Altered expression of insulin-like growth factor-I (IGF-I) and IGF receptor genes after unilateral nephrectomy in immature rats.

0013-7227/92/1301-0249$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine

Vol. 130, No. 1

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Altered Expression of Insulin-Like Growth Factor-I (IGF-I) and IGF Receptor Genes after Unilateral Nephrectomy in Immature Rats* SUSAN E. MULRONEY, CHARLES T. ROBERTS,

AVIAD JR.,

HARAMATI,

AND DEREK

HAIM LEROITH

WERNER,

CAROLYN

BONDY,

Department of Physiology and Biophysics, Georgetown University School of Medicine (S.E.M., A.H.), Washington, D.C. 20007; and the Section on Molecular and Cellular Physiology, Diabetes Branch, National Institute of Diabetes ana’ Kidney and Digestive Diseases (S.E.M., H. W., C.T.R., D.L.), and the Developmental Endocrinology Branch, National Institute of Child Health and Human Development (C.B.), National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT. There is a developmental difference in the initial phase of compensatory renal growth (CRG) following unilateral nephrectomy (UNX), in that CRG is GH-dependent in adult rats and GH-independent in immature rats. Furthermore, CRG in immature rats is associated with an increase in renal IGF-I mRNA, an effect not seen in adult rats. In this study we have examined the age-related differences in expression of the insulin-like growth factor-I (IGF-I) and IGF-II genes as well as in IGF-I and IGF-II receptors and membrane binding after UNX. Immature (22-24 days of age) and adult (4 months of age) male Wistar rats underwent a sham operation or left UNX and were killed 24 or 48 h later. Levels of mRNA for IGF-I and IGFII and their receptors were determined in the left (control) and right (compensated) remnant kidneys using solution hvbridizati&/RNase protection assays. Steady state levels of IGF-I mRNA as well as IGF-I receptor and IGF-II/mannose-6-phosphate receptor mRNAs were increased 3- to 4-fold in immature

G

remnant kidneys, but not in adult kidneys. The findings related to IGF-I gene expression were confirmed by in situ hybridization to immature and adult kidney slices. The increase in IGF-I gene expression in the immature remnant kidneys was localized to the thick ascending limbs of the !OODS of Henle. Furthermore. in concert with thechanges in mRNA levels, membrane binding studies showed significant increases in specific binding to IGFI in cortical membranes and increases in specific binding to IGF-II in whole kidney membranes from immature, but not adult, rats. Thus, these findings demonstrate that the initial phase of CRG in the immature rat is associated with increased renal IGF-I gene expression as well as enhanced specific renal binding of IGF-I and IGF-II to plasma membranes and support the notion that this period of rapid renal growth in the immature UNX rat may involve the paracrine influence of the IGFs. (Endocrinology 130: 249-256,1992)

the prepubertal rat during early development. For any putative growth factor to have a relevant role in CRG, its receptors must be present on the cells that are growing, and there should be an early increase in either the level of the growth factor and/or receptor binding to account for the initiation of a growth response. Previous studies in adult rats have reported mixed results regarding the potential role of insulin-like growth factorI (IGF-I) in the CRG after UNX. Both IGF-I and IGFII/mannose-6-phosphate (M-6-P) receptors (3) have been demonstrated in the proximal tubules of the kidney, a primary growth site during CRG. Renal tissue IGF-I levels have also been shown to rise 3-5 days after UNX in adult rats (4-7), although there is some question as to whether this is associated with prior increases in IGF-I gene expression (4, 5). The delayed increase in tissue IGF-I observed after UNX suggests that IGF-I may play a role in sustaining a long term increase in the renal growth rate. However, it remains unclear whether IGF-I

H HAS been implicated in compensatory renal growth (CRG) on the basis that 24 h after unilateral nephrectomy (UNX), circulating pulsatile GH levels increase 3- to 4-fold in the adult, but not the immature, rat (1). Moreover, when the pulses of GH are suppressed using an antagonist to GH-releasing factor (GRF-AN; [N-Ac-Tyr’-A$]GRF-(l-29)NHz)l CRG is attenuated in the adult, but not the immature, rat (2). This suggests that the initial stage of CRG is GH-dependent in the adult rat and GH-independent in the immature rat. Thus, factors other than GH must be initiating CRG in Received July 11, 1991. Address all correspondence and requests for reprints to: Susan E. Mulroney, Ph.D., Georgetown University School of Medicine, Room 256B, Basic Science Building, 3900 Reservoir Road NW, Washington, D.C. 20007. * This work was supported by NIH Grant DK-36111 and a grant from the National Kidnev Foundation. National Can&al Area Chanter (to A.H.); a National Kidney Foundation Fellowshib (to S.E.M.); and a grant from the Washington Affiliate of the American Diabetes Association (to C.T.R.). 249

