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Am J Physiol Renal Physiol 292: F1681-F1690, 2007. First published February 20, 2007; doi:10.1152/ajprenal.00482.2006
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Testosterone treatment promotes tubular damage in experimental diabetes in prepubertal rats

Jianhong Sun,1 Kay Devish,1 William J. Langer,1 Pamela K. Carmines,2 and Pascale H. Lane1

Departments of 1Pediatrics and 2Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska

Submitted 6 December 2006 ; accepted in final form 12 February 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Puberty unmasks or accelerates progressive kidney diseases, including diabetes mellitus (DM), perhaps through effects of sex steroids. To test the hypothesis that rising androgen levels at puberty permit diabetic kidney damage, we studied four groups of male rats with and without streptozocin-induced DM: adult onset (A), adult onset after castration (AC), juvenile onset (J), and juvenile onset with testosterone treatment (JT). Profibrotic markers were measured after 6 wk with blood glucose levels 300–450 mg/dl. JT permitted increased expression of mRNA for two isoforms of transforming growth factor-beta and connective tissue growth factor compared with J animals with DM; prior castration did not provide protection in adult-onset DM. JT also permitted greater tubular staining for {alpha}-smooth muscle actin and fibroblast-specific protein, two markers of cell damage and potential epithelial mesenchymal transition. Once again, castration was not protective for these effects of DM in the AC group. These data indicate that puberty permits detrimental effects in the tubulointerstitium in the diabetic kidney, an effect mimicked by testosterone treatment of juvenile animals and partially blunted by castration of adults, but damage does not correlate with testosterone levels, suggesting a less direct mechanism.

connective tissue growth factor; transforming growth factor-beta; epithelial mesenchymal transition; glomerulus; castration

A NUMBER OF PROGRESSIVE KIDNEY disorders show age- and sex-related differences in clinical and experimental studies (68, 70). Clinical evidence of the microvascular complications of DM, including microalbuminuria, is unusual before the onset of puberty, even though structural changes are occurring in the eye, kidney, and other organs (33, 47). Loss of renal function accelerates after the age of puberty in nondiabetic kidney disease as well (2). In adults, most kidney diseases progress more quickly in men than in women before menopause (68, 70). Studies on diabetic kidney disease have demonstrated variable results, with many showing male sex as a risk factor for nephropathy and progression (27, 28, 45, 52, 58, 66) while others show no sex differences (8, 14, 65). Some studies support beneficial roles for estrogen in the kidney (15, 32, 59, 67), while others suggest that the detrimental effects of androgens are more important (13, 48, 59–63). These observations suggest a role for sex hormones in the progression of diabetic kidney disease.

Prepubertal onset of experimental DM in male rats suppresses diabetic renal and/or glomerular hypertrophy, depending on the duration of observation and other experimental conditions (3–5, 35, 37). In our earlier work, prepubertal onset of DM did not increase renal cortical production of transforming growth factor-beta1, a major mediator of diabetic renal growth, although it did in adult animals (35). Expression of transforming growth factor-beta-inducible gene H3, an adhesion molecule induced by transforming growth factor-beta, was also suppressed in these experiments, further supporting less function of this growth factor before sexual maturity. In contrast, prepubertal onset of DM in female rats does not suppress kidney or glomerular enlargement, suggesting that there may be different mechanisms for these early processes in the two sexes (36). Once again, these findings could potentially implicate sex steroids in early kidney growth in DM.

The following experiments tested our overall hypothesis that rising androgen levels at puberty permit or accelerate diabetic kidney damage in males. Indices of damage included kidney growth and profibrotic events such as production of transforming growth factor-beta, levels of connective tissue growth factor (a mediator of transforming growth factor-beta-induced fibrosis), and immunostaining for markers of epithelial-mesenchymal transition of tubular cells (EMT; a major source of interstitial fibroblasts in progressive kidney diseases). Administration of testosterone to prepubertal males was expected to promote diabetic kidney growth and damage, while castration of adult-onset males was expected to blunt these processes following 6 wk of streptozocin DM. The results reveal that testosterone treatment permits profibrotic events in the tubules of juvenile rats with DM, although castration was not completely protective for animals with adult-onset DM. The mechanism of pubertal acceleration of diabetic kidney damage appears to be more complex than our hypothesis.


