Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jan;21(1):37-46.
doi: 10.1038/nm.3762. Epub 2014 Dec 1.

Defective fatty acid oxidation in renal tubular epithelial cells has a key role in kidney fibrosis development

Hyun Mi Kang  1 , Seon Ho Ahn  1 , Peter Choi  1 , Yi-An Ko  1 , Seung Hyeok Han  1 , Frank Chinga  1 , Ae Seo Deok Park  1 , Jianling Tao  1 , Kumar Sharma  2 , James Pullman  3 , Erwin P Bottinger  4 , Ira J Goldberg  5 , Katalin Susztak  1
Affiliations

Defective fatty acid oxidation in renal tubular epithelial cells has a key role in kidney fibrosis development

Hyun Mi Kang et al. Nat Med. 2015 Jan.

Abstract

Renal fibrosis is the histological manifestation of a progressive, usually irreversible process causing chronic and end-stage kidney disease. We performed genome-wide transcriptome studies of a large cohort (n = 95) of normal and fibrotic human kidney tubule samples followed by systems and network analyses and identified inflammation and metabolism as the top dysregulated pathways in the diseased kidneys. In particular, we found that humans and mouse models with tubulointerstitial fibrosis had lower expression of key enzymes and regulators of fatty acid oxidation (FAO) and higher intracellular lipid deposition compared to controls. In vitro experiments indicated that inhibition of FAO in tubule epithelial cells caused ATP depletion, cell death, dedifferentiation and intracellular lipid deposition, phenotypes observed in fibrosis. In contrast, restoring fatty acid metabolism by genetic or pharmacological methods protected mice from tubulointerstitial fibrosis. Our results raise the possibility that correcting the metabolic defect in FAO may be useful for preventing and treating chronic kidney disease.

PubMed Disclaimer

Figures

Figure 1
Figure 1. The transcriptional landscape of human tubulointerstitial fibrosis
(a), Gene ontology analysis of human chronic kidney disease (CKD) samples. The graph shows the minus log p-values for the enrichment of a specific pathway. (b), Gene set enrichment analysis highlighted strong enrichment for FAO pathway in CKD human samples. (c), Heatmap analysis of transcripts expression related to fatty acid metabolism. (d), Relative mRNA levels of genes related to FAO in human control (CTL) and CKD samples (CPT1a; carnitine palmitoyl-transferase 1a, CPT2; carnitine palmitoyl-transferase 2, ACOX1; acyl-CoA oxidase 1, ACOX2; acyl-CoA oxidase 2, PPARa; Peroxisome proliferator-activated receptor alpha and PPARGC1a; peroxisome proliferator-activated receptor gamma, coactivator 1 alpha). (e), Representative images of control and CKD human kidney samples stained with periodic acid-schiff (PAS), PPARa and PPARGC1Aa and oil red o (lipid droplets). Scale bar, 20 µm. (f), Quantification of PPARa and PPARGC1a immunostaining in CTL and CKD human kidney samples. (g), Relative transcript levels of enzymes related to carbohydrate metabolism (HK; hexokinase, PFK; phosphofructokinase, GAPDH; Glyceraldehyde 3-phosphate dehydrogenase, PGK1; phosphoglycerate kinase 1, IDH1; isocitrate dehydrogenase 1, PK; pyruvate kinase and GLUT1; Glucose transporter 1). All data are presented as means as ± s.e.m (n=95, 59 of control and 36 of CKD), and * p<0.05 compare to CTL.
Figure 2
Figure 2. Dysregulation of FAO in mouse models of tubulointerstitial fibrosis
(a), Representative images of PAS stained kidney sections of control and Pax8rtTA/TREICNotch1 mice, followed by Ppara immunnostaining images. (b), Gene-ontology analysis of control and Pax8rtTA/TREICNotch1 mice. (c,d), Relative mRNA levels of genes related to FAO (Cpt1a; carnitine palmitoyl-transferase 1a, Cpt2; carnitine palmitoyl-transferase 2, Acox1; acyl-CoA oxidase 1, Acox2; acyl-CoA oxidase 2, Ppara; Peroxisome proliferator-activated receptor alpha and Ppargc1a; peroxisome proliferator-activated receptor gamma, coactivator 1 alpha) and glucose utilization (Hk; hexokinase, Pgk1; phosphoglycerate kinase 1, Gapdh; Glyceraldehyde 3-phosphate dehydrogenase, Idh1; isocitrate dehydrogenase 1, Pk; pyruvate kinase and Glut1; Glucose transporter 1) in control (CTL) and mouse TIF (ICNotch1) samples. All data are presented as means as ± s.e.m (n=6, 3 of control and 3 of Pax8rtTA/TREICNotch1 mice) and * p<0.05 compare to CTL. (e), Relative transcript levels of FAO related enzymes in control (CTL) and FA-induced TIF samples (FAN). All data are presented as means as ± s.e.m (n=12, 6 of control and 6 of FAN mice) and * p<0.05 compare to CTL. (f), Representative images of mouse kidney sections from control and folic acid induced nephropathy (FAN) with PAS staining, immunostaining for Ppara and Ppargc1a and Oil Red O staining. Scale bar, 20 µm. (g), Quantification of Ppara and Ppargc1a images in the CTL and FAN mouse kidney samples. (h), Triglyceride (TG) contents in mouse kidney tissue. N = 10, 4 of CTL and 6 of FAN. * p<0.05 compare to CTL.
