Introduction
Microalbuminuria is an independent predictor of cardiovascular mortality and cardiovascular diseases such as cardiac, cerebrovascular and peripheral arterial disease in patients with diabetes or hypertension, and in the general population. Determination of microalbuminuria is now recommended as a risk stratification strategy in diabetic and hypertensive patients. In this review, methods for the measurement of microalbuminuria and reporting of urinary albumin excretion are described and a question of their standardization is raised. As the association between microalbuminuria, endothelial dysfunction and cardiovascular diseases in the presence of diabetes or hypertension has not yet been fully explained, the authors discuss the recently published studies related to the topic.
Definition and measurement of microalbuminuria
Definition of microalbuminuria
The term “microalbuminuria” first appeared in the literature in 1981 to describe the presence of albumin in the urine below the detection limit of a standard test strip, but at a highly predictive level for future proteinuria in patients with diabetes mellitus (1). According to classic definition, “normal” urinary albumin excretion (UAE) may be defined as:
UAE < 30 mg/24 h in 24-hour collection;
urinary albumin excretion rate < 20 µg/min in timed urine sample;
urinary albumin-to-creatinine ratio (ACR) < 30 mg/g or < 3.4 mg/mmol in women and < 20 mg/g or < 2.5 mg/mmol in men; and
urinary albumin concentration < 20 mg/L in spot urine (2,3).
Recently, de Jong and Curhan have recommended the term “high normal” to be used for the values defined above (4). The diagnostic threshold for normal albuminuria has been decreased to
UAE < 15 mg/24 h;
ACR < 10 mg/g or < 1.25 mg/mmol in men and ACR < 15 mg/g or < 1.75 mg/mmol in women;
and < 10 mg/L of urinary albumin (4-6).
The values defining microalbuminuria are:
UAE 30 to 300 mg/24 h;
20 to 200 µg/min;
ACR 20 to 200 mg/g or 2.5 to 25 mg/mmol in men and 30 to 300 mg/g or 3.5 to 35 mg/mmol in women; and
20 to 200 mg/L of urinary albumin.
Higher UAE levels are classified as macroalbuminuria (5). Klausen et al. examined 2,762 subjects without a history of coronary heart disease and report that microalbuminuria was a strong determinant of coronary heart disease and death (7). In the general population, the risk of cardiovascular diseases rises at albuminuria level below the threshold for microalbuminuria. A continuous relationship exists between the level of urinary albumin concentration and the cardiovascular risk (1). An overnight UAE above only 5 µg/min was strongly predictive of coronary heart disease and death in the general population (7).
Data from several large studies suggest that UAE over 2 mg/day (about 4 µg/min) was associated significantly with cardiovascular events even in subjects without diabetes (1). Assuming the normal range of UAE as 20 µg/min, the risk of cardiovascular disease or death increased by 70% or 50% in hypertensive patients with albuminuria as low as 5–10 µg/min (8).
The presence of microalbuminuria defined as excretion above 5 µg/min in subjects with cardiovascular or cerebrovascular disease increases the risk of death by 100% (9). According to this study, the definition of microalbuminuria as UAE above 5 µg/min should be accepted.
Measurement of microalbuminuria
Testing for microalbuminuria, generally once a year, is recommended for patients with diabetes and hypertension according to the evidence-based guidelines. In patients with hypertension, albuminuria should be assessed every 6 months within the first year of treatment to monitor the impact of antihypertensive therapy (10). However, the US data from 2004 have disclosed that diabetic patients are mainly tested for their lipid profile (91%) and HbA1c levels (86%), and only about 50% for microalbuminuria (11). In 2006, the American Heart Association published recommendations for testing to detect kidney disease, that includes estimated glomerular filtration rate (GFR) using the Modification of Diet in Renal Disease (MDRD) equation and a test for microalbuminuria for detection of chronic kidney disease (12). These recommendations are important developments because they link chronic kidney disease and cardiovascular diseases.
