C5a-C5aR1 axis controls mitochondrial fission to promote podocyte injury in lupus nephritis

Abstract

Podocytes are essential to maintaining the integrity of the glomerular filtration barrier, but they are frequently affected in lupus nephritis (LN). Here, we show that the significant upregulation of Drp1^S616 phosphorylation in podocytes promotes mitochondrial fission, leading to mitochondrial dysfunction and podocyte injury in LN. Inhibition or knockdown of Drp1 promotes mitochondrial fusion and protects podocytes from injury induced by LN serum. In vivo, pharmacological inhibition of Drp1 reduces the phosphorylation of Drp1^S616 in podocytes in lupus-prone mice. Podocyte injury is reversed when Drp1 is inhibited, resulting in the alleviation of proteinuria. Mechanistically, complement component C5a (C5a) upregulates the phosphorylation of Drp1^S616 and promotes mitochondrial fission in podocytes. Moreover, the expression of C5a receptor 1 (C5aR1) is notably upregulated in podocytes in LN. C5a-C5aR1 axis-controlled phosphorylation of Drp1^S616 and mitochondrial fission are substantially suppressed when C5aR1 is knocked down by siRNA. Moreover, lupus-prone mice treated with C5aR inhibitor show reduced phosphorylation of Drp1^S616 in podocytes, resulting in significantly less podocyte damage. Together, this study uncovers a novel mechanism by which the C5a-C5aR1 axis promotes podocyte injury by enhancing Drp1-mediated mitochondrial fission, which could have significant implications for the treatment of LN.

Keywords: C5a-C5aR1 axis; drp1; lupus nephritis; mitochondrial fission; podocyte injury.

Graphical abstract

Figure 1

Drp1-mediated mitochondrial fission is increased in podocytes from both human and mice with LN Transcriptomic data of glomerular tissue were obtained from Berthier Lupus Glom Dataset in the public database Nephroseq, which enrolled renal biopsies derived from HC and LN patients. (A) The DEGs were illustrated by heatmap. Genes with p < 0.005 and |log fold change|>1.2 were regarded as DEGs. (B) Up- and downregulated genes were adopted for gene set enrichment analysis. GO terms and KEGG pathways with p < 0.05 were regarded as enriched gene sets. (C and D) Kidney sections from patients with LN or HCs were stained with antibodies against synaptopodin and p-Drp1^S616. The number of synaptopodin⁺p-Drp1^S616+ double-positive cells per glomerulus was counted. Scale bar: 50 μm. (E and F) The correlations between the number of synaptopodin⁺p-Drp1^S616+ podocytes and SLEDAI or proteinuria levels. (G and H) Fresh urine samples were collected from patients with LN or HCs. Cellular sediments were enriched and stained with antibodies against synaptopodin and p-Drp1^S616. The percentages of synaptopodin⁺p-Drp1^S616+ double-positive cells were summarized from 4 independent samples. Scale bar: 10 μm. (I and J) Kidney sections from MRL/lpr or control mice were stained with antibodies against synaptopodin and p-Drp1S616. The number of synaptopodin⁺p-Drp1^S616+ double-positive cells per glomerular was counted and summarized from 6 independent samples. Scale bar: 20 μm. (K and L) Representative images showed PAS staining of kidney sections. TEM images demonstrated mitochondrial morphology in podocytes. PAS magnification: 400×. Scale bar: 50 μm. Electron microscopy magnification: 5,000×. Scale bar: 1 μm. All of the data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by unpaired Student’s t test.

Figure 2

Drp1 impairs mitochondrial function and reduces podocyte viability in LN Human podocytes were stimulated with serum from patients with LN or HCs in the presence or absence of mdivi-1 (20 μM) for 24 h. (A and B) The expressions of p-Drp1^S616, Drp1, OPA1, and MFN2 were measured by western blot. Representative bands were shown, and relative expression was summarized. (C and D) The level of p-Drp1^S616 in podocytes was measured by flow cytometry. Representative histograms were shown, and data were summarized from 8 serum samples. (E) Podocytes were stained with Hoechst (blue) and MitoTracker Green (green). Representative images by confocal microscopy showed mitochondrial staining in the cells. Scale bar: 10 μm. (F and G) Mitochondrial branch length and mitochondrial footprint per cell were calculated and summarized from 10 independent serum samples, respectively. (H–J) Cells were stained with MitoSox/DCFH-DA and measured by flow cytometry. Mean fluorescence intensity (MFI) was summarized from 6 serum samples. (K and L) Cells were stained with JC1 probe, and mitochondrial membrane potential was measured by flow cytometry. The ratio of JC1 red to JC1 green MFI was calculated and summarized in (L). (M) ATP production in podocytes was measured by ATP assay kit. The level of ATP was normalized by the amount of protein (mg). (N and O) Cells were stained with 7-AAD and annexin V. The apoptosis rate was measured by flow cytometry. Representative dot plots were shown and data from 8 serum samples were summarized. All of the data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001 by unpaired Student’s t test in (B) and 1-way ANOVA with p value adjusted in the remaining panels. ns, not significant.

