Combined lineage tracing and scRNA-seq reveal the activation of Sox9⁺ cells in renal regeneration with PGE₂ treatment

Abstract

Uncovering mechanisms of endogenous regeneration and repair through resident stem cell activation will allow us to develop specific therapies for injuries and diseases by targeting resident stem cell lineages. Sox9⁺ stem cells have been reported to play an essential role in acute kidney injury (AKI). However, a complete view of the Sox9⁺ lineage was not well investigated to accurately elucidate the functional end state and the choice of cell fate during tissue repair after AKI. To identify the mechanisms of fate determination of Sox9⁺ stem cells, we set up an AKI model with prostaglandin E2 (PGE₂) treatment in a Sox9 lineage tracing mouse model. Single-cell RNA sequencing (scRNA-seq) was performed to analyse the transcriptomic profile of the Sox9⁺ lineage. Our results revealed that PGE₂ could activate renal Sox9⁺ cells and promote the differentiation of Sox9⁺ cells into renal proximal tubular epithelial cells and inhibit the development of fibrosis. Furthermore, single-cell transcriptome analysis demonstrated that PGE₂ could regulate the restoration of lipid metabolism homeostasis in proximal tubular epithelial cells by participating in communication with different cell types. Our results highlight the prospects for the activation of endogenous renal Sox9⁺ stem cells with PGE₂ for the regenerative therapy of AKI.

References

[1] ZhangW, GaoC, TsilosaniA, Samarakoon R, PlewsR, HigginsPJ. Potential renal stem/progenitor cells identified by in vivo lineage tracing. Am J Physiol Renal Physiol. 2022;322(4):F379-F391.

[2] WagnerDE, KleinAM. Lineage tracing meets single-cell omics: opportunities and challenges. Nat Rev Genet. 2020;21(7):410-427.

[3] KesterL, van Oudenaarden A. Single-cell transcriptomics meets lineage tracing. Cell Stem Cell. 2018;23(2):166-179.

[4] ZhangK, LiR, ChenX, et al. Renal endothelial cell-targeted extracellular vesicles protect the kidney from ischemic injury. Adv Sci (Weinh). 2023;10(3):e2204626.

[5] HuangH, QianM, LiuY, et al. Genetically engineered mesenchymal stem cells as a nitric oxide reservoir for acute kidney injury therapy. eLife. 2023;12:e84820.

[6] FitzSimonsM, Beauchemin M, SmithAM, et al. Cardiac injury modulates critical components of prostaglandin E(2) signaling during zebrafish heart regeneration. Sci Rep. 2020;10(1):3095.

[7] MiyoshiH, VanDussen KL, MalvinNP, et al. Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium. EMBO J. 2017;36(1):5-24.

[8] LiuY, RenHZ, WangJL, et al. Prostaglandin E-2 secreted by mesenchymal stem cells protects against acute liver failure via enhancing hepatocyte proliferation. FASEB J. 2019;33(2):2514-2525.

[9] ChengH, HuangH, GuoZ, ChangY, LiZ. Role of prostaglandin E2 in tissue repair and regeneration. Theranostics. 2021;11(18):8836-8854.

[10] ReginensiA, Clarkson M, NeirijnckY, et al. SOX9 controls epithelial branching by activating RET effector genes during kidney development. Hum Mol Genet. 2011;20(6):1143-1153.

[11] KumarS, LiuJ, PangP, et al. Sox9 activation highlights a cellular pathway of renal repair in the acutely injured mammalian kidney. Cell Rep. 2015;12(8):1325-1338.

[12] ChenS, HuangH, LiuY, et al. Renal subcapsular delivery of PGE2 promotes kidney repair by activating endogenous Sox9(+) stem cells. iScience. 2021;24(11):103243.

[13] ZhangK, ChenS, SunH, et al. In vivo two-photon microscopy reveals the contribution of Sox9+ cell to kidney regeneration in a mouse model with extracellular vesicle treatment. J Biol Chem. 2020;295(34):12203-12213.

[14] HuangH, ChenS, ChengH, et al. The sustained PGE(2) release matrix improves neovascularization and skeletal muscle regeneration in a hindlimb ischemia model. J Nanobiotechnol. 2022;20(1):95.

[15] SatijaR, Farrell JA, GennertD, SchierAF, RegevA. Spatial reconstruction of single-cell gene expression data. Nat Biotechnol. 2015;33(5):495-502.

[16] AibarS, Gonzalez-Blas CB, MoermanT, et al. SCENIC: single-cell regulatory network inference and clustering. Nat Methods. 2017;14(11):1083-1086.

[17] LuoWJ, Brouwer C. Pathview: an R/Bioconductor package for pathway-based data integration and visualization. Bioinformatics. 2013;29(14):1830-1831.

[18] ChenJ, Crawford R, ChenC, XiaoY. The key regulatory roles of the PI3K/Akt signaling pathway in the functionalities of mesenchymal stem cells and applications in tissue regeneration. Tissue Eng Part B Rev. 2013;19(6):516-528.

[19] MengD, FrankAR, JewellJL. mTOR signaling in stem and progenitor cells. Development. 2018;145(1):dev152595.

[20] Garcia-PratL, Perdiguero E, Alonso-MartinS, et al. FoxO maintains a genuine muscle stem-cell quiescent state until geriatric age. Nat Cell Biol. 2020;22(11):1307-1318.

