Loss of decay-accelerating factor triggers podocyte injury and glomerulosclerosis

Abstract

Kidney glomerulosclerosis commonly progresses to end-stage kidney failure, but pathogenic mechanisms are still poorly understood. Here, we show that podocyte expression of decay-accelerating factor (DAF/CD55), a complement C3 convertase regulator, crucially controls disease in murine models of adriamycin (ADR)-induced focal and segmental glomerulosclerosis (FSGS) and streptozotocin (STZ)-induced diabetic glomerulosclerosis. ADR induces enzymatic cleavage of DAF from podocyte surfaces, leading to complement activation. C3 deficiency or prevention of C3a receptor (C3aR) signaling abrogates disease despite DAF deficiency, confirming complement dependence. Mechanistic studies show that C3a/C3aR ligations on podocytes initiate an autocrine IL-1β/IL-1R1 signaling loop that reduces nephrin expression, causing actin cytoskeleton rearrangement. Uncoupling IL-1β/IL-1R1 signaling prevents disease, providing a causal link. Glomeruli of patients with FSGS lack DAF and stain positive for C3d, and urinary C3a positively correlates with the degree of proteinuria. Together, our data indicate that the development and progression of glomerulosclerosis involve loss of podocyte DAF, triggering local, complement-dependent, IL-1β-induced podocyte injury, potentially identifying new therapeutic targets.

Graphical abstract

Figure 1.

Glomerular DAF downregulation promotes murine ADR-induced FSGS through a complement-mediated mechanism. (A–F) Representative pictures and data quantification of glomerular (A–C) DAF and (D–F) C3b staining of male WT BALB/c mice treated with vehicle or ADR (10 mg/kg, i.v.). Background staining for C3b is present in the periphery of all glomeruli, with higher intraglomerular C3b staining (arrows) in the ADR-treated animals. DAF and C3b glomerular fluorescence intensity was quantified relative to isotype using ImageJ software. At least 30 glomeruli per mouse from two animals were included in the analysis. Each dot represents a glomerulus. (G and H) Urinary A/C at weekly intervals (G) and representative renal histological (PAS stain) lesions (H) of male WT (n = 8) or DAF^−/− (n = 9) BALB/c mice sacrificed at 5 wk after ADR injection (10 mg/kg, i.v.). (I) Urinary A/C at weekly intervals of female WT (n = 5) or DAF^−/− (n = 4) BALB/c mice sacrificed at 5 wk after ADR injection (10 mg/kg, i.v.). (J–M) Urinary A/C at weekly intervals (J) and representative renal histological (PAS) lesions (K) with data quantification (L and M) of male WT (n = 18), DAF^−/− (n = 13), or DAF^−/−C3^−/− (n = 4) B6 mice sacrificed at 5 wk after ADR injection (20 mg/kg, i.v.). All experimental data were verified in at least three independent experiments. *P < 0.05 versus WT at the same time point. n.s., not significant. Scale bars: 50 µm. Error bars are SEM.

Figure S1.

ADR injection associates with glomerular C3b deposition. (A–F) Representative pictures of glomerular DAF (A and B) and C3b staining (D and E) and data quantification (C and F) of male B6 WT mice at 2 wk after treatment with vehicle or ADR (20 mg/kg, i.v., n = 4). (G–I) Representative glomerular C3b deposition (G and H) and data quantification (I) in kidneys from B6 DAF^−/− mice at 2 wk after ADR or vehicle injection (n = 4). All images within the same experiment, including vehicle and ADR, were captured at the same exposure time. n.s., not significant. Scale bars: 50 µm. *P < 0.05. Error bars are SEM.

Figure S2.

