Evaluation of Early Markers of Nephropathy in Patients with Type 2 Diabetes Mellitus.

Aims. T2DM often remains undiagnosed for many years because hyperglycemia develops gradually and may not produce any symptoms. As patients with T2DM are at increased risk of microvascular and macrovascular complications, the preclinical diagnosis of the state is the key point of the disease management. Methods. We evaluated parameters such as GAGs/PGs, NAG, and NGAL in urine samples from 43 normoalbuminuric T2DM patients and 31 apparently healthy control subjects. Results. The total urinary GAG excretion showed no significant differences between patients and controls. The electrophoretic analysis evidenced the presence of UTI and its degradation products (LSC and SM-LSC), CS, and HS. We observed modifications of HS and total UTI (including UTI and its degradation products) relative contents in T2DM patients compared with controls whereas no differences in CS percentage were found. NGAL levels were significantly increased in T2DM patients and were positively correlated with both NAG (r = 0.606, p < 0.0001) and the presence of hypertension (r = 0.352, p < 0.05). Conclusions. These data suggest that the assessed molecules could represent useful markers to detect early renal impairment in patients with T2DM.

Development of a method for urine bikunin/urinary trypsin inhibitor (UTI) quantitation and structural characterization: Application to type 1 and type 2 diabetes.

Bikunin is a plasma proteinase inhibitor often associated with inflammatory conditions. It has a half-life of few minutes and it is rapidly excreted into urine as urinary trypsin inhibitor (UTI). UTI levels are usually low in healthy individuals but they can increase up to tenfold in both acute and chronic inflammatory diseases. This article describes a sensitive method for both direct UTI quantitation and structural characterization. UTI purification was performed by anion exchange micro-chromatography followed by SDS-PAGE. A calibration curve for protein quantitation was set up by using a purified UTI fraction. UTI identification and structural characterization was performed by Nano-LC-MS/MS analysis. The method was applied on urine samples from 9 patients with type 1 diabetes, 11 patients with type 2 diabetes, and 28 healthy controls, matched for age and sex with patients, evidencing higher UTI levels in both groups of patients with respect to controls (p < 0.001 and p = 0.001, respectively). Spearman's correlation tests highlighted no association between UTI levels and age in each group tested. Owing to the elevated sensitivity and specificity, the described method allows UTI quantitation from very low quantities of specimen. Furthermore, as UTI concentration is normalized for creatinine level, the analysis could be also performed on randomly collected urine samples. Finally, MS/MS analysis prospects the possibility of characterizing PTM sites potentially able to affect UTI localization, function, and pathophysiological activity. Preliminary results suggest that UTI levels could represent a useful marker of chronic inflammatory condition in type 1 and 2 diabetes.

A longitudinal evaluation of urinary glycosaminoglycan excretion in normoalbuminuric type 1 diabetic patients.

BACKGROUND: Previously, we found high urinary glycosaminoglycan (GAG) concentration, together with an altered electrophoretic pattern, in normoalbuminuric type 1 diabetic subjects with hemoglobin A(1c) (HbA(1c)) > or =8.0%. The purpose of this study was long-term evaluation of GAG excretion variations in these patients compared to those with HbA(1c) < 8.0% at baseline who maintained better metabolic control over time. METHODS: We enrolled 26 normotensive, normoalbuminuric type 1 diabetic patients and divided them into two groups according to mean HbA(1c) levels during the follow-up period. GAGs were isolated from 24-h urine samples on two separate occasions, at baseline and after a mean (+/-SD) follow-up of 6.8+/-1.1 years. RESULTS: All patients remained normoalbuminuric at follow-up, and had normal urinary alpha(1)-microglobulin levels. In patients with HbA(1c) <8.0%, total GAG levels and low sulfated chondroitin sulfate-proteoglycan/chondroitin sulfate ratio were almost unchanged during the follow-up period. In contrast, these increased in patients with HbA(1c) > or =8.0% and were significantly related to both HbA(1c) levels and the duration of poor glycemic control. CONCLUSIONS: Our results show a strong influence of hyperglycemic environment on GAG metabolism in diabetes and indicate that the distribution pattern of urinary GAGs, besides their total concentration, may be predictive of altered GAG metabolism in type 1 diabetes.

