6-8-week-old C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were used in all experiments. UUO was performed by ligation of the left ureter as described (7), and the mice were euthanized 2, 7, or 14 days following the operation. Kidneys and KLNs were harvested at the time of euthanasia and stored at appropriate temperatures for subsequent analysis.

The UUO release (R-UUO) model was achieved as described by Cochrane et al. (8). Ureteral ligation was performed on the left kidney, and the clamp was removed under general anesthesia 2 days or 7 days after the UUO surgery. The UUO release mice were euthanized at 21 days following UUO. Animal studies were approved and conducted according to the Institutional Animal Care and Use Committee of Brigham and Women’s Hospital, Boston, MA.

CCL19-Cre (CCL19Cre [Tg (CCL19-cre)489Biat] mice were originally a gift from Shannon Turley (Genentech, South San Francisco, CA). C57BL/6-Gt (ROSA)26Sortm1 (HBEGF)Awai/J (C57BL/6-iDTR) mice were purchased from Jackson Laboratories. The C57BL/6-iDTR mice were backcrossed with the CCL19-Cre mice to create CCL19-Cre x iDTR mice, a transgenic mouse strain of C57BL/6 background in which FRCs can be depleted significantly following administration of diphtheria toxin (DT) (3, 9). These mice were injected with 100 ng of diphtheria toxin (DT) intraperitoneally (i.p.) daily for 5 days following UUO (Days 0-4) to deplete FRCs. CCL19-Cre x iDTR mice that did not receive DT were used as the control group.

KLNs were frozen and sectioned with a cryomicrotome. After blocking with 3% (vol/vol) bovine serum albumin in phosphate-buffered saline (PBS) for nonspecific antigens, sections were incubated overnight with primary antibodies at 4°C, followed by secondary conjugated antibodies for 1 hour at room temperature. The following antibodies were used: rat anti-MECA79 (Novus Biologicals, 1:200), goat anti-PDPN (R&D Systems, 1:200), rabbit anti-fibronectin (Abcam, 1:300), rabbit anti-collagen I (Abcam,1:300), rabbit anti-LYVE-1 (Abcam, 1:300), fluorescein (FITC)-conjugated rat anti-LYVE-1 (BioLegend, 1:100), and rat anti-TGFβR1 (Santa Cruz, 1:200). The secondary antibodies used were either FITC-anti-rat, Cy3-anti-rabbit/goat, or aminomethylcoumarin acetate (AMCA)-anti-goat (Jackson ImmunoResearch, 1:200). Images were captured using a EvosFL Auto2 fluorescence microscope. Areas of positive staining were assessed semi-quantitatively by ImageJ software (NIH). Statistical analysis was performed using two-tailed Student’s t tests by Prism (GraphPad Software, Inc).

Kidneys and KLNs were stored at -80°C after harvest from the mice. RNA from KLNs was isolated by Direct-zol RNA Miniprep Plus kit (Zymo research, Irvine, CA), as per the manufacturer’s protocol. The following primers were used: Gapdh forward 5’-AGCCACATCGCTCAGACAC-3’ and reverse 5’-GCCCAATACGACCAAATCC-3’; Tgfb1 forward 5’-CAACAATTCCTGGCGTTACCTTGG-3’ and reverse 5’-GAAAGCCCTGTATTCCGTCTCCTT-3’; Tgfbr1 forward 5’-TGCCATAACCGCACTGTCA-3’ and reverse 5’-AATGAAAGGGCGATCTAGTGATG-3’; Tgfbr2 forward 5’- AGCATCACGGCCATCTGTG-3’ and reverse 5’-TGGCAAACCGTCTCCAGAGT-3’; Smad7 forward 5’-GGGCTTTCAGATTCCCAACTT-3’ and reverse 5’-CACGCGAGTCTTCTCCTCC-3’; Bmp7 forward 5’-CAAGCAGCGCAGCCAGAATCG-3’ and reverse 5’-CAATGATCCAGTCCTGCCAGCCAA-3’.