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IGF mRNA POSTNEPHRECTOMY

plays a role in the initial few days of CRG. The actions of IGF-I on kidney function in uiuo have been well studied. IGF-I administration increases kidney weights in hypophysectomized adult rats (8), and IGF-I infusion also increases the glomerular filtration rate and renal plasma flow (g-11), which are some of the first physiological changes noted during CRG (12, 13). Furthermore, renal hypertrophy has been observed in mice transgenic for IGF-I, again suggesting a possible association between renal growth and IGF-I (14, 15). Thus, there appears to be a potential role for IGF-I in CRG. We have recently reported that renal IGF-I mRNA levels increase 3- to 4-fold 24 and 48 h after UNX in immature, but not adult, rats (16). This supports our initial findings that the stimuli initiating CRG may differ with age (2) and further suggests that CRG in the immature animal may indeed be attributed to changes in renal IGF-I. The objective of the present study was to further investigate the role of IGFs in CRG by examining the time course, cellular localization, and pattern of expression of the genes encoding the IGFs and their receptors in kidneys from immature and adult rats 24 and 48 h post-UNX and to identify whether changes in IGFs and their receptors are associated with the initial renal growth after UNX. Materials

and Methods

Male adult (4-month-old) and immature (22- to 24-day-old) Wistar rats underwent either left nephrectomy (adrenal gland remainedintact) or a shamoperation. Body (and kidney when excised)weights were obtained at the onset of the experiment, and animalswere allowed food and water ad libitum. The rats were killed 24 or 48 h postoperation, and the kidneys were removed, weighed, flash-frozen in liquid nitrogen, and stored at -70 C. Solution

hybridization/RNase

protection

assays

Total RNA from left (control, excised) and right (remnant, compensated)kidneys was prepared by the guanidinium isothiocyanate/cesium chloride technique (17). The RNA was quantified spectrophotometrically by the absorbance at 260 nm, and the integrity of the RNA wasconfirmed by analysisof the ethidium bromide-stained 18s and 28s ribosomal RNA bands.To determine the levels of IGF-I and IGF-II as well as IGF-I receptor and IGF-11/M-6-P receptor mRNA in renal tissue, solution hybridization/RNase protection assayswere performed, as previously described (18-20). Briefly, a 20-g sampleof total RNA washybridized with a “*P-labeledhomologousantisenseRNA probe. After hybridization, RNA samples were digested with RNase-A and -Tl, and the hybrids were extracted with phenol-choloroform, precipitated with ethanol, and electrophoresedon an 8% polyacrylamide-8 M urea denaturing gel. Each samplewas analyzed in multiple assays,and severalautoradiographic exposureswere obtained and quantified by scanning densitometry. Paired Student’s t tests were used to determine significant changes between control and

Endo. Voll30.

1992 No 1

remnant kidneys. Significancebetweengroupswasanalyzed by unpaired Student’s t tests. In situ hybridization In situ hybridization was performed as previously described (21,22). Frozen kidneys were sectioned(16 pm), thaw mounted onto slidesthat were precoated with poly-L-lysine, and stored at -20 C until hybridization. Before hybridization, the sections were fixed in 4% formaldehyde and immersed(10 min) in a solution of 0.25% acetic anhydride, 0.1 M triethanolamine hydrochloride, and 0.9% NaCl. The sectionswere dehydrated by ethanol washes,delipidated with choloroform, rehydrated, and then air dried. The IGF-I antisenseRNA probe wasthe same one previously usedfor the RNaseprotection assays.[35S]CTP and [35S]UTP were usedin the transcription reaction, and after extraction and precipitation, the labeledprobeswere shortened to an averagelength of 150 basesby alkaline hydrolysis. The 35S-labeledprobes (lo7 dpm/ml or -50 rig/ml) were addedto hybridization buffer composedof 50% formamide,0.3 M NaCl, 20 mM Tris-HCl (pH 8), 5 mM EDTA, 500 rg tRNA/ml, 10% dextran sulfate, 10 mM dithiothreitol, and 0.02% eachof BSA, Ficoll, and polyvinylpyrollidone. After the 35S-labeled probe in hybridization buffer was addedto the sections,they were covered with glasscoverslips and placed in humidified chambers for overnight (14- to 16-h) incubation at 55 C. After hybridization, coverslips were removed by washing the slide several times in 4 x SSC (NaCl, Na citrate), followed by dehydration in ethanol and immersionin a solution containing 0.3 M NaCl, 50% formamide,20 mM Tris-HCl, and 1 mM EDTA at 60 C for 15 min. The slides were subsequently treated for 30 min at room temperature with 20 rg/ml RNase-A, followed by 0.1 x SSC for 15 min at 55 C. After air drying, the slideswereexposed to Hyperfilm-beta Max (Amersham,Arlington Heights, IL) for l-3 days, then dipped in Kodak NTB3 nuclear emulsion(Eastman Kodak, Rochester,NY) for l-3 weeks. The slideswere developedand stained with hematoxylin and eosin (H & E) for microscopicevaluation. Membrane

