Kidney Week

Abstract: TH-PO0260

Flow Cytometry Identifies CD45-PDGFRβ+ Stromal Cells as a Collagen I-Enriched Population in Unilateral Ureteral Obstruction Kidney Fibrosis

Session Information

• CKD: Mechanisms of Injury and Fibrosis - 1
  October 22, 2026 | Location: Exhibit Hall A, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

• 2203 CKD (Non-Dialysis): Mechanisms

Authors

• Suwa, Junya, Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Maebashi, Gunma Prefecture, Japan

Junya Suwa, MD

• Kaneko, Yoriaki, Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Maebashi, Gunma Prefecture, Japan

Yoriaki Kaneko, MD, PhD

• Watanabe, Mitsuharu, Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Maebashi, Gunma Prefecture, Japan

Mitsuharu Watanabe, MD

• Imai, Yoichi, Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Maebashi, Gunma Prefecture, Japan

Yoichi Imai, MD, PhD

• Kinoshita, Masato, Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Maebashi, Gunma Prefecture, Japan

Masato Kinoshita, MD

• Hamatani, Hiroko, Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Maebashi, Gunma Prefecture, Japan

Hiroko Hamatani, MD, PhD

• Ikeuchi, Hidekazu, Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Maebashi, Gunma Prefecture, Japan

Hidekazu Ikeuchi, MD, PhD

• Hiromura, Keiju, Gunma Daigaku Daigakuin Igakukei Kenkyuka Igakubu, Maebashi, Gunma Prefecture, Japan

Keiju Hiromura, MD, PhD

Background

Renal fibrosis is a final common pathway of chronic kidney disease, but quantitative approaches for viable non-hematopoietic fibrogenic cells remain limited. Because α-smooth muscle actin (αSMA) is intracellular and not suitable for live sorting, surface markers are needed to isolate fibrogenic stroma. We developed a flow cytometry (FCM)-based workflow to define and isolate fibrosis-associated renal stromal populations in unilateral ureteral obstruction (UUO).

Methods

UUO was induced in C57BL/6J mice. Kidney fibrosis was confirmed by Masson trichrome and elastic van Gieson staining. Kidney single-cell suspensions collected from day 0 to day 28 were analyzed by FCM using CD45.2 compartment gating, intracellular αSMA staining, surface-marker phenotyping, and absolute counting beads. αSMA and PDGFRβ localization was assessed by immunofluorescence. Viable CD45.2−PDGFRβ+ stromal cells and CD45.2+F4/80+ macrophages were sorted for western blotting, quantitative PCR, and chemokine receptor profiling.

Results

UUO markedly increased αSMA+ cells, predominantly in the CD45.2− non-hematopoietic compartment. Within CD45.2− cells, αSMA expression was associated with PDGFRβ and vimentin, whereas CD34 and CD73 were infrequent. Immunofluorescence confirmed patchy αSMA–PDGFRβ co-localization from cortex to deep medulla. Absolute counting showed that CD45.2−αSMA+PDGFRβ+ cells increased from day 2 and peaked at day 14, while CD45.2+F4/80+ macrophages peaked earlier at day 7. Live sorting of CD45.2−PDGFRβ+ cells achieved approximately 90% purity. This stromal fraction showed strong collagen I enrichment by western blotting and increased Col1a1 and Acta2 transcript expression, whereas F4/80+ macrophages showed little collagen I protein. Gene and receptor profiling suggested reciprocal stromal-macrophage signaling: F4/80+ cells were enriched for Pdgfb, Il1b, Ccl2/CCR2, Ccl3/CCR5, and CX3CR1, while PDGFRβ+ stromal cells expressed Pdgfrb, Il1r1, Cx3cl1, Col1a1, and Acta2; Tgfb was increased in both populations.

Conclusion

CD45−PDGFRβ+ stromal cells represent a major collagen I-enriched fibrogenic compartment in UUO kidney fibrosis. This FCM-based platform enables quantitative time-course analysis and viable isolation of renal fibrogenic stroma, providing a practical tool for studying stromal activation and stromal-immune crosstalk in kidney fibrosis.

Funding

• Government Support – Non-U.S.