a positive relationship between GH levels and GHR gene expression (20, 23-26).
In summary, the present study has describeda very distinct anatomical pattern of GHR gene expression in the kidney which has significant implications for GH’s role in renal function. The abundance of GHR mRNA in the PST, and the lack of IGF-I receptor mRNA in this part of the nephron suggeststhat GH has important direct effects on proximal tubule function. The localization of GHR mRNA in the MTAL, where renal IGF-I is synthesized, supports the view that GH has a direct effect on renal IGF-I synthesis. FIG. 4. Comparison
of patterns of gene expression for the IGF-I receptor (IGFR-I), GHR, and IGF-I in the rat kidney. These film autoradiographs are of serial sections hybridized to the respective cRNA probes. IGFR-I and IGF-I films were exposed for 4 days, and the GHR film was exposed for 10 days. Ad, Adrenal. Bar = 1.6 mm.
proposed that GH stimulates renal IGF-I production in response to varying dietary protein loads, and that IGF-I produced in this site serves to augment MTAL ability to reabsorb sodium in the face of the increased solute load imposed by high-protein diets (5). In this setting, renal IGFI could induce an increasein GFR via a reduction in tubuloglomerular feedback. The developmental time course of renal GHR expression demonstrated in this study agrees with that reported in a previous study employing solution hybridization (16) and is consistent with the postnatal time course of maturation of renal function in the rat. GH appears to influence the level of renal GHR gene expression, since GHR mRNA was reduced after hypophysectomy and normalized in responseto
Acknowledgments We are grateful to Drs. Haim Werner, Charles T. Roberts, Jr., Derek LeRoith, and Lawrence S. Mathews for supplying the clones used for probe synthesis and to Ricardo Dreyfuss and John Ward for expert photography.
References 1. Hammerman 2.
3.
MR 1989 The growth
hormone-insulin-like growth Am J Physiol257:F504-F514 Schwander JC, Hauri C, Zapf J, Froesch ER 1983 Synthesis and secretion of insulin-like growth factor and its binding proteins by the perfused rat liver: dependence on growth hormone status. Endocrinology 113:297-302 D’Ercole AJ, Stiles AD, Underwood LE 1984 Tissue concentrations of somatomedin C: further evidence for multiple sites of synthesis and autocrine/paracrine mechanisms of action. Proc Nat1 Acad Sci USA 81:935-939 factor
axis in the kidney.
4. Bortz JD, Rotwein P, DeVol D, Bechtel PJ, Hansen VA, Hammerman MR 1988 Focal expression of insulin-like growth factor I in rat kidney
collecting
duct. J Cell Biol 107:811-819
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3066
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GHR
GENE
5. Chin E, Zhou J, Bondy C 1992 Anatomical
relationships in the and IGF-I receptor gene expression in the 130:3237-3245 Hirschberg R, Kopple JD 1989 Effects of growth hormone and IGF-I on r&al fun%on. Kidney Int 36:S20-556 Guler H-P, Schmid C. Zauf 1. Froesch ER 1989 Effects of recombinant insulin-like growth’ factor I on insulin secretion and renal function in normal human subjects. Proc Natl Acad Sci USA pattern of IGF-I, IGFBP-I rat kidney. Endocrinology
6. 7.
86:2868-2872 8. Hammerman MR, Karl IE, Hruska KA 1980 Regulation renal vesicles phosphate transport thyroid hormone. Biochim Biophys
9. Rogers SA, Karl IE, Hammerman directly stimulates gluconeogenesis Am J Physiol257:E751-E756 10.
on renal phosphate D. Endocrinology
and
plasma
1,25-
4893 Van Buul-Offers
E, Froesch ER 1988 Recombinant growth and has distinct effects on organ rats. F’roc Nat1 Acad Sci USA 85:4889and
weight
hormone
and
IGF-I
mice.
20. 21.
