key: cord-0885080-n6j642w1 authors: Ye, Yinyin; Swensen, Adam C.; Wang, Yang; Kaushal, Madhurima; Salamon, Diane; Knoten, Amanda; Nicora, Carrie D.; Marks, Laura; Gaut, Joseph P.; Vijayan, Anitha; Orton, Daniel J.; Mudd, Philip A.; Parikh, Chirag R.; Qian, Wei-Jun; O’Halloran, Jane A.; Piehowski, Paul D.; Jain, Sanjay title: A Pilot Study of Urine Proteomics in Covid-19-associated Acute Kidney Injury date: 2021-10-07 journal: Kidney Int Rep DOI: 10.1016/j.ekir.2021.09.010 sha: f1af0b8918b018a1ae5217182653d1cc65b2b350 doc_id: 885080 cord_uid: n6j642w1 nan Acute kidney injury (AKI) is a major complication associated with COVID-19 and occurs in up to 76% of intensive care unit patients 1, 2 . The mortality rate of COVID-19 patients who developed AKI (COVID-AKI) is more than 10 times higher than those who did not. While candidate AKI markers exist, the etiology of COVID-AKI is multifactorial requiring agnostic approaches for identification of analytes early in hospital course to provide insights into biomarkers and mechanisms associated with COVID-AKI and COVID-19 infection. Research on COVID-19-associated effects on the urinary proteome is limited, and kidney dysfunction has not been reported 3 . Approximately 70% of the proteins detected in urine are produced in the kidney with a significant amount filtered from blood. We hypothesize that the changes in protein abundances in urine could lead to the discovery of protein markers associated with COVID-19 or COVID-AKI and provide mechanistic insights to improve understanding. We analyzed urine samples from 14 participants (6 COVID-AKI, 3 COVID-NoAKI and 5 NoCOVID-NoAKI) ( Figure 1A , Table S1 ). To account for the large variation in protein content across urine specimens we utilized a bicinchoninic acid (BCA) to measure peptide concentration after protein digestion. All peptide samples are then normalized to the same concentration prior to analysis to allow for relative quantitation of differences in the urinary proteome. After confirming the quality of urine for analyte discovery (Table S2 , Figure S1 ), we examined if underlying variance could distinguish between AKI+ (6 COVID-AKI) from other samples without AKI (8 AKI-); all AKI samples were from COVID-19 patients. The first two principal components accounted for 42.1% of the variance, and clearly separated the two groups ( Figure 1B) ; thereby J o u r n a l P r e -p r o o f suggesting a prominent impact by AKI on the urine proteome of COVID patients. There was a statistically significant abundance increase in 97 proteins and decrease in 140 proteins in the AKI+ group (adjust p < 0.05, Figure 1C , Figure S2A ). Hierarchical clustering and pathway enrichment analysis revealed that the top upregulated pathways were complement activation, coagulation cascades, and regulated exocytosis ( Figure 1D , E). Specifically, complement component C2, C3, C5, C6, and C8G were significantly increased in the AKI+ group affecting complement initiation ( Figure 1D ). Notably, decay-accelerating factor CD55 decreased 2.5-fold in the AKI+ group. This suggests reduced capacity to regulate complement activation thereby exacerbating injury. This finding is supported by exacerbation of AKI in mice with deletion of CD 55 after ischemia reperfusion injury 4 . Activation of the complement system is associated with severe COVID-19. The kidney cell protein sC5b-9, commonly associated with respiratory failure, is upregulated in plasma in COVID-19 patients with moderate to severe kidney injury. Comparison of COVID-19 samples with and without AKI also showed enrichment of proteins associated with the complement pathway (Table S2 columns In addition, we detected several known urinary biomarkers of AKI including NGAL, fatty acid binding proteins (FABP3, FABP4), meprin A (MEP1A), and retinol binding protein (RBP4) increased in the urine of the COVID-AKI patients (Fig. 1D , Table S2 ). However, proteome analysis also showed a significant decrease in UMOD and EGF in this group. Urinary UMOD and EGF have been shown to be inversely correlated with AKI and their decrease is strongly associated with AKI severity. These results further validate our proteome approach