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Susceptibility of podocytes to palmitic acid is regulated by stearoyl-CoA desaturases 1 and 2 - PubMed

Susceptibility of podocytes to palmitic acid is regulated by stearoyl-CoA desaturases 1 and 2

Jonas Sieber et al. Am J Pathol. 2013 Sep.

Abstract

Type 2 diabetes mellitus is characterized by dyslipidemia with elevated free fatty acids (FFAs). Loss of podocytes is a hallmark of diabetic nephropathy, and podocytes are highly susceptible to saturated FFAs but not to protective, monounsaturated FFAs. We report that patients with diabetic nephropathy develop alterations in glomerular gene expression of enzymes involved in fatty acid metabolism, including induction of stearoyl-CoA desaturase (SCD)-1, which converts saturated to monounsaturated FFAs. By IHC of human renal biopsy specimens, glomerular SCD-1 induction was observed in podocytes of patients with diabetic nephropathy. Functionally, the liver X receptor agonists TO901317 and GW3965, two known inducers of SCD, increased Scd-1 and Scd-2 expression in cultured podocytes and reduced palmitic acid-induced cell death. Similarly, overexpression of Scd-1 attenuated palmitic acid-induced cell death. The protective effect of TO901317 was associated with a reduction of endoplasmic reticulum stress. It was lost after gene silencing of Scd-1/-2, thereby confirming that the protective effect of TO901317 is mediated by Scd-1/-2. TO901317 also shifted palmitic acid-derived FFAs into biologically inactive triglycerides. In summary, SCD-1 up-regulation in diabetic nephropathy may be part of a protective mechanism against saturated FFA-derived toxic metabolites that drive endoplasmic reticulum stress and podocyte death.

Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1
Figure 1

Expression of fatty acid metabolism associated enzymes in human DN and cultured podocytes exposed to palmitic acid. A: Microarray data were obtained from isolated glomeruli of type 2 diabetic patients with DN and controls (pretransplantion allograft biopsy specimens). Gene expressions of ACC-2, CPT-1a, CPT-1b, CPT-1c, DGAT1, DGAT2, SCD-1, and SCD-5 were significantly regulated in DN compared with controls with SCD-1 that had the highest up-regulation. Up-regulated enzymes are indicated in red and down-regulated enzymes in blue. The related metabolic pathways of enzymes analyzed are depicted in the bottom panels. B: Immunoperoxidase staining against SCD-1 in a tumor nephrectomy specimen from a middle-aged adult without any known history of medical renal disease. No significant expression of SCD-1 is seen in glomeruli. Mild, granular cytoplasmic staining of renal tubules is present. Representative sample of four nephrectomies analyzed is shown. C: Murine podocytes treated with 200 μmol/L palmitic acid for 14 hours induced mRNA levels of Scd-1, Scd-2, and Cpt-1a, whereas levels of Cpt-1b, Cpt-1c, Dgat1, Dgat2, AccI, and AccII remained unchanged. Bar graph represents fold induction ± SD of enzymes normalized to Gapdh. BSA control treatment was set to 1 (n = 9; ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001). D: Immunoperoxidase staining against SCD-1 in a renal biopsy specimen from a 57-year-old man with type 2 diabetes mellitus revealing early DN and glomerular hypertrophy. Arrows point to podocytes with intense, granular cytoplasmic staining for SCD-1. There is also some cytoplasmic staining of parietal cells. Representative example of four DN samples analyzed. Scale bars: 20 μm (B and D).

Figure 2
Figure 2

TO and GW strongly induce Scd-1 and Scd-2 and ameliorate palmitic acid–induced cell death. A: Quantitative real-time PCR analysis of Scd-1, Scd-2, Lxrα, and Lxrβ after 14 hours of 1 μmol/L TO or GW treatment. Bar graph represents fold inductions ± SD of each gene normalized to Gapdh. Vehicle (dimethyl sulfoxide) treatment was set to 1 (n = 3; ∗∗P < 0.01). B: Western blot analysis of SCD-1 after 14 hours of treatment with TO. GAPDH was used as a control. Bar graph represents relative expression ± SD of SCD-1. Vehicle (dimethyl sulfoxide) treatment was set to 100% (n = 4; ∗∗P < 0.01). C: 14 hours pretreatment with TO or GW attenuated palmitic acid–induced podocyte death. Bar graph represents means ± SD percentages of apoptotic and necrotic cells (n = 3; ∗P < 0.05, ∗∗P < 0.01). D: TO attenuated the palmitic acid–mediated induction of CHOP. Podocytes were pretreated with 1 μmol/L TO or vehicle (dimethyl sulfoxide) for 14 hours and subsequently incubated with 200 μmol/L palmitic acid for 24 hours. CHOP levels were analyzed by Western blot. β-Actin served as a loading control. Bar graph represents the relative means ± SD expressions (n = 3; ∗P < 0.05). Vehicle treated controls were set to 100%.