Figure 3
Figure 3. Role of tubular epithelial cell lipid accumulation in tubular interstitial fibrosis development
(a), Protein expression (Western blot) of human CD36 in control and Pax8rtTA/TRECD36 mouse kidney samples. (b), Representative images of PAS, oil red o and Sirius red stained control and CD36 transgenic animals. Scale bar, 20 µm. (c), Triglyceride (TG) contents in mouse kidney tissue. N = 5 of CTL and n=5 of 20 week old human CD36 transgenic mice. * p<0.05. (d), Fatty acid quantification in the control and CD36 TG mice by GC/FID. (e–g), Quantitative RT-PCR analysis of transcripts of key FAO enzymes and regulators (Cpt1a; carnitine palmitoyl-transferase 1a, Cpt2; carnitine palmitoyl-transferase 2, Acox1; acyl-CoA oxidase 1, Acox2; acyl-CoA oxidase 2 and Ppara; Peroxisome proliferator-activated receptor alpha), markers of fibrosis (Vim; vimentin, Fn; fibronectin, Col1a1; collagen 1a1, Col3a1; collagen 3a1, Col4a1; collagen 4a1 and Acta2; alpha smooth muscle actin) and apoptosis markers (Apaf1; apoptosis inducing factor1, Bax; BCL2 associated X protein, Bcl2; B-cell CLL/ lymphoma 2) in 8 week and 20 week old human CD36 transgenic mice. All data are presented as means as ± s.e.m (n=17, n=5 control, n=5 Pax8rtTA/TRECD36 at 8 weeks, and n=7 of Pax8rtTA/TRECD36 at 20 weeks) and * p<0.05 compared to controls.
Figure 3
Figure 3. Role of tubular epithelial cell lipid accumulation in tubular interstitial fibrosis development
(a), Protein expression (Western blot) of human CD36 in control and Pax8rtTA/TRECD36 mouse kidney samples. (b), Representative images of PAS, oil red o and Sirius red stained control and CD36 transgenic animals. Scale bar, 20 µm. (c), Triglyceride (TG) contents in mouse kidney tissue. N = 5 of CTL and n=5 of 20 week old human CD36 transgenic mice. * p<0.05. (d), Fatty acid quantification in the control and CD36 TG mice by GC/FID. (e–g), Quantitative RT-PCR analysis of transcripts of key FAO enzymes and regulators (Cpt1a; carnitine palmitoyl-transferase 1a, Cpt2; carnitine palmitoyl-transferase 2, Acox1; acyl-CoA oxidase 1, Acox2; acyl-CoA oxidase 2 and Ppara; Peroxisome proliferator-activated receptor alpha), markers of fibrosis (Vim; vimentin, Fn; fibronectin, Col1a1; collagen 1a1, Col3a1; collagen 3a1, Col4a1; collagen 4a1 and Acta2; alpha smooth muscle actin) and apoptosis markers (Apaf1; apoptosis inducing factor1, Bax; BCL2 associated X protein, Bcl2; B-cell CLL/ lymphoma 2) in 8 week and 20 week old human CD36 transgenic mice. All data are presented as means as ± s.e.m (n=17, n=5 control, n=5 Pax8rtTA/TRECD36 at 8 weeks, and n=7 of Pax8rtTA/TRECD36 at 20 weeks) and * p<0.05 compared to controls.
Figure 4
Figure 4. Transforming growth factor beta 1 suppresses FAO in renal TEC
(a), Oxygen consumption rate (OCR) of human tubule epithelial cell line (HKC8); each data-point represents the mean (and S.E.M) of ten independent samples. When indicated palmitate (180 µM), etomoxir (40 µM), and oligomycin (1 µM) was added. (b), OCR and extracellular acidification rate (ECAR) of HKC8 were measured in a Seahorse XF24 analyzer. Each data-point represents the mean of ten independent samples. 10 mM glucose, 2–4 dinitrophenol (2–4 DNP, 100 µM), 2-deoxyglucose (2-DG, 100 mM), and rotenone (1 µM) were injected sequentially at the indicated time points. (c), ATP levels of control (CTL) HKC8 cells, and cells treated with 40 µM etomoxir (CPT1 inhibitor). (d), Bright field, oil red o staining and caspase 3 immunofluorescence images of HKC8 cells, untreated (left) or etomoxir treated (right). (e), Relative transcript amounts of genes related to apoptosis (APAF1; apoptosis inducing factor1, BAX; BCL2 associated X protein, BCL2; B-cell CLL/ lymphoma 2, BCL2L1; BCL2 like 1) of HKC8 cells upon etomoxir treatment (n=6, 3 of control and 3 of etomoxir treated cells). (f), QRT-PCR based relative mRNA levels of markers of cellular dedifferentiation (VIM; vimentin, FN; fibronectin, COL1; collagen1, COL3, collagen3, and ACTA2; alpha smooth muscle actin) of HKC8 cells treated with etomoxir. (g), OCR measurement of HKC8 exposed to 50ng ml−1 TGFβ1 for 48 hrs or controls. Representative traces are shown right, and summary data left analyzed for 16 wells from independent experiments. When indicated palmitate (180 µM), glucose (25mM) and oligomycin (1 µM) were added. (h), Relative mRNA amount of transcripts of FAO enzymes (CPT1a; carnitine palmitoyl-transferase 1a, CPT2; carnitine palmitoyl-transferase 2, ACOX1; acyl-CoA oxidase 1, ACOX2; acyl-CoA oxidase 2, PPARa; Peroxisome proliferator-activated receptor alpha and PPARGC1a; peroxisome proliferator-activated receptor gamma, coactivator 1 alpha) of control and TGFB1 treated cells. All experiments were replicated twice with triplicate repeated measures for each condition. (i), Lipid accumulation was determined by oil red O staining of control and TGFB1 treated cells. Scale bar, 20 µm (j), ATP levels of control (CTL) HKC8 cells, and cells treated with TGFB1. All data are presented as means as ± s.e.m (n=8, 4 of control and 4 of TGFB1) and * p<0.05.
