Elevated Neutrophil Gelatinase-Associated Lipocalin Is Associated With the Severity of Kidney Injury and Poor Prognosis of Patients With COVID-19

Abstract

Introduction: Loss of kidney function is a common feature of COVID-19 infection, but serum creatinine (SCr) is not a sensitive or specific marker of kidney injury. We tested whether molecular biomarkers of tubular injury measured at hospital admission were associated with acute kidney injury (AKI) in those with COVID-19 infection.

Methods: This is a prospective cohort observational study consisting of 444 consecutive patients with SARS-CoV-2 enrolled in the Columbia University emergency department (ED) at the peak of the pandemic in New York (March 2020-April 2020). Urine and blood were collected simultaneously at hospital admission (median time: day 0, interquartile range: 0-2 days), and urine biomarkers were analyzed by enzyme-linked immunosorbent assay (ELISA) and a novel dipstick. Kidney biopsies were probed for biomarker RNA and for histopathologic acute tubular injury (ATI) scores.

Results: Admission urinary neutrophil gelatinase-associated lipocalin (uNGAL) level was associated with AKI diagnosis (267 ± 301 vs. 96 ± 139 ng/ml, P < 0.0001) and staging; uNGAL levels >150 ng/ml had 80% specificity and 75% sensitivity to diagnose AKI stages 2 to 3. Admission uNGAL level quantitatively associated with prolonged AKI, dialysis, shock, prolonged hospitalization, and in-hospital death, even when admission SCr level was not elevated. The risk of dialysis increased almost 4-fold per SD of uNGAL independently of baseline SCr, comorbidities, and proteinuria (odds ratio [OR] [95% CI]: 3.59 [1.83-7.45], P < 0.001). In the kidneys of those with COVID-19, NGAL mRNA expression broadened in parallel with severe histopathologic injury (ATI). Conversely, low uNGAL levels at admission ruled out stages 2 to 3 AKI (negative predictive value: 0.95, 95% CI: 0.92-0.97) and the need for dialysis (negative predictive value: 0.98, 95% CI: 0.96-0.99). Although proteinuria and urinary (u)KIM-1 were implicated in tubular injury, neither was diagnostic of AKI stages.

Conclusion: In the patients with COVID-19, uNGAL level was quantitatively associated with histopathologic injury (ATI), loss of kidney function (AKI), and severity of patient outcomes.

Keywords: COVID-19; KIM1; NGAL; acute kidney injury; acute tubular injury; dialysis; kidney biopsy.

Figure 1

Urine sample collection: Residual urine samples were collected from 444 consecutive patients with COVID-19 by the Columbia University COVID-19 Urine Biobank between March 24, 2020 and April 27, 2020. A total of 4 patients with end-stage kidney disease on dialysis were excluded from the final cohort (N = 440). Patients with complete records of SCr measurements were staged by AKIN criteria (n = 371). A total of 69 patients missing adequate kidney function data were labeled “unknown.” AKIN, Acute Kidney Injury Network; SCr, serum creatinine.

Figure 2

uNGAL is associated with the duration and severity of acute tubular injury in patients with COVID-19: (a) uNGAL, but not uKIM-1, was associated with sAKI (meeting AKIN criteria for ≥72 hours) in patients with COVID-19. No AKI and transient AKI (<72 hours) levels are found for comparison. (b) uNGAL, but not uKIM-1, was associated with the severity of AKI (AKIN stage); bars represent medians. Notably, mean levels of uKIM-1 were equally elevated in all 4 groups, including in patients with COVID-19 with AKIN stage 0 (no elevation of SCr). (c) ROC curves for uNGAL (shades of red) and uKIM-1 (shades of blue), by ascending AKI severity (AKIN 1–3 vs. 0, AKIN 2–3 vs. 0–1, AKIN 3 vs. 0–2). (d) In non–COVID-19 AKI biopsies, NGAL (LCN2) mRNA is expressed in the distal nephron including AQP2+ collecting ducts (top panel), whereas KIM-1 (HAVCR1) is expressed in proximal tubules and not in AQP2⁺ collecting ducts (bottom panel). Bars = 50 μM. (e) COVID-19–positive kidney biopsies with widespread acute tubular injury (80%–100% of tubules) had extensive expression of NGAL in a noncanonical distribution, in LRP2⁺ and KIM-1⁺ proximal tubules (top panels), whereas COVID-19 kidney biopsies with limited ATI (30% of tubules) exhibited limited NGAL-KIM-1 overlap (bottom right panel). KIM-1 was expressed only in AQP2⁻ proximal tubules (bottom left panel). Tissue sections were counterstained with hematoxylin. Bars = 20 μM. AKI, acute kidney injury, AKIN, Acute Kidney Injury Network; ATI, acute tubular injury; NS, not significant; ROC, receiver operating characteristics; sAKI, sustained acute kidney injury; SCr, serum creatinine; uKIM-1, urinary KIM-1; uNGAL, urinary neutrophil gelatinase-associated lipocalin.

Figure 3

Urinary NGAL dipstick: (a) Urinary NGAL dipstick reveals dose responses to increasing concentrations of NGAL in the urine. (b) Correlation between urine dipstick and ELISA measurements. ELISA, enzyme-linked immunosorbent assay; NGAL, neutrophil gelatinase-associated lipocalin.

Figure 4

Higher urinary NGAL levels are associated with critical illness and death in patients with COVID-19: (a) Urinary NGAL levels were associated with AKI and sustained AKI (>72 hours) after adjustment for age, sex, race, and ethnicity (minimally adjusted model, blue) and baseline SCr and preexisting comorbidities (fully adjusted model, red; n = 371). Urinary NGAL levels were also associated with secondary outcomes of death, dialysis, shock, and respiratory failure in both minimally and fully adjusted models, N = 440. In contrast, uKIM-1 level was not associated with AKI or any secondary outcomes except for respiratory failure. ORs and HRs are expressed per 1 unit of SD of biomarker distribution; 95% CI. (b) Kaplan-Meier survival analysis reveals survival differences by tertile of urinary NGAL levels measured by ELISA or (c) by 3 levels of urinary NGAL dipstick test (unadjusted P values provided for both b and c; N = 440). AKI, acute kidney injury; ELISA, enzyme-linked immunosorbent assay; HR, hazard ratio; NGAL, neutrophil gelatinase-associated lipocalin; OR, odds ratio; SCr, serum creatinine; uKIM-1, urinary KIM-1.

Figure 5

The expression level of NGAL (red-brown) and KIM-1 (blue-purple) depended on the dose of arterial ischemia in mouse: (a–e) NGAL expression was found at the corticomedullary junction after 10 minutes of ischemia, but throughout the medulla and papilla after 30 to 40 minutes of ischemia. KIM-1 expression was found in the cortex and throughout the corticomedullary junction. (e–f) Prolonged ischemia (40 minutes) broadened the expression domain of NGAL to include the proximal tubule marked by KIM-1. In contrast to NGAL, KIM-1 expression remained localized to the cortex and corticomedullary junction. Bars a–e = 500 μm; bars f = 20 μm. NGAL, neutrophil gelatinase-associated lipocalin.