We have recently demonstrated that the exposure to hyperglycemia in utero impairs nephrogenesis in rat fetuses (Amri K et al., Diabetes 48:2240–2245, 1999). Diabetic pregnancy is commonly associated with alterations in the IGF system in fetal tissues. It has also been shown that both IGF-I and IGF-II are produced within developing metanephros and promote renal organogenesis. Therefore, we investigated the effect of maternal diabetes on IGFs and their receptors in developing fetal rat kidney. Diabetes was induced in pregnant rats by a single injection of streptozotocin on day 0 of gestation. We measured the amounts of IGF and their receptors, both proteins and mRNAs, in the metanephroi of fetuses issued from diabetic subjects and in age-matched fetuses from control subjects (14–20 days of gestation). IGF-II was produced throughout fetal nephrogenesis, whereas IGF-I protein was not detected, suggesting a critical role of IGF-II in kidney development. Fetal exposure to maternal diabetes caused no change in IGF production in the early stages of nephrogenesis. Similarly, the amounts of IGF-I receptor and insulin receptor were not altered. By contrast, there was an increase in production of IGF-II/mannose-6-phosphate receptor throughout nephrogenesis. Because this receptor plays an essential role in regulating the action of IGF-II, the altered nephrogenesis in fetuses exposed to maternal diabetes may be linked to a decrease in IGF-II bioavailability.

More than 12% of pregnancies in the U.S. occur in diabetic women. The resulting children are at higher-than-normal risk of having congenital malformations, making this a serious endocrine problem (1,2,3,4). These malformations result from defects occurring in early organogenesis (2,5), including failure of neural tube closure, caudal regression syndrome, and urogenital abnormalities, which can be as severe as renal agenesis (2,6,7,8). In a recent study (9), we demonstrated that exposure to hyperglycemia in utero impairs nephrogenesis in the rat, leading to a reduced number of nephrons. An inborn nephron deficit, even a moderate one, is a risk factor for the development of chronic renal disease and hypertension in adulthood (10,11,12,13). The factors underlying these developmental abnormalities remain obscure.

Diabetic pregnancy is commonly associated with alterations in the expression or bioavailability of IGFs in several fetal organs (14,15,16,17,18). However, the impact of maternal diabetes on the expression of IGFs and their receptors in fetal kidney has not yet been examined.

IGF-I and IGF-II are produced within developing metanephros (19,20) and promote renal organogenesis (21,22). The signaling of IGF is mediated by IGF-I receptor (IGF-IR), which is a heterotetrameric transmembrane glycoprotein with tyrosine kinase activity that resembles the insulin receptor (IR) (23). Recent gene-targeting experiments have demonstrated that the IR mediates some of the growth-promoting functions of IGF-II during mouse embryogenesis (24). IGF-II also binds with high affinity to a type 2 receptor (IGF-II/mannose-6-phosphate receptor [M6PR]) that is devoid of tyrosine kinase activity and is identical to M6PR. IGF-II/M6PR is involved in the clearance of IGF-II by receptor-mediated endocytosis (25). Both receptors are present in developing rodent kidneys. In vitro studies in which growth of metanephroi was prevented by the addition of antibodies to IGFs or IGF-II/M6PR and by the addition of antisens oligodeoxynucleotides to IGF-IR suggest that these receptors are involved in renal development (19,20).

Therefore, we investigated the effect of maternal diabetes on IGFs and their receptors in developing fetal kidney. The expression of genes encoding IGF-I, IGF-II, IGF-IR, IGF-II/M6PR, and IR in metanephros issued from control fetuses and fetuses exposed to hyperglycemia during gestation was quantified by Northern and Western blotting. In addition, the intrarenal distribution of IGF-II/M6PR during the early stage of nephrogenesis was localized by in situ hybridization.

Animals.

Female Sprague-Dawley rats, weighing 200–300 g and given free access to water and standard laboratory diet (UAR Laboratory, Villemoison sur Orge, France), were caged overnight with male rats; vaginal smears were collected the following morning and tested for pregnancy. The day a positive result was obtained was designated as day 0 of gestation.

Diabetes was induced in the pregnant female rats by a single injection of 40 mg/kg body wt of streptozotocin (STZ; Sigma, Saint Quentin Fallavier, France) on day 0 of gestation. STZ was diluted in 0.4 mol/l citrate buffer, pH 4.5. Control animals were given an equivalent amount of citrate buffer. Maternal blood samples were taken every 2 days from the cut tip of the tail, and the plasma glucose concentration was determined immediately by the glucose-oxidase technique using a glucose analyzer (Beckman Instruments, Fullerton, CA). The mean maternal plasma glucose concentration of the STZ group remained almost constant until the experimental day and was approximately three times higher (18.7 ± 0.1 mmol/l) than in the controls (5.27 ± 0.05 mmol/l, n = 45 for both groups).

