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Life-Long Suppression of Growth Hormone-Insulin-Like Growth Factor I Activity in Genetically Altered Rats Could Prevent Age-Related Renal Damage Yan Zha, Viet Thang Le, Yoshikazu Higami, Isao Shimokawa, Takashi Taguchi, and M. Shawkat Razzaque Department of Pathology (Y.Z., V.T.L., T.T., M.S.R.), Nagasaki University Graduate School of Biomedical Sciences; and Department of Investigative Pathology (Y.H., I.S.), Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan To determine whether life-long reduction in the GH/IGF-I activity could be renoprotective and attenuate renal damage, we examined the kidneys of transgenic strain of rats whose GH gene was suppressed by an antisense GH transgene. Male rats homozygote for the transgene (tg/tg) had a reduced number of pituitary GH-secreting cells, and 53% less plasma concentration of IGF-I, compared with wild-type (wt/wt) rats at 6 months of age. We compared the kidneys obtained from male wildtype young (6 months) and old (24 months) rats with male homozygote transgenic young (6 months) and old (24 months) rats. The wild-type rats showed features of renal damage as they grew older, including glomerulosclerosis (higher sclerosis index at 24 months; P < 0.0001), tubulointerstitial widening (increased interstitial volume at 24 months; P < 0.0001), and presence of phenotypically altered myofibroblasts and increased accumulation of collagens. Life-long suppression of

GH/IGF-I activity resulted in prevention of age-associated renal diseases in homozygote transgenic rats at 24 months (sclerosis index: 1.65 ⴞ 0.11 in wild-type vs. 0.463 ⴞ 0.061 in transgenic rats; interstitial volume: 34.2 ⴞ 0.82 in wild-type vs. 12.8 ⴞ 0.32 in homozygote transgenic rats at 24 months; P < 0.0001). Such reno-protective effects in transgenic rats were associated with decreased renal accumulation of ED-1-positive macrophages, and less renal expression of pro-fibrogenic factors, including connective tissue growth factor and heat shock protein 47. Our in vivo genetic manipulation study provides direct evidence of reno-protective effects of life-long suppression of GH/IGF-I system, by reducing renal infiltration of inflammatory cells, and by suppressing the synthesis of profibrogenic factors and accumulation of extracellular matrix protein. (Endocrinology 147: 5690 –5698, 2006)

This study tested the hypothesis that life-long suppression of the GH/IGF-I system can prevent age-associated renal damage, including glomerulosclerosis and tubulointerstitial fibrosis. We recently generated a transgenic strain of rats in which GH synthesis and release are suppressed by induction of an antisense GH transgene (11, 12). Here, we used these transgenic rats to study the effects of GH/IGF-I suppression on renal diseases in naturally aging kidney (24 months), a nontoxic model of progressive glomerulosclerosis and tubulointerstitial fibrosis.

LOMERULOSCLEROSIS IS excessive accumulation of extracellular matrix in the kidney glomerulus. It is a feature of many glomerular diseases including glomerulonephritis, diabetes mellitus, and hypertensive nephrosclerosis (1, 2). The glomerulosclerotic process is usually progressive in nature, eventually affecting the function of neighboring structures due to the gradual expansion of scar tissue. The degree of glomerulosclerosis correlates with a progressive loss of renal function, and eventual end-stage renal disease (1, 3– 6). Although the exact molecular mechanisms underlying glomerulosclerosis remain unclear, there is evidence for the involvement of GH; e.g. the development of glomerulosclerosis in GH-overexpressing transgenic mice (7). IGF-I is produced by many tissues, including the kidney, and may induce renal hypertrophy and sclerosis through endocrine, autocrine, or paracrine activities (8). However, because IGF-I is only weakly expressed in the glomerulus, these IGF-I-induced effects are more likely due to circulating peptide(s) that interacts with glomerulus-resident IGF-I receptor-expressing cells (9, 10). First Published Online September 7, 2006 Abbreviations: CTGF, Connective tissue growth factor; HSP47, heat shock protein 47; PAS, periodic acid-Schiff; PCNA, proliferating cell nuclear antigen; ␣-SMA, ␣-smooth muscle actin; tg/tg, male rats homozygote for the transgene; tg/wt, hemizygote; wt/wt, wild-type rats. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

Materials and Methods Animals The transgenic male rats [mini, Jcl:Wistar-TgN(ARGHGEN)1Nts] used in the present study were obtained from the Nippon Institute for Biological Science (Oume City, Tokyo, Japan). The generation of these transgenic strains to express an antisense GH transgene to suppress GH expression has been described previously (11). Wistar rats with a similar genetic background to the transgenic rats were used as wild-type controls. Rats were maintained in accordance with the guidelines for the care and use of laboratory animals and were studied according to the protocols approved by the Ethics Review Committee for Animal Experimentation at Nagasaki University Graduate School of Biomedical Sciences.

