|Year : 2016 | Volume
| Issue : 1 | Page : 32-38
Urinary level of vitamin D-binding protein as a new biomarker for diabetic nephropathy
Samy A Khodeir1, Nesin M Kotb1, Kamal M Okasha1, Kamal A Ahmed1, Hala M Nagy2
1 Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
|Date of Submission||04-Nov-2015|
|Date of Acceptance||15-Dec-2015|
|Date of Web Publication||22-Mar-2016|
Kamal M Okasha
MD, Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta
Source of Support: None, Conflict of Interest: None
Diabetes is now the major cause of end-stage kidney failure, both in developing and developed nations. It is the primary diagnosis causing kidney diseases in 20-40% of patients starting treatment for end-stage renal diseases worldwide. The aim of the study was to evaluate the urinary level of vitamin D-binding protein (UVDBP) as a new biomarker for diabetic nephropathy (DN). Urine samples were obtained from 45 patients with type 2 diabetes mellitus and were classified into three groups (normoalbuminuric, microalbuminuric, and macroalbuminuric). Fifteen healthy participants served as the control group. The excretion levels of UVDBP were quantified with enzyme-linked immunosorbent assay. The results showed that UVDBP levels were significantly elevated in patients of the DN3 and DN4 groups compared with those of the DN2 group and normal controls. In conclusion, the current study demonstrated that UVDBP levels were significantly elevated in patients with DN. Moreover, a strong positive correlation was observed between the expression level of UVDBP and the development of DN. Thus, the findings indicate that UVDBP levels are a potential biomarker for the early detection of DN.
Keywords: Binding protein, diabetic nephropathy, vitamin D
|How to cite this article:|
Khodeir SA, Kotb NM, Okasha KM, Ahmed KA, Nagy HM. Urinary level of vitamin D-binding protein as a new biomarker for diabetic nephropathy. J Egypt Soc Nephrol Transplant 2016;16:32-8
|How to cite this URL:|
Khodeir SA, Kotb NM, Okasha KM, Ahmed KA, Nagy HM. Urinary level of vitamin D-binding protein as a new biomarker for diabetic nephropathy. J Egypt Soc Nephrol Transplant [serial online] 2016 [cited 2018 Jul 18];16:32-8. Available from: http://www.jesnt.eg.net/text.asp?2016/16/1/32/179210
| Introduction|| |
Diabetes mellitus (DM) belongs to a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of different organs, especially the eyes, kidneys, nerves, heart, and blood vessels . In 2013, according to the International Diabetes Federation, an estimated 381 million people had diabetes. Its prevalence is increasing rapidly, and by 2030 this number is estimated to almost double .
Diabetic nephropathy (DN) is one of the most important long-term complications of diabetes. It is characterized by the development of proteinuria with a subsequent decline in glomerular filtration rate (GFR), which progresses over a long period of time, often over 10-20 years [3,4]. Over the past 20 years, the prevalence of DN in the USA has increased in direct proportion to the prevalence of diabetes . Although DN cases vary largely among countries, on average it develops in 30-40% of patients with diabetes .
D-binding protein (DBP), a multifunctional and highly polymorphic plasma protein synthesized primarily in the liver, was identified about half a century ago and characterized as able to bind various forms of vitamin D . DBP (also referred to as Gc-globulin) is a member of the albumin gene family . Vitamin D circulate bound to vitamin D-binding protein (VDBP) (85-90%) and albumin (10-15%), with less than 1% of circulating hormone in its free form. VDBP prolongs the serum half-life of 25-hydroxyvitamin D and protects against vitamin D deficiency by serving as a vitamin D reservoir . The study comprised 45 patients referred to Tanta University Hospital with type 2 diabetes during the period from 13 March 2014 to 9 November 2014. They were examined at a single center (Nephrology Department, Tanta University, Egypt) and compared with 15 healthy controls. Patients were enrolled into the study after their informed written consent and approval of the Ethics Committee of Tanta University Hospitals were obtained. The patients were divided into four groups according to their urinary albumin concentration: group I, healthy controls (15 participants); group II, diabetic patients with normoalbuminuria (15 participants); group III, diabetic patients with microalbuminuria (15 participants); and group IV, diabetic patients with macroalbuminuria (15 controls). Patients with type I DM, collagen diseases (such as rheumatoid arthritis and systemic lupus erythematosus), oncogenic patients, patients with severe renal impairment (GFR < 30 ml/min), those with infections (such as urinary tract infection and chest infections), liver disease patients, and patients without any renal diseases other than diabetes and decompensated heart failure were excluded.
| Patients and methods|| |
All patients and controls were subjected to full history taking, thorough clinical examination, routine laboratory investigations [estimated glomerular filtration rate (eGFR), fasting and 2-h postprandial blood sugar, glycosylated hemoglobin, serum creatinine, blood urea, serum cholesterol, triglycerides, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and C-reactive protein (CRP)], and specific laboratory investigations [urinary vitamin D-binding protein (UVDBP) by enzyme-linked immunosorbent assay and albumin creatinine ratio (ACR)].
