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Am J Physiol Renal Physiol 287: F1011-F1020, 2004. First published July 27, 2004; doi:10.1152/ajprenal.00031.2004
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Dissociation of renal TGF-{beta} and hypertrophy in female rats with diabetes mellitus

Pascale H. Lane, Jianhong Sun, Kay Devish, and William J. Langer

Department of Pediatrics, University of Nebraska Medical Center, Omaha, Nebraska 68198-2169

Submitted 2 February 2004 ; accepted in final form 21 July 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Prepubertal onset of diabetes mellitus (DM) in male rats delays diabetic renal hypertrophy and suppresses renal transforming growth factor-{beta} (TGF-{beta}) compared with onset in adults. Because there are sex differences in normal and pathological renal growth, we performed similar experiments in female rats and examined the effects of prior ovariectomy. As in male rats, adult onset of DM increased renal weight ~35%, total renal TGF-{beta} ~35%, and mRNA for TGF-{beta} inducible gene H3 ({beta}IG-H3) ~200%. TGF-{beta} levels did not increase with DM in prepubertal animals, but renal weight increased ~40%, similar to the enlargement seen in adults. In nondiabetic rats, ovariectomy suppressed renal TGF-{beta} levels by 25–50% in both age groups, but {beta}IG-H3 was stable in younger animals and increased by ~200% in older animals after ovariectomy. Ovariectomy increased kidney weight ~10% in both age groups. DM further increased kidney weight by an additional 40% after ovariectomy with an ~150% increase in {beta}IG-H3, even though TGF-{beta} levels were not significantly increased. Prepubertal (~99% lower), diabetic (~50% lower), and ovariectomized rats (~90% lower) all tended toward lower estradiol levels than intact adults, although not all differences were statistically significant. Both prepubertal onset and ovariectomy suppress TGF-{beta} in the kidneys of female rats with DM compared with adult-onset animals, but these states have no effect on renal enlargement. Production of the extracellular matrix component {beta}IG-H3 is dissociated from TGF-{beta} under these conditions. These observations may help explain some of the sex differences demonstrated in progressive kidney diseases, including DM.

ovarectomy; estrogen; transforming growth factor-{beta}-inducible gene H3

CLINICAL AND EXPERIMENTAL studies suggest that there are age- and sex-related differences in the course of a number of progressive kidney disorders (49, 51). Detection of microalbuminuria or other evidence of the microvascular complications of diabetes mellitus (DM) is unusual before the onset of puberty, even though structural changes are occurring (27, 36). Even in nondiabetic kidney diseases, the rate of loss of renal function accelerates after the age of puberty (2). Once adulthood is reached, most kidney diseases progress more quickly in males than in premenopausal females (49, 51). Studies in diabetic kidney disease have varied in their results, with many showing male sex as a risk factor for nephropathy and progression (20, 21, 34, 40, 44, 46). Other studies have shown no sex differences in diabetic nephropathy (8, 12, 45). Together, these observations suggest a role for sex hormones in these processes.

We and others have demonstrated that prepubertal onset of experimental DM in male rats suppresses diabetic renal and glomerular hypertrophy (3–5, 28, 29). Prepubertal onset of DM did not increase production of transforming growth factor-{beta}1 (TGF-{beta}1), a major mediator of diabetic renal growth, as it did in adult animals (28). Expression of TGF-{beta}-inducible gene H3 ({beta}IG-H3), an adhesion molecule induced by TGF-{beta}, was also suppressed in these experiments, further supporting loss of function of this growth factor.

Many studies show a central role for TGF-{beta} in the initiation and progression of diabetic renal disease (7, 14). Renal mesangial and tubular cells produce TGF-{beta}, as do infiltrating cells. Mesangial and tubular cells respond to this growth factor with hypertrophy and biosynthesis of collagen and other matrix components. Hyperglycemia increases production and activation of TGF-{beta} in the kidney. By acting via an autocrine or paracrine mechanism, this growth factor contributes to diabetic renal and glomerular hypertrophy (50).

