Proximal Tubular Cell–Specific Ablation of Carnitine Acetyltransferase Causes Tubular Disease and Secondary Glomerulosclerosis
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Figures
- Figure 1
Generation of PTC-specific CrAT knockout mouse and baseline whole-body parameters. A: Genetic strategy to generate mice with PTC-specific ablation of CrAT (PT-CrAT mouse). B: Deletion was confirmed by PCR in tail DNA. C: CrAT knockout kidney cortices, but not heart, skeletal muscle, or epididymal white adipose tissue (eWAT), showed a ∼2.6-fold reduced expression of CrAT mRNA (n = 6). *P < 0.006, Gapdh reference gene. D: Western blot analysis of CrAT protein levels in kidney cortices showed successful deletion in the PT-CrAT mice. Kidney and skeletal muscle tissues from mice overexpressing CrAT under the β-actin promoter (BAP-CrAT) were used as positive control. E: Enzyme activity was measured in whole-kidney homogenates (residual CrAT activity derives from cells other than PTCs). PTC-specificity was verified by staining for CRE activity with an anti-Cre antibody (F) and by breeding PT-CrAT female mice with a tdTomato male (G). Red fluorescence around green brush borders stained positive by Lotus tetragonolobus lectin confirms PTC-specific Cre activity. Scale bars = 50 μm. PT-CrAT mice body weights (H), kidney weights (I), fasting blood glucose levels (J), and glucose tolerance test (K) (males, n = 6–8).
- Figure 2
CrAT deletion in PTCs causes kidney disease. A: Representative photomicrographs of PAS and trichrome staining in fixed, paraffin-embedded fl/fl kidneys showing normal histology. B: PT-CrAT kidneys show numerous protein casts in tubular lumen, tubular dilation (*), lipid droplets in tubular cells, trichrome-positive material/fibrosis (yellow arrows), and secondary glomerular disease ($) (≥18 months). C: Tubular injury area was evaluated semiquantitatively, and scores are expressed as minimal 1: <5% of cortex, mild 2: 5–24%, moderate 3: 25–49%, and severe 4: >50% of cortex (n = 6). **P < 0.005. D: Serum creatinine levels in fl/fl and PT-CrAT mice (n = 4–10). *P < 0.05. E: Urine protein analysis by electrophoresis/Coomassie blue staining (arrow points at albumin, ∼67 kDa) in PT-CrAT mice compared with fl/fl controls, with equal amounts of protein (20 μg) loaded and urinary albumin-to-creatinine ratios (ACR) (males, n = 8–12). **P < 0.005. Staining positive for collagen IV (cyan: collagen IV, blue: DAPI) (F) and vimentin (red: vimentin, blue: DAPI) (G) in PT-CrAT tubules and glomeruli. (Photomicrographs are from mice with the highest tubular injury scores.) H: Active caspase-3 staining (red: cleaved caspase-3, green: Lotus tetragonolobus) in fl/fl and PT-CrAT kidneys. Scale bars = 50 μm. I: Percentage of apoptotic PTCs was evaluated by counting cleaved caspase-3–positive, Lotus tetragonolobus counterstained cells (n = 4). *P < 0.05. J: Gene expression analysis in fl/fl and PT-CrAT kidney cortices (n = 6, male mice). *P < 0.05, #P < 0.01, &P < 0.0005 vs. fl/fl.
- Figure 3
Altered acylcarnitine and organic acid profiles in the PT-CrAT model. Kidney cortex (A), plasma (B), and urine samples (C) from PT-CrAT mice and fl/fl controls at 18–24 months of age were analyzed by MS/MS for short- (SCAC), medium- (MCAC), and long-chain acylcarnitines (LCAC), as well as free (D) and total carnitine levels (E). MS/MS analysis was normalized to show mice with the strongest kidney injury histology scores. F: Free-to-total carnitine ratios indicating urinary wasting of free carnitine are shown. G: Kidney cortices were analyzed for organic acid intermediates. α-ketogl., α-ketoglutarate. *P < 0.05, **P < 0.005 vs. fl/fl.
- Figure 4
PT-CrAT mice have increased lipid-derived free radical levels. A: Representative spectra of POBN-lipid radical adducts detected by electron spin resonance spectroscopy from Folch extracts of kidney cortex of PT-CrAT mice and fl/fl littermates (n = 5). Increases in the amplitude of the six line spectrum peaks (arrows) are proportional to an increase in carbon-centered lipid-derived free radical levels. Spectra show an approximately threefold increase of lipid radicals in the PT-CrAT mice vs. fl/fl control. a.u., arbitrary units. B: Western blot analysis detected 4-HNE protein adducts. C: Band intensities (normalized to β-actin as loading control) were measured using ImageJ (n = 4). *P < 0.05 vs. fl/fl.
- Figure 5
The HFD accelerated kidney disease in the PT-CrAT mice. Representative photomicrographs of PAS- and trichrome-stained kidneys of littermate fl/fl mice showing normal histology (A), fl/fl mice fed the HFD (B), and PT-CrAT mice fed the HFD (C) showing tubular damage (*), fibrosis, and trichrome-positive material (yellow arrows), as well as glomerular sclerosis ($) and glomerulomegalia (12–15 months). D: Glomerular tuft size was measured in each group (n = 30–50 glomeruli per group, at 12 months of age). Gene expression analysis in kidney cortices (E), body weights (F), plasma triglyceride (G), and free fatty acid levels (H) in each experimental group (n = 4–6). #P < 0.05 vs. fl/fl on chow diet, *P < 0.05 vs. PT-CrAT on chow diet.
- Figure 6
Primary PTCs from PT-CrAT mice have early alterations in mitochondrial respiratory function. PTCs isolated from fl/fl and PT-CrAT mouse kidneys (9 months) were grown on Seahorse XF24 plates (75,000 cells/well). A: Mitochondrial oxygen consumption rates (OCR) were measured at baseline and after the addition of 2 μmol/L oligomycin, 2 μmol/L FCCP, or 1.5 mg/mL antimycin A (AA). B: Mitochondrial respiratory parameters were calculated and compared from the graph shown on A (n = 10). *P < 0.05 vs. fl/fl. Total acetylated lysine (Acetyl-Lys) levels (C), abundance of mitochondrial ETC complexes (D), and activity of mitochondrial complex I and V in kidney cortices of fl/fl and PT-CrAT mice (n = 4) (E). a.u., arbitrary units. *P < 0.05 vs. fl/fl.
- Figure 7
A proposed scenario of mitochondrial overload in PTCs. Mitochondrial overload modeled by CrAT deletion causes acetyl-CoA/free CoA imbalance and leads to the accumulation of incompletely oxidized products. Such imbalance affects all three major metabolic pathways in PTCs: fatty acid, amino acid, and carbohydrate metabolism. As shown by our mass spectrometry results, overload also affects TCA cycle metabolite levels. Altogether, these metabolic disturbances can contribute to PTC energy deficit. Furthermore, overload can cause mitochondrial redox imbalance through affecting the ETC. Increased ROS and lipid peroxide production then also potentially contributes to PTC apoptosis.
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