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Edison Biotechnology Institute (L.L.B., A.N.H., J.J.K.) and the Department of Biomedical Sciences (J.J.K.), College of Osteopathic Medicine, Ohio University; Athens, Ohio 45701; and the Division of Nephrology, Department of Medicine, University of Miami School of Medicine (S.D., L.J.S., G.E.S.), Miami, Florida 33136
Address all correspondence and requests for reprints to: Dr. John J. Kopchick, Edison Biotechnology Institute, Konneker Research Laboratory, The Ridges, Ohio University, Athens, Ohio 45701. E-mail: kopchick{at}ohio.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Diabetogenic effects of excess GH have been demonstrated in humans with acromegaly as well as in dogs that were given GH injections (2). In acromegaly, progressive insulin resistance and the development of frank diabetes can occur. These problems are usually reversed upon treatment of the disorder. Increased GH secretion has been demonstrated in many individuals with insulin-dependent diabetes (3). As in acromegaly, excessive GH secretion in diabetes has been shown to produce insulin resistance. In diabetes, persistent elevations in GH also foster deterioration of metabolic control (4). Although diabetes-associated abnormalities in GH are most apparent when hyperglycemia is poorly controlled, elevated GH cannot always be normalized even with intensive insulin therapy (3).
The involvement of GH in diabetic end-organ damage was first suggested based on the finding that pituitary ablation could arrest or retard the development of proliferative retinopathy (4, 5). Because these patients received replacement therapy with adrenal steroids, thyroid hormones, and sex hormones, GH reduction appeared to account for the improvement. Renal function did not improve and sometimes deteriorated after pituitary ablation, but this was found to be due to increases in renal plasma flow and glomerular filtration rate subsequent to GH withdrawal. In other words, with regard to the effects of GH reduction on diabetic renal complications, improvements in microvascular disease were offset by hemodynamic changes (3).
Direct evidence for a role of GH in nephropathy has come from studies of transgenic mice expressing GH (6, 7). In addition to displaying a giant phenotype, transgenic mice expressing either bovine (b) GH or human (h) GH-releasing hormone (GHRH) developed progressive glomerulosclerosis. Transgenic mice expressing hIGF-I did not develop renal damage, although their glomeruli were enlarged. Together, these findings argue for a direct role of GH in nephropathy, independent of IGF-I activation.
Several years ago, we carried out site-directed mutagenesis to create
GH analogs in which one or more amino acid was changed. One such analog
involved two mutations in 
-helix III of the bGH gene, such that
amino acid 121 was converted from leucine to proline, and amino acid
126 was converted from glutamic acid to glycine (8). Mice expressing
this mutated bGH (called M11) developed glomerulosclerosis as severe as
that in bGH transgenic mice, yet they had normal body size and normal
IGF-I levels (7, 9). These findings in the M11 mice strengthen the
hypothesis of a direct role of GH in the development of
nephropathy.
Another analog of bGH, in which amino acid 119 was converted from glycine to lysine (G119K), was shown to be a functional GH antagonist, which, when expressed in transgenic mice, produced a dwarf phenotype (10, 11). Similarly, an analog of hGH in which amino acid 120 is converted from glycine to arginine (G120R) produced GH antagonist effects, including a dwarf phenotype (12). Both analogs bound to GH receptors, but failed to activate postreceptor signal transduction.
To determine whether bGH-G119K also could protect mice from GH-associated nephropathy, streptozotocin (STZ) was used to induce diabetes in female mice expressing bGH-G119K (13). Mice expressing another bGH analog, in which amino acid 117 was mutated from glutamic acid to leucine (E117L), but which produced a giant phenotype identical to that of transgenic mice expressing native bGH, were also made diabetic, as were nontransgenic control mice. After 27 weeks, kidneys were examined for evidence of nephropathy. bGH-E117L mice developed severe glomerulosclerosis, whether given STZ or not. Nontransgenic control mice treated with STZ also developed glomerulosclerosis. However, bGH-G119K mice were completely protected from diabetes associated kidney damage.
While these findings in bGH-G119K suggested that the prevention of GH-associated functional activation inhibited the development of diabetes-associated glomerular lesions, it remained possible that some other action of the analog was responsible for the protection. Recently, we generated a new strain of mice in which the gene for the GH receptor/binding protein (GHR/BP) has been disrupted, creating a GHR/BP knockout mouse (GHR/BP-KO) (14). Like bGH-G119K mice, these mice have a dwarf phenotype, although only mice homozygous for the GHR/BP null mutation show reduced growth relative to wild-type controls. Both dwarf strains have significant reductions in IGF-I that are approximately 1 order of magnitude less that those in controls (14). However, whereas bGH-G119K mice have normal levels of endogenous GH, GHR/BP-KO mice have significantly elevated levels of GH. If GH-associated kidney damage depends on signal transduction resulting from GH binding to GHR, then despite elevated circulating GH, the GHR/BP-KO mice, like the bGH-G119K mice, should be protected from diabetes-induced glomerulosclerosis. In the present study we tested this hypothesis by assessing kidney histopathology in homozygous (-/-) and heterozygous (+/-) GHR/BP-KO mice and in their wild-type (+/+) littermates 10 weeks after induction of diabetes with STZ.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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STZ treatment
Daily ip injections of STZ (80 µg/g BW, diluted in 0.1
M citrate buffer in a volume of 10 µl/g) or an equal
volume of citrate buffer were administered to 8-week-old mice until
blood glucose levels exceeded 200 mg/dl or for a maximum of eight
injections. Three mice (two +/- and one -/-) had blood glucose
concentrations less than 200 mg/dl after the eighth STZ injection.
