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Inflammatory PAF Receptor Signaling Initiates Hedgehog Signaling and Kidney Fibrogenesis During Ethanol Consumption.

Latchoumycandane C, Hanouneh M, Nagy LE, McIntyre TM - PLoS ONE (2015)

Bottom Line: We show acute kidney inflammation resulting from chronic ingestion of the common xenobiotic ethanol initiates Gli1 transcription and hedgehog synthesis in kidney pericytes, and promotes renal fibrosis.Shh also was present in urine of patients with acute kidney injury, but not in normal individuals or those with fibrotic liver cirrhosis We conclude neither endogenous PTAFR signaling nor CYP2E1-generated radicals alone are sufficient to initiate hedgehog signaling, but instead PTAFR-dependent neutrophil infiltration with myeloperoxidase activation is necessary to initiate ethanol-induced fibrosis in kidney.We also show fibrogenic mediators escape to urine, defining a new class of urinary mechanistic biomarkers of fibrogenesis for an organ not commonly biopsied.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio, United States of America.

ABSTRACT
Acute inflammation either resolves or proceeds to fibrotic repair that replaces functional tissue. Pro-fibrotic hedgehog signaling and induction of its Gli transcription factor in pericytes induces fibrosis in kidney, but molecular instructions connecting inflammation to fibrosis are opaque. We show acute kidney inflammation resulting from chronic ingestion of the common xenobiotic ethanol initiates Gli1 transcription and hedgehog synthesis in kidney pericytes, and promotes renal fibrosis. Ethanol ingestion stimulated transcription of TGF-ß, collagens I and IV, and alpha-smooth muscle actin with accumulation of these proteins. This was accompanied by deposition of extracellular fibrils. Ethanol catabolism by CYP2E1 in kidney generates local reactive oxygen species that oxidize cellular phospholipids to phospholipid products that activate the Platelet-activating Factor receptor (PTAFR) for inflammatory phospholipids. Genetically deleting this ptafr locus abolished accumulation of mRNA for TGF-ß, collagen IV, and α-smooth muscle actin. Loss of PTAFR also abolished ethanol-stimulated Sonic (Shh) and Indian hedgehog (Ihh) expression, and abolished transcription and accumulation of Gli1. Shh induced in pericytes and Ihh in tubules escaped to urine of ethanol-fed mice. Neutrophil myeloperoxidase (MPO) is required for ethanol-induced kidney inflammation, and Shh was not present in kidney or urine of mpo-/- mice. Shh also was present in urine of patients with acute kidney injury, but not in normal individuals or those with fibrotic liver cirrhosis We conclude neither endogenous PTAFR signaling nor CYP2E1-generated radicals alone are sufficient to initiate hedgehog signaling, but instead PTAFR-dependent neutrophil infiltration with myeloperoxidase activation is necessary to initiate ethanol-induced fibrosis in kidney. We also show fibrogenic mediators escape to urine, defining a new class of urinary mechanistic biomarkers of fibrogenesis for an organ not commonly biopsied.

