Journal: Disease Models & Mechanisms

Article Title: Transforming growth factor β3 deficiency promotes defective lipid metabolism and fibrosis in murine kidney

doi: 10.1242/dmm.048249

Figure Lengend Snippet: Renal parameters in male Tgfb3 +/− and Tgfb3 +/+ mice. (A–C) Relative mRNA expression and western blot quantification in whole kidneys of male mice at 4 months of age of TGFβ3 (A), TGFβ1 (B) and TGFβ2 (C) ( n =4–5; data are shown as mean±s.e.m.; unpaired Student's t -test with Welch's correction). (D) ACR at 1, 4 and 16 months of age ( n =5–6 for the 1-month-old group, n =11–14 for the 4-month-old group and n =7–12 for the 16-month-old group; data are shown as mean±s.e.m.; unpaired Student's t -test was performed with Welch's correction for each age). (E) mGFR at 4 and 16 months of age ( n =5–9; data are shown as median±interquartile range; Mann–Whitney U test was performed). * P <0.05 versus the control. ACR, albumin-creatinine ratio; mGFR, measured glomerular filtration rate.

Article Snippet: Cells (podocytes and MCTs) were transfected with 40 nM of an siRNA specific for Tgfb3 (sc-39805; Santa Cruz Biotechnology) or the corresponding scramble control (sc-37007; Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, Western Blot, MANN-WHITNEY, Control, Filtration

Journal: Disease Models & Mechanisms

Article Title: Transforming growth factor β3 deficiency promotes defective lipid metabolism and fibrosis in murine kidney

doi: 10.1242/dmm.048249

Figure Lengend Snippet: Tgfb3 +/− male mice show renal fibrosis at 4 months, but not 1 month, of age. (A,B) α-SMA immunohistochemistry ( n =5) (A) and Picrosirius Red staining ( n =4–6) (B) in 1-, 4- and 16-month-old mice. Scale bars: 50 µm. (C) Relative mRNA expression of N-cadherin, E-cadherin and β-catenin in whole kidney of 4-month-old mice ( n =5–6). (D) Representative western blot and densitometry of PAI-1 in whole kidney of 4-month-old mice ( n =5–6). (E) GBM thickness and foot process width measured on transmission electron microscopy images in whole kidney of 4-month-old mice ( n =4–5). Images were taken at a magnification of 400×. Black arrows mark positively stained areas of the tissue. White arrows mark thickened areas of the GBM, and white asterisks mark foot process effacement. Scale bars: 1 µm. Data are shown as mean±s.e.m.; unpaired Student's t -test with Welch's correction was performed. * P <0.05 versus the control. GBM, glomerular basement membrane; IHC, immunohistochemistry; PAI-1: plasminogen activator inhibitor-1; α-SMA, α-smooth muscle actin.

Article Snippet: Cells (podocytes and MCTs) were transfected with 40 nM of an siRNA specific for Tgfb3 (sc-39805; Santa Cruz Biotechnology) or the corresponding scramble control (sc-37007; Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen).

Techniques: Immunohistochemistry, Staining, Expressing, Western Blot, Transmission Assay, Electron Microscopy, Control, Membrane

Journal: Disease Models & Mechanisms

Article Title: Transforming growth factor β3 deficiency promotes defective lipid metabolism and fibrosis in murine kidney

doi: 10.1242/dmm.048249

Figure Lengend Snippet: Tgfb3 is involved in renal lipid metabolism. (A) Oil Red O staining in kidney (200× magnification; glomerulus, 400× magnification). Scale bars: 100 µm. (B) Bodipy staining intensity measured by flow cytometry ( n =5–6). (C) Relative mRNA expression of lipid metabolism genes in whole kidney ( n =5–7). (D) Representative western blot and densitometry of phospho-AKT ( n =4–5). (E) Oil Red O staining of siTGFβ3 podocytes. Images were taken at 400× magnification. Scale bars: 50 µm. (F) Relative mRNA expression of lipid metabolism genes in siTGFβ3 podocytes ( n =3). All in vivo experiments were performed in 4-month-old male mice. Black arrows mark red dots representing lipid droplets. Data are shown as mean±s.e.m.; unpaired Student's t -test with Welch's correction was performed. * P <0.05 versus the control. ACC, acetyl-CoA carboxylase; AKT, protein kinase B; A.U., arbitrary units; CPT1, carnitine palmitoyltransferase 1; Ins, insulin; p, phospho; PGC1, peroxisome proliferator-activated receptor gamma coactivator 1; PPAR, peroxisome proliferator-activated receptor; Sal, saline; SREBP1, sterol regulatory element-binding protein 1; t, total.

