Serum Neutrophil Gelatinase-associated Lipocalin Correlates With Kidney Function in Renal Allograft Recipients

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Malyszko J, et al

Abstract: The value of neutrophil gelatinase-associated lipocalin (NGAL) as a novel marker for early detection of acute renal failure has been highlighted recently. The aim of this study was to assess whether serum NGAL correlates with kidney function in kidney allograft recipients. Serum NGAL, creatinine, and estimated glomerular filtration rate (GFR) were evaluated in 100 kidney allograft recipients on triple therapy: calcineurin inhibitor, mycophenolate mofetil or azathioprine, prednisone, and healthy volunteers. Kidney transplant recipients had significantly higher NGAL than the control group. Serum NGAL in univariate analysis was strongly correlated with serum creatinine (r = 0.78). Estimated GFR (r = )0.69), on the other hand, was moderately correlated with white blood cell count (r = 0.43) and only weakly with other parameters. In multiple regression analysis, the best predictor of serum NGAL was eGFR (beta )0.69), with other predictors being white blood cell count (beta 0.25) and high sensitivity C-reactive protein (hsCRP) (beta 0.23) explaining 82% of NGAL concentration. Even successful kidney transplantation is associated with kidney injury as reflected by elevated serum NGAL and lowered eGFR. Therefore, NGAL needs to be investigated as a potential early marker for impaired kidney function/kidney injury, especially in patients with another risk factor for kidney damage, i.e., hypertension or diabetes.

Keywords: kidney function – kidney transplantation – NGAL

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Kidney transplantation is the renal replacement therapy of choice for most patients with end-stage renal disease, not only improving quality of life but also offering extended life expectancy compared with dialysis. Even though kidney transplantation may be successful, its function is not always restored to normal. Impaired kidney function is associated with a higher risk of morbidity and mortality compared with the general population, largely as a result of cardiovascular disease (1). The important problem remains with the assessment of kidney function following renal transplantation. Unfortunately, creatinine is an unreliable parameter describing kidney function (2). First, using serum creatinine to estimate true renal function has well-recognized inaccuracies and limitations (2). A marked reduction in GFR can be present before it is reflected in a rise in serum creatinine (up to 50% of kidney function has already been lost before creatinine might change). Neutrophil gelatinase-associated lipocalin (NGAL) is not only a member of the lipocalin family (3) but also is expressed at a low level in other human tissues including kidneys, prostate, and epithelia of the respiratory and alimentary tracts (4). Mishra et al. (5) highlighted the value of NGAL as a novel marker for the early detection of acute kidney injury. Because of its small molecular size (25 kDa) and resistance to degradation, NGAL is readily excreted and detected in urine. NGAL is highly accumulated in the human kidney cortical tubules, blood, and urine after nephrotoxic and/or ischemic injury (6). In recent publications, the value of urinary NGAL as an early biomarker for tubulointerstitial injury of IgA nephropathy and perhaps other types of renal disease, in general, was highlighted (7, 8). Moreover, serum and urinary NGAL levels were evaluated in patients with adult polycystic kidney disease (9). Both levels were significantly higher in patients and correlations were observed between these parameters and residual renal function (9). In our previous study on hypertensive and normotensive patients, we found positive correlations between NGAL, serum cystatin C, and eGFR in these patients (10). Up to the author's knowledge, there have been no data about simultaneous measurements of serum and urinary NGAL in relation to creatinine and eGFR in kidney diseases to highlight these different but equally important aspects of chronic kidney disease (CKD). Therefore, as creatinine is an unreliable indicator of kidney function, the aim of this study was to assess if NGAL correlates with kidney function in kidney allograft recipients.

