key: cord-0947258-gap0zs33
authors: Xu, Katherine; Shang, Ning; Levitman, Abraham; Corker, Alexa; Kudose, Satoru; Yaeh, Andrew; Neupane, Uddhav; Stevens, Jacob; Sampogna, Rosemary; Mills, Angela M.; D’Agati, Vivette; Mohan, Sumit; Kiryluk, Krzysztof; Barasch, Jonathan
title: Elevated NGAL is Associated with the Severity of Kidney Injury and Poor Prognosis of Patients with COVID-19
date: 2021-10-08
journal: Kidney Int Rep
DOI: 10.1016/j.ekir.2021.09.005
sha: c829373a73bbef1a0a1f30e04913278cd9070464
doc_id: 947258
cord_uid: gap0zs33

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 AKI in those with COVID-19 infection. METHODS: This is a prospective cohort observational study consisting of 444 consecutive SARS-CoV-2 patients enrolled in the Columbia University Emergency Department at the peak of New York’s pandemic (March-April 2020). Urine and blood were collected simultaneously at hospital admission (median time: day 0, IQR 0-2 days) and urine biomarkers analyzed by ELISA and by a novel dipstick. Kidney biopsies were probed for biomarker RNA and for histopathologic acute tubular injury (ATI) scores. RESULTS: Admission uNGAL was associated with AKI diagnosis (267±301 vs. 96±139 ng/mL, P < 0.0001) and staging; uNGAL levels >150ng/mL demonstrated 80% specificity and 75% sensitivity to diagnose AKI-stage 2-3. Admission uNGAL quantitatively associated with prolonged AKI, dialysis, shock, prolonged hospitalization, and in-hospital death, even when admission SCr was not elevated. The risk of dialysis increased almost 4-fold per standard deviation of uNGAL independently of baseline SCr, co-morbidities, and proteinuria [OR(95%CI): 3.59 (1.83-7.45), P < 0.001]. In COVID-19 kidneys, NGAL mRNA expression broadened in parallel with severe histopathological injury (ATI). Conversely, low uNGAL levels at admission ruled out stage 2-3 AKI (NPV 0.95, 95%CI: 0.92-0.97) and the need for dialysis (NPV: 0.98, 95%CI: 0.96-0.99)). While proteinuria and uKIM-1 implicated tubular injury, neither were diagnostic of AKI stages. CONCLUSIONS: In COVID-19 patients, uNGAL quantitatively associated with histopathological injury (ATI), the loss of kidney function (AKI), and the severity of patient outcomes.

Acute loss of kidney function, measured by a rise in serum creatinine (SCr), is common in the setting of acute SARS-CoV-2 infection [1] [2] [3] [4] . Elevated SCr is present in one-third of patients hospitalized with COVID-19 [5] [6] [7] [8] [9] [10] [11] . Yet, the measurement of SCr fails to reflect the full burden of COVID-19 kidney injury. For example, SCr can only detect kidney dysfunction in retrospect, after enough time has elapsed for the accumulation of SCr to a diagnostic threshold. In addition, clinically significant changes in SCr may not occur in subtotal or focal kidney damage as a result of compensatory changes in uninjured nephrons which can attenuate the rise in SCr. Even when elevations in SCr are apparent, evidence of tubular injury may be lacking, for example in the presence of volume depletion 12,13 , a common presentation in patients with COVID-19 associated diarrhea 10,14, . A rise in SCr may also be confounded by the incidence of rhabdomyolysis in COVID-19, which enhances creatinine production 15, 16 .

The burden of kidney injury during surges of COVID-19 has strained hospital resources, including Emergency Medicine, Critical Care and Nephrology, creating an urgent need to limit delays in the identification of individuals at risk for kidney injury, loss of kidney function, and renal replacement therapy. As a result, triage decisions for patients and for resource allocations would benefit from the use of a rapidly responsive, sensitive, and specific non-invasive marker of kidney injury and its attendant outcomes common in COVID-19 infection.