Several methods for urinary albumin measurement have been applied including immunologic detection (with the use of test strips, immunonephelometry, immunoturbidimetry, radioimmunoassay, ELISA, chemiluminescence immunoassay, fluorescence immunoassay), HPLC and spectrophotometry. The most frequently used are immunologic methods, which are sensitive but enable assessment of intact immunoreactive albumin molecules only (13). Albumin fragments are detected by spectrophotometry and non-immunoreactive albumin molecules by high-performance liquid chromatography (HPLC) (14). The clinical significance of non-immunoreactive albumin is not yet understood and remains to be explained. Recently, the resonance scattering spectral assay (RSSA) for microalbumin measurement, based on the immunoreaction and its resonance scattering effect, has been reported as being simple, rapid and sensitive (15). It should be noted that neither HPLC technique nor immunoresonance scatter spectral assay for assessment of urinary albumin can be widely used because of their high cost and technology inaccessibility.
There are few methods of urine sampling: complete 24-hour urine sample or timed urine sample (4 hours or overnight) used mostly in clinical settings, and random sample – spot urine collection used mainly in primary care (www.labstestonline.org). The validity of screening by albumin measurements in spot morning urine sample has been investigated not only to identify subjects with microalbuminuria in the general population but also in patients with hypertension (16,17). Diagnostic performance of albumin concentration measurement in spot urine sample was found to be comparable to that of the albumin-to-creatinine ratio. Moreover, the measurement of the albumin-to-creatinine ratio requires an additional determination of creatinine and the use of gender-specific cut-off values (17). Measuring albumin concentration in the spot morning urine sample has therefore been proposed as a screening method that is more convenient in daily clinical practice.
One should also be aware of the variability of UAE, which is an important limitation of the measurement accuracy. There are several factors and conditions that may temporary rise UAE, such as fever, exercise, heart failure, urinary tract infection and high-protein diet (13,18). UAE decreases at night and varies between days. Because the variability depends on the time of urine collection, the reproducibility of UAE is better for samples collected during the nighttime and for the first morning samples. The individual variability and circadian variability also depend on the type of the disease, being lower in patients with hypertension than in diabetics (13). The reproducibility of albumin measurement can be increased if the measurement is repeated three times in the urine samples collected in the same fashion and if preanalytical errors are excluded.
In the year 2005, the International Federation of Clinical Chemistry founded the Working Group for the Standardization of Microalbumin Assay in Urine, with the aim to establish a reference procedure and reference materials. The standardization has not yet been completed, however, projects on chemical and immunochemical characterization of the various forms of albumin in urine (definition of the analyte) and on the decision on the optimum analyte for the assessment of (micro)albuminuria have just been under way (19).
The standardization for reporting UAE or urine albumin-to-creatinine ratio is also of primary importance as well as indicating the three thresholds for albuminuria on the lab report. The uniform reporting format in the same units would allow for comparability of the results across different countries and avoidance of misinterpretation of the results.
Prevalence of microalbuminuria
Microalbuminuria represents an established risk factor for cardiovascular morbidity and mortality and for end-stage renal disease in individuals with an adverse cardiovascular risk profile. It is common in the general population, particularly in patients with diabetes mellitus or hypertension. Data from large population-based studies in the United States, Europe and Australia show that the prevalence of microalbuminuria is 5%–15% in the general population, 20%–30% in diabetics and 11%–17% in patients with hypertension (20–23).
In earlier studies, an association was found between albuminuria/proteinuria and cardiovascular mortality in patients without diabetes (24,25). Report from the Prevention and Vascular End-Stage Disease Study shows that in 7% out of 40,856 examined subjects, microalbuminuria was associated independently with previous myocardial infarction and stroke. It was concluded that UAE was a predictor of all-cause mortality in the general population (26,27).
Endothelial dysfunction and cardiovascular risk in patients with diabetes
Pathogenesis of endothelial dysfunction
Microalbuminuria can be a cause or a consequence of vascular disease. Endothelial dysfunction could contribute to the pathogenesis of albuminuria directly, by causing increased glomerular pressure and the synthesis of glomerular basement membrane of improper structure leading to transvascular albumin leakage. On the other hand, endothelial dysfunction, in a paracrine fashion, could influence glomerular mesangial and epithelial cell function (28). The association between microalbuminuria and cardiovascular diseases is not clear, however, the endothelial function and chronic inflammation have been suggested as the possible factors to explain the underlying mechanism (29). It is known that low-grade inflammation may be a cause and a consequence of endothelial dysfunction and can be related to the occurrence and progression of microalbuminuria and a higher cardiovascular risk (30). In spite of the existing link between endothelial dysfunction, low-grade inflammation and microalbuminuria, they are independent risk factors for cardiovascular death (31).
