Denby, Laura

Conway, Bryan

Benezech, Cecile

Bell, Rachel Mary Buchanan

British Heart Foundation

2024-03-08T15:17:58Z

2024-03-08T15:17:58Z

2024-03-08

Chronic kidney disease (CKD) represents a global health burden of increasing prevalence. The common pathway in all progressive kidney disease is fibrosis, where scar tissue replaces normal functioning tissue. Macrophages are a major cellular component of the renal mononuclear phagocyte system, with roles in defence against infection, renal injury, and repair. Renal macrophages are derived from foetal precursors and, in adult life, circulating monocytes, with a greater contribution in response to infection or injury. Macrophages possess enormous plasticity and heterogeneity; they are known to adopt different phenotypic and functional characteristics that depend on the local tissue microenvironment and stress signals (due to tissue insult) and perhaps ontogeny. Hence, they can be injurious or may mediate repair by scavenging cell debris, degrading excess extracellular matrix and secreting factors that may promote the regeneration of injured tissue. Using single-cell RNA sequencing, we have recently identified novel myeloid subsets in murine reversible, unilateral ureteric obstruction (UUO), an acute non-functional model of kidney fibrosis and repair. Amongst the subsets, a population of cells was identified exclusively in acute injury (2 days post-UUO); they transcriptomically align to monocytes but express Arginase 1 (Arg1) and C-type lectin Clec4d, and many pro-inflammatory and profibrotic genes. The hypothesis presented in this thesis is that a proportion of monocytes recruited to the kidney in early injury transition into monocyte-derived macrophages (Mo- MΦ) with an Arg1⁺ phenotype and have a role in activating inflammation and fibrosis in kidney disease. Arg1 and Clec4d were detectable in bulk kidney tissue in different models of renal fibrosis, including the 5/6ᵗʰ subtotal nephrectomy, UUO and unilateral ischaemic reperfusion injury (IRI). Further detailed flow cytometry investigations in the UUO and IRI over a seven-day time course showed persistent recruitment of Ly6Cʰᶦ monocytes transitioning to pro-fibrotic inflammatory macrophages in injured kidneys. Arg1⁺ Mo-MΦ persisted in the kidney; across the injury time course, they increased in number and, consistent with a monocyte-tomacrophage transition, there was a decrease in monocyte markers but an increase in activated macrophage marker expression in this subset. Arg1 was not detectable in the blood. In human fibrotic kidney biopsy samples, ARG1 was detected in the classical- and CLEC4D in the classical and intermediate monocyte populations. To confirm that Arg1⁺ Mo-MΦ are derived from monocytes, the Ccr2-CreERᵀ²- TdTomato mouse model was employed, in which tamoxifen administration induces irreversible labelling of Ccr2⁺ cells, including Ly6Cʰᶦ monocytes and their progeny. Tamoxifen was administered under a single or multiple-dose regimen following UUO surgery, showing the Arg1⁺ Mo-MΦ are derived from Ccr2⁺ monocytes in fibrotic injury. To investigate the therapeutic potential of targeting Clec4d⁺ expressed on the Arg1⁺ Mo- MΦ, mice underwent UUO surgery and were given multiple doses of a neutralising anti- Clec4d monoclonal antibody. A decrease in Arg1, Clec4d and fibrosis-related gene expression was evident by day seven post-injury. Taken together, these data confirm the presence of an Arg1⁺ Mo-MΦ population in renal injury and fibrosis development in pre-clinical models of kidney disease and human fibrotic renal biopsies. The Arg1⁺ macrophages in late fibrotic injury are monocyte-derived, and targeting them via Clec4d neutralisation has the potential to reduce the extent of renal fibrosis. These findings could be of clinical relevance with the potential to aid the development of novel therapeutics to limit fibrosis and halt the progression of CKD.

https://hdl.handle.net/1842/41603

http://dx.doi.org/10.7488/era/4335

en

The University of Edinburgh

Rachel M.B. Bell, Elisa Villalobos, Mark Nixon, Allende Miguelez-Crespo, Lee Murphy, Angie Fawkes, Audrey Coutts, Matthew G.F. Sharp, Martha V. Koerner, Emma Allan, Onno C. Meijer, Renè Houtman, Alex Odermatt, Katharina R. Beck, Scott G. Denham, Patricia Lee, Natalie Z.M. Homer, Brian R. Walker, Ruth A. Morgan. Carbonyl reductase 1 amplifies glucocorticoid action in adipose tissue and impairs glucose tolerance in lean mice. Molecular Metabolism. 2021; 48: 101225

Rachel M.B. Bell & Laura Denby. Myeloid Heterogeneity in Kidney Disease as Revealed through Single-Cell RNA Sequencing. Kidney 360. 2021; 2: 1844-1851

Rachel M.B. Bell & Bryan R. Conway. Macrophages in the kidney in health, injury and repair. International Review of Cell and Molecular Biology. 2022; 367

Eoin D. O'Sullivan, Katie J. Mylonas, Rachel M.B. Bell, Cyril Carvalho, David P. Baird, Carolynn Cairns, Kevin Gallagher, Ross A. Campbell, Marie Docherty, Alexander Laird, Neil C. Henderson, Tamir Chandra, Kristina Kirschner, Bryan R. Conway, Gry H. Dihazi, Michael Zeisberg, Jeremy Hughes, Laura Denby, Hassan Dihazi, David A. Ferenbach. Single-cell analysis of senescent epithelia reveals targetable mechanisms promoting fibrosis. JCI Insight. 2022; 7: e154124

Arginase 1+ monocytemacrophage

fibrosis

Chronic kidney disease (CKD)

Renal macrophages

unilateral ureteric obstruction (UUO)

fibrosis in kidney disease

Arg1+ Mo-MΦ

Ccr2+ monocytes

fibrotic injury

Clec4d+

Investigating an Arginase 1⁺ monocyte- macrophage population in driving fibrosis in chronic kidney disease.

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy