Evaluation of the cisplatin nephrotoxicity using the urinary neutrophil gelatinase-associated lipocalin ( NGAL ) in patients with head and neck cancer

Correspondence to: Luis Alberto Batista Peres. State University of Western Paraná (UNIOESTE). Rua Vicente Machado, no 2687, Country. Cascavel, PR, Brasil. CEP: 85813-250. E-mail: peres@certto.com.br Introduction: Acute kidney injury (AKI) in patients receiving cisplatin is common, therefore the evaluation of renal function in patients on use of nephrotoxic drugs is fundamental. Objective: To evaluate the incidence of AKI and the role of lipocalin associated to neutrophil gelatinase (NGAL) in the monitoring of renal function in patients with head and neck cancer (HNC) who received cisplatin. Methods: We prospectively studied 50 patients with HNC treated with three sessions of cisplatin. Blood and urine were collected 24 hours before cisplatin, 24 hours after infusion, 48 hours after each application and 35 days after the end of treatment (urine NGAL, C-reactive protein, creatinine, glomerular filtration rate, plasma lactate dehydrogenase and magnesium). Results: AKI was observed in 78% of patients. There was increase in creatinine, and decrease in GFR after each cycle of cisplatin, and increased urine NGAL. Positive association was observed between the levels of NGAL, creatinine and C-reactive protein. It was observed an increase in creatinine, NGAL, C-reactive protein and decreased GFR in AKI patients compared to patients without AKI. Conclusion: AKI was noted in 78% of patients with HNC treated with cisplatin and showed the correlation of NGAL with creatinine and GFR in demonstrating renal injury. NGAL levels may be elevated compared to baseline levels, even before the use of cisplatin. AbstrAct


IntroductIon
Complications such as impaired renal function and acute kidney injury (AKI) are caused in individuals with cancer by the disease itself or as a consequence of the medications used in oncologic treatment, particularly chemotherapy.Renal function assessment is carried out to allow safe administration of medication and monitor the effects of treatment on the patient.Serum creatinine may not suffice as an indicator of kidney function, given the error it may introduce in the estimation of the glomerular filtration rate (GFR). 1,2isplatin predominantly accumulates and is excreted through the kidneys.Non-toxic blood levels of the drug may be toxic in the kidneys, as concentrations in the tubule epithelial cells are five times higher than in blood.The limited possibility of increasing drug dosage posed by dose-dependent renal toxicity may compromise the effectiveness of treatment.Toxic effects occur primarily in the proximal tubule, particularly in the tubule epithelium cells on segment S-3.Distal tubules and glomeruli are involved at a later stage.Chronic nephrotoxicity is rare, as patients usually recover from acute drug toxicity.The main renal DOI: 10.5935/0101-2800.20140041][5] Several AKI biomarkers have been studied and shown their relevance in finding kidney injury, especially in septic patients, subjects in critical condition, heart surgery patients, and in individuals with contrast-induced nephropathy.The most commonly studied markers are neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18), kidney injury molecule-1 (KIM-1), fatty-acidbinding proteins (FABPs), and cystatin C, 4,6,7 all of which associated with improvements in early kidney injury detection when compared to traditional tests based on creatinine and GFR.
The literature lacks studies on patients with head and neck cancer treated with cisplatin, and few clinical trials have analyzed the role of NGAL as a biomarker of AKI in cancer patients.Enrolled cohorts usually include patients with various tumor types, on different cisplatin protocols, showing mixed results regarding the risk of nephrotoxicity.[10] Murine models of ischemic acute kidney injury have revealed that NGAL is one of the faster proteins to be synthesized by gene expression, with levels detected in the first urine sample within two hours of ischemia.Studies with rodents on cisplatin have also widely reported the presence of urinary NGAL.][13][14][15][16][17] The main purpose of this study was to assess the incidence of AKI and the role of NGAL in the renal function evaluation of patients with head and neck cancer treated with cisplatin, and its potential use in the early diagnosis of acute kidney injury.