The following experiments explored diabetic renal and glomerular enlargement in female rats pre- and postpuberty, with or without prior ovariectomy. Renal TGF-{beta} measurements were lower with prepubertal onset of DM than with adult onset, similar to findings previously demonstrated in male rats; however, prepubertal onset or ovariectomy did not suppress {beta}IG-H3 or delay renal hypertrophy. This study suggests that there are significant sex differences in the control of renal growth in response to the diabetic state.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

All experiments were performed using female Sprague-Dawley rats (Sasco, Wilmington, MA). Animals included intact rats and animals ovariectomized during the second week of life by the supplier. At the age of 4 or 16 wk, rats received streptozocin (65 mg/kg iv) to induce DM. Nondiabetic controls were given an equal volume of saline. Hyperglycemia was confirmed with a glucose meter 3 days after injection. On day 4, an insulin palmitate or vehicle caplet was implanted (LinBit or LinPlant, respectively, Linshin Canada, Scarborough, ONT). In adult-onset animals, the initial dose was about one-third of a LinPlant. Whole LinPlants release ~2 U of insulin/24 h for at least 6 wk. This level of insulin often normalizes blood glucose in our rats. The present dose has been found to keep blood glucose at 300–450 mg/dl for at least 6 wk. With weanling-onset animals, we began with 1 LinBit, which releases ~0.1 U of insulin/24 h. Additional LinBits were added weekly to keep blood glucose levels at 300–450 mg/dl. 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. At least 15 rats were included in each of the 8 groups. All experiments were approved by the Animal Care and Use Committee of the University of Nebraska Medical Center.

During week 6 of the protocol, rats were housed in metabolic cages to collect 24-h urine samples. 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.

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 (ALPCO Diagnostics, Windham, NH). Levels of sex steroids were also measured by EIA (kits from Diagnostic Systems Laboratories, Webster, TX). These included estradiol, dehydroepiandrosterone (DHEA), and testosterone.

Albumin in the 24-h urine collection was measured using a Nephrat ELISA kit (Exocell, Philadelphia, PA).

Renal homogenate studies.
TGF-{beta}1 PROTEIN. Approximately 100 mg of renal cortex were homogenized in a buffer containing Tris-buffered saline (pH 8.0), 1% NP-40, 10% glycerol, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 10 mM PMSF, and 0.5 mM sodium vanadate. Total protein in the extract was measured using the Coomassie method (kit from Pierce, Rockford, IL). TGF-{beta}1 levels were measured before and after acidification using an ELISA kit (EMax, Promega, Madison, WI). Levels of active and total growth factor are reported as picograms per milligram total protein.


REAL-TIME RT-PCR. Using published sequences, standards and primers were designed 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 ~125 bp. The standard was created using a Syber Green RT PCR kit (Qiagen, Valencia, CA) for 35 cycles. The standard was visualized for purity with electrophoresis on a 1% agarose gel, extracted and purified (kits from Qiagen), and then the sequence was confirmed by the University of Nebraska Molecular Biology Core Laboratory. 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 the Rotor-Gene RG-3000 cycler (Corbett Research, Mortlake, NSW, Australia). Standard and target sequences used for these experiments are as follows.

Histology

Formalin-fixed tissue was embedded in paraffin and sectioned at ~5 µm. Tissue was stained with periodic acid-Schiff (PAS). Digital images were captured at a final magnification of x100, and glomerular profile areas were measured using ScionImage Software (Scion, Frederick, MD). At least 15 profiles from each animal were averaged as an index of glomerular size. We have previously shown that this index of glomerular area correlates well with measurements of glomerular volume determined through serial section analysis (30, 31).

Statistics

Data were analyzed using two-factor ANOVA with post hoc Tukey testing or similar nonparametric studies if data were not normally distributed. First, intact females were analyzed with age (prepubertal onset vs. adult onset) and metabolic state (nondiabetic vs. diabetic) as factors. This analysis is comparable to prior experiments in males with age of onset of streptozotocin (STZ) DM (28, 29).

The effects of ovariectomy were then assessed in nondiabetic females of both ages using a similar strategy with age and gonadal state as factors. The effects of ovariectomy on DM in adult-onset females were then studied with gonadal and metabolic states as factors. Intact prepubertal-onset diabetes was compared with adult-onset diabetes in ovariectomized rats using age and metabolic state as factors.

All analyses were performed with SigmaStat 3.0 (SPSS, Chicago, IL). A value of P < 0.05 was considered significant for all comparisons.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of Age of Onset of STZ DM

Weight at the initiation of the protocol (start wt) was influenced by age, whereas final weight was influenced by both age and metabolic state (Table 1). Body weight increased markedly during sexual maturation in the prepubertal onset groups (final wt 173 ± 1% of start wt for all prepubertal animals) and to a lesser extent in the adult-onset groups (103 ± 1% of start wt for all adults; P < 0.001). Diabetic animals of both ages gained less than nondiabetic animals (final wt 130 ± 1% for diabetic rats vs. 146 ± 1% of start wt for nondiabetic rats; P < 0.001), although this difference was more pronounced for the prepubertal animals (Table 1).


