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Growth Hormone and Growth Hormone. Releasing Factor but Not in Those Expressing. Insulinlike Growth Factor-i. TOSHIO DOI, MD,* LILIANEJ. STRIKER, MD,*.
American Joumal of Pathology, Vol. 131, No. 3, June 1988 Copyright © American Association of Pathologists

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Progressive Gbomeruloscerosis Develops in Transgenic Mice Chronically Expressing Growth Hormone and Growth Hormone Releasing Factor but Not in Those Expressing Insulinlike Growth Factor-i From the Renal Cell Biology Group, Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland,* the Howard Hughes Medical Institute, and Department ofBiochemistry, University of Washington, Seattle, Washington, t and the Laboratory of Reproductive Physiology, School of Veterinary Medicine, University ofPennsylvania, Philadelphia, Pennsylvaniat

TOSHIO DOI, MD,* LILIANEJ. STRIKER, MD,* CAROL QUAIFE, PhD,t FRANCESCO G. CONTI, MD,* RICHARD PALMITER, PhD,t RICHARD BEHRINGER, PhD,* RALPH BRINSTER, VMD, PhD,,* and GARY E. STRIKER, MD

An increase in glomerular size occurs in normal maturation after subtotal renal ablation and disease states

remained morphologically normal except for the enlargement. These data suggest that the glomerulosclerosis was due, in part, to disordered mesangial cell growth in response to circulating GH. The mesangial lesions in mice with chronically high plasma GH levels mimicked those in human diabetes mellitus. These models provide a means to study the hormonal regulation of glomerular growth and the role that specific hormones might play in the pathogenesis of glomerulosclerosis. (AmJ Pathol 1988, 131:398-403)

such as diabetes mellitus. The role that growth hormone (GH), growth hormone releasing factor (GHRF), and insulinlike growth factor-i (IGF-1) play in these processes has been investigated using transgenic mice chronically expressing these hormones. The glomeruli were enlarged in all 3 strains of mice. Mesangial proliferation followed by progressive glomerulosclerosis was observed in the GH and GHRF animals only. In the IGF-1 mice the large glomeruli

INCREASED GLOMERULAR size occurs in the presence of normal maturation,' after unilateral nephrectomy in humans and animals,2 and in disease states such as diabetes mellitus.3 The glomeruli are morphologically and functionally normal after nephrectomy in rats unless the remaining renal mass is severely reduced, in which case progressive glomerulosclerosis ensues.4 The hormonal regulation of compensatory hypertrophy is not fully understood, however total kidney insulinlike growth factor- 1 (IGF- 1) mRNA levels are increased following unilateral nephrectomy.2'5 This suggests a role for this hormone in

hypertrophy of the adult kidney as well as in normal development.6 The growth factors operative in the disorderly growth characteristic of some glomerular disease processes have not been elucidated. However, there are abnormalities in the circulating levels of Supported in part by the National Institutes of Health Grants HDO9 172 (RDP) and HD 1 9018 (RLB). Accepted for publication April 8, 1988. Address reprint requests to Liliane J. Striker, MD, Metabolic Diseases Branch, NIDDK, Building 10, Room 3N 1 10, National Institutes of Health, Bethesda, MD 20892.

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growth hormone (GH) in some diseases associated with increases in glomerular extracellular matrix and cell number such as diabetes mellitus. The availability of transgenic mouse strains expressing elevated levels of IGF-I, GH, and growth hormone releasing factor (GHRF) provides an opportunity to study the effects of chronic hormone exposure during development and adulthood.7'8 Herein we report that mice containing an metallothionein- 1 (MT- 1) IGF- 1 fusion gene develop large glomeruli that are normal in appearance, whereas those transgenic for either GH or GHRF have large glomeruli which are abnormal. Initially the glomeruli are hypercellular, later sclerosis becomes apparent, and the end result is progressive glomerulosclerosis.