MAterIAls And Methods
This prospective observational study was approved by the UNIOESTE Research Ethics Committee and given permit 272/2012-CEP.Data collection took place between October of 2012 and November of 2013.Patients deemed able to understand the treatment and its complications were asked to give their informed consent, and allowed the authors to report their information in the study.
Fifty patients aged 18 years and over diagnosed with head and neck cancer treated at the Cascavel Cancer Hospital with three cycles of cisplatin-based chemotherapy at a dosage of 100 mg/m 2 associated with radiotherapy (7000 cGy) were enrolled in the study.
Individuals previously treated with cisplatin, patients with a GFR < 60 ml/min/1.73m 2 , subjects with uncontrolled hyperthyroidism or hypothyroidism, and those who refused to have blood or urine samples taken as per the study protocol were excluded.
The Du Bois body surface area (BSA) formula was used to calculate the dose of chemotherapy. 18The Jaffee reaction without deproteinization was used to determine serum creatinine levels in mg/dL.Subsequently, the GFR in ml/min/1.73m 2 was calculated using the aMDRD. 1,2Other lab tests used the following methods: for LDH (U/L), the enzyme method; for urea (mg/dl), the enzyme/ automated method; for magnesium (mg/dL), the colorimetric method; for calcium (mg/ dL), the colorimetric/automated method; for sodium (mEq/L) and potassium (mEq/L), the selective electrode method; for CRP (mg/dl), the immunoturbidimetric method; for albumin (g/dL), nephelometry; urine was analyzed by sediment qualitative and quantitative analysis; and NGAL (mg/l) chemiluminescence (Abbott Diagnostics) was used to quantify the levels present in urine after storage at -80 °C.
The AKIN criteria was used to define and stage AKI 19 in all observation cycles of the study, as follows: AKIN 1: increases in creatinine greater than 0.3 mg/dL; AKIN 2: increases ≥ 100 % and < 200%; and AKIN 3: increases ≥ 200% from baseline creatinine levels 48 hours into follow-up.Another finding used in the comparative analysis between patients with and without AKI was an increase of 0.3 mg/dl in the baseline creatinine level (D0).Residual kidney disease was defined for subjects with a GFR < 60 ml/min/1.73m 2 on D78.Serum CRP concentrations ranging from zero to 1.0 mg/ DL 20 and magnesium levels from 1.6 to 2.3 mg/ dL were deemed normal. 21n statistical analysis, quantitative variables were expressed as mean ± standard deviation, for normal and non-normal data.Categorical variables were compared using Fisher's exact test or the chi-square test.In comparisons between two independent groups, Student's t-test was used in normal data and the Mann-Whitney U test when the hypothesis of data normality had been rejected.Nonparametric analysis of variance (Dunn's post test following the Kruskal-Wallis test) was employed to compare between three or more groups.Workup results were assessed for each treatment cycle (three days each) and throughout the study.Spearman's rank correlation coefficient was used to evaluate the correlation between the levels of NGAL and other continuous variables with non-normal distributions.
Groups with and without AKI were compared against each other.A significance level of 5% (p < 0.05) was adopted.Data sets were stored in a Microsoft Excel spreadsheet and treated using software program R (SPSS version 13.0).

results clinical characteristics
Fifty patients with head and neck cancer were considered eligible for the study.Their mean age was 58.5 years, and 80% were males.In terms of histopathology, 94% of the patients had squamous cell carcinomas.Forty-four percent had primary tumors in the oropharynx, 20% in the larynx, and 14% in the oral cavity.Seventy percent of the patients had stage IV disease.Seven patients had high blood pressure and four had diabetes, and none had been diagnosed with chronic kidney disease previously.Table 1 shows the clinical characteristics and the baseline workup of enrolled patients.

incidence Of aki
Seventy eight percent of the enrolled patients (n = 50) were diagnosed with AKI during the study.According to the AKIN classification, in the first cycle 26% of the patients had AKI, with 17% on AKIN 1 and 9% on AKIN 2; in the second cycle 42% had AKI, with 21% on AKIN 1, 19% on AKIN 2, and 2% on AKIN 3; and in the third cycle 29% had AKI, with 20% on AKIN 1, 6% on AKIN 2, and 3% on AKIN 3 (data not shown).Drug dosage had to be reduced for nine patients (18%) and treatment had to be suspended for another two subjects (4%) on D43 due to febrile neutropenia and for another three (6%) due to AKI, as per the institution's protocol.

cOMparisOn BetWeen grOups With and WithOut aki
No statistically significant differences were observed (p > 0.05) between the clinical characteristics and baseline workup of patients with and without AKI (see Table 3).When the workup results of patients with and without AKI in the three cycles of treatment were compared, increases were seen in NGAL, CRP and creatinine levels and decreases observed in the GFR.Statistical differences were seen only in the creatinine levels and GFR of patients with AKI against individuals without AKI starting on D21 (p < 0.05), while CRP was significantly increased only on D42 (p < 0.05); serum magnesium was not significantly different between the two groups throughout the study.The analysis of Spearman's rank correlation coefficients between urinary NGAL and creatinine, GFR, CRP, LDH, and magnesium revealed the existence of a positive correlation between NGAL and creatinine and a negative correlation between creatinine and GFR in all cycles (p = 0.000).A negative correlation between NGAL and magnesium was observed on cycle 1 (p = 0.037) and a positive correlation between creatinine and magnesium on cycle 2 (p = 0.020) 48 hours after cisplatin administration.
Positive correlations between urinary NGAL and CRP (p = 0.000) and creatinine and urinary NGAL (p = 0.015) were observed when all values obtained in a single correlation analysis throughout the treatment were included.Graph 1 shows the curve with median levels of NGAL, creatinine and CRP over the course of the study.

