Abstract
Abstract. The growth hormone (GH)/insulin-like growth factor (IGF) axis is involved in diabetic renal disease. The role of a specific GH receptor (GHR) antagonist in the development of early renal changes in nonobese diabetic (NOD) mice was investigated. Female diabetic (nonketotic) NOD mice treated with a polyethylene glycol-treated GHR antagonist (2 mg/kg, every other day) (DA group) or saline (D group) and their nonhyperglycemic age-matched littermates (control animals) were euthanized 3 wk after the onset of diabetes. Body weights at euthanasia were similar among the groups. Serum GH levels were markedly elevated, and serum IGF-I levels were significantly decreased in D and DA animals, compared with controls. The increases in kidney weights and glomerular volumes observed for the D group were absent in the DA group. Albuminuria was increased in the D group but was normalized in the DA group. Extractable renal IGF-I protein levels were increased in the D group but were partially normalized in the DA group. Renal IGF-binding protein 1 mRNA levels were increased in the D group but returned to almost normal levels in the DA animals. Kidney IGF-I and GHR mRNA levels were decreased in both the D and DA groups. Renal GH-binding protein mRNA levels remained unchanged in both diabetic groups. GHR antagonism had a blunting effect on renal/glomerular hypertrophy and albuminuria in diabetic NOD mice. These salutary effects were associated with concomitant inhibition of increased renal IGF-I protein levels and were obtained without affecting either somatic growth or circulating GH and IGF-I levels. Therefore, modulation of GH effects may have beneficial therapeutic implications in diabetic nephropathy.
Nephropathy is one of the major complications of diabetic angiopathy and is one of the leading causes of end-stage renal failure and death among diabetic patients (1). The characteristic renal changes in early human diabetes include an increase in GFR, renal and glomerular hypertrophy, and accumulation of mesangial extracellular matrix (2). Obstruction of the glomerular capillary lumen and loss of glomerular filtration and function appear next (3). Microalbuminuria followed by proteinuria and worsening renal function are the clinical findings (4).
Insulin-like growth factor I (IGF-I) is a potent mitogenic polypeptide under the regulation of growth hormone (GH) (5). Evidence of significant involvement of the GH/IGF system in diabetic nephropathy and other nephropathies has been provided by several studies (6, 7). Kidney tissue expresses receptors not only for IGF-I but also for GH (8), suggesting that although most of the biologic effects of GH are mediated by IGF-I, GH may also act independently of IGF-I.
GH exerts its biologic effects through the activation of a specific membrane receptor (9). GH induces receptor dimerization (10), followed by induction of the intracellular tyrosine protein kinase Janus-activated kinase-2 (11). Chen et al. (12) demonstrated that the glycine residue at position 120 of the human GH molecule is critical for the biologic activity of GH. Indeed, a GH analogue with a substituted amino acid at that position (G120R) has potent inhibitory effects on the activity of the GH receptor (GHR). G120R consistently antagonizes stimulation by wild-type human GH (13, 14).
We recently found increased serum GH levels in nonobese diabetic (NOD) mice (15) and streptozotocin (STZ)-treated mice (16), similar to the changes described for human patients. In addition, Flyvbjerg et al. (16) recently observed a blunting effect of a synthetic GHR antagonist on diabetic renal hypertrophy in mice, using the STZ model. The molecular mechanism that mediates this effect is still unknown, as is the response in other animal models. The aim of the study reported here was to investigate the involvement of the GH/IGF system on the development of nephropathy in NOD mice, by disrupting the biologic effects of GH with a synthetic GHR antagonist.
