Abstracts

INTRODUCTION

Diabetic nephropathy (DN) is a major microvascular complication of diabetes mellitus (DM) becoming the main cause of end-stage renal disease worldwide. It is assumed that a urinary albumin excretion within the range of microalbuminuria constitutes the first clinical evidence of DN and is the most important non-invasive marker of glomerular alteration and disease progression risk. Indeed, in diabetic patients, microalbuminuria commonly precedes both clinical albuminuria and the decline of renal function (¹1 American Diabetes Association. Standards of Medical Care in Diabetes-2013. Diabetes Care. 2013;36(Suppl 1):S11-66.). On the other hand, tubular injury may also be involved in the pathogenesis and progression of DN. A wide variety of causes related with the pathophysiological alterations that develop in the setting of diabetes are contributing factors. Examples are hyperglycemia, alterations in vasoactive hormones, formation of glycation advanced end products, hemodynamic changes, protein kinase C activation, increased oxidative stress and polyol production (²2 Gilbert RE, Cooper ME. The tubulointerstitium in progressive diabetic kidney disease: more than an aftermath of glomerular injury? Kidney Int. 1999;56:1627-37.). Furthermore, persistent albuminuria secondary to glomerular lesions may be directly harmful to renal tubular cells, leading to tubular inflammation and tubulointerstitial fibrosis (³3 Comper WD, Hilliard LM, Nikolic-Paterson DJ, Russo LM. Disease-dependent mechanisms of albuminuria. Am J Physiol Renal Physiol. 2008;295:F1589-600.,⁴4 Abbate M, Zoja C, Remuzzi G. How does proteinuria cause progressive renal damage? J Am Soc Nephrol. 2006;17:2974-84.). One widely used tubular injury marker is N-acetyl-beta-D-glucosaminidase (NAG) whose presence in the urine invariably signifies tubular damage since, due to its high molecular weight, it is not able to filter by the glomerulus, but it is released in the tubular lumen as a consequence of proximal tubular damage (⁵5 Hong CY, Chia KS. Markers of diabetic nephropathy. J Diabetes Complications. 1998;12:43-60.). Studies aimed at evaluating the utilization of urinary NAG as a marker of tubular dysfunction in type 2 DM (T2DM) have not been conclusive. While some authors demonstrated that urinary NAG is elevated in type 2 diabetic patients with normoalbuminuria compared to non-diabetic controls (6‐8), as well as in type 2 diabetic patients with microalbuminuria compared to those with normoalbuminuria (8‐10), others disagree with these findings (¹¹11 Fujita H, Morii T, Koshimura J, Ishikawa M, Kato M, Miura T, et al. Possible relationship between adiponectin and renal tubular injury in diabetic nephropathy. Endocr J. 2006;53:745-52.).

The aim of this study was to evaluate the clinical utility of urinary NAG excretion for the detection of early tubular dysfunction, particularly referred to the possible degree of association with urinary albumin excretion in type 2 diabetic patients.

SUBJECTS AND METHODS

We studied 36 patients with T2DM of more than 5 five years of evolution who were referred from the Medical Clinic Service to the Nephrology Service of the Hospital Provincial del Centenario of Rosario. The exclusion criteria were body mass index (BMI)≥30 kg/m², history of other endocrinopathies, hypertension (median of blood pressure >130/80 mmHg determined as triplicates, recorded in 2 different opportunities), urinary tract infection, renal lithiasis (discarded by kidney ultrasonography), proteinuria, abnormal urinary sediment (presence of hematuria, pyuria and/or casts) or renal failure [estimated glomerular filtration rate (eGFR) <60 mL/min per 1.73 m²] (¹²12 Levey AS, Eckardt KU, Tsukamoto Y, Levin A, Coresh J, Rossert J, et al. Definition and classification of chronic kidney disease: a position statement from Kidney Disease: Improving Global Outcomes (KDIGO). Kidney Int. 2005;67:2089-100.). Normoalbuminuria was defined as urinary albumin to creatinine ratio (ACR)<30 mg/g and microalbuminuria as ACR =30‐300 mg/g (¹²12 Levey AS, Eckardt KU, Tsukamoto Y, Levin A, Coresh J, Rossert J, et al. Definition and classification of chronic kidney disease: a position statement from Kidney Disease: Improving Global Outcomes (KDIGO). Kidney Int. 2005;67:2089-100.,¹³13 Murussi M, Murussi N, Campagnolo N, Pinho Silveiro S. Detecção precoce da nefropatia diabética. Arq Bras Endrocrinol Metab. 2008;52:442-51.). Diabetic patients were categorized into two groups based on their ACR: normoalbuminuria (n=19) and microalbuminuria (n=17). In the normoalbuminuria group, 5 patients were under diet therapy, 10 patients received metformin combined with sulfonylureas and 4 patients were in treatment with insulin. In the microalbuminuria group, the number of patients under diet, metformin combined with sulfonylureas or insulin were 3, 8 and 6, respectively. Ethical approval was obtained from the Ethics Committee of the Faculty of Biochemical and Pharmaceutical Sciences of Rosario.