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  		Table 1. Age of onset of STZ diabetes in female rats

 
Plasma glucose was lower in older animals than younger ones (433 ± 8 for all prepubertal animals vs. 176 ± 8 mg/dl for all adults; P < 0.001). While diabetic animals had higher glucose levels, this effect of DM was most pronounced in the younger age group (Table 1). These age-related differences in glucose control were not related to insulin levels; this hormone was reduced by DM as expected (0.62 ± 0.04 for all nondiabetic rats vs. 0.41 ± 0.03 ng/ml for all diabetic animals; P < 0.001) but did not vary with age. Urine albumin excretion did not differ among these groups (Table 1).

Kidney weight increased with age and with diabetes as expected (Table 1). Kidney weight was greater in the older animals (0.97 ± 0.01 for all adults vs. 0.90 ± 0.02 g for all prepubertal animals; P < 0.001) and in rats with diabetes (1.09 ± 0.01 for all diabetic rats vs. 0.78 ± 0.01 g for all nondiabetic rats; P < 0.001). Absolute kidney weight increased to a similar degree with diabetes in both age groups (Table 1). When examined relative to body weight, this parameter increased relatively more with prepubertal-onset diabetes than with adult-onset disease (Table 1). Glomerular area increased in parallel with kidney weight (Fig. 1). It was larger in older animals (9,909 ± 198 for all adult animals vs. 8,128 ± 188 µm2 for all prepubertal rats; P < 0.001) and in those with diabetes (9,851 ± 188 for all diabetic animals vs. 8,186 ± 198 µm2 for all nondiabetic animals; P < 0.001). Glomerular area increased with DM to a similar degree in both age groups.



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  		Fig. 1. Mean glomerular area in female rats with prepubertal onset (~4 wk of age) and adult onset (~16 wk of age) of experimental diabetes mellitus.

 
Differences in major sex steroids occurred with both age and metabolic state (Table 2). Estradiol was higher in older animals (119.49 ± 21.56 for all adults vs. 0.88 ± 20.40 pg/ml for all prepubertal rats; P < 0.001), whereas testosterone was reduced in this group (0.05 ± 0.02 for adult rats vs. 0.13 ± 0.02 ng/ml for all prepubertal rats; P = 0.006). The adrenal androgen DHEA did not vary with age but was reduced by diabetes (0.02 ± 0.03 for all diabetic animals vs. 0.12 ± 0.03 ng/ml for all nondiabetic animals; P = 0.009). Estradiol levels showed a trend to be lower with STZ DM and did not reach statistical significance (40.02 ± 20.40 for all diabetic animals vs. 80.35 ± 21.56 pg/ml for all nondiabetic animals; P = 0.18, power = 0.14).


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  		Table 2. Sex steroids and age of onset of STZ diabetes

 
Message for major components of the TGF-{beta} system were influenced by these factors as well (Table 3). Older age groups had lower levels of TGF-{beta}1 (8.5 ± 0.4 for all adult rats vs. 12.3 ± 0.7 x 109 copies/100 ng RNA for all younger rats; P < 0.001) and TGF-{beta}3 (0.3 ± 0.8 for all older animals vs. 2.0 ± 1.0 x 109 copies/100 ng RNA for all prepubertal rats; P < 0.001) than younger aged rats. Only mRNA for TGF-{beta}2 increased with age (3.0 ± 0.2 for all adults vs. 0.5 ± 0.3 x 109 copies/100 ng RNA for all prepubertal animals; P < 0.001). Diabetes was associated with reduced levels of mRNA for TGF-{beta}1 (9.4 ± 0.5 for all diabetic rats vs. 11.3 ± 0.6 x 109 copies/100 ng RNA for all nondiabetic rats; P = 0.02). Metabolic state itself had no significant influence on the other two isoforms. {beta}IG-H3 mRNA also decreased with age (0.9 ± 0.1 for all adults vs. 1.6 ± 0.1 x 109 copies/100 ng RNA for all prepubertal rats; P < 0.001), but the effect of DM was age dependent. Interaction analysis showed that {beta}IG-H3 decreased with prepubertal-onset diabetes but increased with adult-onset diabetes (Table 3).


