Deletion of the Mitochondrial Complex-IV Cofactor Heme A:Farnesyltransferase Causes Focal Segmental Glomerulosclerosis and Interferon Response

Abstract

Mutations in mitochondrial DNA as well as nuclear-encoded mitochondrial proteins have been reported to cause tubulointerstitial kidney diseases and focal segmental glomerulosclerosis (FSGS). Recently, genes and pathways affecting mitochondrial turnover and permeability have been implicated in adult-onset FSGS. Furthermore, dysfunctioning mitochondria may be capable of engaging intracellular innate immune-sensing pathways. To determine the impact of mitochondrial dysfunction in FSGS and secondary innate immune responses, we generated Cre/loxP transgenic mice to generate a loss-of-function deletion mutation of the complex IV assembly cofactor heme A:farnesyltransferase (COX10) restricted to cells of the developing nephrons. These mice develop severe, early-onset FSGS with innate immune activation and die prematurely with kidney failure. Mutant kidneys showed loss of glomerular and tubular epithelial function, epithelial apoptosis, and, in addition, a marked interferon response. In vitro modeling of Cox10 deletion in primary kidney epithelium compromises oxygen consumption, ATP generation, and induces oxidative stress. In addition, loss of Cox10 triggers a selective interferon response, which may be caused by the leak of mitochondrial DNA into the cytosol activating the intracellular DNA sensor, stimulator of interferon genes. This new animal model provides a mechanism to study mitochondrial dysfunction in vivo and demonstrates a direct link between mitochondrial dysfunction and intracellular innate immune response.

Figure 1

Cox10 deficiency in the nephron causes severe forms of focal segmental glomerulosclerosis. A and B: Representative photomicrographs taken from periodic acid-Schiff (PAS)–stained kidney cortical sections of Cox10^Δ/Δ (Six2-Cre⁺;Cox10^fl/fl; knockout phenotype) and Cox10^+/+ (Six2-Cre⁻;Cox10^fl/fl; wild-type phenotype) mice (A) and methenamine silver (silver)–stained kidney cortical sections of Cox10^Δ/Δ mice at 3 months of age (B). C: Evaluation of FSGS in Cox10^Δ/Δ and Cox10^+/+ kidneys using an index system and based on the percentage of sclerotic glomeruli and glomeruli with crescent formation. Statistics analyzed using the U-test. D: Representative electron micrographs taken from glomeruli of Cox10^Δ/Δ and Cox10^+/+ kidneys. Arrows indicate podocyte foot processes. Data are expressed as means ± SEM (C). n = 4 to 9 (C). ^∗P ≤ 0.05, ^∗∗P ≤ 0.01. Scale bars: 50 μm (A and B); 2 μm (D).

Figure 2

Cox10 deficiency in the nephron produces prenatal and postnatal lethality with renal failure. A: Left panel: Kaplan-Meier curve showing the survival of Cox10^Δ/Δ compared with Cox10^+/+ mice. Right panel: Breeding scheme to test Cox10 deficiency–associated embryogenic lethality and a pie chart showing genotype distribution of the offspring. B: Body weights of male Cox10^Δ/Δ compared with Cox10^+/+ mice at 2 and 3 months of age. C: Representative photomicrographs and graphs showing kidney weights of male Cox10^Δ/Δ compared with Cox10^+/+ mice at the time of natural death and euthanasia. D: Assessment of kidney function of Cox10^Δ/Δ and Cox10^+/+ mice by serum creatinine, blood urea nitrogen (BUN), and urinary albumin levels. Statistics analyzed using the U-test. Data are expressed as means ± SEM (B–D). n = 55 (A, right panel); n = 4 mice aged 2 months (B); n = 7 to 11 mice aged 3 months (B); n = 8 (C); n = 11 (D). ^∗P ≤ 0.05, ^∗∗∗∗P ≤ 0.0001. Scale bar = 1 cm (C).