However, within 2 weeks, these levels had risen to well over 200. The
mice were maintained for 10 weeks without insulin treatment. Blood
glucose levels in diabetic mice were determined 1 week after the final
STZ injection, 2 weeks after completion of the STZ regimen, and at the
end of the study 10 weeks after the STZ regimen to track changes in
hyperglycemia. Blood glucose concentrations were measured in
nondiabetic mice only once, at the end of the experiment. All mice were
weighed at the beginning and end of the study. At death, the left
kidney was perfused through the aorta, first with 0.15 M
NaCl, then with 4% paraformaldehyde in 0.15 M NaCl.
Thereafter, it was removed, cut in half longitudinally, and stored in
4% paraformaldehyde for at least 24 h before preparation of
sections for examination by light microscopy. The right kidney was
removed without prior perfusion and weighted.
Renal histology
Light microscopy. Blocks of fixed kidney tissue were
embedded in glycol methacrylate. Four-micron sections were stained with
hematoxylin and eosin and periodic acid-Schiff (PAS). The kidney
sections were examined blinded, without prior knowledge of the
group.
Morphometry. For each kidney, digital images of 20 randomly chosen consecutive glomeruli were captured with a 1-chip color CCD camera (Bunton Instruments, Rockville, MD) mounted on an Olympus Corp. light microscope (Bunton Instruments) by moving from cortex to medulla in a serpentine fashion. The glomerular tuft was traced, and the enclosed area was copied to create a new image. Both the total glomerular area (including nuclei and spaces within capillary loops) and the mesangial area (excluding nuclei) were obtained using Adobe Photoshop 4.0 (Adobe Systems, Inc., San Jose, CA) and NIH Image 1.61 (15). Glomerular volume was determined by measuring 50 consecutive, randomly chosen glomeruli for each slide as previously described (16). For each glomerulus, the ratio of mesangial/total glomerular area was calculated. Ratios were averaged for each animal. The result (percent mesangial area) was used to determine the severity of glomerulosclerosis in each animal.
Statistical analysis
The data were analyzed with two-way ANOVA to compare the effects
of STZ treatment [diabetic (DB) vs. nondiabetic (ND)] and
of genotype (+/+ vs. ± vs. -/-) using
Statistics (StatSoft, Tulsa, OK). Blood glucose measurements in the
diabetic groups were compared using a repeated measure design (1, 2,
and 10 weeks after STZ treatment). When significant interactions
occurred, post-hoc pairwise comparisons were made with
Tukey’s highest significant difference test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the mean blood glucose
levels in STZ-treated +/+, +/-, and -/- groups measured 1, 2, and 10
weeks after diabetes induction. Statistical comparisons of the three
blood glucose determinations indicated that hyperglycemia increased
significantly and equivalently over the course of the study period in
the three groups (F = 25.98; P < 0.00001). A
two-way ANOVA comparing final blood glucose levels in DB and ND groups
(Fig. 1B) indicated a significant effect of STZ treatment (F =
243.85; P < 0.000001), verifying that all DB groups
had significantly higher blood glucose levels than ND groups. Separate
one-way ANOVAs indicated there were no differences in final blood
glucose concentration among either the three DB groups or the three ND
groups.
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). There were significant genotype by time
point and STZ treatment by time point interactions (F = 17.2;
P = 0.004 and F = 145.6; P <
0.000001, respectively). Therefore, the time points were further
analyzed with individual two-way ANOVAs. In the initial body weight
measurement, there were no differences between mice receiving STZ and
those receiving vehicle, but as expected, the two -/- groups weighed
significantly less than either +/- or +/+ mice. (F =
84.5,;P < 0.00001). In the final body weight
measurement, there were significant effects of genotype (F = 72.3;
P < 0.00001) and STZ treatment (F = 31.4;
P < 0.0001). DB +/+ and DB -/- weighed significantly
less than their ND counterparts, but the difference between DB ±
and ND +/- failed to reach statistical significance. There were
interactive effects of genotype and STZ treatment on kidney wet weight
(Fig. 3; F = 17.6; P =
0.00001). Mean kidney wet weight of both DB +/+ and DB +/- both were
significantly greater than that of their respective ND control groups,
while kidney wet weight of DB -/- and ND -/- did not differ
statistically.