Article Snippet: Cells (podocytes and MCTs) were transfected with 40 nM of an siRNA specific for Tgfb3 (sc-39805; Santa Cruz Biotechnology) or the corresponding scramble control (sc-37007; Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen).

Techniques: Staining, Flow Cytometry, Expressing, Western Blot, In Vivo, Control, Saline, Binding Assay

Journal: Disease Models & Mechanisms

Article Title: Transforming growth factor β3 deficiency promotes defective lipid metabolism and fibrosis in murine kidney

doi: 10.1242/dmm.048249

Figure Lengend Snippet: Lipidomic and metabolomic analysis reveals a link between TGFβ3 and renal metabolism. (A) Heatmap representations of lipidomic analysis performed in negative ionization mode {left, LC-MS [−]} and positive ionization mode {right, LC-MS [+]}. (B) Metabolomic analysis showing meaningful biological patterns identified in metabolite concentration through enrichment analysis (left) and heatmap representation (right). All experiments were done in whole kidney of 4-month-old male mice ( n =6–7). Peak intensity for each individual was obtained applying univariate and multivariate tests ( P ≤0.05 or VIP>1). DG, diacylglycerol; GalBetaCer, galbetaceramide; GyceroPC, glycerophosphatidylcholine; LC-MS, liquid chromatography–mass spectrometry; LysoPA, lysophosphatidic acid; LysoPC, lysophosphatidylcholine; LysoPE, lysophosphatidylethanolamine; LysoPS, lysophosphatidylserine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; siTGFβ3, siRNA-silenced Tgfb3 ; SM, sphingomyelin; TG, triglyceride; VIP, variable influence of projection.

Article Snippet: Cells (podocytes and MCTs) were transfected with 40 nM of an siRNA specific for Tgfb3 (sc-39805; Santa Cruz Biotechnology) or the corresponding scramble control (sc-37007; Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen).

Techniques: Liquid Chromatography with Mass Spectroscopy, Concentration Assay, Liquid Chromatography, Mass Spectrometry

Journal: Disease Models & Mechanisms

Article Title: Transforming growth factor β3 deficiency promotes defective lipid metabolism and fibrosis in murine kidney

doi: 10.1242/dmm.048249

Figure Lengend Snippet: Tgfb3 deficiency leads to mitochondrial dysregulation. (A) Relative mRNA expression of mitochondrial and mitochondria-related genes ( n =12). (B) Mitochondrial structural alterations by transmission electron microscopy. Scale bars: 1 µm. (C) Relative mtDNA/nDNA ratio ( n =9). (D) Dihydroethidium staining intensity measured by flow cytometry ( n =4–5). (E) Nitrotyrosine immunohistochemistry ( n =6). Scale bars: 50 µm. All experiments were done in whole kidneys of 4-month-old male mice. Data are shown as mean±s.e.m.; unpaired Student's t -test with Welch's correction was performed. * P <0.05, ** P <0.01 and *** P <0.001 versus the control. A.U., arbitrary units; IHC, immunohistochemistry; Mfn1, mitochondrial fusion protein type 1; Mfn2, mitochondrial fusion protein type 2; mtAtp6, ATP synthase membrane subunit 6; mtCo1, cytochrome oxidase subunit 1; mtCo2, cytochrome oxidase subunit 2; mtCytB, cytochrome B; mtDNA, mitochondrial DNA; mtND1, NADH dehydrogenase subunit 1; mt12S, mitochondrial 12S ribosomal gene; nDNA, nuclear DNA; OLS, onion-like shaped; OPA1, mitochondrial dynamin like GTPase; TEM, transmission electron microscopy; TFAM, mitochondrial transcription factor A; 2HD, dihydroethidium.

