The chronic noncommunicable diseases represent one of the most difficult challenges for all health care systems, in both industrialized and developing countries, due to their continuous and relentless growth. The most paradigmatic example is certainly represented by diabetes mellitus. Epidemiological evidences suggest that, without effective prevention and control plans, diabetes will likely continue to increase globally [
The renal impairment in diabetes mellitus affects ~40% of type 1 and type 2 diabetic patients. Diabetic nephropathy is responsible not only for ESRD, but also for a significant increase in cardiovascular risk in this population. Although CKD is a common comorbidity condition of T2DM, in the early stages it is often unrecognized, especially in the elderly, and, therefore, untreated [
Over the past few years we have described quali-quantitative variations of GAGs/PGs in both normoalbuminuric type 1 and 2 diabetic patients with respect to healthy control subjects, suggesting that both levels and relative abundance of urinary GAGs may be predictive of altered GAG metabolism in diabetes mellitus [
It has been shown that tubular damage occurs early in the course of diabetic nephropathy and that it is not merely secondary to glomerular damage as previously thought [
Therefore, the aim of the present study is to assess parameters such as urinary GAGs/PGs, NAG, and NGAL to detect alterations of renal function in normoalbuminuric patients with T2DM.
43 normoalbuminuric patients with type 2 diabetes mellitus, referred to the Unità Operativa di Diabetologia e Malattie del Ricambio, AOU-Sassari, were enrolled. Exclusion criteria were ketoacidosis, fever, infection, surgery as well as evidences of systemic diseases, renal, cardiac, or hepatic diseases, and malignant tumors. Normoalbuminuria was defined as a urinary albumin excretion rate lower than 30 mg/24 h (mean of three different samples over a period of three months). The mean age of the T2DM patients included (16 men and 27 women) was
Demographic and clinical characteristics of both patients and controls.
Parameters | Controls | Patients |
---|---|---|
Age (years) | 61.14 ± 6.65 | 64.21 ± 7.18 |
Sex (men/women) | 10/21 | 16/27 |
BMI (kg/m2) | 26.355 ± 2.247 | 27.583 ± 3.619 |
Duration of disease (years) | 7.02 ± 5.15 | |
Blood glucose (mg/dL) | 88.26 ± 21.21 | 131.59 ± 33.40 |
HbA1c (%) | 6.34 ± 0.73 | |
Total cholesterol (mg/dL) | 165.35 ± 24.38 | 170.71 ± 27.01 |
HDL cholesterol (mg/dL) | 49.22 ± 10.21 | 55.20 ± 11.20 |
LDL cholesterol (mg/dL) | 92.54 ± 13.58 | 96.24 ± 18.91 |
Triglycerides (mg/dL) | 89.65 ± 36.28 | 94.47 ± 42.42 |
Serum creatinine (g/dL) | 0.75 ± 0.12 | 0.78 ± 0.15 |
eGFR (mL/min/1.73 m2) | 89.6 ± 14.5 | |
Microalbuminuria (mg/24 h) | 9.38 ± 6.67 | |
Systolic blood pressure (mmHg) | 132.1 ± 16.2 | |
Diastolic blood pressure (mmHg) | 78.6 ± 9.7 |
Data are mean ± SD. eGFR = estimated glomerular filtration rate calculated using the equation developed in the MDRD (Modification of Diet in Renal Disease) study.
The control group was composed of 31 apparently healthy subjects matched for age, sex, and BMI with T2DM patients.
Informed consent was obtained before enrolment. Institutional Review Board approval was obtained. The study was conducted in accordance with the ethical principles of the current Declaration of Helsinki.
Urinary GAG/PG purification and quali-quantitative analysis were performed by a method previously described [
Urinary NAG was estimated kinetically by using NAG colorimetric kit (FAR DIAGNOSTICS, Verona, ITA), whereas NGAL levels were performed with the Lipocalin-2/NGAL Human ELISA Kit (Abcam, Cambridge, UK). The assays were performed according to the protocols provided by the manufacturers.
Statistical analyses were performed using the software package Sigma Stat 3 (Systat Software). Values are reported as mean (±SD) or median (interquartile range). Differences between groups were assessed by Student’s
The total urinary GAG excretion showed no significant differences between patients and controls (3.757 (2.868–5.234) mg of hexuronate/g creatinine versus 4.40 (3.475–5.635) mg of hexuronate/g creatinine,
Representative cellulose acetate electrophoretic profiles of urine glycosaminoglycans/proteoglycans from T2DM patients (lanes 1–5) and control subjects (lanes 6-7). UTI: urinary trypsin inhibitor. LSC: low sulfate chondroitin sulfate. SM-LSC: slow migration-LSC. HS: heparan sulfate. CS: chondroitin sulfate.