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. Figure 4, A, C, and E, shows kidney
sections of nondiabetic +/+, +/-, and -/- mice, respectively. None
of the nondiabetic mice exhibited any glomerular lesions. Diabetic +/+
mice (Fig. 4B) exhibited diffuse, but moderate, glomerulosclerosis with
a diffuse increase in PAS-positive material, consistent with previous
data from our laboratory (13, 17). The number of cells in the mesangial
areas were not increased in the diabetic mice. The lesions were
somewhat irregular from one glomerulus to the next, but there were no
normal glomerular tufts in the DB +/+ mice. The glomerular vascular
spaces appeared dilated without apparent thickening of the peripheral
glomerular basement membranes. The mesangial sclerosis appeared
distributed equally between the cortical and the juxtamedullary
regions. There was occasional thickening of Bowman’s capsules. Many
tubular epithelial cells contained PAS-positive droplets. Diabetic +/-
mice (Fig. 4D) exhibited the same lesions as DB +/+ mice. In contrast,
there were no glomerular and tubular lesions in DB -/- mice (Fig. 4F).
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) revealed significantly
smaller glomerular volume in ND -/- mice (2.02 ± 0.38) relative
to ND +/- and +/+ mice (4.31 ± 0.83 and 4.23 ± 1.10,
respectively). There was a significant increase in glomerular volume in
DB +/+ (7.85 ± 1.34) and DB +/- (6.04 ± 0.86) mice
compared with ND +/+ (4.23 ± 1.10) and ND +/- (4.31 ±
0.83) mice (P < 0.001 and P < 0.05,
respectively). In contrast, there was no difference in glomerular
volume between DB -/- (2.67 ± 0.27) and ND -/- (2.02 ±
0.37) mice. The ratio of mesangial to total glomerular surface area
(Fig. 6) was also significantly increased in
DB +/+ (151 ± 47.5%) and DB +/- (156 ± 24.2%) mice
compared with those in ND +/+ (100 ± 17.6) and ND +/- (104
± 18.7) mice (P < 0.05), but not in the DB -/- mice
relative to ND -/- animals. The ratio of mesangial to total
glomerular surface area was significantly smaller in DB -/- mice than
in both DB +/+ and DB +/- mice. However, the ratio in ND -/- mice
did not differ statistically from that in either the ND +/+ or the ND
+/- group.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clinically, diabetes mellitus is often accompanied by elevated GH but reduced IGF-I levels, possibly due to hepatic resistance to GH, which is followed by reduced IGF-I production that, in turn, lowers feedback inhibition over further GH production (4). These changes are most evident in poorly controlled diabetes, and it is in poorly controlled diabetes that complications such as nephropathy are most likely to occur. Recently, somatostatin, the hypothalamic peptide that exerts inhibitory control over the GH-IGF-I axis, and a somatostatin analog, octreotide, which has a longer half-life, have been shown to reduce elevated glomerular filtration and increased kidney size in individuals with diabetes (19). Studies of octreotide’s effects in STZ-diabetic rats demonstrated both short term and long term reductions in the development of glomerular hypertrophy when the drug therapy was initiated early after diabetes induction (18). Interestingly, these benefits accrued despite the fact that, unlike humans with diabetes mellitus, STZ-diabetic rats had reduced GH.
A novel GH receptor antagonist, G120K-PEG, has been developed that may offer more specific inhibitory regulation of excessive GH activity. STZ-diabetic mice were treated with G120K-polyethylene glycol for 1 month beginning on the day after inducing diabetes with a single STZ injection (20). Kidney weight and glomerular volume were normalized in the drug-treated mice, whereas kidneys in the placebo-treated diabetic mice showed early changes indicative of developing nephropathy. One important finding in this study was that both groups of diabetic mice had significant elevations in circulating GH relative to levels in nondiabetic controls, making the STZ mouse model more consistent with the human disorder than the STZ rat.
A growing body of evidence has indicated a direct role of GH in the development of glomerulosclerosis (6, 7, 8, 9). Transgenic mice that express GH fusion genes (giant animals) develop glomerulosclerosis, whereas those that express GH antagonists transgenes (dwarf animals) do not (6, 7, 8, 9). Additionally, animals that express IGF-I transgenes do not develop glomerulosclerosis, although their glomeruli are enlarged (6). Whether expression of GH signaling molecules is altered in diabetic kidneys is not known, but is the subject of our current research. In this regard, we are determining the levels of GHR, Jak2 (Janus kinase-2), and Stat5 (signal transducer and activator of transcription-5) in the kidneys of these animals, as these molecules are involved in GH signaling. In our first series of studies, we found no change in the levels of GHR and a decrease in Jak2 in the kidneys of diabetic normal and GHR gene-disrupted animals. Additionally, the levels of Stat5 were increased in the diabetic kidney of GHR gene-disrupted animals relative to controls (Cataldo, L., and J. J. Kopchick, unpublished results).
Because nephropathy is one of the most common complications of diabetes, it is important to find treatment approaches that will minimize or eliminate the progression of kidney damage. The present finding of renal protection in GHR/BP knockout mice taken together with our previous finding of parallel protection in transgenic mice expressing a GH antagonist present a consistent picture of a specific approach for protecting the kidney.
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Acknowledgments |
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Footnotes |
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Received June 22, 1999.
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References |
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-helix of bovine growth
hormone dramatically affect its intracellular distribution in
vitro and growth enhancement in transgenic mice. J Biol Chem 266:2252–2258This article has been cited by other articles:
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