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Inflammation and hypoxia linked to renal injury by CCAAT/enhancer-binding protein δ.

Yamaguchi J, Tanaka T, Eto N, Nangaku M - Kidney Int. (2015)

Bottom Line: CEBPD was induced in the nuclei of tubular epithelial cells in both acute and chronic hypoxic kidneys.In turn, CEBPD induction augmented HIF-1α expression and its transcriptional activity.Mechanistically, CEBPD directly bound to the HIF-1α promoter and enhanced its transcription.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan.

ABSTRACT
Tubulointerstitial hypoxia plays a critical role in the pathogenesis of kidney injury, and hypoxia-inducible factor (HIF)-1 is a master regulator of cellular adaptation to hypoxia. Aside from oxygen molecules, factors that modify HIF-1 expression and functional operation remain obscure. Therefore, we sought to identify novel HIF-1-regulating genes in kidney. A short-hairpin RNA library consisting of 150 hypoxia-inducible genes was derived from a microarray analysis of the rat renal artery stenosis model screened for the effect on HIF-1 response. We report that CCAAT/enhancer-binding protein δ (CEBPD), a transcription factor and inflammatory response gene, is a novel HIF-1 regulator in kidney. CEBPD was induced in the nuclei of tubular epithelial cells in both acute and chronic hypoxic kidneys. In turn, CEBPD induction augmented HIF-1α expression and its transcriptional activity. Mechanistically, CEBPD directly bound to the HIF-1α promoter and enhanced its transcription. Notably, CEBPD was rapidly induced by inflammatory cytokines, such as IL-1β in a nuclear factor-κB-dependent manner, which not only increased HIF-1α expression during hypoxia, but was also indispensable for the non-hypoxic induction of HIF-1α. Thus our study provides novel insight into HIF-1 regulation in tubular epithelial cells and offers a potential hypoxia and inflammation link relevant in both acute and chronic kidney diseases.

No MeSH data available.


Related in: MedlinePlus

CCAAT/enhancer-binding protein δ (CEBPD) is expressed in hypoxic kidney tubular cells in vivo. (a) Mice were exposed to 8% O2 for 6 h using a hypoxia chamber (hypoxia group, n=6). The mice at ambient oxygen were used as the control (control group, n=5). The whole kidney was examined for CEBPD expression by real-time quantitative (q)RT-PCR. (b) The CEBPD expression levels in the renal cortex of acute and chronic rat hypoxic injuries were evaluated by qRT–PCR. (A) The ischemia–reperfusion (I/R) group (control group, n=6; I/R group, n=6). (B) The cisplatin nephrotoxicity group (control group, n=6; cisplatin group, n=7). (C) The remnant kidney (RK) group (control group, n=5; RK-1w group, n=5; RK-4w group, n=5). (D) The RAS group (control group, n=6; RAS group, n=6). (c) Fluorescent images of control kidney tissues for CEBPD and Phaseolus vulgaris Erythroagglutinin (PHA-E), a marker for proximal tubules. (A–C) Cortex and (D–F) outer stripe (OS) of the outer medulla, × 200. Bar=50 μm. CEBPD is stained in the S3 segment of proximal tubules in the OS. (G) Cortex and (H) OS of the outer medulla for further higher magnification, × 400. Bar=30 μm. (d) (A) Control kidney and four hypoxic kidney models ((B) I/R group, (C) cisplatin group, (D) RK group, and (E) RAS group) were stained with an anti-CEBPD antibody. CEBPD is stained in the nuclei of tubular cells in all four models compared with the control kidney. Original magnifications, × 400. Bar=30 μm. Bar graph (combined results from two independent experiments, shown as mean±s.d.) statistics performed using Student's t-test or one-way analysis of variance (ANOVA) with Dunnett's post-hoc tests. *P<0.05 and **P<0.01.
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fig2: CCAAT/enhancer-binding protein δ (CEBPD) is expressed in hypoxic kidney tubular cells in vivo. (a) Mice were exposed to 8% O2 for 6 h using a hypoxia chamber (hypoxia group, n=6). The mice at ambient oxygen were used as the control (control group, n=5). The whole kidney was examined for CEBPD expression by real-time quantitative (q)RT-PCR. (b) The CEBPD expression levels in the renal cortex of acute and chronic rat hypoxic injuries were evaluated by qRT–PCR. (A) The ischemia–reperfusion (I/R) group (control group, n=6; I/R group, n=6). (B) The cisplatin nephrotoxicity group (control group, n=6; cisplatin group, n=7). (C) The remnant kidney (RK) group (control group, n=5; RK-1w group, n=5; RK-4w group, n=5). (D) The RAS group (control group, n=6; RAS group, n=6). (c) Fluorescent images of control kidney tissues for CEBPD and Phaseolus vulgaris Erythroagglutinin (PHA-E), a marker for proximal tubules. (A–C) Cortex and (D–F) outer stripe (OS) of the outer medulla, × 200. Bar=50 μm. CEBPD is stained in the S3 segment of proximal tubules in the OS. (G) Cortex and (H) OS of the outer medulla for further higher magnification, × 400. Bar=30 μm. (d) (A) Control kidney and four hypoxic kidney models ((B) I/R group, (C) cisplatin group, (D) RK group, and (E) RAS group) were stained with an anti-CEBPD antibody. CEBPD is stained in the nuclei of tubular cells in all four models compared with the control kidney. Original magnifications, × 400. Bar=30 μm. Bar graph (combined results from two independent experiments, shown as mean±s.d.) statistics performed using Student's t-test or one-way analysis of variance (ANOVA) with Dunnett's post-hoc tests. *P<0.05 and **P<0.01.

