1 Steno Diabetes Center, Gentofte, Denmark
2 Medical Department M (Immunoendocrine Research Unit) and Medical Research Laboratories, Aarhus University Hospital, Aarhus, Denmark
3 Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark
4 Department of Clinical Immunology, Aalborg Hospital, Aalborg, Denmark
5 Faculty of Health Science, University of Aarhus, Aarhus, Denmark
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ABSTRACT |
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A substantial portion of type 1 diabetic patients develop diabetic nephropathy, whereas others seem to be protected from this complication. Persistent microalbuminuria (urinary albumin excretion rate [UAER] of 30300 mg/24 h) is an established risk factor for the development of overt diabetic nephropathy, characterized by albuminuria >300 mg/24 h (1). Microalbuminuria may be regarded as an early marker of diabetic kidney disease, as early renal structural lesions can be detected at this stage (2). Genetic susceptibility, metabolic abnormalities, hemodynamic changes, upregulated growth factors, and cytokines may all play a part in the development of diabetic glomerulopathy, although the complex pathogenesis of diabetic nephropathy is not fully understood (1). It has been suggested that inflammation and complement activation are involved in the pathogenesis of diabetic microvascular complications (36). Mannose-binding lectin (MBL; also known as mannan-binding lectin) can activate the complement system independent of antibodies via MBL-associated serine proteases, thereby initiating the so-called MBL pathway of complement activation (7). Through this pathway, MBL plays an important role in the innate immune system. The level of MBL varies considerably among individuals, mainly because of frequently occurring polymorphisms within exon 1 as well as in the promoter region of the MBL2 gene on chromosome 10 (8). High levels of MBL are known to protect against invading microorganisms (9,10) and in other situations may mediate detrimental inflammation through exaggerated complement activation (11,12). In a recent cross-sectional study, it was demonstrated that in normoalbuminuric type 1 diabetic patients, circulating MBL levels correlate positively with the UAER (4). Higher levels of MBL have been found to be associated with micro- and macrovascular complications in type 1 diabetic patients (5). However, no prospective studies have evaluated the association between MBL and subsequent development of microvascular complications. In the present study, MBL was measured 3 years after diabetes onset in a large inception cohort of newly diagnosed type 1 diabetic patients. These patients were followed for a median of 18 years to evaluate in a prospective design the association between baseline MBL concentrations and the development of persistent microalbuminuria. The change in MBL levels during 15 years of diabetes was also assessed.
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RESEARCH DESIGN AND METHODS |
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All patients attended the outpatient clinic of the Steno Diabetes Center every 34 months as part of their routine follow-up. Patients were treated by diabetologists and nurses according to set principles and guidelines, as previously described (13,14). No specific intervention was carried out. From 1 January 1980, HbA1c was measured at each visit (14). The method used for measuring HbA1c from venous blood samples has changed over the years; the predominate method has been high-performance liquid chromatography ion-exchange using a normal range of 4.16.4% (13). The patients had their 24-h UAER measured at least once a year. Until 1984, UAER was quantitated using automated immunotopical nephelometric analysis (15); from 1984 to 1990, using radioimmunoassay (sensitivity 0.5 mg/l, coefficient of variation [CV] 9%) (16); and from 1990, using enzyme immunoassay (sensitivity 1.1 mg/l, CV 8%) (17). A very close correlation between radial immunodiffusion and radioimmunoassay (r = 0.98) (18) and radioimmunoassay and enzyme immunoassay (r = 0.99) (17) was documented before changing the methods. From 1997, the DAKO Turbidimetric method was used to measure UAER. This method is closely correlated with enzyme immunoassay (r = 0.99) and has a CV of 5%. Persistent micro- and macroalbuminuria were defined as a UAER of 30300 mg/24 h and >300 mg/24 h in at least two of three consecutive samples, respectively, with at least a 30% increase above baseline (13).
Serum MBL concentrations were measured using an in-house, time-resolved immunofluorometric assay with a lower detection level of 10 µg/l (19). In brief, microtiter wells were coated with mannan then incubated with samples diluted 200-fold. After the samples were washed, monoclonal anti-MBL antibody (1311; Immunolex, Copenhagen, Denmark) labeled with europium using reagents from Wallac (Turku, Finland) was added. After samples were incubated and washed, the amount of bound, labeled antibody was assessed by time-resolved fluorometry (Delfia; Wallac). A number of control serum samples covering different MBL levels were included in all assays. The CVs obtained were 10% for a sample of 3,004 mg/l, 7.5% for a sample of 1,330 mg/l, 4.9% for a sample of 238 mg/l, and 23% for a sample of 66 mg/l. In healthy subjects, the median day-to-day variability in serum MBL concentrations expressed as the CV was 5.6% (20). MBL was measured twice: initially 3 years after diabetes onset in all subjects (i.e., after initial glycemic stabilization) and before the development of persistent microalbuminuria. In patients progressing to persistent microalbuminuria, MBL was measured again 1 year after the development of the disorder. In persistent normoalbuminuric patients, MBL was measured 15 years after diabetes onset.