Plots showing the median (line within box), 25th and 75th percentiles (box), 5th and 95th percentiles (whiskers), and outliers (•) of HS (a), total UTI (UTI plus SM-LSC and LSC) (b), and CS (c) percentages in the controls group and in T2DM patients. Percentages were evaluated by performing image analysis on cellulose acetate electrophoretic profiles using Quantity One software (Bio-Rad Laboratories). Differences with
Plot showing the median (line within box), 25th and 75th percentiles (box), 5th and 95th percentiles (whiskers), and outliers (•) of neutrophil gelatinase-associated lipocalin (NGAL) levels, normalized for creatinine (Cr) content, in the controls group and in T2DM patients. Differences with
The identification of sensitive markers of renal function continues to be of considerable interest. Although UAE is considered the “gold standard” for diagnosing renal dysfunction, other markers, detectable at an earlier stage of the disease, may have diagnostic and/or prognostic value. Moreover, the association between glomerular lesions and MA is less pronounced in T2DM. In this respect, Fioretto et al. [
Several studies have been performed to elucidate the molecular mechanisms underlying glomerular ultrafiltration, suggesting a role of PGs, especially their carbohydrate moieties, in maintaining the glomerular filtration barrier [
The alteration of tubular reabsorption is found almost always in the early stages of kidney disease, including those characterized by predominant glomerular involvement. In this study, we observed an increase of NGAL in normoalbuminuric diabetic patients. Being an iron transporter, NGAL may be expressed by the damaged renal tubule to induce regeneration, since this element is necessary for reepithelialization. Furthermore, it is known that pathological changes in diabetic nephropathy involve accumulation of extracellular matrix, the degradation of which involves mainly MMPs. The activities of these enzymes are dependent on metal ions and are limited by TIMP-1. In particular, it has been reported that a high glucose concentration causes a reduction in the amount of MMPs secreted by the mesangial cells [
In our study, the urinary levels of NGAL show a highly significant relationship with the urinary NAG levels and a positive relationship with the presence of hypertension. We did not find any relationship among these parameters and serum creatinine levels or eGFR, probably because the increase of serum creatinine may not be evident before 50% or more of the nephrons are damaged.
Image analysis of GAG/PG profiles also enabled us to detect a significant increase in the relative content of total UTI in T2DM patients.
The role of inflammation in the pathogenesis of type 2 diabetes and associated complications is now well established. Multiple mechanisms underlie defective insulin secretion and responses in type 2 diabetes. These include glucotoxicity, lipotoxicity, oxidative stress, and endoplasmic reticulum stress. Interestingly, all of these mechanisms are associated with inflammatory responses [
During inflammation, UTI is released from I
In conclusion, we observed an increased level of NGAL, as well as an altered relative content of HS and total UTI in normoalbuminuric T2DM patients with significant correlations among NGAL levels and both NAG excretion and presence of hypertension. It is likely that, rather than a single biomarker, the identification of a panel of strategically selected analytes may represent a useful tool for the accurate prediction and monitoring of the disease. In this perspective, our results, although preliminary, suggest that the assessed markers could represent good candidates to detect early renal alteration in normoalbuminuric patients with T2DM. Further longitudinal studies with a larger sample size are required to clarify these results.
Type 2 diabetes mellitus
Glycosaminoglycans/proteoglycans
N-Acetyl-
Neutrophil gelatinase-associated lipocalin
Urinary trypsin inhibitor
Low sulfate chondroitin sulfate
Slow migration-LSC
Heparan sulfate
Chondroitin sulfate
End-stage renal disease
Chronic kidney disease
Urinary albumin excretion
Glomerular basement membrane
Body mass index
Microalbuminuria
Heparan sulfate proteoglycan
Matrix metalloproteinases
Tissue inhibitor of metalloproteinase
Estimated glomerular filtration rate.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by Regione Autonoma della Sardegna (P.O.R. Sardegna F.S.E. 2007/2013, Asse IV Capitale Umano, Obiettivo Competitività Regionale e Occupazione, Asse IV Capitale Umano, Linea di Attività l.3.1).