[21] ChanYH, LeeYC, HungCY, Yang PJ, LaiPC, FengSW. Three-dimensional spheroid culture enhances multipotent differentiation and stemness capacities of human dental pulp-derived mesenchymal stem cells by modulating MAPK and NF-kB signaling pathways. Stem Cell Rev Rep. 2021;17(5):1810-1826.

[22] BergenV, LangeM, PeidliS, Wolf FA, TheisFJ. Generalizing RNA velocity to transient cell states through dynamical modeling. Nat Biotechnol. 2020;38(12):1408-1414.

[23] TrapnellC, Cacchiarelli D, GrimsbyJ, et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat Biotechnol. 2014;32(4):381-386.

[24] KrizW, Kaissling B, Le HirM. Epithelial-mesenchymal transition (EMT) in kidney fibrosis: fact or fantasy? J Clin Invest. 2011;121(2):468-474.

[25] RudnickiM, PercoP, EnrichJ, et al. Hypoxia response and VEGF-A expression in human proximal tubular epithelial cells in stable and progressive renal disease. Lab Invest. 2009;89(3):337-346.

[26] YoshidaGJ. Emerging roles of Myc in stem cell biology and novel tumor therapies. J Exp Clin Cancer Res. 2018;37(1):173.

[27] SeruggiaD, OtiM, TripathiP, et al. TAF5L and TAF6L maintain self-renewal of embryonic stem cells via the MYC regulatory network. Mol Cell. 2019;74(6):1148-1163.e7.

[28] PengJC, WuT, WuX, et al. Development of mortality prediction model in the elderly hospitalized AKI patients. Sci Rep. 2021;11(1):15157.

[29] PalikarasK, Lionaki E, TavernarakisN. Balancing mitochondrial biogenesis and mitophagy to maintain energy metabolism homeostasis. Cell Death Differ. 2015;22(9):1399-1401.

[30] ShangD, SunD, ShiC, et al. Activation of epidermal growth factor receptor signaling mediates cellular senescence induced by certain pro-inflammatory cytokines. Aging Cell. 2020;19(5):e13145.

[31] DoiK, NoiriE, FujitaT. Role of vascular endothelial growth factor in kidney disease. Curr Vasc Pharmacol. 2010;8(1):122-128.

[32] VillegasG, Lange-Sperandio B, TufroA. Autocrine and paracrine functions of vascular endothelial growth factor (VEGF) in renal tubular epithelial cells. Kidney Int. 2005;67(2):449-457.

[33] GaoL, ZhongX, JinJ, LiJ, MengXM. Potential targeted therapy and diagnosis based on novel insight into growth factors, receptors, and downstream effectors in acute kidney injury and acute kidney injury-chronic kidney disease progression. Signal Transduct Target Ther. 2020;5(1):9.

[34] LiM, ZhengH, HanY, et al. LncRNA Snhg1-driven self-reinforcing regulatory network promoted cardiac regeneration and repair after myocardial infarction. Theranostics. 2021;11(19):9397-9414.

[35] TangSW, ChangWH, SuYC, et al. MYC pathway is activated in clear cell renal cell carcinoma and essential for proliferation of clear cell renal cell carcinoma cells. Cancer Lett. 2009;273(1):35-43.

[36] CleversH, LohKM, NusseR. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. Science. 2014;346(6205):1248012.

[37] VarlakhanovaNV, Cotterman RF, deVriesWN, et al. myc maintains embryonic stem cell pluripotency and self-renewal. Differentiation. 2010;80(1):9-19.

[38] SatoT, Okumura F, ArigaT, HatakeyamaS. TRIM6 interacts with Myc and maintains the pluripotency of mouse embryonic stem cells. J Cell Sci. 2012;125(Pt 6):1544-1555.

[39] Chang-PanessoM, Humphreys BD. Cellular plasticity in kidney injury and repair. Nat Rev Nephrol. 2017;13(1):39-46.

[40] LiM, LiCM, YeZC, et al. Sirt3 modulates fatty acid oxidation and attenuates cisplatin-induced AKI in mice. J Cell Mol Med. 2020;24(9):5109-5121.

[41] BatailleA, Galichon P, ChelghoumN, et al. Increased fatty acid oxidation in differentiated proximal tubular cells surviving a reversible episode of acute kidney injury. Cell Physiol Biochem. 2018;47(4):1338-1351.

[42] EmmaF, Montini G, ParikhSM, SalviatiL. Mitochondrial dysfunction in inherited renal disease and acute kidney injury. Nat Rev Nephrol. 2016;12(5):267-280.

[43] ZhaoY, LiX, YangL, et al. Transient overexpression of vascular endothelial growth factor a in adipose tissue promotes energy expenditure via activation of the sympathetic nervous system. Mol Cell Biol. 2018;38(22):e00242-18.

[44] di SommaM, VlioraM, GrilloE, et al. Role of VEGFs in metabolic disorders. Angiogenesis. 2020;23(2):119-130.

[45] WrightGL, Maroulakou IG, EldridgeJ, et al. VEGF stimulation of mitochondrial biogenesis: requirement of AKT3 kinase. FASEB J. 2008;22(9):3264-3275.

[46] AggarwalS, WangZ, Rincon Fernandez PachecoD, et al. SOX9 switch links regeneration to fibrosis at the single-cell level in mammalian kidneys. Science. 2024;383(6685):eadd6371.

RIGHTS & PERMISSIONS

2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.