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  		Table 3. Renal TGF-{beta} system and age of onset of STZ diabetes

 
TGF-{beta}1 protein was assessed in renal cortex from these animals, both in its nonacidified active form and after sample acidification to measure total levels. Both active (0.38 ± 0.05 for all adults vs. 0.21 ± 0.07 pg/mg total protein for all younger rats; P = 0.04) and total levels (0.65 ± 0.07 for all adult animals vs. 0.32 ± 0.09 pg/mg total protein for prepubertal rats; P = 0.004) were higher in older animals. No effects of diabetes could be demonstrated on protein levels (Table 3).

Effects of Ovariectomy in Nondiabetic Rats

Ovariectomy during the second week of life resulted in increased body weight as the animals went through the period of "adolescence." Thus start weight did not differ with gonadal state in the younger group (4 wk of age at initiation of protocol) but was significantly elevated by ovariectomy in the older group (16 wk of age at the start of protocol; Table 4). Weight differences between intact and ovariectomized animals were more pronounced at the end of the protocol (259 ± 4 for intact animals vs. 323 ± 5 g for ovariectomized animals; P < 0.001). Weight gain during the 6 wk of study was most evident when final weight was assessed as a percentage of start weight (Table 4). Intact animals in the younger group gained ~87% of their initial weight during the 6 wk of study, whereas those without gonads gained 128% during the same period. Intact animals in the older age group gained ~5% from 16 to 22 wk of age; ovariectomy resulted in a gain of 13% during the same period.


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  		Table 4. Effects of ovariectomy in nondiabetic female rats

 
Plasma glucose levels were affected by age and ovariectomy as well. Younger animals had higher levels than adults (159 ± 4 for all younger rats vs. 103 ± 4 mg/dl for all adult animals; P < 0.001) as in the prior analysis, and ovariectomy increased glucose levels as well (148 ± 4 for all ovariectomized rats vs. 115 ± 4 mg/dl for all intact animals; P < 0.001). The effects of ovariectomy on plasma glucose were confined to the older age group (Table 4). Insulin levels in this comparison were significantly influenced by age (0.73 ± 0.05 for younger animals vs. 0.55 ± 0.05 for older animals; P = 0.01). While no statistically significant effect of ovariectomy was demonstrated by ANOVA, this age effect was not demonstrated in the preceding analysis. A trend toward increased insulin levels in the younger ovariectomized rats seems to be driving this effect in the present analysis (Table 4). Neither age nor gonad state influenced urine albumin excretion (Table 4).

Kidney weight was greater in older animals (0.85 ± 0.01 for adults vs. 0.80 ± 0.02 g for all younger animals; P = 0.02) and in ovariectomized rats (0.87 ± 0.02 for all ovariectomized rats vs. 0.78 ± 0.01 g for all intact rats; P < 0.001). Kidney weight as a percentage of body weight was reduced by age (0.27 ± 0.04 for all adults vs. 0.31 ± 0.04% for all prepubertal animals; P < 0.001) and ovariectomy (0.27 ± 0.04 for all ovariectomized rats vs. 0.31 ± 0.04% for all intact rats; P < 0.001). This was obviously an effect of the larger body weights after ovariectomy. Glomerular area was not significantly affected by age in these animals (9,029 ± 197 for younger animals vs. 9,546 ± 194 µm2 for older animals; P = 0.07), but ovariectomy significantly increased this parameter (10,388 ± 197 for all ovariectomized animals vs. 8,186 ± 194 µm2 for all intact animals; P < 0.001). This effect was particularly pronounced in the 10-wk-old group (Fig. 2).



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  		Fig. 2. Effects of ovariectomy and age on mean glomerular area. NS, not significant.

 
Estradiol levels, as expected, were significantly lower in ovariectomized animals (7.00 ± 21.90 after ovariectomy vs. 80.35 ± 21.51 pg/ml for all intact rats; P = 0.02). Levels were comparable in 10-wk-old animals with or without gonads and 22-wk-old animals without ovaries; only older intact animals had higher levels of estradiol (Table 4). DHEA levels did not differ with gonadal state (0.12 ± 0.04 for intact animals vs. 0.12 ± 0.04 ng/ml for ovariectomized animals; P = 0.99). Testosterone levels were also unaffected by ovariectomy (0.07 ± 0.05 for intact animals vs. 0.10 ± 0.05 ng/ml for ovariectomized animals; P = 0.78).