Fetuses were removed from anesthetized pregnant female rats on days 14, 15, 16, 18, and 20 of gestation. Metanephroi were surgically removed from embryos, immediately frozen in liquid nitrogen, and stored at –80°C. For RNA and protein extraction, fetal kidneys were pooled from one to three litters at 14, 15, and 16 days of gestation and from two fetuses at 18 and 20 days of gestation.

Isolation of RNA and protein and probe preparation and labeling.

RNA and proteins were isolated from the same sample by one-step liquid-phase separation using the Trireagent procedure (Gibco BRL, Grand Island, NY). The precipitated RNA was resuspended in sterile H2O and quantified by measuring the absorbance at 260 nm. The protein pellet was dissolved in 1% SDS. The protein content of the tissue extracts was determined using the Bio-Rad DC protein assay (Bio-Rad, Marne la Coquette, France). Dilutions of bovine serum albumin were used as protein standards.

The cDNA probes were labeled with [32P]dCTP using Rediprime DNA labeling system (Amersham, Arlington, IL) and purified on NucTrap Probe Purification columns (Stratagene, Cambridge, U.K.).

Northern blot analysis.

RNA samples (15 μg) were denatured, fractionated by electrophoresis, and then transferred onto nylon membranes as previously described (26). Northern blots were prehybridized and then hybridized with [32P]dCTP-labeled cDNA probes. The blots were exposed to X-ray film (Reflection; DuPont NEN, Boston, MA) for 6 h and up to 7 days at –80°C using intensifying screens. The blots were stripped in between hybridizations with different probes by washing for 1 min at 80°C in 0.1× sodium chloride–sodium citrate and 0.1% SDS. The consistency of the relative amounts of total RNA loaded into each lane was checked by probing the blots with [32P]dCTP-labeled cDNA-encoding 18 S ribosomal RNA. Signal intensity was quantified by densitometric analysis of autoradiograms using image analysis software (NIH Image).

Western blot analysis.

A protein extract (25 μg) was boiled for 5 min with SDS–dissociation buffer (0.125 mol/l Tris-HCl, pH 6.8, 4% SDS, 20% glycerol) with or without β-mercaptoethanol (Table 1). Proteins were separated by electrophoresis in a discontinuous SDS–polyacrylamide gel gradient. Proteins were transferred to nitrocellulose membranes (Hybond–C Extra; Amersham) and the membranes were blocked by incubation for 1 h with 5–10% dried skim milk in Tris-buffered saline with Tween (TBST; 50 mmol/l Tris-HCl, 150 mmol/l NaCl, 0.05% Tween 20, pH = 8). Specific proteins were detected by incubating with antibodies suitably diluted in TBST (Table 1) for 1 h at room temperature. The membranes were then washed three times in TBST, and the bound primary antibody was detected with a peroxidase-antiperoxidase system (Jackson ImmunoResearch Laboratories, West Grove, PA). Antigen-antibody complexes were detected by enhanced chemiluminescence, as recommended by the manufacturer (Amersham). Finally, each membrane was labeled with a monoclonal anti-β-actin antibody to normalize the amount of protein loaded. Bands were quantified by densitometry using image analysis software (NIH Image).

In situ hybridization.

In situ hybridization was performed in kidney of fetuses on day 16 of gestation as previously described (27,28), using 4% paraformaldehyde-fixed, paraffin-embedded tissues and the 35S-labeled IGF-II/M6PR probe. For controls, tissue sections were first treated with ribonuclease A for 30 min and then washed, permeated, and hybridized with the specific probe.

cDNA probes and antibodies.

Monoclonal anti–β-actin antibody was obtained from Sigma (St. Quentin Fallavier, France). Anti–IGF-I and anti–IGF-IR β-subunit antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and anti–IGF-II antibody was obtained from Upstate Biotechnology (Lake Placid, NY). Anti–IGF-II/M6PR was a gift from Dr. MacDonald (University of Nebraska Medical Center, Lincoln, NE). Anti-IR β-subunit was obtained from Chemicon (Temecula, CA).