KLNs were harvested at time of euthanasia, then crushed and filtered through a 70-μm cell strainer. The resulting single-cell suspension was centrifuged at 1,500 rpm for 5 minutes, resuspended in DMEM, then counted manually utilizing 0.4% trypan blue (Thermo Fisher Scientific) to exclude dead cells. The cell suspension was placed in 96-well round-bottom plates for intracellular cytokine staining. The cells were first incubated with phorbol 12-mystirate 13-acetate (100 ng/ml) (Sigma-Aldrich), ionomycin (1 mg/ml) (Sigma-Aldrich), and GolgiStop protein transport inhibitor (BD Biosciences, San Jose, CA) at 37°C for 4 hours. Then the cells were washed and stained according to standard protocols, and flow cytometry was performed via BD FACSCanto II flow cytometer (BD Biosciences). Results were analyzed using FlowJo software (FlowJo LLC). All antibodies were purchased from BD (Becton Dickinson Franklin Lakes, NJ).

KLNs were fixed in Karnovsky fixative and processed as previously described (4). Sections were visualized using an FE-SEM (Zeiss Crossbeam 540), using the aSTEM detector.

Data are presented as means ± SEM. Differences between two groups were analyzed for significance by unpaired two-tailed Student’s t test, and differences among groups were analyzed for significance by analysis of variance (ANOVA). P values less than 0.05 were considered significant. All statistical analysis was performed using GraphPad Prism.

As the prominent cells of the stromal compartment in DLNs, FRCs are PDPN⁺ resident stromal cells that support the integrity of the microarchitecture by producing ECM. We have demonstrated previously the importance of ECM deposition and fibrosis in the KLN to the pathogenesis of two other preclinical kidney disease models: renal ischemia-reperfusion injury (4) and glomerulonephritis (3), In those studies, we found that the activity of FRCs is critical to the fibrogenesis in the KLN. However, the contributions of FRCs and ECM deposition in the KLN to the pathogenesis of UUO have not been investigated. Therefore, we examined the KLNs in the UUO model at different time points following ureteral ligation for histological evidence of these changes. To identify the contribution of FRCs in the KLNs after UUO, we investigated whether PDPN colocalized with the ECM markers collagen 1 and fibronectin through immunofluorescence staining. We noticed that the collagen 1 and fibronectin fibers colocalized with PDPN⁺LYVE-1^- cells, the molecular signature consistent with FRCs, at 2 days, 7 days, and 14 days (Days 2, 7, and 14) following UUO ( Figures 1A, B ). In addition, the density of collagen fibers was significantly higher in the UUO KLN than the KLN draining the unaffected contralateral kidney at all three time points, and the fibronectin fiber density was significantly higher at Days 7 and 14. Moreover, we found through co-staining of PDPN⁺ cells with αSMA, PDGFRβ, and vimentin that FRCs in the UUO KLNs expressed significantly higher levels of these fibrosis markers than those in the contralateral KLNs ( Figures 1C–E and Supplementary Figures 1A–C ). Electron micrographs of the UUO KLN at Day 14 also revealed evidence of extensive ECM deposition within fibrils extending from FRCs ( Supplementary Figure 1D ). This fibrosis was also reflected in a higher overall density of ECM fibers in the UUO KLNs, as indicated by Masson’s trichrome stain ( Supplementary Figure 1E ).

The position of the KLN as the primary secondary lymphoid organ that drains the kidney situates it as the prime site for modulation of adaptive immunity in the kidney, through regulation of the interplay between the innate and adaptive immune responses. Therefore, we sought to investigate this interaction by determining whether macrophage infiltration and T cell activation increase concurrently in the KLN following UUO. We stained the KLNs for CD11b⁺ antigen-presenting cells by immunofluorescence, and we found that they increased progressively through Day 14 ( Figure 2A ). Semiquantitative analysis indicated that the CD11b⁺ macrophage population was higher in the UUO KLN than the contralateral KLN; although the comparison did not quite reach significance (p=0.06) at Day 7, a significant increase (p<0.01) was observed at Day 14 ( Figure 2B ). In addition, the CD11c⁺ dendritic cell population was significantly higher in the UUO KLN, as quantified by flow cytometry ( Figure 2C ). Moreover, flow cytometric analysis revealed that the TNFα-producing CD4⁺ and CD8⁺ T cell populations and IFNγ-producing CD8⁺ T cell population were significantly higher in the UUO KLN ( Figures 2D–F ). The UUO KLN also contained a higher population of CD4⁺IFNγ⁺ cells, although this comparison did not achieve significance ( Figures 2F, G ). Finally, we stained the KLNs for LYVE1⁺ lymphatics, through which antigen-presenting cells likely traffic from the kidney to the KLN (10, 11), and MECA79⁺ high endothelial venules, through which naïve T cells enter the KLN from the systemic circulation. We found that the expansion of LYVE1⁺ lymphatic vessels peaks at Day 14, when the lymphatic vasculature is significantly more extensive than in the contralateral KLN ( Supplementary Figures 2A, B ).

