The loss of renal dendritic cells and activation of host adaptive immunity are long-term effects of ischemia/reperfusion injury following syngeneic kidney transplantation

Abstract

Ischemia/reperfusion injury associated with kidney transplantation induces profound acute injury, influences early graft function, and affects long-term graft outcomes. To determine whether renal dendritic cells play any role during initial innate ischemia/reperfusion injury and the subsequent development of adaptive immune responses, we studied the behavior and function of renal graft and host infiltrating dendritic cells during early and late phases of renal ischemia/reperfusion injury. Wild type to green fluorescent protein (GFP) transgenic rat kidney transplantation was performed with and without 24-h cold storage. Ischemia/reperfusion injury in cold-stored grafts resulted in histopathological changes of interstitial fibrosis and tubular atrophy by 10 weeks, accompanied by upregulation of mRNAs of mediators of interstitial fibrosis and inflammation. In normal rat kidneys, we identified two populations of renal dendritic cells, predominant CD103(-)CD11b/c(+) and minor CD103(+)CD11b/c(+) cells. After transplantation without cold storage, grafts maintained CD103(-) but not CD103(+) GFP-negative renal dendritic cells for 10 weeks. In contrast, both cell subsets disappeared from cold-stored grafts, which associated with a significant GFP-expressing host CD11b/c(+) cell infiltration that included CD103(+) dendritic cells with a TNF-α-producing phenotype. These changes in graft/host dendritic cell populations were associated with progressive infiltration of host CD4(+) T cells with effector/effector-memory phenotypes and IFN-γ secretion. Thus, renal graft ischemia/reperfusion injury caused graft dendritic cell loss and was associated with progressive host dendritic cell and T-cell recruitment. Renal-resident dendritic cells might function as a protective regulatory network.

Figure 1. Renal resident leukocytes in naïve rat kidney

(A) Renal leukocytes were isolated from naïve rat kidneys with collagenase digestion and low-speed centrifugation, and analyzed by three to seven-color flow cytometry. CD45⁺ cells are gated, and kidney resident leukocyte composition was determined by surface marker expressions. Note that ~70% of renal CD45⁺ leukocytes were CD103⁻CD11b/c⁺ and ~10% CD103⁺CD11b/c⁺. (B) Expression of various surface molecules on CD45⁺CD11b/c⁺ renal DC. (C) Immunofluorescent stain of naïve kidney revealed networks of CD11b/c⁺ cells throughout the entire kidney interstitium (red, upper). CD103 expression was limited (red, lower). Representative images of n=5 experiments.

Figure 2. Early kidney graft injury caused by prolonged 24 hrs cold storage

Kidney graft samples were obtained at 3 and 12 hrs after WT to GFP KTx with and without 24 hrs static cold storage in UW solution (24h-CS and no-CS groups). (A) Samples at 3 hrs were analyzed for mRNA levels for proinflammatory mediators with RT-PCR. (B) Neutrophil infiltration (yellow arrows) in kidney grafts at 12 hrs was evaluated in IHC with mAb for neutrophils (red, RP-1). N=3–4 for each group. *: p<.05 vs. no-CS controls. #: P<0.005 vs. no-CS control.

Figure 3. Late kidney graft changes caused by I/R injury

(A) Histopathological findings: WT to GFP KTx was performed with and without 24 hrs static cold storage in UW solution (24h-CS and no-CS groups). Representative histological images of kidneys grafts at 10 weeks are shown. (a) H&E stained section of 24h-CS grafts shows areas of interstitial fibrosis and tubular atrophy (arrow) with chronic interstitial inflammation. Tubular atrophy in the form of dilatation (“thyroidization”) is also seen (arrowhead) (H&E ×100). Glomeruli contained globular material suggestive of protein. (b) In control no-CS grafts, tubules are intact with no significant increase in interstitial fibrous tissue. Glomeruli are unremarkable (H&E ×100). (c) Masson’s trichrome stain reveals significant fibrosis (blue) in 24h-CS grafts. (d) Control no-CS grafts show minimum fibrosis. (e, f) Immunofluorescent stain for α-SMA (red) shows normal arterial α-SMA stain in no-CS grafts. 24h-CS graft shows area of α-SMA positive myofibroblasts with numerous GFP⁺ host infiltrates. Blue: nuclear stain. Representative images from no-CS (n=4) and 24h-CS (n=5) grafts. (B) mRNA levels for inflammatory and pro-fibrotic mediators in kidney grafts: RT-PCR was conducted using kidney graft samples obtained at 10 weeks. n=2–5 for each group. *: p <.05 vs. no-CS controls.

