Acute kidney injury (AKI) is a complex clinical syndrome characterized by a rapid decline in renal excretory function, accumulation of nitrogenous waste products, and dysregulation of fluid and electrolyte homeostasis [1,2]. In intensive care units (ICUs) and emergency settings, AKI affects up to 30% to 50% of critically ill patients and is independently associated with prolonged hospitalization, increased healthcare costs, transition to chronic kidney disease (CKD), and high short-term mortality [3,4]. Despite major advances in supportive critical care and hemodialysis technology, the clinical outcomes of AKI remain suboptimal, largely due to delayed therapeutic intervention [5]. The traditional reliance on functional parameters, specifically serum creatinine (SCr) and urine output, represents a fundamental limitation in current nephrology practice [6]. Serum creatinine is a delayed, non-specific biomarker that fluctuates based on age, sex, muscle mass, nutritional status, fluid administration, and liver function [7]. Crucially, serum creatinine concentrations typically rise only after more than 50% of functional renal parenchymal mass has been damaged, creating a critical diagnostic lag phase of 24 to 48 hours following acute structural tubular insult [8,9]. During this crucial therapeutic window, ongoing cellular injury proceeds undetected and unmitigated. To overcome these limitations, recent research has focused on structural biomarkers of renal tubular injury [10]. Neutrophil gelatinase-associated lipocalin (NGAL), a 25-kDa protein belonging to the lipocalin superfamily, is rapidly synthesized and secreted by distal nephron epithelial cells in response to ischemic, toxic, or inflammatory renal insult [11,12]. While normally expressed at low levels in human tissues, NGAL synthesis is upregulated multi-fold within 2 to 4 hours of tubular injury, making it an ideal candidate for early detection [13,14]. Although preliminary trials in pediatric cardiac surgery cohorts demonstrated exceptional diagnostic efficacy, validation in real-world, heterogeneous adult critical care populations in low- and middle-income clinical settings remains essential [15,16]. Therefore, this study aimed to prospectively evaluate the diagnostic accuracy, temporal kinetics, and prognostic utility of serum and urinary NGAL in early AKI detection among adult ICU patients at a tertiary academic medical center in Karnataka, India.

Study Setting This prospective observational cohort study was conducted over a period of time. Written informed consent was routinely secured from all eligible patients or their legal authorized representatives in accordance with the Declaration of Helsinki. Study Population and Eligibility Criteria A non-probability consecutive sampling strategy was utilized to recruit adult patients admitted to the medical, surgical, and cardiac intensive care units. Inclusion criteria comprised adult patients aged 18 years or older who presented within 12 hours of ICU admission with a high risk of AKI (due to severe sepsis, septic shock, major abdominal/cardiac surgery, hemodynamic instability, or nephrotoxic exposures). Exclusion criteria included pre-existing chronic kidney disease (baseline estimated glomerular filtration rate [eGFR] < 30 mL/min/1.73 m² or Stage 4–5 CKD), end-stage renal disease (ESRD) on maintenance dialysis, prior renal transplantation, active urinary tract infection at admission, urinary tract obstruction, or death within 12 hours of enrollment. Sample Collection and Biomarker Measurements Venous blood samples and fresh mid-stream or catheterized urine specimens were collected concurrently at enrollment (0 hour / admission) and subsequently at 4, 12, 24, and 48 hours post-admission. Blood specimens were centrifuged at 3000 rpm for 10 minutes, and serum was aliquoted. Serum and urinary NGAL concentrations were quantitatively measured using commercially available human NGAL Enzyme-Linked Immunosorbent Assay (ELISA) kits (Abcam, Cambridge, UK) in accordance with the manufacturer's protocol. The assay demonstrated intra-assay and inter-assay coefficients of variation of < 5% and < 8%, respectively. Routine biochemical parameters, including serum creatinine, blood urea nitrogen, serum electrolytes, and blood cell counts, were analyzed using standard automated laboratory platforms. Clinical Endpoints and AKI Definition The primary diagnostic endpoint was the development of AKI within 48 hours of admission, defined according to the Kidney Disease: Improving Global Outcomes (KDIGO) consensus criteria: an absolute increase in SCr by ≥ 0.3 mg/dL within 48 hours, or a relative increase in SCr to ≥ 1.5 times baseline value within 7 days, or oliguria (urine output < 0.5 mL/kg/h for 6 consecutive hours) [3]. AKI severity was categorized into KDIGO Stages 1, 2, and 3. Secondary clinical endpoints evaluated included the need for Renal Replacement Therapy (RRT), ICU length of stay exceeding 7 days, and 30-day all-cause mortality. Statistical Analysis All analytical procedures were performed using SPSS version 28.0 (IBM Corp., Armonk, NY, USA). Descriptive data were presented as mean ± standard deviation (SD) for normally distributed continuous variables, median with interquartile range (IQR) for skewed parameters, and absolute numbers with percentages for categorical variables. Comparison of continuous variables between AKI and non-AKI cohorts was executed using the Independent Samples Student's t-test or the Mann-Whitney U test, as appropriate. Cross-tabulations and categorical comparisons were conducted using the Pearson Chi-square (χ²) test or Fisher's exact test. Diagnostic capacity was evaluated by constructing Receiver Operating Characteristic (ROC) curves, calculating the Area Under the Curve (AUC), optimal threshold values (via Youden's Index), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). A two-tailed p-value < 0.05 was defined as statistically significant for all tests.