Arterial blood pressure was measured at least once a year with a standard mercury sphygmomanometer and an appropriate cuff size. The measurements were performed with the patient in the sitting position after 10 min rest. Smoking history was elicited via a questionnaire, and patients were classified as smokers if they smoked more than one cigarette per day. Retinopathy was assessed through dilated pupils and graded as absent, simplex, or proliferative (14).
The local ethics committee (Copenhagen, Denmark) approved the experimental design, and all patients gave written informed consent to participate in this study.
MBL genotyping.
Of the 270 patients, 216 gave informed consent for genetic risk factors involved in the development of micro- and macroalbuminuria to be evaluated. As previously described, a real-time PCR technique was used to genotype for polymorphisms in the human MBL (MBL2) gene (5). These polymorphisms comprised two variations in the 5' regulatory region at positions 550 (H/L) and 221 (X/Y), one in the 5' untranslated sequence at position +4 (P/Q), and three structural mutations within exon 1 at codons 52, 54, and 57, also known as the D, B, and C variants, respectively. The presence of one of the three structural mutations within exon 1 (designated "O") significantly reduces circulating MBL. Of the promoter polymorphisms, only the X/Y polymorphism influences serum MBL, with the presence of the X promoter variant causing MBL levels similar to that found in individuals with the structural gene variants (5). Genotypes were divided into low (YO/YO, XA/YO, YA/YO, and XA/XA) and high (YA/YA and XA/YA) MBL genotypes.
Statistical analysis.
MBL, UAER, and serum creatinine concentrations had a positively skewed distribution, and consequently values are given as medians with interquartile ranges. All other values are given as means ± SD. Comparisons between groups were performed using the Mann-Whitney U test for nonnormally distributed variables and the unpaired Students t test for normally distributed variables. A 2 test was used for comparing groups of noncontinuous variables. The cumulative incidence of persistent microalbuminuria was calculated based on the entire follow-up period ending in 2000, with a life-table method taking into account differences in the follow-up intervals; this method makes proper allowances for those censored observations and uses information from all subjects during follow-up to the time to event or censoring. Groups were compared using the log-rank test. The Cox proportional hazards regression model was used to evaluate the relative contributions of covariates to the risk of developing persistent microalbuminuria, correcting for different lengths of follow-up. To evaluate MBL as a causal determinant of the development of persistent microalbuminuria, a Cox regression model was used, including variables that either previously had been shown to be associated with the level of MBL or were correlated with MBL in the present study. Statistical significance was assumed for P < 0.05. All statistical calculations were performed with SPSS for Windows (version 12.0; SPSS, Chicago, IL).
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RESULTS |
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In a Cox proportional hazards model with sex and age as fixed covariates, serum MBL measured 3 years after the onset of diabetes was significantly associated with later development of persistent micro- or macroalbuminuria (hazard ratio per 1,000 µg/l increase in serum MBL: 1.21 [CI 1.071.37]; P = 0.003). After adjusting for the confounding effects of smoking status, HbA1c, blood pressure, and serum creatinine, MBL was independently associated with the development of micro- or macroalbuminuria (hazard ratio 1.21 [CI 1.02 1.42] per 1,000 µg/l increase in serum MBL; P = 0.03). To compare the impact of MBL on the development of microalbuminuria, the association of mean arterial blood pressure and UAER with the development of microalbuminuria was 1.39 (CI 1.121.71) per 10- mmHg increase in blood pressure and 3.88 (CI 1.529.92) per 10-fold increase in UAER, respectively.
MBL was measured twice in 203 of the 270 type 1 diabetic patients. In 62 of the 75 patients whose disorder progressed, MBL was measured 3 years after diabetes onset and within 1 year after the development of persistent microalbuminuria. In these patients, MBL was measured after a mean follow-up time of 13.0 ± 4.7 years. In 141 of the 195 patients who did not progress to persistent microalbuminuria, MBL was measured 3 and 15 years after diabetes onset. In the second measurement of MBL, the median level of MBL was 1,131 (3412,253) vs. 1,626 (7852,532) µg/l in those who did not versus those who did progress to persistent microalbuminuria (P = 0.04). Among those who did develop microalbuminuria, the median change in MBL from baseline to 1 year after the development of microalbuminuria was 58 µg/l (609 to 116) and among those who did not develop the disorder, the change in MBL from baseline to 15 years after onset of diabetes was 81 µg/l (397 to 58) (NS). Age at diabetes onset was the only measured variable associated with a slight decrease in MBL over time (r = 0.23, P = 0.001).
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DISCUSSION |
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MBL is a pattern-recognition molecule with the ability to recognize glycosylated surfaces. When it recognizes specific carbohydrate structures, MBL initiates the complement system via activation of associated serine proteases. A large variability in serum concentrations of MBL, which to a large extent are determined by mutations in the promoter region and within exon 1 of the MBL2 gene on chromosome 10, has been found (8). There is growing evidence for a link between complement activation and the development and progression of renal disease (5,6,22,23), and it could be hypothesized that high levels of MBL may contribute to the development of diabetic microvascular complications through the MBL pathway of complement activation.