Prior research in human and mouse models demonstrated that two molecular markers of tubular injury, urinary NGAL (uNGAL) and urinary KIM-1 (uKIM-1) derive from different segments of the kidney and allow for sensitive detection and real time distinction between volume sensitive and volume insensitive intrinsic forms of tubular injury 12, [17] [18] [19] . Prior evidence J o u r n a l P r e -p r o o f

The Columbia COVID-19 Biobank recruited consecutive COVID-19 cases (positive nasopharyngeal SARS-CoV-2 PCR test), regardless of age, sex, or race/ethnicity who received care at Columbia University Irving Medical Center. The Biobank stored residual blood and urine samples after clinical testing from every patient with COVID-19 including an initial urine sample and serum obtained at hospital admission, that were used for biomarker measurements.

For our analysis, we identified 444 consecutive patients who were admitted between 3/24/2020 and 4/27/2020 with COVID-19. This included 371 patients who not only had a urine sample from presentation, but also information on baseline SCr and complete SCr measurements in hospital required to determine the stage and duration of AKI ( Figure 1 ). In addition, 4 patients with ESRD were excluded. Kidney biopsies were accessioned by the CUIMC Renal Pathology Laboratory including 13 kidney biopsies from COVID-19 cases 23 and four non-COVID-19 specimens analyzed by in situ hybridization for NGAL and KIM-1 RNA.

The COVID (+) cohort was compared to a COVID (-) cohort that was recruited in an identical fashion in the CUIMC Emergency Department between 6/2017 and 1/2019 20 . Similar to the COVID (+) cohort, 426 consecutive COVID (-) patients were enrolled at presentation, regardless of age, sex, or race/ethnicity. This included 318 patients with complete SCr measurements in hospital required to determine stage and duration of AKI. Urine samples were collected within 24 hours of arrival in the ED. Patients with ESRD were excluded.

J o u r n a l P r e -p r o o f

Primary outcomes included SCr-based AKI, AKIN stage, and the duration of elevated SCr consistently in both the COVID (+) and the COVID (-) cohorts. In both cases, baseline SCr was defined using a standardized algorithm, as previously described by Stevens et al 20 Patients were classified as "Unknowns" if none of the above criteria were met.

Acute Kidney Injury Network (AKIN) 23 stages were classified as follows:

 AKIN Stage 1: ≥0.3mg/dL increase in SCr within a 48-hour window OR 1.5 to 2-fold increase in SCr compared to baseline.

 AKIN Stage 2: >2 to 3-fold increase in SCr compared to baseline.  AKIN Stage 3: ≥0.5mg/dL increase in SCr within a 48-hour window when SCr ≥4.0mg/dL OR >3-fold increase in SCr compared to baseline.

The peak SCr measured within 48hrs after urine collection was used to diagnose AKIN stage. In select cases, the day 1 AKIN score was imputed when the preceding and subsequent AKIN scores were identical.

Further categorization was based on the duration of SCr elevation above baseline:

 No AKI (AKIN 0)not meeting AKIN criteria within 2 days of presentation (must have SCr values for both days).

J o u r n a l P r e -p r o o f  Transient AKI (tAKI)met AKIN criteria on day 0 or 1 of presentation but normalized below AKIN detection thresholds within 2 days after first detection (total AKI duration <72 hours).

 Sustained AKI (sAKI)met AKIN criteria within 2 days of presentation but normalized below the AKIN detection thresholds only after 2 days from the first detection (total AKI duration >72 hours).

 Unknownmissing baseline SCr, or insufficient SCr measurements to determine SCr kinetics, or missing measurements on day 0 or 1 that could not be imputed due to discrepant AKIN scores (see Figure 1 ). Further categorization of elevated SCr was based on the duration of SCr elevation above baseline, including Transient AKI (tAKI) rapidly normalizing SCr (<72hrs) and Sustained (sAKI) delayed normalization of SCr (>72hrs).

