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The American Journal of Pathology logoLink to The American Journal of Pathology
. 2013 Sep;183(3):735–744. doi: 10.1016/j.ajpath.2013.05.023

Susceptibility of Podocytes to Palmitic Acid Is Regulated by Stearoyl-CoA Desaturases 1 and 2

Jonas Sieber ∗,†, Astrid Weins †,‡, Kapil Kampe ∗, Stefan Gruber §, Maja T Lindenmeyer ¶,‖, Clemens D Cohen ¶,‖, Jana M Orellana ∗, Peter Mundel †, Andreas W Jehle ∗,∗∗,††,∗
PMCID: PMC3763774  PMID: 23867797

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.

Diabetic nephropathy (DN) is the major cause of end-stage renal disease, and most affected patients have type 2 diabetes.1,2 Podocyte injury and loss are critical events in DN3 and precede albuminuria.4–6 Type 2 diabetes mellitus is characterized by hyperglycemia and dyslipidemia with increased plasma levels of free fatty acids (FFAs).7 Intraglomerular lipid deposits in the kidneys of diabetic humans were described in 1936 by Kimmelstiel and Wilson.8 However, the potential role of FFAs and fatty acid metabolism in the pathogenesis of DN is only emerging.

Recently, we reported that podocytes are highly susceptible to the saturated FFA palmitic acid, which induces podocyte death.9 Mechanistically, palmitic acid–induced podocyte death is linked to endoplasmic reticulum (ER) stress that involves the proapoptotic transcription factor C/EBP homologous protein (CHOP).9 In contrast, monounsaturated FFAs (MUFAs), such as palmitoleic or oleic acid, attenuate palmitic acid–induced lipotoxicity in podocytes.9 The cytoprotective actions of MUFAs are incompletely understood. Several studies indicated that MUFAs can induce fatty acid oxidation and increase lipid storage in the form of triglycerides (TGs), thereby reducing cytotoxic metabolites, such as diacylglycerides (DAGs).10 Essential enzymes in the synthesis of TGs are acyl-CoA:diacylglycerolacyltransferases (DGATs), which transfer acyl-CoAs to DAGs,11,12 and stearoyl-CoA desaturases (SCDs), which desaturate saturated FFAs and thereby provide DGATs with their preferential substrates, MUFAs.13,14 Four SCD isoforms have been identified in mice.15–17 Scd-1 and Scd-2 are ubiquitously expressed, whereas Scd-3 is restricted to preputial glands, harderian glands, and the skin. Scd-4 is thought to be solely expressed in the heart.17,18 The predominant isoform in the kidney is Scd-1.18 Scd-1 is positively regulated by liver X receptor (LXR)19 and by LXR-mediated stearoyl regulatory-element binding protein 1c.20 In humans, two SCD isoforms, SCD-1 and SCD-5, are known, of which only SCD-1 has high homology with the murine isoforms.21

We describe changes in the gene expression of key enzymes of fatty acid metabolism in glomeruli of patients with DN. Up-regulation of SCD-1 mRNA expression was the predominant change, which correlated with up-regulation of Scd-1 protein abundance in podocytes in kidney biopsy specimens from patients with type 2 DN. Functionally, Scd-1 can protect against palmitic acid–induced podocyte death, suggesting that SCD-1 up-regulation in DN is a protective response of podocytes to the diabetic milieu.

Experimental Design and Methods

Patients and Microarray Analysis

For microarray analysis, samples from the European Renal cDNA Bank-Kröner-Fresenius Biopsy Bank were used.22 Total RNA was isolated from microdissected glomeruli, reverse transcribed, and amplified as reported.23 Affymetrix Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA) was used. ChipInspector version 2.1 (Genomatix Software GmbH, Munich, Germany) was used for transcript annotation, total intensity normalization, significance analysis of microarrays, and transcript identification based on significantly changed probes.24 No fold change cut-off was applied, but minimum coverage of probes with significant alteration per transcripts was set at five.