IGF-I and IGF-II cDNA probes were a gift from Professor Rotwein (University of Washington Medical Center, Seattle, WA). Dr. Haugel (Endocrinologie Métabolisme et Développement, CNRS UPR 1,524, Meudon, France) kindly provided the IGF-IR cDNA probe. The IGF-II/M6PR cDNA probe was kindly provided by Dr. A. Clément (INSERM U515, Hôpital Trousseau, Paris, France), and the IR cDNA probe was a gift from Dr. B. Maitre (Hôpital Henri Mondor, Créteil, France).

Statistics.

Differences between developmental stages were calculated by analysis of variance. Control and hyperglycemic groups, at the same stage, were compared by Student’s unpaired t test. The threshold of significance was P < 0.05. All values are expressed as means ± SE.

IGFs in fetal kidney.

IGF-I mRNA was detected as a 7.5-kb band by Northern blotting. Densitometric analysis in the control group revealed a constant expression between days 14 and 20 of gestation (Fig. 1). IGF-I protein was under detection limit by Western blotting. The amounts of IGF-I mRNA in the diabetic and control fetuses were the same (Fig. 1).

The IGF-II cDNA probe detected a single major 3.6-kb mRNA band. IGF-II mRNA was abundant on days 14 and 15 of gestation and then declined gradually until day 20 (Fig. 2A). IGF-II protein was found as a specific hybridizing band of ∼12 kDa. Band-densitometric analysis revealed that IGF-II was abundant at the early stage of fetal development (days 14–16 of gestation) and then decreased by ∼50% on day 20 of gestation (Fig. 2B). The amounts of IGF-II mRNA and protein in fetuses from diabetic subjects were unchanged during the first stages of fetal nephrogenesis and then increased on day 20 of gestation by ∼60 and 80%, respectively, as compared with age-matched fetuses from control subjects (Figs. 2A and B).

IGF receptors and IR in fetal kidney.

The IGF-IR cDNA probe detected a single 11-kb mRNA band. Western blot analysis demonstrated a specific labeled band at ∼90 kDa corresponding to the β-subunit of this receptor. Densitometric analysis (Figs. 3A and B) showed that mRNA expression was constant throughout fetal development, whereas the protein expression increased sharply on day 20 of gestation, suggesting a posttranslational regulation. The amounts of IGF-IR mRNA and protein were not altered by maternal diabetes (Figs. 3A and B).

Specific bands of 7 and 9 kb were observed in Northern blot hybridization analysis for IR. The 97-kDa band specific for the β-subunit of the IR was detected by Western blot analysis. The amounts of mRNA and protein increased on day 18 of gestation. Then, between days 18 and 20, mRNA expression remained constant, whereas protein expression increased (Figs. 4A and B). Maternal diabetes did not alter the amounts of IGF-IR mRNA and protein (Figs. 4A and B).

The 9.5-kb IGF-II/M6PR mRNA was abundant in controls on day 14 of gestation and then decreased by ∼50% on day 20 of gestation. Western blot analysis showed a specific band at ∼250 kDa that had the same expression profile as the mRNA (Figs. 5A and B). However, the expression of IGF-II/M6PR mRNA was increased from day 15 of gestation in fetuses from diabetic subjects. IGF-II/M6PR protein also increased throughout the study period (Fig. 5). The greatest increase was on day 20 of gestation (75% for mRNA and 84% for protein as compared with age-matched control fetuses).

In situ hybridization in metanephros on day 16 of gestation showed that IGF-II/M6PR mRNA was localized in the epithelium, the ureteric bud, and in the neighboring domain of committed metanephric mesenchyme, which will differentiate into nephrons. It was also localized in the nonmature glomeruli, the S-shaped bodies (Fig. 6).

We have previously shown that exposure to hyperglycemia in utero leads to a nephron deficit (9). The mechanisms whereby hyperglycemia alters nephrogenesis are unknown. The major finding in this study is an increase in the amount of IGF-II/M6PR in the kidneys of fetuses from diabetic subjects.

Although IGF-II/M6PR has been reported to play a role in transmembrane signal transduction (29,30,31), its function remains unresolved and controversial. The main function of this receptor in mammals is the transport of mannose-6-phosphate–containing lysosomal enzyme to lysosomes and the clearance of IGF-II from serum and tissue fluids by receptor-mediated endocytosis (25). Gene-targeting experiments also indicate that IGF-II/M6PR functions as a scavenger receptor by internalizing and degrading IGF-II (32,33,34). A truncated form of the IFG-II/M6PR cleaved from the cell-surface receptor may also bind a significant amount of IGF-II, thus functioning as an IGF-II specific binding protein regulating its bioavailability (35). This form seems to be most abundant in fetal circulation (35). It is also higher in the plasma of fetuses from diabetic women (35). We show that, at early stages of nephrogenesis, IGF-II/M6PR is localized in the nephrogenic zone, especially in undifferentiated mesenchymal cells. IGF-II mRNA is abundant in developing kidney in mice and humans and is mainly expressed in the mesenchymal cells (36,37). The colocalization of the receptor with the growth factor and the increase in the IGF-II/M6PR protein we observed in the kidneys of fetuses from diabetic mothers may thus reduce the bioavailability of IGF-II in this organ.