Materials and Methods
Animal Experimentation
Twelve-week-old, female, NOD/Alt mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Animal breeding procedures complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The cumulative incidence of overt diabetes among female animals of this strain is >70% by 100 d. Animals were housed in standard laboratory cages and provided free access to unlimited amounts of normal mouse chow and tap water. The onset of diabetes was determined by the appearance and persistence of glycosuria, which was assessed twice each week for all animals, using chemstrips (Ketostix; Bayer Diagnostics, Basingstoke, Hampshire, United Kingdom). When the urine glucose test results were positive, tail capillary blood samples were assayed for glucose using a glucometer (Elite; Bayer Diagnostics, Puteaux, France). The normal range (99% confidence level) of blood glucose levels for NOD mice is 3.0 to 9.9 mM. Diabetes was diagnosed when blood glucose levels were above the normal range on 2 d consecutively; the day that glycosuria was first noted was considered day 1 of diabetes. Only mice with elevated serum glucose levels and no ketonuria were included in the diabetic group. Diabetic mice could be maintained alive without insulin supplementation for up to 1 mo. The mice were divided into three groups, i.e., diabetic mice (D group), diabetic mice treated with GHR antagonist (DA group), and nondiabetic agematched controls. For the DA group, subcutaneous injections of a polyethylene glycol (PEG)-treated GHR antagonist (G120K-PEG, at a dose of 2 mg/kg; Sensus, Austin, TX) (16, 17) were administered every other day, beginning on day 1 of hyperglycemia. The other two groups were given injections with the same frequency, using the same volume of saline. Food intake was assessed twice each week. Somatic growth was measured as body weight and tail length. A proven standardized method of GH stimulation and secretion was used (18); mice were anesthetized with intraperitoneally administered pentobarbital (50 mg/kg), and blood was drawn from the retrobulbar plexus at least 5 min after injection. The serum was separated and frozen at -20°C for later measurement of IGF-I, IGF-binding protein 3 (IGFBP-3), and GH levels. The right kidney and liver were carefully removed and immediately frozen in liquid nitrogen and then at -70°C. A coronal 2-mm slice from the midportion of the left kidney was separated and fixed in a 4% paraformaldehyde solution for histomorphologic assessment.
Immunoassays
Serum GH levels were measured by RIA using a specific rabbit polyclonal anti-rat GH (rGH) antibody, with rGH as the standard. Semilogarithmic linearity of mouse serum and rGH (standard) values was found at multiple dilutions, indicating antigen similarity between mouse GH and rGH. The reagents, including 125I-rGH, were obtained from Amersham International (Bucks, United Kingdom). Serum IGF-I levels were measured after extraction with acid/ethanol. The mixture was incubated for 2 h at room temperature and centrifuged, and 25 μl of the supernatant was diluted 1:200 before analysis. Kidney protein extraction was performed as described previously (19). Briefly, 80 to 100 mg of tissue were homogenized on ice in 1 M acetic acid (5 ml/g of tissue) with an Ultra Turrax TD 25 (Janke-Kunkel, Stafen, Germany) and were further disrupted with a Potter Elvehjelm homogenizer. With this procedure, all IGFBP forms are removed from kidney tissue. After lyophilization, the samples were redissolved in phosphate buffer (pH 8.0) and kept at -80°C until the IGF-I assay was performed with diluted extracts. Serum and kidney IGF-I levels were measured by RIA using a rabbit polyclonal antibody (Nichols Institute Diagnostics, San Capistrano, CA), with recombinant human IGF-I (Amersham International) as the standard. The tissue IGF-I concentrations were corrected for the contribution of entrapped serum IGF-I (20). Monoiodinated IGF-I [125I-(Tyr31)-IGF-I] was obtained from Novo-Nordisk A/S (Bag-Svaerd, Denmark). Intra- and interassay coefficients of variation were <5% and 10%, respectively.
Western Ligand Blotting for Determination of Serum IGFBP-3 Levels
Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western ligand blotting were performed according to the method of Hossenlopp et al. (21), as described previously (22). Two microliters of serum was subjected to SDS-polyacrylamide gel electrophoresis (10% polyacrylamide) under nonreducing conditions. The electrophoresis-separated proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Munich, Germany) by electroelution, and the membranes were incubated overnight at 4°C with approximately 500,000 cpm of 125I-IGF-I (specific activity, 2000 Ci/mmol), in 10 ml of 10 mM Tris-HCl buffer containing 1% bovine serum albumin and 0.1% Tween (pH 7.4). The membranes were washed with 10 mM Tris-HCl buffer and dried overnight. The nitrocellulose membranes were then subjected to autoradiography using Kodak X-AR film and Du Pont-New England Nuclear enhancing screens, at -80°C, for 3 to 7 d. The specificity of the IGFBP bands was ensured by competitive coincubation with unlabeled IGF-I purchased from Bachem (Bubendorf, Switzerland). After Western ligand blotting (with 125I-IGF-I as the ligand), IGFBP-3 appeared as a 38- to 42-kD doublet band, corresponding to the intact, acid-stable, IGF-binding subunit of IGFBP-3. Western ligand blots were quantified by densitometric analysis of the autoradiographs obtained on x-ray-sensitive film, using a Shimadzu CS-9001 PC dual-wavelength flying spot scanner; gray pixel density was measured.