The association of microalbuminuria with the increased synthesis of vascular endothelial growth factor and with C-reactive protein, an acute phase protein and a marker of chronic inflammation, has been described showing that multiple mechanisms are involved in the development and progression of cardiovascular complications in diabetic patients with albuminuria (32). Endothelial dysfunction in both types of diabetes complicated by micro- or macroalbuminuria is generalized and affects many aspects of endothelial function. In both types of diabetes, microalbuminuria is accompanied by a variety of markers of endothelial dysfunction. A significant and independent correlation between microalbuminuria and increased levels of plasma von Willebrand factor (vWF), endothelin, thrombomodulin, tissue plasminogen activator (t-PA), inhibitor of plasminogen activator 1 (PAI-1), soluble adhesion molecules and soluble E-selectin was found in type 2 diabetics (6,29,33).
Latest findings show that microalbuminuria and coronary vasomotor abnormalities are both predictors for cardiac events in type 2 diabetics. These patients had a more severely impaired coronary endothelium-dependent vasodilation in the presence of microalbuminuria (34). Cao et al. showed that in the absence of hypertension or diabetes, microalbuminuria was associated with clinical cardiovascular disease but not with subclinical atherosclerosis. They postulated that the mechanism of association of microalbuminuria with clinical vascular disease involved destabilization of the vasculature, leading to clinical disease (35).
The link between microalbuminuria and cardiovascular events is only partly explained by age, gender, diabetes, hypertension, obesity, dyslipidemia and smoking. Weir suggests that it may result from inadequate measures of endothelial function and inflammation using biochemical estimates (5).
Another explanation has been proposed by de Zeeuw, relating the individual susceptibility to organ damage to inherent variability of the vascular state as determined by albumin excretion (36). According to this hypothesis, microalbuminuria may be a predictor of cardiovascular diseases as well as of new-onset hypertension and diabetes (5). Whatever the mechanism, microalbuminuria identifies an early stage with an increased risk of developing renal and cardiovascular complications in cases with diabetes, hypertension and dyslipidemia.
Hyperhomocysteinemia and endothelial dysfunction in patients with microalbuminuria
Elevated homocysteine was found to be associated with microalbuminuria and retinopathy in both types of diabetes. Hyperhomocysteinemia seems to be one of the main causes of increased mortality in type 2 diabetic patients. Increased homocysteine correlated directly and significantly with the level of microalbuminuria. These results suggest that hyperhomocysteinemia in patients with type 2 diabetes may play a role in the development of vascular complications (37,38).
The underlying mechanism linking homocysteine with endothelial dysfunction in diabetic patients with microalbuminuria is not clear. Whether hyperhomocysteinemia-mediated oxidative stress leading to impaired endothelial function is related to increased cardiovascular risk in patients with diabetes and microalbuminuria remains to be established. A recent study in patients with type 1 diabetes and microalbuminuria has shown the presence of mild hyperhomocysteinemia and reduced antioxidant defense in these patients as compared with normoalbuminuric patients and nondiabetic subjects (39).
Microalbuminuria and the metabolic syndrome
Both microalbuminuria and metabolic syndrome have been associated to cardiovascular diseases. Recently, strong relationship between microalbuminuria, defined as urinary albumin-to-creatinine ratio, and metabolic syndrome was demonstrated in a large population-based study (40). Microalbuminuria was shown to be linked to particular components of the metabolic syndrome such as hyperglycemia and insulin resistance, which are significant predictors of endothelial dysfunction (6).
Obesity is an important component of metabolic syndrome. Excessive fat accumulation and changes in the synthesis and secretion of adipokines may be the causative factors contributing to the development of type 2 diabetes, hypertension and cardiovascular diseases (41). Adiponectin synthesized by fat cells has anti-inflammatory and antidiabetic effects but is decreased in obesity and diabetes with insulin resistance (42). The diminished adiponectin secretion in obesity may contribute to inflammatory response and endothelial dysfunction leading to atherosclerotic changes in the vessels (43). In patients with hypertension, microalbuminuria negatively correlated with adiponectin levels, reflecting progressing atherosclerosis (44). In spite of the increasing evidence, the explanation of the role of adipokines in physiology and disease requires much more studies.