Figure 4
Figure 4. Transforming growth factor beta 1 suppresses FAO in renal TEC
(a), Oxygen consumption rate (OCR) of human tubule epithelial cell line (HKC8); each data-point represents the mean (and S.E.M) of ten independent samples. When indicated palmitate (180 µM), etomoxir (40 µM), and oligomycin (1 µM) was added. (b), OCR and extracellular acidification rate (ECAR) of HKC8 were measured in a Seahorse XF24 analyzer. Each data-point represents the mean of ten independent samples. 10 mM glucose, 2–4 dinitrophenol (2–4 DNP, 100 µM), 2-deoxyglucose (2-DG, 100 mM), and rotenone (1 µM) were injected sequentially at the indicated time points. (c), ATP levels of control (CTL) HKC8 cells, and cells treated with 40 µM etomoxir (CPT1 inhibitor). (d), Bright field, oil red o staining and caspase 3 immunofluorescence images of HKC8 cells, untreated (left) or etomoxir treated (right). (e), Relative transcript amounts of genes related to apoptosis (APAF1; apoptosis inducing factor1, BAX; BCL2 associated X protein, BCL2; B-cell CLL/ lymphoma 2, BCL2L1; BCL2 like 1) of HKC8 cells upon etomoxir treatment (n=6, 3 of control and 3 of etomoxir treated cells). (f), QRT-PCR based relative mRNA levels of markers of cellular dedifferentiation (VIM; vimentin, FN; fibronectin, COL1; collagen1, COL3, collagen3, and ACTA2; alpha smooth muscle actin) of HKC8 cells treated with etomoxir. (g), OCR measurement of HKC8 exposed to 50ng ml−1 TGFβ1 for 48 hrs or controls. Representative traces are shown right, and summary data left analyzed for 16 wells from independent experiments. When indicated palmitate (180 µM), glucose (25mM) and oligomycin (1 µM) were added. (h), Relative mRNA amount of transcripts of FAO enzymes (CPT1a; carnitine palmitoyl-transferase 1a, CPT2; carnitine palmitoyl-transferase 2, ACOX1; acyl-CoA oxidase 1, ACOX2; acyl-CoA oxidase 2, PPARa; Peroxisome proliferator-activated receptor alpha and PPARGC1a; peroxisome proliferator-activated receptor gamma, coactivator 1 alpha) of control and TGFB1 treated cells. All experiments were replicated twice with triplicate repeated measures for each condition. (i), Lipid accumulation was determined by oil red O staining of control and TGFB1 treated cells. Scale bar, 20 µm (j), ATP levels of control (CTL) HKC8 cells, and cells treated with TGFB1. All data are presented as means as ± s.e.m (n=8, 4 of control and 4 of TGFB1) and * p<0.05.
Figure 4
Figure 4. Transforming growth factor beta 1 suppresses FAO in renal TEC
(a), Oxygen consumption rate (OCR) of human tubule epithelial cell line (HKC8); each data-point represents the mean (and S.E.M) of ten independent samples. When indicated palmitate (180 µM), etomoxir (40 µM), and oligomycin (1 µM) was added. (b), OCR and extracellular acidification rate (ECAR) of HKC8 were measured in a Seahorse XF24 analyzer. Each data-point represents the mean of ten independent samples. 10 mM glucose, 2–4 dinitrophenol (2–4 DNP, 100 µM), 2-deoxyglucose (2-DG, 100 mM), and rotenone (1 µM) were injected sequentially at the indicated time points. (c), ATP levels of control (CTL) HKC8 cells, and cells treated with 40 µM etomoxir (CPT1 inhibitor). (d), Bright field, oil red o staining and caspase 3 immunofluorescence images of HKC8 cells, untreated (left) or etomoxir treated (right). (e), Relative transcript amounts of genes related to apoptosis (APAF1; apoptosis inducing factor1, BAX; BCL2 associated X protein, BCL2; B-cell CLL/ lymphoma 2, BCL2L1; BCL2 like 1) of HKC8 cells upon etomoxir treatment (n=6, 3 of control and 3 of etomoxir treated cells). (f), QRT-PCR based relative mRNA levels of markers of cellular dedifferentiation (VIM; vimentin, FN; fibronectin, COL1; collagen1, COL3, collagen3, and ACTA2; alpha smooth muscle actin) of HKC8 cells treated with etomoxir. (g), OCR measurement of HKC8 exposed to 50ng ml−1 TGFβ1 for 48 hrs or controls. Representative traces are shown right, and summary data left analyzed for 16 wells from independent experiments. When indicated palmitate (180 µM), glucose (25mM) and oligomycin (1 µM) were added. (h), Relative mRNA amount of transcripts of FAO enzymes (CPT1a; carnitine palmitoyl-transferase 1a, CPT2; carnitine palmitoyl-transferase 2, ACOX1; acyl-CoA oxidase 1, ACOX2; acyl-CoA oxidase 2, PPARa; Peroxisome proliferator-activated receptor alpha and PPARGC1a; peroxisome proliferator-activated receptor gamma, coactivator 1 alpha) of control and TGFB1 treated cells. All experiments were replicated twice with triplicate repeated measures for each condition. (i), Lipid accumulation was determined by oil red O staining of control and TGFB1 treated cells. Scale bar, 20 µm (j), ATP levels of control (CTL) HKC8 cells, and cells treated with TGFB1. All data are presented as means as ± s.e.m (n=8, 4 of control and 4 of TGFB1) and * p<0.05.