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Patients and methods

This cross-sectional pilot study was performed on 100 renal allograft recipients (48 women, 52 men, age range 26–74 yr), including 15 diabetic patients. The immunosuppressive regimen consisted of CsA (blood trough CsA levels: 100–250 ng/mL), prednisone (5–10 mg daily) and azathioprine (100– 150 mg daily, n = 46) or MMF (mycophenolate mofetil, dose 2 g/d n = 54). All of them maintained sufficient and stable graft function, showed no clinical signs of rejection, no inflammation (Creactive protein below 6 mg/L using standard laboratory method), and no liver dysfunction (prothrombin time, alanine aminotransferase activity within normal range). Meantime after transplantation was 75 months. The control group consisted of 30 healthy volunteers (eGFR over 90 mL/min) recruited mainly from the medical staff, friends, and their families. All subjects gave informed consent, and the protocol was approved by the Medical University Ethics Committee. Blood was drawn in the morning when patients appeared for routine office assessment after an overnight fast. Venous blood samples were collected into vacutainer tubes. The blood was centrifuged at 2500 g for 15 min at room temperature in the clinical laboratory to yield serum. The samples were aliquotted and stored at )40C before assay. Assays were done within one month after sampling. NGAL assessment was performed from the first aliquot (samples were not frozen and thawed). GFR was estimated using simplified Modification of Diet in Renal Disease (MDRD) formula (eGFR = 186.3 · serum creatinine (mg/dL))1.14 · age)0.203 · 0.742 if female · 1.21 if African American) and the Cockcroft–Gault formula [creatinine clearance = (140)age) · body weight/ serum creatinine · 72 if female · 0.85]. The CKD stages were defined according to NKF/DOQI guidelines (11).

Complete blood count, urea, cholesterol, triglycerides, fasting glucose, total protein, and albumin were studied by standard laboratory methods. Serum creatinine was measured by the standard laboratory method (Jaffe) in the central laboratory of the hospital. NGAL was evaluated in serum using commercially available enzyme-linked immunosorbent assay (ELISA) from ANTIBODYSHOP (Gentofte, Denmark). In short, it is a sandwich monoclonal ELISA procedure determination in human urine and serum. Microtiter platelets coated with monoclonal antibodies against human NGAL were coated with samples (urine or serum) or standards (NGAL concentrations ranging from 1 to 1000 lg/L) and incubated with biotinylated monoclonal antibodies against human NGAL followed by streptavidin-conjugated horseradish peroxidase. Tetramethylbenzidine substrate was then added for color development, which was read at 450 nm with a microplate reader. High-sensitivity CRP was measured using a commercially available assay from American Diagnostica (Greenwich, CT, USA) and IL-6 using the commercially available kit from R&D (Abingdon, UK). All tests were performed according to the manufacturer's instructions by the same person.

The data were expressed as means ± SD or median (minimum, maximum values). The examination of the distribution normality of variables was done using the Shapiro–Wilk W-test. The comparisons between groups were done by ANOVA. Correlations between NGAL and other variables were evaluated by the Pearsons or Spearman's test as appropriate. Values of p < 0.05 were taken as statistically significant. Multiple regression analysis was used to determine independent factors affecting the dependent variable. The factors showing linear correlation with NGAL (p < 0.1) were included in the analysis.

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Results

All the data are presented in Table 1. Kidney transplant recipients had significantly higher serum creatinine, urea, cholesterol, triglycerides, fasting glucose, blood pressure, white blood cell count, hsCRP, IL-6, serum NGAL, and lower eGFR than the control group. In kidney transplant recipients only 1 patient had eGFR higher than 90 mL/min (CKD stage 1), 41 patients had stage 2 CKD (eGFR between 90 and 60 mL/min), 49 patients had stage 3 CKD (eGFR between 60 and 30 mL/min) and 9 patients had stage 4 CKD (eGFR between 30 and 15 mL/min). Kidney transplant recipients had significantly higher NGAL than the control group. The mean serum NGAL value in stage 2 CKD was 107.34 ± 36.76 ng/mL, in stage 3 CKD was 141.26 ± 47.54 ng/mL (p < 0.01 vs. stage 2) and in the stage 4 CKD was 198.87 ± 55.43 ng/ mL (p < 0.01 vs. stage 3, p < 0.001 vs. stage 2), respectively.

Univariate analysis

In univariate analysis serum NGAL was strongly correlated, with serum creatinine (r = 0.78, p < 0.001) (Fig. 1), eGFR by MDRD (r = )0.69, p < 0.001) (Fig. 2) and Cockcroft–Gault (r = )0.56, p < 0.001), moderately correlated with white blood cell count (r = 0.43, p < 0.001), urea (r = 0.45, p < 0.001), and only weakly correlated with other parameters: hemoglobin (r = )0.25, p < 0.05), hematocrit (r = )0.27, p < 0.05), duration of hypertension (r = 0.25, p < 0.05), age (r = 0.24, p < 0.05), presence of diabetes (r = 0.27, p < 0.05), albumin r = )0.27, p < 0.05), hsCRP (r = 0.26, p < 0.05), cyclosporine (CSA) concentration (r = 0.25, p < 0.05), and proinflammatory cytokine: IL-6 (r = 0.28, p < 0.05).

