Neutrophil gelatinase-associated lipocalin (NGAL) has been demonstrated to be a novel biomarker in acute and chronic kidney disease. We hypothesized that 24-hour urinary NGAL excretion may be a predictor for renal damage in patients with systemic lupus erythematosus (SLE). Thirty-four SLE patients with renal involvement (SLE-renal group), 8 SLE patients without renal involvement (SLE-nonrenal group), 14 patients with non-SLE autoimmune diseases (disease control or DC group), and 12 healthy volunteers (normal control or NC group) were compared for 24-hour urinary excretion of NGAL and different cytokines. We found that the 24-hour urinary NGAL excretion in the SLE-renal group was higher than that in the SLE-non-renal, DC, and NC groups. However, the excretion of interleukin-10, transforming growth factor-
Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease affecting many organ systems. Lupus nephritis (LN) is a common and serious complication involving a major organ in patients with SLE. From one-third to one-half of SLE patients have different degrees of renal damage [
Recent advances in the diagnosis and treatment of LN have resulted in markedly improved renal function and survival in SLE patients with renal survival rates of 83–92% and 74–84% after 5 and 10 years, respectively [
At present, renal biopsy remains the gold standard in establishing the diagnosis and prognosis of LN that can guide treatment decisions. However, renal biopsy is not routinely performed serially and does not reliably represent the global status of the kidney [
Urine is an ideal source of potential biomarkers for LN, because of its easy accessibility and the fact that it can directly reflect the status of local inflammation/damage in the kidney. In recent years, many studies have proposed different cytokines/chemokines such as transforming growth factor (TGF)-
Neutrophil gelatinase-associated lipocalin (NGAL), a 25-kDa small protein belonging to the lipocalin protein superfamily, is specialized in binding and transporting small hydrophobic molecules including iron [
In the present study, we measured 24-hour urinary NGAL excretion using a commercially available enzyme-linked immunosorbent assay (ELISA) kit in SLE patients with and without renal involvement and compared this to the excretion of the cytokines IL-10, TGF-
Forty-two patients fulfilling the 1997 revised American College of Rheumatology classification criteria for SLE [
Medical records of all patients were reviewed. The demographic, laboratory, and clinical data were collected and are summarized in Table
Demographic, laboratory, and clinical characteristics of the different patient groups.a, b
Clinical parameter | SLE | DC | ||
SLE-active nephritis | SLE-renal | SLE-non-renal | ||
Patients (number) | 24 | 34 | 8 | 14 |
Age (years) | 33 (16) (24) | 33 (12) (34) | 36 (7) (8) | 36 (26) (14) |
Female: male ratio | 18 : 6 (24) | 27 : 7 (34) | 8 : 0 (8) | 14 : 0 (14) |
Leukocyte (109/I) | 7.26 (4.92) (24) | 6.47 (5.47) (34) | 6.73 (5.80) (8) | 9.33 (9.28) (13) |
Neutrophil (109/I) | 5.27 (4.62) (16) | 4.27 (5.52) (23) | 4.48 (5.82) (5) | 7.45 (6.62) (6) |
