Serum Adiponectin Is Increased in Type 1 Diabetic Patients With Nephropathy

RESEARCH DESIGN AND METHODS

A total of 189 type 1 diabetic patients were selected from the ongoing, nationwide, multicenter Finnish Diabetic Nephropathy Study (FinnDiane) and divided into three groups according to their urinary albumin excretion rate (AER) in three consecutive overnight or 24-h urine collections. Normal AER was defined as an AER persistently <20 μg/min or <30 mg/24 h, microalbuminuria as an AER between 20 and 200 μg/min or 30 and 300 mg/24 h, and macroalbuminuria as an AER >200 μg/min or >300 mg/24 h in at least two out of three urine collections.

Type 1 diabetes was defined as an onset of diabetes before the age of 35 years and permanent insulin treatment initiated within 1 year of diagnosis. Patients with normal AER were required to have neither antihypertensive medication nor signs of cardiovascular disease. Patients with micro- and macroalbuminuria were required to receive treatment with ACE inhibitors to be representative of type 1 diabetic patients with micro- or macroalbuminuria in Finland. Thereafter, patients were primarily matched for sex and, secondly, for duration of diabetes. A total of 401 type 1 diabetic patients of the initial study population of 1,616 met these criteria, and from this cohort we randomly selected 189 with a duration of diabetes of at least 10 years. The ethical 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 2000.

Data on medication, cardiovascular status, diabetes complications, hypertension, and cardiovascular disease were obtained using a standardized questionnaire, which was completed by the patient’s attending physician based upon medical files. Blood pressure was measured twice in the sitting position with a mercury sphygmomanometer after at least a 10-min rest. Height and weight were recorded, and blood was drawn for the measurements of HbA_1c, lipids, creatinine, inflammatory markers, and adiponectin.

HbA_1c and lipids were measured by enzymatic methods at the local hospitals. Serum creatinine was determined using a modified Jaffé reaction. We calculated the estimated glucose disposal rate as a surrogate measure of insulin sensitivity with an equation developed by Williams et al. (21) and modified for HbA_1c. In addition to AER, we estimated glomerular filtration rate (GFR) by using the Cockroft-Gault formula (22). CRP was measured by radioimmunoassay and IL-6 by high-sensitivity enzyme immunoassay as previously described (2).

Serum adiponectin was determined by a novel in-house time-resolved immunofluorometric assay based on two monoclonal antibodies and recombinant human adiponectin (R&D Systems, Abingdon, U.K.) as recently described (23). Adiponectin has a molecular weight of ∼30–36 kDa depending on the degree of glycosylation, but the molecule is known to form a wide range of polymers in vivo. The predominant polymers include trimers, hexamers, and highly congregated multimers of ∼300 kDa (24). Both of these antibodies were able to detect several adiponectin polymers in serum, including the three major molecular forms (data not shown). Within and in-between assay coefficients of variation of standards and unknown samples averaged <5 and 10%, respectively.

Statistical analysis

Data are expressed as means ± SD for normally distributed values and median (range) for nonnormally distributed values. Frequencies are given as percentages. Differences among groups for normally distributed variables were tested using ANOVA, and nonparametric data were analyzed using the Kruskal-Wallis and Mann-Whitney U test. Correlations were calculated using the Pearson correlation coefficient. Multiple linear regression analyses with adiponectin, AER, and estimated GFR as the dependent variable were used to assess independent relationships. The distribution of triglycerides, creatinine, AER, CRP, IL-6, and adiponectin were all skewed and therefore logarithmically transformed before analyses. All calculations were performed with a BMDP statistical package (Biomedical Data Processing Statistical Software, Los Angeles, CA). P values <0.05 were considered statistically significant.

RESULTS

The clinical characteristics of the patient population are shown in Table 1. The adiponectin concentrations were higher in women than in men. However, since there was no difference in sex distribution among the groups, data were pooled. Adiponectin concentrations were higher in patients with macroalbuminuria than in patients with microalbuminuria (P < 0.001) or normoalbuminuria (P < 0.0001) (Fig. 1). No difference was observed between patients with normoalbuminuria or microalbuminuria (P = 0.2). Adiponectin was positively associated with creatinine (r = 0.41; P < 0.0001), AER (r = 0.33; P < 0.0001), IL-6 (r = 0.22; P = 0.002), systolic blood pressure (r = 0.21; P = 0.004), HbA_1c (r = 0.17; P = 0.02), total cholesterol (r = 0.16; P = 0.03), and HDL cholesterol (r = 0.16; P = 0.03) and negatively with estimated GFR (r = −0.52; P < 0.0001) and waist-to-hip ratio (WHR, r = −0.16; P = 0.03) in a univariate analysis. No association was observed with age, duration of diabetes, BMI, diastolic blood pressure, estimated glucose disposal rate, triglyceride, or CRP (data not shown). In a multiple linear regression analysis, including all variables that were associated with adiponectin in the univariate analysis, estimated GFR, AER, and WHR were independently associated with adiponectin concentrations (r² = 0.32) (Table 2). On the other hand, in a similar analysis when AER was used as dependent variable, adiponectin, WHR, estimated GFR, and estimated glucose disposal rate were independently associated with AER (r² = 0.46) (Table 3). With estimated GFR as dependent variable, adiponectin, AER, and HbA_1c were independently associated with GFR (r² = 0.26) (Table 4).