binding

studies

Membrane binding studies were performed as previously described(19). Briefly, on the day of surgery, the left excised kidneys from adult and immature rats were weighed, flashfrozen in liquid nitrogen, and stored at -70 C. After 48 h, the animals were killed, and remnant kidneys were excised, weighed, frozen in liquid nitrogen, and stored. To assessany differential changesin binding within the renal segments,a secondgroupof immature rats wasnephrectomized,the kidneys were bisected, and the medullary and cortical regions were separated.The kidney sectionsweresubsequentlyhomogenized (on ice) in a buffer containing NaHC03, leupeptin, aprotinin, and phenylmethylsulfonylfluoride and centrifuged to obtain the membrane pellet. The membraneswere resuspendedin calcium-free Krebs-Ringersphosphate,and protein measurements were madeusing Bio-Rad assayreagents(Richmond,CA). The membraneswere diluted to a final concentration of 1.2 mg/ml and incubated with [‘251]IGF-I or [1251]IGF-II,and either KrebsRingers phosphate buffer alone (for total binding) or with an excessof cold ligand (>500 rig/ml IGF-I or IGF-II; for nonspecific background binding). Sampleswere analyzed in triplicate,


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IGF mRNA

POSTNEPHRECTOMY

autoradiograph of the increase in IGF-I mRNA in immature rat kidneys 48 h after UNX. Both the exon 1 and exon 2 bands increased to the same extent, although it has been suggested that they encode for primarily GHsensitive (exon 2) and GH-independent (exon 1) transcripts of IGF-I. To identify the local site(s) of endogenous renal IGFI mRNA synthesis and determine if there were any changes in the distribution of these sites associated with CRG, in situ hybridization employing the IGF-I cRNA probe was performed on kidney sections from both immature and adult control and remnant kidneys. IGF-I mRNA was detected in the renal tubular epithelial cells composing the medullary thick ascending limbs of the loops of Henle using the H & E stain. As shown in the film autoradiographs (Fig. 2), in the inner stripe of the outer medulla where the medullary thick ascending limbs of Henle are concentrated, the densest hybridization signal was observed. As found in the solution hybridization assays, the IGF-I mRNA hybridization signal was increased in the immature remnant kidneys and decreased in the adult remnant kidneys compared to levels in their respective controls. The cellular distribution of IGF-I mRNA did not change in the remnant kidneys, but became relatively more (immature; Fig. 3, the H & E-stained kidney sections) or less (adult) abundant in the thick ascending limbs. To ascertain whether the increase in steady state levels of IGF-I mRNA was accompanied by changes in IGF-I receptor gene expression, the levels of IGF-I receptor mRNA were measured in the same animals. Again, while kidneys from adult animals had a reduced amount of IGF-I receptor mRNA, the levels in the kidneys from immature rats were increased 3-fold (P < 0.05) over control levels at both 24 and 48 h post-UNX (Fig. 4).

and total specific binding of IGF-I and IGF-II was determined in two assays to confirm the findings. Binding is expressed as the mean + SEM, and paired Student’s t tests were used to determine the significance of the changes. Results

After UNX, the remnant kidney weight in both adult and immature rats was significantly greater than the weight of the excised kidney (control) or the shamoperated kidney. The growth parameters in immature and adult rats are shown in Table 1. Although the remnant kidneys from adult animals grew rapidly, the remnant kidney of immature rats grew a remarkable 68% of its initial kidney weight in 48 h, which resulted in a significant increase in the kidney/body weight ratio. The immature rats continued the rapid gain in body weight despite the surgical stress, whereas the adult rats lost weight over the 48-h period. Although the animals continued to eat and drink after UNX, the differences in growth may reflect metabolic differences between the age groups. The important finding is that despite the reduced whole body weight gain in the adult rats, there was still preferential renal growth over the 48 h after UNX. Protein levels in left and right kidneys were not significantly different per g kidney tissue (59.4 f 5.2 us. 60.4 f 1.4 mg protein/g tissue), indicating that the increase in wet weight reflected a true increase in renal mass. Solution

hybridization/RNase

protection

assays

IGF-I and IGF-I receptor gene expression. Nuclease protection assays were performed on RNA from 8 adult and 12 immature rats. In the initial studies using kidneys from sham-operated rats, there was variability in mRNA levels from animal to animal. Consequently, comparisons were made between RNA from the left (excised, control) kidney and the right (compensated, remnant) kidney for each animal. The remnant kidneys of adult rats had a 2fold decrease in IGF-I mRNA 24 and 48 h after UNX. However, the remnant (compensated) kidneys from immature UNX rats had significant increases in IGF-I mRNA levels 24 (3.2 f 0.3-fold) and 48 h (4.0 k 1.2-fold) after UNX (P < 0.05). Figure 1 shows a representative TABLE