Multiple regression analysis

The parameters which correlated or tended to correlate with NGAL (p < 0.1) were included in the model of multiple regression analysis. When all the parameters assessing kidney function (creatinine, eGFR by either formula) were included in the model of multiple regression analysis, the best predictor of serum NGAL in multiple regression analysis, turned out to be eGFR (beta )0.69, p < 0.00001), with other predictors being white blood cell count (beta 0.25, p < 0.05) and hsCRP (beta 0.23, p < 0.05) explaining 82% of NGAL concentration. When eGFR was substituted by creatinine the results were the same. Adjusted r 2 for variables in the equation = 0.82, F = 7.15, p < 0.00001, SE of estimate = 52.41.

Impact of diabetes, hypertension, and gender of kidney allograft recipients on serum

NGAL Normotensive kidney allograft recipients had significantly lower NGAL (109.54 ± 23.65 vs. 128.5 ± 57.74 ng/mL, p < 0.05) than hypertensives. Despite similar serum creatinine levels, eGFR was significantly higher in normotensives (p < 0.05) than in hypertensives. Serum NGAL was significantly higher in diabetic patients than in non-diabetic patients (132.14 ± 99.34 vs. 93.63 ± 50.05 ng/mL, p < 0.05), whereas creatinine did not differ significantly between these groups. Females had lower eGFR (all formulas) relative to males (MDRD 54.10 ± 20.84 vs. 72.15 ± 21.25 mL/min, p < 0.01, Cockcroft– Gault 47.69 ± 17.81 vs. 60.59 ± 16.61 mL/min, p < 0.05).

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Discussion

Even successful kidney transplantation did not restore normal kidney function. Generally, patients are transferred from stage 5 CKD into stage 2 or 3 CKD. In current practice, creatinine is not a reliable indicator of kidney function, particularly during acute changes. Moreover, calcineurin inhibitors are widely known to be nephrotoxic. Therefore, the administration of cyclosporine or tacrolimus requires careful monitoring of blood through concentration. In this study, we found that serum NGAL correlated with kidney function/ dysfunction in renal allograft recipients. The strongest predictor of serum NGAL was eGFR indicating the utility of NGAL measurements in this population. It has been recently reported that NGAL could represent a marker of renal function in chronic kidney disease in children (12) and adults (13). NGAL is synthesized systemically in response to kidney damage followed by glomerular filtration and tubular uptake, it could be produced locally by injured tubules. As a small molecule, NGAL is freely filtered by the glomerulus. It is largely absorbed in the proximal tubules by efficient megalin-dependent endocytosis (14). Mori et al. (6) showed that a systemic injection of labeled NGAL resulted in an accumulation of NGAL in the proximal tubules without a concomitant appearance in urine. Thus, NGAL excretion in urine, in the case of associated proximal tubule injury, is likely to be due to NGAL reabsorption and/or enhanced de novo NGAL synthesis. Indeed, in the model of acute kidney injury, a massive and rapid upregulation of NGAL mRNA was demonstrated (15). Schmitt-Ott et al. (14) hypothesized that although NGAL was synthesized in the distal nephron, it was also delivered to the proximal tubule from the circulation. It could most likely be explained by glomerular filtration of circulating NGAL and subsequent uptake by proximal tubular epithelia through endocytosis (6). Concerning the serum/plasma pool of NGAL, it was reported that acute kidney injury resulted in a dramatic increase in NGAL mRNA in various organs, mainly the lungs and the liver (16). Thus, NGAL could be released into circulation and present a systemic pool. The third source of NGAL may be activated by neutrophils/macrophages or inflamed vasculature, frequently found in coronary artery disease, hypertension, and chronic kidney disease (17), as NGAL is increased in atherosclerotic plaques (18). It may be also released into the circulation from inflamed/ damaged endothelium in kidney allograft recipients, a common finding in this population (19). In addition, chronic kidney disease is associated with a subclinical inflammatory state and the majority of kidney allograft recipients exhibit stage 2 or 3 CKD. Tonelli et al. (20) reported a significant correlation between CRP and kidney function. The declining renal function may also affect the levels of the inflammatory molecules as they are inversely correlated with creatinine clearance (21). Therefore, the source of serum and urinary NGAL during renal injury could be a sum of three sources: kidney, liver, and leukocytes (from the injured tissues and circulation). Moreover, an impairment in kidney function, as reflected by a decreased GFR, could contribute to the decreased clearance of NGAL and subsequent accumulation in circulation, as shown by elevated NGAL levels in patients with chronic kidney disease (12, 13). It may explain that the best predictor of NGAL was eGFR with other predictors being leukocyte count and hs-CRP.

