Abstract
Male sex is a risk factor for development and progression of diabetic nephropathy; however, the relationship between sex hormone levels and diabetic nephropathy in type 1 diabetic men is unknown. This was a prospective follow-up study as part of the nationwide Finnish Diabetic Nephropathy (FinnDiane) Study; 297 patients were followed for 5.9±1.5 years. Serum total testosterone (Tt) and estradiol (Te), calculated free testosterone (cFt) and estradiol (cFe) and sex hormone binding globulin were measured at baseline and correlated with urinary albumin excretion rate, estimated glomerular filtration rate and markers of metabolic syndrome. Diabetes without renal disease was associated with decreased Tt (p<0.001), Te (p<0.001) and cFt (p=0.001) levels compared with healthy non-diabetic men. With progression of renal disease from micro- to macroalbuminuria, this decrease in serum Tt was even more pronounced. Cox regression showed that cFt and cFe were independent predictors of the progression from macroalbuminuria to end-stage renal disease. Our study shows that men with type 1 diabetes exhibit dysregulated sex hormone levels, which is most pronounced in men with progressive renal disease, suggesting that sex hormones may play a role in the pathogenesis of diabetic nephropathy associated with type 1 diabetes.
Keywords: type 1 diabetes, diabetic nephropathy, albuminuria, testosterone, SHBG, estradiol
1. Introduction
The incidence and the rate of progression of non-diabetic renal disease are far greater in men compared to age-matched women [1,2]. In the setting of diabetes however, the female sex as a protective factor against the development of renal disease is diminished, such as that diabetes narrows the gap between the sexes in terms of development and progression of renal disease [1,2]. However, studies show that the male sex remains a risk factor, and that the rate of progression of diabetic renal disease is still greater in men compared with age-matched women. Specifically, in patients with type 1 diabetes male sex was shown to be a risk factor for the progression of albuminuria in adult men with initially normal renal function or mild renal insufficiency [3]. In patients with established diabetic nephropathy, male sex was associated with a more severe decline in glomerular filtration rate (GFR) over 5 years of patient follow-up [4]. Similarly, several studies have shown a higher incidence of diabetic nephropathy, as evidenced by micro- or macroalbuminuria, in type 2 diabetic adult males compared to age-matched females [5–7]. These observations strongly support the notion that the male sex is a risk factor for the development and progression of diabetic nephropathy, at least in adults.
Based on the fact that the male sex is a risk factor for the development and progression of diabetic nephropathy, one could presume that testosterone, being the predominant sex hormone in males, would be the underlying cause of this risk. Further supporting this concept is the fact that diabetic renal complications very rarely develop before puberty [8], while the onset of puberty, and thus the surge of androgens, greatly accelerates disease development [9,10]. Indeed, increased levels of testosterone have been observed during puberty in patients with type 1 diabetes developing microalbuminuria [11]. In addition, increased serum testosterone levels have been shown in young adult males (average age of subjects 23 years) with type 1 diabetes with proliferative retinopathy compared with diabetic males with minimal or no retinopathy [12]. These studies favor the hypothesis that high testosterone concentrations may be associated with the development of diabetic end-organ complications in younger type 1 diabetic men. In contrast, decreased testosterone levels have been observed in men with type 2 diabetes [13–15]. However, to date, no studies have examined the correlation between testosterone levels and diabetic nephropathy associated with type 1 diabetes in adult men.
Furthermore, emerging evidence suggests that increased estradiol levels are associated with complications, such as atherosclerosis in men with type 2 diabetes mellitus [14]. However, virtually nothing is known about the association between estradiol levels and diabetic nephropathy in men with type 1 diabetes. Thus, the aim of the present study was to examine the correlation between sex hormone levels and progression of diabetic nephropathy in adult men with type 1 diabetes.
2. Experimental
2.1 Data collection
This study is part of the ongoing prospective Finnish Diabetic Nephropathy Study (FinnDiane), which is a nationwide, comprehensive multicenter study, with the aim to identify genetic and environmental risk factors for diabetic complications, with special emphasis on diabetic nephropathy in patients with type 1 diabetes.
At baseline and at follow-up, all patients underwent a thorough clinical exam during a visit to the attending physician. For all patients in the present analysis, all available medical files, including laboratory data were reviewed and changes in renal status and new cardiovascular events were verified. Data on medication and diabetic complications were registered with a standardized questionnaire which was completed by the patient’s attending physician at regular patient visits based upon medical files. Blood pressure was measured twice in the sitting position after a 10 minute rest and the average of these measurements were used in the analysis. Height, weight and waist hip ratio (WHR) were recorded, and blood was drawn for the measurements of HbA1c, lipids, creatinine and sex hormones. Estimated glucose disposal rate (eGDR) was calculated as earlier described as a measure of insulin sensitivity [16]. No patients were receiving testosterone replacement.
The ethics committees of all participating centers approved the study protocol. Written informed consent was obtained from each patient and the study was performed in accordance with the Declaration of Helsinki as revised in the year 2000. The protocol of patient recruitment has previously been published [17]. For this particular study, all data were collected between the years of 1998–2002.
2.2 Participants and definition of renal disease progression
A total of 297 men with type 1 diabetes were included in the present study and were followed for 5.9±1.5 years. Based on their urinary albumin excretion rate (AER) in three consecutive overnight or 24-h urine collections, at baseline, 101 men had normal AER (NORMO; AER<20 μg/min or <30 mg/24h), 96 men microalbuminuria (MICRO; 20≥AER<200 μg/min or 30≥AER<300 mg/24h) and 100 men macroalbuminuria (MACRO; AER≥200 μg/min or AER≥300mg/24h). Men with end-stage renal disease (ESRD), defined as men on dialysis or having received a kidney transplant, were excluded from the study.