Mentions: CEBPD is a member of the highly conserved C/EBP family of basic region leucine zipper transcription factors. Although its basic expression level is low in most normal tissues and cells, CEBPD is rapidly upregulated by a variety of stimuli, including inflammatory signals.20, 21 We investigated whether CEBPD expression in vivo correlates with hypoxia in the kidney. Rodent kidneys were subjected to a series of hypoxic stimuli and tested for CEPBD expression. CEBPD was induced in kidneys exposed to systemic hypoxia (8% O2, 6 h), as well as in models of kidney injuries (ischemia–reperfusion (I/R) injury, cisplatin nephrotoxicity, RAS, and 5/6 nephrectomy (remnant kidney (RK)); Figure 2a and b). Although the pathogenesis of these models is multifactorial, they serve as models of acute and chronic ischemic kidney injury. Cisplatin nephrotoxicity is hypoxic in its outer medulla because of the reduced blood flow caused by the toxin,10 and activation of the renin–angiotensin system results in decreased peritubular capillary flow with subsequent hypoxia in RK.22


Inflammation and hypoxia linked to renal injury by CCAAT/enhancer-binding protein δ.

Yamaguchi J, Tanaka T, Eto N, Nangaku M - Kidney Int. (2015)

CCAAT/enhancer-binding protein δ (CEBPD) is expressed in hypoxic kidney tubular cells in vivo. (a) Mice were exposed to 8% O2 for 6 h using a hypoxia chamber (hypoxia group, n=6). The mice at ambient oxygen were used as the control (control group, n=5). The whole kidney was examined for CEBPD expression by real-time quantitative (q)RT-PCR. (b) The CEBPD expression levels in the renal cortex of acute and chronic rat hypoxic injuries were evaluated by qRT–PCR. (A) The ischemia–reperfusion (I/R) group (control group, n=6; I/R group, n=6). (B) The cisplatin nephrotoxicity group (control group, n=6; cisplatin group, n=7). (C) The remnant kidney (RK) group (control group, n=5; RK-1w group, n=5; RK-4w group, n=5). (D) The RAS group (control group, n=6; RAS group, n=6). (c) Fluorescent images of control kidney tissues for CEBPD and Phaseolus vulgaris Erythroagglutinin (PHA-E), a marker for proximal tubules. (A–C) Cortex and (D–F) outer stripe (OS) of the outer medulla, × 200. Bar=50 μm. CEBPD is stained in the S3 segment of proximal tubules in the OS. (G) Cortex and (H) OS of the outer medulla for further higher magnification, × 400. Bar=30 μm. (d) (A) Control kidney and four hypoxic kidney models ((B) I/R group, (C) cisplatin group, (D) RK group, and (E) RAS group) were stained with an anti-CEBPD antibody. CEBPD is stained in the nuclei of tubular cells in all four models compared with the control kidney. Original magnifications, × 400. Bar=30 μm. Bar graph (combined results from two independent experiments, shown as mean±s.d.) statistics performed using Student's t-test or one-way analysis of variance (ANOVA) with Dunnett's post-hoc tests. *P<0.05 and **P<0.01.
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fig2: CCAAT/enhancer-binding protein δ (CEBPD) is expressed in hypoxic kidney tubular cells in vivo. (a) Mice were exposed to 8% O2 for 6 h using a hypoxia chamber (hypoxia group, n=6). The mice at ambient oxygen were used as the control (control group, n=5). The whole kidney was examined for CEBPD expression by real-time quantitative (q)RT-PCR. (b) The CEBPD expression levels in the renal cortex of acute and chronic rat hypoxic injuries were evaluated by qRT–PCR. (A) The ischemia–reperfusion (I/R) group (control group, n=6; I/R group, n=6). (B) The cisplatin nephrotoxicity group (control group, n=6; cisplatin group, n=7). (C) The remnant kidney (RK) group (control group, n=5; RK-1w group, n=5; RK-4w group, n=5). (D) The RAS group (control group, n=6; RAS group, n=6). (c) Fluorescent images of control kidney tissues for CEBPD and Phaseolus