An association between MBL genotype, actual MBL levels, and diabetic nephropathy in type 1 diabetic patients has recently been reported (5). Because we genotyped only a subset of our patients and found a distribution of high and low genotypes similar to that previously reported (5,21), we divided our cohort into two groups according to the median level of serum MBL in the total cohort to minimize survival and selection bias. The median level of serum MBL coincided with the division between high and low MBL genotypes (5) (Fig. 2). Therefore, we could estimate the differences in the cumulative incidence of persistent micro- and macroalbuminuria between high and low MBL genotypes. Accordingly, our results support previous findings indicating that patients with high MBL genotype and therefore higher circulating MBL levels have a substantially higher risk of developing diabetic kidney disease (5). Using data from only patients who were genotyped reduced the number of patients in this subanalysis, making the confidence intervals wider.
Whether differences in circulating MBL levels are a contributing cause or a consequence of the development of microvascular complications due to diabetes cannot be established from cross-sectional studies. In the present prospective study, we found MBL levels to be elevated even before the development of microalbuminuria, and the association of MBL to the development of microalbuminuria persisted after adjusting for known confounders. Our results suggest that MBL may play a causal role in the development of microvascular complications in diabetes. Furthermore, the longitudinal analysis of MBL in our study showed a small decrease in MBL with increasing age and no difference between patients who progressed to microalbuminuria and patients with persistently normal UAE. This implies that rather than being a consequence of microvascular complications, MBL is more likely to be a contributing factor to the development of diabetic microangiopathy. However, whether MBL is a risk factor and thereby actually involved in the pathogenesis of diabetic nephropathy or merely a risk marker associated with other factors of importance for disease progression is not yet established.
In type 1 diabetes, C-reactive protein (CRP) levels have been found to be elevated and correlate with markers of endothelial dysfunction, suggesting that diabetic patients are in a state of chronic, low-grade inflammation (24). We did not measure CRP in the present study. MBL is an acute-phase reactant, although it reacts much slower than CRP. Previous studies in type 1 diabetic patients have found no correlation between MBL and CRP (5,25) or between MBL and the proinflammatory cytokine interleukin-6 (25). Furthermore, adjustments for differences in CRP levels have had no impact on the association between MBL and microvascular complications (5). Even though CRP and MBL are closely interrelated in inflammation and CRP may inhibit MBL activity (26), the evaluation of these markers of inflammation gives rise to different information in the diabetic patients. The association of blood pressure, endothelial damage, and serum MBL remains to be evaluated.
Oxidative stress leading to changes in cell surface glycosylations may activate the complement system via MBL (11). Assuming that ligands to MBL exist in renal tissue, this can in turn lead to renal MBL deposition, with deleterious effects of MBL causing local inflammation and fibrosis within the kidneys. The presence of MBL depositions in kidneys of diabetic patients has not yet been established; we evaluated only circulating MBL levels in our study as kidney biopsies were not available.
Diabetic kidney disease is strongly associated with cardiovascular mortality (27), and the Steno hypothesis, as proposed by Deckert et al. (28), advocates albuminuria to reflect a more generalized vascular process that involves the glomeruli in the kidneys, retina, and intima of large vessels simultaneously. MBL has been demonstrated to be elevated not only in patients with diabetic nephropathy, but also in diabetic patients with a history of cardiovascular disease irrespective of renal involvement (5). Emerging data indicate that complement activation via MBL may aggravate the effects of myocardial ischemia (29); furthermore, downstream inhibition of the complement system with a C5 inhibitor was recently shown to significantly reduce mortality after percutaneous coronary intervention in patients with myocardial infarction (30). The upregulation of MBL and activation of the complement system in diabetic patients may therefore be a candidate for a common link between micro- and macrovascular disease in these patients.
In conclusion, we have demonstrated for the first time that high levels of MBL early in the course of type 1 diabetes and before the development of microvascular complications is significantly associated with the later development of persistent micro- or macroalbuminuria. This suggests that MBL may be involved in the pathogenesis of microvascular complications in diabetes. Future research must assess whether intervention targeting the complement system in high-risk patients can alter the risk for the development of diabetic micro- and macroangiopathy.
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ACKNOWLEDGMENTS |
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We thank and acknowledge the expert technical assistance of Inga Bisgaard, Berit Ruud Jensen, Birgitte V. Hansen, Ulla M. Smidt, Tina R. Juhl, Lotte Pietraszek, and Inge-Lise Rossing. Christian Binder is especially acknowledged for the design and inclusion of patients in the inception cohort.
Address correspondencereprint requests to Peter Hovind, MD, Steno Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark. E-mail: phovind{at}dadlnet.dk
Received for publication December 16, 2004 and accepted in revised form February 7, 2005
CRP, C-reactive protein; MBL, mannose-binding lectin; UAER, urinary albumin excretion rate
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REFERENCES |
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