Feeding of the animals Four-week-old male rats were kept in a barrier facility (temperature, 24 ⫾ 1 C; 12-h light, 12-h dark cycle), which was housed separately and maintained under specific pathogen-free conditions during the study period. Rats were fed a CR-LPF diet (Oriental Yeast Co., Tsukuba, Japan), which is based on the formula of Charles River Inc. (Wilmington,

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Zha et al. • Antisense GH Transgenic Rats Prevent Renal Injury

MA) (Charles River Formula-1); per 100 g body weight, the diet comprised 18.2 G protein, 4.8 g fat, 6.6 g mineral mixture, 5.0 g fiber, 57.9 g nitrogen-free water-soluble substance, and 7.5 g water. The caloric value of the diet is 348 kcal/100 g. All rats were fed with the diet and water ad libitum (11).

Survival study Wild-type (wt/wt; n ⫽ 30), homozygote (tg/tg; n ⫽ 30), and hemizygote (tg/wt; n ⫽ 30) rats were used for the longevity study and were monitored until spontaneous death occurred (11). Internal organs were removed from the dead animals to determine cause of death and to examine any renal lesions. Kidneys of the spontaneously died rats were not used for histomorphometric analysis described below; kidneys collected immediately after the rats were killed at desired time points were used for histomorphometric and immunohistochemical studies.

Plasma concentrations of IGF-I Plasma samples were collected from young and old wild-type and mutant rats. Enzyme immunoassays were conducted on these samples to determine the concentration of circulating IGF-I (Diagnostic Systems Laboratories, Inc., Webster, TX), as instructed by the manufacturer.

Renal tissue collection The wild-type and transgenic control rats were killed at 6 and 24 months of age. At least five rats from each group at each age point were killed. Both kidneys were removed via a midline abdominal incision, and immediately fixed overnight in 10% formalin for morphological and immunohistochemical analysis.

Histological and morphometric analyses Renal tissues were routinely processed and embedded in paraffin, cut into 4-␮m-thick sections and stained with hematoxylin and eosin, periodic acid-Schiff (PAS), periodic acid-Schiff methenamine silver, and Masson’s trichome. The histological changes were determined by light microscopy. The glomerular sclerosis index was calculated using a standard procedure as detailed in earlier publications (13, 14); 12 glomeruli per rat kidney were used to determine the glomerular sclerosis index. The degree of sclerosis in each glomerulus was graded on a scale of 0 (no change) to 4 (global sclerosis) on PAS-stained sections. The average grade was calculated and registered as the glomerular sclerosis index. Glomerular volume was determined by computer-assisted image analysis, using AxioVision software connected to Zeiss microscope (Carl Zeiss, Jena, Germany). The overall mean values of these parameters for each group were calculated based on individual values. The extent of tubulointerstitial change was determined from the interstitial tissue volumes. A standard point-counting method was used to quantify the volume of the renal interstitium (7, 15, 16) on histological sections stained with Masson’s trichome, which stains collagen fibers in the interstitial spaces. Under high magnification (⫻400), consecutive nonoverlapping fields were photographed from each section of renal cortex. A grid containing 117 (13 ⫻ 9) sampling points was superimposed on each photograph and a total of 1170 points were evaluated in each kidney section. The number of points falling on tubular basement membranes was counted, whereas points falling on Bowman’s capsules or peritubular capillaries were ignored. Points falling on renal glomeruli or on larger vessels were also excluded from the total point counting. For this study, only fields containing one glomerulus were studied quantitatively, and the relative volume was considered as the average value from three kidney sections. Although the used calculations are not an unbiased method, a possible error would most likely to affect all groups equally.

