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ORIGINAL ARTICLE
Year : 2021  |  Volume : 34  |  Issue : 1  |  Page : 135-140

Role of urinary neutrophil gelatinase-associated lipocalin in diabetic and nondiabetic patients with nephropathy


1 Departments Internal Medicine and Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Internal Medicine, Tanta General Hospital, Gharbia, Egypt

Date of Submission08-Mar-2020
Date of Decision05-Apr-2020
Date of Acceptance12-Apr-2020
Date of Web Publication27-Mar-2021

Correspondence Address:
Ghada I. E. A. Dwidar
Tanta, Gharbia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_72_20

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  Abstract 


Objective
The aim was to study the role of neutrophil gelatinase-associated lipocalin (NGAL) in diabetic nephropathy patients with type 2 diabetes.
Background
Due to the high morbidity and mortality associated with diabetic nephropathy, it is important to establish a diagnosis and initiate therapy as soon as possible.
Patients and methods
This observational study was conducted on 88 subjects who were divided into four groups: Group 1 included 22 healthy individuals; group 2 included 22 type 2 diabetic patients with normoalbuminuria;, group 3 included 22 type 2 diabetic nephropathy patients; and group 4 included 22 nondiabetic patients with chronic kidney disease (CKD). All patients were admitted to the Internal Medicine Department at Menoufia University Hospital in the period from 20 December to 13 March 2019. Complete history, examination, and investigations urea, creatinine, fasting blood glucose, postprandial blood glucose, HbA1C, albumin/creatinine ratio, estimated glomerular filtration rate, and urinary NGAL were investigated for all subjects.
Results
Urea, creatinine and albumin/creatinine ratio were statistically significantly increased in diabetic nephropathy groups (145.30 ± 57.93), (3.95 ± 1.57), (201.27 ± 50.04) than in the diabetic group, while estimated glomerular filtration rate was significantly decreased among CKDs (23.64 ± 8.57) and diabetic nephropathy group (30.65 ± 14.34) than the diabetic (174.35 ± 86.13) and control groups (183.27 ± 44.17). Also, the level of NGAL was significantly increased in diabetic nephropathy groups (474.48 ± 308.22) and CKDs (367.37 ± 272.63) than diabetic groups (36.65 ± 28.30) and the control group (22.47 ± 21.16).
Conclusion
Diagnosis of diabetic nephropathy is based on the rise of creatinine and urea, and albumin/creatinine ratio with a decrease in estimated glomerular filtration rate. NGAL is a marker of CKD and any causes affecting the kidney. Cutoff point for NGAL was greater than 45 in patients with diabetes mellitus and greater than 30 in patients without diabetes, which were the best cutoff point for early detection of diabetic nephropathy.

Keywords: diabetic nephropathy, lipocalin, marker, urinary neutrophil gelatinase


How to cite this article:
Gazareen SS, Korani MA, Tawfeek AR, Omar TA, Dwidar GI. Role of urinary neutrophil gelatinase-associated lipocalin in diabetic and nondiabetic patients with nephropathy. Menoufia Med J 2021;34:135-40

How to cite this URL:
Gazareen SS, Korani MA, Tawfeek AR, Omar TA, Dwidar GI. Role of urinary neutrophil gelatinase-associated lipocalin in diabetic and nondiabetic patients with nephropathy. Menoufia Med J [serial online] 2021 [cited 2021 Dec 6];34:135-40. Available from: http://www.mmj.eg.net/text.asp?2021/34/1/135/312055




  Introduction Top


Diabetes mellitus (DM) is a group of metabolic disorders characterized by hyperglycemia resulting from a defect in insulin secretion, insulin action, or both [1]. Chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidney, and blood vessels [1]. Diabetic nephropathy is one of the most serious microvascular complications of diabetes. It is characterized by thickening of the glomerular and tubular basement membrane, and increased amount of mesangial matrix, which progress to glomerulosclerosis and tubulointerstitial fibrosis [2].