Demographic and Baseline Characteristics A total of 150 critically ill adult patients who fulfilled the inclusion criteria were enrolled in the study. During the initial 48-hour observation window, 48 patients (32.0%) met the KDIGO criteria for AKI, whereas 102 patients (68.0%) did not develop AKI. Demographic parameters, baseline clinical variables, and underlying primary diagnoses were well-balanced between the two groups, as presented in Table 1. Sepsis was the single most common primary etiology associated with AKI development (45.8% vs. 23.5%, χ² = 7.82, p = 0.005). The baseline serum creatinine measured at admission was comparable between patients who subsequently developed AKI and those who remained non-AKI (1.02 ± 0.24 mg/dL vs. 0.98 ± 0.21 mg/dL, p = 0.308), confirming that baseline renal functional measures failed to discriminate patients at risk during initial evaluation. Table 1: Demographic and Clinical Characteristics of Study Participants Characteristic Total Cohort (n=150) AKI Group (n=48) Non-AKI Group (n=102) p / χ² Value Age (Years), Mean ± SD 54.8 ± 12.4 57.2 ± 11.8 53.7 ± 12.6 t = 1.63, p = 0.105 Male Gender, n (%) 92 (61.3%) 31 (64.6%) 61 (59.8%) χ² = 0.31, p = 0.578 Diabetes Mellitus, n (%) 45 (30.0%) 18 (37.5%) 27 (26.5%) χ² = 1.84, p = 0.175 Hypertension, n (%) 58 (38.7%) 22 (45.8%) 36 (35.3%) χ² = 1.52, p = 0.218 Primary Sepsis, n (%) 46 (30.7%) 22 (45.8%) 24 (23.5%) χ² = 7.82, p = 0.005* Post-Surgical / Trauma, n (%) 52 (34.7%) 15 (31.3%) 37 (36.3%) χ² = 0.36, p = 0.548 Cardiogenic Shock, n (%) 28 (18.7%) 7 (14.6%) 21 (20.6%) χ² = 0.74, p = 0.389 Baseline SCr (mg/dL) 0.99 ± 0.22 1.02 ± 0.24 0.98 ± 0.21 t = 1.02, p = 0.308 APACHE II Score, Mean ± SD 18.4 ± 4.6 22.1 ± 5.2 16.7 ± 3.8 t = 7.14, p < 0.001* Values are expressed as Mean ± SD or frequency (percentage). Comparison performed using Independent Samples Student’s t-test or Pearson Chi-square (χ²) test as appropriate. *Statistically significant (p < 0.05). SCr: Serum Creatinine; APACHE II: Acute Physiology and Chronic Health Evaluation II Diagnostic Accuracy and ROC Curve Performance Serum and urinary NGAL levels measured at ICU admission (0 hour) demonstrated exceptional discriminatory performance for early AKI prediction. Admission serum NGAL was markedly higher in patients who developed AKI compared to non-AKI controls (214.5 ± 82.3 ng/mL vs. 64.2 ± 18.1 ng/mL, p < 0.001). Similarly, admission urine NGAL levels were dramatically elevated in the AKI group (198.4 ± 76.5 ng/mL vs. 52.1 ± 14.3 ng/mL, p < 0.001). As summarized in Table 2, Receiver Operating Characteristic (ROC) curve analysis revealed an Area Under the Curve (AUC) of 0.92 (95% CI: 0.87–0.96) for serum NGAL at an optimal cutoff of 160 ng/mL, providing a sensitivity of 89.6% and specificity of 85.3%. Urinary NGAL yielded an AUC of 0.89 (95% CI: 0.83–0.94) at a cutoff of 140 ng/mL, with 85.4% sensitivity and 82.4% specificity. In contrast, admission serum creatinine yielded a poor AUC of 0.64 (95% CI: 0.56–0.72), confirming its inadequacy as an early diagnostic indicator. Figure 1 visually illustrates the superior ROC curves of serum and urine NGAL compared to conventional serum creatinine. Table 2: Diagnostic Accuracy Parameters of Biomarkers at Admission for Predicting AKI Biomarker Cutoff Value AUC (95% CI) Sensitivity (%) Specificity (%) PPV (%) NPV (%) p-Value Serum NGAL > 160 ng/mL 0.92 (0.87–0.96) 89.6% 85.3% 74.1% 