Progression of renal disease was defined as follows: all urinary AER data between baseline and the follow-up visit (5.9±1.5 years) were reviewed and based on the AER in any two-out of three consecutive urine collections during the follow-up period, the patient’s renal status was classified as in the baseline examination (Note: In patients without kidney disease (i.e. normal AER) measurements were performed at least once yearly. If patients had micro- or macroalbuminuria the measurements were more frequent, sometimes up to 4 times per year). Progression was defined as a change from one level to a higher level of albuminuria or the development of ESRD (Note: We used the second “positive” collection out of any three consecutive urine collections during the follow-up period as the time point for progression). Men without progression of renal disease were classified as non-progressors. Type 1 diabetes was defined as the onset of diabetes before the age of 35 years and permanent insulin treatment initiated within one year of diagnosis. The study also included a group of non-diabetic men (n=96) as a control for comparing changes in sex hormone levels between healthy, non-diabetic subjects and men with type 1 diabetes. The healthy controls were enrolled from the staff of the research center and their spouses. They underwent the same physical examination as well as the same blood and urine sampling as the diabetic patients. None of them had a family history of diabetes or kidney disease or reported any other serious medical conditions. The group of non-diabetic men were reproductively normal males without significant comorbidities.
2.3 Laboratory measures
HbA1c was determined by standardized assays at each participating center. Serum lipid and lipoprotein concentrations were measured at the research laboratory of Helsinki University Central Hospital, Division of Cardiology, Helsinki, Finland, by automated enzymatic methods using the Cobas Mira analyzer (Hoffmann-LaRoche, Basel, Switzerland). Serum creatinine was measured by enzymatic methods at a central laboratory. Urinary AER was determined in one 24-h or overnight urine collection at a central laboratory by immunoturbidimetry. The glomerular filtration rate (eGFR) was calculated using the Cockroft-Gault formula.
Serum testosterone, estradiol and SHBG levels were measured in blood samples collected at baseline using commercially available enzyme immunoassay kits (Alpco Diagnostics, Salem, NH) according to the manufacturers’ protocol. Duplicate measures for each hormone were performed for each sample. The sensitivity and intra- and inter-assay coefficients of variation, respectively, were as follows: testosterone, 0.02 ng/ml, 8.0 and 8.3%; estradiol, 10 pg/ml, 7.7 and 8.7%; SHBG, 0.1 nmol/L, 5.3 and 9.6%. Serum cFt and cFe levels were calculated from Tt, Te and SHBG and serum albumin based on mass action laws with Vermeulen’s formula [18,19].
2.4 Data analysis
All data shown are baseline data and are expressed as means ± SD for normally distributed values and as medians with interquartile range for non-normally distributed values. Differences between groups for normally distributed variables were tested using ANOVA and variables that were not normally distributed with the Kruskal-Wallis test. Categorical variables were analyzed with a χ2-test. Baseline risk factors to the progression of diabetic nephropathy were assessed using Cox regression analysis. All calculations were performed with SPSS 15.0.1 (SPSS Inc., Chicago, IL). Values P<0.05 were considered as statistically significant.
3. Results
The clinical characteristics at baseline of the men enrolled in the study are given in Table 1. The study included 96 non-diabetic, healthy men, 101 men with type 1 diabetes with normal uAER, 96 with microalbuminuria and 100 with macroalbuminuria.
TABLE 1.
Baseline clinical characteristics of male patients with type 1 diabetes and healthy controls
| NORMO | MICRO | MACRO | CONTROL | P-value Within DM | P-value DM vs CONTROL | |
|---|---|---|---|---|---|---|
| N | 101 | 96 | 100 | 96 | ||
| Age (yrs) | 35.5 ± 10.3 | 36.6 ± 9.8 | 41.2 ± 9.0 | 34.3 ± 10.2 | <0.001 | 0.386 |
| Duration of diabetes (yrs) | 21.3 ± 9.1 | 25.2 ± 9.3 | 27.7 ± 7.0 | NA | <0.001 | NA |
| BMI (kg/m2) | 25.3 ± 3.3 | 25.4 ± 3.4 | 26.0 ± 4.3 | 24.5 ± 2.9 | 0.275 | 0.087 |
| WHR | 0.90 ± 0.07 | 0.92 ± 0.06 | 0.94 ± 0.07 | 0.92 ± 0.07 | <0.001 | 0.033 |
| Systolic BP (mmHg) | 129 ± 13 | 136 ± 15 | 145 ± 20 | 131 ± 11 | <0.001 | 0.263 |
| Diastolic BP (mmHg) | 80 ± 10 | 82 ± 10 | 85 ± 9 | 78 ± 9 | 0.002 | 0.168 |
| AHT (%) | 14.0 | 69.8 | 93.9 | 3.1 | <0.001 | 0.007 |
| HbA1c (%) | 8.7 ± 1.5 | 9.0 ± 1.7 | 9.1 ± 1.7 | 5.5 ± 0.3 | 0.223 | <0.001 |
| Triglycerides (mmol/l) | 1.00 (0.33 – 7.04) | 1.22 (0.57 – 8.82) | 1.71 (0.55 – 8.38) | 0.98 (0.31 – 11.75) | <0.001 | 0.250 |
| Total cholesterol (mmol/l) | 4.80 ± 1.03 | 4.96 ± 0.91 | 5.44 ± 1.06 | 4.70 ± 0.93 | <0.001 | 0.478 |
| HDL-cholesterol (mmol/l) | 1.23 ± 0.35 | 1.22 ± 0.34 | 1.04 ± 0.33 | 1.39 ± 0.25 | <0.001 | 0.001 |
| eGFR (ml/min/1.73m2) | 102.7 ± 19.6 | 98.0 ± 22.9 | 56.2 ± 27.2 | 109.1 ± 19.0 | <0.001 | 0.022 |
| uAER (mg/24h)* | 12 (2 – 76) | 79 (6 – 483) | 861 (10 – 5088) | 7 (2 – 25) | <0.001 | <0.001 |
| eGDR (mg/kg/min) | 6.41 ± 2.29 | 4.09 ± 1.63 | 3.54 ± 1.45 | 8.38 ± 1.84 | <0.001 | <0.001 |
Data are presented as means ± SD, medians with ranges or percentages where appropriate.