Renal Histology

Paraffin-embedded normal kidney sections from tumor nephrectomy specimens and renal biopsy specimens were obtained from the Renal Pathology Service at Brigham and Women’s Hospital (Boston, MA; institutional review board protocol 2011-P-002692/3). Sections (4 μm) were deparaffinized and rehydrated. Endogenous peroxidase activity was quenched, and antigen retrieval was performed. The sections were incubated with anti–SCD-1 (sc-14719; 1:100, Santa Cruz, Dallas, TX) for 40 minutes at room temperature. Application of the secondary antibodies was followed by incubation with biotinylated rabbit anti-goat at 1:200 dilution (Vector Laboratory, Burlingame, CA). Sections incubated with ABC reagent were visualized with 3,3′-diaminobenzidine plus substrate-chromogen followed by counterstaining with gill hematoxylin. Images were taken in a masked manner by a renal pathologist (A.W.).

Compounds, Cell Culture, Fatty Acid Preparation, and Cell Death Analysis

TO901317 and GW3965 were obtained from Sigma (Buchs, Switzerland). Podocytes were cultured as described previously.9 FFAs were prepared as previously described.9 Annexin V and PI staining was performed as reported.9 Annexin V–positive/propidium iodide–negative podocytes were considered apoptotic, whereas annexin V–positive/propidium iodide–positive podocytes were considered (late apoptotic) necrotic cells.

Vectors and Lentivirus Production

For gene silencing, Scd-1 (5′-GCCTTTAATCAACCCAAGAAA-3′) and Scd-2 (5′-GAACATTAGCTCTCGGGAGAA-3′) shRNAs in a PLKO.1 puro vector were obtained from Sigma. A 21-nt scrambled sequence (5′-GACCGCGACTCGCCGTCTGCG-3′ or 5′-GAACATTAGCTCTCGGGAGAA-3′) served as a control. For overexpression studies, murine Scd-1 cDNA under a cytomegalovirus promoter in an EZ-Lv153 vector was from GeneCopoeia (Rockville, MD). Green fluorescent protein in a pLVX-puro vector, served as a control. Lentivirus production and podocyte transduction were performed as previously reported.9

Western Blot

Western blotting was performed as previously described.9 Antibodies against SCD-1 and cleaved caspase 3 (Nos. 2794 and 9661; Cell Signaling Technology, Danvers, MA), CHOP (sc-7351; Santa Cruz), GAPDH (EMD Millipore, Billerica, MA), and β-actin (Sigma) were applied at dilutions of 1:100, 1:1000, 1:5000, and 1:100,000, respectively.

Quantitative Real-Time PCR

Total RNA extraction, cDNA synthesis, and real-time PCR were performed as previously reported.9 Primer sequences: AccI forward 5′-GCCTCTTCCTGACAAACGAG-3′, reverse 5′-TGACTGCCGAAACATCTCTG-3′, AccII forward 5′-ACAGAGATTTCACCGTCGCGT-3′, reverse 5′-CGCAGCGATGCCATTGT-3′,25 Cpt-1a forward 5′-CGCACGGAAGGAAAATGG-3′, reverse 5′-TGTGCCCAATATTCCTGG26-3′, Cpt-1b forward 5′-CAAGTTCAGAGACGAACGCC-3′, reverse 5′-TCAAGAGCTGTTCTCCGAACTG-3′,26 Cpt-1c forward 5′-AGAAGTAGAGCTCAGCTCGCCA-3′, reverse 5′-CCAGAGATGCCTTTTCCAGGAG-3′,27 Dgat1 forward 5′-GTGCACAAGTGGTGCATCAG-3′, reverse 5′-CAGTGGGACCTGAGCCATC-3′,28 Dgat2 forward 5′-AGTGGCAATGCTATCATCATCGT-3′, reverse 5′-AAGGAATAAGTGGGAACCAGATCA-3′29), Gapdh forward 5′-CTGCACCACCAACTGCTTAGC-3′, reverse 5′-GGCATGGACTGTGGTCATGAG-3′, Lxrα forward 5′-CGACAGAGCTTCGTCCACAA-3′, reverse 5′-GCTCGTTCCCCAGCATTTT-3′,30 Lxrβ forward 5′-AAGCAGGTGCCAGGGTTCTT-3′, reverse 5′-TCAATGGTGGACGCCTTCA-3′,30 Scd-1 forward 5′-TCTTGTCCCTATAGCCCAATCCAG-3′, reverse 5′-AGCTCAGAGCGCGTGTTCAA-3′31 and Scd-2 forward 5′-AGTGTTGCTCGTGAGCCTGTG-3′, reverse 5′-CCTGCAGATCCATGTCCAGCTA-3′.31