mRNA Studies
Total RNA was prepared from frozen tissues with Tri-ReagentTM (Molecular Research Center, Cincinnati, OH) and was quantified by measurement of absorbance at 260 nm. The integrity of the RNA was assessed by visual inspection of the ethidium bromide-stained 28S and 18S RNA bands after electrophoresis through 1.25% agarose/2.2 M formaldehyde gels. For Northern blot analysis, 20 μg of total RNA was subjected to electrophoresis through 1.3% agarose/2.2 M formaldehyde gels, in 3-morpholinopropanesulfonic acid buffer. The RNA was then transferred to MagnaGraph nylon membranes (Micron Separation, Inc., Westboro, MA) and cross-linked to the membranes with an ultraviolet crosslinker (Hoefer Scientific Instrument, San Francisco, CA). Two probes, i.e., rat IGFBP-1 and rat GHR (a gift from L. Mathews, Department of Biochemistry, University of Washington, Seattle, WA), were used and radiolabeled with [32P]dCTP (3000 Ci/mmol; Amersham) using a random-primer DNA labeling kit (Boehringer, Mannheim, Germany).
RNA hybridization was performed in a hybridization oven (Micro-4; Hybaid, Middlesex, United Kingdom) at 65°C for 20 h, using a hybridization solution containing 0.2 mM Na2HPO4 (pH 7.2), 7% (vol/vol) SDS, 1% (wt/vol) bovine serum albumin, and 1 mM ethylenediaminetetraacetate. Washes were performed with 0.4 × SSC/0.1% SDS at 65°C. Gels were exposed to Kodak X-Omat AR film (Eastman Kodak, Rochester, NY) at - 70°C with two intensifying screens. The autoradiographs were quantified with a PhosphorImager (Imagequant; Molecular Dynamics, Sunnyvale, CA).
IGF-I mRNA levels were determined using the solution hybridization/RNase protection assay, as described elsewhere (23). Briefly, 25 μg of total RNA were hybridized with 400,000 cpm of 32P-labeled riboprobe for 16 h at 45°C, in 75% formamide/0.4 M NaCl, followed by digestion with 40 mg/ml RNase A and 2 mg/ml RNase T1. Protected hybrids were precipitated, denatured, and subjected to electrophoresis through 8% urea/polyacrylamide gels. Gels were exposed to Kodak X-Omat AR film (Eastman Kodak) at -70°C, with two intensifying screens, and the protected band (231 bp) in the autoradiographs that corresponded to IGF-I was analyzed. The autoradiographs were quantified using a PhosphorImager (Imagequant; Molecular Dynamics).
Estimation of Glomerular Volume
A 2-mm-thick, horizontally cut slice from the middle of the right kidney (containing the papilla) was fixed in 4% paraformaldehyde and embedded in Technovit (Heraeus Kulzer, Wehrheim, Germany). Sections of 2-μm thickness were cut on a rotation microtome and stained with periodic acid-Schiff stain and hematoxylin. The mean glomerular tuft volume (VG) was determined from the mean glomerular cross-sectional area (AG) by light microscopy, as described previously (24, 25). Profile areas were traced with a computer-assisted morphometric unit (Image Tool; The University of Texas Health Science Center, San Antonio, TX). AG was determined as the average area of a total of 40 to 80 glomeruli (tufts, omitting the proximal tubular tissue within the Bowman's capsule), and VG was calculated as VG = β/κ × (AG)3/2, where β = 1.38 is the shape coefficient for spheres (the idealized shape of glomeruli) and κ = 1.1 is a size distribution coefficient.
Urinary Albumin Excretion
The albumin concentrations in spot urine samples obtained before euthanasia were determined by RIA, as described previously (19), using anti-rat albumin antibody and standards. Semilogarithmic linearity of mouse and rat (standard) urine albumin values was confirmed at multiple dilutions, indicating antigen similarity between mouse albumin and rat albumin. The urine samples were stored at -20°C until assayed. Rabbit anti-rat antibody was purchased from Nordic Pharmaceuticals and Diagnostics (Tilburg, The Netherlands). For standard use and iodination, globulin-free rat albumin was obtained from Sigma Chemical Co. (St. Louis, MO). Urine creatinine values were assessed simultaneously, to calculate albumin/creatinine ratios.
Statistical Analyses
One-way ANOVA was used to evaluate differences between groups, in combination with the Tukey “honestly significant difference” (HSD) test for multiple comparisons. P values <0.05 were considered significant. Results are presented as mean ± SEM.