Endocrinology
25.
hormone receptor and serum binding protein: purification, and expression. Nature 330:537-543 Green H, Morikawa M, Nixon T 1985 A dual effector
cloning theory
of
J Biol Chem
86:7451-7455 S 1989 Developmental and physiological regulation of aldose reductase mRNA expression in renal medulla. Mol Endocrinol 3:1409-1416 Tiong TS, Herington AC 1991 Tissue distribution, characterization, and regulation of the mRNA for growth hormone receptor and serum binding protein in the rat. Endocrinology 129:1628-1634 Mathews LS 1991 Molecular biology of growth hormone receptors. Trends in Endocrinology and Metabolism 1:179-l 80
S, Lightman
22. Sadeghi H, Wang BS, Lumanglas AL, Logan JS, Baumbach WR
of Snell
14. Leung DW, Spencer SA, Cachianes G, Hammonds GT, Collins C, Henzel WJ, Barnard R, Waters MJ, Wood WI 1987 Growth
gene expression.
gene. Proc Nat1 Acad Sci USA
24. in transgenic
29:195-198
of rat growth 264:9905-9910 Lowe Jr WL, Lasky SR, LeRoith D, Roberts Jr CT 1988 Distribution and regulation of rat IGF-I mRNA encoding alternative carboxyterminal E-peptides: evidence for differential processing and regulation in liver. Mol Endocrinol 2:528-535 receptor
19. Bandy C, Lightman
23. the length
Differentiation
18. Werner H, Woloschek M, Adamo M, Shen-Orr Z, Roberts Jr CT, LeRoith D 1989 Developmental regulation of the rat IGF-I receptor
Quaife CJ, Mathews LS, Pinkert CA, Hammer RE, Brinster RL, Palmiter RD 1989 Histopathology associated with elevated levels of growth 124:40-48
15.
17.
action.
1992 No 6
Mathews LS, Enberg 8, Norstedt G 1989 Regulation hormone
S, Ueda I, Van den Brande JL 1986 Biosynthetic
somatomedin C (IGF-I) increases dwarf mice. Pediatr Res 20:825-833 13.
transport 127:453-459
Guler H-P, Zapf J, Scheiwiller human IGF-I stimulates size in hypophysectomized
12.
growth-hormone 16.
Caverzasio J, Faundez R, Fleisch H, Bonjour JP 1990 Stimulatory effect of IGF-I dihydroxyvitamin
11.
of canine by growth hormone and paraActa 603:322-335 MR 1989 Growth hormone in canine renal proximal tubule.
Endo. Voll31.
EXPRESSION
1990 Identification of the origin of the growth hormone binding protein in rat serum. Mol Endocrinol4:1799-1805 Gullans SR, Hebert SC 1991 Metabolic basis of ion transport. In: The Kidney, Brenner 8, Rector FC (eds) WB Saunders, Philadelphia, pp 76-109 Vikman K, Carlsson B, Billig H, Eden S 1991 Expression and regulation of GH receptor mRNA in rat adipocytes-and adipose tissue: GH rezulation of GH receotor mRNA. Endocrinoloev Y, 1 129:1155-1161” Frick GP, Leonard JL, Goodman H 1990 Effect of hypophysectomy on GH receptor gene expression in rat tissues. Endocrinology
126:3076-3082 26. Nilsson A, Carlsson B, Mathews hormone regulation seal chondrocytes.