Collection and handling of blood and urine samples: Two blood samples were collected: the first after the patient had fasted for 8-10 h for determination of blood glucose, and the second under complete aseptic conditions after the patient had fasted for another 2-4 h. The collected samples were divided into two aliquots. For urine samples the second morning urine voided after rising was preferred.
Determination of UVDBP: This was carried out by means of the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for VDBP was precoated onto a microplate. Standards and samples were pipetted into the wells, and any VDBP present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked monoclonal antibody specific for VDBP was added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells and color was developed in proportion to the amount of VDBP bound in the initial step. The color development was stopped and the intensity of the color was measured.
Statistical presentation and analysis was conducted using the mean, SD, and χ2 -test. A P-value less than 0.05 was considered statistically significant.
| Results|| |
Comparison between studied groups with respect to ACR : ACR was significantly increased in group IV (mean ± SD = 1449.333 ± 643.456) compared with group III, group II, and group I (mean ± SD = 247.267 ± 36.654, 20.000 ± 5.720, and 16.400 ± 4.881, respectively), whereas there was no significant difference when comparing group III with group II and group I (P > 0.05). There was no significant difference between group II and group I either (P > 0.05) [Table 1] and [Figure 1].
|Figure 1: Comparison between studied groups regarding albumin creatinine ratio (ACR).|
Click here to view
|Table 1: Comparison between studied groups regarding albumin creatinine ratio|
Click here to view
Comparison between the studied groups regarding UVDBP: UVDBP was significantly increased in group IV (mean ± SD = 159.920 ± 19.608) compared with group III, group II, and group I (mean ± SD = 141.720 ± 7.540, 132.574 ± 8.757, and 123.347 ± 8.452, respectively). It was also significantly increased in group III compared with group I (P < 0.05), whereas there was no significant difference between group III and group II (P > 0.05), nor between group II and group I (P > 0.05) [Table 2] and [Figure 2].
|Figure 2: Comparison between studied groups regarding urinary vitamin D-binding protein (UVDBP).|
Click here to view
|Table 2: Comparison between studied groups regarding urinary vitamin D-binding protein|
Click here to view
Correlation between ACR and all variables in all groups: ACR correlated positively with UVDBP, systolic blood pressure, diastolic blood pressure, fasing blood glucose (FBS), HbA1c, creatinine, blood urea, cholesterol, triglycerides, LDL, and duration of diabetes but negatively with HDL and eGFR. There was no correlation between ACR and age, sex, PPBS, CRP, and smoking [Table 3].
|Table 3: Correlation between albumin creatinine ratio and all variables in all groups|
Click here to view
Correlation between UVDBP and all variables in all groups: UVDBP correlated positively with ACR, FBS, PPBS, HbA1c, creatinine, blood urea, cholesterol, triglycerides, LDL, and duration of diabetes but negatively with HDL and eGFR. There was no correlation between UVDBP and age, sex, systolic blood pressure, diastolic blood pressure, CRP, and smoking [Table 4] and [Figure 3],[Figure 4],[Figure 5] and [Figure 6].
|Figure 3: Correlation between urinary vitamin D-binding protein (UVDBP) and albumin creatinine ratio (ACR).|
Click here to view
|Figure 4: Correlation between urinary vitamin D-binding protein (UVDBP) and estimated glomerular filtration rate (eGFR).|
Click here to view
|Figure 5: Correlation between urinary vitamin D-binding protein (UVDBP) and high-density lipoprotein (HDL).|
Click here to view
|Figure 6: Correlation between urinary vitamin D-binding protein (UVDBP) and HbA1c.|
Click here to view
|Table 4: Correlation between urinary vitamin D-binding protein and all variables in all groups|
Click here to view
Receiver operating characteristic curves were used to assess the potential utility of UVDBP detection in our patients.
Cutoff value greater than 135 differentiated between diabetic patients and controls with a sensitivity of 90% and specificity of 76.7%.