residual disease after treatMent
At the end of the study (D78), 32% of the patients (n = 16) still had a GFR below 60 ml/min, a statistically significant difference in relation to the number of individuals without kidney disease (Table 3).The other variables failed to show statistically significant differences between groups with and without residual renal disease.The mean NGAL in patients with residual disease was 97.34 g/L (Table 3), while the mean NGAL for the samples collected at the end of the study was 63.3 mg/l.

dIscussIon
3][24] Seventy eight percent of the patients had increases greater than 0.3 mg/dl from their baseline creatinine levels.Based on the AKIN classification, 62% had AKI during the study, 26% had AKI in the first cycle of treatment, 42% on the second cycle, and 29% on the third cycle.The reported incidence of AKI in individuals treated with cisplatin based on creatinine levels and GFR revolves around 20%-40%. 3 Although reports in the literature have described renal function recovery for most patients treated with cisplatin, 25 at the end of our study (D78) 32% of the patients (n = 16) still had a GFR below 60 ml/min and were at risk of developing chronic kidney disease.2.
Many studies have shown that elevation of serum creatinine alone does not correlate with kidney injury and may delay clinical diagnosis.The AKIN 19 and RIFLE 26 classifications have been used to redefine and prevent the occurrence of AKI with parameters based on serum creatinine and urine output.The AKIN classification indicates that elevations of 0.3 mg/dL from baseline creatinine levels within 48 hours are suggestive of subclinical kidney injury (AKIN 1), a finding often overlooked by general practitioners and oncologists, as this scale is more commonly used in critically ill patients and could lead to higher incidences of AKI related to medication, cisplatin in particular, than when creatinine or GFR is used alone.
Our patients had decreases in GRF and increases in serum creatinine and urea 48 hours after each treatment session with cisplatin, thus validating the use of these tests to verify altered kidney function only after and never before exposure to the drug (second and third cycles).
Elevations in mean NGAL levels were observed 48 hours after the first, second, and third cycles of cisplatin administration.NGAL levels were elevated prior to the days of subsequent cycles (before the second and third cycles) and remained high at the end of treatment, possibly indicating the early and persistent increases in NGAL levels before the second and third dose of cisplatin (after the initial insult with the first dose).NGAL levels were high even before creatinine levels had increased 48 hours after cisplatin administration.This may suggest kidney injury occurred before the creatinine levels of patients with head and neck cancer had increased, as they were given high dosages of a potentially nephrotoxic drug with cumulative toxicity.
Preclinical studies have shown that increased urinary NGAL levels were correlated with increased drug dosage three hours after cisplatin infusion in murine models of drug-induced nephrotoxicity. 14Moreover, NGAL was easily detected in urine within three hours of drug administration, against the 96 hours it took for serum creatinine levels to change. 27GAL has been used to identify cisplatininduced acute kidney injury in humans, despite the limitations related to the varied selection of patients and treatment protocols.A study 8 was carried out in 2010 with 24 patients given cisplatin for various tumor types.Twelve subjects with AKI were compared to individuals with stable creatinine levels.Urinary NGAL levels were significantly higher in patients with AKI than in controls on days 1, 2, 3 and 15 after cisplatin infusion.Among patients with AKI, increased urinary NGAL levels appeared to predict residual kidney disease within 15 days. 4,8In a more recent study with 34 patients with various tumor types and treated with different doses of cisplatin (50 mg/m 2 to 80 mg/m 2 ), the authors showed that serum NGAL levels could not predict drug-induced nephrotoxicity. 10ur study showed patients had increased NGAL levels in the days preceding the administration of cisplatin in the second and third cycles (D21 and D42) before their creatinine levels had increased, despite the lack of a difference when patients with and without AKI were compared.Moreover, a positive correlation was observed between the levels of NGAL and CRP, suggesting that higher NGAL levels in patients with head and neck tumors may also be seen as markers of inflammation, in addition to early nephrotoxicity.Evidence strongly suggests the involvement of inflammatory mechanisms in the pathogenesis of cisplatin nephrotoxicity. 