CONCLUSIONS

The major new finding in this study is the demonstration of markedly increased serum concentrations of adiponectin in type 1 diabetic patients with overt nephropathy and further that adiponectin is associated with renal insufficiency. On the contrary, there was no independent association between adiponectin and inflammatory markers in this patient population, which is in contrast to the findings in predialytic patients with ESRD (19) but in line with the findings in ESRD patients on dialysis (25). This may be due to the fact that patients with ESRD presumably have more confounding factors, like polypharmacy and uremic toxins, that can have a more profound impact on adiponectin and inflammatory markers than a patient population with mildly impaired kidney function. Notably, a recent study (26) in patients with early stages of chronic nondiabetic kidney disease did not observe any relationship between adiponectin and CRP. Interestingly, we also found an association between adiponectin and WHR, a strong feature of insulin resistance, but not between adiponectin and more direct indices of insulin resistance.

Antihypertensive medication itself has been suggested to have an effect on adiponectin (27). In a small group of patients (n = 16) with essential hypertension, treatment with an ACE inhibitor or an angiotensin II receptor blocker was associated with an increase in adiponectin. In our study, all micro- and macroalbuminuric patients were on an ACE inhibitor, whereas normoalbuminuric patients did not have any antihypertensive medication at all. If the increase in adiponectin would have been solely due to the ACE inhibition, a significant increase in adiponectin concentrations could have been expected in the patients with microalbuminuria as well.

The collagenous domain of the adiponectin molecule has four conserved lysines that can be hydroxylated and glycosylated. Both hydroxylation and glycosylation are suggested to be critical for the three-dimensional structure of the biologically active adiponectin molecule (28). It is likely that glycosylation is one of the major posttranslational modifications of adiponectin (28). In diabetic patients with constant hyperglycemia, the glycosylation process is probably altered, and this could lead to an altered adiponectin function. Consequently, a modified adiponectin molecule could lead to a diminished negative feedback, a mechanism that is an essential part of hormonal systems, and thus to increased adiponectin concentrations in diabetes.

An essential question to be answered is why advanced diabetic nephropathy is associated with increased concentrations of adiponectin. The opposite would be expected, since a typical feature of nephropathy is insulin resistance, which in type 2 diabetic patients has been associated with suppressed/low serum adiponectin concentrations (8). One possibility could be that the renal insufficiency per se further stimulates adiponectin production or alternatively leads to a defect in the clearance of adiponectin. The latter suggestion is supported by the finding that succesful kidney transplantation is followed by decreased plasma adiponectin concentrations (29). Although the true mechanisms responsible for the increase in circulating adiponectin in diabetic nephropathy are still unclear, one can speculate that adiponectin itself may have a role in mitigating the micro- and macrovascular burden in diabetic nephropathy, like it has been suggested to do in ESRD (25). Interestingly, adiponectin has been shown to be associated with endothelium-dependent vasodilatation (30) and, further, to modulate endothelial function by inhibiting the deleterious effects of tumor necrosis factor-α (10).

Proteinuria has been shown to be positively associated with adiponectin in patients with the nephrotic syndrome, although that study could not detect any association between GFR and adiponectin (18). In another study (19) including both type 1 and type 2 diabetic patients with ESRD, there was neither any association between AER and adiponectin nor between GFR and adiponectin. Our results show that not only AER but also estimated GFR is independently associated with increased adiponectin concentrations in type 1 diabetic patients with nephropathy. The question arises as to why our results differ from previous studies. The most plausible explanation is the inclusion of different patient populations as well as the number of type 1 diabetic patients, which was significantly higher in our study than in the previous studies. Notably, in a recent study in type 2 diabetic Pima Indians, adiponectin was associated with elevated serum creatinine and macroalbuminuria (31).

In conclusion, serum adiponectin concentrations are increased in type 1 diabetic patients with nephropathy, and levels are further associated with renal insufficiency. The reason why insulin-resistant conditions like proteinuria and advanced nephropathy are associated with high circulating adiponectin levels is still unclear.