Figure 3. Late kidney graft changes caused by I/R injury

(A) Histopathological findings: WT to GFP KTx was performed with and without 24 hrs static cold storage in UW solution (24h-CS and no-CS groups). Representative histological images of kidneys grafts at 10 weeks are shown. (a) H&E stained section of 24h-CS grafts shows areas of interstitial fibrosis and tubular atrophy (arrow) with chronic interstitial inflammation. Tubular atrophy in the form of dilatation (“thyroidization”) is also seen (arrowhead) (H&E ×100). Glomeruli contained globular material suggestive of protein. (b) In control no-CS grafts, tubules are intact with no significant increase in interstitial fibrous tissue. Glomeruli are unremarkable (H&E ×100). (c) Masson’s trichrome stain reveals significant fibrosis (blue) in 24h-CS grafts. (d) Control no-CS grafts show minimum fibrosis. (e, f) Immunofluorescent stain for α-SMA (red) shows normal arterial α-SMA stain in no-CS grafts. 24h-CS graft shows area of α-SMA positive myofibroblasts with numerous GFP⁺ host infiltrates. Blue: nuclear stain. Representative images from no-CS (n=4) and 24h-CS (n=5) grafts. (B) mRNA levels for inflammatory and pro-fibrotic mediators in kidney grafts: RT-PCR was conducted using kidney graft samples obtained at 10 weeks. n=2–5 for each group. *: p <.05 vs. no-CS controls.

Figure 4. CD11b/c⁺ cells in kidney grafts

After WT to GFP KTx with or without 24 hrs cold storage in UW solution (24h-CS, no-CS groups), kidney grafts were obtained at 3 hrs, 12 hrs, 4 weeks, and 10 weeks (n=2–4 for each time point in no-CS and n=4–6 in 24h-CS groups) for flow cytometry of CD45⁺ leukocytes isolated from kidney grafts. (A) Percentages of GFP⁻ cells in graft CD45⁺ leukocytes. Significant decreases of GFP⁻ graft cells n 24h-CS grafts compared to no-CS grafts. * p<0.05, # p<0.005 (B) Percentages of GFP⁻CD11b/c⁺ renal DC in graft CD45⁺ leukocytes. Graft resident GFP⁻CD11b/c⁺ DCsignificantly decreased due to I/R injury in 24h-CS than in no-CS grafts. * p<0.05, # p<0.005 (C) Flow cytometry of CD45⁺CD11b/c⁺ cells in kidney grafts. Donor GFP⁻CD103⁺ DC quickly disappeared from both 24h-CS and no-CS grafts within 1 week. In contrast, donor GFP⁻CD103⁻ DC disappeared from 24h-CS, but not from no-CS, grafts. At the same time, host GFP⁺CD11b/c⁺ cells increased with time, and GFP⁺CD11b/c⁺CD103⁺ DC significantly increased in 24h-CS grafts. Representative histograms (upper) and bar graph showing percentages in CD45⁺CD11b/c⁺ gated cells. * p<0.05 (D) In 24h-CS grafts, both GFP⁻CD11b/c⁺ renal DC and GFP⁺CD11b/c⁺ infiltrates produced TNF-α at 12 hrs, but the frequencies were significantly higher in the host population. At 10 weeks, TNF-α was detected mostly in GFP⁺CD11b/c⁺ cells. * p<0.05