Figure 3

Cox10 deficiency in the nephron leads to tubulointerstitial damage. A: Representative photomicrographs taken from periodic acid-Schiff (PAS)–stained kidney sections of Cox10^Δ/Δ and Cox10^+/+ mice at 3 months of age. Using PAS-stained kidney sections, tubular damage was evaluated on the basis of the percentage of dilated, atrophic tubules (T); tubules were in Cox10^Δ/Δ compared with Cox10^+/+ kidney. B–F: Evaluation of fibrosis (B), activated fibroblasts (C), proximal tubular injury (D), apoptosis (E), and macrophage infiltration in kidneys (F) from Cox10^Δ/Δ and Cox10^+/+ mice at 3 months of age using immunostaining. All statistics analyzed using the U-test. Data are expressed as means ± SEM (A–F). n = 4 to 9 (A and B); n = 3 (C–F). ^∗P ≤ 0.05, ^∗∗P ≤ 0.01. Scale bar = 100 μm (A). Original magnification, ×20 (B–F). KIM-1, kidney injury molecule-1; LTL, lotus tetragonolobus lectin; α-SMA, α-smooth muscle actin.

Figure 4

Cox10 deficiency in the nephron up-regulates nephrotoxicity genes and interferon-stimulated genes (ISGs). Comparison of gene expression profile in Cox10^Δ/Δ versus Cox10^+/+ kidneys using RNA sequencing. A: A volcano plot visualizing differentially expressed genes in Cox10^Δ/Δ versus Cox10^+/+ kidneys with fold changes significantly (P < 0.001) >1.4. Significance versus fold change is plotted on the y and x axes, respectively. B: Pathway enrichment analysis using DAVID. C: A heat map featuring differently expressed mtDNA-encoded genes. D: Clustering and annotation of differentially expressed genes associated with nephrotoxicity using Ingenuity Pathway Analysis (IPA) Software version 01-10. E: Identification of ISGs among differentially expressed genes using INTERFEROME version 2.01. F: Expression and clustering of all differentially expressed ISGs graphically presented as a heat map. G: Western blot analysis for selected ISGs in Cox10^Δ/Δ versus Cox10^+/+ kidneys. n = 4 (A–F). DEG, differentially expressed genes; ECM, extracellular matrix; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HTLV-1, human T-lymphotropic virus; IRF, interferon regulatory factor; KEGG, Kyoto Encyclopedia of Genes and Genomes; OAS, 2'-5'-oligoadenylate synthetase 1; STING, stimulator of interferon genes; TNF, tumor necrosis factor.

Figure 5

Cox10 silencing in kidney tubular epithelial cells (TECs) results in mitochondrial dysfunction and release of mtDNA into the cytosol and activates stimulator of interferon genes (STING). A: Schematic representation: TECs isolated from Cox10^Δ/Δ mice are not viable in vitro. Thus, Cox10-targeted (siCox10) and control (siControl) siRNAs were introduced into cultured wild-type (WT) TECs by transfection (each at a final concentration of 100 nmol/L) to study the role of Cox10 in TECs. B: At day 6 of transfection, cells were harvested and analyzed for Cox10 mRNA expression. C: The oxygen consumption rate (OCR) was measured at baseline and in response to oligomycin (Oligo), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and antimycin plus rotenone (Ant + Rot) using the Seahorse XFe extracellular flux analyzer. D: Confocal microscopy images were taken from TECs stained with anti-DNA (DNA) and anti–superoxide dismutase (Mito) at days 2 and 5 of transfection. Arrowheads indicate mitochondria not colocalizing with DNA staining. E: Nuclear-encoded Tert gene expression was quantitated via real-time quantitative PCR in cytosolic and whole-cell extracts of untreated TECs. F: Cytosolic mtDNA content in transfected TECs. G and H: Western blot analysis for phosphorylation of STING (G) and TANK-binding kinase-1 (TBK1; H) in transfected TECs. Dashed lines indicate untreated cells (B, C, and F). Data are expressed as means ± SEM (B, C, E, and F). n = 3 to 4 (B, C, E, and F). ^∗P ≤ 0.05 (U-test). Scale bars = 10 μm (D). Original magnification, ×63 (D). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 6

Cox10 silencing or introduction of mtDNA into the cytosol up-regulates apoptotic, proinflammatory, and interferon transcripts in tubular epithelial cells (TECs). Wild-type TECs were transfected with 100 nmol/L of siCox10 and control siRNA (siControl). A–C: At day 6 after silencing, cells were harvested and analyzed for mRNA expression of oxidative stress-related (A), apoptosis-related (B), and proinflammatory (C) genes. Dashed lines indicate untreated cells. D: Transcriptional expression of Ifna4 and Ifnb1 in TECs transfected with 2.6- or 3.6-kb fragments of synthesized oxidized mtDNA at 4 hours. Data are expressed as means ± SEM (A–D). n = 3 to 4 (A–D). ^∗P ≤ 0.05 (U-test). ND, not detected.