Expression of TGFβ has been linked to fibrosis in secondary lymphoid organs in preclinical HIV models, suggesting a possible signaling pathway that could be responsible for the fibrosis observed in the KLN (12). Hence, we interrogated whether the TGFβ/TGFβR1 pathway is activated in the KLN following UUO. We found through co-staining of PDPN⁺LYVE-1^- cells with TGFβR1 that FRCs in the UUO KLNs expressed higher amounts of TGFβR1 than those of contralateral KLNs ( Figure 3A ), indicating a possible route for the increase in fibrosis. Gene expression levels of TGFβ, TGFβR1, TGFβR2, the GFβ regulator Smad7, and the TGFβ family member BMP-7 were all significantly higher in the UUO KLNs at Day 7 as compared to the contralateral KLNs ( Figure 3B ). These data suggest that the TGFβ signaling pathway could function as a chief mediator of ECM production by FRCs in the KLN during UUO.

A major question within the field of chronic kidney disease is whether the fibrosis associated with impaired kidney function can be reversed. Previous studies of UUO mouse models have sought to answer this question by releasing the ligated ureter at various time points following UUO. These experiments have demonstrated mixed results— the fibrosis that occurs in the kidney following UUO may or may not reverse after the obstruction has resolved (8, 13, 14). However, whether the fibrosis in the KLN resolves following release of ureteral ligation has never been published. Here, we released the ureter from ligation at Day 7 and euthanized the mice at Day 21. Then, we performed immunofluorescence staining to compare the fibrosis in both the kidney and the KLN to the corresponding organs of the mice in which the ureter remained ligated for 21 days. First, we found no significant difference in the extent of fibrosis between the two kidneys, as shown by both collagen I and fibronectin ( Figures 4A, B ). Then, we sought to determine whether this persistence in fibrosis is reflected in the KLN, and we found by immunofluorescence staining that the density of ECM fibers in the KLN was also similar between the UUO and R-UUO groups ( Figure 4C ). Then, we investigated whether releasing the ureteral ligation at an earlier time point could halt progression of fibrosis by releasing the ureters at Day 2 and euthanizing the mice at Day 21. The fibrosis in the R-UUO KLN, as determined by collagen 1 and fibronectin staining, was significantly less dense than the UUO KLN ( Figures 4D, E ). Semiquantitative analysis indicated that the density of collagen I fibers in the R-UUO KLN also was significantly lower, as compared to the UUO KLN ( Figure 4F ).

As demonstrated here in Figure 1 , FRCs colocalize closely with the nidus of fibrogenesis that appears in the KLN following UUO. Therefore, we investigated whether FRCs are the major source of fibrosis in the UUO KLN, through the use of CCL19-Cre x Rosa26-diphtheria toxin receptor (CCL19-Cre x iDTR) mice, a transgenic mouse strain of C57BL/6 background in which FRCs can be depleted significantly following administration of diphtheria toxin (DT) (3, 9). We demonstrated in previous studies of renal IRI and CGN in mouse models that depletion of FRCs was associated with lower kidney damage (3, 4). Here, we sought to determine whether depletion of FRCs would exert similar amelioration of kidney injury following UUO. In this model, we injected C57BL/6 mice with 100 ng of DT daily on 5 days following UUO and euthanized the mice at Day 14. Semiquantitative analysis of immunofluorescence staining of the KLNs revealed significantly lower density of collagen I ( Figure 5A ) and fibronectin fibers ( Figure 5B ) following depletion of FRCs in comparison to the CCL19-Cre x iDTR mice that did not receive DT. Interestingly, this reduction in fibrosis in the KLN was accompanied by a similar significant decrease in fibrosis in the kidneys of the mice that underwent FRC depletion, as reflected by immunofluorescence staining of collagen I and fibronectin ( Figures 5C, D ). CCL19⁺ cells were non-existent in the control (contralateral) kidney, and they were present in sparse amounts in the UUO kidney. However, these few CCL19⁺ cells did not co-stain with PDPN ( Supplementary Figure 3 ). These observations reflect our earlier findings in models of renal IRI and CGN (3, 4), and they reinforce the concept that the FRCs in the KLN function as prominent arbiters of the direction of the immune response and fibrogenesis observed in the kidney following renal injury.