Abbreviations: BMI, body mass index; WHR, waist-hip ratio; AHT, antihypertensive therapy; eGFR, estimated glomerular filtration rate; uAER, urinary albumin excretion rate; eGDR, estimated glucose disposal rate;
NOTE: The urinary AER presented is the last measurement of each patient. Because the classification was based on two out of three consecutive samples, a single value may be higher. A low value may be due to the effect of treatment.
Uncomplicated type 1 diabetes was associated with an overall reduction in serum Tt, cFt, Te and cFe, but not in SHBG levels compared with non-diabetic men (Table 2). When further corrected for age, BMI, WHR, eGDR and SHBG, serum Tt (p=0.003) and cfT (p=0.002) remained significantly reduced in men with type 1 diabetes in comparison with healthy men, while Te (0.088) and cFe (0.094) levels were only of borderline significance (data not shown). Interestingly, in men with established diabetic nephropathy (macroalbuminuria), all these variables were higher compared to men with either normal uAER or microalbuminuria; however, the values were still reduced in these men with macroalbuminuria compared with non-diabetic men.
TABLE 2.
Baseline sex hormone levels in male patients with type 1 diabetes and healthy controls
| NORMO | MICRO | MACRO | CONTROL | P-value Within DM | P-value DM vs CONTROL | |
|---|---|---|---|---|---|---|
| Tt (ng/ml) | 5.26 (1.68 – 14.19) | 4.86 (0.91 – 11.42) | 6.40 (2.02 – 32.92) | 7.20 (2.60 – 23.56) | <0.001 | <0.001 |
| cFt (ng/ml) | 0.050 (0.018 – 0.190) | 0.047 (0.016 – 0.229) | 0.057 (0.023 – 0.749) | 0.063 (0.021 – 0.465) | 0.003 | 0.001 |
| Te (pg/ml) | 47.0 (9.3 – 1416) | 51.5 (11.3 – 2096) | 63.2 (25.2 – 1228) | 69.5 (25.1 – 741) | <0.001 | <0.001 |
| cFe (pg/ml) | 0.429 (0.122 – 15.600) | 0.446 (0.115 – 19.650) | 0.489 (0.201 – 7.935) | 0.553 (0.139 – 4.895) | 0.043 | 0.088 |
| SHBG (nmol/l) | 108.2 ± 49.2 | 102.0 ± 50.5 | 106.9 ± 44.7 | 119.3 ± 49.9 | 0.638 | 0.116 |
Data are presented as means ± SD, medians with ranges or percentages where appropriate.
Abbreviations: Tt, total testosterone; cFt, calculated free testosterone; Te, total estradiol; cFe, calculated free estradiol.
When the data was analyzed according to disease progression (i.e. progressors vs non-progressors) by univariate analysis, we found that in men with incident (i.e. new onset) microalbuminuria (n=50), baseline Tt, cFt, Te or cFe levels were no different from those in men that did not develop microalbuminuria (n=51); however, SHBG levels were decreased with new onset microalbuminuria (Table 3). This decrease in SHBG levels was associated with WHR, serum triglyceride concentrations and HbA1c and a trend in eGDR (Table 3).
TABLE 3.
Baseline clinical characteristics of male patients with type 1 diabetes and normal AER with and without progression to microalbuminuria
| Non-progressors | Progressors | P-value | |
|---|---|---|---|
| N | 51 | 50 | |
| Age (yrs) | 35.6 ± 10.5 | 35.4 ± 10.2 | 0.915 |
| Duration of diabetes (yrs) | 23.4 ± 7.0 | 19.1 ± 10.4 | 0.015 |
| BMI (kg/m2) | 25.0 ± 3.2 | 25.5 ± 3.5 | 0.518 |
| WHR | 0.88 ± 0.08 | 0.91 ± 0.06 | 0.022 |
| Systolic BP (mmHg) | 130 ± 12 | 129 ± 14 | 0.517 |
| Diastolic BP (mmHg) | 81 ± 9 | 78 ± 10 | 0.257 |
| AHT (%) | 13.7 | 14.3 | 0.936 |
| HbA1c (%) | 8.1 ± 1.0 | 9.4 ± 1.6 | <0.001 |
| Triglycerides (mmol/l) | 0.91 (0.33 – 5.47) | 1.15 (0.55 – 7.04) | 0.004 |
| Total cholesterol (mmol/l) | 4.65 ± 0.86 | 4.97 ± 1.16 | 0.118 |
| HDL-cholesterol (mmol/l) | 1.22 ± 0.33 | 1.24 ± 0.37 | 0.839 |
| eGFR (ml/min/1.73m2) | 99.9 ± 18.7 | 105.4 ± 20.2 | 0.158 |
| uAER (mg/24h) | 7 (3 – 32) | 19 (2 – 76) | <0.001 |
| eGDR (mg/kg/min) | 6.83 ± 2.64 | 5.99 ± 1.82 | 0.069 |
| Tt (ng/ml) | 5.26 (1.98 – 14.19) | 5.39 (1.68 – 14.11) | 0.537 |
| cFt (ng/ml) | 0.047 (0.020 – 0.190) | 0.053 (0.018 – 0.162) | 0.166 |
| Te (pg/ml) | 47.1 (18.6 – 1416) | 47.0 (9.3 – 1028) | 0.817 |
| cFe (pg/ml) | 0.384 (0.122 – 15.600) | 0.589 (0.123 – 8.485) | 0.141 |
| SHBG (nmol/l) | 118.9 ± 48.3 | 97.2 ± 48.1 | 0.025 |
Data are presented as means ± SD, medians with range or percentages when appropriate.