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Chronic ethanol ingestion is fibrogenic in rat kidney.A) Fibrotic protein expression in kidney. Rats were fed the standard Lieber-deCarli liquid ethanol diet, or its isomaltose control, for 28 days before kidneys were excised, perfused with saline, fixed and sectioned as described in “Methods.” Sections were rehydrated and stained with anti-TGF-ß1, anti- α-smooth muscle actin, anti-collagen IV or anti-collagen 1 antibodies, and SP-streptavidin-conjugated secondary antibody for anti-TGF-ß1, or fluorescent secondary antibodies for the other sections as stated in “Methods.” TGF-ß sections were developed with horseradish peroxidase and DAB/metal chromogenic solution. B) mRNAs encoding fibrotic proteins were induced in kidneys of rats ingesting ethanol. Total RNA was isolated from ethanol and pair-fed kidneys from rats at the end of the feeding trial, and mRNA was quantified by SYBR Green one-step Reverse Transcription-PCR for the stated mRNAs and ribosomal 18S with the Bio-Rad MyiQ real-time PCR detection system. mRNA expression was normalized to 18S RNA content and 2-ΔΔCT was used to calculate the fold changes. Data are expressed as mean±SEM (n = 4), and p<0.05 (*) was considered as significant. C) Ethanol induced extracellular fibril deposition in kidneys of rats chronically metabolizing ethanol. Kidneys of control pair-fed or ethanol fed rats were isolated, fixed, sectioned and prepared for electron microscopy as described in “Methods.” The micrograph from the control animal shows erythrocytes within a capillary separating three epithelial cells, in contrast to the disorganized milieu with extracellular fibrils found in the kidney of ethanol-fed rats. The inset to the right is an expanded micrograph with extracellular fibrils highlighted by an arrow.
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pone.0145691.g001: Chronic ethanol ingestion is fibrogenic in rat kidney.A) Fibrotic protein expression in kidney. Rats were fed the standard Lieber-deCarli liquid ethanol diet, or its isomaltose control, for 28 days before kidneys were excised, perfused with saline, fixed and sectioned as described in “Methods.” Sections were rehydrated and stained with anti-TGF-ß1, anti- α-smooth muscle actin, anti-collagen IV or anti-collagen 1 antibodies, and SP-streptavidin-conjugated secondary antibody for anti-TGF-ß1, or fluorescent secondary antibodies for the other sections as stated in “Methods.” TGF-ß sections were developed with horseradish peroxidase and DAB/metal chromogenic solution. B) mRNAs encoding fibrotic proteins were induced in kidneys of rats ingesting ethanol. Total RNA was isolated from ethanol and pair-fed kidneys from rats at the end of the feeding trial, and mRNA was quantified by SYBR Green one-step Reverse Transcription-PCR for the stated mRNAs and ribosomal 18S with the Bio-Rad MyiQ real-time PCR detection system. mRNA expression was normalized to 18S RNA content and 2-ΔΔCT was used to calculate the fold changes. Data are expressed as mean±SEM (n = 4), and p<0.05 (*) was considered as significant. C) Ethanol induced extracellular fibril deposition in kidneys of rats chronically metabolizing ethanol. Kidneys of control pair-fed or ethanol fed rats were isolated, fixed, sectioned and prepared for electron microscopy as described in “Methods.” The micrograph from the control animal shows erythrocytes within a capillary separating three epithelial cells, in contrast to the disorganized milieu with extracellular fibrils found in the kidney of ethanol-fed rats. The inset to the right is an expanded micrograph with extracellular fibrils highlighted by an arrow.

Mentions: The early steps in alcohol-induced liver damage are modeled in the Lieber-deCarli liquid ethanol diet where rats ingest a third of their caloric intake as ethanol and then are compared to control animals pair fed an isocaloric diet with isomaltose substituted for ethanol to equalize weight gain. Chronic ethanol ingestion in this model generates only mild liver inflammation by 4 weeks. Kidney, however, is exquisitely sensitive to exogenous ethanol, developing inflammation, loss of filtration, and acute kidney injury syndrome [10,28]. We determined whether inflammation progressed to renal fibrosis, despite the lack of fibrosis in liver, and so stained sections of kidney from rats ingesting a control and ethanol diet for fibrogenic proteins (Fig 1A). Chronic ethanol catabolism, in contrast to a control diet, induced expression in kidney of the fibrotic signaling cytokine TGF-ß1 (top left), α-smooth muscle actin of myofibroblasts (top right) and collagen IV (bottom left) and collagen I (bottom right). Quantitative analysis of mRNA for TGF-ß1 confirmed ethanol feeding increased transcripts for this cytokine, that the level of induction was significant, and that the increase in message was nearly 3 fold (Fig 1B). Alpha-smooth muscle actin protein was increased by ethanol ingestion, with a significant 2-fold increase in its mRNA. Similarly, collagen IV mRNA also was significantly increased by 3 fold and collagen I mRNA increased by 75% in the kidneys of ethanol fed rats. Electron micrographs (Fig 1C) showed an organized epithelium abutting an erythrocyte-containing vessel in control kidney, while specimens from ethanol-fed rats showed disorganized structures with accumulation of extracellular fibrils. We conclude kidney, in contrast to liver, responds to chronic ethanol metabolism with accumulation of fibrotic proteins and fibrils in the standard model of chronic ethanol ingestion.