Lymphocyte (109/I)†, §§ | 0.76 (0.71) (16) | 0.64 (0.65) (23) | 1.45 (1.30) (5) | 2.12 (1.65) (6) |
Hemoglobin (g/I)††, §§ | 95 (24) (24) | 99 (24) (34) | 115 (33) (8) | 117 (18) (13) |
Platelet count (109/I) | 210 (180) (24) | 199 (145) (34) | 252 (334) (8) | 198 (159) (13) |
Serum creatinine ( | 124 (283) (24) | 106 (160) (34) | 71 (9) (8) | 62 (88) (14) |
Serum albumin(g/I)**, ††, &, §§ | 28 (8) (23) | 30 (8) (31) | 43 (3) (7) | 41 (7) (12) |
24-hour Ccr (mL/sec) | 0.72 (1.32) (20) | 0.89 (1.29) (24) | 1.09 (—) (3) | 4.91 (0.27) (7) |
24-hour urine protein (g/day)**, ††, &, §§ | 2.82 (7.59) (23) | 2.26 (4.64) (33) | 0.06 (0.05) (8) | 0.04 (0.11) (13) |
ESR 1 hour (mm) | 44 (32) (16) | 49 (30) (22) | 31 (46) (5) | 30 (46) (10) |
C3 (g/I)*, ††, #, §§ | 0.56 (0.47) (23) | 0.60 (0.54) (31) | 0.80 (0.37) (8) | 1.24 (0.41) (12) |
C4 (g/I) | 0.13 (0.16) (18) | 0.14 (0.15) (26) | 0.13 (0.13) (8) | 0.19 (0.10) (13) |
Positive anti-dsDNA†, § | 12/24 (50) | 15/32 (46.9) | 3/8 (37.5) | 0/7 (0) |
Total SLEDAI**, & | 15 (10) (23) | 14 (10) (29) | 8 (5) (8) | — |
Extrarenal SLEDAI | 4 (4) (23) | 4 (7) (29) | 8 (5) (8) | — |
Renal SLEDAI**, & | 12 (4) (23) | 8 (8) (32) | 0 (0) (8) | — |
Proteinuria >0.5 g/day**, & | 19/23 (82.6) | 29/33 (87.9) | 0/8 (0) | — |
Hematuria**, & | 20/24 (83.3) | 20/33 (60.6) | 0/8 (0) | — |
Pyuria* | 10/24 (41.7) | 10/33 (30.3) | 0/8 (0) | — |
Urinary casts | 9/24 (37.5) | 9/33 (27.3) | 0/8 (0) | — |
Active nephritis | 24/24 (100) | 24/34 (70.6) | 0/8 (0) | 0/14 (0) |
Leukopenia | 1/24 (4.2) | 5/34 (14.7) | 1/8 (12.5) | 0/13 (0) |
Lymphopenia†, §§ | 13/16 (81.3) | 19/23 (82.6) | 3/5 (60) | 1/6 (16.7) |
Thrombocytopenia | 2/24 (8.3) | 3/34 (8.8) | 0/8 (0) | 0/13 (0) |
Low complement†, § | 20/24 (83.3) | 27/33 (81.8) | 5/8 (62.5) | 6/14 (42.9) |
Arthritis | 1/24 (4.2) | 2/34 (5.9) | 0/8 (0) | — |
Rash | 5/24 (20.8) | 8/34 (23.5) | 2/8 (25) | — |
Serositis | 2/24 (8.3) | 2/34 (5.9) | 0/8 (0) | — |
CNS involvement | 3/24 (12.5) | 5/34 (14.7) | 2/8 (25) | — |
Vasculitis | 1/24 (4.2) | 3/34 (8.8) | 2/8 (25) | — |
Disease duration (years) | 3.5 (7.5) (24) | 5.0 (9.5) (34) | 3.1 (3.8) (8) | — |
Steroids††, §§ | 24/24 (100) | 34/34 (100) | 8/8 (100) | 9/14 (64.3) |
Daily prednisolone or equivalent dosage (mg/day)c †, § | 18.4 (17) (24) | 18 (14) (34) | 13 (20) (8) | 7 (20) (13) |
Hydroxychloroquine | 22/24 (91.7) | 30/34 (88.2) | 8/8 (100) | 12/14 (85.7) |
Azathioprine | 6/24 (25) | 11/34 (32.4) | 0/8 (0) | 3/14 (21.4) |
Mycophenolate mofetil | 1/24 (4.2) | 2/34 (5.9) | 0/8 (0) | 0/14 (0) |
Cyclophosphamide | 4/24 (16.7) | 4/34 (11.8) | 2/8 (25) | 1/14 (7.1) |
Aspirin or NSAIDs | 9/24 (37.5) | 14/34 (41.2) | 6/8 (75) | 7/14 (50) |
ACEIs or ARBs*, ††, § | 10/24 (41.7) | 12/34 (35.3) | 0/8 (0) | 0/14 (0) |
Pulse therapyd | 11/24 (45.8) | 12/34 (35.3) | 3/8 (37.5) | 3/14 (21.4) |
aIn continuous variables, data are shown as median (IQR) (number). In categorical variables, data are shown as number (percent).
b*
cThe mean daily dosage of prednisolone or its equivalent during the preceding 30 days.
dNumbers of patients who were treated by methylprednisolone or cyclophosphamide pulse therapy during the preceding 30 days.
