Serum Neutrophil Gelatinase-Associated Lipocalin as a Marker of Renal Function in Non-Diabetic Patients with Stage 2–4 Chronic Kidney Disease

Abstract

The current Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines advocate creatinine-based equations for estimating GFR to identify patients with potential kidney disease and classify them into different stages due to the fact that serum creatinine is very insensitive to changes in the glomerular filtration rate. Very few biomarkers exist for monitoring chronic kidney disease. The aim of the study was to assess whether NGAL could represent a novel, sensitive marker of kidney function in adult patients with CKD. The study was performed on 92 non-diabetic patients with CKD stages 2–4. Serum and urinary NGAL as well as serum cystatin C were measured using commercially available kits. Serum NGAL was related, in univariate analysis, to serum creatinine, urinary NGAL, hemoglobin, hematocrit, leukocyte count, eGFR, and cystatin C. Urinary NGAL correlated with age, hemoglobin, hematocrit, serum creatinine, and eGFR. In multiple regression analysis, predictors of serum NGAL were creatinine (beta value = 0.97, p = 0.005), cystatin C (beta = 0.34, p = 0.01), and eGFR (beta value = 1.77, p = 0.001). In the healthy volunteers, serum NGAL correlated with age, serum creatinine, eGFR, leukocyte count, and cystatin C. Taking into consideration the fact that the recent DOQI (Dialysis Outcomes Quality Initiative) states that individuals with reduced GRF (glomerular filtration rate) are at greater risk for CVD and cardiac deaths, precise evaluation of renal function is important in order to select the appropriate strategy to reduce the cardiovascular risk. NGAL should be investigated as a potential early and sensitive marker of kidney impairment/injury.

Keywords:

• NGAL
• cystatin C
• GFR
• kidney function
• chronic kidney disease

INTRODUCTION

The current Kidney Disease Outcomes Quality Initiative (K / DOQI) guidelines advocate creatinine-based equations for estimating GFR to identify patients with potential kidney disease and classify them into different stages on the basis of these values[1] because serum creatinine is very insensitive to changes in the glomerular filtration rate. In our previous study, we found a very high prevalence of clinically significant CKD (as defined by a GFR<60mL/min) up to 33.8%, depending on the formula used to estimate GFR in patients with coronary artery disease and normal serum creatinine undergoing percutaneous coronary interventions.[2] Very few biomarkers exist for monitoring chronic kidney disease. Serum cystatin C was proposed as a new marker of GFR, even in CKD.[3] On the other hand, neutrophil gelatinase-associated lipocalin (NGAL), a member of lipocalin family, has been recently prove useful in the quantitation of CKD in a small pilot study of 45 children with GFR 15–89 mL/min.[4] NGAL is readily excreted and detected in urine, due to small molecular size (25kDa) and resistance to degradation as cystatin C. Therefore, the aim of the study was to assess whether NGAL could represent a novel, sensitive marker of kidney function in adult patients with CKD. We also studied possible relations between serum, urinary NGAL, and serum cystatin C and estimated GFR.

PATIENTS AND METHODS

The study was performed on 92 non-diabetic patients with CKD stages 2–4, recruited prospectively from an invasive cardiology outpatient unit (evaluation for a possible coronary angiography). The CKD stages were defined according to NKF/DOQI guidelines.[1] All the patients were informed about the aim of the study and gave their consent. The study was approved by Medical University Ethic Committee. The control group consisted of 34 healthy volunteers (eGFR over 90 mL/min) recruited mainly from the medical staff and their friends and families. Hemoglobin, hematocrit, cholesterol, and serum creatinine were studied by the standard laboratory methods in the one central laboratory at the University Hospital. We assessed kidney function according to the simplified MDRD formula.[5] NGAL was evaluated using commercially available ELISA from ANTIBODYSHOP (Gentofte, Denmark). Serum cystatin C was measured using commercially available kits from Dade Behring, Marburg, Germany. All tests were performed according to the manufacturer instructions by the same person. Comparisons between groups were done by ANOVA (Statistica 6.0, StatSoft, Krakow, Poland). Correlations between NGAL and other variables were evaluated by Pearson's or Spearman's test as appropriate.

