Kidney-Draining Lymph Node Fibrosis Following Unilateral Ureteral Obstruction

Abstract

Although the primary organ has been the subject of intense investigation in the field of organ fibrosis over the past several decades, the presence of lymph node fibrosis due to persistent activation of the immune response in its partner organ remains largely unknown. Previously, we demonstrated that activation of the immune response following ischemia-reperfusion injury (IRI) and crescentic glomerulonephritis (CGN) in the kidney was associated with extracellular matrix (ECM) production by fibroblastic reticular cells (FRCs) of the kidney-draining lymph node (KLN). Here, we sought to determine whether FRCs in the KLN become similarly fibrogenic following unilateral ureteral obstruction (UUO) of the kidney. We subjected 6-8-week-old C57BL/6J mice to UUO for 2, 7, and 14 days. We examined the microarchitecture of the kidney and KLN by immunofluorescence staining at each timepoint, and we quantified immune cell populations in the KLN by flow cytometry. The contralateral kidney unaffected by UUO and its partner KLN were used as controls. We found through immunofluorescence staining that FRCs increased production of ECM fibers and remodeled the microarchitecture of the UUO KLN, contributing to fibrosis that mirrored the changes in the kidney. We also observed by flow cytometry that the populations of CD11b⁺ antigen-presenting cells, CD11c⁺ dendritic cells, and activated CD4⁺ and CD8⁺ T cells were significantly higher in the UUO KLN than the KLN draining the unaffected contralateral kidney. Expression of the TGFβ/TGFβR signaling pathway was upregulated and colocalized with FRCs in the UUO KLNs, suggesting a possible mechanism behind the fibrosis. Both release of ureteral ligation at 2 days following UUO and depletion of FRCs at the time of injury onset halted the progression of fibrosis in both the kidney and the KLN. These findings for the first time highlight the association between fibrosis both in the kidney and the KLN during UUO, and they lay the groundwork for future studies that will investigate more deeply the mechanisms behind the connection between FRCs and KLN fibrosis.

Keywords: acute kidney injury; chronic kidney disease; fibroblastic reticular cell (FRC); kidney fibrosis; lymph node; renal immune homeostasis; unilateral ureteral obstruction (UUO).

Figure 1

Contribution of FRCs to increase in ECM density in the KLN over time. (A) Fluorescence micrographs of Lyve-1⁺ lymphatic vessels (green), collagen 1 fibers (red), and PDPN⁺ cells (blue) demonstrate increased density of collagen I fibers and colocalization with Lyve-1^-PDPN⁺ FRCs through Day 14. Semiquantitative analysis and comparison of collagen I⁺-stained area between the UUO and contralateral (control) KLNs show significantly higher density of collagen 1⁺ fibers normalized to DAPI-positive area in the UUO KLN at all time points (n=15 random fields of sections from 3 mice). (B) Fluorescence micrographs of Lyve-1^-PDPN⁺ FRCs (blue) and fibronectin fibers (red) show increased density of fibronectin fibers and colocalization with FRCs through Day 14. Semi-quantitative analysis and comparison of fibronectin⁺-stained area shows significantly higher density of fibronectin fibers at Days 7 and 14 in UUO KLN as compared to contralateral (control) KLN (n=15 random microscopic fields of sections from 3 mice). (C–E) Fluorescence micrographs of UUO KLN and contralateral (control) KLN sections at Day 14 demonstrate increased expression of (C) α-SMA (green), (D) PDGFRβ (green), and (E) vimentin (green), and increased colocalization (yellow) with PDPN⁺ FRCs (red) in UUO KLN at Day 14 (DAPI nuclear stain, blue). Data represented by means ± SEM. *p < 0.05, **p < 0.01.

Figure 2

Infiltration of antigen-presenting cells and activation of lymphocytes in the UUO KLN. (A, B) (A) Fluorescence micrographs of CD11b⁺ antigen-presenting cells (green) and (B) semiquantitative analysis of CD11b⁺ staining normalized to DAPI⁺ area (blue) indicate that the density of CD11b⁺ antigen-presenting cells increases in the UUO KLN over time, and it is significantly higher than the contralateral (control) KLN at Day 14. (C–G) Flow cytometric analysis of KLNs at Day 14 indicate significantly higher percentages of (C) CD45⁺CD11c⁺ dendritic cells, (D) CD4⁺TNFα⁺ T cells, (E) CD8+TNFα+ T cells, (F) CD8⁺IFNγ⁺ T cells, and higher percentage of (G) CD4⁺IFNγ⁺ T cells in the UUO KLN than the contralateral (control) KLN at Day 14 (n=3-4 mice). Data represented by means ± SEM. *p < 0.05, **p < 0.01. NS, not significant.

Figure 3

TGFβ/TGFβR signaling pathway is active in the UUO KLN. (A) Fluorescence micrographs of UUO and contralateral (control) KLNs demonstrate co-staining (yellow) of TGFβR1 (green) with PDPN⁺ FRCs (red) through Day 14. (B) RT-PCR shows significantly higher expression of TGFβ, TGFβR1, TGFβR2, Smad7, and BMP-7 in UUO KLN than contralateral KLN at Day 7. Each sample was performed in duplication (n=3). Data represented by means ± SEM. *p < 0.05, **p < 0.01.

Figure 4

Release of UUO (R-UUO) at Day 2 halts fibrosis in the kidney and KLN but has no effect at Day 7. (A, B) Fluorescence micrographs of collagen 1 fibers (green) and fibronectin fibers (green) reveal no difference in fibrosis between the UUO and R-UUO kidneys at Day 21 following release of ureteral ligation at Day 7 (PDPN glomerular stain, red; DAPI nuclear stain, blue). (C) Fluorescence micrographs of collagen 1 fibers (red) demonstrate no difference in fibrosis between the UUO and R-UUO KLNs at Day 21 following release of ureteral ligation at Day 7 (DAPI nuclear stain, blue). (D, E) Fluorescence micrographs and semiquantitative analysis show significantly higher density of (D) collagen 1 fibers (green) and (E) fibronectin fibers (green) normalized to DAPI-stained area (blue) in UUO kidneys than R-UUO kidneys at Day 21 following release of ureteral ligation at Day 2 (PDPN glomerular stain, red) (n=15 random microscopic fields of sections from 3 mice). (F) Fluorescence micrographs and semiquantitative analysis show significantly higher density of collagen 1 fibers (green) normalized to DAPI-stained area (blue) in UUO KLNs than R-UUO KLNs at Day 21 following release of ureteral ligation at Day 2 (n=15 random microscopic fields of sections from 3 mice). Data represented by means ± SEM. *p < 0.05, **p < 0.01.

Figure 5

Depletion of FRCs reduces fibrosis in KLNs and kidneys following UUO. (A, B) Fluorescence micrographs and semiquantitative analysis reveal significantly lower density of (A) collagen 1 fibers (green) and (B) fibronectin fibers (green) normalized to DAPI-stained area (blue) in KLNs from CCL19-Cre x iDTR mice following FRC depletion (+DT) at Day 14 (n=5 random microscopic fields of sections from 3 mice). (C, D) Fluorescence micrographs and semiquantitative analysis reveal significantly lower density of (C) collagen 1 fibers (green) and (D) fibronectin fibers (green) normalized to DAPI-stained area (blue) in kidneys from CCL19-Cre x iDTR mice following FRC depletion (+DT) at Day 14 (PDPN glomerular stain, red) (n=15 random microscopic fields of sections from 3 mice). Data represented by means ± SEM. *p < 0.05, **p < 0.01.