vulgaris Erythroagglutinin (PHA-E), a marker for proximal tubules. (A–C) Cortex and (D–F) outer stripe (OS) of the outer medulla, × 200. Bar=50 μm. CEBPD is stained in the S3 segment of proximal tubules in the OS. (G) Cortex and (H) OS of the outer medulla for further higher magnification, × 400. Bar=30 μm. (d) (A) Control kidney and four hypoxic kidney models ((B) I/R group, (C) cisplatin group, (D) RK group, and (E) RAS group) were stained with an anti-CEBPD antibody. CEBPD is stained in the nuclei of tubular cells in all four models compared with the control kidney. Original magnifications, × 400. Bar=30 μm. Bar graph (combined results from two independent experiments, shown as mean±s.d.) statistics performed using Student's t-test or one-way analysis of variance (ANOVA) with Dunnett's post-hoc tests. *P<0.05 and **P<0.01.
Mentions: CEBPD is a member of the highly conserved C/EBP family of basic region leucine zipper transcription factors. Although its basic expression level is low in most normal tissues and cells, CEBPD is rapidly upregulated by a variety of stimuli, including inflammatory signals.20, 21 We investigated whether CEBPD expression in vivo correlates with hypoxia in the kidney. Rodent kidneys were subjected to a series of hypoxic stimuli and tested for CEPBD expression. CEBPD was induced in kidneys exposed to systemic hypoxia (8% O2, 6 h), as well as in models of kidney injuries (ischemia–reperfusion (I/R) injury, cisplatin nephrotoxicity, RAS, and 5/6 nephrectomy (remnant kidney (RK)); Figure 2a and b). Although the pathogenesis of these models is multifactorial, they serve as models of acute and chronic ischemic kidney injury. Cisplatin nephrotoxicity is hypoxic in its outer medulla because of the reduced blood flow caused by the toxin,10 and activation of the renin–angiotensin system results in decreased peritubular capillary flow with subsequent hypoxia in RK.22

Bottom Line: CEBPD was induced in the nuclei of tubular epithelial cells in both acute and chronic hypoxic kidneys.In turn, CEBPD induction augmented HIF-1α expression and its transcriptional activity.Mechanistically, CEBPD directly bound to the HIF-1α promoter and enhanced its transcription.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology and Endocrinology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan.

ABSTRACT
Tubulointerstitial hypoxia plays a critical role in the pathogenesis of kidney injury, and hypoxia-inducible factor (HIF)-1 is a master regulator of cellular adaptation to hypoxia. Aside from oxygen molecules, factors that modify HIF-1 expression and functional operation remain obscure. Therefore, we sought to identify novel HIF-1-regulating genes in kidney. A short-hairpin RNA library consisting of 150 hypoxia-inducible genes was derived from a microarray analysis of the rat renal artery stenosis model screened for the effect on HIF-1 response. We report that CCAAT/enhancer-binding protein δ (CEBPD), a transcription factor and inflammatory response gene, is a novel HIF-1 regulator in kidney. CEBPD was induced in the nuclei of tubular epithelial cells in both acute and chronic hypoxic kidneys. In turn, CEBPD induction augmented HIF-1α expression and its transcriptional activity. Mechanistically, CEBPD directly bound to the HIF-1α promoter and enhanced its transcription. Notably, CEBPD was rapidly induced by inflammatory cytokines, such as IL-1β in a nuclear factor-κB-dependent manner, which not only increased HIF-1α expression during hypoxia, but was also indispensable for the non-hypoxic induction of HIF-1α. Thus our study provides novel insight into HIF-1 regulation in tubular epithelial cells and offers a potential hypoxia and inflammation link relevant in both acute and chronic kidney diseases.

No MeSH data available.


Related in: MedlinePlus