Article Snippet: Cells (podocytes and MCTs) were transfected with 40 nM of an siRNA specific for Tgfb3 (sc-39805; Santa Cruz Biotechnology) or the corresponding scramble control (sc-37007; Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, Transmission Assay, Electron Microscopy, Staining, Flow Cytometry, Immunohistochemistry, Control, Membrane

Journal: Disease Models & Mechanisms

Article Title: Transforming growth factor β3 deficiency promotes defective lipid metabolism and fibrosis in murine kidney

doi: 10.1242/dmm.048249

Figure Lengend Snippet: Depletion of Tgfb3 leads to higher phosphorylation of TIR–TIIR-mediated pathways. (A–C) Representative western blots and densitometry of the ERK1/2 pathway ( n =8–9) (A), SMAD2/3 pathway ( n =8-9) (B) and JNK pathway ( n =4-7) (C) in whole kidney. (D,E) Relative mRNA expression of SMAD7 ( n =5) (D) and TIR and TIIR ( n =4–6) (E) in whole kidney. (F) Relative mRNA expression of TIR and TIIR in siTGFβ3 podocytes. (G,H) Representative western blots and densitometry of the ERK1/2 pathway (G) and SMAD2/3 pathway (H) in siTGFβ3 podocytes. All in vivo experiments were done in whole kidney of 4-month-old male mice. Data are shown as mean±s.e.m.; unpaired Student's t -test with Welch's correction was performed. * P <0.05 versus the control. C−, negative control; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; p, phospho; siTGFβ3, siRNA-silenced Tgfb3 ; SMAD, mothers against decapentaplegic homolog; t, total; TIR, TGFβ type I receptor; TIIR, TGFβ type II receptor; WT, wild type.

Article Snippet: Cells (podocytes and MCTs) were transfected with 40 nM of an siRNA specific for Tgfb3 (sc-39805; Santa Cruz Biotechnology) or the corresponding scramble control (sc-37007; Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen).

Techniques: Phospho-proteomics, Western Blot, Expressing, In Vivo, Control, Negative Control

Journal: Disease Models & Mechanisms

Article Title: Transforming growth factor β3 deficiency promotes defective lipid metabolism and fibrosis in murine kidney

doi: 10.1242/dmm.048249

Figure Lengend Snippet: TGFβ3 plays a key role in the physiopathology of the kidney. Reduced TGFβ3 levels lead to a higher TGFβ3/TGFβ3 ratio and lower TIR levels. Because TGFβ1 and TGFβ3 share their receptors, lower levels of TGFβ3 mean higher bioavailability of the TIR and TIIR receptors to TGFβ1. Without TGFβ3 present to counteract the actions of TGFβ1, TGFβ1 overactivates their downstream pathways – SMAD2/3, ERK1/2 and JNK – leading to renal damage. The decrease in TIR levels leads to a preference in the activation of the non-canonical pathways over the canonical SMAD2/3 pathway. As a result, Tgfb3 +/− male mice show albuminuria, loss of renal function, lipid accumulation, insulin resistance, fibrosis, oxidative stress and mitochondrial alterations. Note that the podocyte image is reproduced from Fig. 3 . ECM, extracellular matrix; EMT, epithelial–mesenchymal transition; MCT, mouse proximal tubular renal epithelial cells; MMP, metalloproteinase; TIR, TGFβ type I receptor; TIIR, TGFβ type II receptor.

Article Snippet: Cells (podocytes and MCTs) were transfected with 40 nM of an siRNA specific for Tgfb3 (sc-39805; Santa Cruz Biotechnology) or the corresponding scramble control (sc-37007; Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen).

Techniques: Activation Assay