ASN's Mission

ASN leads the fight to prevent, treat, and cure kidney diseases throughout the world by educating health professionals and scientists, advancing research and innovation, communicating new knowledge, and advocating for the highest quality care for patients.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on Twitter

Kidney Week

Abstract: PO0221

Pericyte and Macrophage C5aR1 Mediate Renal Fibrosis

Session Information

  • AKI Mechanisms - 3
    October 22, 2020 | Location: On-Demand
    Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Sahu, Ranjit K., University of Virginia, Charlottesville, Virginia, United States
  • Xavier, Sandhya, University of Virginia, Charlottesville, Virginia, United States
  • Chauss, Daniel, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, United States
  • Taylor, Ronald P., University of Virginia, Charlottesville, Virginia, United States
  • Kazemian, Majid, Purdue University System, West Lafayette, Indiana, United States
  • Afzali, Behdad, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, United States
  • Portilla, Didier, University of Virginia, Charlottesville, Virginia, United States
Background

We reported that kidney complement plays an important role in the pathogenesis of renal scarring. Here we used a floxed green fluorescent protein (GFP)-C5aR1 reporter mouse and the model of Folic acid (FA)-mediated fibrosis to examine mechanisms by which C5aR1 contributes to fibrosis.

Methods

We used flow cytometry, confocal microscopy and image stream analysis of kidney tissue cell suspensions from vehicle and FA-treated GFP-C5ar1fl/fl reporter mice to characterize cell types where C5ar1 mRNA and protein levels were expressed. To address the functional role of C5aR1 during fibrosis we generated pericyte-specific and myeloid-specific C5aR1 KO mice by crossing GFP-C5ar1fl/fl mice with mice expressing Cre-recombinase under the control of the Foxd1 or LyzM promoter, respectively. We established primary cell cultures of PDGFRß+ kidney pericytes isolated from GFP-C5ar1fl/fl mice and FoxD1-C5aR1 KO mice and performed qPCR and luminex analysis.

Results

Immunofluorescence confocal microscopy demonstrated increased GFP+ (C5aR1+) cells in tubulo-interstitium of FA-treated mice, which co-stained with F4/80 antibody. ImageStream analysis confirmed surface expression of C5aR1 in GFP+PDGFRB+ cells and CD45+F4/80+ cells. Flow cytometry showed increased GFP in CD45+F4/80+ cells. Mice with pericytes-specific deletion of C5aR1 had reduced fibrosis, measured by picrosirious red staining, and reduced α-SMA, fibronectin and collagen1A1, compared to wild-type mice. Pericytes isolated from Foxd1-C5aR1 KO mice treated with FA showed reduced expression and secretion of IL6, TNF, macrophage inflammatory proteins, and MMP-9, when compared to pericytes isolated from GFP-C5aR1fl/fl mice. Myeloid-specific deletion of C5aR1 resulted in significant reduction of C5aR1 in kidney homogenates, confirming macrophages as the major population expressing C5aR1 following FA injury. These mice also showed reduced kidney fibrosis by picrosirious red staining, and qPCR analysis of fibronectin and collagen.

Conclusion

Our findings demonstrate that pericytes and macrophages play important roles in the pathogenesis of renal scarring and implicate C5aR1 as a key mediator. Conditional deletion of C5aR1 in these two interstitial cell types reduces inflammation and extracellular matrix protein formation in pericytes as well as macrophage migration and profibrotic phenotype of kidney macrophages.

Funding

  • NIDDK Support