Several studies have reported that IGF-II is critical during fetal nephrogenesis. The development of the metanephros in vitro is prevented by adding anti–IGF-I and anti–IGF-II antibodies to the culture medium (19). However, using metanephric organ cultures and specific glomerular labeling, we showed that IGF-II stimulates the growth of the metanephros and the number of nephrons formed, whereas IGF-I only stimulates growth of the metanephros (38). It has also recently been shown that exogenous IGF-I does not increase the number of nephrons that has been reduced before the end of nephrogenesis by unilateral ureteral obstruction in neonatal rats (39). The present study shows that IGF-II mRNA and IGF-II protein are developmentally regulated and that their expressions decrease at the end of gestation, as the fetal kidney matures. Together, these data suggest that IGF-II is an autocrine/paracrine growth factor for mesenchymal cell differentiation.

The expression of IGF-II in the kidneys of fetuses from diabetic subjects increased at the end of gestation. This increase may be due, in part, to an increase in plasma IGF-II, which has been reported to occur in fetuses from diabetic rats and humans at the end of gestation (17,35,40). Such an increase in IGF-II would not be sufficient to restore the reduced number of nephrons caused by exposure to diabetes in utero (9), because it occurs concomitantly with the greatest increase in the IGF-II/M6PR and only at the end of nephrogenesis.

An increase in IGF-II/M6PR expression related to increased mRNA expression has also been shown in the kidney of diabetic adult rats (41,42,43). This might be of importance because the kidney is one of the most affected organs in respect to diabetic complications, and changes in the IGF-II/M6PR expression might be relevant to the development of such complications.

The mechanism whereby diabetes increases the expression of IGFs and IGF receptors is unknown. However, glucose per se has been shown to regulate the expression of the IGF system in some in vitro studies. Goya et al. (44) have shown that glucose affects the transcription of the IGF-II gene in fetal rat hepatocytes. Similarly, increased IGF-II mRNA and IGF-IR mRNA, as well as an increased number of IGF-II/M6PR, have been found in mesangial cells cultured in high-glucose medium (45). The same results were obtained with the insulin-secreting cell line RINm5F (46). These data support the idea that IGFs are encoded by genes belonging to the family of genes that are regulated by glucose. Daniel and Kim (47) have reported that transcriptional activator Sp1 binding is required for activation of the acetyl-CoA carboxylase promoter by glucose. But, the CACGTG-type E boxes are critical for the glucose response of the liver-type pyruvate kinase (48). Either the Sp1 binding sites or the CACGTG-type E boxes were found within the proximal promoter of the IGF-II/M6PR. This suggests that glucose regulates the transcription of this receptor.

We have shown for the first time that maternal diabetes causes an increase in the concentration of IGF-II/M6PR in the fetal kidney. Because the receptor is crucial for regulating the levels of free IGF-II ligand, this increase may lead to a decrease in IGF-II action. Because IGF-II plays a critical role in renal development, this may explain the altered nephrogenesis in fetuses of diabetic subjects.

FIG. 1.

Patterns of IGF-I mRNA in the kidneys of fetuses (14–20 days of gestation) from normal (plain line) and diabetic (broken line) rats. Values are means ± SE of five to six experiments. Densitometric results are expressed as percentage of the control (day 14) value after normalization for RNA loading on the basis of hybridization of 18 S rRNA.

FIG. 1.

Patterns of IGF-I mRNA in the kidneys of fetuses (14–20 days of gestation) from normal (plain line) and diabetic (broken line) rats. Values are means ± SE of five to six experiments. Densitometric results are expressed as percentage of the control (day 14) value after normalization for RNA loading on the basis of hybridization of 18 S rRNA.

FIG. 2.