Immunohistochemical studies Immunohistochemical staining was performed as described previously (17, 18). Briefly, paraffin-embedded tissue sections were deparaffinized with xylene, rinsed thoroughly with 95% ethanol, and then soaked in 0.3% hydrogen peroxide in methanol for 30 min at room temperature to inactivate endogenous peroxidase activity. After a 5-min

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treatment with 0.05% trypsin (T4799; Sigma, St. Louis, MO), the tissue sections were incubated with either 10% goat serum or 10% rabbit serum for 30 min, and then with one of the following primary antibodies: anticonnective tissue growth factor (CTGF) (dilution, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA), proliferating cell nuclear antigen (PCNA) (1:100; Dako, Glostrup, Denmark), ED-1 (1:100; Serotec, Oxford, UK), ␣-smooth muscle actin (␣-SMA) (1:100; Dako), heat shock protein 47 (HSP47) (1:100; Stress Gene Biotechnologies, Victoria, British Columbia, Canada) and IGF-I (1:100; Abcam plc, Cambridge, UK). The slides were washed with PBS and processed further using a Histofine streptavidin-peroxidase kit (Nichirei, Tokyo), as recommended by the manufacturer. Antibody binding was visualized by reaction with 3.3⬘ diaminobenzindine and H2O2. Quantitative analysis was conducted by counting the numbers of interstitial-infiltrating macrophages (ED-1-stained), proliferating cells (PCNA stained), phenotypically altered cells (␣-SMA stained), fibrogenic molecule-expressing cells (CTGF and HSP47 stained) and IGF-Iexpressing cells in five randomly selected fields of the renal cortex (⫻400 magnification). The average number of each cell type was then calculated separately in the glomerular and tubulointerstitial compartments.

Statistical analysis Statistically significant differences between groups were evaluated using Student’s t test or Fisher’s test for comparison between two groups, or by one-way ANOVA followed by Tukey’s test for multiple comparisons. Survival was analyzed using Kaplan-Meier’s estimates and was compared using the log-rank test. All values were expressed as mean ⫾ sem. A P value less than 0.05 was considered statistically significant. All analyses were performed using Microsoft Excel, or StatView 5.0 software (SAS Institute Inc., Cary, NC).

Results Survival and plasma levels of IGF-I

The survival rate of the homozygote rats was significantly less (P ⬍ 0.01) than that of the wild-type rats. This was predominantly due to the appearance of several tumors in association with the severely reduced GH/IGF-I activity in the homozygote rats. The homozygote life span was decreased by around 7 wk (5%, 50th percentile) and 14 wk (10%, 25th percentile), compared with wild-type rats (Table 1). In contrast, the hemizygote rats showed significantly higher survival rates (P ⬍ 0.03) than wild-type rats; the life span was around 12 wk (7%, 50th percentile) and 14 wk (10%, 25th percentile) longer in hemizygote rats than in wild-type controls (Fig. 1) (11). Plasma levels of IGF-I in 6-month-old mutant rats (Table 1) were significantly decreased (P ⬎ 0.001) compared with the wild-type rats (1613.6 ⫾ 57.3 ng/ml) in both hemizygote (1172.2 ⫾ 73.3 ng/ml) and homozygote (768.9 ⫾ 12.6 ng/ml) animals. Similar reductions were also detected in homozygote (649 ⫾ 155 ng/ml) rats at 24 months of age, compared with their age-matched wild types (1230.2 ⫾ 121 ng/ml). Gross kidney morphology and food intake

The average body weights of the transgenic rats were significantly (P ⬎ 0.001) lower than those of age-matched wild-type rats (494 ⫾ 17 g and 624 ⫾ 29 g at 6 and 24 months, respectively, in wild-type rats) vs. (192 ⫾ 2 g and 240 ⫾ 9 g at 6 and 24 months, respectively, in transgenic homozygote rats). Kidneys were collected from wild-type and homozygote transgenic rats at 6 and 24 months of age, and examined macroscopically. No significant gross abnormalities were detected in any of the kidneys. The average weights of the

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TABLE 1. Survival analysis and IGF-I concentrations in wild-type and mutant rats Genotype

Age of 50th percentile (wk)

Age of 25th percentile (wk)

Maximum survival (wk)

Plasma IGF-I (ng/ml) (young)