L, Issakson OGP 1990 Growth
of GH receptor mRNA in cultured Mol Cell Endocrinol 70:237-246
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rat epiphy-
Renal Growth Hormone Receptor Relationship to Renal Insulin-Like System EDWARD
CHIN,
JIAN ZHOU,
AND
CAROLYN
Gene Expression: Growth Factor
A. BONDY
Developmental Endocrinology Branch, National Institute of Child Health and Human National Institutes of Health, Bethesda, Maryland 20892
Development,
ABSTRACT In order to elucidate potential sites of direct GH action on the kidnev. we used in situ hvbridization to localize GH receutor (GHR) gene expression during the course of development and in the adult rat. In order to illuminate potential interactions between GH and insulinlike growth factor-I (IGF-I) in regulating renal function, we compared the anatomical localization of GHR messenger RNA (mRNA) with that for the IGF-I receptor and for IGF-I in the rat kidney. Low levels of GHR mRNA were present in the kidney from before birth and increased in abundance until postnatal day 40. Hypophysectomy resulted in a decrease and GH treatment resulted in an increase in renal GHR mRNA levels. Renal GHR mRNA was most abundant in the proximal straight tubule, with lesser levels present in the medullary
thick ascending limb (MTAL), and it was not detected in the glomerulus or inner medulla. In contrast, IGF-I receptor mRNA was concentrated in the glomerulus, distal nephron, and collecting system. The only point of convergence for GHR and IGF-I receptor mRNAs was in the MTAL, where IGF-I mRNA was localized. This segregation of GHR and IGF-I receptor gene expression in the kidney suggests that each hormone has distinct spheres of action along the nephron, with GH acting directly on the proximal straight tubule, whereas IGF-I may act on the glomerulus, distal nephron, and collecting duct. GHR expression in the MTAL, which is the site of renal IGF-I synthesis, supports the view that GH has a direct effect on renal IGF-I synthesis. Finally, it appears that in the kidney, as in other GH-sensitive tissues, GH may regulate its receptor levels. (Endocrinology 131: 3061-3066, 1992)
G
renal and glomerular enlargement without evidence of sclerosis (11-13). The recent cloning of the GH receptor (GHR; Ref. 14) has made it possible to begin dissecting the direct and indirect aspectsof GH action in a given tissueby meansof mapping the distinctive sites of GHR, IGF-I, and IGF-I receptor gene expression. Underlying this approach is the view that GHR gene expressionin the absenceof evidence of IGF-I receptor gene expression suggeststhat GH acts directly, i.e. without the mediation of IGF-I, in that site. Conversely, the localization of IGF-I receptor messengerRNA (mRNA) in a site without evidence of local GHR gene expressionwould indicate that effects of GH on this tissuewould likely be mediated by IGF-I. Moreover, the presence of GHR mRNA in cells which also contain IGF-I mRNA indicates that stimulation of IGF-I synthesis is a likely feature of GH action in these particular cells. Finally, coexpression of GHR and IGF-I receptors would support the possibility of combined or complementary GH and IGF-I action, i.e. the dual effector hypothesis(15).
H HAS important effects on a number of aspects of renal function, including renal plasma flow, glomerular filtration rate, renal gluconeogenesis,and tubular phosphate reabsorption (reviewed in Ref. 1). It is unclear, however, to what extent these parameters are affected by GH directly and to what extent by the GH-stimulated production of insulin-like growth factor-I (IGF-I). GH stimulates the synthesis and secretion of hepatic IGF-I, which contributes largely to the circulating IGF-I pool (2). GH also stimulates the local synthesis of IGF-I in specific peripheral tissues, including the kidney (3-5). Given the observation that there is a time lag of several hours between GH administration and the ensuing renal hemodynamic changes, and the fact that administration of IGF-I produces a prompt increase in renal plasma flow and glomerular filtration rate, it has been inferred that GH’s hemodynamic effects are largely mediated by IGF-I (6, 7). Some metabolic effects, however, may be due to GH itself, since GH has been shown to induce phosphate absorption and gluconeogenesisby renal tubules in vitro (8, 9). It is possible, however, that GH acts by stimulating IGF-I production in these assays,and since IGF-I has been shown to have similar in vitro effects (9, lo), the issueremainsunclear. GH and IGF-I also have significant effects on renal size and morphology. Excess GH levels are associated with renal hypertrophy and glomerular sclerosis,whereas excesscirculating IGF-I in the absenceof elevated GH levels results in Received July 14, 1992. Address all correspondence and requests for reprints to: Dr. Edward Chin, National Institutes of Health, Building 10, Room lON262, Bethesda, Maryland 20892.