Urine output was not utilized for AKI definition because of the variable use of Foley catheters and incomplete recording of urine output in the Emergency Department.

Secondary outcomes included dialysis, shock, respiratory failure, length of hospital stay and in-hospital death. Shock was defined by the need for vasopressors, and respiratory failure was defined by the need for either invasive or non-invasive positive pressure ventilation. Staging of chronic kidney disease (CKD) was determined using the CKD-EPI eGFR formula and the baseline creatinine as described above 24 . J o u r n a l P r e -p r o o f

All urinary measurements were blinded to clinical data. NGAL and uKIM-1 were measured by ELISA (KIM-1: Enzo, ADI-900-226-0001; NGAL: BioPorto, KIT036). Proteinuria was detected with Chemstrip 10 SG (Roche Diagnostics). Urine was centrifuged (12,000rpm; 10min) and applied to NGAL gRAD dipsticks (BioPorto) and color development was compared to a color scale marking NGAL (ng/mL) concentration by two independent readers. Urinary cell pellets were analyzed for LRP2 and UMOD by immunoblot with SDS-PAGE (Bio-rad Laboratories), rabbit anti-LRP2 

Histologic features of acute tubular injury (ATI) included loss of brush border, epithelial simplification, intracytoplasmic vacuolization, overt necrosis, apoptosis, and cell shedding. A semi-quantitative scale was used to measure the extent of ATI: none (<5% of tubules involved), mild (<25%), moderate (25-50%), severe (>50%).

In Situ Hybridization (ISH) on formalin-fixed paraffin-embedded human 

Male and female wild-type C57Bl/6 mice, aged 8-10 weeks (Jackson Labs) were anesthetized with isoflurane and placed on a warming table (rectal temperature: 37°C). 

Normality of continuous variables was tested using Shapiro-Wilk test. Normally distributed continuous variables were compared using a two-sample t-test and summarized as mean ± SD. Non-normally distributed continuous variables were summarized as medians and ranges, and compared using non-parametric Mann-Whitney U test. To improve interpretability of effect estimates from multivariate regression models, all non-normally distributed predictors (including uNGAL and uKIM-1 levels) were log-transformed and standard-normalized before statistical testing. The effect sizes for biomarkers were expressed per standard deviation unit of normalized predictors. Categorical variables were compared using Chi-squared or Fisher's Exact test. For testing binary outcomes, we used logistic regression. Ordinal outcomes, such as AKIN stage, were tested using ordinal logistic regression. Ordinal predictors, such as urine dipstick category or proteinuria grade, were tested under the assumption of linear effects using a slope J o u r n a l P r e -p r o o f test within the framework of a generalized linear model tailored to the outcome of interest (e.g. logistic or ordinal logistic for binary or ordinal outcomes, respectively). We used Cox proportional hazards model for the time-to-event analyses for death outcome. We used competing risks regression model for the analysis of the hospital length of stay, with death as a competing risk 25, 26 . The proportional hazards assumption was verified by testing scaled Schoenfeld residuals for each predictor against observation time. Associations of urinary biomarkers with clinical outcomes were adjusted for the following covariates: age, sex, race, ethnicity (minimally-adjusted model), baseline serum creatinine and pre-existing obesity, diabetes, hypertension, transplant (any organ), cancers, cardiovascular disease (coronary artery disease, heart failure, cerebral infarction), pulmonary disease (asthma, chronic obstructive pulmonary disease, interstitial pulmonary disease, primary pulmonary hypertension, idiopathic pulmonary fibrosis) (fully-adjusted model-1), as well as proteinuria (fully-adjusted model-2). In the analysis of primary outcomes, we considered two-sided P<0.05 as statistically significant. In the analysis of secondary outcomes, we considered P<0.01 as statistically significant (Bonferroni-corrected for 5 independent outcomes tested). We additionally stratified patients by both uNGAL (>150 ng/mL) and SCr-based AKI into four groups: NGAL−AKI−, NGAL+AKI−, NGAL−AKI+, and NGAL+AKI+. We 

The Columbia University Biobank COVID-19 studies were approved by Columbia University Medical Center Institutional Review Board (IRB: AAAS7370 and AAAS7948). A subset of patients was included under a public health crisis IRB waiver of consent specifically for COVID-19 studies if patients were deceased, not able to consent, or for loss of contact.