In the post-ischemic kidney, NGAL is upregulated in proximal tubules and distal nephron segments (22). Nephrotoxic injury after cisplatin administration in mice resulted in a similar pattern of NGAL changes (23). Parikh et al. (24) in their study in a small group of kidney transplant recipients reported that urinary NGAL may represent an early predictive biomarker of delayed graft function, another model of ischemia-reperfusion injury. In their previous study, NGAL staining intensity in early protocol biopsies was suggested to be a novel predictive biomarker of acute kidney injury following transplantation (25). In a recent study, Kusaka et al. (26) suggested that monitoring of serum NGAL levels may make it possible to predict graft recovery and the need for hemodialysis after kidney transplantation from donors after cardiac death.

In this study, in univariate analysis, we observed a correlation between cyclosporine concentration and serum NGAL. The studies have demonstrated that cyclosporine causes vasoconstriction of the afferent and efferent glomerular arterioles (27) and reductions in renal blood flow and GFR. The exact mechanism of vasoconstriction is unclear. Cyclosporine administration is associated with transient reductions in renal plasma flow and glomerular filtration rate, which correlate both with dose and with peak cyclosporine levels reached 2–4 h after the oral dose (28). In the recent study of Schaub et al. (29), the stable transplant with subclinical tubulitis group had slightly higher levels of NGAL (p = 0.06) than the stable transplant with normal tubular histology group with substantial overlap. The clinical tubulitis Ia/Ib and the other clinical tubular pathology groups had significantly higher levels of NGAL than stable transplants with normal tubular histology or stable transplants with subclinical tubulitis (p < 0.002). We did not perform the protocol biopsy in our patients. As cyclosporine is known to be nephrotoxic, it affects GFR and is responsible for changes in kidney histology. We may hypothesize that the observed correlation may be due to these associations.

The best predictor of NGAL was eGFR, thus we should take into account the usefulness of NGAL estimation in clinical practice. In the perspective of the development of urinary (30) or whole blood/plasma assays (31) for NGAL estimation. This method would be available as a new tool for the assessment of kidney function. The assay for the whole blood or plasma is easy with quantitative results available in 15 min and requires only a microliter sample (31). The urinary assay, on the other hand, requires only 150 lL of urine and results are available within 35 min (30).

According to Mori and Nakao (32), at least two aspects should be considered to evaluate the severity of renal failure: the ratio of functional versus atrophic nephrons and the severity of ongoing damage. They proposed that NGAL indicates the extent of kidney lesions. Nowadays, a lot of randomized control trials are ongoing to validate NGAL as a potential biomarker of kidney injury in different clinical settings (22 RCT on the website ClinicalTrials.gov). However, at this moment creatinine still remains a gold standard to assess kidney function. Some promising biomarkers are on the horizon and awaiting clinical implementation.

Therefore, NGAL should be investigated as a potential early and sensitive marker of kidney impairment/injury. Moreover, due to its sensitivity, NGAL may uncover nephrotoxicity in new medications and might help in monitoring immunosuppressive therapy with calcineurin inhibitors (known to be nephrotoxic) in this vulnerable population of kidney allograft recipients

From: ' Serum neutrophil gelatinase-associated lipocalin correlates with kidney function in renal allograft recipients' by Malyszko J, et al

---Clin Transplant 2009: 23: 681–686 DOI: 10.1111/j.1399-0012.2009.01034.x

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