Incorporation of Palmitic Acid into DAGs and TGs and β-Oxidation

Pretreatment with TO901317 was performed for 14 hours. The experiment was performed in serum-free medium supplemented with 0.5% FFA-free bovine serum albumin (BSA) containing 200 μmol/L palmitic acid or palmitic plus oleic acid (100 μmol/L each) in the presence of 0.5 μCi/mL of [3H]-palmitic acid (PerkinElmer, Schwerzenbach, Switzerland). 32 For DAG and TG analysis, cells were washed three times with PBS and scraped in PBS. Lipids were extracted in chloroform/methanol/5N HCl (2:1:0.05, v/v). The organic phase was dried under N2 and redissolved in chloroform. Lipids were separated by unidimensional thin layer chromatography on 20 × 20-cm silica plates (Sigma, Buchs, Switzerland) in n-hexane/diethyl ether/methanol (45:10:1, v/v). Lipid standards (glyceryl tripalmitate, glyceryl trioleate, and 1,2 dipalmitoyl-sn-glycerol from Sigma) were visualized by spraying with KMnO4 stain. The spots corresponding to markers were scraped, and after addition of 150 μL of methanol and 2 mL of scintillation buffer radioactivity was measured. DAGs and TGs were normalized to total lipid. For β-oxidation, 1 mL of culture medium was transferred to 5 mL of chloroform/methanol/5N HCl (2:1:0.05, v/v). Aqueous phase (500 mL; containing 3H2O) was added to 2 mL of scintillation buffer before measuring radioactivity.

Statistical Analysis

Data are expressed as means ± SD. Significance of differences was calculated with a two-sided, unpaired t-test. The description of the significance analysis of microarray data is given in Patients and Microarray Analysis.

Results

Differential Regulation of Genes Involved in Fatty Acid Metabolism in Glomeruli of Patients with Established DN

Microarray analysis of enzymes involved in fatty acid metabolism in glomeruli of patients with type 2 diabetes mellitus compared with pretransplantation living donors revealed significantly altered expression levels of enzymes involved in fatty acid oxidation and TG synthesis (Figure 1A; for patient characteristics see Supplemental Table S1). First, the up-regulation of all three isoforms of carnitine palmitoyltransferase (CPT)-1, the rate-limiting enzyme for fatty acid oxidation, and the down-regulation of acetyl-CoA carboxylase (ACC)-2, which catalyzes the formation of the CPT-1 inhibitor malonyl-CoA, was observed, suggesting disposition for increased fatty acid oxidation (Figure 1A). Second, the prominent induction of SCD-1, which provides DGATs with their preferential substrates, MUFAs, together with the positive regulation of DGAT1, which catalyzes the incorporation of exogenous FFAs into TG, implies a disposition toward increased TG synthesis (Figure 1A).

Figure 1.

Figure 1

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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).

To localize expression of SCD-1 in human glomeruli and to investigate whether glomerular up-regulation of SCD-1 mRNA in DN is also reflected on the protein level, renal biopsy specimens were stained for SCD-1. Normal glomeruli from tumor nephrectomy specimens revealed minimal glomerular staining for SCD-1 (Figure 1B). In contrast, in biopsy specimens from type 2 diabetic patients with DN a clear signal for SCD-1 could be observed in glomeruli, predominately in podocytes (Figure 1D).

Palmitic Acid Induces Scd-1, Scd-2, and Cpt-1a in Podocytes

To address the potential contributory role of FFAs to the altered gene expression profile in glomeruli and podocytes of patients with DN, we treated cultured podocytes with palmitic acid as previously reported.9 Exposure for 14 hours to 200 μmol/L palmitic acid complexed to BSA, compared with uncomplexed BSA, increased Scd-1 (1.7- ± 0.7-fold, P < 0.01) (Figure 1C) and Scd-2 (1.9- ± 0.6-fold, P < 0.001) (Figure 1C), the most abundant SCD isoforms in murine kidneys18 (Supplemental Figure S1A) and murine podocytes (Supplemental Figure S1B). Also, Cpt-1a was significantly increased (2.9- ± 0.9-fold, P < 0.0001) (Figure 1C). No differences were found for Cpt-1b, Cpt1c, AccI, AccII, and Dgat1, which could be due to the incubation with palmitic acid only, instead of the more complex diabetic milieu.