Inflammatory PAF Receptor Signaling Initiates Hedgehog Signaling and Kidney Fibrogenesis During Ethanol Consumption.

Latchoumycandane C, Hanouneh M, Nagy LE, McIntyre TM - PLoS ONE (2015)

Chronic ethanol ingestion is fibrogenic in rat kidney.A) Fibrotic protein expression in kidney. Rats were fed the standard Lieber-deCarli liquid ethanol diet, or its isomaltose control, for 28 days before kidneys were excised, perfused with saline, fixed and sectioned as described in “Methods.” Sections were rehydrated and stained with anti-TGF-ß1, anti- α-smooth muscle actin, anti-collagen IV or anti-collagen 1 antibodies, and SP-streptavidin-conjugated secondary antibody for anti-TGF-ß1, or fluorescent secondary antibodies for the other sections as stated in “Methods.” TGF-ß sections were developed with horseradish peroxidase and DAB/metal chromogenic solution. B) mRNAs encoding fibrotic proteins were induced in kidneys of rats ingesting ethanol. Total RNA was isolated from ethanol and pair-fed kidneys from rats at the end of the feeding trial, and mRNA was quantified by SYBR Green one-step Reverse Transcription-PCR for the stated mRNAs and ribosomal 18S with the Bio-Rad MyiQ real-time PCR detection system. mRNA expression was normalized to 18S RNA content and 2-ΔΔCT was used to calculate the fold changes. Data are expressed as mean±SEM (n = 4), and p<0.05 (*) was considered as significant. C) Ethanol induced extracellular fibril deposition in kidneys of rats chronically metabolizing ethanol. Kidneys of control pair-fed or ethanol fed rats were isolated, fixed, sectioned and prepared for electron microscopy as described in “Methods.” The micrograph from the control animal shows erythrocytes within a capillary separating three epithelial cells, in contrast to the disorganized milieu with extracellular fibrils found in the kidney of ethanol-fed rats. The inset to the right is an expanded micrograph with extracellular fibrils highlighted by an arrow.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4697844&req=5

pone.0145691.g001: Chronic ethanol ingestion is fibrogenic in rat kidney.A) Fibrotic protein expression in kidney. Rats were fed the standard Lieber-deCarli liquid ethanol diet, or its isomaltose control, for 28 days before kidneys were excised, perfused with saline, fixed and sectioned as described in “Methods.” Sections were rehydrated and stained with anti-TGF-ß1, anti- α-smooth muscle actin, anti-collagen IV or anti-collagen 1 antibodies, and SP-streptavidin-conjugated secondary antibody for anti-TGF-ß1, or fluorescent secondary antibodies for the other sections as stated in “Methods.” TGF-ß sections were developed with horseradish peroxidase and DAB/metal chromogenic solution. B) mRNAs encoding fibrotic proteins were induced in kidneys of rats ingesting ethanol. Total RNA was isolated from ethanol and pair-fed kidneys from rats at the end of the feeding trial, and mRNA was quantified by SYBR Green one-step Reverse Transcription-PCR for the stated mRNAs and ribosomal 18S with the Bio-Rad MyiQ real-time PCR detection system. mRNA expression was normalized to 18S RNA content and 2-ΔΔCT was used to calculate the fold changes. Data are expressed as mean±SEM (n = 4), and p<0.05 (*) was considered as significant. C) Ethanol induced extracellular fibril deposition in kidneys of rats chronically metabolizing ethanol. Kidneys of control pair-fed or ethanol fed rats were isolated, fixed, sectioned and prepared for electron microscopy as described in “Methods.” The micrograph from the control animal shows erythrocytes within a capillary separating three epithelial cells, in contrast to the disorganized milieu with extracellular fibrils found in the kidney of ethanol-fed rats. The inset to the right is an expanded micrograph with extracellular fibrils highlighted by an arrow.
Mentions: The early steps in alcohol-induced liver damage are modeled in the Lieber-deCarli liquid ethanol diet where rats ingest a third of their caloric intake as ethanol and then are compared to control animals pair fed an isocaloric diet with isomaltose substituted for ethanol to equalize weight gain. Chronic ethanol ingestion in this model generates only mild liver inflammation by 4 weeks. Kidney, however, is exquisitely sensitive to exogenous ethanol, developing inflammation, loss of filtration, and acute kidney injury syndrome [10,28]. We determined whether inflammation progressed to renal fibrosis, despite the lack of fibrosis in liver, and so stained sections of kidney from rats ingesting a control and ethanol diet for fibrogenic proteins (Fig 1A). Chronic ethanol catabolism, in contrast to a control diet, induced expression in kidney of the fibrotic signaling cytokine TGF-ß1 (top left), α-smooth muscle actin of myofibroblasts (top right) and collagen IV (bottom left) and collagen I (bottom right). Quantitative analysis of mRNA for TGF-ß1 confirmed ethanol feeding increased transcripts for this cytokine, that the level of induction was significant, and that the increase in message was nearly 3 fold (Fig 1B). Alpha-smooth muscle actin protein was increased by ethanol ingestion, with a significant 2-fold increase in its mRNA. Similarly, collagen IV mRNA also was significantly increased by 3 fold and collagen I mRNA increased by 75% in the kidneys of ethanol fed rats. Electron micrographs (Fig 1C) showed an organized epithelium abutting an erythrocyte-containing vessel in control kidney, while specimens from ethanol-fed rats showed disorganized structures with accumulation of extracellular fibrils. We conclude kidney, in contrast to liver, responds to chronic ethanol metabolism with accumulation of fibrotic proteins and fibrils in the standard model of chronic ethanol ingestion.