Patterns of IGF-II mRNA (A) and protein (B) in the kidneys of fetuses (14–20 days of gestation) from normal (plain line) and diabetic (broken line) rats. Values are means ± SE of five to six experiments. Densitometric results are expressed as percentage of the control (day 14) value after normalization for RNA loading on the basis of hybridization of 18 S rRNA and for protein loading by labeling with anti–β-actin antibody. †, ††, and †††: P < 0.05, 0.01, and 0.001 as compared with control (day 14) value. * and **: P < 0.05 and 0.01 as compared with age-matched control fetuses.

FIG. 2.

Patterns of IGF-II mRNA (A) and protein (B) in the kidneys of fetuses (14–20 days of gestation) from normal (plain line) and diabetic (broken line) rats. Values are means ± SE of five to six experiments. Densitometric results are expressed as percentage of the control (day 14) value after normalization for RNA loading on the basis of hybridization of 18 S rRNA and for protein loading by labeling with anti–β-actin antibody. †, ††, and †††: P < 0.05, 0.01, and 0.001 as compared with control (day 14) value. * and **: P < 0.05 and 0.01 as compared with age-matched control fetuses.

FIG. 3.

Patterns of IGF-IR mRNA (A) and protein (B) in the kidneys of fetuses (14–20 days of gestation) from normal (plain line) and diabetic (broken line) rats. Values are means ± SE of five to six experiments. Densitometric results are expressed as percentage of the control (day 14) value after normalization for RNA loading on the basis of hybridization of 18 S rRNA and for protein loading by labeling with anti–β-actin antibody. †††P < 0.001 as compared with control (day 14) value.

FIG. 3.

Patterns of IGF-IR mRNA (A) and protein (B) in the kidneys of fetuses (14–20 days of gestation) from normal (plain line) and diabetic (broken line) rats. Values are means ± SE of five to six experiments. Densitometric results are expressed as percentage of the control (day 14) value after normalization for RNA loading on the basis of hybridization of 18 S rRNA and for protein loading by labeling with anti–β-actin antibody. †††P < 0.001 as compared with control (day 14) value.

FIG. 4.

Patterns of IR mRNA (A) and protein (B) in the kidney of fetuses (14–20 days of gestation) from normal (plain line) and diabetic (broken line) rats. Values are means ± SE of five to six experiments. Densitometric results are expressed as percentage of the control (day 14) value after normalization for RNA loading on the basis of hybridization of 18 S rRNA and for protein loading by labeling with anti–β-actin antibody; mRNA values are the addition of the bands corresponding to the two transcripts. †† and †††: P < 0.01 and 0.001 as compared with control (day 14) value.

FIG. 4.

Patterns of IR mRNA (A) and protein (B) in the kidney of fetuses (14–20 days of gestation) from normal (plain line) and diabetic (broken line) rats. Values are means ± SE of five to six experiments. Densitometric results are expressed as percentage of the control (day 14) value after normalization for RNA loading on the basis of hybridization of 18 S rRNA and for protein loading by labeling with anti–β-actin antibody; mRNA values are the addition of the bands corresponding to the two transcripts. †† and †††: P < 0.01 and 0.001 as compared with control (day 14) value.

FIG. 5.

Patterns of IGF-II/M6PR mRNA (A) and protein (B) in the kidneys of fetuses (14–20 days of gestation) from normal (plain line) and diabetic (broken line) rats. Values are means ± SE of five to six experiments. Densitometric results are expressed as percentage of the control (day 14) value after normalization for RNA loading on the basis of hybridization of 18 S rRNA and for protein loading by labeling with anti–β-actin antibody. †P < 0.05 as compared with control (day 14) value. * and **: P < 0.05 and 0.01 as compared with age-matched control fetuses.

FIG. 5.

Patterns of IGF-II/M6PR mRNA (A) and protein (B) in the kidneys of fetuses (14–20 days of gestation) from normal (plain line) and diabetic (broken line) rats. Values are means ± SE of five to six experiments. Densitometric results are expressed as percentage of the control (day 14) value after normalization for RNA loading on the basis of hybridization of 18 S rRNA and for protein loading by labeling with anti–β-actin antibody. †P < 0.05 as compared with control (day 14) value. * and **: P < 0.05 and 0.01 as compared with age-matched control fetuses.

FIG. 6.

Silver grains labeling of IGF-II/M6PR mRNA in the kidneys of fetuses on day 16 of gestation. In situ hybridization with 35S-labeled IGF-II/M6PR probe followed by autoradiography hematoxylin-eosin counterstaining. UB, ureteric bud; S, S-shaped body.

FIG. 6.