251

and IGF-II/M-6-P receptor gene expression. IGFII and IGF-11/M-6-P receptor mRNA levels were determined by cohybridization with the two riboprobes, since the bands were easily differentiated on the autoradiographs. IGF-II is prevalent prenatally and falls to very low levels after birth in the rat. Accordingly, IGF-II

IGF-II

1. Renal growth in immature and adult rats 48 h postsurgery Adult

Immature ABW 64 AKW k) AKW (%) Control KW/BW (%I Remnant KW/BW (%I

Sham 15.2 f 0.9 0.10 + 0.01 12.2 + 0.8 0.55 + 0.01

UNX 14.0 f 1.1 0.26 + 0.02” 68.6 f 3.4” 0.55 + 0.01 0.77 + 0.02”,*

BW, Body weight; KW, kidney weight; A, change. The control KW/BW “P c 0.01, by unpaired t tests (compared with sham control). *P < 0.01, by paired t test (compared to own control).

Sham 3.70 0.04 2.90 0.33

+ 2.5 f 0.01 + 0.2 3z 0.01

UNX -6.3 Y!Z2.4 0.27 + 0.02” 20.2 * 1.2” 0.34 2 0.01 0.41 + o.ol”**

in sham-operated animals is from the left kidney.


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252 YrixstlAM~~

CR

C

RC

R

C

R

IGF mRNA

M

f+)

(-)

POSTNEPHRECTOMY

Endo. 1992 Vol130. No 1

creases) in mRNA at 24 and 48 h, so the results from both periods were combined. Membrane binding studies

FIG. 1. Representative autoradiograph of a solution hybridization/ RNase protection assay on immature rat kidneys using an IGF-I mRNA probe. The IGF-I mRNA signal is enhanced in the remnant kidneys of immature UNX rats. (+), Native probe with RNase; (-), probe without RNase; M, mol wt marker; C, control (excised) kidney; R, remnant (compensated) kidney.

mRNA was not observed in RNA samples from either the adult or immature rat kidneys. However, mRNA to the IGF-11/M-6-P receptor was readily detectable, and again, while there was a decrease in the remnant kidney of adult animals, the levels were elevated 3-fold (P < 0.05) in the remnant kidneys from immature rats after UNX (Fig. 5). Figure 6 summarizes the quantitative relative changes in mRNA levels in control and remnant kidneys from immature and adult UNX rats. There were no significant differences between the increases (or de-

To determine whether the increased IGF receptor gene expression was associated with a demonstrable increase in receptor activity, membrane binding studies were performed on control and compensated kidneys from adult and immature rats 48 h post-UNX. In plasma membranes from whole kidney homogenates of adult rats, specific binding of IGF-I and IGF-II was slightly decreased in the remnant compared with control kidneys (IGF-I, 3.4 f 0.4% us. 3.7 * 0.5% in control kidneys; IGF-II, 10.4 + 3.5% us. 11.7 f 2.4% in control kidneys; P = NS). In contrast, specific binding of both IGF-I and IGF-II was increased in the immature rat 48 h postUNX; however, only the increase in IGF-II binding in whole kidney preparations was statistically significant (20.5 & 1.6% us. 15.5 f 0.5% in control kidneys; P < 0.05). To further examine and localize the increase in IGF-I binding in the immature rat kidneys, membrane homogenates from dissected cortical and medullary segments were used in the studies. There was a 47.9 f 12.4% increase in IGF-I binding to cortical membranes, while membranes from the medullary region showed no change in IGF-I binding. Table 2 illustrates IGF-I membrane

FIG. 2. IGF-I mRNA localization in control (C) and remnant (R) kidneys of immature and adult rats, shown by in situ hybridization and film autoradiography. The autoradiographs shown in A and B are from an immature rat, with IGF-I mRNA localized in the inner stripe of the outer medulla (IS/OM). The hybridization signal is increased in the remnant (B) kidney compared to itse control (A). C and D are autoradiographs from control and remnant adult kidneys, respectively, where a decrease in the IGF-I mRNA hybridization signal is seen in the remnant kidney. No IGF-I mRNA is detected in the inner medulla (IM), and the only hybridization in the cortex (CX) arises from the cortical continuation of the thick ascending limbs. E shows a film autoradiograph of a comparable kidney section (the outlines of which are indicated by arrowheads) hybridized to a sense control probe. This film was exposed longer than the others in order to be able to detect the image for photography. F shows a H & E stain of the kidney that was used to obtain the autoradiograph in D. Bar = 1 mm.