Results
Body Weights and Metabolic Parameters
There was no significant between-group difference in body weight at euthanasia (D and DA groups, 95.3 and 92.5% of control values, respectively). Blood glucose levels were markedly elevated in the two diabetic groups, with mean values of approximately 20 mM throughout the study period, regardless of G120K-PEG administration. A previous study (16) showed that G120K-PEG administration to normal mice at the dosage used here was not associated with impairment of somatic growth or glycemic control.
Serum GH, IGF-I, and IGFBP-3 Levels
Serum GH levels were markedly elevated in the D group (125 ± 44 μg/L, compared with 11.2 ± 3.6 μg/L for control animals; P < 0.05) and remained elevated and unchanged in the DA group (112.6 ± 44 μg/L, P = NS versus D group) (Figure 1a). An opposite trend was observed for serum IGF-I levels, which were significantly decreased in both diabetic groups compared with controls (169 ± 26 and 183 ± 66 μg/L for the D and DA groups, respectively, compared with 498 ± 21 μg/L for controls; P < 0.05) (Figure 1b). There was no significant difference between D and DA serum IGF-I levels. The decrease in circulating IGF-I levels in diabetic mice was accompanied by a decrease in serum IGFBP-3 levels, which were measured in arbitrary densitometric units (161 ± 33 and 144 ± 39 pixel density units for the D and DA groups, respectively, compared with 271 ± 25 units for controls; P < 0.05). There was no significant difference noted between the D and DA groups (Figure 1c).
Mean levels of serum growth hormone (GH) (a), insulin-like growth factor-I (IGF-I) (b), and IGF-binding protein 3 (IGFBP-3) (c) on day 21 in control (C), untreated diabetic (D), and G 120K-polyethylene glycol (PEG)-treated diabetic (DA) mice. Values are mean ± SEM (n = 6 in each group). *P < 0.05 versus controls.
Kidney Weights and Glomerular Volumes
Figure 2a shows the kidney weights on day 21 of diabetes for the three groups. Kidney weights were increased in the D group (by a mean of 57 ± 5%) compared with the nondiabetic controls (256 ± 8 versus 163 ± 5 mg, P < 0.05), as well as in the DA group (209 ± 8 mg, 128 ± 5% of control values; P < 0.05 versus controls). However, the increase in kidney weight in the DA group was less than the increase in the D group (P < 0.05, by Tukey HSD test). Glomerular volume (Figure 2b) was increased by 27% in the D group compared with controls (2.21 ± 0.11 × 105 versus 1.75 ± 0.1 × 105 μm3, P < 0.05), but not in the DA group, where it remained similar to control values (1.84 ± 0.09 × 105 μm3).
Mean absolute kidney weight (a), glomerular volume (b), urinary albumin excretion (c), and extractable kidney IGF-I protein levels (d) on day 21 in control (C), untreated diabetic (D), and G 120K-PEG-treated diabetic (DA) mice. Values are mean ± SEM (n = 6 in each group). *P < 0.05 versus controls; †P < 0.05 versus D group.
Urinary Albumin Excretion
The urinary albumin excretion (UAE) values derived from spot urine samples before euthanasia for the three experimental groups are depicted in Figure 2c. At 21 d after diabetes onset, the untreated animals (D group) exhibited marked elevations in UAE (to 1.00 ± 0.14 mg/mmol of creatinine, compared with 0.47 ± 0.03 mg/mmol of creatinine in nondiabetic control mice; P < 0.05). The UAE value for the DA group (0.29 ± 0.07 mg/mmol of creatinine) was significantly lower (P < 0.05) than that for the D group but was unchanged from control values. G120K-PEG treatment did not affect diurnal urine production in diabetic animals (data not shown).
Kidney IGF-I Levels
Kidney IGF-I protein levels were increased in the D group compared with control values (402 ± 21 versus 211 ± 13 ng/g tissue, P < 0.05) (Figure 2d). This increase was abolished in the DA animals (273 ± 22 ng/g, 129 ± 10% of control values; P < 0.05, by Tukey HSD test, versus the D group but NS versus controls). In contrast, kidney IGF-I mRNA levels were decreased in both diabetic groups compared with controls (70 ± 9 and 57 ± 7% of control values for the D and DA groups, respectively; P < 0.05) (Figure 3). Kidney IGFBP-1 mRNA levels were increased in the D group (to 282 ± 25% of control values, P < 0.05) (Figure 4) but were not significantly increased in the DA group (160 ± 7% of control values, P = NS).