   METHODS
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 METHODS
RESULTS
 DISCUSSION
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Animals

All experiments were performed with male Sprague-Dawley rats (Sasco, Wilmington, MA), either juvenile (4 wk of age) or adult (14 wk of age) at the onset of the experiment. Castrated rats were surgically altered by the vendor during week 2 of life. On the start date, rats received streptozotocin (65 mg/kg iv). Nondiabetic controls were given an equal volume of vehicle. Hyperglycemia was confirmed by whole-blood tail-vein glucose measurement 3 days after injection. On day 4, an insulin palmitate or vehicle caplet was implanted subcutaneously via trochar (LinBit or LinPlant, Linshin Canada, Scarborough, ON). For juveniles, we began with 1 LinBit, which releases ~0.1 U of insulin/24 h. Additional LinBits were added weekly to keep blood glucose levels within the 300–450 mg/dl range. Adults received about one-third of a LinPlant initially, a dose previously demonstrated to keep blood sugars in our target range for 6 wk. Tail-vein blood glucose levels were monitored at least weekly through the remainder of the protocol, and additional implants were administered as needed to keep blood glucose levels below 450 mg/dl.

Testosterone treatment began 3–4 days after injection of streptozotocin or vehicle, at the same time as insulin placement. Pellets releasing 0.6 mg/day of testosterone or vehicle (Innovative Research of America, Sarasota, FL) were placed subcutaneously into each rat.

During week 6 of the protocol, rats were housed for 24 h in metabolic cages to collect urine. Animals were then anesthetized with isoflurane, and plasma was collected by cardiac puncture. The kidneys were excised, weighed, and then flash-frozen for later study or fixed in formalin for histology.

At least 7 rats were included in each of the groups [adult (A), adult castrated (AC), juvenile (J), juvenile testosterone-treated (JT)]. All experiments were approved by the Animal Care and Use Committee of the University of Nebraska Medical Center.

Biochemical Studies

Plasma and urine chemistries. Plasma collected at the time of euthanasia was used to measure glucose (kit from Sigma, St. Louis, MO). Insulin levels were assessed using a commercial ELISA for rat insulin (ALPCO Diagnostics, Windham, NH). Estradiol, dehydroepiandrosterone (DHEA), and testosterone levels were measured by EIA (kits from Diagnostic Systems Laboratories, Webster, TX). Albumin in the 24-h urine collection was measured using a Nephrat ELISA kit (Exocell, Philadelphia, PA). Urine creatinine was measured with a colorimetric method (kit from Sigma).

Renal Homogenate Studies

Real-time RT-PCR. Standards and primers were designed using published sequences via the Primer 3 web site (www.broad.mit.edu/cgi-bin/primer/primer3.cgi). Standards were designed to be ~450 bp; target sequences for quantification of copy number were nested within the standard sequences at a length of ~115–125 bp. The standard was created using the Syber Green RT PCR kit (Qiagen, Valencia, CA) for 35 cycles. The standard was visualized for purity with electrophoresis on a 1% agarose gel, then extracted and purified (kits from Qiagen), and the sequence was confirmed by the University of Nebraska Molecular Biology Core Lab. The absorbance was assessed at 260 nm to determine RNA concentration, and the number of copies of standard per microliter of solution was calculated. This solution was then serially diluted in RNAse/DNase-free water to generate a standard curve appropriate for the tissue and mRNA of interest. Tissue samples were prepared from previously frozen renal cortex with homogenization in TRIzol (GIBCO BRL, Rockville, MD). One hundred nanograms of total RNA were used for each PCR sample. Standards and tissue samples were then subjected to real-time RT-PCR using a Rotor-Gene RG-3000 cycler (Corbett Research, Mortlake, NSW, Australia). Standard and target sequences used for these experiments are as follows:

Formula

Results are reported as the number of copies per 100 ng RNA, confirmed by the standard curve for the transcript-specific standard. The number of transcripts of {alpha}-tubulin was used as an mRNA loading control; no differences were noted in this housekeeping gene, so only the data for absolute copy number are reported.

Histology

Formalin-fixed tissue was embedded in paraffin and sectioned at ~5 µm. Separate sections were stained for transforming growth factor-beta1, connective tissue growth factor, {alpha}-smooth muscle actin, and fibroblast-specific protein using polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and immunoperoxidase staining. After deparaffination and rehydration, endogenous peroxidase activity was quenched and the slide was blocked with normal serum appropriate for the antibodies. Slides were incubated overnight with primary antibodies and then exposed to peroxidase-conjugated secondary antibodies. Peroxidase labeling was localized with 3-amino-9-ethylcarbazole. Negative controls were run for each substance by omitting the primary antibody, and these showed no immunostaining for any of the antibodies of interest. Results were scored by a single observer masked to the identity of the tissue on images captured at a magnification of x100. A scale of 0–4 was used to score tubular compartments of the renal cortex for transforming growth factor-beta1, connective tissue growth factor, {alpha}-smooth muscle actin, and fibroblast-specific protein as previously described (44). The tubular area was further divided into proximal tubule profiles and distal nephron. The latter cortical compartment included distal convoluted tubules, cortical collecting tubules, cortical thick ascending limb, and other tubular components which could not be reliably separated under the conditions used here. Additional fibroblast-specific protein micrographs were captured at a magnification of x200 to count cells in the interstitial area of the cortex stained for fibroblast-specific protein. Results are expressed as the number of cells per 10 fields of cortex.

Statistics

Data were analyzed using two-factor ANOVA with post hoc Holm-Sidak testing or similar nonparametric methods if data were not normally distributed. Factors included age and treatment (adult, adult castrated, juvenile, or juvenile testosterone) and metabolic state (nondiabetic vs. DM). All analyses were performed with SigmaStat 3.1.1 (Systat Software, Richmond, CA). P < 0.05 was considered significant for most comparisons, or the critical P value was corrected for multiple comparisons in post hoc studies.


   RESULTS
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DISCUSSION
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Age and DM influenced body weight and weight maintenance as expected (all data in this paragraph are shown in Table 1). Terminal glucose values were greater in all DM groups than in the corresponding nondiabetic groups; among the DM groups, the JT group's values were significantly greater than those in the A or J groups. Among nondiabetic groups, insulin levels were lower in both the J and JT than A or AC groups. All DM groups except J were lower than the corresponding nondiabetic group, and no differences in insulin levels were noted among these diabetic groups. DM reduced testosterone levels in the A and J groups, while testosterone treatment gave diabetic JT levels similar to those in nondiabetic animals. Nondiabetic A rats had very low levels of circulating estradiol, while AC, J, and JT rats showed levels 10- to 20-fold higher. With DM estradiol increased in the A and JT groups, while DM reduced levels in the AC and J groups. DHEA levels were not altered by DM in the A, AC, or J groups. Testosterone treatment suppressed levels of DHEA in nondiabetic JT rats and increased levels in diabetic JT rats. Urine output was increased by DM in all groups; when differences in body weight were factored in, only adults showed an increase in output with DM. Albumin excretion did not differ significantly with metabolic or hormonal status. Kidney weight, normalized to body weight, increased with DM in all groups. Diabetic renal hypertrophy was exaggerated in JT animals compared with the other diabetic groups.