Materials and Methods Transgenic mice carrying genes for human IGF- 1,9 bovine GH,'° or human GHRF" were analyzed. The expression of these genes was put under the control of the mouse MT- 1 promoter." The founder mice used to generate the bovine GH (MT-GH line, #896-3) and human GHRF line (MT-GHRF, #765-2) had circulating GH levels of 500 ng/ml and 415 ng/ml respectively, compared with normal levels of 10 ng/ml.""3 The IGF- 1 (MT-IGF- 1, #1219-6) line had circulating IGF- 1 levels of 1.5 ± 0.3 U/ml compared with normal values of 1.0 ± 0.3 U/ml.9 Controls were litter mates that did not inherit the foreign DNA. The total number of mice examined was as follows: IGF- 1, 11; GHRF, 7; GH, 26; and age-matched controls, 19. An equal number of male and female mice were examined. There was no sex-related difference in the appearance of the kidneys. At sacrifice, organs were removed, blotted dry, weighed, and sections were placed in Carnoy's or Karnovsky's fixatives. Sections from paraffin and methacrylate embedded kidneys were stained with hematoxylin and eosin (H & E), PAS, and silver methenamine and were examined without knowledge of the experimental conditions (Figure 1). Methacrylate-embedded tissues were sectioned at 1 , and were stained with PAS for morphometric analysis. More than 30 glomeruli were examined in each animal. The glomeruli were selected randomly by scanning the cortex from the cortico-medullary junction to the outer surface in a serpentine fashion. Thus no glomerulus was counted more than once and every glomerulus encountered was examined. In addition, all levels of the cortex were evenly sampled. The glomerular surface was delineated by tracing the outline of the glomerular tuft.'4 The mean surface area was calculated. It was

399

found that there was a normal distribution of glomerular size in each animal. Statistical significance was assessed using analysis of variance (ANOVA).

Results Mice transgenic for GH were available sequentially from 1-37 weeks of age. At 4 weeks there was detectable mesangial proliferation which increased with age (Table 1, Figure 1B). Synechiae consisting of localized epithelial cell proliferation with adhesions between the glomerular tuft and Bowman's capsule and epithelial cell proliferation involving more than one half of the urinary space (crescents) were observed after 6 weeks. At 37 weeks there was diffuse mesangial sclerosis. Glomerular obsolescence (complete sclerosis) was noted in a few animals. GHRF mice were only examined at 14 weeks and showed severe lesions with mesangial proliferation and sclerosis comparable to those present in the GH mice (Figure 1D). In both groups the glomerular lesions bore strong resemblances to those observed in human diabetic glomerulosclerosis. Tubulointerstitial lesions were present in both groups of mice. They were of the type and intensity expected with the observed glomerular lesions. In GH mice arterial and/or arteriolar lesions were not present before 37 weeks at which time minimal, focal intimal thickening was noted in arterioles. By way of contrast no glomerular or tubulointerstitial lesions were observed in the IGF- 1 or control mice at any time point examined (Figure lA, C). Morphometric analysis revealed that the glomeruli of GH and GHRF mice were similar in size (Table 2). The absolute surface area and that corrected for body weight were greater than normal in the GH mice. The range of sizes in the IGF- 1 mice was large, therefore even though the mean size corrected to body weight was greater, the difference from control did not reach statistical significance. There was no significant difference in glomerular size between the GH and IGF- 1 mice when corrected for body size even though the absolute surface area of the glomeruli in GH mice was greater than that in IGF- 1 mice. Finally, glomerular size continued to increase, absolutely and relatively to body size, in the GH mice when assessed at 37 weeks.

Discussion Significant glomerular enlargement was seen in all 3 strains of transgenic mice. Progressively severe glomerular lesions characterized by initial mesangial hypercellularity followed by the appearance of severe sclerosis were seen in the GH or GHRF transgenic mice. In sharp con-

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., . ;.