Plasma IGF-I (ng/ml) (old)

Wild-type Hemizygote Homozygote

126 ⫾ 7 138 ⫾ 14 119 ⫾ 2

140 ⫾ 3 154 ⫾ 5 126 ⫾ 3

158 171 148

1613.6 ⫾ 57.3 1172.2 ⫾ 73.3b 768.9 ⫾ 12.6c

1230.2 ⫾ 121a 1362.4 ⫾ 104.7 649 ⫾ 155.9d

Hemizygote rats showed the highest overall survival. The maximum life span was 158 wk in wild-type rats, 148 wk in homozygote rats (6% less than wild type), and171 wk in hemizygote rats (8% more than wild type). The plasma levels of IGF-1 were decreased by 53% in homozygote rats and by 28% in hemizygote rats, compared with wild-type rats at 6 months (n ⫽ 4 –11). Note that plasma IGF-1 levels remain low throughout the life span of mutant rats. There was 48% less plasma IGF-1 levels in old homozygote rats, compared with wild-type rats of similar age. a P ⬍ 0.05; compared with 6-month-old wild-type rats. b P ⬍ 0.001; compared with age-matched wild-type rats. c P ⬍ 0.001; compared with 6-month-old wild-type rats. d P ⬍ 0.01; compared with age-matched wild-type rats.

transgenic kidneys were significantly (P ⬎ 0.01) lower than those of age-matched wild-type rats (1.4 ⫾ 0.03 g and 1.8 ⫾ 0.07 g at 6 and 24 months, respectively, in wild-type rats) vs. (0.56 ⫾ 0.02 g and 0.68 ⫾ 0.04 g at 6 and 24 months, respectively, in transgenic rats); however, the differences were not significant when the kidney weights were corrected for total body weight (data not shown). The pattern food intake in homozygote and hemizygote rats was some what similar to that in wild-type rats. The food intake did not change significantly in wild-type rats with increasing age after 12 wk but was slightly reduced after 108 wk. The homozygote and hemizygote rats consumed about 50% and 70 – 80% of the mean intake of wild-type rats, respectively. In addition, there were relatively reduced weight gain and reduced food efficiency in homozygote and hemizygote rats compared with wild-type rats (Fig. 2). We have provided time course data on the body weight and the measurements of food intake in various genotypes in our earlier publication (11).

Figures 4 show the histomorphometric changes in the kidneys of all wild-type and transgenic rats. Compared with 6-month-old wild-type rats, the sclerotic glomeruli index was significantly (P ⬍ 0.0001) higher at 24 months (0.35 ⫾ 0.06 at 6 months vs. 1.65 ⫾ 0.11 at 24 months) (Fig. 4A). The glomerular volume of nonsclerotic glomeruli of 24-monthold wild-type rats was higher than that of 6-month-old rats, whereas the glomerular volume of kidneys of the 24-month-old transgenic rats was significantly (P ⬍ 0.001) less than wild-type rats of the same age (Table 2). In general, glomeruli in the aged wild-type kidneys showed infiltration of inflammatory cells, glomerulosclerosis, and interstitial fibrotic changes, but no such changes were detected in the transgenic rats with suppressed GH/IGF-I activity, even at 24 months (0.287 ⫾ 0.006 at 6 months and 0.463 ⫾ 0.061 at 24 months in homozygote rats) (Fig. 4A). Similar improvement was also detected in the interstitia of aged transgenic homozygote rats based on interstitial volume (34.2 ⫾ 0.8 in wild-types vs. 12.8 ⫾ 0.3 in transgenic rats at 24 months; P ⬍ 0.0001) (Fig. 4B).

Histomorphometric analysis of the kidney

Unlike the wild-type rat kidneys at 6 months of age (Fig. 3A), we found glomerulosclerosis, interstitial inflammatory cell infiltration, and fibrosis in the kidneys of 24month-old control rats (Fig. 3B). No obvious histological changes were observed in kidneys of the transgenic rats at either 6 months (Fig. 3C) or 24 months (Fig. 3D) of age.

FIG. 1. Survival curve of various genotypes of rats, showing higher overall survival in hemizygote (tg/wt) transgenic rats, compared with homozygote (tg/tg) and wild-type (wt/wt) rats. [Reprinted with permission from I. Shimokawa et al.: Am J Pathol 160:2259 –2265, 2002 (11). © American Society for Investigative Pathology.]