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  		Table 1. General characteristics

 
The renal transforming growth factor-beta system was examined at the message and protein level (Fig. 1). Transcription of all isoforms of this growth factor was lower in J than in A rats, regardless of metabolic state or treatment (Fig. 1A). With DM, both the A and JT groups showed increased levels of mRNA for transforming growth factor-beta1, although in the A group this did not achieve statistical significance (P = 0.09). Levels did not differ with metabolic state in AC or J rats. Transforming growth factor-beta2 did not change with DM in the A group as it did in the AC, J, and JT groups, but levels in A rats were similar to those of J and JT rats with DM. Transforming growth factor-beta3 was higher in the A and AC groups than in J and JT rats, and it increased with DM only in A and AC animals. Immunostaining for transforming growth factor-beta1 revealed very low levels in proximal tubules of nondiabetic A rats, and DM did not change this parameter (Fig. 1B). In nondiabetic AC, J, or JT rats, staining was greater than in the A group. Only AC rats showed a significant increase in staining with DM. Distal nephron segments showed intense staining for this growth factor, regardless of age or metabolic state (scores not shown; micrographs in Fig. 1C).


Figure 1
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  		Fig. 1. Transforming growth factor-beta (TGF-beta) message (A) and TGF-beta1 immunostaining scores (B) and representative micrographs (C) in male rats with juvenile (4 wk of age) onset of diabetes or adult (16 wk) onset of diabetes. A, adult; AC, adult castrated; J, juvenile; JT, juvenile testosterone. Error bars show means ± SE. *P < 0.001 vs. age- and treatment-matched nondiabetic group. aP < 0.05 vs. diabetic A group. acP < 0.05 vs. diabetic AC group. jP < 0.05 vs. diabetic J group. jtP < 0.05 vs. diabetic JT group.

 
Two molecules expressed in response to transforming growth factor-beta were examined. The transforming growth factor-beta-inducible gene H3 showed no increase with DM in A, AC, or JT rats, although it dropped significantly in the J group (Fig. 2A). Among DM groups, in both A and JT rats levels were significantly greater than in J rats. Connective tissue growth factor is also stimulated by transforming growth factor-beta, but its expression did not change with DM in A or J rats (Fig. 2B). Expression increased two- to fourfold with DM in the AC and JT groups. Among the diabetic groups, JT expression was significantly greater than in J rats, and in AC rats expression was greater than in all other diabetic groups. Connective tissue growth factor protein was then localized with immunostaining (Fig. 3). Almost no staining was present in nondiabetic A rats in any cortical compartment studied, while nondiabetic AC, J, and JT groups showed minimal staining that was not statistically different from that in A rats. Immunostaining increased significantly with DM in all areas in all groups.


Figure 2
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  		Fig. 2. Expression of TGF-beta-inducible gene H3 (A) and connective tissue growth factor (CTGF; B) in male rats with juvenile (4 wk of age) onset of diabetes or adult (16 wk) onset of diabetes. Error bars show means ± SE. *P < 0.001 vs. age- and treatment-matched nondiabetic group. aP < 0.05 vs. diabetic A group. acP < 0.05 vs. diabetic AC group. jP < 0.05 vs. diabetic J group. jtP < 0.05 vs. diabetic JT group.

 

Figure 3
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  		Fig. 3. Immunohistochemistry scores (A) and representative micrographs (B) for CTGF in male rats with juvenile (4 wk of age) onset of diabetes or adult (16 wk) onset of diabetes. Error bars show means ± SE. *P < 0.001 vs. age- and treatment-matched nondiabetic group.

 
{alpha}-Smooth muscle actin staining was very rare in proximal tubules of nondiabetic A group rats and increased with DM in this group (Fig. 4). AC and JT rats also showed significant increases with DM in the proximal tubule. In contrast, there was a trend toward more cells staining in the proximal tubule in nondiabetic J than in nondiabetic A or JT rats, but DM decreased the proportion of cells stained in J rats. In the distal nephron, DM increased staining in all groups except J, an effect exaggerated in JT rats.