Silver grains labeling of IGF-II/M6PR mRNA in the kidneys of fetuses on day 16 of gestation. In situ hybridization with 35S-labeled IGF-II/M6PR probe followed by autoradiography hematoxylin-eosin counterstaining. UB, ureteric bud; S, S-shaped body.

TABLE 1

Specific conditions used for electrophoresis and immunodetection in Western blot experiments

Electrophoresis
Immunodetection
Reducing conditionGradientHost antibodyDilutionMilk
IGF-I — 10–30% Rabbit 1/500 5% 
IGF-II — 10–30% Mouse 1/500 5% 
IGF-IR β-mercaptoethanol (143 mmol/l) 6–10% Rabbit 1/2,000 5% 
IGF-II/M6PR — 6–10% Rabbit 1/10,000 10% 
IR β-mercaptoethanol (143 mmol/l) 6–10% Rabbit 1/500 5% 
Electrophoresis
Immunodetection
Reducing conditionGradientHost antibodyDilutionMilk
IGF-I — 10–30% Rabbit 1/500 5% 
IGF-II — 10–30% Mouse 1/500 5% 
IGF-IR β-mercaptoethanol (143 mmol/l) 6–10% Rabbit 1/2,000 5% 
IGF-II/M6PR — 6–10% Rabbit 1/10,000 10% 
IR β-mercaptoethanol (143 mmol/l) 6–10% Rabbit 1/500 5% 

We thank Drs. MacDonald, Haugel, Clément, and Maitre and Professor Rotwein for kindly providing antibody and specific probes; J. Vilar and C. Lalou for critically reading the manuscript; and M.F. Belair for technical assistance with in situ hybridization.

