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Cell Profiling of Mouse Acute Kidney Injury Reveals Conserved Cellular Responses to Injury

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Cell Profiling of Mouse Acute Kidney Injury Reveals Conserved Cellular Responses to Injury Cell profiling of mouse acute kidney injury reveals conserved cellular responses to injury Yuhei Kiritaa,b, Haojia Wua, Kohei Uchimuraa, Parker C. Wilsonc, and Benjamin D. Humphreysa,d,1 aDivision of Nephrology, Department of Medicine, Washington University in Saint Louis School of Medicine, St. Louis, MO 63110; bDepartment of Nephrology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; cDivision of Anatomic and Molecular Pathology, Department of Pathology and Immunology, Washington University in Saint Louis School of Medicine, St. Louis, MO 63110; and dDepartment of Developmental Biology, Washington University in Saint Louis School of Medicine, St. Louis, MO 63110 Edited by Martin R. Pollak, Beth Israel Deaconess Medical Center, Brookline, MA, and approved May 20, 2020 (received for review March 25, 2020) After acute kidney injury (AKI), patients either recover or alterna- Results tively develop fibrosis and chronic kidney disease. Interactions We performed single nucleus RNA-sequencing (snRNA-seq) on between injured epithelia, stroma, and inflammatory cells de- cryopreserved mouse kidney (7). Mice were euthanized at 4 and termine whether kidneys repair or undergo fibrosis, but the 12 h and 2, 14, and 42 d after bilateral ischemia–reperfusion molecular events that drive these processes are poorly under- injury (IRI) (Fig. 1A). Both histologic changes (SI Appendix, Fig. stood. Here, we use single nucleus RNA sequencing of a mouse S1) and blood urea nitrogen (BUN) (Fig. 1B) levels confirmed model of AKI to characterize cell states during repair from acute acute injury and its resolution in mouse. injury. We identify a distinct proinflammatory and profibrotic After quality control filtering, we obtained 26,643 cells from proximal tubule cell state that fails to repair. Deconvolution of healthy mouse kidneys. Visualization of single nucleus tran- bulk RNA-seq datasets indicates that this failed-repair proximal scriptomes in Uniform Manifold Approximation and Projection tubule cell (FR-PTC) state can be detected in other models of kid- (UMAP) space resolved 26 separate clusters (SI Appendix,Fig.S2A ney injury, increasing during aging in rat kidney and over time in and Dataset S1). Subclustering of both epithelial (descending loop human kidney allografts. We also describe dynamic intercellular of Henle, thin ascending limb) and nonepithelial cells (immune, communication networks and discern transcriptional pathways endothelial, stromal) revealed additional cell clusters (SI Appendix, driving successful vs. failed repair. Our study provides a detailed Fig. S2B). For example, five separate endothelial clusters were description of cellular responses after injury and suggests that the identified, including arterial, lymphatic, descending vasa recta and FR-PTC state may represent a therapeutic target to improve repair. cortical vs. medullary endothelium. Eight stromal clusters were de- tected, including mesangium and Ren1-positive juxtaglomerular appa- AKI | injury | transcriptomics | epithelia ratus cells (SI Appendix,Fig.S2B). Major cell types and subclusters were identified based on cell type-specific markers (SI Appendix,Fig. idneys maintain fluid and electrolyte balance, excrete waste S2 C and D and Dataset S1)(8–10). Each nephron segment performs Kproducts, and regulate blood pressure. Composed of ap- unique reabsorptive and secretory functions to transform filtrate into proximately one million functional units called nephrons, the urine, and this is reflected by segment-specific expression of all detected kidneys receive ∼20% of cardiac output. Nephrons have high solute-linked carriers, ATPases, and channels (SI Appendix,Fig.S3). metabolic activity rendering them susceptible to injury from toxins or reduced blood flow. These insults cause acute kidney injury Proximal Tubule Responses to Acute Injury. We generated 99,935 mouse AKI single cell transcriptomes (Fig. 1C) and integrated (AKI) characterized by decreased kidney function. In early stages of these with the healthy datasets using the Harmony algorithm to AKI, epithelial cells die, and surviving epithelia dedifferentiate, reduce batch effects (Fig. 1D) (11). We could define unique accompanied by inflammation. Dedifferentiated epithelial cells then anchor genes for all clusters in the integrated datasets and de- proliferate and redifferentiate to repair the damaged nephron (1, fined the relative abundance of each cluster in healthy vs. injured 2). There are no specific treatments for AKI, but, with supportive ’ care, the kidney s intrinsic repair capacity usually allows functional Significance recovery over a period of days to weeks. After repair, kidney