Figure 3

Knockdown of Drp1 preserves mitochondrial function and protects podocytes from cell death in LN Human podocytes were treated with Drp1 siRNA (siDrp1) or scramble siRNA (siNC). Cells were then cultured with serum from patients with LN for 24 h. (A–C) Knockdown efficiency of Drp1 was confirmed by qPCR (A) and western blot (B and C). (D) Cells were stained with MitoTracker Green and Hoechst. Mitochondrial staining was visualized by confocal microscopy, and representative images were shown. Scale bar: 10 μm. (E and F) Mitochondrial branch length and mitochondrial footprint per cell were calculated and summarized. (G–I) Cells were stained with MitoSox/DCFH-DA and measured by flow cytometry. MFI was summarized from 8 serum samples. (J and K) Mitochondrial membrane potential in podocytes was measured by flow cytometry using JC1 probe. The ratio of JC1 red to JC1 green MFI was calculated and summarized. (L) ATP production in podocytes was measured by ATP assay kit. The level of ATP was normalized by the amount of protein (mg). (M and N) Apoptosis rate was measured by flow cytometry. Representative dot plots were shown and data from 8 serum samples were summarized. All of the data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗<0.0001 by 1-way ANOVA with p value adjusted.

Figure 4

Pharmacological inhibition of Drp1 reduces podocyte injury and preserves renal function in lupus-prone mice (A) Scheme of the mouse experiment. Female MRL/lpr mice (12 weeks old) were treated with Drp1 inhibitor mdivi-1 (30 mg/kg) or vehicle intraperitoneally every 2 days for 6 weeks. (B) The level of p-Drp1^S616 in podocytes from mice treated with mdivi-1 or vehicle was measured by immunofluorescent staining. Representative confocal microscopy images were shown. Scale bar: 20 μm. (C–G) Single-cell suspension was prepared from kidneys treated with mdivi-1 or vehicle. (C) Cells were stained with antibodies against CD45, CD26, and nephrin and measured by flow cytometry. Gating strategy: CD45⁻CD26⁺nephrin⁺ cells were identified as podocytes. (D and E) The levels of p-Drp1^S616 in podocytes were measured by flow cytometry. (F and G) The levels of MitoSOX/DCFH-DA in podocytes were measured by flow cytometry. (H) Podocyte integrity were visualized by electron microscopy. Representative images were shown. Magnification 5,000×. Scale bar: 10 μm (left); 2 μm (right). Fluorescent images showed nephrin staining in the glomeruli from mice treated with mdivi-1 or vehicle. Scale bar: 100 μm. (I and J) Representative images of PAS staining were shown. Magnification 400×. Scale bar: 50 μm. Renal pathology score was calculated and summarized. (K) Proteinuria was scored, and cumulative incidence of severe proteinuria was recorded over the course of treatment. (L and M) The levels of creatinine and BUN in the serum of mice treated with mdivi-1 or vehicle. n = 7. All of the data are presented as mean ± SEM. ∗p < 0.05 and ∗∗∗p < 0.001 by unpaired Student’s t test.

Figure 5

C5a-C5aR1 modulates podocyte viability in LN (A) RNA-seq data were acquired from Nephroseq. C5aR1 expression level in the kidney of patients with LN or HCs was summarized. (B and C) Kidney sections were stained with antibodies against synaptopodin and C5aR1. The expression of C5aR1 in synaptopodin⁺ podocytes were measured by immunofluorescence. The percentages of C5aR1⁺ podocytes were calculated and summarized, respectively. Scale bar: 50 μm. (D) Correlation between the percentages of C5aR1⁺ podocytes and proteinuria was examined. (E and F) Urine cellular sediments from LN or HCs were stained with antibodies against synaptopodin and C5aR1. The percentages of C5aR1⁺ podocytes were calculated and summarized from 4 independent samples. Scale bar: 10 μm. (G and H) C5a concentration in the serum was measured by ELISA. The correlation of C5a concentration with SLEDAI was assessed. (I–L) Podocytes were stimulated with serum from patients with LN or HCs. C5aR1 expression was measured by flow cytometry and western blot, respectively. Representative plots were shown, and C5aR1 expression was summarized. (M–R) Podocytes were cultured with LN serum or HC serum. C5aR1 inhibitor avacopan (1 μM) was included in some of the experiments. (M and N) MitoSOX and DCFH-DA were used to detect ROS in podocytes and measured by flow cytometry. Representative histograms were shown, and MFI was summarized, respectively. (O and P) Podocytes were stained with JC1 probe and measured by flow cytometry. The ratio of JC1 red to JC1 green MFI was calculated and summarized. (Q and R) Apoptosis rate was quantified by flow cytometry. Representative dot plots were shown. Data were derived from 6 independent serum samples. All of the data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗<0.0001 by unpaired Student’s t test in (A), (C), (F), (J), and (L) and by 1-way ANOVA with p value adjusted in (G), (N), (P), and (R).

