Abstract
Urinary N-acetyl-β-d-glucosaminidase (NAG) has been suggested as a marker for early diabetic nephropathy. This study aimed to prospectively investigate the relationship between asymptomatic leukocyturia (ASL) and NAG in women. One hundred and five female outpatients aged 31–86 years were selected for a 10-year follow-up study. We regarded ASL to be present if two consecutive samples were found to have 10 or more leukocytes/high-power field at 400× magnifications in a centrifuged midstream urine sample both at baseline and 10 years later. The urinary activities of NAG to creatinine ratios (NAG index) were measured in random spot urine samples. Patients without ASL at the beginning of the study were followed. The patients with ASL had diabetes mellitus more frequently than those without ASL at baseline and after 10 years. Residual urine volume and the NAG index were significantly higher in the former than in the latter (p = 0.014 and p = 0.002, respectively) at baseline. During the observation period, 15 patients had ASL (30.6%). Although a gradual increase in the NAG index was found during the study in both patients who had ASL and those who did not, the mean NAG index was significantly higher in the latter during study period (6.4 ± 3.0 vs. 9.8 ± 5.5, p = 0.004, 9.4 ± 5.2 vs. 11.5 ± 6.4, p = 0.328, respectively). On multiple logistic regression analysis, the NAG index at the beginning of the study was an independent predictor of ASL. These results demonstrate that the NAG index may serve as an indicator of ASL in women.
INTRODUCTION
Urinary tract infection (UTI) is widely considered to be more common in patients with diabetes mellitus (DM) than in nondiabetics. Although diabetic patients are more prone to infection in general, local factors (e.g., glycosuria or autonomic neuropathy affecting the bladder) may render patients particularly prone to urinary infections.Citation1 Many UTIs are asymptomatic, and it is unknown whether symptomatic UTIs are preceded by asymptomatic bacteriuria (ASB). Various risk factors for ASB in women with diabetes have been suggested, including sexual intercourse, age, and disease duration, metabolic control, and complications of diabetes.Citation2
A recent meta-analysis showed that the prevalence of ASB is higher in all patients with diabetes than in control subjects.Citation3 In addition, diabetic subjects with ASB more often had albuminuria and symptomatic UTIs. We have shown that the prevalence of asymptomatic leukocyturia (ASL) is higher in women with diabetes than in those without diabetes, and these diabetic complications, especially neuropathy, are factors contributing to the development of ASL in diabetic women.Citation4 In addition, we recently demonstrated a relationship between ASL and impairment of the parasympathetic nervous system in women.Citation5
On the other hand, N-acetyl-β-d-glucosaminidase (NAG) is a widely distributed lysosomal enzyme with a molecular weight of 130,000–140,000 Da, located predominantly in renal proximal tubules.Citation6 It acts as a marker of tubular dysfunction as this enzyme cannot be filtered from blood through an intact glomerular membrane. We have shown that the NAG index may serve as an early functional indicator of diabetic nephropathy.Citation7
ASL is frequent in patients with diabetes, and increased urinary NAG excretion has been reported in patients with type 2 diabetes.Citation7 However, little is known about the temporal changes in urinary NAG activity in relation to newly developing ASL. Therefore, by investigating ASL, we could possibly predict the increase of NAG, which is a marker of tubular dysfunction, and be able to predict early stage of renal dysfunction. A 10-year prospective study was performed to elucidate the relationship between the development of ASL and urinary NAG activities.
MATERIALS AND METHODS
The subjects were 105 women aged 31–86 years who were outpatient of our hospital. Of these patients, 49 subjects who did not have ASL at the start of observation were selected for a 10-year follow-up study (10 ± 0.2 years). Patients with organic abnormalities of the urinary tract, those who had undergone recent urinary tract instrumentation, who showed symptoms of a UTI (i.e., dysuria, pollakiuria, stranguria, and/or increased urgency of urination), and who used antimicrobial drugs within the previous 30 days were excluded. Prior to the study, the purpose of the examination was explained to all subjects, and informed consent was obtained. The study was designed in compliance with the ethics regulations set out by the Helsinki Declaration.