3,5,28,29he positive correlation observed between NGAL and CRP at end of treatment (D78) and for sample totals probably relates to exposure to high dosages of cisplatin and cumulative dose effects in the third treatment cycle.Considering renal injury as an inflammatory mechanism, NGAL and CRP could be seen as inflammation markers of kidney injury.However, the disease itself and treatments such as radiotherapy and chemotherapy may be involved in the inflammatory process and in the increased levels of CRP. 25,30ur study also showed that the mean NGAL of patients with a GFR below 60 ml/min at the end of the study was higher (97.34 g/L), although not statistically different from patients without renal injury (56.0 mg/L).However, the mean NGAL value of the samples collected in D78 (63.3 mg/L) was statistically different from baseline values (D0).Most studies reporting NGAL levels used ELISA assays, which are not practical in a clinical environment. 31hemiluminescence magnetic microparticle immunoassay (CMIA), commercially available and performed through automated platform ARCHITECT (Abbott Diagnostics), was used in our study to measure urinary NGAL.2][33] Different cutoff levels of urinary NGAL have been described (over 10 g/L, over 60 mg/L, over 100 g/L) to identify patients at a higher risk of developing AKI. 34,35aleena et al. 36 (2012) designed a study to assess the efficacy of urinary enzymes α-glutathione-Stransferase (α-GST) and γ-GT as predictors of kidney injury in patients with head and neck cancer treated with cisplatin.The mean urinary levels of α-GST increased along different time intervals, especially within two hours of cisplatin administration, suggesting that urinary levels of proximal tubular enzymes α-GST and γ-GT may be useful in predicting early cisplatin-induced kidney injury.
Hypomagnesemia is one of the side effects seen in patients given cisplatin-based chemotherapy.However, the correlation between cisplatininduced hypomagnesemia and nephrotoxicity has not been completely elucidated. 37,38Our study showed decreased levels of serum magnesium even in patients without renal injury, although without statistically significant differences between AKIN groups, showing that hypomagnesemia is an important effect of cisplatin even in patients without renal injury. 3,25lves et al. 39 (2013) have recently described increased presence of hypomagnesemia in patients who did not recover renal function (70% vs. 31%), and multivariate analysis identified hypomagnesemia as an independent risk factor for non-recovery of renal function.Rat model studies have suggested that hypomagnesemia could cause dehydration and up-regulation of the cisplatin receptor, the organic cation transporter (OCT2), increasing the renal accumulation of cisplatin and worsening AKI. 40cute kidney injury biomarkers have been extensively studied in the description of AKI, 41 particularly ischemic AKI, both experimentally and in clinical settings in which ischemia is common (e.g.: sepsis and cardiopulmonary bypass).Although few clinical studies have looked into cancer patients, several authors have shed light on the cellular mechanism of cisplatin nephrotoxicity and a considerable number of biomarkers of AKI secondary to nephrotoxicity, particularly in preclinical studies in which cisplatin nephrotoxicity has been included. 42ew publications in the literature 36 have assessed the clinical toxicity of cisplatin and the objective measurement of toxicity using biomarkers to detect early kidney injury in patients with head and neck cancer, in whom the use of high doses of cisplatin significantly increases the incidence of AKI and nephrotoxicity, which combined represent a major limitation to the use of cisplatin in the treatment of solid tumors, including tumors of the head and neck.
6,43 Therefore, new randomized studies are needed to determine the true predictive and prognostic value of NGAL for patients with head and neck cancer treated with cisplatin and the cutoff points for each NGAL level in this specific disease and each clinical outcome.conclusIons Seventy-eight percent of the patients with head and neck cancer enrolled in our study treated with three cycles of cisplatin had acute kidney injury verified by NGAL, creatinine, and GFR within 48 hours of cisplatin administration.Their NGAL levels might also be higher than in baseline conditions even before chemotherapy with cisplatin, which may indicate the presence of kidney injury before increases in serum creatinine were observed.These findings suggest that the identification of patients at risk for acute kidney injury induced by cisplatin could stimulate the development of strategies for the treatment and prevention of nephrotoxicity or lead to a drug ban.The findings described herein have to be replicated and validated in prospective randomized trials.

Graph 1 .
Main median workup results throughout the duration of the study.Cr: Creatinine; NGAL: Neutrophil gelatinase-associated lipocalin; CRP: C-reactive protein.Note: See the p-value of each test on Table

tAble 3
Baseline and d78 clinical and WOrkup data Of head and neck cancer patients treated With cisplatin accOrding tO Occurrence Of aki * p-value from Fisher's exact test; † p-value from Student's t-test; ‡ p-value from Mann-Whitney U test.