Urinary single-cell sequencing captures kidney injury and repair processes in human acute kidney injury

• DOI: 10.1016/j.kint.2022.07.032
• Publication Date: 2022-08-29T16:11:30Z
• Authors: Jan Klocke; Seung Joon Kim; Christopher M. Sk...; Christian Hinze; Anastasiya Bolten...; Diana Metzke; Emil Grothgar; Luka Prskalo; Leonie Wagner; Paul Freund; Nina Görlich; Frédéric Muench; Kai M. Schmidt-Ott; Mir-Farzin Mashreghi; Christine Kocks; Kai-Uwe Eckardt; Nikolaus Rajewsky; Philipp Enghard
• Keywords: Cell Sorting

Acute kidney injury (AKI) is a major health issue, the outcome of which depends primarily on damage and reparative processes tubular epithelial cells. Mechanisms underlying AKI remain incompletely understood, specific therapies are lacking monitoring course in clinical routine confined to measuring urine output plasma levels filtration markers. Here we demonstrate feasibility potential novel approach assess cellular molecular dynamics by establishing robust urine-to-single cell RNA sequencing (scRNAseq) pipeline for excreted cells via flow cytometry sorting. We analyzed 42,608 single transcriptomes 40 samples from 32 patients with compared our data reference material human post-mortem biopsies published mouse data. that mirror pathology reflect distinct repair processes, including oxidative stress, inflammation, tissue rearrangement. also describe an AKI-specific abundant urinary excretion adaptive progenitor-like Thus, transcriptomics provides noninvasive, unprecedented insight into AKI, thereby opening opportunities target identification, sub-categorization, natural disease interventions. see commentary page 1219 concern associated significant morbidity mortality.1Lafrance J.P. Miller D.R. associates increased long-term mortality.J Am Soc Nephrol. 2010; 21: 345-352Crossref PubMed Scopus (441) Google Scholar,2Sawhney S. Marks A. 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By analyzing individuals observed different injury-related tissue. collected defined Disease: Improving Global Outcomes (KDIGO) criteria (Supplementary Figure S1 Supplementary Table S1). Patients sampled at variable time point within 21 days onset. Seven underwent cardiac surgery maximum 48 hours before onset, 15 admitted intensive care units because pneumonia (all fulfilling sepsis criteria) developed 5 their unit stay; most these (14/15) had coronavirus 2019 (COVID-19). An additional 10 other, mostly prerenal, causes gastrointestinal bleeding (n = 1), diarrhea 2), exsiccosis 4), decompensated heart failure 3). For detailed patient characteristics, Methods. Samples morning void catheter (using pooled 4 hours). Viable sorted using cytometric S2). Some barcoded, pooled, later demultiplexed S3). Single sequenced following 10x Genomics protocol Chromium Next GEM Cell 3' v3.1 chemistry (10x Genomics). details, All R.24R: R project statistical computing.https://www.r-project.org/Date accessed: 12, 2022Google Normalization, logarithmic transformation, identification highly features, scaling, principal analysis, extraction differentially expressed features done Seurat. description, Significant differences tested 2-tailed unpaired 2-sample Wilcoxon test. ethics committee Charité University Hospital approved (Charité EA2/141/19). Informed consent was obtained next kin participation. goal understand what types occur sediment AKI. performed scRNAseq derived fresh points 0 onset due 7), 15; among them 14 cases COVID-19), prerenal 10). After quality control filtering, total median 472 per sample well 1436 genes 3991 unique transcripts cell. Data (uniform manifold approximation projection visualization, unbiased clustering, annotation based known marker genes) revealed type categories main (Figure 1 S2): parenchymal composed small, homogeneous fraction podocytes ("PDCs"; NPHS2 PODXL) many tubules ("TECs"; CRYAB EPCAM) various reactive traits, further below. Immune included dominant myeloid signal multiple monocyte/macrophage subsets showing residency25Clatworthy M.R. find resident macrophage: solution.J 30: 715-716Crossref (5) ("MO_kdnrs"; C1QB CD74), proinflammatory ("MO_infl"; IL1B TIMP1), profibrotic26Morse Tabib T. Sembrat al.Proliferating SPP1/MERTK-expressing macrophages idiopathic pulmonary fibrosis.Eur Respir 541802441Crossref (204) Scholar,27Montford J.R. Bauer Dobrinskikh al.Inhibition 5-lipoxygenase decreases progression disease.Am Physiol Physiol. 