Figure 3
Figure 3

Scd-1 single knockdown only partially prevents the protective effect of TO on palmitic acid–induced podocyte death, whereas combined silencing of Scd-1 and Scd-2 completely abrogates the TO effect. A: Knockdown of Scd-1 and/or Scd-2 by shRNA suppresses the TO-mediated up-regulation of Scd-1 and Scd-2. Bar graph indicates means ± SD quantitative real-time PCR mRNA levels of Scd-1 and Scd-2 normalized to Gapdh [control (scrambled) levels were set to 1; ∗P < 0.05, ∗∗∗P < 0.001]. B–I: Scd-1 single- and Scd-1/Scd-2 double-silenced podocytes were pretreated with 1 μmol/L TO for 14 hours before addition of 200 μmol/L palmitic acid for 48 hours. B and F: Means ± SD percentages of apoptotic and necrotic cells (n = 3; ∗P < 0.05, ∗∗P < 0.01). C–E and G–I: Relative means ± SD percent changes of apoptotic (C and G), necrotic (D and H), and apoptotic and necrotic cells (E and I). Vehicle-treated (dimethyl sulfoxide) controls are set to 100% (n = 3; ∗P < 0.05, ∗∗P < 0.01).

Figure 4
Figure 4

Overexpressing SCD-1 partially protects from palmitic acid–induced apoptosis. A: Western blot analysis of SCD-1 levels in green fluorescent protein or SCD-1 overexpressing podocytes. β-Actin served as a loading control. B: SCD-1 reduced palmitic acid–induced apoptosis and necrosis in podocytes. Bar graph represents means ± SD percentages of apoptotic and necrotic cells after exposure to 200 μmol/L palmitic acid for 48 hours (n = 3; ∗P < 0.05, ∗∗P < 0.01; representative experiment of five independent experiments).

Figure 5
Figure 5

Oleic acid and TO) increase palmitic acid incorporation into the triglycerides (TG) fraction but only oleic acid reduces palmitic acid containing DAG levels in palmitic acid–treated podocytes. Podocytes were incubated for 5 hours in serum-free medium containing 0.5% FFA-free BSA and supplemented with 200 μmol/L palmitic acid (±1 μmol/L TO) or oleic and palmitic acid (100 μmol/L each) in the presence of 0.5μCi/mL of [3H]-palmitic acid. TG and DAG fractions were separated by thin layer chromatography and analyzed by a liquid scintillation counter. A and B: Bar graphs represent the means ± SD ratios of [3H]-palmitic acid incorporated into TG versus DAG (n = 9; ∗∗∗P < 0.001). C and D: Bar graphs represent the means ± SD incorporation of [3H]-palmitic acid into TG (C) and DAG (D) normalized to total lipids (n = 9; ∗∗P < 0.01). Vehicle treatment was set to 1.

Figure 6
Figure 6

Co-treatment with oleic acid but not TO increases palmitic acid β-oxidation. Podocytes were incubated for 5 hours in serum-free medium containing 0.5% FFA-free BSA and supplemented with 200 μmol/L palmitic acid (±1 μmol/L TO) or oleic and palmitic acid (100 μmol/L each) in the presence of 0.5 μCi/mL of [3H]-palmitic acid. β-Oxidation was determined by counting [3H2O] as a product of β-oxidation in the aqueous phase of the incubation medium. Bar graph represents means ± SD β-oxidation (n = 9; ∗∗∗P < 0.001). Vehicle treatment was set to 100%.

Figure 7
Figure 7

A working model for the prosurvival effects TO/GW-induced Scd-1/2 and oleic acid on palmitic acid–induced podocyte death. Palmitic acid increases the generation of toxic metabolites, which leads to ER stress and podocyte death. TO and GW increase Scd-1/2 expression, which in turn elevates the TG safe pool and reduces injurious metabolites and subsequent podocyte death. Similarly, oleic acid ameliorates palmitic acid–induced podocyte death by shifting palmitic acid and toxic metabolites to the TG safe pool. In addition, oleic acid but not TO/GW also increases β-oxidation.

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