94.6% < 0.001* Urine NGAL > 140 ng/mL 0.89 (0.83–0.94) 85.4% 82.4% 69.5% 92.3% < 0.001* Serum Creatinine > 1.2 mg/dL 0.64 (0.56–0.72) 45.8% 72.5% 44.0% 74.0% 0.008* AUC: Area Under Receiver Operating Characteristic Curve; CI: Confidence Interval; PPV: Positive Predictive Value; NPV: Negative Predictive Value. Optimal cutoff determined via Youden's Index. *Statistically significant (p < 0.05). NGAL Levels Across KDIGO AKI Severity Stages Admission serum and urine NGAL levels correlated strongly with the ultimate severity of AKI graded according to KDIGO staging. Patients who eventually developed KDIGO Stage 1 AKI (n=28) exhibited mean admission serum NGAL levels of 185.2 ± 32.4 ng/mL, while those progressing to Stage 2 (n=12) and Stage 3 (n=8) demonstrated mean concentrations of 324.6 ± 48.1 ng/mL and 512.8 ± 65.2 ng/mL, respectively. A clear, stepwise gradient in biomarker concentration was observed across severity groups (ANOVA F = 142.6, p < 0.001). Post-hoc Tukey HSD comparisons confirmed statistically significant differences between non-AKI and all individual KDIGO stages (p < 0.001 for all). Figure 2 illustrates the distribution of serum NGAL concentrations categorized by KDIGO severity stages. Temporal Dynamics of Biomarker Elevation Serial profiling of biomarkers over 48 hours revealed marked differences in expression kinetics between NGAL and serum creatinine. In patients developing AKI, serum NGAL and urinary NGAL demonstrated a sharp, significant rise within 4 hours post-admission, achieving a mean 3.8-fold and 4.2-fold increase over baseline, respectively (p < 0.001). NGAL concentrations peaked between 12 and 24 hours. Conversely, serum creatinine displayed minimal change during the initial 12 hours (1.1-fold and 1.3-fold increase) and exceeded the diagnostic KDIGO threshold (1.5-fold increase) only at 24 to 48 hours post-admission. Thus, NGAL provided a diagnostic lead time of 20 to 24 hours over traditional serum creatinine measurements, Prognostic Value of Admission NGAL for Clinical Outcomes Elevated admission NGAL levels strongly stratified patients at high risk for secondary adverse clinical outcomes. Among the 48 AKI patients, 14 patients (29.2%) required acute Renal Replacement Therapy (RRT), 26 patients (54.2%) experienced prolonged ICU stays (> 7 days), and 12 patients (25.0%) died within 30 days. As shown in Table 3, high admission serum NGAL (> 160 ng/mL) was significantly associated with RRT requirement (χ² = 18.42, p < 0.001), prolonged ICU length of stay (χ² = 24.15, p < 0.001), and 30-day all-cause mortality (χ² = 11.28, p < 0.001). Admission NGAL concentrations maintained independent prognostic significance for 30-day mortality even after adjusting for baseline age and APACHE II score in multivariate logistic regression analysis (Adjusted Odds Ratio = 3.82, 95% CI: 1.64–8.91, p = 0.002). Table 3: Prognostic Association of High Admission Serum NGAL (> 160 ng/mL) with Adverse Clinical Outcomes Clinical Outcome High NGAL (>160 ng/mL) (n=54) Low NGAL (≤160 ng/mL) (n=96) Chi-Square (χ²) p-Value Renal Replacement Therapy (RRT), n (%) 13 (24.1%) 1 (1.0%) 18.42 < 0.001* Prolonged ICU Stay (> 7 Days), n (%) 24 (44.4%) 11 (11.5%) 21.15 < 0.001* 30-Day All-Cause Mortality, n (%) 11 (20.4%) 2 (2.1%) 12.84 < 0.001* Categorical comparisons performed using Pearson Chi-Square (χ²) test. *Statistically significant (p < 0.05). ICU: Intensive Care Unit.