In those men that progressed from microalbuminuria to macroalbuminuria, SHBG levels were reduced even further (Table 4). Tt was also recued and there was a trend towards increased cFe levels compared with men that remained microalbuminuric (Table 4). These changes were associated with a significant increase in serum triglyceride concentrations and HbA1c, a decrease in eGDR and a trend towards an increase in WHR and systolic blood pressure as well as lower HDL-cholesterol (Table 4).
TABLE 4.
Baseline clinical characteristics of male patients with type 1 diabetes and microalbuminuria with and without progression to macroalbuminuria
| Non-progressors | Progressors | P-value | |
|---|---|---|---|
| N | 59 | 37 | |
| Age (yrs) | 37.0 ± 9.5 | 36.0 ± 10.4 | 0.629 |
| Duration of diabetes (yrs) | 24.5 ± 8.4 | 26.4 ± 10.5 | 0.350 |
| BMI (kg/m2) | 25.3 ± 3.3 | 25.5 ± 3.6 | 0.763 |
| WHR | 0.91 ± 0.06 | 0.93 ± 0.06 | 0.070 |
| Systolic BP (mmHg) | 133 ± 15 | 139 ± 14 | 0.063 |
| Diastolic BP (mmHg) | 81 ± 10 | 84 ± 10 | 0.135 |
| AHT (%) | 71.2 | 67.6 | 0.707 |
| HbA1c (%) | 8.6 ± 1.4 | 9.7 ± 2.0 | 0.003 |
| Triglycerides (mmol/l) | 1.05 (0.57 – 4.10) | 1.72 (0.60 – 8.82) | <0.001 |
| Total cholesterol (mmol/l) | 4.73 ± 0.79 | 5.34 ± 0.96 | 0.001 |
| HDL-cholesterol (mmol/l) | 1.27 ± 0.34 | 1.14 ± 0.32 | 0.083 |
| eGFR (ml/min/1.73m2) | 96.7 ± 18.4 | 100.0 ± 28.8 | 0.496 |
| uAER (mg/24h) | 46 (6 – 184) | 152 (59 – 483) | <0.001 |
| eGDR (mg/kg/min) | 4.41 ± 1.51 | 3.55 ± 1.69 | 0.012 |
| Tt (ng/ml) | 5.30 (0.91 – 11.42) | 4.26 (1.45 – 9.23) | 0.043 |
| cFt (ng/ml) | 0.047 (0.020 – 0.229) | 0.049 (0.016 – 0.175) | 0.625 |
| Te (pg/ml) | 52.1 (11.3 – 2096) | 48.3 (21.2 – 943) | 0.530 |
| cFe (pg/ml) | 0.344 (0.115 – 19.650) | 0.504 (0.193 – 10.010) | 0.062 |
| SHBG (nmol/l) | 112.9 ± 52.2 | 84.6 ± 43.0 | 0.007 |
Data are presented as means ± SD, medians with range or percentages when appropriate.
Interestingly, in men that progressed from macroalbuminuria to ESRD, a trend towards an increase in Tt and cFt, as opposed to a decrease that was observed in men that progressed from microalbuminuria to macroalbuminuria, was observed (Table 5). Te and cFe levels were twice as high in men that progressed from macroalbuminuria to ESRD compared with non-progressors. No differences in SHBG levels were observed in these men. eGFR in he progressors was 39.8±25.3 compared with 68.5±21.7 in non-progressors (P=0.001). In these men, there was a significant correlation between eGFR and cFe (R=−0.332; P=0.001) and cFt (R=−0.258; P=0.005), but there was no correlation between eGFR and SHBG.
TABLE 5.
Baseline clinical characteristics of male patients with type 1 diabetes and macroalbuminuria with and without progression to end-stage renal disease
| Non-progressors | Progressors | P-value | |
|---|---|---|---|
| N | 57 | 43 | |
| Age (yrs) | 41.5 ± 9.2 | 40.8 ± 8.9 | 0.706 |
| Duration of diabetes (yrs) | 27.7 ± 6.6 | 27.6 ± 7.7 | 0.960 |
| BMI (kg/m2) | 26.0 ± 3.3 | 26.1 ± 5.3 | 0.853 |
| WHR | 0.93 ± 0.07 | 0.94 ± 0.08 | 0.601 |
| Systolic BP (mmHg) | 143 ± 19 | 148 ± 21 | 0.260 |
| Diastolic BP (mmHg) | 83 ± 9 | 86 ± 9 | 0.166 |
| AHT (%) | 94.7 | 92.9 | 0.698 |
| HbA1c (%) | 8.9 ± 1.6 | 9.4 ± 2.0 | 0.219 |
| Triglycerides (mmol/l) | 1.64 (0.55 – 4.51) | 1.93 (0.76 – 8.38) | 0.011 |
| Total cholesterol (mmol/l) | 5.32 ± 0.85 | 5.60 ± 1.28 | 0.187 |
| HDL-cholesterol (mmol/l) | 1.07 ± 0.31 | 0.99 ± 0.35 | 0.192 |
| eGFR (ml/min/1.73m2) | 68.5 ± 21.7 | 39.8 ± 25.3 | <0.001 |
| uAER (mg/24h) | 619 (10 – 4777) | 1561 (70 – 5088) | <0.001 |
| eGDR (mg/kg/min) | 3.70 ± 1.51 | 3.32 ± 1.36 | 0.209 |
| Tt (ng/ml) | 6.19 (2.02 – 17.05) | 7.77 (2.82 – 32.92) | 0.171 |
| cFt (ng/ml) | 0.051 (0.023 – 0.211) | 0.067 (0.026 – 0.749) | 0.066 |
| Te (pg/ml) | 52.1 (25.2 – 1228) | 96.9 (33.4 – 594) | <0.001 |
| cFe (pg/ml) | 0.417 (0.201 – 7.935) | 0.913 (0.252 – 5.130) | <0.001 |
| SHBG (nmol/l) | 110.3 ± 43.4 | 102.3 ± 46.5 | 0.377 |
Data are presented as means ± SD, medians with range or percentages when appropriate.