Mice were utilized according to the Institutional Animal Care and Use Committee (AC-AAAY7464) and adhere to NIH Guide for the Care and Use of Laboratory Animals.

We analyzed urine samples from 444 COVID-19 ED patients admitted for inpatient care.

The samples were collected prospectively at a median time of day 0 (hospital admission, IQR 0-2 days), within 1 day of a positive SARS-CoV-2 test in 70% of patients ( Figure 1 ). The cohort was diverse in age, sex, race, ethnicity (43.9% female, 20.5% African American, 54.1% Latinx), and pre-existing comorbidities ( Table 1) . Four ESRD patients were excluded from the study and 69 patients had incomplete SCr records, and were called "Unknown" and analyzed separately ( Figure 1 ).

Admission uNGAL levels were elevated among patients who subsequently met SCr based AKI criteria (uNGAL: 267±301 vs. 96±139 ng/mL, P < 0.0001) as well as among patients demonstrating sustained AKI (lasting ≥72 hours; sAKI; uNGAL: 332±324 vs. 96±139 ng/mL, P Similarly, the area under the receiver operating characteristics (ROC) curve for uNGAL progressively increased with higher AKIN stages (0.70-0.93; Figure 2c ). uNGAL had 80% specificity and 75% sensitivity to diagnose AKIN stage 2 or 3 at a cutoff level of 150ng/mL ( 96±139 ng/mL, P < 0.01) compared to those without elevation of SCr (Figure 2a The association of uNGAL with the primary outcomes was independent of age, sex, race and ethnicity, baseline creatinine and other comorbidities, and was also independent of proteinuria measured in the same urine sample (Supplementary Table S1 ).

We performed a subgroup analysis of 198 patients with COVID-19 who did not have evidence of AKI on presentation to the Emergency Department (AKIN 0 Stage). uNGAL levels were higher in AKIN 0 patients who subsequently developed AKIN stages 1-3 within 7 days of admission (N=51, mean 158±237 ng/mL) compared to those who did not develop AKI (N=147, mean 74±65 ng/mL, P < 0.05). In this subgroup, the association of admission uNGAL with subsequent recognition of AKI remained significant in our multivariable model [adjusted OR 

In addition to AKI metrics, admission uNGAL was associated with the subsequent In Supplementary Figure S3 and Table S2 , we summarized secondary outcome comparisons for patient subgroups defined by a combination of uNGAL (marker of tubular injury) and SCr (marker of functional AKI). These analyses show that high uNGAL levels (≥150ng/mL) provide additional prognostic information beyond AKIN AKI criteria, consistent with our primary analysis.

In contrast to uNGAL, uKIM-1 levels were not associated with primary outcomes of AKI, sAKI or AKIN stage in the COVID-19 cohort ( Table S1 ).

To determine whether our findings were specific to COVID-19, we examined a second cohort of comparable size (426 patients) admitted through the same Emergency Department (Table 1 ). The COVID-19 cohort was older and enriched in Latinx patients, but the burden of chronic kidney disease was similar in both cohorts. Notably, COVID-19 patients were 2.6-times more likely to present with AKI (35.2% vs. 13.6%, P < 0.0001), 3.9times more likely to have sustained AKI (17.5% vs. 4.5%, P < 0.0001) and 1.8-times more likely to have more severe disease (AKIN 2-3, 12.5% vs. 6.8%, P < 0.01) compared to our historical cohort (Table 1) , similar to published data 28 . implying that severity of ATI drives NGAL RNA patterning.