An interesting theory that provides a plausible physiological pathway for neutrophil gelatinase-associated lipocalin (NGAL) in chronic kidney disease (CKD) and its relationship with other indicators of renal function validates the reliability of NGAL as a marker of chronic renal disease [3]. This hypothesis called the forest fire theory assumes that the increased blood and urine NGAL levels during chronic renal disease are the consequence of sustained production by inflamed but viable tubular cells, whereas the increase in serum creatinine level and decrease in glomerular filtration rate (GFR) were the mere passive result of a general loss of indicator of how much active kidney damage was occurring within the overall condition of chronic renal impairment [3]. However, patients with proteinuria have been observed to show increased levels of urinary NGAL (uNGAL) even if clinical signs of overt chronic renal impairment were not yet present [4].

NGAL is filtered through the glomeruli and almost totally reabsorbed in the proximal tubular ducts. This reabsorption was mediated by binding with the protein-transporter complex Megalin–Cubilin, which is particularly expressed in the brush border of tubular cells. NGAL is then incorporated by endocytosis and successfully removed from the tubular fluid. So that only small quantities (5 ng/ml) can finally be found in the urine of healthy individuals [3]. Owing to the role of tubular injury in the progression of kidney damage in diabetes, several tubular factors have been recognized as a marker for CKD. These include monocyte chemoattractant protein-1, N-acetyl-B-d glycosaminidase, cathepsin, and few others [5]. The aim of this study was to study the role of NGAL in diabetic nephropathy patients with type 2 diabetes.

Patients and methods

This case- control study study was conducted on 88 patients who were admitted to the Internal Medicine Department at Menoufia University Hospital during the period from 20 December to 13 March 2019. All participants were dived into four groups: group 1: included 22 healthy individuals as the control group. Group 2 included 22 type 2 diabetic patients with normoalbuminuria; group 3 included 22 type 2 diabetic nephropathy patients; and group 4 included 22 nondiabetic patients with CKD. Ethical consideration: the study was approved by the Ethics Committee of Menoufia Faculty of Medicine. An informed consent was obtained from all participants before the study started. Inclusion criteria included patients with diabetic nephropathy and CKD. Exclusion criteria included other causes of diabetic nephropathy, such as heart failure, liver disease, pregnancy, those with recent infection, uncontrolled hypertension, and urinary tract infection. All patients were subjected to full history with particular emphasis on the duration of diabetes, urinary symptoms, symptoms of microangiopathic complications of diabetes, history of any other associated disease and therapeutic history age, sex, and diabetic treatment. General examination included blood pressure pulse, edema of lower limbs, diabetic foot, and soft tissue infection. Chest and heart examination were included for exclusion of any other causes of albuminuria such as chronic obstructive lung disease. Assessment of anthropometric data included weight, height, and body mass index. Routine laboratory investigations included Fasting blood sugar (FBS), 2-HppG, serum,urea. creatinine and urine creatinine were measured on AU680 automatic auto-analyzer (Beckman Coulter. United State of America (USA). Quantitative colorimetric measurement of glycated hemoglobin (HbA1c) was done as a percent of total hemoglobin using kits supplied by BIOTEC (London W1G9QR, UK). Urinary albumin was determined in fresh urine samples by an immunoturbidimetric method using HEALES microalbumin test kit on HEALES (Shenzhen Huisong Technology Development, China). Urinary albumin/creatinine ratio (ACR milligram/gram (mg/g) was calculated. Estimated glomerular filtration rate (eGFR) calculated by the Cockcroft-Gault formula as eGFR = [140−age (years)]×weight (kg)×0.85 if female/serum creatinine (mg/dl)×72, and uNGAL using ELISA kit was made on Glory Science Co. Ltd (Del Rio, Texas, USA).

Test principle: the kit is for the quantitative level of NGAL in the blood sample that adopts purified human NGAL to coat the microtiter plate to make a solid-phase antibody, then we added GAL to wells, combining NGAL antibody with labeled HRP to form an antibody–antigen enzyme–antibody complex, after washing completely, we added the TMB substrate solution. The TMB substrate becomes blue color as the HRP enzyme is catalyzed. The reaction was terminated by the addition of the stop solution and the color change was measured at a wavelength of 450 nm. The concentration of NGAL in the samples was then determined by comparing the optical density of the samples to the standard curve.