In Cox regression analyses, including the duration of diabetes, WHR, HbA1c, triglycerides, eGDR, cFt, cFe and SHBG, only the increases in WHR (P=0.033) and HbA1c (P=0.008) were predictors of incident microalbuminuria (Table 6). None of the sex steroids or the SHBG predicted new onset microalbuminuria. We replaced the duration of diabetes with systolic blood pressure in the Cox regression analyses for progression from microalbuminuria to macroalbuminuria and found that increased serum triglyceride concentrations (P=0.004) and cFt (P=0.036) were significant predictors from microalbuminuria to macroalbuminuria. Importantly, while cFe was not a predictor of progression from microalbuminuria to macroalbuminuria, it was an independent predictor of the progression from macroalbuminuria to ESRD (P<0.001), together with the decline in eGFR (P<0.001) and increase in cFt (P=0.038). This model included serum triglycerides, cFt, cFe, SHBG and eGFR.
TABLE 6.
Cox regression models for progression in renal status: A) from normal AER to microalbuminuria, B) from microalbuminuria to macroalbuminuria, and C) from macroalbuminuria to end-stage renal disease (ESRD).
| Model A | ||
|---|---|---|
| Variable | Hazard ratio (95%CI) | P-value |
| Duration of diabetes (yrs) | 0.97 (0.93 – 1.02) | 0.213 |
| WHR | 557.96 (1.65 – 188561.26) | 0.033 |
| HbA1c (%) | 1.37 (1.09 – 1.72) | 0.008 |
| ln(Triglycerides) | 0.95 (0.61 – 1.71) | 0.982 |
| eGDR (mg/kg/min) | 1.15 (0.92 – 1.44) | 0.214 |
| ln(cFt) | 0.89 (0.41 – 1.96) | 0.776 |
| ln(cFe) | 1.01 (0.68 – 1.49) | 0.945 |
| SHBG (nmol/l) | 1.00 (0.99 – 1.01) | 0.666 |
| Model B | ||
|---|---|---|
| Variable | Hazard ratio (95%CI) | P-value |
| Systolic blood pressure (mmHg) | 1.02 (1.00 – 1.05) | 0.096 |
| WHR | 0.04 (0.01 – 726.67) | 0.529 |
| HbA1c (%) | 1.17 (0.91 – 1.51) | 0.230 |
| ln(Triglycerides) | 3.44 (1.48 – 8.04) | 0.004 |
| eGDR (mg/kg/min) | 0.93 (0.65 – 1.33) | 0.690 |
| ln(cFt) | 0.43 (0.19 – 0.95) | 0.036 |
| ln(cFe) | 1.18 (0.77 – 1.81) | 0.445 |
| SHBG (nmol/l) | 1.00 (0.99 – 1.01) | 0.822 |
| Model C | ||
|---|---|---|
| Variable | Hazard ratio (95%CI) | P-value |
| ln(cFe) | 3.03 (1.67 – 5.50) | <0.001 |
| eGFR (ml/min/1.73m2) | 0.95 (0.93 – 0.97) | <0.001 |
| ln(cFt) | 0.46 (0.22 – 0.96) | 0.038 |
| ln(Triglycerides) | 1.68 (0.93 – 3.02) | 0.085 |
| SHBG (nmol/l) | 1.01 (1.00 – 1.01) | 0.221 |
4. Discussion
The present study highlights three major observations: 1. Type 1 diabetes is men is associated with an overall reduction in sex hormone levels; 2. A decrease in serum testosterone is predictive of the progression from microalbuminuria to macroalbuminuria alongside increased serum triglycerides, a marker of insulin resistance; 3. Increases in serum estradiol and testosterone are independent predictors of the progression from macroalbuminuria to ESRD. These data implicate sex hormones in the development and progression of diabetic nephropathy associated with type 1 diabetes.
While several studies have reported reduced Tt and/or cFt levels in men with type 2 diabetes [13–15], to our knowledge, this is the first report on differences in sex hormone levels in type 1 diabetic compared with normal, healthy adult men. A recent report [20] showed no differences in Tt and cFT levels in type 1 diabeteic men, while there was a reduction in Tt and cFt in patients with type 2 diabetes. However, this study did not include an age- and weight-matched non-diabetic control group and only compared testosterone levels to the reported physiological reference range for healthy men. The reduction in testosterone levels, at least in type 2 diabetic men has largely been ascribed to hypogonadism [14] associated with reduced gonadotropin secretion [21], reduced levels of both LH and FSH [21,22], cytokine-mediated inhibition of testosterone production [23] and reduced SHBG levels [24]. While similar mechanisms are likely to contribute to the reduced testosterone levels in men with type 1 diabetes as well, further studies are necessary to determine the precise mechanisms underlying the reduced testosterone levels and type 1 diabetes.