SLE: systemic lupus erythematosus; DC: disease control; Ccr: creatinine clearance; ESR: erythrocyte sedimentation rate; C3: complement 3; C4: complement 4; anti-dsDNA: anti-double stranded DNA; SLEDAI: SLE Disease Activity Index; CNS: central nervous system; NSAIDs: nonsteroidal anti-inflammatory drugs; ACEIs: angiotensin-converting enzyme inhibitors; ARBs: angiotensin II receptor antagonists; IQR: interquartile range.
Disease activity of the SLE patients was assessed by the SLE Disease Activity Index-2000 (SLEDAI-2 K) [
The 24-hour urine amount was measured after thorough mixing. Ten mL of each urine sample was then centrifuged within 2 hours after collection at 3,000 rpm for 10 minutes to remove cellular debris. The cleaned urine samples were stored at −80°C until measurement. The concentration of NGAL, IL-10, TGF-
Statistical analysis was conducted using Statistical Package for Social Sciences version 13.0 for Window software (SPSS Inc.). Because the data of continuous variables were mostly not normally distributed, they were presented in terms of median with interquartile range (IQR), which represents central tendency and spread. For continuous variables, testing between 2 groups was performed by the Mann-Whitney
The present study comprised 42 SLE patients (ratio of females to males, 35 : 7; median age, 34 years; IQR, 11) including 24 SLE patients with active LN, 10 SLE patients with proteinuria only, and 8 SLE patients without renal involvement. In addition, the study included 14 patients without SLE but with other autoimmune diseases (4 had Sjogren’s syndrome; 3, anti-phospholipid syndrome; 1, vasculitis; 1, autoimmune hemolytic anemia; 1, rheumatoid arthritis with systemic sclerosis overlap; 1, polymyositis; 1, adult-onset Still’s disease; 1, pemphigus; 1, acute urticaria) as the DC group (ratio of females to males, 14 : 0; median age, 36 years; IQR, 26) and 12 healthy volunteers as the NC group (ratio of females to males, 10 : 2; median age, 32 years; IQR, 12). There was no significant difference in age or female-male ratio among SLE, DC, and NC groups. The demographic, laboratory, and clinical characteristics of the different patient groups are summarized in Table
Urinary NGAL excretion in the SLE-active nephritis and SLE-renal groups was significantly higher than that in the SLE-non-renal, DC, and NC groups (Figure
24-hour urinary excretion of NGAL, IL-10, TGF-
SLE patients | Controls | ||||
Active nephritis | Renal | Non-renal | DC | NC | |
Number | 24 | 34 | 8 | 14 | 12 |
NGAL (mg/day) | 50.41 (199.93) | 49.05 (114.45) | 21.78 (12.42) | 13.94 (12.53) | 7.65 (7.64) |
IL-10 (ng/day) | 2.34 (16.81) | 3.45 (21.06) | 10.03 (18.27) | 13.12 (15.99) | 5.40 (24.09) |
TGF- | 41.54 (75.59) | 23.82 (69.08) | 16.17 (16.13) | 15.57 (14.94) | 7.83 (6.39) |
TNF- | 6.01 (15.23) | 5.85 (17.63) | 20.49 (24.26) | 26.68 (39.04) | 0 (13.65) |
aData are shown as median (IQR).
NGAL: neutrophil gelatinase-associated lipocalin; IL-10: interleukin-10; TGF-
Comparison of 24-hour urinary excretion of neutrophil gelatinase-associated lipocalin (NGAL) (a), interleukin (IL)-10 (b), transforming growth factor (TGF)-
Although urinary NGAL excretion was higher in the SLE-renal group, it was not correlated with the excretion of IL-10 (Figure
Correlations among 24-hour urinary excretion of neutrophil gelatinase-associated lipocalin (NGAL), interleukin (IL)-10, transforming growth factor (TGF)-
In SLE patients with renal involvement, urinary NGAL excretion was positively correlated with serum creatinine levels (
Correlations of 24-hour urinary excretion of neutrophil gelatinase-associated lipocalin (NGAL) with renal functional parameters and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores in the systemic lupus erythematosus (SLE)-renal group. (a) Correlation between NGAL and serum creatinine levels. (b) Correlation between NGAL and 24-hour creatinine clearance (Ccr). (c) Correlation between NGAL and SLEDAI scores. *
Using a logistic regression univariate model with renal involvement as the outcome and 24-hour urinary NGAL excretion as a predictor, we found that urinary NGAL excretion was a significant predictor for the SLE-renal group in all study patients (
To quantify the diagnostic value of 24-hour urinary NGAL excretion in patients with renal involvement, we performed the ROC curve analysis. When the study population was confined to the SLE patients, the AUC for urinary NGAL excretion was 0.755 (
Sensitivity, specificity, positive predictive values, and negative predictive values of 24-hour urinary NGAL excretion at different cut-off values for the diagnosis of renal involvement in SLE patients.