After 12 hours of overnight fasting, venous blood samples were obtained and first morning urine samples were collected from each individual. Aliquots of urine were then immediately frozen at ‐80 °C until further analysis for no longer than 1 month. Urinary excretion of NAG was measured by colorimetric assay following manufacturer’s instructions (Roche Diagnostics GmbH) and results were expressed as Units/g creatinine (⁹9 Piwowar A, Knapik-Kordecka M, Fus I, Warwas M. Urinary activities of cathepsin B, N-acetyl-beta-D-glucosaminidase, and albuminuria in patients with type 2 diabetes mellitus. Med Sci Monit. 2006;12:CR210-14.). The tests were run in duplicates. Intra-assay variation coefficient was <5%. Urinary concentration of albumin was assessed by an immunoturbidimetric method (Roche Diagnostics GmbH) and ACR was expressed as mg/g creatinine (¹²12 Levey AS, Eckardt KU, Tsukamoto Y, Levin A, Coresh J, Rossert J, et al. Definition and classification of chronic kidney disease: a position statement from Kidney Disease: Improving Global Outcomes (KDIGO). Kidney Int. 2005;67:2089-100.,¹³13 Murussi M, Murussi N, Campagnolo N, Pinho Silveiro S. Detecção precoce da nefropatia diabética. Arq Bras Endrocrinol Metab. 2008;52:442-51.). Microalbuminuria was established when two out of three ACR determinations were found to be within the range 30‐300 mg/g in a six-month period. Creatinine concentration in urine and serum was measured by a kinetic method based on Jaffe reaction. Serum creatinine values were used to calculate an eGFR, by means of the abbreviated Modification of Diet in Renal Disease formula (⁸8 Nauta FL, Boertien WE, Bakker SJ, van Goor H, van Oeveren W, de Jong PE, et al. Glomerular and tubular damage markers are elevated in patients with diabetes. Diabetes Care. 2011;34: 975-81.,¹³13 Murussi M, Murussi N, Campagnolo N, Pinho Silveiro S. Detecção precoce da nefropatia diabética. Arq Bras Endrocrinol Metab. 2008;52:442-51.). Glycemic control was evaluated by assessing fasting plasma glucose and glycated hemoglobin (HbA_1c). Glucose was determined by an enzymatic method in a plasma sample obtained with EDTA/fluoride. HbA_1c was measured by ion-exchange cromatography (Biosystem S. A.) in a whole blood sample obtained with EDTA.

Normally distributed values were expressed as means ± SD and compared using the independent-samples t test, whereas non-normally distributed values were given as median (range) and compared by the Mann-Whitney U test. After non-normally distributed values were log-transformed to better approximate normal distributions, correlations were calculated by Pearson’s correlation analysis. Categorical data were compared by chi-square test. P values of less than 0.05 were considered statistically significant.

RESULTS

Clinical characteristics of type 2 diabetic patients are summarized in table 1. Microalbuminuria group showed higher levels of ACR (by design) and urinary NAG compared to normoalbuminuria group (P<0.001). We found no statistically significant differences between both groups in the time of evolution of DM, nor in blood pressure, BMI, fasting glucose, HbA_1c, serum creatinine levels or eGFR.

Urinary NAG levels were positively correlated with ACR levels in all diabetic patients (r=0.628; P<0.0001) (Figure 1), while no correlation was found between urinary NAG and age, blood pressure, BMI, parameters of glycemic control, serum creatinine and eGFR (Table 2).