RESULTS

All of the results are presented in Table 1. Serum NGAL was related, in univariate analysis, to serum creatinine (r = 0.39, p < 0.001), urinary NGAL (r = 0.25, p < 0.05), hemoglobin (p = −0.44, p < 0.001), hematocrit (r = −0.41, p < 0.001), leukocyte count (r = 0.29, p < 0.05), eGFR (r = −0.48, p < 0.001), and cystatin C (r = 0.46, p < 0.001). Urinary NGAL correlated with age (r = 0.34, p < 0.01), hemoglobin (r = −0.36, p < 0.01), hematocrit (r = −0.32, p < 0.01), serum creatinine (r = 0.31, p < 0.05), and eGFR (r = −0.36, p < 0.01). Then, we calculated serum NGAL/cystatin C and serum NGAL/urinary NGAL ratios and found that they were positively related to eGFR (r = 0.41, p < 0.001 and r = 0.24, p < 0.05, respectively). To the model of multiple regression analysis, parameters that correlated or tended to correlate with NGAL (p < 0.1) were included. When all of the parameters assessing kidney function (creatinine, cystatin C, eGFR) were included into the model of multiple regression analysis, predictors of serum NGAL were creatinine (beta value = 0.97, p = 0.005), cystatin C (beta = 0.34, p = 0.01), and eGFR (beta value = 1.77, p = 0.001). The equation explained 47% of the variation of serum NGAL in CKD group, F = 3.59, SE of estimate = 36.56, p < 0.0007.

Table 1 Basal clinical and biochemical characteristics of the studied groups

	Healthy volunteers (n = 34)	eGFR, 60–89 ml/min (n = 52)	eGFR, 30–59 ml/min (n = 25)	eGFR, 15–29 ml/min (n = 15)
Age (years)	43.15 ± 16.05	43.97 ± 13.12	46.60 ± 12.06	48.10 ± 9.13
eGFR (mL/min)	108.67 ± 18.14	74.27 ± 8.12^‡	49.53 ± 7.84^‡	21.43 ± 6.54^‡
Creatinine (mg/dL)	0.77 ± 0.18	1.03 ± 0.19^‡	1.47 ± 0.87^‡	2.77 ± 1.14^‡
Ht (%)	42.67 ± 2.97	41.97 ± 5.03	39.44 ± 3.73^†	35.85 ± 5.48^‡
Hb (g/dL)	14.75 ± 2.67	14.06 ± 1.66	13.66 ± 1.29^†	11.48 ± 2.22^‡
Leukocyte count (× 10^3/L)	5.45 ± 1.95	6.87 ± 1.98	7.67 ± 1.85	8.53 ± 2.96^†
hsCRP (mg/mL)	0.31 ± 0.10	0.89 ± 0.98	2.16 ± 2.08^‡	3.77 ± 2.52^‡
Cholesterol (mg/dL)	164.26 ± 38.25	188.20 ± 36.43	206.64 ± 39.44	179.80 ± 41.52
Serum NGAL (ng/mL)	76.34 ± 27.87	115.05 ± 40.03^†	154.19 ± 49.25^‡	258.19 ± 73.37^‡
Urinary NGAL (ng/mL)	5.98 ± 3.79	19.98 ± 13.17^†	36.18 ± 27.73^‡	79.85 ± 54.55^‡
Cystatin C (mg/L)	0.81 ± 0.19	1.24 ± 0.47	1.96 ± 1.28^†	3.14 ± 2.98^‡

Values given are means ± SD.

*p < 0.05, ^†p < 0.01, ^‡p < 0.001 vs. healthy volunteers.

In the healthy volunteers, serum NGAL correlated with age (r = 0.37, p < 0.05), serum creatinine (r = 0.45, p < 0.01), eGFR (r = −0.58, p < 0.001), leukocyte count (r = 0.38, p < 0.05), and cystatin C (r = 0.43, p < 0.01). Similarly, serum NGAL/cystatin C and serum NGAL/urinary NGAL ratios were positively related to eGFR (r = 0.49, p < 0.001 and r = 0.29, p < 0.05, respectively).