Figure 5. Immunohistochemistry of CD11b/c⁺ cells in kidney grafts

Representative images of CD11b/c stain (red) in kidney grafts at 10 weeks are shown as merged (left panels), red channel (middle panels), and green channel (right panels). In the peritubular area, no-CS grafts show numerous CD11b/c⁺GFP⁻ renal DC (red), while 24h-CS grafts show GFP⁺ host infiltrates (green) without CD11b/c expressing renal DC. Instead, 24h-CS grafts show many GFP⁺CD11b/c⁺ host infiltrating DC/monocytes. (A) No-CS grafts at 10 weeks show persistence of CD11b/c⁺GFP⁻ renal DC (red, blue arrows). (B) High magnification image of CD11b/c⁺GFP⁻ renal DC (blue arrow) in peritubular area. (C) High magnification image of CD11b/c⁺GFP⁻ renal DC (blue arrowhead) in the area of GFP⁺ infiltrates. (D) In contrast, 24h-CS grafts at 10 weeks had nearly no donor renal DC (GFP⁻CD11b/c⁺) but had numerous CD11b/c⁺ GFP⁺ infiltrates (double positive yellow, yellow arrowhead). (E) High magnification image of GFP⁺ infiltrates and loss of CD11b/c⁺ DC in peritubular area (white circled area in D). (F) High magnification image of GFP⁺CD11b/c⁺ cells among host infiltrates (yellow arrowhead).

Figure 6. Host (GFP⁺) cell infiltrations into kidney grafts

WT to GFP KTx was performed with or without 24 hrs cold storage in UW solution (24h-CS, no-CS groups). Kidney grafts were obtained at 3 hrs, 12 hrs, 4 weeks, and 10 weeks after KTx (n=2–4 for each time point in no-CS and n=4–6 in 24h-CS groups) for immunohistochemistry and flow cytometry. (A) Percentages of GFP⁺ host cells in isolated CD45⁺ leukocytes from kidney grafts. Significantly more GFP⁺ cells were seen in 24h-CS than in no-CS grafts. * p<0.05, # p<0.005 (B) Kidney graft sections are examined for GFP⁺ cells with nuclear Hoechst stain (blue). GFP⁺ host cells increased with time and numerous GFP⁺ infiltrates were seen in 24h-CS grafts at 10 weeks. (C) Percentages of CD3⁺ T cells in graft CD45⁺ leukocytes. T cells increased with time and were significantly more in 24h-CS than in no-CS grafts. * p<0.05, # p<0.005 (D) Immunohistochemistry for CD3 (red) with nuclear Hoechst stain (blue). Significant host (GFP⁺) T cell infiltration in 24h-CS grafts at 10 weeks after KTx. Control no-CS grafts showed few CD3⁺ T cells. Lower panels show images of green GFP channel and red CD3 channel.

Figure 7. Characterization of host (GFP⁺) T cells in kidney grafts with I/R injury

(A) Analysis of host infiltrating T cells (GFP⁺CD45⁺CD3⁺) for CD4/CD8 and CD62L/CD45RC. CD4⁺ T cells gradually increased in 24h-CS grafts. Host T cells were mainly CD4⁺ CD62L⁻CD45RC⁻ effector-memory phenotype T cells. (B) CD4/CD8 ratios were significantly higher in 24h-CS than in no-CS grafts at 4 and 10 weeks (n=2–4 for each time point in no-CS and n=4–6 in 24h-CS groups). * p<0.05 (C) Intracellular IFN-γ expression on CD45⁺ leukocytes from kidney grafts. Early after KTx at 3 hrs, IFN-γ was detected in GFP⁻CD3⁺ graft T cells. At 10 weeks, GFP⁺ host T cells produced IFN-γ. In GFP⁺CD3⁺ cells in 24h-CS grafts at 10 weeks, both CD4⁺ and CD8⁺ T cells were able to produce IFN-γ. N=2–4 for each time point in no-CS and n=4–6 in 24h-CS groups.