Podocyte-specific DAF-KO mice show increased glomerular C3b deposition in the absence of C1q or C4b deposits in response to ADR. (A) Schematic demonstrating the breeding of the DAF^fl/fl mice with the podocin-Cre mice to generate DAF^fl/fl podocin-Cre^POS animals. (B–E) Representative pictures of renal staining of DAF expression in the glomeruli and tubules (B and C) and data quantification (D and E) from 8-wk-old male DAF^fl/fl podocin-Cre^POS and podocin-Cre^NEG mice. DAF glomerular and tubular fluorescence intensity were quantified as in Fig. 1. (F–J) Representative glomerular C1q (F), C4 (G), and C3b (H) staining in B6 DAF^fl/fl podocin-Cre^NEG and podocin-Cre^POS mice at 2 wk after ADR injection. Synaptopodin is stained in red. As positive controls for C1q and C4b, we used MRL-lpr lupus-prone mice at 4 mo of age (I and J). All experimental data were verified in at least three independent experiments. n.s., not significant. Scale bars: 50 µm. *P < 0.05. Error bars are SEM.

Figure 2.

Podocyte-specific removal of DAF from podocytes increases susceptibility to ADR-induced injury through C3a/C3aR signaling. (A–F) Urinary A/C at weekly intervals (A) and representative renal histological (PAS) lesions (B–D) with data quantification (E and F) of male DAF^fl/fl podocin-Cre^NEG (n = 10), DAF^fl/fl podocin-Cre^POS (n = 19), and DAF^fl/fl CD11c-Cre^POS (n = 5) mice injected with ADR (20 mg/kg, i.v.) and sacrificed after 5 wk. (G and H) Representative electron micrographs (G) and quantification (H) of podocyte effacement in 8-wk-old DAF^fl/fl podocin-Cre^POS or DAF^fl/fl podocin-Cre^NEG mice at 5 wk after treatment with saline or ADR (20 mg/kg, i.v.). (I and J) Urinary A/C at weekly intervals (I) and representative renal histological (PAS) lesions (J) of WT (n = 16), DAF^−/− (n = 13), and DAF^−/−C3aR^−/− (n = 4) male B6 mice injected with ADR (20 mg/kg, i.v.). (K and L) Urinary A/C at weekly intervals (K) and representative renal histological (PAS) lesions (L) of male BALB/c mice given ADR (10 mg/kg, i.v.) and treated with C3aR-A (1 mg/kg/d, s.c.; n = 5) or saline (n = 5). (M and N) Urinary A/C at weekly intervals (M) and representative renal histological (PAS) lesions (N) of male B6 DAF^fl/fl podocin-Cre^POS mice given ADR (20 mg/kg, i.v.) and treated with C3aR-A (1 mg/kg/d, s.c.; n = 5) or saline (n = 5). *P < 0.05 versus podocin-Cre^NEG, WT, or C3aR-A. All experimental data were verified in at least three independent experiments. n.s., not significant. Scale bars: 50 µm. Error bars are SEM.

Figure S3.

Mouse and immortalized human podocytes express C3aR and C5aR. (A–C) Representative pictures of glomerular C3aR (A and B) and data quantification (C) of male WT BALB/c mice at 2 wk after treatment with vehicle or ADR (10 mg/kg, i.v., n = 4). Scale bars: 20 µm. (D and E) Representative images (D) and densitometric analysis (E) of C3aR expression over isotype in hiPod. Scale bars: 100 µm. All experimental data were verified in at least three independent experiments. (F–H) Representative pictures of glomerular C5aR (F and G) and data quantification (H) of male WT BALB/c mice at 2 wk after treatment with vehicle or ADR (n = 4). Scale bars: 20 µm. ***P < 0.001. Error bars are SEM.

Figure 3.

