1. Volume 94, Issue 3, Pages 536-550 (September 2018)
Palmitate deranges erythropoietin production via transcription factor ATF4 activation of unfolded protein response 
Thitinun Anusornvongchai, Masaomi Nangaku, Tzu-Ming Jao, Chia-Hsien Wu, Yu Ishimoto, Hiroshi Maekawa, Tetsuhiro Tanaka, Akira Shimizu, Masayuki Yamamoto, Norio Suzuki, Ryoji Sassa, Reiko Inagi 
Kidney International 
Volume 94, Issue 3, Pages (September 2018)
DOI: /j.kint
Copyright © 2018 International Society of Nephrology Terms and Conditions

2. Figure 1
Effect of palmitate on EPO expression. (a) Palmitate did not change the basal level of erythropoietin (EPO) mRNA expression. HepG2 was treated with various concentration of PAL-BSA or BSA under normoxic conditions for 24 hours, and then EPO mRNA expression was measured by real-time polymerase chain reaction (PCR). EPO mRNA expression was not changed by palmitate-conjugated bovine serum albumin (PAL-BSA) or BSA under normoxic conditions. (b) Palmitate suppressed EPO mRNA production when EPO production was stimulated by hypoxia (1% O2) or pseudo-hypoxia (100 μM CoCl2). HepG2 was treated with 120 μM of PAL-BSA or BSA for 12 hours, followed by exposure to hypoxia or normoxia for 12 hours. EPO mRNA expression was then measured by real-time PCR. Each group was subject to 2 or 3 assays, which were repeated 3 or 4 times. *P < 0.05, **P < Plus and minus signs indicate addition and without addition, respectively.
Kidney International  , DOI: ( /j.kint )
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3. Figure 2
Effect of palmitate on hypoxia-inducible factor (HIF) activity. (a) Palmitate did not change nuclear accumulation of HIF even on stimulation with pseudo-hypoxia (100 μM CoCl2). HepG2 was treated with palmitate-conjugated bovine serum albumin (PAL-BSA) or BSA for 24 hours under both normoxic and pseudo-hypoxic conditions, and nuclear accumulation of HIF-1α and -2α protein was then measured by Western blotting followed by densitometry. (b) Palmitate did not change mRNA expression level of other representative HIF target genes, such as adrenomedullin (AM) or heme oxygenase-1 (HO-1). HepG2 was treated with PAL-BSA or BSA for 24 hours under both normoxic and pseudo-hypoxic conditions, then mRNA expression of AM or HO-1 was measured by real-time PCR. Each group was subject to 2 or 3 assays, which were repeated 3 or 4 times. Plus and minus signs indicate addition and without addition, respectively. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
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4. Figure 3
Induction of endoplasmic reticulum (ER) stress responses by palmitate. (a) Palmitate increased expression of UPR molecules, such as GRP78, ATF6, ATF4, CHOP (a downstream gene of ATF4), and spliced XBP-1 at the mRNA level. HepG2 was treated with palmitate-conjugated bovine serum albumin (PAL-BSA) or BSA for 24 hours under both normoxic and pseudo-hypoxic (100 μM CoCl2) conditions, then mRNA expression of UPR molecules was measured by real-time polymerase chain reaction. (b) Palmitate increased protein expression of GRP78 and nuclear accumulation of ATF4. HepG2 treated with PAL-BSA or BSA for 24 hours under both normoxic and pseudo-hypoxic conditions was analyzed for GRP78 or ATF4 expression by Western blotting followed by densitometry. (c) Palmitate did not affect hypoxia response element (HRE) activity but suppressed reporter activity of the 3′-enhancer region of the erythropoietin (EPO) gene under pseudo-hypoxia. HepG2 (1 × 104) were transfected with pHRE-luc or pEPO3′-luc and cotransfected with pTK-Renilla luciferase. After transfection for 24 hours, the cells were exposed to the indicated stimuli for 12 hours, and then luciferase activity was measured. Transfection efficiency was corrected by dividing the relative value of the firefly luciferase light unit by that of the Renilla luciferase. Upper schemas indicate the sequences that were used for the luciferase assay. (d) ATF4 knockdown significantly attenuated the suppressive effect of palmitate in EPO production. ATF4-knocked down HepG2 by siRNA (1 × 104, left) were treated with PAL-BSA or BSA for 24 hours under both normoxic and pseudo-hypoxic conditions. EPO mRNA expression was then measured by real-time PCR (right). Each group consisted of 3 assays, and the experiments were independently repeated 3 or 4 times. *P < 0.05, **P < 0.01, ***P < Plus and minus signs indicate addition and without addition, respectively. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
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5. Figure 3