Materials Experimental
and Methods
animals
Sprague-Dawley rats were obtained from Taconic Farms (Germantown, NY) and used in a protocol approved by the NlCHD Animal Use Committee. Embryonic day 20 (E20) fetuses were removed from timed pregnant dams by hysterotomy, their kidneys removed and frozen over dry ice. Kidneys were also obtained from male rats 5, 16, 40, and approximately 100 days after birth (n = 3 at each age group). Frozen sections were cut at a thickness of 10 pm, thaw-mounted on poly-Llysine-coated slides, and stored at -70 C until use.
3061
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RENAL
3062
GHR
GENE
Endo * 1992 Voll31. No 6
EXPRESSION
FIG. 1. Ontogeny
of renal GHR gene expression as shown by in situ hybridization. Hematoxylin and eosin (HE)-stained sections are shown on the left side of the figure, and film autoradiographs of the sections hybridized to the GHR cRNA probe on the right. All sections were hybridized in the same experiment and exposed for the same time. Ad, Adrenal; IM, inner medulla; Cx, cortex; arrowheads, medullary ray. Bar = 1.6 mm.
Hypophysectomy
and GH treatment
Female Sprague-Dawley rats, hypophysectomized (Hx) or shamoperated at 20 days of age, were obtained from Taconic Farms. Hx rats demonstrating a weight gain 10% or less that gained by sham-operated rats for the first 5 postoperative days were judged to have undergone successful pituitary ablation and were apportioned into groups receiving diluent or GH beginning on the sixth day after surgery. Hx rats received 150 rg GH (NIDDK-Rat GH-B-12; AFP-10478C obtained from the National Hormone and Pituitary Program, Baltimore, MD) ip, or an equal volume of diluent twice a day. Rats were given water containing 5% sucrose and rat chow ad libitum. Rats were killed in groups of three (1 sham + diluent, 1 Hx + diluent, 1 Hx + GH) after 1, 2, and 3 days of treatment, at 7, 8, and 9 days post surgery, respectively.
extracellular region, the transmembrane domain, and a portion of the intracellular region (16). The rat IGF-I probe contained 376 bases complementary to part of the IGF-I coding region as well as 3’-untranslated region sequences (17). The rat IGF-I receptor probe contained 265 bases complementary to 15 bases of 5’-untranslated region and to the region encoding the signal peptide and the first 53 amino acids of the a-subunit of the rat type-1 IGF receptor (18). A control sense probe was made by transcribing this receptor clone insert in the reverse direction. The details of the in situ hybridization protocol have been published previously (5). Controls in the form of parallel tissue sections hybridized to a sense probe were hybridized, washed, and exposed in the same experiments. The background or nonspecific signal from these sections was minimal.
Densitometry In situ hybridization
and complementary
The rat GHR probe was a 900-base the region encoding the signal peptide
RNA (cRNA) probes
BglII fragment
complementary to and the coding sequence for the
Results were quantified ously described (19). Two were selected for analysis,
by computer-assisted densitometry as previsections from the left kidney of each animal and all sections were anatomically matched
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RENAL
GHR GENE EXPRESSION
3063
to include papilla, outer medulla, and cortex. All sections from sham, Hx, and GH-treated animals were prepared, hybridized, washed, exposed, and analyzed together. Density readings from the outer stripe of the outer medulla (OS/OM) were taken, and an average value was determined.
Results
FIG. 2. Paired bright and darkfield photomicrographs showing the cellular localization of GHR mRNA in the renal cortex. In the emulsion-coated sections the mRNA hybridization signal appears as black grains in the brightfield and as white grains in the darkfield illumination. GHR mRNA is abundant in the tubular epithelial cells of the PSTs (hollow arrows). GHR mRNA is not detected in the glomerulus (G, arrowheads) or in the proximal convoluted tubules (solid arrows). G, Glomerulus. Bar = 50 ym.