Recently, the term “cardiometabolic syndrome” has become popular defining a constellation of insulin resistance/hyperinsulinemia, obesity and dyslipidemia, hypertension and microalbuminuria, low-grade inflammation and oxidative stress (45). Hayden et al. have presented important observational findings regarding proximal tubule microvilli remodeling and oxidative stress, which may help explain microalbuminuria in the cardiometabolic syndrome. It has been suggested that albuminuria is associated with proximal tubule injury and loss of integrity of the glomerular filtration barrier in association with obesity and insulin resistance (46).
Dyslipidemia and microalbuminuria
The relationship between dyslipidemia and microalbuminuria is inconsistent. Atherosclerotic vascular disease is associated with increased endothelial permeability that leads to transvascular albumin leakage and enhanced lipid accumulation in the vessel wall (21). Increased UAE in parallel with dyslipidemia was found in diabetic patients (28). In both types of diabetes, dyslipidemia and low HDL-cholesterol levels may impair endothelial function. It was demonstrated that hypertriglyceridemia and low HDL-cholesterol were associated with microalbuminuria (39,47). Molitch et al. tried to determine whether high levels of HDL-cholesterol accompanied lower prevalence of albuminuria (48). They found that type 1 diabetic patients with higher HDL-cholesterol concentrations were much less likely to have albuminuria. In another study, a significant association was found between albuminuria and duration of diabetes, hypertension and HDL-cholesterol concentration (49). The protective role of higher HDL-cholesterol level against the development of albuminuria in patients with type 1 diabetes still needs explanation (48).
Microalbuminuria and hypertension
Microalbuminuria has long been associated with hypertension (1,34,48,50-52). The prevalence of microalbuminuria in patients with essential hypertension varies from 4% to 46%, which may be explained by differences in age and ethnicity of study populations, intra-individual variability, measurement method and definition used. UAE has been associated with left ventricular diastolic dysfunction and left ventricular hypertrophy in patients with hypertension (53,54).
Glomerular endothelial dysfunction has been postulated as an early feature of essential hypertension that may lead to elevation of blood pressure, and albuminuria reflects systemic dysfunction of vascular endothelium. It has been found that the presence of microalbuminuria allows for identification of individuals most likely to develop hypertension (52). Microalbuminuria may be an indicator of early vascular complications of hypertension, a predictor of cardiovascular diseases in patients with essential hypertension (55). However, it can also predict cardiovascular diseases independently of the degree of blood pressure in hypertensive cases without previous vascular complications (56).
Renin-angiotensin system and albuminuria
Impaired endothelial function and disturbed vascular remodeling may be related to activation of the renin-angiotensin system (RAS) occurring in kidney disease. The action of angiotensin II on the angiotensin II type 1 receptor, which leads to the synthesis and release of inflammatory interleukin 6, increased the generation of reactive oxygen species, induction of receptors for oxidized LDL and induction of adhesion molecules, has an essential role in the development of endothelial damage and atherosclerosis (57). Recently, several series of data have been presented on the effect of drugs influencing RAS. Reduction of arterial blood pressure diminishes albuminuria but RAS-blocking drugs were able to reduce UAE more than could be expected from lowering blood pressure alone (58). It may be suggested that RAS is involved in the pathogenesis of albuminuria and plays an important role as a cardiovascular risk factor. The so-called dual blockade of the RAS, with ACE inhibitor and angiotensin II receptor antagonist, reducing albuminuria is dissociated from the blood pressure lowering effect (10). However, the protective effect of this type of medication needs further explanation (59).
Conclusion
The prospective observation trials provide evidence that low-grade microalbuminuria, well below the current threshold, is associated with an increase in cardiovascular events and all-cause mortality. As sensitive and reliable methods for assessment of UAE are commercially available, the screening for microalbuminuria may be of clinical importance comparable to that of blood pressure control and lipid screening in the preventive strategies.
