#5466 CSF-1R SYSTEM ACTIVATES PARIETAL EPITHELIAL CELLS LEADING TO FOCAL SEGMENTAL GLOMERULOSCLEROSIS (FSGS)
Authors: Garrido Ana Amaya , Doladé Nuria , Varela Cristian , Manonelles Anna , Rayego-Mateos Sandra , Ruiz-Ortega Marta , Codina Sergi , Cruzado Josep , Sola Anna 
Published: June 2023

Abstract

Background and Aims
Focal segmental glomerulosclerosis (FSGS) is the most common glomerular cause leading to end-stage kidney disease. Actual treatment of primary FSGS by immunosuppressive agents presents inconsistent results. Thus, new innovative strategies different from those used to date by taking into consideration podocyte renewal and maintenance or even strategies complementary to immunosuppression are needed. In FSGS, parietal epithelial cells (PECs) switch to an activated phenotype (aPECs) in response to podocyte damage promoting glomerulosclerosis. Colony-stimulating factor (CSF-1) is a hematopoietic growth factor that acts via its specific receptor, the tyrosine kinase receptor CSF-1R. CSF-1 has been detected in sera and renal biopsies from patients with different renal complications, including FSGS, attributing their role and presence to macrophage but not to glomerular cells. The potential cellular and molecular mechanisms involved are also unknown. In this work, we evaluated the implication of CSF-1/CSF-1R axis in the pathogenesis of FSGS and its potential as new pharmacological target point by using specific CSF-1R inhibitors. Specifically, we focused on the modulation of PECS activation by de novo production of CD44 and the preservation of podocyte loss.

Method
We evaluated the role of the CSF-1/CSF-1R axis as a driver of glomerular damage in FSGS in adriamycin-induced nephropathy (ADR) in mice, the main experimental model to study human FSGS. To this end, we treated or not ADR-animals with CSF-1R specific inhibitors, GW2580 or Ki20227 (n = 5-7 group). We determined the expression and localization of CSF-1R in the glomerulus in tissue by triple immunofluorescence (WT-1, SSeCks and CSF-1R) and their relevance in glomerulosclerosis (PAS and pro-fibrotic genes), the determination of de novo CD44 formation and its correlation with ERK1/2 pathway by immunohistochemistry (IH). We detected podocyte in the glomerulus by WT-1 IH and the localization of aPECS in the glomerular tuff by Claudin-1 and SSeCkS markers. Finally, we used isolated human kidney progenitor cells with and without CSF-1 treatment (n = 6/group) to identify potential key interactors of CSF-1 by RNAseq. We validated genes of interest in the FSGS experimental model.

Results
We observed a constitutively expression of CSF-1R in the glomerulus of control mice that significantly increased in ADR-treated mice, specifically in podocytes (WT1) and PECs (SSeCks), confirmed with mRNA expression. ADR-treated mice showed important events of sclerotic glomerulus and pro-fibrotic genes as collagen that was significantly reversed by the treatment with CSF-1R inhibitors (p&lt;0.001). Results reflected a CSF-1R inhibitor-dependent renal function recovery with the use of the inhibitors with a reduction of proteinuria and an increase of glomerular filtration rate (p&lt;0.001). We found a positive CD44 staining both in Bowman's capsule and inner the tuff of ADR-treated mice, accompanied by an increase of ERK1/2 activation. CSF-1R inhibitors significantly reduced the percentage of glomeruli with de novo CD44 production. Remarkably, podocyte depletion was also preserved with very similar levels to non-treated animals (p&lt;0.001). For RNAseq results, 227 differentially expressed genes (considering a criterion of a probability of differential expression &gt;0.9 and a |M| &gt; 1) were subjected to enrichment analysis. The top significant gene ontology terms were mainly involved with interferon-induced genes. Genes involved in this pathway were validated in the FSGS model, showing an increase in mice treated with ADR compared to controls and alleviated with CSF-1R inhibitors (p&lt;0.001).

Conclusion
In this study we propose a novel therapeutic strategy to FSGS-associated pathology based on the inhibition of CSF1-R activity having an impact on reducing aPECs, glomerulosclerosis, proteinuria, improving renal function and preserving the podocyte-progenitor phenotype, thus, in podocyte preservation against damage.