RENAL


IGF-I

IGF-I-R IMMATURE

FIG. 3. Bright- and darkfield micrographs showing IGF-I mRNA hybridization in thick ascending limbs in the outer medulla of control (A and B) and remnant (C and D) kidneys from immature rats. Higher levels of IGF-I mRNA hybridization signal (white grains in the dark field) are present in thick ascending limb epithelia from remnant kidneys. A few collecting ducts are seen (arrows), but were not sites of IGF-I mRNA hybridization. vb, Medullary vascular bundles. Bar = 100. M

(9

(+)

C

R

C

R

C

IGF-II-R

IGF-I

IGF-I-A

UNX

ADULT

IGF-II-R

UNX

FIG. 6. A summary of the quantitative changes in IGF-I, IGF-I receptor, and IGF-11/M-6-P receptor mRNA levels in the kidneys of immature and adult rat control and remnant kidneys. There is an increase in gene expression in the immature remnant kidneys and a decrease in gene expression in the adult remnant kidneys. *, P < 0.05 compared to control. TABLE 2. IGF-I membrane binding from immature rats 48 h after UNX

in whole and dissected kidneys

Whole immature kidneys Control 3.9 f 0.3

R

Remnant 4.8 rfr 0.4

Dissected immature kidneys Medulla

Cortex receptor

+IGF-I

FIG. 4. Representative autoradiograph of a solution hybridization/ RNase protection assay on immature rat kidneys using an IGF-I receptor mRNA probe. The IGF-I receptor mRNA signal is enhanced in the remnant kidneys of immature UNX rats. (+), Native probe with RNase; (-), probe without RNase; M, mol wt marker; C, control (excised) kidney; R, remnant (compensated) kidney. ADULT M

P

,"",,"",

ADULT

IMM. G-R

IMM. CUNX

R

-mt

FIG. 5. Representative autoradiograph of a solution hybridization/ RNase protection assay on adult and immature kidneys using an IGFII receptor mRNA probe. The IGF-II receptor mRNA signal is decreased in the adult remnant kidneys, and enhanced in the remnant kidneys of immature UNX rats. M, Mol wt marker; P, native probe; C, control (excised) kidney; R, remnant (compensated) kidney.

binding rats.

in whole and dissected kidneys from immature

Control 3.3 & 0.2

Remnant 4.9 3~ 0.6”

Control 9.4 f 0.9

Remnant 9.1 * 1.2

Values given are percentages. a P < 0.01, by paired t test (us. control).

Discussion The findings of the present study confirm our initial report that renal IGF-I mRNA is elevated after UNX in immature, but not adult, rats (16) and extend that information by determining that IGF-I and IGF-11/M-6-P receptor mRNA levels are also significantly elevated in the immature remnant kidney. Also in support of these changes, in situ hybridization confirmed that IGF-I mRNA is reduced in the adult remnant kidney and increased in the immature kidney. The site of this increase in the immature remnant kidneys was in the thick ascending limbs of the loops of Henle, where IGF-I mRNA localization has recently been documented (22). Other investigators have reported immunostainable IGFI and abundance of IGF-I mRNA in suspensions of papillary collecting ducts (5, 22a) from adult rats. The present findings could reflect differences in either the age of the animals or the methods used, i.e. staining intact sections of renal tissue (Ref. 22 and this paper)


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vs. using cell suspensions composed of similar sized tubular segments (5, 22a). The increase in IGF-I receptor mRNA in the immature remnant kidney was associated with a concomitant increase in plasma membrane binding to IGF-I. Interestingly, although the medulla had higher IGF-I binding than the cortex in control kidneys, the increased binding was exclusive to the cortical region. In situ hybridization studies have localized the steady state IGF-I receptor mRNA signal primarily to the medulla and medullary rays, with considerably less observed in the cortex (22). Thus, the findings of the present study, demonstrating greater IGF-I binding in the medulla than the cortex of the control kidney, are consistent with the localization by in situ hybridization. The fact that increased IGF-I binding was seen only in the cortex of the remnant kidney suggests that the cortical IGF-I receptors may be differentially up-regulated in the early phase of CRG. Compensatory renal growth elicited an increase in both exon 1 and 2 IGF-I mRNA in the immature remnant kidney. Although exon 2 IGF-I mRNA is thought to be more sensitive to the presence of GH (while exon 1 is thought to code for a predominantly GH-independent transcript), it is probably responsive to other factors as well. Indeed, changes in nutrition as well as diabetes can elicit concordant increases in exon 1 and 2 IGF-I mRNA levels (19, 23). Thus, the present data provide further support for a GH-independent induction of exon 2 mRNA. In addition to the increase in IGF gene expression, a significant increase in IGF-I and IGF-II binding was observed in immature, but not adult, remnant kidneys. Thus, these data support the notion that an increase in expression of the IGF-I gene as well as the IGF-I receptor and IGF-11/M-6-P receptor genes may indeed be an initiating step, which could facilitate the increases in hemodynamic parameters and renal growth following UNX in the immature animal. Our finding that the steady state levels of IGF-I mRNA do not increase after UNX in adult animals is consistent with the previous findings of Lajara et al. (5). Furthermore, the age-dependent differences in IGF-I gene expression may explain the increase in IGF-I mRNA observed by Fagin and Melmed (4), since the animals they used were young (4-5 weeks of age), which is in keeping with the present findings. Our results from the nuclease protection assays also show that levels actually decrease in the adult remnant kidneys, possibly as a result of a dilution of the IGF mRNA by an increase in total RNA commensurate with the renal growth. However, in situ hybridization data confirmed the decrease in adult remnant kidney IGF-I mRNA, suggesting that the reduction in IGF-I mRNA is an absolute decrease, since the in situ hybridization signal is not affected by

