Albuminuria (i.e., urinary albumin, or UA) is defined as an abnormally high amount of albumin in the urine and is a strong sign of early renal damage [
Inflammation and endothelial dysfunction may be key mechanisms in the development of albuminuria [
Absolute neutrophil count (ANC) is also a sensitive marker of inflammation, and the test for it is simple and inexpensive. Recently, clinical studies have shown a link between neutrophils and the development of diabetic retinopathy [
We hypothesized an association between ANC and UA levels. Like the world’s human population in general, China’s is aging and we chose to study the elderly. Here, the UA creatinine ratio (UACR) was used as a measure of albumin excretion. Since overnight UACR correlates well with UA excretion [
This community-based, cross-sectional study focused on the clinical risk factors of albuminuria. We performed the study with outpatients of our hospital in Shanghai, from March to August of 2010. All community-dwelling participants aged ≥60 years, including those residing in private residences and personal care homes, were recruited to a Platform of Chronic Disease (PCD) prevention program for a routine medical checkup. Participants who could not come to the prevention center were interviewed in their place of residence by trained personnel. Those who refused to join the study were not included in this analysis. The participants provided informed consent.
All questionnaires and procedures were approved by the Institutional Review Board in our hospital for research with human subjects. The identification of diabetes was based on the diagnostic criteria recommended by the American Diabetes Association in 1997. Potential subjects included those with a history of past diabetes or who had newly received a diagnosis by oral glucose tolerance test [
In total, 3053 subjects aged between 60 and 91 years (median age: 70 y) were considered for the study. The following subjects were excluded: 28 subjects presenting with painful urination, fever, cough, or skin damage; 5 subjects who were incontinent and not capable of caring for themselves; and 12 subjects with white blood cell (WBC) counts ≥10 × 109/L or serum creatinine (SCr) ≥141
Therefore, 2265 subjects were included in the final analysis.
Blood samples were collected at 8:15 a.m. after a 12-hour fast. The subjects underwent a glucose tolerance test with a 75 g oral glucose overload. Only water was permitted during the test period and no physical exercise was taken. The blood samples were collected by EDTA tube before and at 2 h after the tolerance test. After centrifugation, samples were stored at −80°C for subsequent analysis. Plasma biochemical parameters were measured in duplicate by standard enzymatic methods. Glucose, cholesterol, triglycerides, SCr, and alanine aminotransferase (ALT) levels were measured using a Dimension Vista analyzer (Siemens AG). WBC, ANC, and ANC/WBC ratio values were measured using an automated 5-category hematology analyzer (Japanese Sysmex XS1000i).
Participants were asked to collect a random morning spot urine sample in a clean, lidded container. All urine samples were assessed centrally in a single reference laboratory at Fifth Hospital, Shanghai, China. Samples were centrifuged at 1500 ×g for 10 min and then stored at −80°C prior to analysis. Further analysis was performed with a Beckman Coulter Array 360 chemistry analyzer. Test results were considered positive for albuminuria (UA+) at an albumin-to-creatinine ratio (ACR) >0 mg/g; otherwise the results were accepted as negative (UA−; ACR = 0 mg/g). Microalbuminuria, or a moderate increase in UA, was identified as an ACR from 30 to 299 mg/g. Macroalbuminuria was defined as ACR ≥300 mg/g.
Blood pressure was measured twice at about 8:00 a.m., with the subject seated and with an interval of 5 min between measurements. Blood pressure measurements were taken on the right arm, which was relaxed and supported by a table, at an angle of 45° from the trunk (sphygmomanometer, Yutu, Shanghai, China). Hypertension was defined as systolic blood pressure (SBP) ≥140 mmHg or diastolic blood pressure (DBP) ≥90 mmHg.
The following measurements were recorded for all subjects: height, weight, waist circumference (horizontal position of the anterior superior iliac crest and twelfth rib edge midpoint), and hip measurement (measuring horizontally from the front of the pubic symphysis to the most convex point behind the gluteus maximus) by experienced nurses in the PCD program. Body mass index (BMI) was calculated as weight/height2 (kg/m2).
Mann-Whitney
The least significant difference was used for intergroup comparisons. All subjects were categorized into 5 groups according to the quintile of ANC levels (I–V). Multivariate logistic regression analysis was used to investigate an independent association between ANC and UA. Differences were considered significant at
Receiver operating characteristic (ROC) curves were plotted for factors predicting macroalbuminuria. Areas under the ROC (AUC) curve ≥0.5 were considered statistically significant (SPSS software version 17.0; SPSS, Chicago, IL, USA).