We regarded ASL to be present if two consecutive samples were found to have 10 or more leukocytes/high-power field (HPF) at 400× magnifications in a centrifuged midstream urine sample.Citation5 In addition, the residual urine volume, urinary NAG activity, and urinary albumin level were measured.
All samples were collected during urological examinations at room temperature and immediately taken to the laboratory under refrigerated condition. Urinary WBC (u-WBC) counts were obtained by flow cytometry. The device used for measurement was the UF-100i (Sysmex Corporation, Kobe, Japan). Post-voiding residual urine volume was measured with an automated, compact 3D ultrasonography (BladderScan BVI 6100; Diagnostic Ultrasound, Bothell, WA, USA). Ultrasonography was carried out by one examiner in the supine position immediately after voiding and with the scanheads held suprapubically over the bladder. Bladder volumes measured with BladderScan and urethral catheter volumes were closely correlated across a range of 0–1015 cc (y = 1.02x + 12.6, R2 = 0.90, p < 0.001). Urinary activity of NAG to creatinine ratios (NAG index) and albumin to creatinine ratios [albumin index (AI)] in spot urine samples were measured. This assay is specific for NAG, has no detectable reaction with other glycosidases, and has excellent sensitivity, with a linear range up to 200 U/L and excellent precision (coefficient of variation < 5%). The ratio of the urinary activity of NAG to Cr (NAG index) was determined in spot urine samples. Urinary NAG levels were measured spectrophotometrically with sodio-meta-cresolsulfonphthaleinyl-N-acetyl-β-d-glucosaminide as a substrate (NAG assay kit, Shionogi & Co. Ltd., Osaka, Japan).Citation8
After an overnight fast, blood pressure was measured in the sitting position, and blood was taken to determine WBC, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), Cr, and HbA1c [Japanese Diabetes Society (JDS)] levels. Serum C-reactive protein (CRP), TC, TG, HDL-C, TG, and Cr were measured with an automated analyzer. HbA1c (JDS) was measured by high-performance liquid chromatography (HPLC). The estimated glomerular filtration rate (eGFR) was calculated using the Cockcroft–Gault equation. In addition, body weight was measured, and body mass index (BMI) was calculated as the ratio of body weight (kg) to the square of height (m). We defined diabetes as a fasting plasma glucose concentration of 126 mg/dL or more, a 2-h plasma glucose concentration of 200 mg/dL or more, accompanied by HbA1c over 6.1% (JDS), or the use of glucose-lowering medications (oral agents or insulin or both). A subject was defined as having hypertension (HT) if systolic blood pressure was ≥140 mmHg, or diastolic blood pressure ≥90 mmHg, or if a subject was receiving drug treatment for HT. A subject was defined as having hypercholesterolemia if serum TC was ≥220 mg/dL, and/or HDL-C < 40 mg/dL or if a subject was receiving drug treatment for hypercholesterolemia. A subject was defined as having hypertriglyceridemia if serum TG was ≥150 mg/dL, or if a subject was receiving drug treatment for hypertriglyceridemia.
Statistical Methods
Statistical analysis was carried out using the χ2 test, Student’s t-test, and one-way analysis of variance. Multiple logistic regression analysis was used to identify associations between ASL development and explanatory variables. For multiple logistic regression analysis, six sets of explanatory variables were used, without adjustment (Model I); adjusted for age (Model II); for age, DM, HT, and dyslipidemia (DL) (Model III); for age, DM, HT, DL, BMI, and Cr (Model IV); for age, DM, HT, and DL, BMI, Cr, and AI (Model V); and for age, DM, HT, DL, BMI, Cr, Al and u-WBC (Model VI). Data in the text, tables, and figures are expressed as means ± SD.