Statistical analysis

The results were tabulated and statistically analyzed using a personal computer using Microsoft Excel 2016 and SPSS version 21 (SPSS Inc., Chicago, Illinois, USA). Statistical analysis was done using descriptive: for example, percentage, mean, and SD. Analytical: that includes analysis of variance F test, post-hoc test, and receiver operating characteristic curve analysis. A value of P less than 0.05 was considered statistically significant.


  Results Top


Our study included 88 patients, 55 (60%) men and 33 (40%) women with a mean age of 53.64 ± 9.38 years. There was statistically significant difference between the studied groups regarding urea level and creatinine level (P < 0.001). Also, the mean value of albumin/creatinine ratio was 20.59 ± 3.87 in group I, 17.86 ± 4.31 in group II, 201.27 ± 50.04 in group III, and 330.55 ± 97.19 in group IV. Also, there was statistically significant difference between the studied groups regarding eGFR with a mean of 174.35 ± 86.13 in group II, 30.65 ± 14.34 in group III, 23.64 ± 8.57 in group VI, and 183.27 ± 44.17 in the control group I. Also, there was significant difference between the studied groups regarding uNGAL (P < 0.05) with a mean value of 231.59 ± 39.11 in group II, 398.86 ± 45.46 in group III, 778.18 ± 118.52 in group VI, and 91.36 ± 25.41 in control group I [Table 1].
Table 1: Comparison between the studied groups regarding urea and creatinine, albumin/creatinine ratio, estimated glomerular filtration rate and neutrophil gelatinase-associated lipocalin

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In this study, there was statistically significant difference between the studied groups regarding fasting blood glucose with a mean value of 172.13 ± 50.16 in group II, 186.55 ± 74.29 in group III, 82.82 ± 12.10 in group IV, and 86.05 ± 9.34 in control group I. Also, postprandial blood sugar was statistically significantly different among the studied groups with a mean value of 329.82 ± 74.48 in group II, 377.77 ± 90.60 in group III, 151.11 ± 26.98 in group IV, and 116.95 ± 12.41 in the control group. Furthermore, mean HbA1C was 8.08 ± 1.40 in group II, 8.93 ± 2.05 in group III, 3.23 ± 0.74 in group IV, and 3.54 ± 0.78 in the control group [Table 2].
Table 2: Comparison between the studied groups regarding fasting blood glucose, postprandial blood glucose, and glycated hemoglobin

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Receiver operating characteristic curve analysis showed that cutoff point, sensitivity, and specificity of NGAL in groups II and III was more than greater than 45 for uNGAL with a sensitivity of 95%, and specificity of 82% and the positive and negative predictive values were 84 and 94% with an accuracy of 89%. Cutoff point, sensitivity, and specificity of NGAL in groups I and IV was more than greater than 30 for uNGAL with a sensitivity of 91%, specificity of 77% with positive and negative predictive values being 80 and 89% with an accuracy of 85% [Table 3], [Figure 1] and [Figure 2].
Table 3: Cutoff point, sensitivity, and specificity of neutrophil gelatinase-associated lipocalin in the studied groups

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Figure 1: Cutoff point, sensitivity, and specificity of neutrophil gelatinase-associated lipocalin in diabetic with and without nephropathy groups.

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Figure 2: Cutoff point, sensitivity, and specificity of neutrophil gelatinase-associated lipocalin in the control group and chronic kidney disease group.

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  Discussion Top


Diabetic nephropathy is one of the most common microvascular complications of DM, greatly affecting the quality of life and survival of patients. As a global prevalence of type 2 diabetes is steadily increasing, the number of patients with diabetic nephropathy is expanding day by day. Diabetic nephropathy is now the leading cause of end-stage renal disease, a disease that is described as a medical catastrophe of worldwide dimensions [6]. Albumin excretion is the primary result of glomerular damage, but lack of tubular reabsorption of albumin also contributes to albuminuria [7].

The present study showed a statistically significant difference among all groups as regards the duration of DM. Similar to this study, Joslin et al. [8] suggested a link between the development and progression of diabetic nephropathy and the duration of diabetes in type 2 diabetic patients. Also, Kondaveeti et al. [9] noticed that there was a direct correlation between the duration of diabetes and the development of microalbuminuria, because of prolonged exposure to hyperglycemia as well as deposition of advanced glycated end products. However, Faglia et al. [10] found that there is no difference in the duration of diabetes among the stages of nephropathy.