The data analysis included 2265 participants aged between 60 and 91 years (median: 70 y) with a male-to-female ratio of 56.8 to 43.1 (Table
Demographic and clinical data of patients
Diabetes | Albuminuria | |||||
---|---|---|---|---|---|---|
Diabetic | Nondiabetic |
|
UA+ | UA− |
| |
Subjects, |
1254 | 1011 | 641 | 1624 | ||
Mean age, y | 71 (65–76) | 70 (65–75) | 0.021 | 70 (65–75) | 70 (65–75) | 0.0028 |
Gender, male/female | 721/932 | 566/784 | NS | 266/375 | 717/906 | NS |
BMI, kg/m2 | 23.7 (21.4–26.0) | 23.9 (21.6–26.3) | NS | 24.0 (21.8–26.5) | 23.6 (21.2–25.8) | 0.0012 |
|
||||||
Waist-hip ratio | 0.88 (0.84–0.91) | 0.88 (0.84–0.91) | NS | 0.88 (0.84–0.91) | 0.87 (0.84–0.90) | 0.0002 |
SBP, mmHg | 132 (127–147) | 130 (125–142) | 0.0066 | 133 (128–148) | 130 (124–140) | <0.0001 |
DBP, mmHg | 80 (75–83) | 80 (76–83) | NS | 80 (76–84) | 80 (74–81) | 0.0019 |
Hypertension | 774 (1254) | 568 (1011) | 0.0045 | 384 (641) | 924 (1624) | NS |
|
||||||
ACE inhibitor | 73 (1254) | 39 (1011) | 0.0007 | 31 (641) | 60 (1624) | NS |
ARB | 197 (1254) | 128 (1011) | 0.0218 | 91 (641) | 222 (1624) | NS |
Total cholesterol, mmol/L | 5.8 (5.0–6.5) | 5.8 (5.1–6.5) | NS | 5.8 (5.0–6.5) | 5.8 (5.1–6.5) | NS |
Triglycerides, mmol/L | 1.8 (1.3–2.4) | 1.8 (1.3–2.4) | NS | 1.8 (1.3–2.4) | 1.7 (1.2–2.3) | NS |
|
||||||
FBG, mmol/L | 5.7 (5.3–7.4) | 5.8 (5.5–6.2) | <0.0001 | 5.9 (5.4–6.6) | 5.8 (5.4–6.4) | NS |
Two-hour glucose, mmol/L | 8.4 (6.8–12.9) | 8.5 (7.9–9.3) | 0.0032 | 8.5 (7.2–10.7) | 8.4 (7.1–10.3) | NS |
SCr, |
69 (58–90) | 70 (60–81) | NS | 70 (59–2) | 68.5 (60–78) | 0.0038 |
ALT, U/L | 21 (16–27) | 21 (17–28) | NS | 21 (16–28) | 21 (16–27) | NS |
|
||||||
WBC, 109/L | 6.0 (5.1–7.0) | 5.9 (5.1–6.8) | 0.0138 | 6.0 (5.1–7.0) | 5.8 (5.0–6.8) | 0.0131 |
ANC, 109/L | 3.5 (2.8–4.3) | 3.3 (2.8–4.3) | 0.0001 | 3.4 (2.8–4.2) | 3.3 (2.7–4.0) | <0.0001 |
ANC/WBC, % | 57.6 (50.8–63.9) | 56.3 (49.8–62.3) | 0.0007 | 58.0 (51.9–64.0) | 56.2 (50.8–62.3) | 0.0006 |
ACR, mg/g | 6.4 (0–20.8) | 3.2 (0–12.6) | 0.0002 | 9.2 (27–19.2) | 0 | <0.0001 |
ACE, angiotensin-converting enzyme; ARB, angiotensin receptor blocker; NS, not significant.
As described in Subjects and Methods, all subjects were classified as follows: nondiabetic; diabetic without albuminuria; diabetic with low-level albuminuria; diabetic with microalbuminuria; or diabetic with macroalbuminuria (Figure
ANC and ANC/WBC ratios of groups stratified by UA severity. The ANC value and ANC/WBC ratio of the group with diabetes and macroalbuminuria were much higher than those of the other 4 groups. A: compared with nondiabetes group,
Regarding the results of the multivariate logistic regression analyses, after adjustments for age, BMI, gender, SBP, DBP, and SCr, the odds ratios (ORs) indicated that ANC levels significantly correlated with risk of macroalbuminuria (
ANC quintiles in relation to the risk of macroalbuminuria and positive UA, OR (95% CI).
ANC, 103/ |
Macroalbuminuria |
|
|
---|---|---|---|
I | ≤2.6 | Referent level | Referent level |
II | 2.6 to 3.2 | 0.996 (0.691–1.435) | 1.271 (0.963–1.677) |
III | 3.2 to 3.7 | 1.458 (1.012–2.099) | 1.333 (0.993–1.791) |
IV | 3.7 to 4.4 | 1.351 (0.932–1.958) | 1.352 (1.000–1.827) |
V | >4.4 | 2.311 (1.608–3.321) | 1.573 (1.148–2.154) |
ROC curves were plotted to evaluate the diagnostic value of age, BMI, waist-to-hip ratio, gender, SBP, DBP, ALT, SCr, total cholesterol, triglycerides, FBG, 2-hour glucose, and ANC for macroalbuminuria. ANC was the strongest predictive factor for macroalbuminuria (AUC = 0.6001) followed closely by FBG (AUC = 0.5876) and SBP (AUC = 0.5767; Table
AUCs for predicting macroalbuminuria.