Figure 4
Figure 4. Transforming growth factor beta 1 suppresses FAO in renal TEC
(a), Oxygen consumption rate (OCR) of human tubule epithelial cell line (HKC8); each data-point represents the mean (and S.E.M) of ten independent samples. When indicated palmitate (180 µM), etomoxir (40 µM), and oligomycin (1 µM) was added. (b), OCR and extracellular acidification rate (ECAR) of HKC8 were measured in a Seahorse XF24 analyzer. Each data-point represents the mean of ten independent samples. 10 mM glucose, 2–4 dinitrophenol (2–4 DNP, 100 µM), 2-deoxyglucose (2-DG, 100 mM), and rotenone (1 µM) were injected sequentially at the indicated time points. (c), ATP levels of control (CTL) HKC8 cells, and cells treated with 40 µM etomoxir (CPT1 inhibitor). (d), Bright field, oil red o staining and caspase 3 immunofluorescence images of HKC8 cells, untreated (left) or etomoxir treated (right). (e), Relative transcript amounts of genes related to apoptosis (APAF1; apoptosis inducing factor1, BAX; BCL2 associated X protein, BCL2; B-cell CLL/ lymphoma 2, BCL2L1; BCL2 like 1) of HKC8 cells upon etomoxir treatment (n=6, 3 of control and 3 of etomoxir treated cells). (f), QRT-PCR based relative mRNA levels of markers of cellular dedifferentiation (VIM; vimentin, FN; fibronectin, COL1; collagen1, COL3, collagen3, and ACTA2; alpha smooth muscle actin) of HKC8 cells treated with etomoxir. (g), OCR measurement of HKC8 exposed to 50ng ml−1 TGFβ1 for 48 hrs or controls. Representative traces are shown right, and summary data left analyzed for 16 wells from independent experiments. When indicated palmitate (180 µM), glucose (25mM) and oligomycin (1 µM) were added. (h), Relative mRNA amount of transcripts of FAO enzymes (CPT1a; carnitine palmitoyl-transferase 1a, CPT2; carnitine palmitoyl-transferase 2, ACOX1; acyl-CoA oxidase 1, ACOX2; acyl-CoA oxidase 2, PPARa; Peroxisome proliferator-activated receptor alpha and PPARGC1a; peroxisome proliferator-activated receptor gamma, coactivator 1 alpha) of control and TGFB1 treated cells. All experiments were replicated twice with triplicate repeated measures for each condition. (i), Lipid accumulation was determined by oil red O staining of control and TGFB1 treated cells. Scale bar, 20 µm (j), ATP levels of control (CTL) HKC8 cells, and cells treated with TGFB1. All data are presented as means as ± s.e.m (n=8, 4 of control and 4 of TGFB1) and * p<0.05.
Figure 5
Figure 5. Transforming growth factor beta (TGFB1) suppresses FAO in a SMAD3 and PPARGC1A dependent manner
(a) The human PPARGC1A locus followed by SMAD2/3 ChIP-Seq, human kidney H3K4me1 and H3K4me3 ChIPSeq, human kidney specific gene regulatory region annotation (red promoter, yellow enhancer) followed by GERP-based conservation scoring. (b), OCR were measured in primary TEC from wild type (WT) and Pax8rtTA/TREPPARGC1A (TG) mice treated or not with TGFB1. Representative traces are shown right and summary data on left analyzed for 12 wells for independent experiments. All data are presented as means as ± s.e.m and *p<0.05 compare to WT and ** p<0.05 compare to WT-TGF. When indicated palmitate (180 µM), glucose (25mM) and oligomycin (1 µM) were added. (c–d), Expression of key enzymes critically related to FAO and glucose utilization in primary renal tubule epithelial cell from Pax8rtTA/TREPPARGC1A transgenic mice (TG) or control (CTL) exposed with 50ng ml−1 TGFB1 for 48h (Cpt1a; carnitine palmitoyl-transferase 1a, Cpt2; carnitine palmitoyl-transferase 2, Acox1; acyl-CoA oxidase 1, Acox2; acyl-CoA oxidase 2, Ppara; Peroxisome proliferator-activated receptor alpha and Ppargc1a; peroxisome proliferator-activated receptor gamma, coactivator 1 alpha, Hk; hexokinase, G6pc; glucose-6-phosphatase, catalytic subunit, Pfk1; phosphofructokinase 1, Idh2; isocitrate dehydrogenase 2, Pkm2; pyruvate kinase, muscle2, Glut1; glucose transporter 1). (e–f), Protection effects of Ppargc1a transgenic cells from TGFB1 induced fibrosis development including profibrotic, (Vim; vimentin, Fn; fibronectin, Col1a1; collagen 1a1, Col3a1; collagen 3a1, Col4a1; collagen 4a1 and Acta2; alpha smooth muscle actin) and apoptosis related genes (Apaf1; apoptosis inducing factor1 and Bcl2; B-cell CLL/ lymphoma 2). All data are presented as means as ± s.e.m (n=12, 3 of each condition), * p < 0.05 compare to WT and ** p<0.05 compare to WT-TGF. (g), OCR in HKC8 exposed to 50ng ml−1 TGFB1 for 48hrs in the presence or absence of 1 µM fenofibrate. When indicated cells were incubated in palmitate (180 µM), glucose (25mM), and oligomycin (1 µM). (h–i), Relative mRNA expression of transcripts related to FAO, (CPT1a; carnitine palmitoyl-transferase 1a, CPT2; carnitine palmitoyl-transferase 2, ACOX1; acyl-CoA oxidase 1, ACOX2; acyl-CoA oxidase 2, and PPARa; Peroxisome proliferator-activated receptor alpha), fibrosis and apoptosis (VIM; vimentin, FN; fibronectin, COL1A1; collagen1a, COL3A1; collagen3a1, COL4A1; collagen4a1, ACTA2; alpha smooth muscle actin, APAF1; apoptosis inducing factor1, and BCL2; B-cell CLL/ lymphoma 2) in HKC8 cells treated with or without TGFB1 and fenofibrate. All data are presented as means as ± s.e.m (n=9, 3 of control, 3 of TGFB1 and 3 of TGFB1 with fenofibrate treated group), *p<0.05 compare to CTL and ** p<0.05 compare to TGFB1.