FIG. 2. Food efficiency of various genotypes of rats, showing relatively less food efficiency in homozygote and hemizygote rats, compared wild-type rats of similar age. The food efficiency was estimated by the following formula: body weight gain at age A ⫽ (body weight gain between age A and age A⫹4 wk)/4; food intake at age A (g/ d); food efficiency ⫽ (food intake at age A)/(body weight gain at age A) [(g/d)/(g/wk)]. Two-factor ANOVA analysis indicated that rat effect, P ⬍ 0.0001; age effect, P ⫽ 0.0252; rat ⫻ age, P ⫽ 0.0005.

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FIG. 3. Histological features of kidneys from 6-month-old (A and C) and 24-month-old (B and D) wild-type and homozygote transgenic rats. Severe glomerular and tubulointerstitial damage, with massive infiltration of inflammatory cells and interstitial fibrosis were consistently noted in 24-month-old wild-type rat kidneys (B). Life-long suppression of the GH/IGF-I system seemed to significantly reduce age-associated renal damage in the 24-month-old homozygote rats (D). There were no significant changes in the kidneys of 6-month-old control (A) and transgenic (C) rats. A–D, PAS staining; original magnification, ⫻20.

Renal expression of IGF-I

Renal expression of ␣-SMA, CTGF, and HSP47

The immunostaining of IGF-I in 6-month-old wild-type rat kidneys was very weak, compared with the increased renal expression of IGF-I observed in the 24-month-old wild types (Fig. 5A). The renal expression of IGF-I in 24-month-old transgenic homozygote rats was much less (Fig. 5B) than wild-type animals of the same age. The expression of IGF-I in 6-month-old transgenic rat kidneys was similar to the wild-type rat kidneys of the same age (data not shown). Chronic suppression of systemic GH/IGF-I activity appears to affect renal expression of IGF-I.

Phenotypically altered ␣-SMA-positive myofibroblasts in the kidney are the major source of abnormal production of extracellular matrix, which facilitates the progression of renal fibrogenesis. There was no significant expression of ␣-SMApositive myofibroblasts in the 6-month-old wild-type rat kidneys (data not shown), but significantly increased numbers of ␣-SMA-positive cells were detected in 24-month-old wildtype rat kidneys (Fig. 7A), implicating phenotypically altered ␣-SMA-positive renal cells as the source of increased matrix proteins that might eventually contribute to the age-associated renal fibrosis observed in the 24-month-old wild-type rat kidneys. The ␣-SMA-positive cells in the kidneys of homozygote rats at 24 months (Fig. 7B) were significantly lower than that in wild-type rats of similar age. CTGF is a potent transcriptional regulator of collagen synthesis (19, 20), whereas HSP47 is a posttranslational regulator of collagen synthesis (21–23). We studied the expression of these two profibrogenic molecules in the sections of wildtype and transgenic rat kidneys at 6 and 24 months of age. The expression of CTGF in the kidneys of wild-type rats at 24 months was significantly higher (Fig. 7C) than that in transgenic rats of similar age (Fig. 7D); a higher renal expression of CTGF is associated with a higher sclerotic glomerular index, and interstitial widening noted in the 24month-old wild-type rat kidneys (Fig. 4). HSP47 immunostaining was weakly positive in the kidneys of 6-month-old transgenic and wild-type rats (data not

Renal expression of ED-1 and PCNA

Infiltration of ED-I-positive macrophages was significantly higher in the kidneys of 24-month-old wild-type rats (Fig. 6A) than transgenic animals of the same age (Fig. 6B), suggesting that life-long suppression of GH/IGF-I system could exert a renoprotective effect by suppressing inflammatory responses. We found an increased number of PCNApositive cells, mostly located in the tubular epithelial cells of kidneys of 24-month-old wild-type rats (Fig. 6C), compared with the age-matched transgenic kidneys (Fig. 6D), suggesting that life-long suppression of GH/IGF-I system affected the proliferative activity of renal tubular epithelial cells. There was no significant difference in the number of proliferating cells in the kidneys of 6-month-old transgenic vs. control rats (data not shown).