1.
Lowy C: Diabetes in pregnancy. In
Textbook of Diabetes
. Oxford, J.C. Pickup & Williams,
1991
, p.
835
–850
2.
Fuhrmann K, Reiher H, Semmler K, Fischer F, Fischer M, Glockner E: Prevention of congenital malformations in infants of insulin-dependent diabetic mothers.
Diabetes Care
6
:
219
–223,
1983
3.
Pedersen LM, Tygstrup I, Pedersen J: Congenital malformations in newborn infants of diabetic women.
Lancet
1
:
1124
–1126,
1964
4.
Martinez-Friaz ML: Epidemiological analysis of outcomes of pregnancy in diabetic mothers: identification of the most frequent congenital anomalies.
Am J Med Genet
51
:
108
–113,
1994
5.
Mills J, Baker L, Goldman A: Malformations in infants of diabetic mothers occur before the seventh gestational week: implications for treatment.
Diabetes
28
:
292
–293,
1979
6.
Soler N, Walsh C, Malins J: Congenital malformations in infants of diabetic mothers.
Q J Med
45
:
303
–313,
1976
7.
Kitzmiller JL, Gavin LA, Gin GD, Jovanovic-Peterson L, Main EK, Zigrang WD: Preconception care of diabetes: glycemic control prevents congenital anomalies.
JAMA
265
:
731
–736,
1991
8.
Lynch S, Wright C: Sirenomelia, limb reduction defects, cardiovascular malformation, renal agenesis in an infant born to a diabetic mother.
Clin Dysmorphol
6
:
75
–80,
1997
9.
Amri K, Freund N, Vilar J, Merlet-Benichou C, Lelievre-Pegorier M: Adverse effects of hyperglycemia on kidney development in rats: in vivo and in vitro studies.
Diabetes
48
:
2240
–2245,
1999
10.
Brenner BM, Garcia DL, Anderson S: Glomeruli and blood pressure: less of one, more of the other?
Am J Hypertens
1
:
335
–347,
1988
11.
Gilbert T, Lelièvre-Pégorier M, Merlet-Bénichou C: Long-term effects of mild oligonephronia induced in utero by gentamicin in the rat.
Pediatr Res
30
:
450
–456,
1991
12.
He C, Zalups RK, Henderson DA, Striker GE, Striker LJ: Molecular analysis of spontaneous glomerulosclerosis in Os/+ mice, a model with reduced nephron mass.
Am J Physiol
269
:
F266
–F273,
1995
13.
Lelièvre-Pégorier M, Merlet-Bénichou C: The number of nephrons in the mammalian kidney: environmental influences play a determining role.
Exp Nephrol
8
:
63
–65,
2000
14.
Ramsay TG, Wolverton CK, Steele NC: Alteration in IGF-I mRNA content of fetal swine tissues in response to maternal diabetes.
Am J Physiol
267
:
R1391
–R1396,
1994
15.
Rajaratnam VS, Webb PJ, Fishman RB, Streck RD: Maternal diabetes induces upregulation of hepatic insulin-like growth factor binding protein-1 MRNA expression, growth retardation and developmental delay at the same stage of rat fetal development.
J Endocrinol
152
:
R1
–R6,
1997
16.
Streck RD, Rajaratnam VS, Fishman RB, Webb PJ: Effects of maternal diabetes on fetal expression of insulin-like growth factor and insulin-like growth factor binding protein mRNAs in the rat.
J Endocrinol
147
:
R5
–R8,
1995
17.
Rivero F, Goya L, Alaez C, Pascual-Leone AM: Effects of undernutrition and diabetes on serum and liver mRNA expression of IGFs and their binding proteins during rat development.
J Endocrinol
145
:
427
–440,
1995
18.
Chernicky CL, Redline RW, Tan HQ, Gwatkin RB, Johnson TR, Ilan J: Expression of insulin-like growth factors I and II in conceptuses from normal and diabetic mice.
Mol Reprod Dev
37
:
382
–390,
1994
19.
Rogers SA, Ryan G, Hammerman MR: Insulin-like growth factors I and II are produced in the metanephros and are required for growth and development in vitro.
J Cell Biol
113
:
1447
–1453,
1991
20.
Liu ZZ, Wada J, Alvares K, Kumar A, Wallner EI, Kanwar YS: Distribution and relevance of insulin-like growth factor-I receptor in metanephric development.
Kidney Int
44
:
1242
–1250,
1993
21.
Hammerman MR: Growth factors in renal development.
Semin Nephrol
15
:
291
–299,
1995
22.
Liu ZZ, Kumar A, Ota K, Wallner EI, Kanwar YS: Developmental regulation and the role of insulin and insulin receptor in metanephrogenesis.
Proc Natl Acad Sci U S A
94
:
6758
–6763,
1997
23.
LeRoith D, Werner H, Neuenschwander S, Kalebic T, Helman LJ: The role of the insulin-like growth factor-I receptor in cancer.
Ann N Y Acad Sci
766
:
402
–408,
1995
24.
Louvi A, Accili D, Efstratiadis A: Growth-promoting interaction of IGF-II with the insulin receptor during mouse embryonic development.
Dev Biol
189
:
33
–48,
1997
25.
Braulke T: Type-2 IGF receptor: a multi-ligand binding protein.
Horm Metab Res
31
:
242
–246,
1999
26.
Chailley-Heu B, Chelly N, Lelievre-Pegorier M, Barlier-Mur AM, Merlet-Benichou C, Bourbon JR: Mild vitamin A deficiency delays fetal lung maturation in the rat.
Am J Respir Cell Mol Biol
21
:
89
–96,
1999
27.
Bruneval P, Fournier JG, Soubrier F, Belair MF, Da Silva JL, Guettier C, Pinet F, Tardivel I, Corvol P, Bariety J: Detection and localization of renin messenger RNA in human pathologic tissues using in situ hybridization.
Am J Pathol
131
:
320
–330,
1988
28.