Figure 5
Figure 5. Transforming growth factor beta (TGFB1) suppresses FAO in a SMAD3 and PPARGC1A dependent manner
(a) The human PPARGC1A locus followed by SMAD2/3 ChIP-Seq, human kidney H3K4me1 and H3K4me3 ChIPSeq, human kidney specific gene regulatory region annotation (red promoter, yellow enhancer) followed by GERP-based conservation scoring. (b), OCR were measured in primary TEC from wild type (WT) and Pax8rtTA/TREPPARGC1A (TG) mice treated or not with TGFB1. Representative traces are shown right and summary data on left analyzed for 12 wells for independent experiments. All data are presented as means as ± s.e.m and *p<0.05 compare to WT and ** p<0.05 compare to WT-TGF. When indicated palmitate (180 µM), glucose (25mM) and oligomycin (1 µM) were added. (c–d), Expression of key enzymes critically related to FAO and glucose utilization in primary renal tubule epithelial cell from Pax8rtTA/TREPPARGC1A transgenic mice (TG) or control (CTL) exposed with 50ng ml−1 TGFB1 for 48h (Cpt1a; carnitine palmitoyl-transferase 1a, Cpt2; carnitine palmitoyl-transferase 2, Acox1; acyl-CoA oxidase 1, Acox2; acyl-CoA oxidase 2, Ppara; Peroxisome proliferator-activated receptor alpha and Ppargc1a; peroxisome proliferator-activated receptor gamma, coactivator 1 alpha, Hk; hexokinase, G6pc; glucose-6-phosphatase, catalytic subunit, Pfk1; phosphofructokinase 1, Idh2; isocitrate dehydrogenase 2, Pkm2; pyruvate kinase, muscle2, Glut1; glucose transporter 1). (e–f), Protection effects of Ppargc1a transgenic cells from TGFB1 induced fibrosis development including profibrotic, (Vim; vimentin, Fn; fibronectin, Col1a1; collagen 1a1, Col3a1; collagen 3a1, Col4a1; collagen 4a1 and Acta2; alpha smooth muscle actin) and apoptosis related genes (Apaf1; apoptosis inducing factor1 and Bcl2; B-cell CLL/ lymphoma 2). All data are presented as means as ± s.e.m (n=12, 3 of each condition), * p < 0.05 compare to WT and ** p<0.05 compare to WT-TGF. (g), OCR in HKC8 exposed to 50ng ml−1 TGFB1 for 48hrs in the presence or absence of 1 µM fenofibrate. When indicated cells were incubated in palmitate (180 µM), glucose (25mM), and oligomycin (1 µM). (h–i), Relative mRNA expression of transcripts related to FAO, (CPT1a; carnitine palmitoyl-transferase 1a, CPT2; carnitine palmitoyl-transferase 2, ACOX1; acyl-CoA oxidase 1, ACOX2; acyl-CoA oxidase 2, and PPARa; Peroxisome proliferator-activated receptor alpha), fibrosis and apoptosis (VIM; vimentin, FN; fibronectin, COL1A1; collagen1a, COL3A1; collagen3a1, COL4A1; collagen4a1, ACTA2; alpha smooth muscle actin, APAF1; apoptosis inducing factor1, and BCL2; B-cell CLL/ lymphoma 2) in HKC8 cells treated with or without TGFB1 and fenofibrate. All data are presented as means as ± s.e.m (n=9, 3 of control, 3 of TGFB1 and 3 of TGFB1 with fenofibrate treated group), *p<0.05 compare to CTL and ** p<0.05 compare to TGFB1.