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FIG. 4. Quantitative histomorphometric analysis showed that compared with 6-month-old rat kidneys, the glomerular sclerosis index (A) and interstitial volume (B) was significantly higher in 24-monthold wild-type rat kidneys. Note that life-long suppression of GH/IGF-I system could significantly (P ⬍ 0.0001) reduce both glomerular sclerosis index (A) and interstitial volume (B) in 24-month-old homozygote transgenic (Tg) rats.

shown). In contrast, the expression of HSP47 was significantly increased in the kidneys of 24-month-old wild-type rats (Fig. 7E). Life-long suppression of GH/IGF-I system, therefore, markedly suppressed the expression of HSP47 in the 24-month-old transgenic rats (Fig. 7F). Discussion

Serum creatinine levels are the gold standard marker of renal function; levels are elevated only when renal function has deteriorated to less than half normal capacity. A recent National Health and Nutrition Examination Survey revealed elevated serum creatinine levels in nearly 3% of the U.S. population. This survey also revealed that 11% of people over the age of 65, without obvious renal disease, had 60% less renal function when compared with normal individuals (24). Considering that the incidence of age-associated renal diseases is generally underestimated, the underlying causes of and factors involved in the progression of age-related diseases need to be more rigorously studied.

FIG. 5. Immunohistochemical staining of IGF-I in kidney sections prepared from 24-month-old wild-type rats (A), and 24-month-old homozygote transgenic rats (B). The renal expression of IGF-I was significantly induced in 24-month-old wild-type rats, both in the glomeruli and tubules (A). Compared with 24-month-old wild-type rats (A), life-long suppression of the GH/IGF-I system reduced the renal expression of IGF-I in 24-month-old homozygote rats (B). When primary antibody was replaced with rabbit serum, no specific staining was noted in the kidney sections (C). Original magnification, ⫻20.

IGF-I is a major component of the broader GH system, which includes GH and IGF-I-binding proteins; all of these have independent and interdependent effects on cell function and growth (25). IGF-I signaling is an evolutionarily conserved mechanism of controlling the aging process that has been well documented in organisms from Caenorhabditis elegans to Drosophila, to mice. Mutation of the chico gene, a mammalian homolog of the insulin receptor substrate, resulted in a dwarf phenotype and life span extension in Drosophila melanogaster, possibly due to reduced insulin/IGF-I

TABLE 2. Body weight and mean glomerular volume in wild-type and homozygote rats at 6 and 24 months Genotype

Body weight (g) (6 months)

Body weight (g) (24 months)

Glomerular volume (␮m2) (6 months)

Glomerular volume (␮m2) (24 months)

Wild type Homozygote

494 ⫾ 17 192 ⫾ 2

624 ⫾ 29a 240 ⫾ 9c

49099 ⫾ 1189.3 29329 ⫾ 989.3

68865 ⫾ 3277.7b 37343 ⫾ 1066.2d

P ⬍ 0.001; compared with 6-month-old wild-type rats. P ⬍ 0.001; compared with 6-month-old wild-type rats. c P ⬍ 0.001; compared with age-matched wild-type rats. d P ⬍ 0.001; compared with age-matched wild-type rats. a

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FIG. 6. Immunohistochemical staining of ED-1 (A and B) and PCNA (C and D) in kidney sections prepared from 24-month-old wild-type (A and C) and homozygote transgenic (B and D) rats. Compared with 24month-old wild-type rats (arrows) (A and C), life-long suppression of GH/IGF-I significantly reduced ED-1-positive macrophage infiltration (B) and the proliferative ability of the renal tubular epithelial cells (D) in the 24-month-old homozygote rats. Original magnification, ⫻20.