All measurements of the renal TGF-{beta} system were affected by ovariectomy (Table 5). This procedure reduced message levels for all isoforms of TGF-{beta}, as well as protein levels for TGF-{beta}1, in 22-wk-old rats. Similar differences were seen for TGF-{beta}1 and TGF-{beta}3 in 10-wk-old animals; however, TGF-{beta}2 mRNA was not significantly decreased by gonadectomy in this age group. {beta}IG-H3 was stable after gonadectomy in 10-wk-old females and increased after gonadectomy in the older groups (Table 5).


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  		Table 5. Renal TGF-{beta} system and ovariectomy in nondiabetic rats

 
Effects of Ovariectomy on STZ DM in Adults

The effects of ovariectomy on the renal response to diabetes were then analyzed. Ovariectomy increased both start and final weight, regardless of metabolic state (Table 6). STZ DM reduced the percentage of weight gained during the 6-wk protocol (102 ± 1 for all diabetic rats vs. 109 ± 1% of start wt for all nondiabetic rats; P < 0.001), and the magnitude of the reduction was greater in ovariectomized animals (Table 6).


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  		Table 6. Effects of ovariectomy in STZ DM adults

 
Glucose levels were higher after gonadectomy (278 ± 8 for all ovarariectomized rats vs. 176 ± 8 for intact animals; P < 0.001) and with diabetes (351 ± 8 for all diabetic animals vs. 103 ± 8 mg/dl for nondiabetic rats; P < 0.001). Ovariectomy had no significant effect on insulin levels (0.52 ± 0.04 for intact animals vs. 0.43 ± 0.04 ng/ml for ovariectomized animals; P = 0.10), but diabetes, as expected, reduced insulin levels (0.41 ± 0.04 for diabetic rats vs. 0.55 ± 0.04 ng/ml for all nondiabetic animals; P = 0.01). Once again, albumin excretion did not differ among these groups.

Like body weight, kidney weight was increased by ovariectomy (1.07 ± 0.02 after ovariectomy vs. 0.97 ± 0.01 g for intact animals; P < 0.001). As expected, diabetes also increased kidney weight (1.19 ± 0.02 for diabetic rats vs. 0.85 ± 0.02 g for nondiabetic animals; P < 0.001). A similar pattern of relationships was seen for kidney weight when expressed as a percentage of final body weight (Table 6). Effects of DM and ovariectomy on glomerular area paralleled those for kidney weight, with both processes increasing this parameter (Fig. 3).



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  		Fig. 3. Effects of ovariectomy and experimental diabetes mellitus on mean glomerular area in adult (~22 wk of age) female rats.

 
Levels of estradiol were reduced by ovariectomy (6.69 ± 23.03 after ovariectomy vs. 119.49 ± 22.63 pg/ml for intact rats; P < 0.001) but were not significantly affected by diabetes in this analysis (86.16 ± 22.63 for nondiabetic rats vs. 40.02 ± 23.03 pg/ml for diabetic rats; P = 0.16, power = 0.16). DHEA was increased in animals after ovariectomy (0.22 ± 0.03 for ovariectomized rats vs. 0.06 ± 0.03 ng/ml for all intact rats; P < 0.001). In this analysis, diabetes had no effect on this horomone (0.14 ± 0.03 for diabetic animals vs. 0.14 ± 0.03 ng/ml for nondiabetic animals; P = 1.00). Testosterone levels were not significantly affected by ovariectomy (0.15 ± 0.06 after ovariectomy vs. 0.05 ± 0.05 ng/ml for all intact animals; P = 0.18, power = 0.14) nor by diabetes (0.10 ± 0.06 for nondiabetic animals vs. 0.10 ± 0.05 pg/ml for diabetic rats; P = 0.95).

