Elevated Neutrophil Gelatinase-Associated Lipocalin Is Associated With The Severity Of Kidney Injury And Poor Prognosis Of Patients With COVID-19--Part II

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

Katherine Xu1 et al

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RESULTS

Subjects We analyzed urine samples from 444 ED patients with COVID-19 admitted for inpatient care. The samples were collected prospectively at a median time of day 0 (hospital admission, interquartile range: 0–2 days), within 1 day of a positive result from the SARS-CoV-2 test in 70% of the patients (Figure 1). The cohort was diverse in age, sex, race, ethnicity (43.9% female, 20.5% African American, 54.1% Latinx), and preexisting comorbidities (Table 1). There were 4 patients with end-stage renal disease who were excluded from the study, and 69 patients had incomplete SCr records and were called “unknown” and analyzed separately (Figure 1).

CISTANCHE CAN BE ANTI-BACTERIAL AND COVID-19

Association of uNGAL With AKI

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) and among patients having sAKI (lasting $72 hours; sAKI; uNGAL: 332- 324 vs. 96 - 139 ng/ml, P < 0.0001) compared with those who did not have evidence of AKI (Figure 2a, log-transformed data in Supplementary Figure S1). uNGAL levels increased in a stepwise manner with increasing AKIN stage (P < 0.0001) (Figure 2b, log-transformed data in Supplementary Figure S1). Similarly, the area under the receiver operating characteristics 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 150 ng/ml (Tables 2 and 3). In contrast, uNGAL was found to have smaller elevations in patients who experienced transient AKI (187 -257 vs. 96 - 139 ng/ml, P < 0.05) or the AKIN 1 stage (162 - 219 vs. 96 -139 ng/ml, P < 0.01) compared with those without elevation of SCr (Figure 2a and b). In fact, uNGAL (<150 ng/ml) had a negative predictive value of 95% (95% CI: 92%–97%) for the subsequent development of AKIN stages 2 to 3.

The association of uNGAL with the primary outcomes was independent of age, sex, race, 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 ED (AKIN 0 stage). uNGAL levels were higher in patients with AKIN 0 who subsequently developed AKIN stages 1 to 3 within 7 days of admission (n ¼ 51, mean 158 - 237 ng/ml) compared with those who did not develop AKI (n ¼ 147, mean 74 - 65 ng/ml, P < 0.05). In this subgroup, the association of admission uNGAL level with subsequent recognition of AKI remained significant in our multivariable model (adjusted OR [95% CI]: 1.68 per SD of uNGAL [1.06–2.80], P < 0.05)

uNGAL level measured using a novel rapid point-of-care semiquantitative dipstick20 deployed to the bedside correlated with ELISA measurements (Spearman's correlation r ¼ 0.84, P < 0.0001) and reproduced all of the associations of uNGAL with AKI, sAKI, and AKIN stages (Figure 3a and b and Supplementary Figure S2).

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 ¼ 50 mM. (e) COVID-19–positive kidney biopsies with widespread acute tubular injury (80%–100% of tubules) had an extensive expression of NGAL in a noncanonical distribution, in LRP2þ and KIM-1þ 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 ¼ 20 mM. 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

EFFECTS OF CISTANCHE: IMPROVE KIDNEY FUNCTION

Association of uNGAL With Critical Illness

In addition to AKI metrics, admission uNGAL level was associated with the subsequent initiation of acute dialysis (adjusted OR [95% CI]: 3.59 [1.83–7.45] P < 0.001), the occurrence of shock, in-hospital death (adjusted OR [95% CI]: 1.51 [1.10–2.11], P < 0.05) and increased overall length of hospital stay (Figure 4a), independently of demographics, comorbidities, baseline renal function, and proteinuria (Supplementary Table S1). A similar association between the level of admission uNGAL and subsequent dialysis initiation and death (minimally adjusted OR [95% CI]: 2.43 [1.47– 4.29], P < 0.01 and fully adjusted OR [95% CI]: 2.35 [1.39–4.23], P < 0.01) was evident even among patients with normal SCr level on presentation (AKIN 0). In contrast, patients with lower admission uNGAL levels (<173 ng/ml) were unlikely to need dialysis (negative predictive value [95% CI]: 0.98 [0.96–0.99]).