signaling activity; female flies homozygous for chico mutations had a life span increase of up to 48%, and heterozygotes up to 36% (26). In a separate study, IGF-I receptor-knockout heterozygous mutants mice lived 26% longer than the wildtype cohorts (mice homozygous for this mutation die at birth), although there was a gender variation with female mutants living 33% longer than wild-type females, whereas male mutant mice lived approximately 16% longer compared with wild-type males (27). The role of IGF-I in mediating GH function has been reported previously; for instance, in vivo genetic manipulation studies showed that a delayed induction of IGF-I gene expression could delay the onset of GHresponsive events in mice (28, 29). The widespread biological functions of GH, however, may at times be independent of IGF-I. A role for GH in developing renal lesions was elegantly demonstrated in transgenic mice overexpressing human and bovine GH genes; these mice exhibited a giant phenotype and developed diffuse progressive glomerulosclerosis (30). In contrast, transgenic mice expressing GH antagonists (bGH-G119R and hGH-G120R) had a dwarf phenotype and did not produce renal lesions, perhaps due to a block in endogenous GH activity (31). Although reduction of GH/ IGF-I activity can affect the aging process, the possible outcome of life-long suppression of GH/IGF-I signaling on kidney function remains unknown. In the present in vivo genetic engineering study, we have shown that chronic suppression of GH/IGF-I activity for the entire life span of a rat (about 3 yr) prevented the development of naturally occurring, ageassociated renal damage. It is presumed that most of the effects of GH are mediated through IGF-I; however, evidence also exists for GH-independent effects of IGF-I in the kidney. In one study, IGF-I transgenic and GH transgenic mice developed glomerular hypertrophy, but the IGF-I mutants did not develop glomer-

FIG. 7. Immunohistochemical staining of ␣-SMA (A and B), CTGF (C and D) and HSP47 (E and F) in kidney sections prepared from 24month-old wild-type (A, C, and E) and homozygote transgenic (B, D, and F) rats. Compared with 24-month-old wild-type rats (arrows) (A, C, and E), life-long suppression of GH/IGF-I significantly reduced ␣-SMA-positive phenotypically altered cells (B), CTGF expression (D) and HSP47 expression (F) in the 24-month-old homozygote rats. Original magnification, ⫻20.

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FIG. 8. Schematic diagram showing possible effect of suppressing the GH/ IGF-I axis on kidney health and overall survival.

ular sclerosis, even though circulating IGF-I levels in the IGF-I transgenic mice were higher than in the GH transgenic mice (32). Furthermore, in mice transgenically expressing human IGF-binding protein-1, a progressive glomerular sclerosis developed despite low plasma levels of bioactive IGF-I, which would negate a negative feedback of IGF-I on GH secretion from the pituitary (33, 34). In our study, the hemizygote transgenic rats lived longer than their homozygote counterparts (maximum survival 171 and 148 wk, respectively). The pathological analysis revealed that neoplasms including leukemia caused earlier death in the homozygote rats, suggesting that a severely reduced GH/IGF-I axis could promote tumorigenesis. Moreover, the natural killer cell numbers and activity were decreased in homozygote rats (11, 12). Because GH and IGF-I are both required for normal development of the immune system, which intrinsically fights tumorigenesis, the severely reduced GH/IGF-I activity in the homozygote rats might enhance tumorigenesis by reducing immune function, particularly natural killer cell activity. The reduced survival in homozygote transgenic rats, despite markedly reduced plasma IGF-I levels, might suggest that severe reduction in GH/IGF-I activity during early development could adversely affect organogenesis, which manifest later in life as age-related pathologies to affect overall survival. In contrary to homozygote rats, hemizygote transgenic rats survived longer (than wild-type rats), even though their plasma IGF-I level was moderately reduced, supports the notion that controlled reduction of the GH/IGF-I activities can increase life span by delaying age-associated pathologies, whereas uncontrolled severe reduction of GH/IGF-I activities may accelerate some of the age-associated pathologies. Antagonistic pleiotropy usually refers to a situation in which involved factors are able to produce multiple competing effects: both beneficial and deteriorating effects. From relatively decreased longevity of homozygote rats and increased longevity of hemizygote rats, it is tempting to assume that our genetically altered rats, whose GH gene was