STZ DM in ovariectomized rats produced levels of mRNA for TGF-{beta}1 similar to those seen in intact animals (Table 7). Total and active protein levels for this growth factor were reduced by ovariectomy with no significant effects or interactions with the diabetic state (Table 7). Message for TGF-{beta}2 was reduced by ovariectomy with no significant effects or interactions with the diabetic state. TGF-{beta}3 mRNA was increased by ovariectomy (0.5 ± 0.0 for ovariectomized animals vs. 0.3 ± 0.0 x 109 copies/100 ng RNA for intact animals; P < 0.001) and by STZ DM (0.7 ± 0.0 for diabetic rats vs. 0.2 ± 0.0 x 109 copies/100 ng RNA for nondiabetic rats; P < 0.001). Ovariectomized animals showed a greater increase with diabetes than intact animals (Table 7). {beta}IG-H3 also increased with ovariectomy (115.8 ± 7.9 after ovariectomy vs. 0.9 ± 6.7 x 109 copies/100 ng RNA for intact rats; P < 0.001) and diabetes (115.6 ± 7.3 for diabetic rats vs. 1.0 ± 7.3 x 109 copies/100 ng RNA for nondiabetic animals; P < 0.001). The increase in diabetic ovariectomized animals was particularly significant (Table 7).


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  		Table 7. Renal TGF-{beta} system and ovariectomy in diabetic rats

 
Effects of Low-Estrogen States: Prepubertal Onset Compared with Adult Onset After Ovariectomy

Prepubertal animals had smaller start weights (119 ± 4 for prepubertal animals vs. 325 ± 4 g for adults; P < 0.001) and final weights (207 ± 5 for younger rats vs. 352 ± 5 g for adult rats; P < 0.001) than ovariectomized adult-onset animals (Table 8). Prepubertal animals gained more weight during the 6-wk protocol (173 ± 2 for prepubertal rats vs. 109 ± 2% start wt for adult animals; P < 0.001), but both diabetic groups gained less than their nondiabetic counterparts (Table 8). Blood glucose measurements were similar for the two nondiabetic groups, whereas hyperglycemia was greater in prepubertal-onset diabetes than in adult ovariectomy diabetes (Table 8). Insulin levels differed with metabolic state but were not influenced by age and gonadal state. Once again, no differences in albumin excretion were demonstrated (Table 8).


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  		Table 8. Prepubertal onset vs. adult onset after ovariectomy of STZ diabetes

 
Kidney weight was greater in the older ovariectomized animals (1.07 ± 0.02 for adult animals vs. 0.90 ± 0.02 g for younger rats; P < 0.001) and was proportionately increased by diabetes in both age groups (Table 8). When kidney weight was expressed relative to body weight, renal size was much greater in prepubertal diabetic animals than in adult ovariectomized animals (Table 8). Glomerular area paralleled kidney weight, with gonadectomized adults having greater values than prepubertal animals (10,822 ± 229 for adults vs. 8,128 ± 213 µm2 for prepubertal rats; P < 0.001). STZ DM increased glomerular area within each age group to a similar degree (Fig. 4).



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  		Fig. 4. Effect of experimental diabetes mellitus on mean glomerular area in 2 low-estrogen states: prepubertal onset of protocol (~4 wk of age) and adult onset (~16 wk of age) after ovariectomy at 2 wk of age.

 
Levels of estradiol were similar in nondiabetic prepubertal animals (1.77 ± 3.16 pg/ml) and ovariectomized adults (13.39 ± 3.16 pg/ml; P = 0.07). STZ DM reduced estradiol levels in all animals (0.00 ± 2.16 for all diabetic animals vs. 7.58 ± 2.24 pg/ml for all intact animals; P = 0.02). Levels of this estrogen were similar in diabetic prepubertal rats (0.00 ± 2.81 pg/ml) and diabetic adults after ovariectomy (0.00 ± 3.27 pg/ml; P = 0.07). DHEA was greater in the adult animals (0.22 ± 0.04 for all adults vs. 0.07 ± 0.04 ng/ml for all younger rats; P = 0.008), but no significant effect of STZ DM was demonstrated in either age group (0.14 ± 0.04 for all diabetic rats vs. 0.16 ± 0.04 ng/ml for all nondiabetic animals; P = 0.62). No differences in testosterone levels were demonstrated (data not shown).

The renal TGF-{beta} system did show significant differences (all values shown in Table 9). All parameters measured differed significantly between prepubertal-onset and adult ovariectomized animals, and all groups showed significant differences in response to STZ DM. TGF-{beta}1 mRNA was lower in prepubertal animals with diabetes compared with nondiabetic controls, whereas adult ovariectomized animals showed higher values than in diabetic animals. TGF-{beta}2 mRNA was also diminished in the prepubertal diabetes group, although no differences could be demonstrated in the adult ovariectomized animals. TGF-{beta}3 message increased ~10-fold in the adult ovariectomized animals with STZ DM; it was not significantly different with diabetes in the prepubertal-onset group, although levels were about twice those of the older animals. {beta}IG-H3 was similar in all groups except the adult ovariectomized animals with diabetes. TGF-{beta}1 protein levels were lower in older animals, but diabetes brought levels in this age group back to those seen with prepubertal onset.


