Kidney Dendritic Cells Become Pathogenic during Crescentic Glomerulonephritis with Proteinuria

Abstract

It is unclear why kidney dendritic cells attenuate some models of kidney disease but aggravate others. Kidney dendritic cells ameliorate the early phase of nonaccelerated nephrotoxic nephritis, a murine model of crescentic glomerulonephritis, but their effect on the later phase is unknown. Here, we report that kidney dendritic cells at later stages of nephrotoxic nephritis expressed higher levels of costimulatory molecules but lower levels of the cosuppressor molecule ICOS-L and started production of IL-12/23p40 and TNF-α. Furthermore, we noted that kidney dendritic cells captured more filterable antigen in proteinuric mice at late time points of nephrotoxic nephritis and started to capture molecules that were too large for filtration by a healthy kidney. They presented filtered antigen to Th cells, which responded by producing the proinflammatory cytokines IL-2, IFN-γ, TNF-α, IL-6, and IL-17. Notably, production of the suppressive cytokine IL-10 further increased in late nephrotoxic nephritis. Depletion of kidney dendritic cells at a late stage attenuated nephrotoxic nephritis, in contrast to the exacerbation observed with depletion at an early stage, indicating that their acquired proinflammatory phenotype adversely affected disease. These findings indicate that the intrarenal inflammatory microenvironment determines how kidney dendritic cells affect nephritis. In addition, proteinuria may harm the kidney by providing dendritic cells with more antigens to stimulate potentially pathogenic Th cells.

Figure 1.

KDCs mature during the course of NTN. (A and B) Single cell suspensions were obtained from kidneys of healthy (day 0), early-nephritic (day 4), and late-nephritic (day 10) mice. DCs, defined as CD45⁺CD11c⁺MHCII⁺ cells, were analyzed for expression levels of CD40, CD86, and CD80 (A) and ICOS-L (B) by flow cytometry. Shown are representative histograms and statistical analysis (MFI, mean fluorescence intensity; n = 3 mice per group). (C) Expression of Gr1 and CD11b by KDCs. Contour plots show representative profiles for healthy 5- and 10-day nephritic mice. Percentages of Gr1⁺ KDCs are shown in the upper left corner of each plot. Results are representative for three separate experiments. (D) Total number of DCs per kidney in healthy 4- and 10-day nephritic mice (n = 6 to 9 per group, combined from two separate experiments). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2.

Cytokine production by KDCs increases during the course of NTN. (A and B) Percentage of KDCs producing TNF-α (A) or IL-12/23p40 (B) in healthy 4- and 10-day nephritic mice, as determined by intracellular cytokine staining and flow cytometry. Results are representative for two separate experiments. (n = 3 to 4 mice per group; **P < 0.01). (C and D) Representative dot plots of kidney CD45⁺ cells from 4-day nephritic mice showing TNF-α (C) and IL-12/23p40 (D) production as a function of CD11c expression. (E and F) Representative dot plots of kidney CD11c⁺ cells from 4-day nephritic mice showing TNF-α (E) and IL-12/23p40 (F) production as a function of Gr1 expression.

Figure 3.

KDCs capture more filterable antigen in late NTN. (A) Mice were intravenously injected with 500 ng fluorescently labeled OVA (45 kD) per gram body weight on day 0, 4, or 10 after NTN induction. Thirty minutes later, single cell suspensions gated for CD45⁺ cells were analyzed for OVA uptake via flow cytometry. (B) The same experiment as shown in A, except that mice received 10 μg fluorescence-labeled 500-kD dextrane per gram body weight instead of OVA and that cells were analyzed after 60 minutes. (C) Percentage of CD45⁺CD11c⁺MHCII⁺ KDCs containing OVA in kidney single cell suspensions from healthy (day 0), early-nephritic (day 4), and late-nephritic (day 10) mice 30 minutes after intravenous injection of OVA (n = 3). (D) Mean fluorescence intensity (MFI) of OVA⁺ KDCs, indicating the amount of antigen taken up per DC (n = 3). (E and F) Percentage (E) and MFI (F) of CD45⁺ CD11c⁺ MHCII⁺ KDCs positive for 500-kD dextrane 1 hour after intravenous injection (n = 3 to 4). (G) Mice were intravenously injected with 500 ng fluorescence-labeled OVA (45 kD) per gram body weight on day 10 after NTN induction, and expression of CD40, CD80, CD86, and ICOS-L was determined. Representative contour plots show the expression of costimulatory molecules as a function of antigen uptake. *P < 0.05; **P < 0.01.

Figure 4.

KDCs activate Th cells specific for filtrated antigen more effectively. KDCs (10⁵) from healthy (0 days), 4-day, and 10-day nephritic mice, which had been injected with 700 μg of OVA 1 hour before DC isolation, were cultured alone (DC-culture: white bars) or cocultured with 2 × 10⁵ OT-II cells (Coculture: black bars) for 24 hours in 250 μl RPMI (10% FCS). As a control for antigen specificity, KDCs from 10-day nephritic mice that had not received OVA were cocultured with OT-II cells (dashed lines). Concentrations of IL-2 (A), TNF-α (B), IFN-γ (C), IL-17 (D), IL-6 (E), and IL-10 (F) in the supernatants were measured by Th1/Th2 multiplex bead assay. Results are representative for three separate experiments (n = 3). **P < 0.01; ***P < 0.001.

Figure 5.

Late DC depletion attenuates NTN. (A and B) DCs were depleted on day 7 after NTN induction. Numbers of DCs (A) and of macrophages and Th cells (B) in the kidneys were determined by flow cytometry on days 7, 8, 9, and 10. (C and D) Numbers of CD3⁺ T cells per high power field (hpf) in the tubulointerstitium (C) or per glomerulus (D) in control (ctr) and DC-depleted mice (DTR) on day 10 after disease induction (=3 days after DC depletion), determined by histologic analysis (n = 4). (E and F) Numbers of MAC-2⁺ macrophages per hpf in the tubulointerstitium (E) or per glomerulus (F) in control (ctr) and DC-depleted mice (DTR) on day 10 of disease (=3 days after DC depletion), determined by histologic analysis (n = 4). (G) Albuminuria normalized to creatinine excretion by ctr and DTR mice 3 days after DC depletion. Urine was collected for 17 hours before mice were killed (n = 4). (H) Percentage of crescentic glomeruli in periodic acid-Schiff–stained kidney sections from ctr and DTR mice 3 days after DC depletion (n = 4). Results are representative for two individual experiments.

Figure 6.

Late DC depletion attenuates structural kidney damage in NTN. Representative kidney sections of control (A and B) and CD11c-DTR (C and D) mice on day 10 after NTN induction and day 3 after DT injection. Magnification, 100- (A and C) or 400-fold (B and D).