Figure 7

Cox10 silencing elevates the expression levels of interferon-stimulated genes (ISGs) in tubular epithelial cells (TECs). A: Wild-type (WT) TECs transfected with 100 nmol/L of siCox10 and control siRNA (siControl). At day 6 of transfection, cells were harvested and analyzed for mRNA expression of differentially expressed ISGs. Dashed lines indicate untreated cells. B: Comparison of ISG expression in Cox10^Δ/Δ versus Cox10^+/+ kidneys using RNA sequencing. Select ISGs with fold changes >1.4 are highlighted in red in the volcano plot (left panel) and visualized in a heat map (right panel). Data are expressed as means ± SEM (A). n = 3 (A); n = 4 (B). ^∗P ≤ 0.05 (U-test).

Supplemental Figure S1

Principal component analysis (PCA) and functional annotation analysis of cell death–associated genes. A: Western blot analysis for COX10 and COX1 protein expression in Cox10^Δ/Δ and Cox10^+/+ kidneys. B: Body weights of female Cox10^Δ/Δ compared with Cox10^+/+ mice at 2 and 3 months of age. C: Kidney weights of female Cox10^Δ/Δ compared with Cox10^+/+ mice at the time of natural death and euthanasia. D: Urinary albumin/creatinine ratio of mice at 2.5 to 3 months of age. E: PCA analysis of RNA sequencing data from kidneys of Cox10^Δ/Δ versus Cox10^+/+ mice. F: Clustering and annotation of differentially expressed genes associated with cell death using Ingenuity Pathway Analysis software version 01-10. n = 4 mice aged 2 months (B); n = 9 to 10 mice aged 3 months (B); n = 8 (C); n = 3 to 7 (D). ^∗P ≤ 0.05, ^∗∗P ≤ 0.01, and ^∗∗∗P ≤ 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Supplemental Figure S2

Functional protein association networks among differentially expressed genes in Cox10^Δ/Δ versus Cox10^+/+ kidneys. Differentially expressed genes in Cox10^Δ/Δ versus Cox10^+/+ kidneys were subjected to functional protein association network analysis using STRING database version 10.5.

Supplemental Figure S3

Cox10 silencing up-regulates apoptotic and proinflammatory genes in proximal tubular epithelial cells (PTECs). A: Schematic representation: 100 nmol/L of siCox10 and control siRNA (siControl) was introduced into cultured wild-type (WT) PTECs by transfection. B–E: At days 2 and 6 of transfection, cells were harvested and analyzed for mRNA expression of Cox10 (B) and genes related to oxidative stress (C) and apoptosis (D) or proinflammatory genes (E). Dashed lines indicate untreated cells. Data are expressed as means ± SEM (B–E). n = 3 (B–E). ^∗P ≤ 0.05 (U-test). ND, not detected.

Supplemental Figure S4

Oxidative stress and apoptosis-related genes are not up-regulated during the first 2 days of siCox10 transfection in tubular epithelial cells (TECs). A: Schematic representation: wild-type (WT) TECs were transfected with 100 nmol/L of siCox10 and control siRNA (siControl). B–E: At day 2 of transfection, cells were harvested and analyzed for mRNA expression of Cox10 (B) as well as genes related to oxidative stress (C) and apoptosis (D) or proinflammatory genes (E). Dashed lines indicate untreated cells. Data are expressed as means ± SEM (B–E). n = 3 to 4 (B–E). ^∗P ≤ 0.05 (U-test).

Supplemental Figure S5

Cox10 silencing increases ATP production but does not influence cell viability. Cells were analyzed for cell viability (A), ATP production at day 5 of transfection (B), and mitochondrial membrane potential normalized by mitochondrial mass by costaining with MitoHealth and MitoTracker Green FM (C). Dashed lines indicate untreated cells. Data are expressed as means ± SEM (A–C). n = 4 (A–C). ^∗P ≤ 0.05 (U-test).

Supplemental Figure S6

Nuclear-encoded Tert gene expression in cytosolic and whole-cell extracts of transfected tubular epithelial cells (TECs) and TEC transfection with mtDNA fragments. Nuclear-encoded Tert gene expression was quantitated via real-time quantitative PCR in cytosolic and whole-cell extracts of TECs transfected with siCox10 or control siRNA (siControl) at days 2 and 5. Data are expressed as means ± SEM. n = 3. ^∗P ≤ 0.05 (U-test).