TO901317 (TO) and GW3965 (GW) Induce Scd-1 and Scd-2 and Ameliorate Palmitic Acid–Induced Podocyte Death

Previously, we reported that MUFAs protect podocytes from palmitic acid–induced death.9 We found in this study that human SCD-1, which shares high homology to murine Scd-1 and Scd-2,33 is up-regulated in podocytes of DN patients. To determine the functional relevance of SCD expression in podocytes, we tested the effect of SCD induction on palmitic acid–induced podocyte death. We first took advantage of TO901317 (TO) and GW3965 (GW), both LXR agonists, which are known Scd-1 inducers.19 Having confirmed the presence of LXRs in podocytes (Figure 2A), we examined the effect of TO and GW on Scd mRNA expression. TO and GW induced Scd-1 (31- ± 3-fold and 33- ± 11-fold, P < 0.01) (Figure 2A) and Scd-2 mRNA levels (19- ± 9-fold and 22- ± 8-fold, P < 0.01) (Figure 2A). In contrast, Scd-3 and Scd-4 mRNA levels were not increased by TO (data not shown). The changes in mRNA expression were also accompanied by changes in Scd-1 protein abundance (Figure 2B). Functionally, TO and GW prevented palmitic acid–induced podocyte death assessed by flow cytometry after staining for annexin V and propidium iodide to a comparable degree (Figure 2C). Specifically, apoptosis was inhibited by 40% to 60% and necrosis by 20% to 30%. To confirm the protective effect of TO and GW with a second, independent approach, we examined the activation of effector caspase 3 by Western immunoblotting. Consistent with the annexin V/propidium iodide data (Figure 2C), caspase 3 was less activated in the presence of TO and GW (Supplemental Figure S2). For the reason of the equivalent effects observed by TO and GW, all further experiments were performed with TO only. Because CHOP gene silencing attenuates palmitic acid–induced podocyte death and MUFAs can prevent CHOP up-regulation,9 we next tested whether TO can also attenuate CHOP induction. TO significantly reduced CHOP induction by almost 50% (n = 3; P < 0.05) (Figure 2D), which may explain, at least in part, the prosurvival effect of TO.

Figure 2.

Figure 2

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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%.

Combined Gene Silencing of Scd-1 and Scd-2 Abrogates the Protective Effect of TO on Palmitic Acid–Induced Podocyte Death

To investigate the role of desaturases in the protective action of TO, we first generated Scd-1 knockdown podocytes by lentiviral knockdown of Scd-1. A scrambled shRNA served as a control. By real-time PCR, we found suppression of Scd-1 expression (Figure 3A). Scd-1 silenced podocytes had comparable overall apoptosis and necrosis (Figure 3B). However, as indicated in Figure 3C, the effect of TO on palmitic acid–induced apoptosis was reduced in Scd-1–deficient cells (34% ± 2%) compared with controls (56% ± 5%, P < 0.01). Similar findings were observed for apoptosis and necrosis combined (Figure 3E), but no difference was detected for necrosis (Figure 3D). Because Scd-2 is the other dominant SCD isoform in podocytes, we also generated podocytes deficient in Scd-2. Knockdown of Scd-2 was comparable to Scd-1 (Figure 3A). Overall apoptosis and necrosis tended to be slightly increased in Scd-2–deficient podocytes (Supplemental Figure S3A). In contrast to Scd-1 knockdown podocytes (Figure 3, B–E), the protective effect of TO was preserved in Scd-2–deficient podocytes (Supplemental Figure S3, A–D). Next, we generated Scd-1/Scd-2 double-deficient podocytes that were highly susceptible to palmitic acid–induced cell death. Palmitic acid–induced apoptosis increased from 8.5% ± 1.0% in control cells to 21.6% ± 0.9% in Scd-1/Scd-2 double-deficient cells (Figure 3F). Moreover, the protective effect of TO was almost completely lost in the Scd-1/Scd-2 double-deficient cells (Figure 3F). The protective effect of TO on apoptosis was reduced from 40.3% ± 4.0% in control cells to 11.1% ± 7.7% (P < 0.01) in Scd-1/Scd-2 double-silenced cells (Figure 3G). Similarly, the protective effect of TO on apoptosis and necrosis combined was reduced from 30.6% ± 1.1% in controls to 11.6% ± 7.9% (P < 0.05) in Scd-1/Scd-2 double-silenced cells (Figure 3I), whereas the TO effect was not significantly different for necrosis (Figure 3H). Together, these data indicate that Scd-1 and Scd-2 induction is responsible for the protective effect of TO. Furthermore, Scd-1 and Scd-2 are presumably compensating for each other because single knockdown of Scd-1 (Figure 3, B–E) had only a limited effect on the protective effect of TO and single knockdown of Scd-2 did even not affect the protective effect of TO (Supplemental Figure S3, A–D).