Induction of endoplasmic reticulum (ER) stress responses by palmitate. (a) Palmitate increased expression of UPR molecules, such as GRP78, ATF6, ATF4, CHOP (a downstream gene of ATF4), and spliced XBP-1 at the mRNA level. HepG2 was treated with palmitate-conjugated bovine serum albumin (PAL-BSA) or BSA for 24 hours under both normoxic and pseudo-hypoxic (100 μM CoCl2) conditions, then mRNA expression of UPR molecules was measured by real-time polymerase chain reaction. (b) Palmitate increased protein expression of GRP78 and nuclear accumulation of ATF4. HepG2 treated with PAL-BSA or BSA for 24 hours under both normoxic and pseudo-hypoxic conditions was analyzed for GRP78 or ATF4 expression by Western blotting followed by densitometry. (c) Palmitate did not affect hypoxia response element (HRE) activity but suppressed reporter activity of the 3′-enhancer region of the erythropoietin (EPO) gene under pseudo-hypoxia. HepG2 (1 × 104) were transfected with pHRE-luc or pEPO3′-luc and cotransfected with pTK-Renilla luciferase. After transfection for 24 hours, the cells were exposed to the indicated stimuli for 12 hours, and then luciferase activity was measured. Transfection efficiency was corrected by dividing the relative value of the firefly luciferase light unit by that of the Renilla luciferase. Upper schemas indicate the sequences that were used for the luciferase assay. (d) ATF4 knockdown significantly attenuated the suppressive effect of palmitate in EPO production. ATF4-knocked down HepG2 by siRNA (1 × 104, left) were treated with PAL-BSA or BSA for 24 hours under both normoxic and pseudo-hypoxic conditions. EPO mRNA expression was then measured by real-time PCR (right). Each group consisted of 3 assays, and the experiments were independently repeated 3 or 4 times. *P < 0.05, **P < 0.01, ***P < Plus and minus signs indicate addition and without addition, respectively. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
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6. Figure 4
Selective suppression of renal erythropoietin (EPO) production without an effect on the HIF pathway in palmitate-injected mice. C57BL/6 mice were i.p. injected with 0.5 ml (0.3 mg/g body weight) of palmitate-conjugated bovine serum albumin (PAL-BSA), oleic acid- conjugated BSA (OLE-BSA), or BSA once daily for 11 days. CoCl2 (60 mg/kg) was then subcutaneously injected 12 hours before animals were killed (n = 5 for each group) in the pseudo-hypoxia group. (a,b) Injection of PAL-BSA as well as BSA did not induce histological changes in the kidney, as estimated by periodic acid–Schiff staining and plasma creatinine or albuminuria levels under normoxic and pseudo-hypoxic conditions. (c) Injection of PAL-BSA, but not OLE-BSA, or BSA, suppressed the pseudo-hypoxia–induced increase in both renal EPO mRNA level and plasma EPO level. (d) PAL-BSA injection did not affect the induction of other hypoxia-induced hypoxia-inducible factor target genes, such as adrenomedullin (AM) and heme oxygenase-1 (HO-1). (e,f) Suppression of pseudo-hypoxia–induced renal EPO production by PAL-BSA was associated with endoplasmic reticulum stress, as estimated by expression of GRP78, ATF4, and its downstream genes CHOP and GADD34, and with the accumulation of lipid droplets, which were detected by Oil red O staining, in tubulointerstitial cells. (g) Electron microscopy revealed lipid foam cell formation in peritubular cells, including renal EPO producing cells, in PAL-BSA–injected mice. Bar = 100 μM in (a) and (f) and 4 μM in (g). *P < Plus and minus signs indicate addition and without addition, respectively. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
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7. Figure 4
Selective suppression of renal erythropoietin (EPO) production without an effect on the HIF pathway in palmitate-injected mice. C57BL/6 mice were i.p. injected with 0.5 ml (0.3 mg/g body weight) of palmitate-conjugated bovine serum albumin (PAL-BSA), oleic acid- conjugated BSA (OLE-BSA), or BSA once daily for 11 days. CoCl2 (60 mg/kg) was then subcutaneously injected 12 hours before animals were killed (n = 5 for each group) in the pseudo-hypoxia group. (a,b) Injection of PAL-BSA as well as BSA did not induce histological changes in the kidney, as estimated by periodic acid–Schiff staining and plasma creatinine or albuminuria levels under normoxic and pseudo-hypoxic conditions. (c) Injection of PAL-BSA, but not OLE-BSA, or BSA, suppressed the pseudo-hypoxia–induced increase in both renal EPO mRNA level and plasma EPO level. (d) PAL-BSA injection did not affect the induction of other hypoxia-induced hypoxia-inducible factor target genes, such as adrenomedullin (AM) and heme oxygenase-1 (HO-1). (e,f) Suppression of pseudo-hypoxia–induced renal EPO production by PAL-BSA was associated with endoplasmic reticulum stress, as estimated by expression of GRP78, ATF4, and its downstream genes CHOP and GADD34, and with the accumulation of lipid droplets, which were detected by Oil red O staining, in tubulointerstitial cells. (g) Electron microscopy revealed lipid foam cell formation in peritubular cells, including renal EPO producing cells, in PAL-BSA–injected mice. Bar = 100 μM in (a) and (f) and 4 μM in (g). *P < Plus and minus signs indicate addition and without addition, respectively. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
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8. Figure 5