In this study, there was a statistically significant difference among all studied groups as regards body mass index. In the Wahl et al. [11] study, there was a statistically significant difference among all studied groups as regards systolic blood pressure. This study showed an increase in glycated hemoglobin (HbA1c), FBG fasting blood sugar, and postprandial 2hpp in DM with proteinuria patients than DM without proteinuria. This finding agrees with that of Modebe and Massomi [12]. Also, Wahl et al. [11] recorded that poor glycemic control is a risk factor for most diabetic complications not only diabetic nephropathy.

Our results indicated that there was an increase in urea and creatinine and urinary albumin/creatinine ratio and decreased eGFR in DM with nephropathy (proteinuria patients compared with DM without nephropathy. This coincided with Dent et al. [13] who reported higher level of blood urea and creatinine to indicate a fall of GFR as a result of decreased capability of the kidneys to excrete waste products. Also, an increased urea level was observed in the studied CKD Patients, while Bennett et al. [14] found no elevation in urea in the studied CKD and found that blood urea was only elevated above the normal when more than 60% of kidney cells are no longer functioning and blood urea as an independent indicator of renal function was affected by other nonrenal factors such as gastrointestinal hemorrhage, mild dehydration, high-protein diet, and decreased perfusion of the kidneys. The present study showed a significant increase in the serum level of creatinine in diabetic patients with microalbuminuria and macroalbuminuria (group III). In comparison to the control group these results were in agreement with Ethridge et al. [15] and Kosieradzki et al. [16].

This study showed that there was statistically significant difference among the studied groups as regards fasting blood sugar it being significantly higher in patients with advanced stage of diabetic nephropathy compared with diabetic patients without nephropathy. There was also a significant increase in levels of fasting blood glucose in diabetic patients with microalbuminuria compared with nondiabetic patients. The results of fasting blood glucose among the studied groups were in agreement with Hinoda et al. [17], who found that FBS and HbA1C were significantly higher in patients with diabetic nephropathy compared with controls and patients without diabetes with the CKD group. Also, HbA1c was significantly higher in macroalbuminuric diabetic patients compared with macroalbuminuric diabetic patients. In diabetic patients, glucose reacts with amino groups on proteins to form covalently bound glycated products, an example of such a product was the HbA1c used to monitor the long-term control of glucose in diabetic patients. Moreover, Thorn et al. [18] confirm that the uncontrolled state of diabetes reflected by elevated fasting blood sugar HbA1c with a long duration of diabetes facilitate the progression of diabetic nephropathy. Also, Lehmann and Schleicher [19] demonstrated that type 2 diabetic patients have a state of chronic hyperglycemia and glucose-dependent processes are likely to be involved in the pathogenesis of diabetic complications, including nephropathy glucose-induced tissue injury may be mediated by the generation of advanced glycated proteins which have been implicated in nephropathy.

In the current study, there was a statistically significant difference between the studied groups regarding eGFR level; it was significantly lower in diabetic nephropathy and CKD than diabetic without nephropathy and control groups. Another study by Wahl et al. [11] found that eGFR was decreased in CKD patients and diabetic nephropathy than those diabetic patients without nephropathy and control groups. In this study, there was a statistically significant difference among the studied groups regarding uNGAL. Another study conducted by Bolignano et al. [20] who studied 33 patients with proteinuria secondary to CKD and diabetes found greater uNGAL values in diabetic nephropathy and CKDs patients than diabetic and control groups. Also, there was a significant correlation between uNGAL with serum creatinine and daily protein loss.


  Conclusion Top


Diagnosis of diabetic nephropathy is based on the rise of creatinine, urea, and albumin/creatinine ratio with a decrease in eGFR. NGAL is a new marker for the diagnosis of diabetic nephropathy. It is a marker of CKD and any causes affecting the kidneys. The cutoff point for NGAL was greater than 45 in patients with d DM and greater than 30 in patients without diabetes, and were the best cutoff values for early detection of diabetic nephropathy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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