In a time-to-event analysis, a dose-dependent association of uNGAL with 90-day mortality was observed in both ELISA and dipstick measurements. Within 30 days of admission, the highest uNGAL ELISA tertile (>128 ng/ml) and the highest uNGAL dipstick category (>150 ng/ml) both revealed lower survival probability, from 86.3% to 71.2% and from 81.8% to 66.7%, respectively, comparing the lowest and highest tertiles (Figure 4b and c).

In Supplementary Figure S3 and Supplementary Table S2, we summarized secondary outcome comparisons for patient subgroups defined by a combination of uNGAL levels (a marker of tubular injury) and SCr levels (a marker of functional AKI). These analyses reveal that high uNGAL levels ($150 ng/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 (Figure 2, log-transformed data in Supplementary Figure S1) nor with secondary outcomes including 90-day mortality (Figure 4a and Supplementary Table S1).

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 ¼ 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 ¼ 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 ¼ 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

Comparison of COVID-19 (-) and COVID-19 (+) ED Cohorts

To find whether our findings were specific to COVID19, we evaluated a second cohort of comparable size (426 patients) admitted through the same ED (June 2017–January 2019)20 before the COVID-19 pandemic in New York City and analyzed using identical methods (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, patients with COVID-19 were 2.6 times more likely to present with AKI (35.2% vs. 13.6%, P < 0.0001), 3.9 times more likely to have sAKI (17.5% vs. 4.5%, P < 0.0001), and 1.8 times more likely to have the more severe disease (AKIN 2–3, 12.5% vs. 6.8%, P < 0.01) compared with our historical cohort (Table 1), similar to published data.28

Proximal Tubule Injury in COVID-19

Without AKI at Hospital Presentation Urinary findings differed in the COVID-19 (-) and COVID-19 (+) cohorts even in the absence of AKI. KIM1 was elevated in COVID-19 (+) AKIN 0 cases compared with the COVID (-) AKIN 0 historical cohort (uKIM-1: 2.57 - 2.44 in COVID-19 [+] vs. 1.96 - 2.51 ng/ml in COVID-19 [-], P < 0.01; Supplementary Figure S4). Proteinuria was also increased in COVID-19 (+) compared with COVID (-) AKIN 0 cases (P < 0.0001; Supplementary Figure S5). In addition, evaluation of the cellular pellets of the AKIN 0 urine samples revealed that the shedding of proximal tubule cells (marked by proximal tubule gene LRP2) was more prominent in COVID-19 (+) than in the COVID-19 (-) cohort (n ¼ 40; 2.61-fold increase in urinary LRP2+ cells, P < 0.01), whereas UMOD+ cells were present regardless of COVID-19 status (Supplementary Figure S6). In sum, AKIN 0 COVID-19 urine was enriched for KIM-1, proteinuria, and proximal tubule cells. As a result, KIM-1 was not substantially increased with progressive AKIN stages in the COVID (+) cohort, whereas uNGAL was associated with AKIN stages in both COVID (+) and COVID (-) cohorts (Supplementary Figure S4).

NGAL RNA Expression Correlates With Histopathology

To explore the responses of different nephron segments, we evaluated the transcriptomic patterning of the biomarkers in kidney biopsies from 13 patients with COVID-19 23 and 4 controls without COVID-19 with ATI. In both COVID-19 and non–COVID-19 biopsy findings, KIM-1 was found to be expressed in the proximal tubule whereas NGAL was prominently expressed in the limbs of Henle and collecting ducts. The distributions were confirmed by simultaneously probing for segment-specific markers, LRP2 (proximal tubule), and AQP2 (collecting duct). Surprisingly, in addition to its canonical distribution, NGAL transcripts were expressed in additional nephron segments in COVID-19 biopsy samples. At maximum extent of ATI (>50% of tubules), KIM-1 was expressed in 27% (3322 of 12,123), whereas NGAL was expressed in 65.7% (6580 of 10,111) of the tubules, including significant coexpression with KIM-1 in 85% of COVID-19 kidneys and with proximal marker LRP2 in 77% of the kidneys (Figure 2d and e). Furthermore, 62.5% of the tubules in high ATI biopsy samples and 20% of the tubules in low ATI biopsy samples (P < 0.05) were found to have coexpression of KIM-1 and NGAL, implying severity of ATI drives NGAL RNA patterning. Confirmation That NGAL Expression Correlates With Histopathology in Models of Injury To confirm that the patterning of NGAL RNA in COVID (þ) biopsy samples reflected ATI, we evaluated a classical model of ischemia-reperfusion injury in the mouse. Similar to human kidneys, increasing degrees of ATI also resulted in the broadening of NGAL RNA expression in mouse kidneys (Figure 5a–f). In the setting of prolonged arterial ischemia, the entirety of the corticomedullary junction, medulla, papilla, and KIM-1+ proximal tubules expressed NGAL RNA. These findings are consistent with a recent report describing NGAL expression in the proximal tubule cells by single-cell sequencing after ischemic injury29 and revealing the expansion of NGAL RNA in COVID (+) biopsies is a reproducible consequence of severe kidney injury.