Figure 3.

Figure 3

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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).

Overexpression of Scd-1 Reduces Palmitic Acid–Induced Podocyte Apoptosis

To further explore the beneficial role of desaturases on palmitic acid–induced apoptosis in podocytes, we overexpressed Scd-1. Overexpression of Scd-1 was confirmed by Western immunoblotting (Figure 4A). Scd-1 overexpression significantly reduced palmitic acid–induced apoptosis (7.0% ± 0.3% versus 10.3% ± 0.7%, P < 0.01) and necrosis (5.7% ± 0.4% versus 7.3% ± 0.5%, P < 0.05) (Figure 4B). The protective effect of Scd-1 overexpression is consistent with the Scd-1– and Scd-2–dependent protective effect of TO (Figure 3, F–I).

Figure 4.

Figure 4

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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).

MUFAs and TO Shift Palmitic Acid into TGs, and MUFAs Induce Fatty Acid β-Oxidation

To examine changes in lipid storage and fatty acid oxidation induced by MUFAs (oleic acid) and TO, we used tritium-labeled palmitic acid to trace the incorporation of palmitic acid–derived FFAs into DAGs and TGs and to estimate levels of β-oxidation of palmitic acid. Analysis of the TG to DAG ratio revealed a shift of palmitic acid toward TGs in the presence of oleic acid and TO. Coincubation with oleic acid strongly shifted the ratio toward the TG fraction (2.8- ± 0.6-fold, P < 0.001) (Figure 5A). TO also increased the proportion of fatty acids derived from tritium-labeled palmitic acid incorporated in TGs in relation to DAGs but to a lower extent (1.3- ± 0.2-fold, P < 0.001) (Figure 5B). Looking at the single fractions, the presence of oleic acid significantly increased the radioactive-labeled TG fraction (Figure 5C) but even more prominently reduced the radioactivity in the DAG fraction (0.5- ± 0.1-fold of control cells, P < 0.01) (Figure 5D), whereas TO had no significant effect on incorporation in DAGs (Figure 5D) but mainly effected the incorporation in TGs (Figure 5C). We did not measure [3H]-palmitic acid integrated into ceramides because fumonisin B1, a ceramide synthetase inhibitor, had no protection from palmitic acid–induced lipotoxicity in podocytes (Supplemental Figure S4). Finally, we investigated the effect of oleic acid and TO on palmitic acid β-oxidation. Podocytes were incubated with 200 μmol/L palmitic acid along with 0.5 μCi/mL of tritiated palmitic acid. The formation of [3H2O] is used as a read out of palmitic acid β-oxidation, and oxidation of palmitic acid is expressed as disintegrations per minute normalized to the total protein of cell lysates. Five hours of co-treatment with oleic acid elevated β-oxidation by 30% to 40% (P < 0.001) when compared with palmitic acid–treated podocytes (Figure 6). In contrast, the presence of TO revealed no detectable change in palmitic acid β-oxidation (Figure 6).

Figure 5.

Figure 5

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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

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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%.

Discussion

Type 2 diabetes is characterized by dyslipidemia and elevated FFAs,7 and podocytes are highly susceptible to saturated FFAs but not MUFAs.9 We found that in the glomeruli of type 2 diabetic patients with DN mRNA expression levels of several key enzymes involved in fatty acid metabolism are altered. The most prominent change is the up-regulation of SCD-1, which results mainly from increased expression in podocytes. Functionally, the LXR agonists TO and GW protect podocytes against palmitic acid–induced cell death in an Scd-1– and Scd-2–dependent fashion. Moreover, oleic acid or TO promotes the incorporation of palmitic acid into TGs, suggesting that the protective effect of TO results at least in part from compartmentalization of palmitic acid in safe lipid pools.10

The microarray data revealing increased expression of all three CTP-1 isoforms and decreased expression of ACC-2 suggest an increased capacity for FFA oxidation. This finding is particularly relevant because increased fatty acid oxidation may protect against DN. Consistently, two recent genomewide association studies in type 2 diabetic patients found a polymorphism in a noncoding region of ACC-2, with a strong association with proteinuria.34,35 Importantly, the DN-risk single-nucleotide polymorphism of ACC-2 results in a higher ACC-2 expression,34 which favors impairment of fatty acid oxidation. On the basis of these results, the higher expression levels of all CPT-1 isoforms and the lower ACC-2 expression observed in the present study may be part of a protective response against lipotoxicity.