Cutoff value greater than 151.2 differentiated between macroalbuminuric and microalbuminuric groups with a sensitivity of 66.7% and specificity of 100% [Table 5] and [Table 6] and [Figure 7] and [Figure 8].
|Figure 7: The sensitivity, specificity, and accuracy of urinary vitamin D-binding protein (UVDBP) (patients and controls).|
Click here to view
|Figure 8: The sensitivity, specificity, and accuracy of urinary vitamin D-binding protein (UVDBP) (microalbuminuric and macroalbuminuric groups).|
Click here to view
|Table 5: The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of urinary vitamin D-binding protein (patients and controls)|
Click here to view
|Table 6: The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of urinary vitamin D-binding protein (microalbuminuric and macroalbuminuric groups)|
Click here to view
| Discussion|| |
About 246 million people worldwide had diabetes in 2007, which is projected to increase to 550 million in 2030 . Increased risk for vascular complications, including both macrovascular and microvascular complications, accounts for the major cause of morbidity and mortality in diabetic patients .
VDBP is a 58-kDa glycoprotein and is present in the serum at a concentration of 300-600 mg/ml . It serves as the main carrier protein for vitamin D in the bloodstream. VDBP is important for the bioavailability of active 1,25-dihydroxyvitamin D (1,25 (OH) 2 D) and its precursor 25-hydroxyvitamin D . The transportation of vitamin D by VDBP is important for the functioning of a wide variety of tissues, and changes in VDBP activity result in the development of a number of diseases .
In addition to its transport function, VDBP is the parent molecule of VDBP-MAF (macrophage activating factor). VDBP-MAF is the product of the selectively deglycosylated form of VDBP and has been demonstrated to be a potent antiangiogenic and antitumorigenic molecule. Such functions would greatly benefit the regulation of the growth of cancer cells and protection against certain immune disorders. These important and diverse properties of VDBP have been suggested in previous studies with regard to a number of tumor types. Moreover, VDBP is important in inflammation processes .
The aim of the present work was to assess the UVDBP as a new biomarker for DN and evaluate its clinical significance in diagnosis as well as assessment of disease severity. In the present study, following quantitative measurements of 60 urine samples with enzyme-linked immunosorbent assay, diabetic patients were categorized into three groups depending on their ACR (normoalbuminuria, microalbuminuria, and macroalbuminuria). Receiver operating characteristic analysis rendered that an optimum cutoff value of UVDBP corresponding to 90.0% sensitivity and 76.7% specificity is appropriate for detecting DN.
The reasons underlying the enhanced excretion of UVDBP in patients with DN may be associated with renal tubular damage in DN patients . It has been increasingly documented that renal tubular injury plays an integral role in the pathogenesis of diabetic kidney disease. In addition, tubulointerstitial lesions were found to be the early and independent features of diabetic kidney disease . In a previous study, increased excretion of UVDBP was observed following long-term cadmium exposure (significant positive correlations between urinary levels of DBP and renal tubular dysfunction), and it was suggested that the marked loss of VDBP in the urine may be linked to renal tubular dysfunction and bone lesions in the inhabitants of cadmium-polluted areas . Clinically, a study by Zoidakis and colleagues (2012) identified that the reduction in VDBP levels in the urine of patients with invasive bladder cancer was significant, which is consistent with the findings by Li and colleagues (2012). Moreover, Li and colleagues also demonstrated that the expression levels of UVDBP were positively associated with the pathological classification of bladder cancer. Their results suggested that UVDBP may be a potential noninvasive biomarker for the early diagnosis and effective surveillance of bladder cancer . Mirkoviæ et al . (2013)  indicated that the urinary excretion of VDBP may be a novel urinary biomarker of tubulointerstitial damage. They also indicated that damaged tubular epithelial cells in areas of tubulointerstitial fibrosis may no longer be able to handle VDBP, resulting in gross VDBP loss into the urine, and that it can be modulated by antiproteinuric treatment in patients. Although the combination of the renin-angiotensin-aldosterone system blockade and dietary sodium restriction, an intervention considered optimal for renoprotection, considerably reduced VDBP excretion, they demonstrated that UVDBP excretion is increased early after renal injury and is associated with tubulointerstitial inflammation and fibrosis independently of albuminuria. In humans, UVDBP increased with increasing severity of renal damage, and responded to renoprotective therapy. Yet, persisting UVDBP above normal suggested persistent tubular interstitial damage .
Concerning UVDBP our results were in agreement with those of Tian and colleagues (2014), who studied 105 Chinese individuals with diabetes and 45 healthy volunteers. The patients were divided into three groups according to the value of ACR: the DM group without nephropathy and albuminuria; the early DN group with microalbuminuria; and the overt DN group with macroalbuminuria. They demonstrated that UVDBP levels were significantly elevated in patients with DN compared with patients without nephropathy. Moreover, strong positive correlation was observed between expression levels of UVDBP and the parameters of kidney dysfunction. The analysis of their result rendered an optimum cutoff value of 552.243 ng/mg corresponding to 92.86% sensitivity and 85% specificity. They concluded that UVDBP levels are a potential biomarker for early detection and prevention of DN .