In addition to reduced Tt and cFt levels, our data show that men with type 1 diabetes also exhibit reduced Te and cFe levels compared with non-diabetic men. One of the possible mechanisms for this observation on estradiol levels may be reduced levels of testosterone and thus lack of substrate for aromatization to estradiol [25]. In addition, reduced aromatase activity may also contribute to reduced estradiol synthesis [25]. Indeed, a mutation in the aromatase gene, resulting in aromatase deficiency, was first identified in a man with type 2 diabetes [26]; however, it is unknown whether changes in aromatization contributes to reduction in estradiol levels in type 1 diabetics. Interestingly, no differences in SHBG levels compared with normal, healthy controls were observed in our study. Although SHBG levels have been reported to be decreased during puberty in boys, as well as in young men with type 1 diabetes [27], SHBG levels have shown to be increased in adult type 1 diabetic men [28]. Although portal insulin levels have been shown to regulate SHBG levels [29], the study by van Dam and colleagues showing increased SHBG levels in type 1 diabetic men concluded that estimated portal insulin levels did not influence SHBG [28]. Future studies are needed to determine whether no changes in SHBG found in our patient population is related to portal insulin levels.
While our study does not show that incipient microalbuminuria is associated with any changes in sex hormone levels, increased testosterone levels have been observed in patients with type 1 diabetes developing microalbuminuria during puberty [11], but this effect is more pronounced in women than men. This study suggested that increased testosterone levels during puberty could be related to reduced insulin-like growth factor levels, poor glycemic control and also growth hormone levels [11]. Lower growth hormone levels in the adult population may potentially explain the absence of an association between incipient microalbuminuria and testosterone levels observed in our study.
Our data show that the progression from microalbuminuria to macroalbuminuria is associated with a further reduction in testosterone levels. This reduction appears to be associated with components of the metabolic syndrome and insulin resistance. These observations are consistent with the previous reports that low testosterone is associated with insulin resistance [30,31] and is predictive of development of metabolic syndrome and type 2 diabetes in middle-aged men [13,32]. In addition to reduced testosterone levels, there was a concomitant decrease in SHBG levels. Reduced levels of SHBG have been shown to predict the metabolic syndrome and type 2 diabetes in middle-aged men [13,24,32]. Indeed, SHBG production is known to be regulated by insulin concentration or insulin resistance (higher insulin levels or insulin resistance are associated with lower levels of SHBG) [29,33].
The present study shows that men that progressed from macroalbuminuria to ESRD exhibit an 86% increase in Te, 119% increase in Fe and 31% in cFt compared with the men that did not progress. In contrast, Grossmann et al showed a reduction in cFT levels in men with type 1 diabetes [30]. The discrepancy in these findings may partly be explained by the fact that their study included a mix of men with and without chronic kidney disease and the results were not compared to a non-diabetic control group, but rather to a published reference range of testosterone in healthy men. In our study, the Cox model showed that cFe was an independent predictor of the progression from macroalbuminuria to ESRD together with the decline in renal function and increase in cFt. Part of the reason for the relative increase in sex hormone levels in the progresses (note: the overall hormone levels in these men were still decreased compared with non-diabetic men) can possibly be explained by their reduced clearance due to the decrease in GFR. However, given the fact that cFe and cFt were both predictors of the disease progression independently of eGFR suggests that the increase in cFe and cFt observed with the progression from macroalbuminuria to ESRD may be unrelated to the decline in eGFR.
The increased production of sex hormones may be a result of either increased hormone biosynthesis and/or decreased degradation. It is conceivable that increased levels of cFe observed with progression from macroalbuminuria to ESRD may occur due to increased testosterone aromatization at tissue and peripheral levels. Similarly, the increased levels of cFt may potentially also be explained by increased testosterone production in response to progressive target organ injury. However, further studies are needed to test these hypotheses.
The strength of our study is that it included a larger number of men with type 1 diabetes than previous studies. Furthermore, the men included in the study were part of the nationwide, multicenter FinnDiane study, as opposed to studies in which patients were recruited from a single center. In addition, our study included an age and weight-matched non-diabetic control group. While previous studies have reported limitations in comparing sex hormone levels in blood drawn at varying times of the day, in our study, the samples were collected in the early morning, according to a standardized protocol. Moreover, sex hormones were measured in all samples at the same time. The study is limited by the fact that we did not include measures of testosterone secretion, such as levels of luteinizing and follicle stimulating hormones, so we can not explain the mechanisms of reduced hormone levels. In addition, we did not examine the relationship between insulin and sex hormone levels in our patients. One of the caveats of the study is that sex hormones were measured by commercial ELISA without extrication by chromatography [34]. While extraction followed by mass spectrometry is emerging as the gold standard for measurement of sex hormones, the cost associated with this methodology make it virtually impossible to apply in studies that include a large number of samples.
In conclusion, our study shows that men with type 1 diabetes exhibit dysregulated sex hormone levels. This dysregulation is most pronounced in men with progressive renal disease, suggesting that sex hormones may play a role in the pathogenesis of diabetic kidney disease.
Acknowledgments
We acknowledge all of the physicians and nurses at each of the FinnDiane center participating in patient recruitment and characterization (see online supplement). The authors would like to acknowledge the technical help of Ms. Anjali Prabhu and Ms. Qin Xu (Department of Medicine, Georgetown University Medical Center, Washington DC, USA) in measuring serum hormone levels. This research was supported by grants from the Folkhälsan Research Foundation, Wilhelm and Else Stockmann Foundation, Liv och Hälsa Foundation, Sigrid Juselius Foundation and the European Commission (QLG2-CT-2001-01669, LSHB-CT-2003-503364 and LSB-CT-2006-037681) to P-H.G and the National Institutes of Health grant (RO1DK075832) to C.M.