AUC (95% CI) | Fold change |
| ||
---|---|---|---|---|
ANC, 103/ |
0.6001 (0.5689–0.6313) | — | — | |
FBG, mmol/L | 0.5876 (0.5561–0.6191) | — | — | |
SCr, |
0.5545 (0.5225–0.5869) | — | — | |
SBP, mmHg | 0.5767 (0.5457–0.6077) | — | — | |
|
||||
Model 1 | ANC × FBG | 0.6179 (0.5872–0.6486) | — | — |
Model 2 | ANC × FBG × SBP | 0.6282 (0.6068–0.6676) | 1.67 | 0.5188 |
Model 3 | ANC × FBG × SBP × SCr | 0.6372 (0.5978–0.6586) | 3.12 | 0.5338 |
To investigate interactions among ANC, FBG, SBP, and SCr, they were grouped in separate analyses. ANC × FBG × SBP (Model 2; variables are multiplied) and ANC × FBG × SBP × SCr (Model 3) indicated 1.67% and 3.12% increase, respectively, in the predictive value for macroalbuminuria, compared with Model 1 (ANC × FBG), but these increases were not statistically significant (
This study investigated an association between systemic ANC and albuminuria in elderly Chinese people. We found that ANCs significantly increased in diabetes with macroalbuminuria, and ANC was the strongest factor predicting macroalbuminuria. These results indicate that neutrophil-mediated inflammation may be involved in the exacerbation of albuminuria.
To the best of our knowledge, this is the first report describing an association between UA and ANC levels in a population-based study. It is perplexing that the systemic neutrophil count, a very simple and inexpensive laboratory parameter, has not been reported previously in association with UA, although data from a study of 30 793 people conducted in Korea showed an association between ANC and diabetic retinopathy, after adjusting for the effects of blood pressure, blood glucose, and SCr [
In our present study, patients with albuminuria or those with diabetes had higher ANC levels. To study further the association between diabetes, UA, and ANC, subjects were stratified into a nondiabetic group and 4 groups with diabetes at different levels of severity of albuminuria. We found that patients with diabetes with macroalbuminuria had a notably higher mean ANC than did the other 4 groups. There were no statistical differences among the other 4 groups. In addition, ANC was an independent indicator of macroalbuminuria, but it was not an independent indicator of UA+. Thus, ANC might have an important role in accelerating the progression of albuminuria but not the formation of urinary protein.
Although establishing a causal mechanism for the association between ANC and albuminuria was beyond the scope of this study, the underlying link may concern vascular damage. In clinical and laboratory studies, elevated neutrophil counts were found associated with increased levels of tumor necrosis factor- (TNF-)
In the present study, compared with the other 4 quintile ANC groups, the highest level of the ANC group had the highest OR for the risk of macroalbuminuria and not quintiles II or III. This shows that the higher the ANC level, the more likely the presence of macroalbuminuria, reflecting an overtly inflammatory status in patients with macroalbuminuria [
There are several limitations to our study. First, the study design was cross-sectional and therefore we were not able to confirm a causal link between ANC and macroalbuminuria. Second, the inclusion of participants attending a routine health checkup in one community represents a potential source of selection bias, which should be considered when interpreting the results. Third, information regarding other traditional risk factors for albuminuria, such as heavy exercise [
This clinical study revealed an association between elevated systemic neutrophil counts and macroalbuminuria. High levels of UA may indicate an overt inflammatory state. The potential effect of neutrophil-mediated inflammation in the progression of macroalbuminuria warrants further research.
Absolute neutrophil count
Alanine aminotransferase
Albumin-to-creatinine ratio
Area under the curve
Body mass index
Chemotactic factor 2 alpha/beta
Confidence interval
Diastolic blood pressure
Fasting blood glucose
Interleukin
Odds ratio
Platform of Chronic Disease
Receiver operating characteristic
Serum creatinine
Systolic blood pressure
Tumor necrosis factor
Urinary albumin
Urinary albumin creatinine ratio
White blood cell.
Jun Liu is the guarantor and takes full responsibility for the work as a whole, including the study design, data access, and the decision to submit and publish the paper.
The authors declare that they have no competing interests.
The authors are grateful to the nurses of the Clinical Research Center for their expert nursing assistance. They also thank the Minhang District PCD and most importantly the commitment of the participants. This study was funded by grants from the Science and Technology Commission of Shanghai (no. 08411962500) and the Scientific Research Plan Project of Health and Family Planning Commission of Shanghai (201440514).