Karam H, Valdenaire O, Belair MF, Prigent-Sassy C, Rakotosalama A, Clozel M, Itskovitz J, Bruneval P: The endothelin system in human and monkey ovaries: in situ gene expression of the different components.
Cell Tissue Res
295
:
101
–109,
1999
29.
Nissley P, Lopaczynski W: Insulin-like growth factor receptors.
Growth Factors
5
:
29
–43,
1991
30.
Rogers SA, Hammerman MR: Mannose 6-phosphate potentiates insulin-like growth factor II-stimulated inositol trisphosphate production in proximal tubular basolateral membranes.
J Biol Chem
264
:
4273
–4276,
1989
31.
Nishimoto I, Hata Y, Ogata E, Kojima I: Insulin-like growth factor II stimulates calcium influx in competent BALB/c 3T3 cells primed with epidermal growth factor: characteristics of calcium influx and involvement of GTP-binding protein.
J Biol Chem
262
:
12120
–12126,
1987
32.
Lau MM, Stewart CE, Liu Z, Bhatt H, Rotwein P, Stewart CL: Loss of the imprinted IGF2/cation-independent mannose 6-phosphate receptor results in fetal overgrowth and perinatal lethality.
Genes Dev
8
:
2953
–2963,
1994
33.
Wang ZQ, Fung MR, Barlow DP, Wagner EF: Regulation of embryonic growth and lysosomal targeting by the imprinted Igf2/Mpr gene.
Nature
372
:
464
–467,
1994
34.
Ludwig T, Eggenschwiler J, Fisher P, D’Ercole AJ, Davenport ML, Efstratiadis A: Mouse mutants lacking the type 2 IGF receptor (IGF2R) are rescued from perinatal lethality in Igf2 and Igf1r null backgrounds.
Dev Biol
177
:
517
–535,
1996
35.
Gelato MC, Rutherford C, San-Roman G, Shmoys S, Monheit A: The serum insulin-like growth factor-II/mannose-6-phosphate receptor in normal and diabetic pregnancy.
Metabolism
42
:
1031
–1038,
1993
36.
Lindenbergh-Kortleve DJ, Rosato RR, van Neck JW, Nauta J, van Kleffens M, Groffen C, Zwarthoff EC, Drop SL: Gene expression of the insulin-like growth factor system during mouse kidney development.
Mol Cell Endocrinol
132
:
81
–91,
1997
37.
Matsell DG, Bennett T: Evaluation of metanephric maturation in a human fetal kidney explant model.
In Vitro Cell Dev Biol
34
:
138
–148,
1998
38.
Gilbert T, Amri K, Lelièvre-Pégorier M, Moreau M, Riveau B, Vilar J, Merlet-Bénichou C: Insulin-like growth factor II (IGF-II) but not IGF-I stimulates in vitro nephron formation.
J Am Soc Nephrol
9
:
361
,
1998
39.
Chevalier RL, Goyal S, Kim A, Chang AY, Landau D, LeRoith D: Renal tubulointerstitial injury from ureteral obstruction in the neonatal rat is attenuated by IGF-1.
Kidney Int
57
:
882
–890,
2000
40.
Liu YJ, Tsushima T, Onoda N, Minei S, Sanaka M, Nagashima T, Yanagisawa K, Omori Y: Expression of messenger RNA of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBP1–6) in placenta of normal and diabetic pregnancy.
Endocr J
43 (Suppl.):S89–S91,
1996
41.
Flyvbjerg A, Kessler U, Kiess W: Increased kidney and liver insulin-like growth factor II/mannose-6-phosphate receptor concentration in experimental diabetes in rats.
Growth Regul
4
:
188
–193,
1994
42.
Kiess W, Hoeflich A, Yang Y, Groenbaek H, Flyvbjerg A: Streptozotocin induction of diabetes in rats leads to increased insulin-like growth factor-II/mannose-6-phosphate receptor mRNA expression in kidney but not in lung or heart.
Growth Regul
6
:
66
–72,
1996
43.
Werner H, Shen-Orr Z, Stannard B, Burguera B, Roberts CT Jr, LeRoith D: Experimental diabetes increases insulinlike growth factor I and II receptor concentration and gene expression in kidney.
Diabetes
39
:
1490
–1497,
1990
44.
Goya L, de la Puente A, Ramos S, Martin MA, Escriva F, Pascual-Leone AM: Regulation of insulin-like growth factor-I and -II by glucose in primary cultures of fetal rat hepatocytes.
J Biol Chem
274
:
24633
–24640,
1999
45.
Pugliese G, Pricci F, Locuratolo N, Romeo G, Romano G, Giannini S, Cresci B, Galli G, Rotella CM, Di Mario U: Increased activity of the insulin-like growth factor system in mesangial cells cultured in high glucose conditions: relation to glucose-enhanced extracellular matrix production.
Diabetologia
39
:
775
–784,
1996
46.
Zhang Q, Berggren PO, Tally M: Glucose increases both the plasma membrane number and phosphorylation of insulin-like growth factor II/mannose 6-phosphate receptors.
J Biol Chem
272
:
23703
–23706,
1997
47.
Daniel S, Kim KH: Sp1 mediates glucose activation of the acetyl-CoA carboxylase promoter.
J Biol Chem
271
:
1385
–1392,
1996
48.
Koo SH, Towle HC: Glucose regulation of mouse S(14) gene expression in hepatocytes: involvement of a novel transcription factor complex.
J Biol Chem
275
:
5200
–5207,
2000

Address correspondence and reprint requests to Dr. Martine Lelièvre-Pégorier, INSERM U319, Université Paris 7, 2 Place Jussieu, 75251 Paris Cedex 05, France. E-mail: pegorier@paris7.jussieu.fr.

Received for publication 8 August 2000 and accepted in revised form 16 January 2001.

IGF-IR, IGF-I receptor; IR, insulin receptor; M6PR, mannose-6-phosphate receptor; STZ, streptozotocin; TBST, Tris-buffered saline with Tween.