Footnotes
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
References
- 1.Neugarten J. Gender and the progression of renal disease. J Am Soc Nephrol. 2002;13:2807–2809. doi: 10.1097/01.asn.0000035846.89753.d4. [DOI] [PubMed] [Google Scholar]
- 2.Silbiger SR, Neugarten J. The impact of gender on the progression of chronic renal disease. Am J Kidney Dis. 1995;25:515–533. doi: 10.1016/0272-6386(95)90119-1. [DOI] [PubMed] [Google Scholar]
- 3.Mangili R, Deferrari G, Di Mario U, Giampietro O, Navalesi R, Nosadini R, Rigamonti G, Spezia R, Crepaldi G. Arterial hypertension and microalbuminuria in IDDM: the Italian Microalbuminuria Study. Diabetologia. 1994;37:1015–1024. doi: 10.1007/BF00400465. [DOI] [PubMed] [Google Scholar]
- 4.Jacobsen P, Rossing K, Tarnow L, Rossing P, Mallet C, Poirier O, Cambien F, Parving HH. Progression of diabetic nephropathy in normotensive type 1 diabetic patients. Kidney Int Suppl. 1999;71:S101–S105. doi: 10.1046/j.1523-1755.1999.07125.x. [DOI] [PubMed] [Google Scholar]
- 5.Savage S, Nagel NJ, Estacio RO, Lukken N, Schrier RW. Clinical factors associated with urinary albumin excretion in type II diabetes. Am J Kidney Dis. 1995;25:836–844. doi: 10.1016/0272-6386(95)90565-0. [DOI] [PubMed] [Google Scholar]
- 6.Parving HH, Gall MA, Skott P, Jorgensen HE, Lokkegaard H, Jorgensen F, Nielsen B, Larsen S. Prevalence and causes of albuminuria in non-insulin-dependent diabetic patients. Kidney Int. 1992;41:758–762. doi: 10.1038/ki.1992.118. [DOI] [PubMed] [Google Scholar]
- 7.Gall MA, Hougaard P, Borch-Johnsen K, Parving HH. Risk factors for development of incipient and overt diabetic nephropathy in patients with non-insulin dependent diabetes mellitus: prospective, observational study. BMJ. 1997;314:783–788. doi: 10.1136/bmj.314.7083.783. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Mauer M, Drummond K. The early natural history of nephropathy in type 1 diabetes: I. Study design and baseline characteristics of the study participants. Diabetes. 2002;51:1572–1579. doi: 10.2337/diabetes.51.5.1572. [DOI] [PubMed] [Google Scholar]
- 9.Schultz CJ, Konopelska-Bahu T, Dalton RN, Carroll TA, Stratton I, Gale EA, Neil A, Dunger DB. Microalbuminuria prevalence varies with age, sex, and puberty in children with type 1 diabetes followed from diagnosis in a longitudinal study. Oxford Regional Prospective Study Group. Diabetes Care. 1999;22:495–502. doi: 10.2337/diacare.22.3.495. [DOI] [PubMed] [Google Scholar]
- 10.Svensson M, Nystrom L, Schon S, Dahlquist G. Age at onset of childhood-onset type 1 diabetes and the development of end-stage renal disease: a nationwide population-based study. Diabetes Care. 2006;29:538–542. doi: 10.2337/diacare.29.03.06.dc05-1531. [DOI] [PubMed] [Google Scholar]
- 11.Amin R, Schultz C, Ong K, Frystyk J, Dalton RN, Perry L, Orskov H, Dunger DB. Low IGF-I and elevated testosterone during puberty in subjects with type 1 diabetes developing microalbuminuria in comparison to normoalbuminuric control subjects: the Oxford Regional Prospective Study. Diabetes Care. 2003;26:1456–1461. doi: 10.2337/diacare.26.5.1456. [DOI] [PubMed] [Google Scholar]
- 12.Haffner SM, Klein R, Dunn JF, Moss SE, Klein BE. Increased testosterone in type I diabetic subjects with severe retinopathy. Ophthalmology. 1990;97:1270–1274. doi: 10.1016/s0161-6420(90)32428-4. [DOI] [PubMed] [Google Scholar]
- 13.Stellato RK, Feldman HA, Hamdy O, Horton ES, McKinlay JB. Testosterone, sex hormone-binding globulin, and the development of type 2 diabetes in middle-aged men: prospective results from the Massachusetts male aging study. Diabetes Care. 2000;23:490–494. doi: 10.2337/diacare.23.4.490. [DOI] [PubMed] [Google Scholar]
- 14.Fukui M, Soh J, Tanaka M, Kitagawa Y, Hasegawa G, Yoshikawa T, Miki T, Nakamura N. Low serum testosterone concentration in middle-aged men with type 2 diabetes. Endocr J. 2007;54:871–877. doi: 10.1507/endocrj.k07-077. [DOI] [PubMed] [Google Scholar]
- 15.Barrett-Connor E. Lower endogenous androgen levels and dyslipidemia in men with non-insulin-dependent diabetes mellitus. Ann Intern Med. 1992;117:807–811. doi: 10.7326/0003-4819-117-10-807. [DOI] [PubMed] [Google Scholar]
- 16.Thorn LM, Forsblom C, Fagerudd J, Thomas MC, Pettersson-Fernholm K, Saraheimo M, Waden J, Ronnback M, Rosengard-Barlund M, Bjorkesten CG, et al. Metabolic syndrome in type 1 diabetes: association with diabetic nephropathy and glycemic control (the FinnDiane study) Diabetes Care. 2005;28:2019–2024. doi: 10.2337/diacare.28.8.2019. [DOI] [PubMed] [Google Scholar]
- 17.Pettersson-Fernholm K, Karvonen MK, Kallio J, Forsblom CM, Koulu M, Pesonen U, Fagerudd JA, Groop PH. Leucine 7 to proline 7 polymorphism in the preproneuropeptide Y is associated with proteinuria, coronary heart disease, and glycemic control in type 1 diabetic patients. Diabetes Care. 2004;27:503–509. doi: 10.2337/diacare.27.2.503. [DOI] [PubMed] [Google Scholar]