The most prominent change in the microarray analysis was the up-regulation of glomerular SCD-1, which could be confirmed in four independent patients at the protein level. Of note, on renal biopsy specimens, SCD-1 expression was mainly observed in podocytes. In contrast, a previous study in mice found a reduction of Scd-1 expression in mice.36 However, the results of this murine study were obtained from whole kidney extracts in a model of type 1 diabetes. Thus, the changes in the type 1 model may come from a different compartment but not from glomeruli, as done in the present study.

Several lines of evidence indicate a protective role of SCDs against saturated FFAs, such as palmitic acid. Specifically, the LXR agonists TO and GW, which increase levels of desaturases, also reduce palmitic acid–induced cell death. Similarly, overexpression of Scd-1 attenuates palmitic acid–induced cell death. Moreover, the protective effect of TO was lost after gene silencing of Scd-1/-2, thereby further confirming that the protective effect of TO is mediated by Scd-1/-2. Although we cannot rule out that the protective effect of TO or GW involves other targets, including other enzymes involved in FFA metabolism,37 the loss of TO-mediated protection in Scd-1/-2 double-deficient podocytes, suggests a major role of SCDs.

The mechanisms whereby MUFAs protect against saturated FFAs are incompletely understood. MUFAs, such as oleic acid, can increase fatty acid incorporation into TGs and increase fatty acid oxidation, thereby reducing biologically active saturated FFAs and avoiding the formation of cytotoxic metabolites, such as DAGs and ceramides.10 We found that TO favors palmitic acid–derived FFA incorporation into TG. The effect was even more pronounced in podocytes co-incubated with oleic acid. TO has no significant effect on palmitic acid–derived FFA incorporated into DAGs. Previously, it has been suggested that DAG-mediated lipotoxicity may depend on the saturation of fatty acids incorporated in DAGs.38 Thus, the beneficial action of TO on palmitic acid–induced podocyte death may still be in part due to an increase in palmitic acid–derived MUFAs incorporated in DAGs. In addition, the increased amount of palmitic acid recovered from TGs after treatment with TO is likely protective because palmitic acid is stored away in a safe lipid pool.10 It is also worth mentioning that oleic acid but not TO increased palmitic acid β-oxidation. These differences in the synthesis of TGs and the up-regulation of β-oxidation after oleic acid versus TO treatment may account for the more protective effect of oleic acid.

MUFAs can prevent the up-regulation of CHOP, which likely contributes further to their protective effect because gene silencing of CHOP attenuates palmitic acid–induced cell death.9 Interestingly, TO also significantly reduces the induction of CHOP in podocytes exposed to palmitic acid, which may result from the TO-induced increase in Scd-1/-2, thereby promoting a shift from saturated FFAs as palmitic acid to MUFAs. Clearly, more experiments are needed to clarify this interesting observation.

A limitation of our study is that the relevance of CHOP and the regulation of podocyte cell death by saturated FFAs and MUFAs in kidneys of patients with DN require further exploration. By immunohistochemistry no obvious CHOP induction was seen in glomeruli of DN patients (unpublished observation). Although podocyte loss or death clearly contributed to the progression of DN,4,5 we did not observe apoptosis in our patient biopsy specimens. Both the absence of CHOP staining and apoptosis detection may be due to the slow progression of DN, which makes it challenging to capture these events. Future studies are needed to confirm or refute this hypothesis.