Cutoff value (mg/day) | Sensitivity (%) | Specificity (%) | Positive predictive value (%) | Negative predictive value (%) |
---|---|---|---|---|
24.00 | 70.8 | 62.5 | 85 | 41.7 |
27.00 | 70.8 | 75 | 84.5 | 46.2 |
31.00 | 70.8 | 87.5 | 94.4 | 50 |
36.00 | 66.7 | 87.5 | 94.1 | 46.7 |
NGAL: neutrophil gelatinase-associated lipocalin; SLE: systemic lupus erythematosus.
Detection of sensitivity and specificity of 24-hour urinary neutrophil gelatinase-associated lipocalin (NGAL) excretion (a) and antidouble stranded DNA (anti-dsDNA) titers (b) for the diagnosis of renal involvement in systemic lupus erythematosus (SLE) patients by receiver operating characteristic curve (ROC) analysis.
Our study demonstrates that urinary NGAL, rather than IL-10, TGF-
NGAL was originally identified as a protein associated with purified gelatinase from the supernatant of activated neutrophils [
Although the SLE-non-renal group exhibited relatively lower disease activity (median SLEDAI score, 8) than the SLE-renal group (median SLEDAI score, 14), we observed no correlation between urinary NGAL excretion and the SLEDAI score. Furthermore, no correlation was observed with IL-10, TGF-
It is interesting to identify the source of NGAL in the urine of SLE patients with renal involvement. We believe that plasma NGAL is unrelated to urinary NGAL in SLE patients based on the observation that plasma NGAL did not differ significantly between SLE patients with or without active global or renal disease [
NGAL is normally expressed at very low levels in human renal tubular cells [
Cell types other than renal tubular cells in the body, such as various types of epithelial cells, endothelial cells, or immune cells, might contribute to urinary NGAL levels in patients even without nephritis. It is not surprising that those patients without renal involvement, both in the SLE-non-renal and non-SLE autoimmune disease groups, exhibited higher urinary NGAL excretion than did normal controls.
The role of NGAL in the pathogenesis of renal damage remains to be elucidated. In a murine model of ischemic kidney injury, administration of recombinant NGAL before ischemia or within 1 hour after reperfusion significantly decreased serum creatinine levels and apoptotic cell numbers, but increased the numbers of proliferating tubules cells [
Drawbacks of the present study include the following. (1) The patient numbers were relatively small. (2) Rather than early morning spot urine, we collected 24-hour urine, which was hard to maintain in a sterile condition; this might affect the accuracy of NGAL determination. (3) All SLE and non-SLE patients expressed diverse clinical manifestations, and disease activity of SLE patients was uniformly assessed by SLEDAI; this might complicate the statistical analysis. (4) We used stringent selection criteria for active nephritis, including the presence of cellular urine sediments, a >30% increase in serum creatinine levels within 3 months, or biopsy-proven nephritis, which reflected active renal inflammation. In consideration of possible irreversible damage to the glomerular basement membrane and lack of active renal inflammation, we classified a group of SLE patients with only proteinuria (>0.5 g/day) as the SLE-renal group along with those that were included in the SLE-active nephritis group. However, the diagnosis of active nephritis in our study was primarily based on laboratory data and rarely on renal pathological findings. Therefore, we could not analyze the relationship of urinary NGAL excretion with histopathological changes in active nephritis. (5) We used the commonly used SLEDAI scoring system to assess the disease activity of the SLE patients. It has the advantage of facilitating global evaluation of the patients, but lacks a severity grading that reduces its sensitivity [
Our study demonstrated that urinary NGAL is a potential biomarker for renal damage in SLE patients. Direct demonstration of NGAL mRNA and protein expression in the lupus kidney by
The authors declare that they have no conflict of interests.
This study was supported by a grant from the National Sciences Council (NSC99-2628-B-002-020-MY3). The authors are indebted to the Clinical Immunology and Transplantation Research Center, the Eighth Core Lab, Department of Medical Research, and National Taiwan University Hospital for providing space and laboratory facilities.