GHR mRNA is detected in the rat kidney as early as embryonic day 20 (E20) and gradually increasesin abundance until postnatal day 40 when stable adult levels are attained (Fig. 1). These changes were seen in all animals from each time period. From E20 GHR mRNA is concentrated in the outer stripe of the outer medulla (OS/OM) and in the medullary rays which penetrate the cortex. By postnatal day 16, GHR mRNA is also detected in the inner stripe of the outer medulla (IS/OM, Fig. 1). GHR mRNA is localized in the straight portion of the proximal tubule (PST), a part of the nephron which is concentrated in medullary rays and OS/OM (Fig. 2). PSTs are identified not only by their distinctive localization in medullary rays and OS/OM but also by the presence of a brush border and linear appearance (Fig. 2). Hybridization is also detected in some proximal tubule segmentslocated in the renal cortex (i.e. not in the OS/OM or medullary rays; Fig. 2) but was not above background levels in glomeruli. Becausethe developmental time course demonstrated by renal GHR gene expression was parallel to the ontogeny of GH-stimulated growth in the rat, we examined the effect of hypophysectomy (Hx) and GH replacement treatment on renal GHR gene expression. Figure 3 shows the results of hypophysectomy in the absenceand presence of GH treatment. Renal GHR mRNA levels were reduced after hypophysectomy and were normalized by GH treatment. The effect of GH on renal GHR mRNA levels was maximal after just 1 day of GH treatment and was maintained after 2 and 3 days of treatment. GHR mRNA levels as measured by computer-assisteddensitometry were similar after 1, 2, and 3 days of GH treatment, so these values were pooled. Mean values for each group, expressedin arbitrary densitometric units were: sham, 155.38 + 8.57 (n = 4); Hx, 116.77 + 1.57 (n = 4); and GH-treated, 137.55 + 4.03 (n = 3). The value for the Hx group was significantly lower than either the sham or GH-treated groups. (P c 0.005 by one-way analysis of variance). We have previously shown that IGF-I mRNA is localized in medullary thick ascendinglimbs (MTAL) and IGF-I receptor mRNA in glomeruli and distal nephron, including MTALs and collecting ducts (5). In order to directly compare anatomical patterns of GHR, IGF-I, and IGF-I receptor gene expression, sequential sections from a single kidney were hybridized to cRNA probes for these different mRNAs, and the results are shown in Fig. 4. The GHR-hybridized sections were deliberately over-exposed (with respect to the proximal tubule signal) in order to bring up the pattern in the inner stripe. GHR, IGF-I receptor, and IGF-I mRNAs are colocalized in TALs occupying the IS/OM. GHR and IGF-I receptor gene expression are both present in the OS/OM and medullary ray but arise from different nephron segments.The IGF-I receptor hybridization signal in the medullary rays arisesfrom distal nephron segmentsand collecting ducts, not
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FIG. 3. Effects of hypophysectomy with and without GH treatment on renal GHR mRNA. Rats were Hx or sham-operated at 20 days of age. GH or diluent treatment commenced 5 days afterwards (Hx and Hx/GH, respectively). This figure shows representative film autoradiographs from kidney sections taken from littermates after 3 days of GH or diluent computer-assisted densitometry, after just 1 day of treatment; however, the area demonstrating GHR gene expression was reduced, as was the size of the entire kidney-compared to the sham controls. Bar = 1.26 mm.
from proximal tubules (5). Note that GHR and IGF-I mRNAs are localized in the perinephric fat, and GHR and IGF-I receptor mRNAs are abundant in the adrenal cortex (Fig. 4). The segmental distributions of GHR, IGF-I receptor, and IGF-I mRNAs in the nephron are diagrammed in Fig. 5.