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  		Table 9. Renal TGF-{beta} system and prepubertal onset vs. adult onset after ovariectomy of STZ diabetes

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
As in our previous work in male rats (28, 29), STZ DM did not increase TGF-{beta}1 mRNA with prepubertal onset as it did in adult animals, even though hyperglycemia was more severe in younger rats. Unlike males, prepubertal females develop renal and glomerular hypertrophy in response to the diabetic state. These data suggest that the factors that promote diabetic renal and glomerular growth may differ between males and females, with production and activation of TGF-{beta} of less importance in females.

Blood glucose levels differed with many of the factors examined in these experiments. Both nondiabetic and diabetic rats in the younger age group tended to have higher blood glucose levels than their adult-onset counterparts. Prepubertal-onset animals were 10 wk of age at the end of the protocol, the approximate midpoint of rat puberty (26). Several studies have demonstrated decreased insulin sensitivity in nondiabetic and diabetic children (1, 6, 11, 17, 38, 42). This probably explains the age-related difference in blood glucose demonstrated in our nondiabetic animals. Insulin dosing may also have contributed in the diabetic groups. As the animals grew during the study, additional insulin became necessary and was added almost weekly; we did achieve levels similar to those in diabetic adults, even though our glycemic control was worse in the prepubertal groups.

Our study differs from others in its TGF-{beta} findings. Most studies of the kidney disease of DM have been performed in male rats because they develop microalbuminuria more rapidly than females. In a longitudinal study of streptozocin DM in female rats, mRNA for TGF-{beta}1 increased by day 3 and remained elevated through 2 wk of DM (19). By 30 days, levels of message had returned to control values, consistent with the results of our present study. TGF-{beta}1 protein in the renal cortex did not increase until 30 days of DM and remained elevated for 90 days; by 180 days, levels had returned to control values (19). There were important differences between the above-mentioned study and our present one. Rats in the earlier study were not treated with insulin and had greater blood glucose values than those seen in the present experiment. Different methods were also used for protein measurement in these studies. Six other studies at least 6 wk in duration have been performed in female rats. Two of these used transgenic Ren-2 rats, so results cannot be compared directly (24, 25). Three of these assessed TGF-{beta} only through immunohistochemical studies (23, 43, 53), whereas one measured only urine levels of the growth factor (37). Direct comparisons among these studies are difficult, although the others all suggested increased TGF-{beta}.

In nondiabetic females, ovariectomy promotes somatic and renal growth while producing modest increases in blood glucose levels. Steroid hormones, including estrogen, are well-established modulators of body mass (54, 55). Ovariectomy has been shown to increase weight gain in females, even as it reduces food intake (55). Estrogen replacement reduced weight gain in this earlier work, as did tamoxifen treatment (55). The present study did not assess food intake, but it did confirm greater weight gain after ovariectomy. We also found greater weight gain in ovariectomized than in intact diabetic rats. Others have shown that these metabolic effects of ovariectomy are also present in spontaneously hypertensive rats (54).

TGF-{beta} production in the kidney is generally suppressed by ovariectomy, but {beta}IG-H3 actually increases with the renal growth demonstrated in this state. This extracellular matrix molecule is produced in a number of cell types in the kidney, including endothelial cells and tubular epithelial cells (16). It contains an integrin recognition sequence (arg-gly-asp) at its COOH terminus and functions in cell adhesion (15, 16, 32, 39, 48, 52). {beta}IG-H3 was first identified through differential hybridization of cell lines exposed to TGF-{beta}1. Proximal tubular cells in vitro show dose-dependent production of {beta}IG-H3 in response to TGF-{beta}1 (16). {beta}IG-H3 message is not produced in response to either insulin-like growth factor-I or epidermal growth factor in this system (16). {beta}IG-H3 has been used extensively as a bioassay for TGF-{beta} activity, although it is still possible that other growth factors may induce its expression (16, 48).