Bottom Line: We show acute kidney inflammation resulting from chronic ingestion of the common xenobiotic ethanol initiates Gli1 transcription and hedgehog synthesis in kidney pericytes, and promotes renal fibrosis.Shh also was present in urine of patients with acute kidney injury, but not in normal individuals or those with fibrotic liver cirrhosis We conclude neither endogenous PTAFR signaling nor CYP2E1-generated radicals alone are sufficient to initiate hedgehog signaling, but instead PTAFR-dependent neutrophil infiltration with myeloperoxidase activation is necessary to initiate ethanol-induced fibrosis in kidney.We also show fibrogenic mediators escape to urine, defining a new class of urinary mechanistic biomarkers of fibrogenesis for an organ not commonly biopsied.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio, United States of America.

ABSTRACT
Acute inflammation either resolves or proceeds to fibrotic repair that replaces functional tissue. Pro-fibrotic hedgehog signaling and induction of its Gli transcription factor in pericytes induces fibrosis in kidney, but molecular instructions connecting inflammation to fibrosis are opaque. We show acute kidney inflammation resulting from chronic ingestion of the common xenobiotic ethanol initiates Gli1 transcription and hedgehog synthesis in kidney pericytes, and promotes renal fibrosis. Ethanol ingestion stimulated transcription of TGF-ß, collagens I and IV, and alpha-smooth muscle actin with accumulation of these proteins. This was accompanied by deposition of extracellular fibrils. Ethanol catabolism by CYP2E1 in kidney generates local reactive oxygen species that oxidize cellular phospholipids to phospholipid products that activate the Platelet-activating Factor receptor (PTAFR) for inflammatory phospholipids. Genetically deleting this ptafr locus abolished accumulation of mRNA for TGF-ß, collagen IV, and α-smooth muscle actin. Loss of PTAFR also abolished ethanol-stimulated Sonic (Shh) and Indian hedgehog (Ihh) expression, and abolished transcription and accumulation of Gli1. Shh induced in pericytes and Ihh in tubules escaped to urine of ethanol-fed mice. Neutrophil myeloperoxidase (MPO) is required for ethanol-induced kidney inflammation, and Shh was not present in kidney or urine of mpo-/- mice. Shh also was present in urine of patients with acute kidney injury, but not in normal individuals or those with fibrotic liver cirrhosis We conclude neither endogenous PTAFR signaling nor CYP2E1-generated radicals alone are sufficient to initiate hedgehog signaling, but instead PTAFR-dependent neutrophil infiltration with myeloperoxidase activation is necessary to initiate ethanol-induced fibrosis in kidney. We also show fibrogenic mediators escape to urine, defining a new class of urinary mechanistic biomarkers of fibrogenesis for an organ not commonly biopsied.

Show MeSH
Related in: MedlinePlus