EFFECTS OF CISTANCHE: BOOST IMMUNITY

DISCUSSION

We have identified a quantitative association between uNGAL and both functional kidney failure (AKI) and ATI in patients admitted to the hospital with COVID-19infection. The level of uNGAL was associated in a stepwise fashion with clinical metrics and outcomes of AKI, such as dialysis, whereas renal biopsy findings correlated NGAL RNA with more severe forms of ATI. Consequently, admission uNGAL measurements provided prognostic data relevant to kidney injury and dysfunction in the COVID (+) patients.

As diagnostic tools, uNGAL and SCr have many different characteristics.12 NGAL is expressed within 2 to 3 hours of injury,13,30,31 whereas the elevation of SCr level is delayed by 24 to 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 infarctions13 and unilateral kidney disease,35 whereas SCr is insensitive to focal or subtotal injury. In fact, we found that elevated uNGAL level was associated with AKI and clinical outcomes, even when patients with COVID-19presented at admission without AKI (AKIN 0), confirming admission SCr level underestimated the evolution of COVID-19–associated kidney disease, revealing the differential sensitivity of the 2 analytes.

The stepwise association of an injury marker, NGAL, and a functional marker of glomerular filtration, SCr, can be explained by the progressive induction of NGAL RNA in the kidney with greater degrees of histopathologic injury (Figure 2). Increasing severity of injury broadened the classical patterning of NGAL RNA expression in the limb of Henle and collecting ducts to encompass multiple segments of the nephron from the proximal tubule to the papilla. The dose responsiveness of NGAL RNA and its patterning were reproducible beyond human biopsies to include classical models of tissue damage created by ischemia-reperfusion injury in mice (Figure 5). Indeed, elevated uNGAL level 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) that induce little, if any, the response by different injury biomarkers.12,32,36 Hence, we reveal for the first time that the level of NGAL mirrors the severity of ATI in human biopsies, including severe forms of ATI in COVID-19 kidneys. In light of this, 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 receiver operating characteristics 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 reveal that accurate testing for COVID-19–associated kidney injury is possible in the ED using rapid point-of-care dipsticks.20,37 The NGAL dipstick correlated closely with ELISA-based measurements (r ¼ 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 EDs 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 the criteria for AKIN staging.

Our study has a number of limitations. Similar to previous studies,38,39 we were limited by the use of SCr as the gold standard for AKI. Notably, many patients with COVID-19 did not have previous 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 patients (15.5%) 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 illness, precluding comparative studies of the kinetics of urinary biomarkers, and it remains possible that subsequent measurements of uNGAL, uKIM-1, and proteinuria could have provided additional prognostic information, suggested being important by Nugent et al.40 in understanding the impact of COVID-19.

In conclusion, a large cohort of patients with COVID-19 had a dose-responsive relationship of uNGAL with ATI and AKI and severe clinical outcomes, independently of other established risk factors. These relationships were found even in patients who did not meet the SCr-based AKIN criteria in the ED. Conversely, the absence of elevated uNGAL levels essentially ruled out the need for dialysis and identified patients at lower risk for death. The utility of a dipstick further underscores the value of uNGAL for rapid triage decisions. Given the recent resource challenges that the COVID-19 pandemic has created for Emergency Departments, Nephrology, and Critical Care services,41 uNGAL can aid in triage and disposition of patients without waiting for repetitive measurements of SCr or kidney biopsies

EFFECTS OF CISTANCHE: BOOST KIDNEY FUNCTION

DISCLOSURE

Columbia University owns and licensed patents involving NGAL to BioPorto and Abbott. Dr. Mohan reports receiving grant funding and personal fees from Angion Biomedica and personal fees from KI Reports as deputy editor. The authors have nothing else to disclose.