Renal IGF-I mRNA levels on day 21 in control (C), untreated diabetic (D), and G 120K-PEG-treated diabetic (DA) mice. Measurements were performed using solution hybridization/RNase protection assays. Quantification of IGF-I transcripts was performed using a PhosphorImager. The mRNA levels for the experimental groups (n = 5 in each group) are expressed as percentages of control values. *P < 0.05 versus controls.
(a) Northern blot analysis of kidney IGFBP-1 mRNA levels, using 50 μg of total RNA, on day 21 in control (C), untreated diabetic (D), and G 120K-PEG-treated diabetic (DA) mice. (Top) The 1.6-kb bands corresponding to the IGFBP-1 transcript. The film was exposed for 24 h at -70°C, with two intensifying screens. (Bottom) Ethidium bromide staining of the 18S rRNA of the samples before transfer to the membrane. (b) Quantification of kidney IGFBP-1 mRNA levels in the autoradiograph shown in Panel a, using a PhosphorImager. The data are expressed as percentages of control values (n = 5 in each group). *P < 0.05 versus controls.
Kidney GHR mRNA Levels
A 4.4-kb transcript encoding the GHR and a 1.2-kb transcript encoding the GH-binding protein (GHBP) were detected using a 964-bp fragment of the GHR cDNA (Figure 5). Renal GHR mRNA levels were significantly decreased at 3 wk after the onset of diabetes in both the D and DA groups (30 ± 10 and 24 ± 12% of control values, respectively; P < 0.05). GHBP mRNA levels were unchanged in both the D and DA groups compared with control values.
Northern blot analysis of kidney GH receptor (GHR) and GH-binding protein (GHBP) levels, using 25 μg of total RNA, on day 21 in control (C), untreated diabetic (D), and G 120K-PEG-treated diabetic (DA) mice. The GHR (▪) and GHBP (□) levels were quantified from autoradiographs using a PhosphorImager. The data are expressed as percentages of controls (n = 5 in each group). *P < 0.05 versus controls.
Discussion
This study shows that exogenous administration of a GHR antagonist to mice with spontaneous insulin-dependent diabetes has a positive effect on markers of early diabetic nephropathy (renal hypertrophy, glomerulomegaly, and albuminuria). GH and IGF-I may have pathogenic roles in diabetic nephropathy and other nephropathies. Mice transgenic for the bovine GH gene develop a disproportionate increase in glomerular volume, followed by severe glomerulosclerosis, uremia, and death (26). Renal hypertrophy and albuminuria in diabetic rats can be prevented by administration of long-acting somatostatin analogues (e.g., octreotide and lanreotide), which, on the other hand, are thought to be less specific inhibitors of the GH/IGF axis (19, 27, 28). We previously described a persistent increase in extractable kidney IGF-I levels, in association with renal hypertrophy, in NOD mice up to 4 wk after diabetes onset (29). In addition, kidneys of transgenic diabetic mice that expressed GHR antagonists (bGH-G119R and hGH-G120R) showed less glomerular hypertrophy and damage, no proteinuria, and no increase in glomerular α1 type IV collagen mRNA levels, compared with transgenic diabetic mice that expressed wildtype bovine GH (30, 31, 32). However, mice transgenic for the GHR antagonists may be inherently less susceptible to diabetic renal changes because of inborn effects of GHR blockade and low circulating IGF-I levels (resulting in a dwarf phenotype) before the induction of diabetes. In contrast, our data are based on a model of spontaneously acquired insulin-dependent diabetes (33, 34), which closely resembles human disease (35). Like human subjects, NOD mice develop proteinuria and glomerular lesions, including increases in glomerular surface area and mesangial sclerosis (36). The data presented here are the first to show the important role of GHR antagonism in this model.