To confirm that the patterning of NGAL RNA in COVID (+) biopsies reflected ATI, we examined a classical model of ischemia reperfusion injury in the mouse. Similar to human kidneys, increasing degrees of ATI also resulted in broadening of NGAL RNA expression in mouse kidneys ( Figure 5 ). In the setting of prolonged arterial ischemia, the entirety of the cortico-medullary junction, the medulla, and the papilla, as well as the KIM-1+ proximal tubules expressed NGAL RNA. These findings are consistent with a recent report describing NGAL expression in proximal tubule cells by single cell sequencing after ischemic injury 29 As diagnostic tools, uNGAL and SCr have many different characteristics 12 . NGAL is expressed within 2-3 hours of injury 13, 30, 31 , while the elevation of SCr is delayed by 24-48 hours 32, 33 , depending on mechanisms that enhance its excretion (the renal reserve) or limit its production 34 . In addition, NGAL is detected in the urine after small wedge infarctions 13 and unilateral kidney disease 35 , while SCr is insensitive to focal or subtotal injury. In fact, we demonstrated that elevated uNGAL was associated with AKI and clinical outcomes, even when COVID-19 patients presented at admission without AKI (AKIN 0), confirming that admission SCr underestimated the evolution of COVID-19 associated kidney disease, demonstrating the differential sensitivity of the two analytes. Figure 5) . Indeed, elevated uNGAL is associated with inflammatory, ischemic, toxic and obstructive uropathies, which injure the tubule, rather than reversible hemodynamic challenges (e.g. volume depletion, diuretics, and heart failure) which induce little, if any, response by different injury biomarkers 12, 32, 36 . Hence, we demonstrate for the first time that the level of NGAL mirrors the severity of ATI in human kidney biopsies, including severe forms of ATI in COVID-19 kidneys. In this light, the association of uNGAL with functional stages of AKI is likely due to its quantitative association with ATI, which in severe cases limits the clearance of SCr. The progressive increase in the area under the ROC curves for uNGAL, from 0.70 to 0.93 with increasing AKIN stage, highlights that severe dysfunction (AKI) is found in cases of severe injury (ATI).

The association of uNGAL with AKI and ATI provides the possibility of a sensitive diagnostic strategy that bypasses the delays and insensitivity of SCr. We show that accurate testing for COVID-19 associated kidney injury is possible in the Emergency Department using rapid point-of-care dipsticks 20, 37 . The NGAL dipstick correlated closely with ELISA-based measurements (ρ=0.84, P < 0.0001), but the dipstick limits the risk of handling infectious body fluids. The dipstick may be particularly helpful in the setting of high patient volumes witnessed in Emergency Departments during COVID-19 surges, providing prognostic information in real time.

We also suggest that our diagnostic strategy may be further enhanced by measuring proteinuria, which is indicative of kidney injury even without elevation of SCr (AKIN 0) and is associated with a number of adverse clinical outcomes independently of NGAL. When measured together, NGAL and proteinuria may offer a comprehensive evaluation of kidney injury in COVID-19 especially in patients who have not reached criteria for AKIN staging.

Our study has a number of limitations. Similar to prior studies 38,39 , we were limited by the use of SCr as the gold standard for AKI. Notably many COVID-19 patients did not have prior health records, making it difficult to establish their baseline SCr values and to detect AKI and calculate its stage. As a consequence, we were unable to define AKI in 69 (15.5%) patients with missing SCr measurements at baseline (no records) or follow up (early death or discharge).

We were also not able to collect urine samples on subsequent days because of the severity of Supplementary information is available at KI Report's website. 

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We salute the steadfast service of the Department of Emergency Medicine at Columbia University Irving Medical Center during the ongoing surges of the COVID-19 pandemic. We would like to thank all patients for their participation, and the following members of the