Endo. Voll30.

1992 No 1

an increase in nontarget RNAs. These findings combined with our previous observations (1,2, 16) suggest that the effects of GH on CRG in the adult rat may be through direct GH action on the renal tubule (and/or glomerulus), via circulating IGF-I, or through a mediator other than IGF-I. The physiological role of IGF-II is unclear. It is considered to be primarily a prenatal growth factor, because it is present in fetal serum and tissues in large quantities (which decrease rapidly after birth) and has cell multiplication-stimulating properties (24-26). Although IGFII is virtually undetectable in adult serum, IGF-11/M-6P receptors are found in some adult brain tissue. Hammerman and Rogers (3) identified IGF-11/M-6-P receptors on both basolateral and brush border membranes from canine proximal tubular cells. They postulated that there may be different mechanisms for the IGF-II-mediated activation of luminal vs. apical receptors and also that the presence of luminal receptors may indicate glomerular or proximal synthesis of IGF-II and, hence, an autocrine or paracrine action of the growth factor. Although IGF-II is a far less potent mitogen than IGFI, it does promote growth in hypophysectomized animals (27) and cell cultures (28, 29). Polychronakos et al. (30) reported an increase in both IGF-I and IGF-II membrane binding in remnant kidneys of adult rats, which occurred 4-5 days after UNX. In the present study membrane binding to IGF-I and IGF-II 48 h after UNX was significantly enhanced only in the immature remnant kidneys and not in the adult remnant kidneys. This data supports the work of Polychronakos et al., in that they did not find increased binding in adults until 4 days after UNX. Of further interest is that the magnitude of the early increase in IGF-I and IGF-II binding in our immature remnant kidneys corresponds to the increase in binding observed in the adult remnant kidney 4 days after UNX (30). Thus, while enhanced IGF binding after CRG may contribute to the sustained growth in the adult remnant kidney, early stimulation of the IGF axis may be associated with the relatively rapid initial growth and increased hyperplasia observed in the immature remnant kidney. In the present studies membrane binding of IGF-II as well as the levels of IGF-11/M-6-P receptor mRNA were enhanced in immature UNX rats. This suggests that, as previously stated, although it may be a small component of the CRG, IGF-11/M-6-P receptors may be turned on to facilitate the growth process. Although the levels of IGF-II peptide mRNA were virtually undetectable, 2week autoradiographic exposures revealed that IGF-II mRNA tended to increase in the immature remnant kidney; however, this could only be assessed qualitatively due to the increase in background on the film. Thus, there may have been a slight increase in renal IGF-II synthesis in addition to the increase in receptors. A more


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likely possibility is that IGF-I may be binding to the IGF-II receptors, albeit at a greatly reduced rate and affinity. The IGF-II receptor may be important for the intracellular transport and packaging of lysosomal enzymes and may play an active role in the remodelling processes that occur during CRG. The differential findings in immature us. adult compensatory renal growth may be indicative of alternate underlying mechanisms regulating CRG during development. Other important differences in CRG between ages include renal growth rates (16, 31-33) and the type of growth occurring (i.e. hypertrophy us. hyperplasia) (34-37). Although growth of the remnant adult kidney after UNX is accelerated (20-25% increase in wet weight 48 h after UNX), the relative increase in growth of the immature remnant kidney is twice the adult rate (>60% increase in wet weight). Measurements of protein concentration confirm that the growth reflects an increase in renal mass. Thus, the immature animal has an astounding capacity to increase renal mass after UNX. Previous reports have also identified different patterns of CRG in immature and adult animals. In the adult animal, CRG results primarily from hypertrophy of the glomerulus and proximal tubular cells, while in the immature animal (~40 days of age) hyperplasia occurs, resulting in a greater number of renal cells (34, 35, 37). Thus, our findings of separate mechanisms regulating CRG in immature and adult rats may reflect the different rates and patterns of growth observed with age after UNX. In summary, our findings support a potential role for renal IGFs, particularly IGF-I, in the initial phase of compensatory renal growth after UNX in the immature rat. The developmental differences in IGF gene expression and GH dependency at various ages in this model of renal growth will be an important focus of future work. Acknowledgment