Discussion Previous studies have shown that GHR mRNA is most abundant in the rat liver, but significant expression is also found in other rat tissues,including the kidney (16, 20). Two major transcripts, representing alternatively spliced RNAs (reviewed in Ref. 21) are found in most tissues.A high mol wt transcript (4.5 kilobases)representsthe membrane-bound GH receptor, and a lower weight species (1.2 kilobases) encodes the extracellular portion of this receptor, which is apparently secretedfrom hepatocytes into the circulation and functions as a GH binding protein (22). The liver contains a high percentage of the low mol wt soluble binding protein transcript, whereas in other tissues,including the kidney, the high mol wt receptor transcript is the predominant species (20). The probe used in this study correspondsto the extracellular portion of the GH receptor protein and thus hybridizes to both transcripts. The anatomical pattern of GHR gene expression in the kidney has distinct implications with respect to GH’s physiological effects on renal function. GHR mRNA is most abundant in the straight portion of the proximal tubule, where IGF-I receptor mRNA is not detected, suggestingthat GH directly regulates metabolic functions in this portion of the nephron. The proximal tubule is the site of renal gluco-
neogenesis,and the kidney may produce as much as 50% of the glucose synthesized in the counter-regulatory response (23). Since GH stimulates gluconeogenesisin its capacity as a counter-regulatory hormone, it seemslikely that its direct action on these renal tubules may be an important part of the hypoglycemic response. In support of this view is the observation that GH stimulates gluconeogenesisfrom proximal tubules in vitro (8). The proximal tubule is also a major site of renal phosphate reabsorption, which is stimulated by GH (9). IGF-I has also been reported to stimulate phosphate reabsorption from proximal tubules in vitro (10). This observation could be explained by a low level of IGF-I receptor expression in proximal tubules, perhaps up-regulated by in vitro conditions, or by the contamination of preparations by cortical thick ascendinglimbs. Renal phosphate reabsorption is necessaryto maintain the high serum phosphate levels required during skeletal growth, hence GH’s stimulation of renal phosphate reabsorption is harmonious with its role in promoting somatic growth. It is notable that although both gluconeogenesisand phosphate reabsorption take place in all divisions of the proximal tubule, GHR mRNA is concentrated predominantly in the straight portion. This finding suggeststhat GH may have some additional role in proximal tubule function, which is specific to this latter domain, GHR mRNA is also found in the MTALs, which are concentrated in the IS/OM. Since this is the site of renal IGF-I mRNA localization, it is likely that GH may act here to stimulate renal IGF-I production. In support of this view, hypophysectomy has been shown to reduce and GH treatment to increaserenal IGF-I mRNA content (4, 5). We have
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CD
IGFR- I
IGF-I
FIG. 5. Schematic
outline of the distributions of GHR, IGF-I receptor IGF-I mRNAs in the rat nephron. PCT, Proximal convolutedtubule;TAL, thick ascending limb of the loop of Henle; DCT, distal convoluted tubule; CD, collecting duct. (IGFR-I),
and
GH treatment. Other studies have also reported a positive relationship between GH levels and GHR gene expression (20, 23-26).
In summary, the present study has describeda very distinct anatomical pattern of GHR gene expression in the kidney which has significant implications for GH’s role in renal function. The abundance of GHR mRNA in the PST, and the lack of IGF-I receptor mRNA in this part of the nephron suggeststhat GH has important direct effects on proximal tubule function. The localization of GHR mRNA in the MTAL, where renal IGF-I is synthesized, supports the view that GH has a direct effect on renal IGF-I synthesis. FIG. 4. Comparison
of patterns of gene expression for the IGF-I receptor (IGFR-I), GHR, and IGF-I in the rat kidney. These film autoradiographs are of serial sections hybridized to the respective cRNA probes. IGFR-I and IGF-I films were exposed for 4 days, and the GHR film was exposed for 10 days. Ad, Adrenal. Bar = 1.6 mm.
proposed that GH stimulates renal IGF-I production in response to varying dietary protein loads, and that IGF-I produced in this site serves to augment MTAL ability to reabsorb sodium in the face of the increased solute load imposed by high-protein diets (5). In this setting, renal IGFI could induce an increasein GFR via a reduction in tubuloglomerular feedback. The developmental time course of renal GHR expression demonstrated in this study agrees with that reported in a previous study employing solution hybridization (16) and is consistent with the postnatal time course of maturation of renal function in the rat. GH appears to influence the level of renal GHR gene expression, since GHR mRNA was reduced after hypophysectomy and normalized in responseto
Acknowledgments We are grateful to Drs. Haim Werner, Charles T. Roberts, Jr., Derek LeRoith, and Lawrence S. Mathews for supplying the clones used for probe synthesis and to Ricardo Dreyfuss and John Ward for expert photography.
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rat epiphy-