RESULTS
The clinical characteristics of the subjects with and without ASL are shown in . At baseline, residual urine volume and the NAG index were significantly higher in the group with ASL than in the group without ASL (18.7 ± 49.9 vs. 48.1 ± 62.5, p = 0.014; 7.3 ± 4.0 vs. 10.7 ± 6.5, p = 0.002). However, there was no significant intergroup difference at baseline in age, BMI, systolic blood pressure, diastolic blood pressure, CRP, WBC, TC, HDL-C, TG, serum Cr, eGFR, or urinary AIs. The prevalence of DM was significantly higher in the group with ASL than those without ASL at baseline (p = 0.001). However, no significant differences between the two groups were evident in the prevalence of HT or DL.
Table 1. Baseline characteristics of study subjects with and without ASL.
illustrates the NAG index (logarithmically converted) and the u-WBC counts (logarithmically converted) at baseline. At baseline, the NAG index showed a significant positive correlation with the u-WBC counts (r = 0.273, p = 0.008). Among subjects who did not have ASL at baseline, 30.6% were ASL positive 10 years later.
Figure 1. Correlation between logarithmic transformation of u-WBC counts and logarithmic transformation of the NAG index at baseline. u-WBC, urinary white blood cells; NAG, N-acetyl-β-d-glucosaminidase.
The clinical characteristics between the subjects without ASL at 10 years (n = 34) and those with ASL at 10 years (n = 15) are shown in .
Table 2. Longitudinal changes of NAG index and other parameters at baseline and after 10 years in the subject group without ASL at baseline.
Table 3. Odds ratio between developing ASL after 10 years and NAG index.
Among the subjects without ASL at 10 years, the NAG index after 10 years was significantly higher than that at baseline (6.4 ± 3.0 vs. 9.8 ± 5.5, p = 0.004), whereas no significant differences were noted in TC, HDL, TG, Cr, u-WBC counts, or urinary AIs. Among the subjects with ASL at 10 years, the NAG index after 10 years was higher than that at baseline although this difference was not statistically significant (9.4 ± 5.2 vs. 11.5 ± 6.4, p = 0.328). The NAG index at baseline tended to be higher in the group with ASL at 10 years than in the group without ASL at 10 years, whereas this difference did not reach statistical significance (6.4 ± 3.0 vs. 9.4 ± 5.2, p = 0.067). Among the subjects with ASL at 10 years, u-WBC counts after 10 years were significantly higher than that at baseline (11.2 ± 10.7 vs. 535.6 ± 1804, p = 0.017), whereas no other variable showed a significant change.
shows the odds ratio between ASL after 10 years and NAG index after adjustment for different clinical variables among the subjects who did not have ASL at baseline. Even after adjustment for age, DM, HT, DL, BMI, Cr, urinary AIs, and u-WBC counts, the NAG index remained an independent risk factor for ASL.
DISCUSSION
In this study, the prevalence of DM, the residual urine volume, and the NAG index were significantly higher in patients with ASL at baseline than in those without ASL at baseline. At baseline, the NAG index showed a significant positive correlation with the u-WBC counts. Patients who remained ASL negative until the end of the 10-year follow-up period showed significant elevation of the NAG index. The NAG index was identified as an independent risk factor for ASL by multiple logistic regression analysis.