Figure 5
Figure 5. Transforming growth factor beta (TGFB1) suppresses FAO in a SMAD3 and PPARGC1A dependent manner
(a) The human PPARGC1A locus followed by SMAD2/3 ChIP-Seq, human kidney H3K4me1 and H3K4me3 ChIPSeq, human kidney specific gene regulatory region annotation (red promoter, yellow enhancer) followed by GERP-based conservation scoring. (b), OCR were measured in primary TEC from wild type (WT) and Pax8rtTA/TREPPARGC1A (TG) mice treated or not with TGFB1. Representative traces are shown right and summary data on left analyzed for 12 wells for independent experiments. All data are presented as means as ± s.e.m and *p<0.05 compare to WT and ** p<0.05 compare to WT-TGF. When indicated palmitate (180 µM), glucose (25mM) and oligomycin (1 µM) were added. (c–d), Expression of key enzymes critically related to FAO and glucose utilization in primary renal tubule epithelial cell from Pax8rtTA/TREPPARGC1A transgenic mice (TG) or control (CTL) exposed with 50ng ml−1 TGFB1 for 48h (Cpt1a; carnitine palmitoyl-transferase 1a, Cpt2; carnitine palmitoyl-transferase 2, Acox1; acyl-CoA oxidase 1, Acox2; acyl-CoA oxidase 2, Ppara; Peroxisome proliferator-activated receptor alpha and Ppargc1a; peroxisome proliferator-activated receptor gamma, coactivator 1 alpha, Hk; hexokinase, G6pc; glucose-6-phosphatase, catalytic subunit, Pfk1; phosphofructokinase 1, Idh2; isocitrate dehydrogenase 2, Pkm2; pyruvate kinase, muscle2, Glut1; glucose transporter 1). (e–f), Protection effects of Ppargc1a transgenic cells from TGFB1 induced fibrosis development including profibrotic, (Vim; vimentin, Fn; fibronectin, Col1a1; collagen 1a1, Col3a1; collagen 3a1, Col4a1; collagen 4a1 and Acta2; alpha smooth muscle actin) and apoptosis related genes (Apaf1; apoptosis inducing factor1 and Bcl2; B-cell CLL/ lymphoma 2). All data are presented as means as ± s.e.m (n=12, 3 of each condition), * p < 0.05 compare to WT and ** p<0.05 compare to WT-TGF. (g), OCR in HKC8 exposed to 50ng ml−1 TGFB1 for 48hrs in the presence or absence of 1 µM fenofibrate. When indicated cells were incubated in palmitate (180 µM), glucose (25mM), and oligomycin (1 µM). (h–i), Relative mRNA expression of transcripts related to FAO, (CPT1a; carnitine palmitoyl-transferase 1a, CPT2; carnitine palmitoyl-transferase 2, ACOX1; acyl-CoA oxidase 1, ACOX2; acyl-CoA oxidase 2, and PPARa; Peroxisome proliferator-activated receptor alpha), fibrosis and apoptosis (VIM; vimentin, FN; fibronectin, COL1A1; collagen1a, COL3A1; collagen3a1, COL4A1; collagen4a1, ACTA2; alpha smooth muscle actin, APAF1; apoptosis inducing factor1, and BCL2; B-cell CLL/ lymphoma 2) in HKC8 cells treated with or without TGFB1 and fenofibrate. All data are presented as means as ± s.e.m (n=9, 3 of control, 3 of TGFB1 and 3 of TGFB1 with fenofibrate treated group), *p<0.05 compare to CTL and ** p<0.05 compare to TGFB1.
Figure 5
Figure 5. Transforming growth factor beta (TGFB1) suppresses FAO in a SMAD3 and PPARGC1A dependent manner
(a) The human PPARGC1A locus followed by SMAD2/3 ChIP-Seq, human kidney H3K4me1 and H3K4me3 ChIPSeq, human kidney specific gene regulatory region annotation (red promoter, yellow enhancer) followed by GERP-based conservation scoring. (b), OCR were measured in primary TEC from wild type (WT) and Pax8rtTA/TREPPARGC1A (TG) mice treated or not with TGFB1. Representative traces are shown right and summary data on left analyzed for 12 wells for independent experiments. All data are presented as means as ± s.e.m and *p<0.05 compare to WT and ** p<0.05 compare to WT-TGF. When indicated palmitate (180 µM), glucose (25mM) and oligomycin (1 µM) were added. (c–d), Expression of key enzymes critically related to FAO and glucose utilization in primary renal tubule epithelial cell from Pax8rtTA/TREPPARGC1A transgenic mice (TG) or control (CTL) exposed with 50ng ml−1 TGFB1 for 48h (Cpt1a; carnitine palmitoyl-transferase 1a, Cpt2; carnitine palmitoyl-transferase 2, Acox1; acyl-CoA oxidase 1, Acox2; acyl-CoA oxidase 2, Ppara; Peroxisome proliferator-activated receptor alpha and Ppargc1a; peroxisome proliferator-activated receptor gamma, coactivator 1 alpha, Hk; hexokinase, G6pc; glucose-6-phosphatase, catalytic subunit, Pfk1; phosphofructokinase 1, Idh2; isocitrate dehydrogenase 2, Pkm2; pyruvate kinase, muscle2, Glut1; glucose transporter 1). (e–f), Protection effects of Ppargc1a transgenic cells from TGFB1 induced fibrosis development including profibrotic, (Vim; vimentin, Fn; fibronectin, Col1a1; collagen 1a1, Col3a1; collagen 3a1, Col4a1; collagen 4a1 and Acta2; alpha smooth muscle actin) and apoptosis related genes (Apaf1; apoptosis inducing factor1 and Bcl2; B-cell CLL/ lymphoma 2). All data are presented as means as ± s.e.m (n=12, 3 of each condition), * p < 0.05 compare to WT and ** p<0.05 compare to WT-TGF. (g), OCR in HKC8 exposed to 50ng ml−1 TGFB1 for 48hrs in the presence or absence of 1 µM fenofibrate. When indicated cells were incubated in palmitate (180 µM), glucose (25mM), and oligomycin (1 µM). (h–i), Relative mRNA expression of transcripts related to FAO, (CPT1a; carnitine palmitoyl-transferase 1a, CPT2; carnitine palmitoyl-transferase 2, ACOX1; acyl-CoA oxidase 1, ACOX2; acyl-CoA oxidase 2, and PPARa; Peroxisome proliferator-activated receptor alpha), fibrosis and apoptosis (VIM; vimentin, FN; fibronectin, COL1A1; collagen1a, COL3A1; collagen3a1, COL4A1; collagen4a1, ACTA2; alpha smooth muscle actin, APAF1; apoptosis inducing factor1, and BCL2; B-cell CLL/ lymphoma 2) in HKC8 cells treated with or without TGFB1 and fenofibrate. All data are presented as means as ± s.e.m (n=9, 3 of control, 3 of TGFB1 and 3 of TGFB1 with fenofibrate treated group), *p<0.05 compare to CTL and ** p<0.05 compare to TGFB1.