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B-GH mouse, mesangial proliferation with A-Normal (WT). Figure 1-Methacrylate embedded sections, PAS stain, original magnification x1300. D-GHRF mouse, mesangial C-IGF-1 mouse, enlarged but otherwise normal appearing, 14 weeks. sclerosis and a nodular pattem, 14 weeks. proliferation with sclerosis, 14 weeks. ...

els were found to be maximal following the administration of 0.1 mg GH and did not increase further at substantially higher administered doses of GH.6 GH is known to induce multiple species of mRNA.'5 Therefore, it seems likely that the lesions in GH and GHRF mice were related to some action of GH in addition to its induction of IGF- 1 synthesis and release. However, because the IGF- 1 levels in the GH and GHRF mice were 2-3-fold higher than controls, compared with a 1.5-fold increase in the IGF- 1 mice, it remains possible that some of the pathologic glomerular changes could have been due to this hormone. We postulate that in GH and GHRF mice the increase in glomerular size may be related to the increased levels of IGF-1, but that the pathologic increase in glomerular cell number and the sclerosis result from other effects of GH. Furthermore, the data suggest that IGF- 1 plays a role in glomerular development similar to its role in other tissues, namely that it is responsible for the normal, coordinated development of tissues and it may be synthesized and act lo-

trast to these observations, IGF- 1 mice had no histologic

lesions other than their large size. The plasma levels of IGF- were reported to be elevated in all 3 strains: 1.5 times higher in IGF- 1 mice and 2-3 times higher in GH and GHRF mice.9" 0"'3 Glomerular surface area as a function of total kidney size remained relatively constant in all mouse strains examined in the current study (data not shown). The fact that the GHRF mouse glomerular lesions were similar to those in the GH mice is evidence that the renal response in both is due to an endocrine rather than paracrine or autocrine effect of GH. It also suggests that the glomerular lesions are not due to an abnormal or disproportionate local glomerular production ofGH. The plasma levels of GH and GHRF were high, but the IGF- 1 levels and growth response never exceeded 2-3-fold normal values. In fact, in other transgenic strains, GH levels as much as 10-fold higher induced no additional skeletal growth or body weight gain.9"'0 3 These data are consistent with that from hypophysectomized rats in which circulating IGF- 1 lev-

Table 1 -Morphologic and Morphometric Analysis (GH) Age Mean surface (wks) No./sex 273/NA 274/NA 276/M 277/M 279/F 285/F 391/M 280/M 282/F 284/M 392/M 389/M 327/M 328/M 329/M

333/F 334/F 335/F 390/M 412/F 409/F 41 0/M

402/F 403/F 405/M 407/M

1 1 4 5 5 6

6 10 10 10 10 12 14 14 14 14 14 14 24

30 31 35 36 36 37 37

2341 1883 4378 4013 3973

Mesangial hypercellularity

NA NA 6145

6026

-

NA NA NA 6741 6104 6116 5426 6104

+ ++

NA

8867 8734 9001 6679 8164 8904 8308

Synechiae

Crescents

-

-

_ _

_ _ _

+ + + + + +

5734

Mesangial sclerosis

+ + +

+ +

+ + + + -

-

obs 10% obs 10% obs 4%

-

-

NA, not available; obs, obsolescent; Mean surface area is expressed in Mm2.

obs 2%

++ ++ ++ ++ + ++ +++ ++ ++ ++ +++ + ++++ +++ ++++ ++++ +++

+ ++

_ +

_ +

_

_

+ + + +

-

-

-

+

++

++ + + + + ++ ++ + +

+++ +++

+

+ +

+

+ +

_

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Table 2-Glomerular Surface in Normal (WT) and Transgenic Mice Surface area

Age

GH IGF-I WT

GH WT

(weeks)

N

Mean

14 13 14

6 6 5 7 6

6038 4557 2708 8380 3506

30-37 30-37

SE 180 1 214 J J 153 307 * 250

Surface area/body weight SE Mean 140.8 150.8 117.6 231.7 131.0

6.3 15.3 t 2.1 27.15 t 5.5

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