In this study, we used leukocyturia as an indicator of ASB.Citation5 Although both urine cultures and urinary leukocyte may be suitable diagnostic methods for ASB, the urinary leukocyte counts are strongly recommended because it is easier and faster to perform, more convenient and more acceptable to patients, more reproducible, and less expensive.Citation9
Our results indicate that the prevalence of DM was significantly higher in patients with ASL at baseline than in those without ASL at baseline, and that among patients without ASL at baseline, the prevalence of DM was significantly higher in the group with ASL at the end of the follow-up period than in the group without ASL at the end of the follow-up period. According to previous reports, the prevalence of ASL was higher in diabetic patients and increases progressively with age, including urogenital disease, in both sexes, regardless of whether the patients have diabetes. Some studies demonstrated no differences in the prevalence of ASB between diabetic and nondiabetic women, but the majority of investigators reported a prevalence of ASB two to three times as high in diabetic women.Citation2,10,11 In addition, in our previous study, the patients with ASL had a higher prevalence of DM and higher residual urine volume than the patients without ASL.Citation5
The observation that age is associated with ASB is consistent with all ambulatory populations, regardless of whether they have diabetes.Citation1,2,12,13 The average age at the start of follow-up is 67 years in this study. Thus, the effect of age on ASB may also be explained by an increased prevalence of mechanical or functional bladder outlet disorders in the elderly.Citation1,14 Furthermore, Ellenberg and WeberCitation15 conducted urological investigations on diabetic patients with and without diabetic neuropathy and nondiabetic controls, all of whom were completely free of symptoms and signs referable to the urinary tract. These results are consistent with their findings, because the ASL positive group had DM more frequent and a higher residual urine volume. A number of factors may precipitate ASB, including age-related changes in urological function, multiple comorbid chronic illnesses, and interventional procedures on the urinary tract because of incontinence or because of neurogenic bladder of various pathologies.Citation13
We previously demonstrated that parasympathetic nervous system dysfunction is associated with ASL.Citation5 Recently, Chiodini et al.Citation16 revealed that type 2 diabetic patients with asymptomatic parasympathetic derangement show increased hypothalamic-pituitary-adrenal (HPA) axis activity related to the degree of neuronal dysfunction. Given the deleterious role of glucocorticoids on the immune system, it is possible that an increased cortisol secretion contributes to deterioration of the immune system, consequently inducing a higher frequency of ASL.Citation5 These findings suggest that parasympathetic dysfunction can stimulate the HPA axis and increase insulin resistance and that ASL is closely associated with blood glucose levels.
On the other hand, increased urinary NAG excretion is considered to reflect subtle degrees of renal injury.Citation17 NAG index for random specimens provides a useful, convenient measurement of daily NAG excretion and avoids many of the problems of 24-h collections.Citation18 In this study, NAG was significantly higher in the group with ASL at baseline than in the group without ASL at baseline. Among the patients without ASL at baseline, NAG rose significantly during the follow-up period in those without ASL at the end of the follow-up period and showed an increase (not statistically significant) in the group with ASL at the end of the follow-up period. It is well known that urinary NAG excretion increases with age. In our previous study, we demonstrated that the NAG index rose by 0.65 U/g Cr during a 10-year period. The results in this study also seem to reflect the influence of aging. During follow-up, the NAG index rose in both the group without ASL after 10 years (from 6.4 ± 3.0 to 9.8 ± 5.5) and the group with ASL after 10 years (from 9.4 ± 5.2 to 11.5 ± 6.4).
According to previous reports, NAG rises in the presence of proximal tubular disorders and serves as an indicator of early diabetic nephropathy.Citation7,19–22 Our other study provides evidence for similar deterioration of glomerular and tubular renal function in diabetic patientsCitation7 and Brito et al.Citation23 revealed that characteristic structural changes are found in the kidneys of diabetics, both in the glomerular basement membrane and in the tubular cells. In a report of the UK Prospective Diabetes Study Group, the decrease in urinary albumin and urinary NAG excretion was associated with a reduction in the fasting plasma glucose level in response to dietary treatment.Citation24 The association with blood glucose level was stronger for urinary NAG than for urinary albumin, as only urine NAG reduction correlated with the degree of fall in the glucose levels. In patients with type 2 DM, it seems likely that blood glucose levels have a great effect on renal excretion of NAG than on albumin. In our previous study, significantly higher mean HbA1c was found at the beginning of the study in patients who later developed diabetic nephropathy in comparison with those who did not.Citation7
This study has several limitations. First, it was a hospital-based study, involving only a small number of cases in which very long-term follow-up was possible. Furthermore, we did not examine the relationship between ASL and NAG in men. Because the number of men with underlying prostatic disease increases with aging, we considered that we could not detect the accurate ASL in men. We have no study associated with interventional consequences. Further investigation will elucidate the effect of interventional consequences on ASL in the future. Regarding the relationship underlying changes in NAG and ASL, the degree of glycemia seems very likely to play a role. Further studies are needed to elucidate this issue.
Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.
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