- 18.Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab. 1999;84:3666–3672. doi: 10.1210/jcem.84.10.6079. [DOI] [PubMed] [Google Scholar]
- 19.Rinaldi S, Geay A, Dechaud H, Biessy C, Zeleniuch-Jacquotte A, Akhmedkhanov A, Shore RE, Riboli E, Toniolo P, Kaaks R. Validity of free testosterone and free estradiol determinations in serum samples from postmenopausal women by theoretical calculations. Cancer Epidemiol Biomarkers Prev. 2002;11:1065–1071. [PubMed] [Google Scholar]
- 20.Tomar R, Dhindsa S, Chaudhuri A, Mohanty P, Garg R, Dandona P. Contrasting testosterone concentrations in type 1 and type 2 diabetes. Diabetes Care. 2006;29:1120–1122. doi: 10.2337/diacare.2951120. [DOI] [PubMed] [Google Scholar]
- 21.Vermeulen A, Kaufman JM, Deslypere JP, Thomas G. Attenuated luteinizing hormone (LH) pulse amplitude but normal LH pulse frequency, and its relation to plasma androgens in hypogonadism of obese men. J Clin Endocrinol Metab. 1993;76:1140–1146. doi: 10.1210/jcem.76.5.8496304. [DOI] [PubMed] [Google Scholar]
- 22.Strain GW, Zumoff B, Kream J, Strain JJ, Deucher R, Rosenfeld RS, Levin J, Fukushima DK. Mild Hypogonadotropic hypogonadism in obese men. Metabolism. 1982;31:871–875. doi: 10.1016/0026-0495(82)90175-5. [DOI] [PubMed] [Google Scholar]
- 23.Kalyani RR, Dobs AS. Androgen deficiency, diabetes, and the metabolic syndrome in men. Curr Opin Endocrinol Diabetes Obes. 2007;14:226–234. doi: 10.1097/MED.0b013e32814db856. [DOI] [PubMed] [Google Scholar]
- 24.Chubb SA, Hyde Z, Almeida OP, Flicker L, Norman PE, Jamrozik K, Hankey GJ, Yeap BB. Lower sex hormone-binding globulin is more strongly associated with metabolic syndrome than lower total testosterone in older men: the Health in Men Study. Eur J Endocrinol. 2008;158:785–792. doi: 10.1530/EJE-07-0893. [DOI] [PubMed] [Google Scholar]
- 25.Sharpe RM. The roles of oestrogen in the male. Trends Endocrinol Metab. 1998;9:371–377. doi: 10.1016/s1043-2760(98)00089-7. [DOI] [PubMed] [Google Scholar]
- 26.Maffei L, Murata Y, Rochira V, Tubert G, Aranda C, Vazquez M, Clyne CD, Davis S, Simpson ER, Carani C. Dysmetabolic syndrome in a man with a novel mutation of the aromatase gene: effects of testosterone, alendronate, and estradiol treatment. J Clin Endocrinol Metab. 2004;89:61–70. doi: 10.1210/jc.2003-030313. [DOI] [PubMed] [Google Scholar]
- 27.Danielson KK, Drum ML, Lipton RB. Sex hormone-binding globulin and testosterone in individuals with childhood diabetes. Diabetes Care. 2008;31:1207–1213. doi: 10.2337/dc07-2169. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.van Dam EW, Dekker JM, Lentjes EG, Romijn FP, Smulders YM, Post WJ, Romijn JA, Krans HM. Steroids in adult men with type 1 diabetes: a tendency to hypogonadism. Diabetes Care. 2003;26:1812–1818. doi: 10.2337/diacare.26.6.1812. [DOI] [PubMed] [Google Scholar]
- 29.Yki-Jarvinen H, Makimattila S, Utriainen T, Rutanen EM. Portal insulin concentrations rather than insulin sensitivity regulate serum sex hormone-binding globulin and insulin-like growth factor binding protein 1 in vivo. J Clin Endocrinol Metab. 1995;80:3227–3232. doi: 10.1210/jcem.80.11.7593430. [DOI] [PubMed] [Google Scholar]
- 30.Grossmann M, Thomas MC, Panagiotopoulos S, Sharpe K, Macisaac RJ, Clarke S, Zajac JD, Jerums G. Low testosterone levels are common and associated with insulin resistance in men with diabetes. J Clin Endocrinol Metab. 2008;93:1834–1840. doi: 10.1210/jc.2007-2177. [DOI] [PubMed] [Google Scholar]
- 31.Haffner SM, Valdez RA, Mykkanen L, Stern MP, Katz MS. Decreased testosterone and dehydroepiandrosterone sulfate concentrations are associated with increased insulin and glucose concentrations in nondiabetic men. Metabolism. 1994;43:599–603. doi: 10.1016/0026-0495(94)90202-x. [DOI] [PubMed] [Google Scholar]
- 32.Laaksonen DE, Niskanen L, Punnonen K, Nyyssonen K, Tuomainen TP, Valkonen VP, Salonen R, Salonen JT. Testosterone and sex hormone-binding globulin predict the metabolic syndrome and diabetes in middle-aged men. Diabetes Care. 2004;27:1036–1041. doi: 10.2337/diacare.27.5.1036. [DOI] [PubMed] [Google Scholar]
- 33.Pugeat M, Crave JC, Elmidani M, Nicolas MH, Garoscio-Cholet M, Lejeune H, Dechaud H, Tourniaire J. Pathophysiology of sex hormone binding globulin (SHBG): relation to insulin. J Steroid Biochem Mol Biol. 1991;40:841–849. doi: 10.1016/0960-0760(91)90310-2. [DOI] [PubMed] [Google Scholar]
- 34.Rosner W, Auchus RJ, Azziz R, Sluss PM, Raff H. Position statement: Utility, limitations, and pitfalls in measuring testosterone: an Endocrine Society position statement. J Clin Endocrinol Metab. 2007;92:405–413. doi: 10.1210/jc.2006-1864. [DOI] [PubMed] [Google Scholar]