It has been demonstrated that the presence of vitamin D deficiency or insufficiency in patients with diabetes is independently associated with the development of DN. This can be explained by decreased UVDBP secondary to tubular damage .
Our results revealed that eGFR, based on the MDRD study equation, was significantly lower in diabetic patients than in healthy controls. Also, serum creatinine and blood urea nitrogen (BUN) were significantly higher in microalbuminuric and macroalbuminuric patients when compared with healthy controls. This is in agreement with the findings of Tian et al. (2014) , who reported that higher levels of BUN and creatinine indicate a failing of GFR as a result of decreased capability of the kidney to excrete waste products.
However, in chronic kidney disease, BUN will be elevated above the normal level only when more than 60% of kidney cells are no longer functioning. Hence, a more accurate measure of renal function is generally preferred to assess the clearance. Israni and Kasiske (2011)  reported that the usefulness of BUN as an independent indicator of renal function is limited to the variability of its blood levels as a result of nonrenal factors such as gastrointestinal hemorrhage, mild dehydration, high protein diet, and decreased perfusion of the kidneys.
Concerning parameters of kidney function our results revealed that ACR correlated positively with creatinine and BUN and negatively with eGFR. These results were in agreement with those of Hoefield and colleagues (2011), who reported that individuals with diabetes and macroalbuminuria have an estimated 19 times more rapid decline in renal function compared with those without albuminuria (5.7% per annum vs. 0.3% per annum). Individuals with microalbuminuria also have a significantly increased rate of eGFR decline (five times greater) compared with people with normoalbuminuria .
Our findings are comparable to recent studies demonstrating that albuminuria is independently associated with increased risk for declining GFR in people with diabetes. In particular, data from a follow-up study (n = 1303) in a hospital-based cohort of patients with type 2 diabetes in Japan showed that those with normoalbuminuria at baseline had a relatively benign rate of progression, with less than 0.2% reaching eGFR below 30 ml/min compared with 24% of those with macroalbuminuria after 3 years ,.
An important limitation of the present study regarding the specificity of this biomarker should be considered when UVDBP detection is used for early prevention of DN. It has been demonstrated that UVDBP levels are closely associated with renal dysfunction. In the present study, urine samples were collected from patients with DN only but not from patients with additional nephropathies. This may have caused an overestimation of the specificity of VDBP as a biomarker for the detection of DN. Therefore, further studies including a larger sample and analyses of patients with various types of non-DN are required to clarify this issue.
| Conclusion|| |
Our findings indicate that UVDBP levels are a potential biomarker for early detection of DN, but an important limitation in terms of the specificity of this biomarker should be considered when UVDBP detection is used for early prevention of DN. In the present study, urine samples were collected from patients with DN only but not from patients with additional nephropathies. This may have caused an overestimation of the specificity of VDBP as a biomarker for the detection of DN. Therefore, we recommend that further studies including a larger sample and analyses of patients with various types of non-DN are required to clarify this issue.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Inzucchi S, Bergenstal R, Fonseca V, et al.
Diagnosis and classification of diabetes mellitus, American Diabetes Association. Diabetes 2010; 33
King H, Rewers M. Global estimates for prevalence of diabetes mellitus and impaired glucose tolerance in adults. WHO Ad Hoc Diabetes Reporting Group. Diabetes Care 1993; 16
Collins AJ, Foley RN, Herzog C, Chavers B, Gilbertson D, Herzog C, et al.
DN complication of diabetes. Am J Kidney Dis 2013; 61
De Boer IH, Rue TC, Hall YN, Heagerty PJ, Weiss NS, Himmelfarb J. Temporal trends in the prevalence of diabetic kidney disease in the United States. JAMA 2011; 305
Papale M, Di Paolo S, Magistroni R, Lamacchia O, Di Palma AM, De Mattia A, et al.
Urine proteome analysis may allow noninvasive differential diagnosis of diabetic nephropathy. Diabetes Care 2010; 33
American Diabetes Association. Standards of medical care in diabetes. Diabetes Care 2013; 36
Powe CE, Ricciardi C, Berg AH, Erdenesanaa D, Collerone G, Ankers E, et al.
DBP, a multifunctional and highly polymorphic plasma protein. J Bone Miner Res 2011; 26
Antoniades, CG, et al.