The role of SCDs and increased TG content in the development of type 2 diabetes is under debate.39 Scd-1 deficiency in mice attenuates high fat diet–induced obesity and insulin resistance40 but at the same time promotes inflammation and atherosclerosis.41 The favorable effect of Scd-1 deficiency on diet-induced obesity is associated with altered skin lipid composition, leading to decreased skin insulation and a substantial increase in energy expenditure, which may at least in part explain the increased insulin sensitivity in this model.42 The role of TG storage in nonadipose tissue may be context dependent. Insulin-resistant obese rodents have increased intramuscular TG content and synthesis rates.43 In contrast, in humans the so-called athletes paradox indicates that ectopic fat accumulation in muscles per se is not negative because endurance-trained humans have higher insulin sensitivity and increased intramuscular TG stores.44 Furthermore, transgenic mice overexpressing the TG-synthesizing enzyme DGAT1 in the heart have increased TG content and improved cardiac function.45

In conclusion, our results suggest that the glomerular SCD-1 up-regulation in DN may be part of a protective mechanism against saturated FFAs. The following working model can be suggested based on our functional studies for the prosurvival effects of Scd-1 and/or Scd-2 and oleic acid on podocytes (Figure 7). Palmitic acid increases the generation of toxic metabolites, which leads to ER stress and podocyte death.9 Induction of Scd-1/-2 expression by TO or GW elevates the TG safe pool, which in turn reduces injurious metabolites and prevents podocyte death. Similarly, oleic acid mitigates palmitic acid–induced podocyte death by shifting palmitic acid and toxic metabolites to the TG safe pool. In addition, oleic acid but not TO or GW increase β-oxidation as an additional protective mechanism. Our findings have potential therapeutic implications because shifting of the FFA balance toward MUFAs and/or activation of SCD-1 in podocytes may help to prevent DN in type 2 diabetic patients.

Figure 7.

Figure 7

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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.

Acknowledgments

We thank all participating centers of the European Renal cDNA Bank-Kroener-Fresenius Biopsy Bank and their patients for their cooperation. Active members at the time of the study were as follows: Clemens David Cohen, Holger Schmid, Michael Fischereder, Lutz Weber, Matthias Kretzler, Detlef Schlöndorff (from Munich, Germany; Zurich, Switzerland; Ann Arbor, MI; New York, NY); Jean Daniel Sraer, Pierre Ronco (Paris, France); Maria Pia Rastaldi, Giuseppe D’Amico (Milano, Italy); Peter Doran, Hugh Brady (Dublin, Ireland); Detlev Mönks, Christoph Wanner (Würzburg, Germany); Andrew Rees (Aberdeen, UK); Frank Strutz, Gerhard Anton Müller (Göttingen, Germany); Peter Mertens, Jürgen Floege (Aachen, Germany); Norbert Braun, Teut Risler (Tübingen, Germany); Loreto Gesualdo, Francesco Paolo Schena (Bari, Italy); Jens Gerth, Gunter Wolf (Jena, Germany); Rainer Oberbauer, Dontscho Kerjaschki (Vienna, Austria); Bernhard Banas, Bernhard Krämer (Regensburg, Germany); Moin Saleem (Bristol, UK); Rudolf Wüthrich (Zurich, Switzerland); Walter Samtleben (Munich, Germany); Harm Peters, Hans-Hellmut Neumayer (Berlin, Germany); Mohamed Daha (Leiden, The Netherlands); Katrin Ivens, Bernd Grabensee (Düsseldorf, Germany); Francisco Mampaso (deceased) (Madrid, Spain); Jun Oh, Franz Schaefer, Martin Zeier, Hermann-Joseph Gröne (Heidelberg, Germany); Peter Gross (Dresden, Germany); Giancarlo Tonolo (Sassari, Italy); Vladimir Tesar (Prague, Czech Republic); Harald Rupprecht (Bayreuth, Germany); Hermann Pavenstädt (Münster, Germany); and Hans-Peter Marti (Bern, Switzerland).

J.S., K.K., S.G., M.T.L, and A.W. performed experiments; J.S. and A.W.J. designed experiments; J.S., P.M., and A.W.J. wrote the manuscript; and C.D.C. and J.M.O. reviewed the manuscript.

Footnotes

Supported by Swiss National Science Foundation grants 31003A-119974 and 31003A-144112/1 (A.W.J.), Novartis Foundation for Medical Biological Research10C57 (A.W.J.), Swiss National Science Foundation FellowshipPBBSP3-144160 (J.S.), and National Institutes Health Grants DK62472 and DK57683 (P.M.), NIH/N IDDKK08 DK0933783-01 (A.W.), and the Else-Kröner-Fresenius Foundation (C.D.C.).