Figure 4
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  		Fig. 4. Immunohistochemistry scores (A) and representative micrographs (B) for {alpha}-smooth muscle actin ({alpha}SMA) in male rats with juvenile (4 wk of age) onset of diabetes or adult (16 wk) onset of diabetes. Error bars show means ± SE. *P < 0.001 vs. age- and treatment-matched nondiabetic group. aP < 0.05 vs. diabetic A group. acP < 0.05 vs. diabetic AC group. jP < 0.05 vs. diabetic J group. jtP < 0.05 vs. diabetic JT group.

 
Fibroblast-specific protein staining in proximal tubules did not vary among nondiabetic rats, while JT rats showed more staining in this cortical compartment than did the other diabetic groups (Fig. 5A). DM increased distal nephron staining in both A and AC animals but did not significantly increase scores in either J group. Diabetic J rats showed lower scores than any other diabetic group studied. The number of fibroblast-specific, protein-positive cells within the cortical interstitium did not differ with age, treatment, or metabolic state (Fig. 5C); however, numbers trended up with DM in the A and JT groups but remained stable or dropped in J and AC rats.


Figure 5
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  		Fig. 5. Immunohistochemistry scores (A), representative micrographs (B), and interstitial cells counts (C) for fibroblast-specific protein (FSP) in male rats with juvenile (4 wk of age) onset of diabetes or adult (16 wk) onset of diabetes. Error bars show means ± SE. *P < 0.001 vs. age- and treatment-matched nondiabetic group. aP < 0.05 vs. diabetic A group. acP < 0.05 vs. diabetic AC group. jP < 0.05 vs. diabetic J group. jtP < 0.05 vs. diabetic JT group.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Two markers of tubular damage and EMT show age- and treatment-related responses to 6 wk of DM. Both {alpha}-smooth muscle actin and fibroblast-specific protein increased in cortical tubules of diabetic adults but not animals with juvenile onset of DM. JT rats showed increased tubular {alpha}-smooth muscle actin with DM, and castration blunted this increase in staining with DM in adults. Fibroblast-specific protein staining was also blunted in J rats with DM. These results suggest that hormonal changes in puberty may modulate the tubular response to DM and promote EMT.

EMT represents dedifferentiation of damaged epithelial cells to myfibroblasts that promote the fibrosis characteristic of progressive nephropathies (reviewed in Refs. 30 and 80). A related process is called scatter, a temporary expression of the EMT phenotype that reverses when its stimulus is removed (30). Scatter and true EMT can be difficult to differentiate in the earliest phases that precede frank fibrosis, before myofibroblast migration (11, 30). Two markers of the EMT phenotype were chosen for the present study. {alpha}-Smooth muscle actin is the most commonly employed indicator of EMT, and its staining was minimal in nondiabetic kidneys as previously reported (11, 30). Fibroblast-specific protein, also known as S100A4, p9Ka, and calvasculin (6, 64, 75, 79), was initially felt to be fibroblast specific (30). Indeed, initial mouse studies suggested that tubular epithelial cells did not express this protein (75); however, subsequent studies in the rat have shown low-level staining of proximal and distal tubules in the kidney, vascular smooth muscle, endothelial cells, and circulating mononuclear leukocytes (23, 43). Our data confirm low-level staining for fibroblast-specific protein in tubules of the renal cortex of normal rats, as well as a few positive cells within the interstitial space. The latter may represent fibroblasts, infiltrating leukocytes, or endothelial cells. Staining for {alpha}-smooth muscle actin or fibroblast-specific protein would not conclusively indicate EMT, since some normal cells can express these proteins. However, staining for both {alpha}-smooth muscle actin and fibroblast-specific protein suggests that the tubules of these diabetic animals are undergoing profibrotic damage that is modulated in our model by age and treatment.