ACKNOWLEDGMENTS

We salute the steadfast service of the Department of Emergency Medicine at the Columbia University Irving Medical Center during the ongoing surges of the COVID-19pandemic. We thank all patients for their participation and the following members of the Columbia University COVID-19 Biobank: Sheila O’Byrne, Renu Nandakumar, Amritha Menon, Yat So, Danielle Pendrick, Eldad Hod, Soumitra Sengupta, Wendy Chung, and Muredach Reilly. The Columbia University COVID-19 Biobank is supported by the Vagelos College of Physicians & Surgeons Office for Research, Precision Medicine Resource, and Biomedical Informatics Resource of the Columbia University Irving Institute for Clinical and Translational Research (Columbia CTSA).

XK is supported by the National Institutes of Health-National Institute of Diabetes and Digestive and Kidney Diseases T32 Research in Nephrology Training Grant (5T32DK108741- 05). KK, JB, NS, SK, and VD are supported by the KidneyPrecision Medicine Project of the National Institutes of Health-National Institute of Diabetes and Digestive and Kidney Disease (4UH3DK114926-03). SM and JS are supported by the National Institutes of Health-National Institute of Diabetes and Digestive and Kidney Diseases (R01- DK126739). SM is also supported by National Institutes of Health grants R01DK114893, R01MD014161, and U01DK116066. Columbia Clinical and Translational Science Award are funded by the National Center for Advancing Translational Sciences, National Institutes of Health, through grant number UL1TR001873. The funders had no role in formulating the hypotheses, collecting or analyzing samples, interpretation of data, or the preparation and revision of the manuscript and data sets.

AUTHOR CONTRIBUTIONS KK, KX, and JB conceived and designed the study. The Columbia University COVID-19 Biobank collected residual blood and urine samples after clinical testing from every COVID-19 patient diagnosed at the Columbia University Irving Medical Center. KX, JB, and UN conducted laboratory measurements including urinary biomarker and protein measurements and in situ hybridizations. NS implemented electronic AKI definitions and analyzed the Electronic Health Records to define clinical outcomes and covariates. AL, AC, AY, KX, and JB quantified the distribution of RNA expression. RVS performed mouse experiments. VDA and SK analyzed human kidney biopsy tissue to diagnose AKI and quantify tubular injury. JS and SM contributed to the comparative analysis of COVID-19–negative data set. KX and KK performed statistical analyses. KX, KK, SM, JB, and AMM wrote the manuscript draft. All authors reviewed and edited the manuscript. Drs. Kiryluk, Xu, and Barasch had full access to the data and take responsibility for the integrity and accuracy of the results. All data analyses and results are described in this article and Supplementary files. The primary de-identified data are available from the corresponding authors on reasonable request if approved by the Institutional Review Board of Columbia University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or Columbia University.

SUPPLEMENTARY MATERIAL Supplementary File (PDF)

Table S1. Urinary biomarker associations with primary and secondary outcomes (PDF).

Table S2. Associations of uNGAL-based stratification of AKI with clinical outcomes (PDF).

Figure S1. Log-transformed levels of uNGAL and uKIM-1 in patients with COVID-19 (PDF).

Figure S2. Comparison of urine NGAL measurements by ELISA or dipstick (PDF).

Figure S3. Comparison of clinical outcomes in patients stratified by uNGAL and SCr-based AKI (PDF).

Figure S4. Urinary NGAL and KIM-1 in COVID-19–negative and COVID-19–positive cohorts (PDF).

Figure S5. Comparison of proteinuria in COVID-19– negative and COVID-19–positive cohorts (PDF).

Figure S6. Shedding of LRP2þ proximal tubule cells into the urine in COVID-19 cases (PDF). STROBE Statement (PDF).

CISTANCHE AND CISTANCHE EXTRACT

From: 'Elevated Neutrophil gelatinase-associated Lipocalin Is Associated With the Severity of Kidney Injury and Poor Prognosis of Patients With COVID-19'--by Katherine Xu1 et al

---Kidney International Reports (2021) 6, 2979–2992 CLINICAL RESEARCH

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