Urinary transforming growth factor-beta 1 in various types of nephropathy.

Transforming growth factor-beta1 (TGF-beta1) is a potent multifunctional polypeptide that is involved in normal renal function and in the development of glomerular sclerosis. It is also an important mediator of the immune and anti-inflammatory responses. The purpose of this study was to examine whether the measurement of urinary TGF-beta1 excretion in patients with different types of renal diseases and in newly diagnosed type 1 diabetes mellitus represents a non-invasive tool to evaluate disease activity and to monitor response to therapy. We studied the urinary excretion of TGF-beta1 in 57 nephropathic patients divided in different groups according to the underlying disease: 15 had mesangial glomerulonephritis (IgAGN), 9 membranous glomerulonephritis (MGN), 7 rapidly progressive glomerulonephritis (RPGN), 8 systemic lupus erythematosus (SLE), 9 interstitial nephritis (IN), 9 chronic renal failure (CRF). TGF-beta1 was also measured in 38 patients with type 1 (insulin-dependent) diabetes mellitus (12 with newly diagnosed diabetes, 26 long-standing diabetes) and 31 healthy controls. Total urinary TGF-beta1 concentration was assayed by enzyme-linked immunoassay (ELISA), and expressed as a ratio to urinary creatinine concentration. The urinary TGF-beta1 levels were compared with the findings of biopsy and clinical parameters. Urinary TGF-beta1 excretion was significantly increased in all groups except MGN, IN and CRF. In non-diabetic patients, urinary TGF-beta1 levels correlated with crescent formation, floccular adhesion and mesangial proliferation, but not with the degree of tubulo-interstitial fibrosis. Urinary TGF-beta1 levels did not correlate with indices of renal function (serum creatinine, glomerular filtration rate (GFR), albumin excretion rate [AER]). Among diabetic patients, HbA(1C) significantly correlated with TGF-beta1 urinary excretion. Urinary TGF-beta1 levels may represent a valid indicator of acute glomerular flogosis associated with mesangial proliferation in glomerulonephrities. In newly diagnosed diabetic patients, hyperglycaemia seems to represent the principal factor leading to TGF-beta1 overproduction. Follow-up studies of urinary TGF-beta1 levels measured during optimal glycaemic control are necessary to clarify the relationship between hyperglycaemia and TGF-beta1 excretion.

Urinary glycosaminoglycan and proteoglycan excretion in normoalbuminuric patients with type 1 diabetes mellitus.

BACKGROUND: Diabetic nephropathy may be related to an abnormal metabolism of glycosaminoglycans (GAG) in the glomerular basement membrane (GBM). The first manifestation of nephropathy is microalbuminuria, whose appearance indicates a loss of GBM selectivity. The present study evaluated whether GAG excretion becomes abnormal in parallel with microalbuminuria, and whether such abnormalities are also present in normoalbuminuric diabetic patients. METHODS: We measured urinary GAG excretion in 60 patients with type 1 (insulin-dependent) diabetes mellitus and in 22 healthy subjects. GAG were isolated from 24-h urine using ion-exchange chromatography on DEAE Sephacel. GAG composition was determined by cellulose acetate electrophoresis and expressed as percentages by densitometric scanning of Alcian Blue stained strips. RESULTS: On subgrouping for albuminuric status and glyco-metabolic control, we found high urinary GAG concentrations in all except the normoalbuminuric patients with good glyco-metabolic control. The urinary GAG electrophoretic pattern showed alterations in chondroitin sulphate (CS) and heparan sulphate (HS) relative contents. A higher frequency of low sulphated chondroitin sulphate-proteoglycan (LSC-PG) was observed in all patients, including those with normoalbuminuria and good glyco-metabolic control. CONCLUSIONS: This urinary pattern may be indicative of an abnormal GBM metabolism. Since GAG play an important role in GBM permeability, these anomalies might consequently represent a first step towards selective changes of GBM in type 1 diabetes mellitus.