Supplemental Data

Supplemental Figure S1

The expression pattern of murine Scd isoforms in the whole kidney differs from podocytes. Scd-1 is predominant in the kidney (A), whereas in podocytes (B) SCD-2 has the highest expression levels. Compared with Scd-1 and Scd-2, mRNA levels of Scd-3 and Scd-4 are low. Bar graphs represent relative means ± SD expression in the kidney (A) and in podocytes (B). Each isoform was normalized to Gapdh, and Scd-1 expression was set to 100% (kidneys: n = 4; podocytes: n = 9)

mmc1.pdf (78.7KB, pdf)
Supplemental Figure S2

TO and GW prevent palmitic acid–induced caspase 3 activation. Podocytes were pretreated with 1 μmol/L TO, 1 μmol/L GW, or vehicle (dimethyl sulfoxide) for 14 hours and subsequently incubated with 200 μmol/L palmitic acid in the presence of 1 μmol/L TO, GW, or vehicle for 24 hours. Western blot analysis of cleaved caspase 3 from whole cell lysates. GAPDH was used as a control.

mmc2.pdf (131.9KB, pdf)
Supplemental Figure S3

Scd-2 silencing does not affect the protective effect of TO on palmitic acid–induced cell death. Scd-2 single-silenced podocytes were pretreated with 1 μmol/L TO (14 hours) before addition of 200 μmol/L palmitic acid for 48 hours. A: Bar graph represents means ± SD percentages of apoptotic and necrotic cells (n = 3; *P < 0.05, **P < 0.01). B–D: Bar graphs represent relative means ± SD percent changes of apoptosis (B), necrosis (C), and apoptosis plus necrosis (D). Vehicle-treated (dimethyl sulfoxide) controls are set to 100%. n = 3, *P < 0.05, **P < 0.01.

mmc3.pdf (81KB, pdf)
Supplemental Figure S4

Fumonisin B1 (Fumo) does not prevent podocytes from palmitic acid–induced cell death. Podocytes were pretreated with 10 μmol/L Fumo for 1 hour and incubated with 200 μmol/L palmitic acid for 48 hours. Bar graph represents means ± SD percentages of apoptotic and necrotic cells (n = 3).

mmc4.pdf (78.5KB, pdf)
Supplemental Table S1
mmc5.doc (59KB, doc)

Supplemental Data

Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2013.05.023.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Figure S1

The expression pattern of murine Scd isoforms in the whole kidney differs from podocytes. Scd-1 is predominant in the kidney (A), whereas in podocytes (B) SCD-2 has the highest expression levels. Compared with Scd-1 and Scd-2, mRNA levels of Scd-3 and Scd-4 are low. Bar graphs represent relative means ± SD expression in the kidney (A) and in podocytes (B). Each isoform was normalized to Gapdh, and Scd-1 expression was set to 100% (kidneys: n = 4; podocytes: n = 9)

mmc1.pdf (78.7KB, pdf)
Supplemental Figure S2

TO and GW prevent palmitic acid–induced caspase 3 activation. Podocytes were pretreated with 1 μmol/L TO, 1 μmol/L GW, or vehicle (dimethyl sulfoxide) for 14 hours and subsequently incubated with 200 μmol/L palmitic acid in the presence of 1 μmol/L TO, GW, or vehicle for 24 hours. Western blot analysis of cleaved caspase 3 from whole cell lysates. GAPDH was used as a control.

mmc2.pdf (131.9KB, pdf)
Supplemental Figure S3

Scd-2 silencing does not affect the protective effect of TO on palmitic acid–induced cell death. Scd-2 single-silenced podocytes were pretreated with 1 μmol/L TO (14 hours) before addition of 200 μmol/L palmitic acid for 48 hours. A: Bar graph represents means ± SD percentages of apoptotic and necrotic cells (n = 3; *P < 0.05, **P < 0.01). B–D: Bar graphs represent relative means ± SD percent changes of apoptosis (B), necrosis (C), and apoptosis plus necrosis (D). Vehicle-treated (dimethyl sulfoxide) controls are set to 100%. n = 3, *P < 0.05, **P < 0.01.

mmc3.pdf (81KB, pdf)
Supplemental Figure S4

Fumonisin B1 (Fumo) does not prevent podocytes from palmitic acid–induced cell death. Podocytes were pretreated with 10 μmol/L Fumo for 1 hour and incubated with 200 μmol/L palmitic acid for 48 hours. Bar graph represents means ± SD percentages of apoptotic and necrotic cells (n = 3).

mmc4.pdf (78.5KB, pdf)
Supplemental Table S1
mmc5.doc (59KB, doc)

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