suppressed by an antisense GH transgene, are consistent with the model of antagonistic pleiotropy of aging; life-long severe reduction of IGF-I level in homozygote rats has adverse affect on the survival due to appearance of accelerated age-related pathologies, whereas moderate reduction of IGF-I in hemizygote rats has not only delayed such agerelated pathologies as tumorigenesis, but also exerted beneficial effect on overall survival. It is, therefore, reasonable to speculate that uncontrolled reduction of GH/IGF-I activities does not always necessarily increase life span. It is possible that the rate of progressive decline in GH/ IGF-I activity may dictate the course of the aging process. In our homozygote rats, there was severe reduction of plasma IGF-I level throughout the life span (53% reduction in young age and 48% reduction in old age, compared with the agematched wild-type rats); such reduction of IGF-I level has actually deleterious effects on overall survival (maximum survival 148 wk). On the other hand, hemizygote rats have moderate reduction of plasma IGF-I level in early age (28% reduction, compared with the age-matched young wild-type rats); interestingly plasma IGF-I level in aged 2 yr old hemizygote rats was actually increased by 10%, compared with the age-matched wild-type rats; such increase of IGF-I level has shown to be associated with the increased overall survival of the hemizygote rats (maximum survival 171 wk). It is, however, worthwhile to mention that although compared with the age-matched wild-type controls, we have noted 10% increase in the plasma IGF-I level in 2-yr-old hemizygote rats, the plasma IGF-I level in these old hemizygote rats remained 16% lower than the 6-month-old wild-type rats (Table 1). Our genetically altered rat model, therefore, provides the in vivo tool to study the differential effects of GH/IGF-I system in the progression of aging and age-related pathologies. Recently, Sonntag et al. (35) showed that a specific and limited reduction of GH and IGF-I in a rodent model, initiated in adulthood and continued thereafter throughout life, increased life span in the male rats (35). Such prolonged survival was also associated with reduced neoplasms, and

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Zha et al. • Antisense GH Transgenic Rats Prevent Renal Injury

the amelioration of age-associated nephropathy (35). It is interesting to note that, unlike suppression in adulthood, the life-long suppression of GH and IGF-I in rats of the same genetic background failed to measurably prolong life span; again providing the in vivo evidence of beneficial effects of controlled and selective reduction of the GH/IGF-I system to increase life span by delaying age-associated organ damage. One obvious positive aspect of reduced GH/IGF-I activity was reno-protection in the transgenic rats. The transgenic homozygote rats in this study showed a significant reduction in plasma IGF-I levels by 6 months of age (wild-type 1613.6 ⫾ 57.3 ng/ml vs. homozygote 768.9 ⫾ 12.6 ng/ml) (11), which remain low throughout the life span (wild-type 1230.2 ⫾ 121 ng/ml vs. homozygote 649 ⫾ 155.9 ng/ml at old age), whereas life-long suppression of GH/IGF-I activity delayed or prevented the development of age-associated glomerulosclerosis and interstitial damage in transgenic rats. This delay in the GH/IGF-I-suppressed rats was accompanied by a lower proliferative activity (as determined by PCNA staining) and less phenotypic alterations (as determined by ␣-SMA staining) of resident renal cells. Furthermore, renal infiltration by macrophages (as determined by ED-1 staining), as well as renal expression of CTGF (a potent transcriptional regulator of collagen synthesis) and HSP47 (a posttranslational regulator of collagen synthesis) were reduced in the GH/IGF-I-suppressed rats, resulting in less collagen accumulation in the glomeruli and interstitium. Thus, it seems that prevention of age-associated renal fibrogenesis by controlled inhibition of GH/IGF-I activities could influence overall survival (Fig. 8) (36). This delayed renal fibrogenesis due to the GH/IGF-I suppression increases the feasibility of pharmacologically modifying fibrogenic responses in commonly encountered renal diseases (37), including glomerulonephritis, hypertensive nephrosclerosis, and diabetic nephropathy (1, 2, 38, 39). Acknowledgments We thank Ms. Kanako Egashira for kindly preparing paraffin sections used in this study. Received March 8, 2006. Accepted August 30, 2006. Address all correspondence and requests for reprints to: M. Shawkat Razzaque, M.D., Ph.D., Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4, Sakamoto, Nagasaki 852-8523, Japan. E-mail: sasebo@gmail.com. M.S.R. received institutional support from Nagasaki University Graduate School of Biomedical Sciences (Nagasaki, Japan) and Harvard School of Dental Medicine (Boston, MA). Current address for M.S.R.: Department of Developmental Biology, Harvard School of Dental Medicine, Research and Educational Building, Room 312, 190 Longwood Avenue, Boston, Massachusetts 02115. E-mail: mrazzaque@hms.harvard.edu. The disclosure of the manuscript by authors: Y.Z., V.T.L., Y.H., I.S., T.T., and M.S.R. have nothing to declare.

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