DBP (also referred to as Gc-globulin) is a member of the albumin gene family. Liver Transplant 2007; 13
Speeckaert, MM, et al.
Vitamin D circulate bound to vitamin D-binding protein and albumin. Clin Chem Lab 2008; 46
Yamagishi S, Matsui T. Advanced glycation end products, oxidative stress and diabetic nephropathy. Oxid Med Cell Longev 2010; 3
Dihazi H, Müller GA. Urinary proteomics: a tool to discover biomarkers of kidney diseases. Expert Rev Proteomics 2007; 4
Chun RF, Peercy BE, Adams JS, Hewison M. Vitamin D binding protein and monocyte response to 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D: analysis by mathematical modeling. PLoS One 2012; 7
Christakos S, Ajibade DV, Dhawan P, Fechner AJ, Mady LJ. Vitamin D metabolism. Endocrinol Metab Clin North Am. 2010; 39
Li F, Chen DN, He CW, Zhou Y, Olkkonen VM, He N, et al.
Identification of urinary Gc-globulin as a novel biomarker for bladder cancer by two-dimensional fluorescent differential gel electrophoresis (2D-DIGE). J Proteomics 2012; 77
Lau GJ, Godin N, Maachi H, Lo CS, Wu SJ, Zhu JX, et al
. Bcl-2-modifying factor induces renal proximal tubular cell apoptosis in diabetic mice. Diabetes 2012; 61
Uchida M, Teranishi H, Aoshima K, Katoh T, Kasuya M, Inadera, H. Elevated urinary levels of vitamin D-binding protein in the inhabitants of a cadmium polluted area, Jinzu River basin, Japan. Tohoku J Exp Med. 2007; 211
Doorenbos CR, de Cuba MM, Vogt L, Kema IP, van den Born J, Gans RO, et al.
Antiproteinuric treatment reduces urinary loss of vitamin D-binding protein but does not affect vitamin D status in patients with chronic kidney disease. Mol Biol 2012; 128
Thrailkill KM, Jo CH, Cockrell GE, Moreau CS, Fowlkes JL. Enhanced excretion of vitamin D binding protein in type 1 diabetes: a role in vitamin D deficiency?. J Clin Endocrinol Metab 2011; 96
Zoidakis J, Makridakis M, Zerefos PG, Bitsika V, Esteban S, Frantzi M, et al
. Profi lin 1 is a potential biomarker for bladder cancer aggressiveness. Mol Cell Proteomics 2012; 11:M111.009449.
Mirkoviæ K, Doorenbos CR, Dam WA, Lambers Heerspink HJ, Slagman MC, et al
. Urinary vitamin D binding protein: a potential novel marker of renal interstitial inflammation and fibrosis. PLoS One 2013; 8
:e55887. doi: 10.1371/journal.pone.0055887. Epub 2013 Feb 11.
Vegter S, Perna A, Postma MJ, Navis G, Remuzzi G, Ruggenenti P. Sodium intake, ACE inhibition, and progression to ESRD. Am J Soc Nphrol 2012; 23
X-Q Tian, L-M Zhao, J-P Ge, Y Zhang. UVDBP levels were significantly elevated in patients with DN compared to patients without nephropathy. Exp Med 2014; 7
Yamamoto N, Suyama H, Nakazato H, Yamamoto N. Immunotherapy of metastatic colorectal cancer with DBP derived macrophage activating factor. Cancer Immunol Immunother 2008; 57
Israni AK, Kasiske BL. Laboratory assessment of kidney disease: filtration rate, urinalysis, and proteinuria. Chapter 25. In Taal MW, Chertow GM, Marsden PA, et al.
, editors. Brenner and Rector′s the Kidney
, 9th ed. Philadelphia: Elsevier Saunders; 2011.
Hoefield RA, Kalra PA, Baker PG, Sousa Ines, Diggle PJ, Gibson MJ, et al.
Individuals with microalbuminuria also have a significantly increased rate of decline of eGFR. New Nephrol Dial Transplant 2011; 26
Meguro S, Shigihara T, Kabeya Y, Tomita M, Atsumi Y. Increased risk of renal deterioration associated with low e-GFR in type 2 diabetes mellitus only in albuminuric subjects.
Intern Med 2009; 48
Ninomiya T, Perkovic V, de Galan BE, Zoungas S, Pillai A, Jardine M, et al
., ADVANCE Collaborative Group Albuminuria and kidney function independently predict cardiovascular and renal outcomes in diabetes. J Am Soc Nephrol 2009; 2:1813-1821.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]