Poorly controlled diabetes in human subjects is characterized by GH hypersecretion (37, 38). On the other hand, there are decreases in circulating GHBP levels (mostly hepatic in origin) and hepatic GHR number (39, 40). In contrast, only a few reports on renal GHR and GHBP expression in experimental diabetes are available. In a previous study, renal GHR mRNA expression was unchanged 4 d after STZ induction of diabetes in rats, despite decreasing levels of hepatic GHR mRNA (41). In another study (42), there were no changes in renal cortical GHR mRNA levels for diabetic animals during an observation period of 6 mo. However, there were significant increases in cortical GHBP mRNA and peptide levels, beginning 1 mo after the induction of diabetes and persisting thereafter. In the study presented here, downregulation of renal GHR mRNA levels was observed at 3 wk of diabetes, but renal GHBP mRNA levels were unchanged in control diabetic and G120K-PEG-treated diabetic NOD mice, compared with control nondiabetic animals. The lack of difference between the groups with respect to GHBP gene expression, as measured in whole-tissue homogenates, could be attributable to more subtle region-specific changes that might have been detected if in situ hybridization techniques had been used (6). Therefore, in type I diabetes, the high circulating GH levels in the presence of normal renal GHBP levels might be involved in renal hypertrophy and nephropathy by enhancing GH availability to the tissue. However, this hypothesis must be clarified in future studies.
Theoretically, the use of a GHR-blocking agent in diabetes could produce deterioration of the metabolic balance. In poorly controlled diabetes in human subjects (and in mice, as shown in this study), hyperglycemia and insulinopenia do not suppress GH secretion. Serum IGF-I levels are reduced in hyperglycemic diabetic subjects, despite elevated GH levels. This phenomenon has been explained by inhibition of hepatic IGF-I synthesis, resulting from decreases in hepatic GHR expression and binding (41). The metabolic consequences of these alterations produce a “vicious cycle,” wherein the hyperglycemia/insulinopenia induce decreases in serum IGF-I levels, which in turn induce GH hypersecretion (8, 37, 38), making optimal metabolic control more difficult to achieve. Therefore, a potential negative effect of GHR antagonists in diabetes could be a further lowering of circulating IGF-I levels, with associated deterioration of metabolic control. However, with the dose of GHR antagonist used in this study, no such effects on metabolic control or on endogenous GH/IGF-I levels were observed.
Our data support findings in previous reports that suggest that renal hypertrophy in type I diabetes is mediated by the increase in renal IGF-I bioavailability, despite the decrease in circulating levels of IGF-I. The increase in kidney IGF-I protein levels in untreated diabetes was abolished by G120K-PEG treatment. However, renal IGF-I mRNA levels were decreased in both diabetic and G120K-PEG-treated groups, suggesting that the G120K-PEG effect is not mediated entirely by reductions in IGF-I gene expression. Interestingly, G120K-PEG treatment was associated with a decrease in renal IGFBP-1 mRNA levels, which are usually elevated in diabetic mice (6, 30). We previously speculated that renal IGF-I accumulation is attributable to IGF-I binding to IGFBP-1 in kidney tissue. It is therefore possible that GHR blockade can prevent the accumulation of IGF-I protein in kidney tissue by effects on IGFBP-1 message levels.
This study, like a previous one with STZ-treated mice (16), demonstrates no toxic effects of G120K-PEG treatment on diabetic animals and no effects on UAE in nondiabetic mice. These safety characteristics make G120K-PEG a potential therapeutic agent for use with human patients. The chronic administration of this GHR antagonist to diabetic mice has inhibitory effects on diabetic renal/glomerular hypertrophy and UAE but no effects on metabolic control or circulating levels of GH, IGF-I, or IGFBP-3. Therefore, we speculate that the mechanism underlying the renal effects of this GHR antagonist involves renal GHR inhibition of renal IGF-I (and IGFBP-1) protein accumulation. This study demonstrates that the GH/IGF axis plays a central role in the pathogenesis of early diabetic renal changes, and it suggests specific GHR blockade as a new concept in the treatment of diabetic kidney disease.
Acknowledgments
Acknowledgments
This study was supported in part by the Sarah Lea and Jesse Z. Shafer Trust. The study was also supported by grants from the Danish Medical Research Council (Grant 9700592), the Danish Kidney Foundation, the Ruth König Petersen Foundation, the Novo Foundation, the Aage Louis-Hansen Memorial Foundation, the Nordic Insulin Foundation, the Eva and Henry Fränkels Memorial Foundation, and the Aarhus University-Novo Nordisk Center for Research in Growth and Regeneration (Grant 9600822). The GHR antagonist (G 120K-PEG) was a generous gift from Sensus Drug Development Corp. (Austin, TX). We thank Prof. Dina Pilpel (Department of Epidemiology, Ben Gurion University Faculty of Health Sciences) for help with statistical data analysis. We are grateful to Karen Mathiassen, Kirsten Nyborg, and Ninna Rosenqvist for excellent technical assistance.
Footnotes
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- © 1999 American Society of Nephrology
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