The authors would like to thank John Ladas for his excellent assistance. References 1. Haramati A, Mulroney SE, Lumpkin MD 1990 Early rise in pulsatile growth hormone release following unilateral nephrectomy. J Am Sot Nephrol 1:456 (Abstract) 2. Mulroney SE, Lumpkin MD, Haramati A 1990 Suppression of growth hormone release attenuates compensatory renal growth following unilateral nephrectomy in adult but not immature rats. J Am Sot Nephrol 1:637 (Abstract) 3. Hammerman MR, Rogers S 1987 Distribution of IGF receptors in the plasma membrane of proximal tubular cells. Am J Physiol 253:F481-487 4. Fagin JA, Melmed S 1987 Relative increase in insulin-like growth factor-I messenger ribonucleic acid levels in compensatory renal hypertrophy. Endocrinology 120:718-724 5. Lajara R, Rotwein P, Bortz JD, Hansen VA, Sadow JL, Betts CR, Rogers SA, Hammerman MR 1989 Dual regulation of insulin-like

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growth factor-I expression during renal hypertrophy. Am J Physiol 257:F252-F261 6. Flyvbjerg A, Thorlacius-Ussing 0, Naerra R, Ingerslev J, Orskov H 1988 Kidney tissue somatomedin C and initial renal arowth in diabetic and uninephrectomized rats. Diabetologia 31:310-314 7. Stiles AD, Sosenko IR, D’Ercole AJ, Smith BT 1985 Relation of kidney tissue somatomedin C/insulin-like growth factor I to postnephrectomy renal growth in the rat. Endocrinology 117:2397-2401 8. Guler H-P, Zapf J, Schweiller E, Froesch ER 1988 Recombinant human insulin-like growth factor I stimulates growth and has distinct effects on organ size in hypophysectomized rats. Proc Nat1 Acad Sci USA 85:4889-4893 9. Guler HP, Eckard KU, Zapf J, Bauer C, Froesch ER 1989 Insulinlike growth factor I increases glomerular filtration rata and renal plasma flow in man. Acta End&in01 (Copenh) 121:101-106 10. Hirschberg R. Kounle JD 1989 Evidence that insulin-like erowth factor I in&eases renal plasma flow and glomerular filtratiin rata in fasted rats. J Clin Invest 83:326-330 11. Kopple JD, Hirschberg R 1990 Physiological effects of growth hormone and insulin-like growth factor I on the kidney. Mineral Electrolyte Metab 16:82-88 12. Fine L 1986 The biology of renal hypertrophy. Kidney Int 29:619634 13. Kujubu DA, Fine LG 1989 Physiology and cell biology update: polypeptide growth factors and their relation to renal disease. Am J Kidney Dis 1461-73 14. Quaife CJ, Mathews LS, Pinkert CA, Hammer RE, Palmiter RD 1989 Histopathology associated with elevated levels of growth hormone and insulin-like growth factor I in transgenic mice. Endocrinology 12440-48 15. Doi T, Striker LJ, Quaife C, Conti F, Palmiter R, Behringer R, Brinster R, Stricker GE 1988 Progressive glomerulosclerosis develops in transgenic mice chronically expressing growth hormone and growth hormone releasing factor but not in those expressing insulin-like growth factor I. Am J Path01 131:398-403 16. Mulroney SE, Haramati A, Roberts Jr CT, LeRoith D 1991 Renal IGF-I mRNA levels are enhanced followine unilateral nenhrectomv in immature but not adult rata. Endocringlogy 1282666-2662 ” 17. Chirgwin JM, Przybla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry l&5294-5299 18. Lowe WL, Roberts Jr CT, Lasky SR, LeRoith D 1987 Differential expression of alternative 5’-untranslated regions in mRNAs encoding rat insulin-like growth factor I. Proc Nat1 Acad Sci USA 848946-8950 19. Werner H, Shen Orr Z, Stannard B, Burguera B, Roberts Jr CT, LeRoith D 1990 Experimental diabetes increases insulin-like growth factor-1 and II receptor concentration and gene expression in the kidney. Diabetes 391490-1497 20. Adamo ML, Ben-Hur H, LeRoith D, Roberts Jr CT 1991 Transcription initiation in the two leader exons of the rat IGF-I gene occurs from disperse versus localized sites. B&hem Biophys Res Commun 176887-893 21. Bondy CA, Werner H, Roberta Jr CT, LeRoith D 1990 Cellular pattern of insulin-like growth factor-I (IGF-I) and type I IGF receptor gene expression in early organogenesis: comparison with IGF-II gene expression. Mol En&&no1 4:1386-1398 22. Chin E. Zhou J, Bondv C, Gene exnression for insulin-like mowth factors.and their receptors in nephrogenesis. Program of the 73rd Annual Meeting of The Endocrine Society, Washington, DC, 1991, p 143 (Abstract) PPa.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 23. Lowe WL, Adamo M, Werner H, Roberta Jr CT, LeRoith D 1989 Regulation by fasting of rat insulin-like growth factor I and its receptor. J Clin Invest 84:619-626 24. Lund PK, Moats-Staats BM, Hynes MA 1986 Somatomedin C/ insulin-like growth factor I and insulin-like growth factor II mRNA in rat fetal and adult tissues. J Biol Chem 261:14539-14543 25. Brown AL, Graham DE, Nissley SP 1986 Developmental regulation of insulin-like growth factor II mRNA in different rat tissues. J


