Regenerative potential of colony-stimulating factor-1 therapy and macrophages in renal disease
Creators: name: Alikhan, Maliha
Publisher: name: Monash University
Year: 2016
Subjects: Uncategorized

description: Kidney disease is a growing public health concern, and current therapies inflict substantial strain on both patients and the health care system. Fortunately, the adult kidney can repair itself following transient damage. While the cellular events that lead to kidney regeneration are well defined, less is understood about the endogenous repair mechanisms that control these pathways. In adult organs, macrophages display significant heterogeneity during disease progression. The surrounding microenvironment ultimately influences disease outcome via polarisation of macrophages toward a classically activated M1-like state or an alternatively activated M2-like phenotype. M1-like macrophages are a pro-inflammatory population that are involved in host defence and tissue destruction, whereas M2-like macrophages secrete anti-inflammatory mediators that induce inflammatory resolution and wound healing. In the mouse model of renal ischemia-reperfusion (IR) injury, M2-like macrophages play a fundamental role in repair by resolving inflammation and mediating the restoration of injured renal epithelial cells. Colony-stimulating factor (CSF)-1 is a hematopoietic growth factor that primarily regulates the development, proliferation and survival of macrophages. In addition, CSF-1 exhibits important pleiotropic roles during development, homeostasis, injury and cancer. Studies also demonstrate that CSF-1 can foster epithelial cell repair and tissue remodelling in animal models of acute injury. The overall aim of this thesis was to investigate the regenerative potential of CSF-1 therapy in an acute renal IR injury model, with particular attention to the functional role of M2-like macrophages. Chapter 2 reports the structural and functional kidney recovery following CSF-1 administration to mice with established IR injury. Five days after injection of CSF-1 to IR mice, increased epithelial cell proliferation and reduced intratubular cast formation and interstitial matrix expansion was evident compared to vehicle-treated mice. Structural recovery at day 7 also coincided with reduced collagen accumulation and improved functional recovery as evident by reduced urinary albumin compared to vehicle-treated mice. In Chapter 3, extensive characterisation of macrophage phenotype and function was performed to determine the functional contribution of CSF-1-mediated renal regeneration. CSF-1 influenced renal macrophage number and maturity following IR injury. The number of CSF-1-responsive macrophages increased at day 5 and then declined at day 7 when recovery was complete. This coincided with macrophage maturity with increased expression of F4/80 and MHC II at day 5 when damage was still evident followed by reduced expression at day 7. Flow cytometry and real-time PCR analysis of the expression of known M1- and M2-associated genes provided further insight into the influence of CSF-1 therapy on macrophage phenotype and function. CSF-1-responsive macrophages displayed increased expression of common M2-like genes including arginase, CCL17, scavenger receptor and macrophage mannose receptor. To further corroborate these findings, microarray analysis was performed in order to investigate the specific macrophage signalling pathways and genes involved in the CSF-1-accelerated repair response. The most significant differences in macrophage gene expression occurred at day 5 post-IR injury, where the genes differentially expressed in CSF-1-responsive macrophages were primarily associated with M2-like signalling pathways. These included the glucocorticoid receptor, IL-10 and insulin-like growth factor (IGF)-1 signalling pathways, which are important in inflammatory resolution and wound healing. Of significant interest was the differential expression of genes associated with the IGF-1 signalling pathways. IGF-1 is known to play important reparative roles in post-ischemic kidneys, and CSF-1 can also control the expression of macrophage-derived IGF-1. Further analysis and validation was therefore performed using real-time PCR analysis, which revealed increased IGF-1 expression in CSF-1-responsive macrophages at both time-points compared to vehicle-treated mice. These findings suggest that CSF-1 accelerates renal regeneration following acute IR injury via alteration of macrophage phenotype, and in part by macrophage-derived IGF-1 production. However, reports from other investigators also indicate that CSF-1 can signal to epithelial cells in the body by direct autocrine and paracrine actions. In Chapter 4, the precise contribution of macrophages, macrophage-derived IGF-1 and renal epithelial cells following CSF-1 therapy was therefore examined in vitro using co-culture assays with bone marrow-derived macrophages and the immortalised renal mouse cortical tubular cell line. While the findings from the functional assays did not provide further insight into the mechanisms underlying the renoprotective effects of CSF-1 therapy, they did support our hypothesis that CSF-1 regulates macrophage-derived IGF-1 production. However, additional mechanistic experiments in a variety of animal models of renal disease in combination with blocking antibodies are needed to fully elucidate the functional contribution of CSF-1 therapy. A better understanding of the role of macrophages and the factors that control their phenotype and function may ultimately lead to the development of therapies that attenuate acute renal disease and prevent the progression to chronic renal failure for patients with renal disease.