function may not return back to baseline Single nucleus RNA sequencing revealed gene expression due to residual subclinical inflammation and fibrosis. Survivors changes during repair after acute kidney injury. We describe a of AKI are at high risk of developing future chronic kidney small population of proximal tubule cells that fail to repair disease (CKD) and even kidney failure (3). The mechanisms for (FR-PTCs). Since this subpopulation expresses abundant proin- failed repair are not well understood, but a subpopulation of flammatory and profibrotic genes, it may represent a new ther- injured proximal tubule (PT) epithelia (the segment most sus- apeutic target to improve repair and reduce fibrosis after AKI. ceptible to injury) are proposed to become arrested at the G2/M cell cycle phase and adopt a senescence-associated secretory Author contributions: Y.K. and B.D.H. designed research; Y.K., H.W., and K.U. performed phenotype (4). This may prevent complete repair, driving in- research; Y.K. contributed new reagents/analytic tools; Y.K., H.W., K.U., P.C.W., and B.D.H. analyzed data; and B.D.H. wrote the paper. flammation and fibrosis, and mouse ischemia–reperfusion injury The authors declare no competing interest. (IRI) models this process well (5). The aim of our study was to understand the cellular events underlying both recovery from This article is a PNAS Direct Submission. This open access article is distributed under Creative Commons Attribution-NonCommercial- AKI as well as the transition to CKD. Bulk transcriptional pro- NoDerivatives License 4.0 (CC BY-NC-ND). filing has successfully characterized kidney injury and recovery Data deposition: All relevant data have been deposited in the Gene Expression Omnibus (5, 6), but these approaches describe a transcriptional average under accession number GSE139107. across cell populations, which may hide or skew signals of in- See online for related content such as Commentaries. terest. We hypothesized that understanding transcriptional 1To whom correspondence may be addressed. Email: [email protected]. changes in single cell types over the course of AKI, repair, and This article contains supporting information online at https://www.pnas.org/lookup/suppl/ fibrosis would provide unique insights into disease pathogenesis doi:10.1073/pnas.2005477117/-/DCSupplemental. and potentially identify new therapeutic strategies. First published June 22, 2020. 15874–15883 | PNAS | July 7, 2020 | vol. 117 | no. 27 www.pnas.org/cgi/doi/10.1073/pnas.2005477117 Downloaded by guest on September 30, 2021 AB200 C Control 4 hours 12 hours 2 days 14 days 6 weeks 150 Rep. 1 15444 7053 11232 14339 16295 16106 100 Rep. 2 5745 1970 4699 4520 2294 2212 BUN (mg/dl) 50 Rep. 3 5465 5463 4529 1944 2113 5155 Subtotal 26643 14486 20460 20803 20702 23473 (n = 3) 0 l s s s s r r y y ks Total 126578 tro u a a n ou o d d ee o h h w C 4 2 2 4 Single Nucleus RNA-seq 1 1 6 Nucleus 10X Chromium system Computational analysis isolation Comp. E by group PT-S1 PT-S2 PT-S3 New PT1 New PT2 DTL-ATL MTAL CTAL1 Group CTAL2 D Control 1 MD 4hours 12hours 2 1. PT S1 14. CPC DCT 2days 2. PT S2 15. MPC 14days DCT-CNT 6weeks 25 3. PT S3 16. ICA 26 4 CNT 4. New PT1 17. ICB CPC Average Expression 3 5 2.5 5. New PT2 18. Uro MPC 2.0 20 6. DTL-ATL 19. Pod 1.5 22 ICA 1.0 19 7. MTAL 20. PEC ICB 0.5 21 6 0.0 23 8. CTAL1 21. EC1 Uro Percent Expressed 24 18 7 9. CTAL2 22. EC2 Pod 25 10. MD 23. Fib PEC 50 75 16 10 9 11. DCT 24. PER EC1 25. Mø EC2 15 12. DCT-CNT 17 8 13. CNT 26. T cell Fib Per 14 13 12 Mø MEDICAL SCIENCES 11 T cell 2 3 1 1 it b b t1 r 47 1 a a d4 1 K m1 c Eln 2 a 3 2 s os1 k Fl gfr a gam v Lrp2 1 1 d h nn1g t N UMAP dim. 2 Aqp2 d a I c16a9 c5 T c Enox1 C Epha7 Up Nphs2 l l Myh11 em207 Slc8a1 P Vcam1 Nc H S Slc12a3 Slc S Slc Slc26a4 S UMAP dim. 1 m T Fig. 1. Single nucleus RNA-seq atlas of mouse IRI kidney. (A) Summary of experimental strategy. n = 3 mice per group. (B) BUN (mg/dL) after sham and IRI. Data are shown as the mean ± SEM. (C) Table with details of group, replicates, and cell numbers of mouse IRI datasets present in this figure. (D) UMAP plots of all mouse IRI kidney datasets integrated with Harmony. ATL, thin ascending limb of loop of Henle; Bil, bilateral; CNT, connecting tubule; CPC, principle cells of collecting duct in cortex; CTAL, thick ascending limb of loop of Henle in cortex; DCT, distal convoluted tubule; DTL, descending limb of loop of Henle; EC, endothelial cells; Fib, fibroblasts; ICA, type A intercalated cells of collecting duct; ICB, type B intercalated cells of collecting duct; MD, macula densa; Mø, macrophages; MPC, principle cells of collecting duct in medulla; MTAL, thick ascending limb of loop of Henle in medulla; PEC, parietal epithelial cells; Per, pericytes; Pod, podocytes; PT-S1, S1 segment of proximal tubule; PT-S2, S2 segment of proximal tubule; PT-S3, S3 segment of proximal tubule; Uro, uro- thelium.
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