Relative protection from tubulopathy before puberty could help explain why microalbuminuria is rare in this age group. In rats, kidney weight increases within the first 72 h of streptozocin-induced DM, mostly due to hypertrophy and proliferation of proximal tubular cells (77). Ultimately, cortical tubulointerstitial lesions correlate with renal dysfunction at least as well as glomerular changes (39). Tubular dysfunction early in DM may contribute to the development and progression of diabetic kidney disease. Alterations in tubuloglomerular feedback promote glomerular hyperfiltration, while disordered protein processing in the tubule probably contributes to albuminuria (77). Clinical studies confirm that tubular lesions develop in the clinically silent period of diabetic nephropathy as suggested by our model (77). Children and young adults with type 1 DM showed subtle morphometric differences in the baseline kidney biopsies of patients who developed persistent microalbuminuria over 5 yr compared with patients who remained normoalbuminuric throughout the study period (74). Both groups were 17 yr old, on average, at the time of initial biopsy, had been diabetic for 8–9 yr, and had normal blood pressure and albumin excretion rates. Glomerular basement membrane width and interstitial volume fraction in the cortex were significantly greater in patients developing persistent microalbuminuria over 5 yr than in those whose albumin excretion remained normal throughout the study period. Patients who developed microalbuminuria also had greater hyperfiltration at baseline than normoalbuminuric patients. No differences in blood pressure or glycemic control were demonstrated between these groups of patients. These clinical data support early concurrent development of tubulopathy and glomerulopathy, albeit at a very subtle level, in progressive diabetic kidney disease (74).

Many studies show a central role for transforming growth factor-beta in the initiation and progression of diabetic renal disease (7, 18). Acting via an autocrine or paracrine mechanism, this growth factor contributes to diabetic tubular and glomerular hypertrophy in DM (69). It probably plays a role in normal kidney growth as well (34). Transforming growth factor-beta is commonly used in vitro to induce scatter or EMT in epithelial cells (17). DM generally increased transforming growth factor-beta in adults in the present study; juvenile-onset rats were relatively protected from increases in transforming growth factor-beta1 and -beta3 with DM. While testosterone treatment allowed isoform 1 to increase in this age group, castration was not protective in adults, suggesting that androgens are not directly responsible for these tissue responses.

Some effects of transforming growth factor-beta are not directly mediated by this growth factor but through its induction of connective tissue growth factor (1, 9, 41, 42). Connective tissue growth factor is a cystein-rich peptide that modulates fibroblast cell growth and the production of extracellular matrix components by mesangial cells and fibroblasts (25). In addition to mediating downstream effects of transforming growth factor-beta, this growth factor may potentiate or prolong the profibrotic activities of transforming growth factor-beta (12, 24, 25, 55). Connective tissue growth factor also promotes EMT independently of transforming growth factor-beta in diabetes (11). Connective tissue growth factor seems to be necessary for sustained fibrosis to occur (42), but it is expressed under normal conditions by some tissues. In the adult mouse kidney, mRNA for connective tissue growth factor is constitutively expressed within arterioles and central, most likely mesangial, areas of glomeruli, but not in epithelial cells (22). The present experiments using cortical homogenate suggest low-level expression of mRNA for connective tissue growth factor in the normal rat kidney that is higher in adults than in younger animals. Immunohistochemistry shows minimal localization in glomeruli and proximal tubules of nondiabetic animals, similar to mouse findings, with greater staining in distal nephron profiles. DM increased the intensity of staining in all areas, with no change in the relative localization. Increased expression of mRNA was permitted by testosterone treatment in diabetic J animals. DM produced the most dramatic increases in AC animals, so testosterone itself is probably not the driving force for these effects of our treatments.