DM potentially drives {beta}IG-H3 production in several ways. First, diabetes stimulates production and activity of TGF-{beta} at many levels, including its receptor complex and transcription factors involved downstream in the signaling process (Fig. 5) (9, 10, 14, 19, 22, 33, 41). TGF-{beta} signaling proceeds via phosphorylation of Smad3 by the TGF-{beta} receptor complex (10, 33, 41). Smad3 then complexes with Smad4 and interacts with DNA in the nucleus to promote transcription of a number of molecules implicated in diabetic nephropathy, including {beta}IG-H3 and extracellular matrix components such as collagen IV (10, 33, 41). Other transcription factors may be involved in these nuclear events as well.



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  		Fig. 5. Interactions of diabetes mellitus, transforming growth factor-{beta} (TGF-{beta}), advanced glycation end products (AGE), the Smad signaling system, and estrogen. LTBP, latent TGF-{beta} binding protein; RAGE, receptor for AGEs; {beta}IG-H3, TGF-{beta}-inducible gene H3.

 
Smad-mediated nuclear events may also be stimulated by advanced glycation end products (AGE), independently of TGF-{beta} (Fig. 5) (33). AGEs are produced during hyperglycemia, and their receptor (RAGE) is upregulated during the diabetic state (22, 33). The AGE-RAGE complex phosphorylates ERK/p38 MAP kinase, which, in turn, phosphorylates Smad2/3, resulting in similar downstream events to those stimulated by TGF-{beta} (33).

Estrogen signaling demonstrates cross talk with Smad signaling pathways (47). Estrogen inhibits transcription of Smad3, and its complex with the estrogen receptor (ER) binds to Smad3 (35). These processes make Smad3 less available for phosphorylation and signaling. TGF-{beta} receptor binding also activates casein kinase 2, which then translocates to the nucleus. There it phosphorylates the transcription factor Egr-1, freeing Sp1 to bind to a response element in the collagen IV gene. Estrogen-ER binding blocks the activation and translocation of casein kinase 2 (57). Egr-1 and Sp1 remain complexed, so less Sp1 transcription factor is available to promote collagen IV transcription.

Conditions associated with increases in {beta}IG-H3 included prepubertal onset of the experimental protocol, DM, and ovariectomy. Estrogen levels tended to be lower in all of these conditions, suggesting that loss of estrogen opposition to Smad signaling may be a key component of renal growth and {beta}IG-H3 production in these states. Further experiments will be necessary to test this possibility directly.

While it did not achieve statistical significance, there was a strong trend toward reduced estrogen levels in our intact diabetic adult females. Our experiments were not designed with steroid levels as a primary end point, and no attempt was made to time our protocols to the estrus cycle. Power for these observations was very low; however, the trend is intriguing for a number of reasons. Menstrual irregularities, including delayed menarche, amenorrhea, and oligomenorrhea, have been noted in women with type 1 diabetes since the 1950s, and more recent studies still show menstrual dysfunction is increased two- to fourfold compared with in nondiabetic women (18). Evidence from clinical and animal studies suggests that the gonadotropin-releasing hormone pulse generator is abnormal, but other abnormalities of the hypothalamic-pituitary-gonadal axis may be involved as well (18). Prior animal studies have demonstrated impaired steroidogenesis in rodents with diabetes, but abnormalities were shown for progesterone, not estrogen, levels (18). No attempt was made to time the present experiments to the estrus cycle, perhaps explaining the wide variability in estrogen levels in the intact animals. Estradiol was the only form of estrogen measured. Other forms of estrogen may play a role in modulating renal growth and fibrosis, in particular the estrogen metabolites hydroxyestradiol and methoxyestradiol (13, 56). These metabolites suppress proliferation of cultured mesangial cells via estrogen receptor-independent mechanisms. Once again, more detailed study of the reproductive neuroendocrine axis is necessary to explore these issues.

In summary, prepubertal onset and ovariectomy both prevent increased TGF-{beta} production in the kidneys of female rats with streptozocin DM, but these states have no effect on renal or glomerular enlargement. Production of the extracellular matrix component {beta}IG-H3 is dissociated from measurements of TGF-{beta} in this system, possibly because of other stimuli to Smad signaling pathways and estrogen’s opposition to Smad pathways. These observations may help to explain some of the sex differences seen in progressive kidney diseases, including diabetes.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-59689.


   ACKNOWLEDGMENTS
 
This work was presented at the Annual Meeting of the American Society of Nephrology, San Diego, CA, 2003, and published in abstract form in J Am Soc Nephrol 14: 126A, 2003.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. H. Lane, Dept. of Pediatrics, 982169 University of 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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