ADR induces PLAD-dependent cleavage of DAF. (A and B) Representative images (A) and distribution of DAF expression (B) quantified in hiPod exposed to vehicle, ADR (0.3 µg/ml), PLADi (1 µM), or ADR + PLADi (0.3 µg/ml in 1 µM) for 24 h. DAF IF signal was normalized to actin expression pixel by pixel, and the MFI for each cell was computed. Results are representative of two independent experiments with similar results. (C and D) Representative blots (C) and densitometric analysis (D) of PLAD expression in hiPod cell lysates previously exposed to vehicle or ADR for 24 h. (E and F) Representative blots (E) and densitometric analysis (F) of DAF in the supernatants of hiPod exposed to ADR for 24 h with or without PLADi (WB). (G) Representative blot of DAF in the urine from BALB/c male mice at 2 wk after treatment with vehicle or ADR compared with recombinant mouse DAF (rDAF). In each group, we pooled and concentrated urine samples from eight mice (see Materials and methods). All experimental data were verified in at least three independent experiments. *P < 0.05; n.s., not significant. Scale bars: 50 µm. Error bars are SEM.

Figure 4.

Glomerular DAF downregulation promotes murine STZ-induced diabetic kidney disease. (A and B) Representative pictures (A) and data quantification (B) of glomerular C3b in BALB/c WT mice at 5 wk after vehicle or STZ injection. ***P < 0.001. (C and D) Urinary A/C at weekly intervals (C) and representative renal histological (PAS) lesions (D) of male WT (n = 8) or DAF^−/− (n = 5) BALB/c mice sacrificed at 5 wk after STZ injection (50 mg/kg, i.p., for five consecutive days). *P < 0.05 versus WT at the same time point. (E) Glycemic levels of mice injected with STZ shown in A–D. (F and G) Representative pictures (F) and data quantification (G) of glomerular C3b in DAF^fl/fl podocin-Cre^NEG/POS B6 mice at 5 wk after STZ injection. ****P < 0.001. (H and I) Urinary A/C at weekly intervals (H) and representative renal histological (PAS) lesions (I) of male DAF^fl/fl podocin-Cre^NEG (n = 9) or podocin-Cre^POS (n = 8) B6 mice sacrificed at 20 wk after STZ injection (50 mg/kg, i.p., for five consecutive days). *P < 0.05 versus DAF^fl/fl podocin-Cre^NEG at the same time point. (J) Glycemic levels of mice injected with STZ shown in H and I. Only mice that reached glycemia >300 mg/dl at 2 wk after STZ injection were included in this set of experiments. All experimental data were verified in at least three independent experiments. Scale bars: 50 µm. Error bars are SEM.

Figure 5.

FSGS in humans is associated with DAF down-regulation and complement activation. (A–D) C3 (A), C3aR (B), C5aR (C), and DAF mRNA (D) expression in glomeruli of human biopsy specimens with pathological diagnosis of FSGS or diabetic kidney disease compared with normal kidneys. Data are from previously published microarray studies by Ju et al. (2013) and were subjected to further analysis using Nephroseq. (E–H) Representative renal staining and data quantification for C3d (IF; E and F) and DAF (immunohistochemistry; G and H) in patients with FSGS (n = 18) and in kidneys from healthy renal donors (n = 10). (I) Correlation between protein and C3a in urine samples from 27 patients with FSGS taken at the time of kidney biopsy (before therapy). (J and K) Differences in proteinuria (J) and urinary C3a (K) measured before versus 3–6 mo after steroid therapy in a subset of 13 patients with FSGS. (L) Correlation between the change in proteinuria and change in urinary C3a before and after therapy for each of the same 13 patients. (M) Representative blot of DAF in the urine from healthy control individuals and patients with FSGS compared with recombinant human DAF (rDAF). In each group, we pooled and concentrated urine samples from five and five subjects, respectively (see Materials and methods). *P ≤ 0.05. Scale bars: 25 μm. Error bars are SEM.

Figure 6.

