Kidney Week

Abstract: SA-PO586

Establishment of a Preparative Method and Gating Strategy for Renal Mononuclear Phagocytes (rMophs) and Analysis of rMophs in CD11c-Specifc Shp-1 Knockout Mice

Session Information

• Glomerular Diseases: Immunology, Inflammation - II
  November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1202 Glomerular Diseases: Immunology and Inflammation

Authors

• Watanabe, Mitsuharu, Gunma University Graduate School of Medicine, Maebashi, Japan

Mitsuharu Watanabe,

• Kaneko, Yoriaki, Gunma University Graduate School of Medicine, Maebashi, Japan

Yoriaki Kaneko,

• Kinoshita, Masato, Gunma University Graduate School of Medicine, Maebashi, Japan

Masato Kinoshita,

• Shrestha, Shreya, Gunma University Graduate School of Medicine, Maebashi, Japan

Shreya Shrestha,

• Ohishi, Yuko, Gunma University Graduate School of Medicine, Maebashi, Japan

Yuko Ohishi,

• Sakairi, Toru, Gunma University Graduate School of Medicine, Maebashi, Japan

Toru Sakairi,

• Ikeuchi, Hidekazu, Gunma University Graduate School of Medicine, Maebashi, Japan

Hidekazu Ikeuchi,

• Nojima, Yoshihisa, Inoue Hospital, Takasaki, Japan

Yoshihisa Nojima,

• Hiromura, Keiju, Gunma University Graduate School of Medicine, Maebashi, Japan

Keiju Hiromura,

Background

Recent studies demonstrated the existence of renal mononuclear phagocytes (rMophs), which express both macrophage markers, CD11b and F4/80, and dendritic cell marker, CD11c. However, standardized procedures to isolate and characterize rMophs have not been adopted; therefore, the function of rMophs is not fully understood. In this study, we aimed to provide a preparative method of rMophs and optimize gating strategy for flow cytometry (FCM). We further examined the contribution of rMophs in renal injury by inducing nephritis in CD11c-specific Shp-1 knockout mice (Shp-1 CKO), in which certain population of rMophs lack Shp-1, a negative regulator of hematopoietic cell signaling.

Methods

C57BL/6J mice were used to establish the preparative method of rMophs. To induce nephritis, Shp-1 CKO and control littermates mice (Ctrl) at the age of 8-10 weeks were immunized by subcutaneous injection with bovine serum albumin (BSA) at 2-week intervals.

Results

We first determined the efficacy of renal cell collections from the mouse kidney. Higher yield of CD45.2⁺ cells and F4/80^high cells was achieved by collagenase digestion compared to simple mechanical dissociation. We then characterized the isolated cells by FCM. A significant number of neutrophils, which expressed Ly6G, mixed up into F4/80^low cells. Therefore, isolated renal cells expressing CD45.2 and CD11b, but not Ly6G, could be referred as rMophs. These CD45.2⁺CD11b⁺Ly6G^- rMophs were, then, divided into 3 subpopulations: F4/80^highCD11c^int rMophs, F4/80^lowCD11c^high rMophs and F4/80^lowCD11c^low rMophs. In animal experiments, Shp1-CKO developed proteinuria at 6-8 weeks after the first BSA injection, when Ctrl did not develop proteinuria yet. Proliferative glomerulonephritis was also developed at this early time point in Shp-1 CKO. Finally using the isolation and gating methods, the FCM analysis revealed that proportion as well as absolute number of F4/80^high rMophs increased in Shp-1 CKO treated with BSA.

Conclusion

We found that the use of collagenase digestion is necessary to collect rMophs and the gating strategy of eliminating neutrophils is important for the analysis of rMophs. We also showed that Shp-1 may regulate rMophs and protect against renal injury.