DISCUSSION

Our study in CKD patients demonstrated elevated serum and urinary NGAL together with a rise in serum creatinine and cystatin C. Moreover, serum NGAL closely correlates with serum cystatin C, creatinine, and eGFR, and could serve as a marker of impaired kidney function/kidney injury. There are a few existing biomarkers for quantitation or monitoring of CKD. Ravani et al.[6] reported that plasma asymmetric dimethylarginine (ADMA) was found be to negatively correlated with eGFR (using the MDRD formula) in adults with CKD. A similar correlation between CRP and kidney function was observed by Tonelli et al.[7] However, both biomarkers are produced outside of the kidney, and their levels were dependent on kidney function rather than degree of kidney injury. Deteriorating renal function may enhance overall inflammatory responses because of the decreased renal clearance of factors that are directly or indirectly involved in inflammation. In humans, declining renal function may also affect the levels of additional inflammatory molecules, as serum C-reactive protein (CRP) and interleukin-6 levels are inversely correlated with creatinine clearance.[8] In our study, we also observed a rise in hsCRP together with a fall in eGFR. NGAL belongs to the lipocalin family of proteins. NGAL was originally isolated from the supernatant of activated human neutrophils, but it is also expressed at a low level in other human tissues, including the kidney.[9] NGAL is synthesized systemically in response to kidney damage followed by glomerular filtration and tubular uptake, and it could be produced locally by injured tubules. A third source of NGAL may be activated neutrophils/macrophages or inflamed vasculature,[9] frequently found in chronic kidney disease. However, at present, we have very little information concerning this pool. Understanding the contribution of individual pools of NGAL synthesized in response to renal damage is important in defining the role of endogenous NGAL. So far, there are no knockout models (genetic deletion of NGAL from infiltrating leukocytes) to study this pool of NGAL. In recent studies, attempts to investigate neutrophil activation markers (i.e., NGAL) did not help explain the role of endogenous neutrophil/macrophage NGAL. Aström-Olsson et al.[10] found no significant changes in 8-isoprostane-prostaglandin F₂alpha and serum NGAL in forty-nine patients with acute myocardial infarction treated with primary percutaneous coronary interventions and infusion of abciximab. On the other hand, Paulsson et al.[11] observed elevated systemic IL-8 and MMP-9/NGAL concentrations in 13 with angiographically verified CAD, indicating a primed state in circulating neutrophils. In our study, serum NGAL correlated in univariate analysis with leukocyte count, but in multiple regression analysis, leukocyte count was not associated with serum NGAL in patients with chronic kidney disease. Taking all these data into consideration, it seems plausible that serum NGAL is rather renal dysfunction than leukocyte-dependent. Moreover, Ding et al.[12] demonstrated the upregulation of tubular expression of NGAL in biopsies from Lee grade III IgA nephropathy patients as well as increased urinary excretion of NGAL in these subjects (even in Lee grade II). In addition, the strict correlation between NGAL levels and residual renal function were reported in 26 APKD patients by Bolignano et al. [13] In our study, we confirmed a recent finding by Mitsnefes et al.,[5] who showed that NGAL could represent a marker of renal function in children with chronic kidney disease. They also found out that eGFR calculated using the Schwartz formula correlated very significantly with both cystatin C and NGAL. In their study, NGAL clearly outperformed cystatin C and eGFR at GFR levels below 30 mL/min. They indicated that serum NGAL might provide an additional accurate measure of kidney impairment in CKD, particularly at advanced stages. However, they did not provide any correlations with other parameters, except markers of kidney function, in studied children. Even subtle deteriorations in kidney function, as reflected by small changes in serum creatinine, may have devastating consequences in CKD patients; therefore, the ability to identify chronic renal insufficiency may allow early implementation of treatments that could arrest or delay the progression of renal damage, enabling the effective treatment of its complications. The strength of our study is the simultaneous measurement of urinary, serum NGAL, and cystatin C in non-diabetic patients. However, there are also some limitations. This is a single-center, cross-sectional study, without measured GFR. Taking into consideration the fact that the recent DOQI (Dialysis Outcomes Quality Initiative) states that individuals with a reduced GRF (glomerular filtration rate) is at greater risk for CVD and cardiac deaths,[1] precise evaluation of renal function is important in order to select the appropriate strategy to reduce the cardiovascular risk. NGAL should be investigated as a potential early and sensitive marker of kidney impairment/injury.