316: F732-F742Crossref (8) ("MO_SPP1+"; SPP1 ALOX5AP), antioxidative ("MO_MT+"; metallothionein genes), severely ("MO_dmg") phenotypes. Granulocytes excluded sort S2) featured herein only small residual cluster ("GRAN"; CSF3R MNDA). Lymphocytes regularly featured, being more frequent than B Finally, urogenital tract ("UGECs") epithelia reproductive system (KRT13 PSCA) urothelial expressing uroplakins (UPK2). Interestingly, detectable across Figures S4 S5), despite diverse overall quantity captured high-quality transcriptomes, (range, 8–5900) cells/sample S4D). UGECs prominently female 1b d S5). asked whether other factors influence composition examined entities separately public sets. Briefly, (GSE157640)18Abedini consisted almost entirely UGECs, whereas like sclerosis, varying degrees. Severe respiratory syndrome 2 (SARS-CoV-2) infection own (for information cross-disease comparisons, Results S15–S20). Overall, immune importantly kidney, reflecting severity damage. 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Taken together, seemed determined lesser extent origin. important investigate pathophysiology. used single-nuclei postmortem generated previously12Hinze 3a ). contained 106,971 nuclei tubular, leukocyte, endothelial, fibrocyte identities. Hinze phenotypes, notably signs transition, PT_New, TL_New, TAL_New, DCT_New, CNT_New, CD-PC_New, CD-IC_New.12Hinze biopsy construct symphony37Kang J.B. Nathan Weinand al.Efficient precise mapping Symphony.Nat Commun. 5890Crossref (27) mapped onto b; conservative integration sets harmony found S9). This overlap validated prior findings, few CD. Injury-related ("New_"), especially TAL, (TL), Map query low abundance fibrocytes 3b) previously clustered nonintegrated 1). Encouraged Correlations profiles similarity corresponding 3c), better correlation "New" counterparts. Wanting validate approach, automated (SingleR) yielded similar results: Aside healthy, segment-specific appeared phenotypes loop Henle S10). Comparing location (based mapping) bias toward medullary distal 3d). influenced loss integrity and/or detachment basal membrane, viable TL, CD prone final excretion. Further comparisons bladder origin suggested impact exposure transcriptome information, S21 S22). conclusion, urine, while about pathophysiology described models. These states, "injury," "repairing" "failed-repair"5Kirita Scholar,9Ide samples.8Legouis cross-species mouse/human comparative (see Methods), trained multinomial ischemia-reperfusion set Kirita al.,5Kirita who initially 4a b indicated "injured" 4c). portion clusters, assigned "healthy." TEC_VCAM1 state, overlapped "repair" "healthy" 4c d). trajectory states. Urinary displayed evaluate investigated monocle3, algorithm computational trajectories 5a TAL (SLC12A1+, UMOD+, LCN2–) (LCN2+) end adaptive, (SOX4+, PAX2+) (VCAM1+, PROM1+) 5c). graph, comparatively samples. Nevertheless, separate, differentiated (LRP2+, GPX3+) injured/adaptive (HAVCR1+, VCAM1+, S11). interpreted cautiously, starting needs chosen investigator knowledge assumption. observations suggest state: Phospholipase C γ (PLCG2), together FOS JUN, S12). Although PLCG2 cells,38Lampson B.L. Are BTK mutations necessary sufficient ibrutinib resistance lymphocytic leukemia?.Expert Rev Hematol. 185-194Crossref (51) recent evidence