DISCUSSION

Although glomerular injury has classically been considered as the cause of early DN, studies in the last two decades have focused on the role of a concomitant or perhaps earlier occurrence of tubular injury in DN (²2 Gilbert RE, Cooper ME. The tubulointerstitium in progressive diabetic kidney disease: more than an aftermath of glomerular injury? Kidney Int. 1999;56:1627-37.,³3 Comper WD, Hilliard LM, Nikolic-Paterson DJ, Russo LM. Disease-dependent mechanisms of albuminuria. Am J Physiol Renal Physiol. 2008;295:F1589-600.). In the present study we measured urinary NAG excretion, as a marker of tubular damage, in type 2 diabetic patients with normo- and microalbuminuria. We found that urinary NAG levels were significantly increased in patients with microalbuminuria compared to those with normoalbuminuria, thus corroborating previous studies performed in type 2 diabetic patients (8‐10). We also observed a significant positive correlation between urinary NAG excretion and ACR which is consistent with the data reported by other studies in patients with T2DM (¹⁴14 Udomah FP, Ekpenyong Ekrikpo U, Effa E, Salako B, Arije A, Kadiri S. Association between Urinary N-Acetyl-Beta-D-Glucosaminidase and Microalbuminuria in Diabetic Black Africans. Int J Nephrol. 2012;2012:235234.) as well as in type 1 diabetic patients (¹⁵15 Gibb D, Tomlinson P, Dalton N, Turner C, Shah V, Barratt T. Renal tubular proteinuria and microalbuminuria in diabetic patients. Arch Dis Child. 1989;64:129-34.). These findings suggest that tubular dysfunction, evidenced by an increase in urinary NAG excretion, is already present in the microalbuminuria stage of DN in T2DM and that it becomes exacerbated with the degree of albuminuria. The association between the regression of microalbuminuria and the decrease in urinary NAG observed by Vaidya and cols. (¹⁶16 Vaidya V, Niewczas M, Ficociello L, Johnson A, Collings F, Warram J, et al. Regression of microalbuminuria in type 1 diabetes is associated with lower levels of urinary tubular injury biomarkers, kidney injury molecule-1,and N-acetyl-b-D-glucosaminidase. Kidney Int. 2011;79:464-70.) in a prospective analysis performed in type 1 diabetic patients would support our hypothesis.

The lack of correlation between urinary NAG excretion and glycemic state are consistent with those results reported by other authors in type 2 diabetic patients (⁹9 Piwowar A, Knapik-Kordecka M, Fus I, Warwas M. Urinary activities of cathepsin B, N-acetyl-beta-D-glucosaminidase, and albuminuria in patients with type 2 diabetes mellitus. Med Sci Monit. 2006;12:CR210-14.,¹⁴14 Udomah FP, Ekpenyong Ekrikpo U, Effa E, Salako B, Arije A, Kadiri S. Association between Urinary N-Acetyl-Beta-D-Glucosaminidase and Microalbuminuria in Diabetic Black Africans. Int J Nephrol. 2012;2012:235234.). However, data from the literature referred to T2DM are controversial (¹⁰10 Farvid MS, Djalali M, Siassi F, Farvid S. Association of glomerular and tubular dysfunction with glycaemic control, lipid, lipoprotein, apolipoprotein and antioxidant status in type 2 diabetes mellitus. Singapore Med J. 2007;48:840-6.,¹⁷17 Yamanouchi T, Kawasaki T, Yoshimura T, Inoue T, Koshibu E, Ogata N, et al. Relationship between serum 1,5-anhydroglucitol and urinary excretion of N-acetylglucosaminidase and albumin determined at onset of NIDDM with 3-year follow-up. Diabetes Care. 1998;21:619-24.), while in type 1 diabetic patients such a relation is more clearly evidenced (¹⁵15 Gibb D, Tomlinson P, Dalton N, Turner C, Shah V, Barratt T. Renal tubular proteinuria and microalbuminuria in diabetic patients. Arch Dis Child. 1989;64:129-34.).

Despite the relatively small size of the sample and the cross sectional design of this study, we are able to state that urinary NAG levels are elevated in type 2 diabetic patients with microalbuminuria and that these levels correlate with ACR. These findings would suggest a possible clinical application of urinary NAG as a complementary marker for the detection of early renal disease in T2DM.

Acknowledgments

the authors would like to thank Professor Dr. Aldo Mottino for his critical review of the corrected manuscript and Dr. Cecilia Basiglio for language revision.

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