C3a–C3aR interaction disrupts actin cytoskeleton in human cultured podocytes. (A and B) Representative images (A) and quantification (B) of cell injury of hiPod exposed to vehicle, C3a (50 nM), or C3a + C3aR-A (50 nM) for 24 h and stained for F-actin and nephrin. (C) Representative images of caspase-3 staining of the same cells pictured above. (D and E) Representative images (D) and quantification (E) of cell injury of amniotic fluid–derived human podocytes exposed to vehicle, C3a (50 nM), or C3a + C3aR-A (50 nM) for 24 h and stained for F-actin and nephrin. *P < 0.05. (F and G) Representative blots and densitometric analysis of Snail (F) and TGF-β (G) expression in hiPod treated with vehicle, C3a, or C3a + C3aR-A for 24 h. Snail was measured in cell lysates; TGF-β was measured in cell supernatants. *P < 0.05; **P < 0.01. (H) Differentially expressed proteins in the supernatants of hiPod exposed for 24 h to vehicle, C3a (50 nM), or C3a + C3aR-A (50 nM; Proteome Profiler Human XL Cytokine Array). The cytokines represented here are the only ones among the 105 analyzed (see Materials and methods) whose expression levels significantly differed in C3a-treated podocytes versus both vehicle- and C3a + C3aR antagonist–treated cells. *P < 0.05 versus vehicle and C3a + C3aR antagonist. (I) Functional network showing the relationship between C3aR, IL-1β, and nephrin in renal tubular cells and in podocytes (https://hb.flatironinstitute.org/gene/; query genes C3aR, IL1B, and NPHS1; tissue, renal tubules or podocytes; maximum number of genes, 15). All experimental data were verified in at least two independent experiments. Scale bars: 20 µm. Error bars are SEM.

Figure 7.

IL-1β mediates complement-induced podocyte injury in vitro. (A) IL1B gene expression in hiPod at serial time points after C3a stimulation (50 nM). (B) IL-1β levels in the supernatants of hiPod at 24 h after LPS (5 ng/ml) with or without C3a stimulation. (C) Nephrin expression in hiPod at 24 h after stimulation with isotype, C3a + anti–IL-1β–neutralizing antibody, and C3a + isotype (WB). *P < 0.05 versus 0 h. (D and E) Representative images (D) and quantification (E) of cell injury of hiPod exposed to isotype control, IL-1β (50 ng/ml) + isotype control, or IL-1β (50 ng/ml) + anti–IL-1β–neutralizing antibody (0.5 µg/ml; upper row). In the bottom row, the same cells were exposed to C3a + isotype control or C3a + anti–IL-1β–neutralizing antibody for 1 h. All experimental data were verified in at least two independent experiments. *P < 0.05. Scale bars: 100 µm. Error bars are SEM.

Figure 8.

IL-1β mediates complement-induced podocyte injury in vivo. (A and B) Urinary A/C (A) and representative renal histological changes (B) in BALB/c mice injected with 10 mg/kg of ADR (i.v.) and rat anti-mouse IL-1β–neutralizing mAb (50 µg/mice twice per week, i.p.; n = 4) or isotype control (n = 4). Scale bars: 50 µm. *P < 0.05 versus anti–IL-1β–neutralizing antibody at the same time point. (C and D) Representative images of C3b deposition in the glomeruli of mice treated with (C) anti–IL-1β–neutralizing antibody or (D) isotype control. (E and F) Urinary A/C (E) and representative renal histological changes (F) in B6 DAF^fl/fl podocin-Cre^POS male mice injected with 10 mg/kg of ADR (i.v.) and anakinra (25 mg/kg/d through s.c. pumps; n = 4) or vehicle control (n = 3). Scale bars: 50 µm. *P < 0.05 versus anakinra at the same time point. (G and H) IL1B (G) and IL1R1 mRNA expression (H) in glomeruli of human biopsy specimens with pathological diagnosis of FSGS compared with normal kidneys. Data are from previously published microarray studies by Ju et al. (2013) and were subjected to further analysis using Nephroseq. *P < 0.05. All experimental data were verified in at least two independent experiments. Error bars are SEM.