and are indicative of tubular injury affecting secretion (NGAL, UMOD, EGF) or defective absorption by proximal tubules (FABP3, 4) . A novel observation was a more than 30-fold increase in IGFBP6 in COVID-AKI patients. Relatively little is known about IGFBP6 in AKI but it has previously been shown to correlate with reduced kidney function and renal failure 5 . Additional studies using larger cohorts of urine samples across conditions are needed to confirm changes in these biomarkers and to compare with other causes of AKI, covariates, and outcomes. Table S2 columns AA-AC). Additionally, five proteins were significantly decreased in COVID+, including granulin precursor (GRN), cAMP responsive element binding protein 3 like 3 (CREB3L3), mucin 1 (MUC1), CD320, and dihydrolipoamide S-succinyl transferase (DLST) ( Figure 2C , Table S2 columns AA-AC). These newly identified proteins could potentially be used as more sensitive biomarkers to assess the COVID-19 infection. Moreover, the data reveals that several markers that are extrarenal in COVID+ patients (Fig. 2) including 5.68-fold increase in myoglobin (heart specific), 3.92-fold increase in zymogen pepsinogen C (lung, esophagus and stomach injury); promising targets for non-invasive urine proteome profiling to detect and monitor kidney-independent organ damage. The SARS-CoV-2 virion is not typically shed in urine but has tropism to kidney cell types 6, 7, 8 . We examined if urine proteomics can detect SARS-CoV-2 proteins. Interestingly, peptides from SARS-CoV-2 nucleoprotein were detected in urine samples collected from two severely ill COVID-AKI patients who died ( Figure 1A , Table S1 ). The peptide identification was confirmed by searching with both MaxQuant and MS-GF+ software. Near-complete sequence coverage of the RNA binding domain was achieved by 1D-LC-MS/MS analysis. Sequences near the Nterminus of the SARS-CoV-2 nucleoprotein were also observed ( Figure 2D ). Due to the relatively low-count peptide-spectrum matches (PSMs) of SARS-CoV-2 peptides observed, the concentration of SARS-CoV-2 nucleoprotein is predicted to be low in these urine samples (Table S3) . Although our study is a proof-of-concept investigation with a relatively small number of patient samples, we were able to establish methods for high quality urine proteomics of COVID-19 patients in a clinical setting. We report potential biomarkers of COVID-19 infection and of COVID-AKI that could be useful for detecting and monitoring the development of COVID infection or AKI. In particular, the complement and coagulation cascade pathways, and exocytosis pathway were highly up-regulated in COVID-AKI patients. Further, proteins associated with antigen presentation were significantly correlated with COVID-19 infection. We propose a list of potential novel biomarker proteins (i.e., MB, CA1, MANSC1, ABRACL, GRN, CREB3L3, DEFA1, MUC1, DLST, IGFBP6) and of previously reported AKI biomarker proteins Supplementary information is available at KI Report's website. Includes information about the methods used for sample acquisition and patient inclusion, urine processing and storage, proteomics sample preparation, LC-MS/MS-based proteomics analysis, proteomics data processing, correlation analysis of COV-AKI patients compared to non-AKI patients, and where the data is available to download. Supplemental Figures (PDF) Figure S1 : Pearson correlation of urinary proteomics across the studied samples. Renal Involvement and Early Prognosis in Patients with COVID-19 Pneumonia SARS-CoV-2 in the kidney: bystander or culprit? Urine proteome of COVID-19 patients Critical protection from renal ischemia reperfusion injury by CD55 and CD59 Circulating levels of human insulin-like growth factor binding protein-6 (IGFBP-6) in health and disease as determined by radioimmunoassay Infectious SARS-CoV-2 in Feces of Patient with Severe COVID-19 Isolation of infectious SARS-CoV-2 from urine of a COVID-19 patient Multiorgan and Renal Tropism of SARS-CoV-2 MHC peptides and the sensory evaluation of genotype This study utilized samples obtained from the Washington University School of Medicine's COVID-19 biorepository, which is supported by: the Barnes