While testosterone permitted tubular damage to occur with juvenile onset of DM, castration was not completely protective for adults. Androgen may not be a direct cause of tubular damage or EMT but may exert its effects indirectly, as the experimental manipulations employed affected the complex hormonal milieu of these rats. DM has previously been shown to reduce testosterone levels in male rats, similar to the values in the present study (26, 29, 49, 53, 72, 73). In addition to treatment-related changes in plasma testosterone levels, other circulating gonadal steroids also changed. Estradiol levels remained well below those of nondiabetic adult female rats (159 ± 31 pg/ml) (36) in all male groups, although nondiabetic AC, J, and JT rats had elevated levels compared with nondiabetic A rats. Among diabetic animals, only AC rats had virtually undetectable levels similar to those of nondiabetic A rats. Since estradiol may have beneficial effects on the growth of kidney structures in DM (46), these changes may be confounding our model. Circulating DHEA was also affected by these hormonal manipulations, but levels were not associated with structural changes and remained well below values that would be considered pathological. Moreover, many enzymes that metabolize gonadal steroids have been identified in the kidney, including 17beta-hydroxysteroid dehydrogenase (HSD), 3{alpha}-HSD, and 5{alpha}-reductase (31, 57). The kidney can thus convert circulating DHEA to testosterone and testosterone to 5{alpha}-dihydrotestosterone. One weakness of the present study is that effects of these hormones at the tissue level cannot be directly assessed. The use of hormone receptor agonists and antagonists and/or genetically modified animals may allow this issue to be examined in future studies.

Manipulations employed in these experiments altered normal growth and development of the rats, perhaps indirectly affecting their kidneys. Puberty is a period of complex physiological changes triggered by pulsatile secretion of releasing hormones from the hypothalamus (54, 71). Sexual maturation is dependent on gonadotropin-releasing hormone while linear growth occurs in response to growth hormone-releasing hormone and ghrelin, the endogenous ligand for the growth hormone-releasing peptide receptor (54, 71, 76). The somatotropic and gonadotropic axes feed back on each other (78), producing an incredibly complex system of circulating hormones which impact a number of peripheral tissues, including the kidney (19, 50, 51). The gonadotropin-luteinizing hormone has been shown to directly promote kidney hypertrophy (50, 51), as do most androgens (10, 16, 56, 60). Insulin-like growth factor I mediates many growth hormone effects and promotes kidney growth, although growth hormone may have direct renal effects, independent of insulin-like growth factor I. Somatostatin treatment, which opposes the growth hormone-insulin-like growth factor I system, has been shown to alter a number of nephropathy models in a beneficial way (19–21). Because castration and testosterone treatment alter many of these processes and their feedback loops, these indirect effects may promote increased susceptibility to tubular damage in DM.

In summary, our data suggest that puberty permits detrimental effects in the tubulointerstitium in diabetic kidney disease, promoting tubular damage that ultimately results in EMT. While testosterone treatment of prepubertal animals mimics the postpubertal state, castration is only partially protective for adults. These effects do not correlate with circulating testosterone levels, suggesting a less direct effect on the kidney. Indirect alterations of the neuroendocrine axis that controls puberty, sexual function, and growth may ultimately be implicated in these phenomena. Further study is needed to define the mechanism of these effects of puberty in the diabetic kidney, as well as identifying other age-related factors that may promote tubular damage and EMT.


   GRANTS
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 ABSTRACT
 METHODS
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 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-59689. Portions of these studies were presented at the Annual Meeting of the American Society of Nephrology, November 10, 2005, Philadelphia, PA, and were published in abstract form (J Am Soc Nephrol 16: 202A–203A, 2005).


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. H. Lane, Dept. of Pediatrics, 982169 Nebraska Medical Center, Omaha, NE 68198-2169 (e-mail: phlane{at}unmc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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Q. Xu, C. C. Wells, J. H. Garman, L. Asico, C. S. Escano, and C. Maric
Imbalance in Sex Hormone Levels Exacerbates Diabetic Renal Disease
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