Figure 6

C5a-C5aR1 controls mitochondrial fission through calcium signaling in podocytes in LN (A–G) Human podocytes were cultured with HC serum or LN serum. Avacopan (1 μM) was included in some of the experiments. (A–C) The levels of p-Drp1^S616 and total Drp1 in podocytes was measured by western blot. Representative bands of 3 repeated experiments were shown. (D–G) Podocytes were first labeled with MitoTracker Deep Red (DR) and then stained with antibodies against p-Drp1^S616. Images were generated using fluorescent confocal microscopy. Representative images were shown. Scale bar: 10 μm. Mitochondrial branch length (E), mitochondrial footprint per cell (F), and p-Drp1^S616/MitoTracker colocalization (Manders’ coefficient) (G) were summarized for each group. (H) Podocytes were labeled with calcium probe Fluo-3. Calcium mobilization in podocytes stimulated by C5a were recorded by fluorescent microscopy. (I and J) Podocytes were cultured in the presence of C5a for the indicated time. The levels of NFATc1, p-Drp1^S616, total Drp1, and C5aR1 in podocytes were measured by western blot. The experiment was repeated 3 times, and representative bands were shown. (K–N) Podocytes were cultured in the presence of C5a for 24 h. FK506 was included in some of the experiments. (K and L) The levels of NFATc1, p-Drp1^S616, total Drp1, and C5aR1 in podocytes were measured by western blot. Representative bands of 3 repeated experiments were shown. (M and N) Podocytes were labeled with MitoTracker DR and then stained with antibodies against p-Drp1^S616. Images were generated using fluorescent microscopy. Representative images are shown. Scale bar: 10 μm. Mitochondrial branch length, mitochondrial footprint per cell, and p-Drp1^S616/MitoTracker colocalization (Manders’ coefficient) were summarized for each group. All of the data are presented as mean ± SEM. ∗∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by 1-way ANOVA with p value adjusted in (B)–(L) and unpaired Student’s t test in (N). ns, not significant.

Figure 7

Blocking C5a-C5aR1 interaction inhibits mitochondrial fission through calcium signaling in podocytes (A–H) Human podocytes were treated with C5aR1 siRNA (siC5aR1) or scramble siRNA (siNC). Cells were then cultured with serum from LN or HCs for 24 h. (A and B) Knockdown efficiency of C5aR1 was confirmed by western blot. (C and D) Podocytes were labeled with calcium probe Fluo-3 measured by fluorescent microscopy. Representative images were shown and Fluo-3 MFI was summarized, respectively. (E–H) The levels of p-Drp1^S616, total Drp1, and C5aR1 in podocytes were measured by western blot. Representative bands of 3 repeated experiments were shown, and relative expressions were summarized, respectively. (I–T) Human podocytes were treated with siC5aR1 or scramble siNC. Cells were then cultured in the presence of C5a or vehicle for 24 h. (I) Calcium probe Fluo-3 was used to detect calcium mobilization, and Fluo-3 MFI was summarized from 3 samples. (J–M) Western blot was used to measure the expression of NFATc1, p-Drp1^S616, and total Drp1, respectively. Representative bands of western blot are shown. The levels of NFATc1 (K), p-Drp1^S616 (L), and total Drp1 (M) were summarized from 3 samples. (N–P) Podocytes were harvested and stained with MitoTracker Green and Hoechst. Mitochondrial staining was visualized by confocal microscopy. Representative images were shown. Scale bar: 10 μm. Mitochondrial branch length (O) and mitochondrial footprint per cells (P) were calculated and summarized. (Q and R) MitoSOX in podocytes was measured by flow cytometry. Representative histograms are shown, and MFI were summarized. (S and T) Apoptosis rate of podocytes was measured by flow cytometry. Representative dot plots were shown. Data were derived from 8 independent serum samples. All of the data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by 1-way ANOVA with p value adjusted.

Figure 8

Inhibition of C5a-C5aR1 axis reduces Drp1-mediated podocyte injury in lupus-prone mice (A) Scheme of the mouse experiment. MRL/lpr mice (12 weeks old) were injected subcutaneously with C5aR1 antagonist PMX-53 (1 mg/kg) or vehicle for 6 weeks. Kidneys were harvested for further experimentation. (B) The expression of p-Drp1^S616 in podocytes was measured by immunofluorescent staining. Representative confocal microscopy images are shown. Scale bar: 20 μm. (C–H) Single-cell suspension was prepared from kidneys treated with PMX-53 or vehicle. The levels of p-Drp1^S616, mitochondrial ROS, and cellular ROS in podocytes were measured by flow cytometry. Representative histograms were shown, and MFI of p-Drp1^S616 (D), mitochondrial ROS (F), and cellular ROS (H) were summarized, respectively. (I) Podocyte integrity was visualized by electron microscopy. Representative images are shown. Foot process width was measured and summarized for 10 samples. Magnification 5,000×. Scale bar: 1 μm. (K and L) Representative images of PAS staining are shown. Magnification 400×. Scale bar: 50 μm. Renal pathology score was calculated and summarized. (M) Cumulative incidence of severe proteinuria was calculated. (N and O) The levels of creatinine and BUN in the serum of mice treated with PMX-53 or vehicle. All of the data are presented as mean ± SEM. ∗p < 0.05 and ∗∗p < 0.01 by unpaired Student’s t test.