Figure 6
Figure 6. Genetic or pharmacological restoration of FAO protects animals from fibrosis development
(a), Representative PAS, cleaved caspase 3 and Sirius Red stained kidney sections of control and FAN fibrosis model (day 7). We used wild type and single transgenic animals as controls (CTL, or FAN) and Pax8rtTA/TRE-PPARGC1A transgenic mice with folic acid (Ppargc1a FA). Scale bar, 20 µm. (b), Markedly lower expression of fibrosis related transcripts in PPARGC1A overexpressed mice followed by folic acid administration (Vim; vimentin, Fn; fibronectin, Col1a1; collagen 1a1, Col3a1; collagen 3a1, Col4a1; collagen 4a1, Acta2; alpha smooth muscle actin, and Kim-1; kidney injury molecule-1). (c–e), Relative mRNA levels of FAO enzymes (Cpt1a; carnitine palmitoyl-transferase 1a, Cpt2; carnitine palmitoyl-transferase 2, Acox1; acyl-CoA oxidase 1, Acox2; acyl-CoA oxidase 2 and Ppara; Peroxisome proliferator-activated receptor alpha), apoptosis associated markers (Apaf1; apoptosis inducing factor1, Bcl2; B-cell CLL/ lymphoma 2, and glucose utilization (Hk; hexokinase, G6pc; glucose-6-phosphatase, catalytic subunit, Pfk1; phosphofructokinase 1, Idh2; isocitrate dehydrogenase 2, Pkm2; pyruvate kinase, muscle2, Glut1; glucose transporter 1). All data are presented as means as ± s.e.m (n=18, 6 of control, 6 of FAN and 6 of PPARGC1a FA group), * p<0.05 compare to CTL and ** p<0.05 compare to FAN. (f), Relative mRNA levels of FAO enzymes in control (CTL) folic acid treated (FAN) and folic acid and fenofibrate treated (FAN+fenofibrate) mice. (g), Ppara agonist fenofibrate ameliorated fibrosis development in the FA induced fibrosis model. Representative PAS, cleaved caspase 3, oil red o and sirius red stained kidney sections. Scale bar, 20 µm. (h,i), Relative mRNA levels of fibrosis, apoptosis and glycolysis associated markers. All data are presented as means as ± s.e.m (n=14, 5 of control, 5 of FAN and 4 of FAN with fenofibrate treated group) and * p<0.05. (j), Serum creatinine levels in CTL, FAN and FAN+fenofibrate mice. N = 15, 5 of CTL, 5 of FAN and 5 of FAN with fenofibrate mice. * p<0.05 compare to CTL and **p<0.05 compare to FAN.
Figure 6
Figure 6. Genetic or pharmacological restoration of FAO protects animals from fibrosis development
(a), Representative PAS, cleaved caspase 3 and Sirius Red stained kidney sections of control and FAN fibrosis model (day 7). We used wild type and single transgenic animals as controls (CTL, or FAN) and Pax8rtTA/TRE-PPARGC1A transgenic mice with folic acid (Ppargc1a FA). Scale bar, 20 µm. (b), Markedly lower expression of fibrosis related transcripts in PPARGC1A overexpressed mice followed by folic acid administration (Vim; vimentin, Fn; fibronectin, Col1a1; collagen 1a1, Col3a1; collagen 3a1, Col4a1; collagen 4a1, Acta2; alpha smooth muscle actin, and Kim-1; kidney injury molecule-1). (c–e), Relative mRNA levels of FAO enzymes (Cpt1a; carnitine palmitoyl-transferase 1a, Cpt2; carnitine palmitoyl-transferase 2, Acox1; acyl-CoA oxidase 1, Acox2; acyl-CoA oxidase 2 and Ppara; Peroxisome proliferator-activated receptor alpha), apoptosis associated markers (Apaf1; apoptosis inducing factor1, Bcl2; B-cell CLL/ lymphoma 2, and glucose utilization (Hk; hexokinase, G6pc; glucose-6-phosphatase, catalytic subunit, Pfk1; phosphofructokinase 1, Idh2; isocitrate dehydrogenase 2, Pkm2; pyruvate kinase, muscle2, Glut1; glucose transporter 1). All data are presented as means as ± s.e.m (n=18, 6 of control, 6 of FAN and 6 of PPARGC1a FA group), * p<0.05 compare to CTL and ** p<0.05 compare to FAN. (f), Relative mRNA levels of FAO enzymes in control (CTL) folic acid treated (FAN) and folic acid and fenofibrate treated (FAN+fenofibrate) mice. (g), Ppara agonist fenofibrate ameliorated fibrosis development in the FA induced fibrosis model. Representative PAS, cleaved caspase 3, oil red o and sirius red stained kidney sections. Scale bar, 20 µm. (h,i), Relative mRNA levels of fibrosis, apoptosis and glycolysis associated markers. All data are presented as means as ± s.e.m (n=14, 5 of control, 5 of FAN and 4 of FAN with fenofibrate treated group) and * p<0.05. (j), Serum creatinine levels in CTL, FAN and FAN+fenofibrate mice. N = 15, 5 of CTL, 5 of FAN and 5 of FAN with fenofibrate mice. * p<0.05 compare to CTL and **p<0.05 compare to FAN.