Suppression of renal erythropoietin (EPO) production induced by an high-palmitate diet (HPD) associated with endoplasmic reticulum stress in mice. C57/BL6 mice were fed with normal chow (Chow), or with a high-fat diet with low-palmitate (LPD) or high-palmitate (HPD) for 16 weeks (n = 6 for each). CoCl2 (100 μM) was then injected subcutaneously to induce pseudo-hypoxia, and after 12 hours renal EPO mRNA expression, plasma EPO level, and ER stress status were measured. (a) The mice fed with HPD for 16 weeks showed increased body weight (*P < 0.05 vs. Chow or LPD). (b,c) Periodic acid–Schiff staining of the renal cortex (b) and albuminuria (c) in HPD-fed mice showed that HPD-fed mice (for 16 weeks) did not show histological or functional changes as compared with findings in Chow-fed mice. Bar = 100 μM. (d) HPD, but not Chow or LPD, significantly suppressed EPO production under pseudo-hypoxia. ND, not detectable. (e) Suppression of pseudo-hypoxia–induced EPO production by HFD was associated with ER stress, as estimated by mRNA expression of ATF4 and its downstream genes CHOP and GADD34. *P < To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
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9. Figure 6
ISAM showed suppression of erythropoietin (EPO) expression by palmitate without affecting hypoxia-inducible factor (HIF) activity. Inherited super-anemic mice (ISAM), in which the EPO gene is replaced with green fluorescent protein (GFP) and chronic hypoxia (HIF activation) is induced by severe anemia, were i.p. injected with 0.5 ml (0.3 mg/g body weight) of palmitate-conjugated bovine serum albumin (PAL-BSA) or BSA once daily for 11 days (n = 6 for each). (a) PAL-BSA–injected ISAM showed significant lowering of GFP production in the kidney. Bar = 100 μM. (b) The number of GFP-positive cells in the peritubular area of the renal cortex was significantly decreased by PAL-BSA. *P < 0.05. (c) PAL-BSA decreased the GFP mRNA expression level in the renal cortex by real-time PCR. (d) PAL-BSA–injected ISAM did not show a change in the nuclear accumulation level of HIF-2α by Western blotting. (e) PAL-BSA did not change the mRNA expression levels of other HIF target genes, such as vascular endothelial growth factor (VEGF), adrenomodullin (AM), glucose transporter (GLUT) 1, or GLUT3. *P < 0.05 versus wild-type mice. Plus and minus signs indicate addition and without addition, respectively. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
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10. Figure 7
Suppression of erythropoietin (EPO) production by ATF4 activated by palmitate-conjugated bovine serum albumin (PAL-BSA) in REP cells. Both inherited super-anemic mice (ISAM), in which the EPO gene is replaced with green fluorescent protein (GFP), and ISAM-REC mice, whose REP cells were lineage-labeled with tdTomato, show chronic hypoxia (hypoxia-inducible factor activation) caused by severe anemia. These were separately injected i.p. with 0.5 ml (0.3 mg/g body weight) of PAL-BSA or BSA once daily for 11 days (n = 2–4 for each). (a) Suppression of EPO expression (green) by PAL-BSA was correlated with an increased number of interstitial cells positive for ATF4 (red) in ISAM. (b) The signal for EPO (green) was not colocalized with that for ATF4 (blue) in REP cells (red) of PAL-BSA–treated ISAM-REC mice. (c) Analysis of 300 interstitial cells of each tissue isolated by the laser capture microdissection technique showed that the interstitial area was equally positive for tdTomato in each sample, whereas ATF4 activation was detected only in the interstitial area of PAL-BSA–treated ISAM-REC mice in association with a decrease in GFP (EPO) mRNA expression. Bar = 100 μM. ND, not detectable. Plus and minus signs indicate addition and without addition, respectively. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
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11. Figure S1
Oil red O staining showed intracellular lipid accumulation in HepG2 treated with PAL-BSA for 12 hours followed by pseudo-hypoxia (100 μM CoCl2) for 12 hours, as compared with control BSA. Bar = 200 μM in (A), (B), and 50 μM in (C), (D).
Kidney International  , DOI: ( /j.kint )
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12. Figure S2
Lipid metabolism, estimated by peroxisome proliferator-activated receptor-α and cluster of differentiation 36 gene expressions, or mitochondrial biogenesis, estimated by PGC1α gene expression, was not significantly affected in HepG2 treated with BSA or PAL-BSA for 12 hours, followed by exposure to normoxia (untreated) or pseudo-hypoxia (100 μM CoCl2) for 12 hours.
Kidney International  , DOI: ( /j.kint )
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13. Figure S3
The plasma lipid profiles, estimated by triglyceride and free fatty acid, showed no significant difference between mice injected with palmitate or oleic acid for 11 days, followed by exposure to normoxia (untreated) or pseudo-hypoxia (CoCl2 injection) for 12 hours.
Kidney International  , DOI: ( /j.kint )
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