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satory renal hypertrophy following unilateral nephrectomy. J Exp Med 56:255-265 32. Galla JH, Klein-Robbenhaar T, Hayslett JP 1974 Influence of age on the compensatory response in growth and function to unilateral nephrectomy. Yale J Biol Med 47:218-226 33. Larsson L, Aperia A, Wilton P 1986 Effect of normal development on compensatory renal growth. Kidney Int 18:29-35 34. Karp R, Brasel JA, Winick M 1972 Compensatory kidney growth after uninephrectomy and adult and infant rata. Am J Dis Child 121:186-192

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Secreting

Adenomas

Satellite Symposium to the Ninth International Congress of Endocrinology, Barcelona, September 7-8, 1992. A monographical meeting on basic and clinical advances in pituitary hormone-secreting adenomas. Further information: Dr. I. Halpeiin, Servicio de Endocrinologia, Hospital Clinic, Villarroel 170, E-08036 Barcelona, Spain, FAX (34)3.4546691; Tel. (34)3.3231414, Ext. 2116.


Effect of a growth hormone-releasing factor antagonist on compensatory renal growth, insulin-like growth factor-I (IGF-I), and IGF-I receptor gene expression after unilateral nephrectomy in immature rats.

Renal IGF-1 mRNA levels are enhanced following unilateral nephrectomy in immature but not adult rats.

Expression of insulinlike growth factor (IGF) and IGF-binding protein genes in human lung tumor cell lines.

Expressions of receptor gene for hepatocyte growth factor in kidney after unilateral nephrectomy and renal injury.

Insulin and IGF-I actions on IGF-I receptor in seminiferous tubules from immature rats.

Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.

Gene expression of growth-related proteins and ECM constituents in response to unilateral nephrectomy.

Morphometric and enzyme histochemical behaviour of the kidney of young rats before and after unilateral nephrectomy.

Granularity of cells in the juxtaglomerular apparatus in fetal rats after maternal nephrectomy or fetal unilateral nephrectomy.

Adrenal insufficiency after unilateral radical nephrectomy.

Long-term results after partial and unilateral nephrectomy in childhood.

Renal function before and after unilateral nephrectomy in renal donors.

Transient increase in renal epidermal growth factor content after unilateral nephrectomy in the mouse.

Growth of rats' kidneys after unilateral uretero-caval anastomosis.

Expression of the insulin-like growth factor (IGF)-I and -II and the IGF-I and -II receptor genes during postnatal development of the rat ovary.

Expression of growth hormone receptor, insulin-like growth factor 1 (IGF-1) and IGF-1 receptor mRNA and proteins in human skin.

Adaptability of the Immature Ocular Motor Control System: Unilateral IGF-1 Medial Rectus Treatment.

Regulation of insulin-like growth factor (IGF) binding proteins in transgenic mice with altered expression of growth hormone and IGF-I.

Autometallographic localization of inorganic mercury in the kidneys of rats: effect of unilateral nephrectomy and compensatory renal growth.

The effects of infusion of insulinlike growth factor (IGF) I, IGF-II, and insulin on glucose and protein metabolism in fasted lambs.

A unique receptor-independent mechanism by which insulinlike growth factor I regulates the availability of insulinlike growth factor binding proteins in normal and transformed human fibroblasts.

IGF-I and the truncated analogue des-(1-3)IGF-I enhance growth in rats after gut resection.

IGF-I binding and IGF-I expression in regenerating muscle of normal and hypophysectomized rats.

Anatomical relationships in the patterns of insulin-like growth factor (IGF)-I, IGF binding protein-1, and IGF-I receptor gene expression in the rat kidney.