Figure 6
Figure 6. Genetic or pharmacological restoration of FAO protects animals from fibrosis development
(a), Representative PAS, cleaved caspase 3 and Sirius Red stained kidney sections of control and FAN fibrosis model (day 7). We used wild type and single transgenic animals as controls (CTL, or FAN) and Pax8rtTA/TRE-PPARGC1A transgenic mice with folic acid (Ppargc1a FA). Scale bar, 20 µm. (b), Markedly lower expression of fibrosis related transcripts in PPARGC1A overexpressed mice followed by folic acid administration (Vim; vimentin, Fn; fibronectin, Col1a1; collagen 1a1, Col3a1; collagen 3a1, Col4a1; collagen 4a1, Acta2; alpha smooth muscle actin, and Kim-1; kidney injury molecule-1). (c–e), Relative mRNA levels of FAO enzymes (Cpt1a; carnitine palmitoyl-transferase 1a, Cpt2; carnitine palmitoyl-transferase 2, Acox1; acyl-CoA oxidase 1, Acox2; acyl-CoA oxidase 2 and Ppara; Peroxisome proliferator-activated receptor alpha), apoptosis associated markers (Apaf1; apoptosis inducing factor1, Bcl2; B-cell CLL/ lymphoma 2, and glucose utilization (Hk; hexokinase, G6pc; glucose-6-phosphatase, catalytic subunit, Pfk1; phosphofructokinase 1, Idh2; isocitrate dehydrogenase 2, Pkm2; pyruvate kinase, muscle2, Glut1; glucose transporter 1). All data are presented as means as ± s.e.m (n=18, 6 of control, 6 of FAN and 6 of PPARGC1a FA group), * p<0.05 compare to CTL and ** p<0.05 compare to FAN. (f), Relative mRNA levels of FAO enzymes in control (CTL) folic acid treated (FAN) and folic acid and fenofibrate treated (FAN+fenofibrate) mice. (g), Ppara agonist fenofibrate ameliorated fibrosis development in the FA induced fibrosis model. Representative PAS, cleaved caspase 3, oil red o and sirius red stained kidney sections. Scale bar, 20 µm. (h,i), Relative mRNA levels of fibrosis, apoptosis and glycolysis associated markers. All data are presented as means as ± s.e.m (n=14, 5 of control, 5 of FAN and 4 of FAN with fenofibrate treated group) and * p<0.05. (j), Serum creatinine levels in CTL, FAN and FAN+fenofibrate mice. N = 15, 5 of CTL, 5 of FAN and 5 of FAN with fenofibrate mice. * p<0.05 compare to CTL and **p<0.05 compare to FAN.
Figure 6
Figure 6. Genetic or pharmacological restoration of FAO protects animals from fibrosis development
(a), Representative PAS, cleaved caspase 3 and Sirius Red stained kidney sections of control and FAN fibrosis model (day 7). We used wild type and single transgenic animals as controls (CTL, or FAN) and Pax8rtTA/TRE-PPARGC1A transgenic mice with folic acid (Ppargc1a FA). Scale bar, 20 µm. (b), Markedly lower expression of fibrosis related transcripts in PPARGC1A overexpressed mice followed by folic acid administration (Vim; vimentin, Fn; fibronectin, Col1a1; collagen 1a1, Col3a1; collagen 3a1, Col4a1; collagen 4a1, Acta2; alpha smooth muscle actin, and Kim-1; kidney injury molecule-1). (c–e), Relative mRNA levels of FAO enzymes (Cpt1a; carnitine palmitoyl-transferase 1a, Cpt2; carnitine palmitoyl-transferase 2, Acox1; acyl-CoA oxidase 1, Acox2; acyl-CoA oxidase 2 and Ppara; Peroxisome proliferator-activated receptor alpha), apoptosis associated markers (Apaf1; apoptosis inducing factor1, Bcl2; B-cell CLL/ lymphoma 2, and glucose utilization (Hk; hexokinase, G6pc; glucose-6-phosphatase, catalytic subunit, Pfk1; phosphofructokinase 1, Idh2; isocitrate dehydrogenase 2, Pkm2; pyruvate kinase, muscle2, Glut1; glucose transporter 1). All data are presented as means as ± s.e.m (n=18, 6 of control, 6 of FAN and 6 of PPARGC1a FA group), * p<0.05 compare to CTL and ** p<0.05 compare to FAN. (f), Relative mRNA levels of FAO enzymes in control (CTL) folic acid treated (FAN) and folic acid and fenofibrate treated (FAN+fenofibrate) mice. (g), Ppara agonist fenofibrate ameliorated fibrosis development in the FA induced fibrosis model. Representative PAS, cleaved caspase 3, oil red o and sirius red stained kidney sections. Scale bar, 20 µm. (h,i), Relative mRNA levels of fibrosis, apoptosis and glycolysis associated markers. All data are presented as means as ± s.e.m (n=14, 5 of control, 5 of FAN and 4 of FAN with fenofibrate treated group) and * p<0.05. (j), Serum creatinine levels in CTL, FAN and FAN+fenofibrate mice. N = 15, 5 of CTL, 5 of FAN and 5 of FAN with fenofibrate mice. * p<0.05 compare to CTL and **p<0.05 compare to FAN.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Brosius FC, 3rd, et al. Mouse models of diabetic nephropathy. J Am Soc Nephrol. 2009;20:2503–2512. - PMC - PubMed
    1. Nath KA, Salahudeen AK, Clark EC, Hostetter MK, Hostetter TH. Role of cellular metabolites in progressive renal injury. Kidney Int Suppl. 1992;38:S109–S113. - PubMed
    1. Humphreys BD, et al. Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis. Am J Pathol. 2010;176:85–97. - PMC - PubMed
    1. Zeisberg M, et al. BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med. 2003;9:964–968. - PubMed
    1. Bielesz B, et al. Epithelial Notch signaling regulates interstitial fibrosis development in the kidneys of mice and humans. J Clin Invest. 2010;120:4040–4054. - PMC - PubMed

MeSH terms

  NODES
admin 6
twitter 2