The results of this prospective study demonstrate that serum and urinary NGAL serve as highly accurate, early diagnostic biomarkers for acute kidney injury in critically ill adult patients. The principal finding of our investigation is that both serum NGAL (AUC = 0.92) and urine NGAL (AUC = 0.89) detect acute renal tubular injury approximately 24 hours before conventional serum creatinine criteria are met [4,6]. These clinical findings corroborate landmark experimental and clinical studies establishing NGAL as an indispensable real-time indicator of tubular stress and structural injury [11,13]. Under physiological conditions, NGAL is filtered freely by the glomerulus and reabsorbed almost completely in the proximal tubule via endocytosis [12]. However, following ischemic, nephrotoxic, or inflammatory insults, gene expression of NGAL is dramatically induced in the thick ascending limb of Henle's loop and collecting ducts, leading to massive luminal and systemic release [11,14]. In our cohort, NGAL levels rose within 4 hours of critical illness onset, reflecting active cellular transcription and translation prior to any detectable decline in glomerular filtration rate. This dynamic response fills the diagnostic gap inherent to functional markers like creatinine, which depend on accumulation kinetics rather than cellular injury signaling [7,8]. When comparing serum and urinary NGAL, serum NGAL demonstrated slightly superior sensitivity (89.6% vs. 85.4%) and overall diagnostic accuracy (AUC 0.92 vs. 0.89). While urinary NGAL specifically reflects localized tubular secretion, systemic serum NGAL reflects both renal synthesis and systemic inflammatory responses associated with critical illness and sepsis [13,16]. Nevertheless, both biofluids provided excellent diagnostic precision at clinical cutoffs of 160 ng/mL and 140 ng/mL, respectively. These cutoffs closely align with international literature reporting optimal diagnostic thresholds between 150 ng/mL and 200 ng/mL in adult ICUs [6,10]. Furthermore, our study demonstrated a progressive, stepwise correlation between admission NGAL concentrations and KDIGO injury severity. Patients with higher initial NGAL levels subsequently experienced severe tubular dysfunction, requiring dialysis and suffering higher 30-day mortality. This risk-stratification capacity reinforces NGAL's clinical value not merely as a diagnostic tool, but as a prognostic biomarker capable of identifying high-risk patients who require targeted hemodynamic optimization, nephrotoxin stewardship, and early nephrology consultation [17,18]. Several study limitations merit consideration. First, as a single-center study conducted at an academic institution in South India, external generalizability to broader primary care settings requires further multi-center validation. Second, systemic inflammation or severe sepsis may independently elevate serum NGAL levels, necessitating careful clinical interpretation alongside urine NGAL and clinical status [16,20]. Finally, cost-effectiveness studies regarding point-of-care NGAL testing in resource-limited settings remain warranted.

In conclusion, serum and urinary NGAL exhibit outstanding diagnostic performance, superior sensitivity, and high prognostic value for early detection of acute kidney injury in critically ill patients. Elevated NGAL concentrations precede significant serum creatinine rises by 24 hours, providing clinicians with a vital window of opportunity to initiate nephroprotective strategies. Routine implementation of NGAL testing in critical care units can facilitate early risk stratification and optimize clinical management of AKI.

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