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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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Ethanol induces expression of Sonic hedgehog and its Gli1 transcription factor in murine kidney.A) Ethanol-induced accumulation of Sonic hedgehog and Gli1 mRNA is dependent on PTAFR. Kidneys of wild-type BL6 or ptafr-/- mice ingesting ethanol or control diets were harvested and mRNA was extracted for qPCR quantitation and analyzed as in Fig 1. SEM (n = 4); *, p<0.05. B) Shh induction by chronic ethanol ingestion is dependent on PTAFR. Kidneys of wild-type BL6 or ptafr-/- mice fed ethanol or their pair-fed controls were harvested and stained by fluorescent immunohistochemistry for Shh and Fluor 568 secondary antibody. C) Gli1 induction by chronic ethanol ingesting is dependent on PTAFR. Sections of kidneys of mice with the stated genotype and diet were stained with anti-Gli1 and Fluor 488-conjugated secondary antibody as in the preceding panel. D) Shh and Gli1 co-localize in the kidneys of ethanol-fed mice. Image overlay of Panels B and C show Shh and Gli1 co-localize, and that the doubly positive cells are positioned to surround vascular lumens as shown by inclusion of a panel reporting DAPI (fluorescing blue) counterstained nuclei.
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pone.0145691.g003: Ethanol induces expression of Sonic hedgehog and its Gli1 transcription factor in murine kidney.A) Ethanol-induced accumulation of Sonic hedgehog and Gli1 mRNA is dependent on PTAFR. Kidneys of wild-type BL6 or ptafr-/- mice ingesting ethanol or control diets were harvested and mRNA was extracted for qPCR quantitation and analyzed as in Fig 1. SEM (n = 4); *, p<0.05. B) Shh induction by chronic ethanol ingestion is dependent on PTAFR. Kidneys of wild-type BL6 or ptafr-/- mice fed ethanol or their pair-fed controls were harvested and stained by fluorescent immunohistochemistry for Shh and Fluor 568 secondary antibody. C) Gli1 induction by chronic ethanol ingesting is dependent on PTAFR. Sections of kidneys of mice with the stated genotype and diet were stained with anti-Gli1 and Fluor 488-conjugated secondary antibody as in the preceding panel. D) Shh and Gli1 co-localize in the kidneys of ethanol-fed mice. Image overlay of Panels B and C show Shh and Gli1 co-localize, and that the doubly positive cells are positioned to surround vascular lumens as shown by inclusion of a panel reporting DAPI (fluorescing blue) counterstained nuclei.

Mentions: The hedgehog signaling pathway induces fibrosis in acutely damaged kidney [25,31], and we find mRNA for Sonic hedgehog (Shh) also was significantly induced in the kidney when the insult was chronic ethanol metabolism (Fig 3A). As with inflammation [28], genetic abolition of ptafr abolished the increase in Shh mRNA. Similarly, the transcription factor Gli1, which is induced by hedgehog signaling [22], also was induced in the kidneys of ethanol-fed mice, and loss of PTAFR signaling abolished the increase in Gli1 mRNA. Fate mapping shows kidney pericytes are the precursors of kidney myofibroblasts when injury is acutely induced by unilateral ureteral obstruction [25,31], and that this response is through Shh signaling [32]. Fluorescent immunohistochemistry showed expression of both Shh (Fig 3B) and Gli1 (Fig 3C) proteins were increased in the kidneys of ethanol-fed mice and that the two proteins were specifically associated with cells surrounding microvessels. Overlaying the images of fluorescent anti-Shh and anti-Gli1 showed these two proteins were extensively co-localized (Fig 3D). These data show ethanol induces Shh along with Gli1 in the kidney during chronic ethanol feeding and, like the ethanol-induced inflammatory response, that Gli1 and Shh induction require PTAFR.


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

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

Ethanol induces expression of Sonic hedgehog and its Gli1 transcription factor in murine kidney.A) Ethanol-induced accumulation of Sonic hedgehog and Gli1 mRNA is dependent on PTAFR. Kidneys of wild-type BL6 or ptafr-/- mice ingesting ethanol or control diets were harvested and mRNA was extracted for qPCR quantitation and analyzed as in Fig 1. SEM (n = 4); *, p<0.05. B) Shh induction by chronic ethanol ingestion is dependent on PTAFR. Kidneys of wild-type BL6 or ptafr-/- mice fed ethanol or their pair-fed controls were harvested and stained by fluorescent immunohistochemistry for Shh and Fluor 568 secondary antibody. C) Gli1 induction by chronic ethanol ingesting is dependent on PTAFR. Sections of kidneys of mice with the stated genotype and diet were stained with anti-Gli1 and Fluor 488-conjugated secondary antibody as in the preceding panel. D) Shh and Gli1 co-localize in the kidneys of ethanol-fed mice. Image overlay of Panels B and C show Shh and Gli1 co-localize, and that the doubly positive cells are positioned to surround vascular lumens as shown by inclusion of a panel reporting DAPI (fluorescing blue) counterstained nuclei.
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getmorefigures.php?uid=PMC4697844&req=5

pone.0145691.g003: Ethanol induces expression of Sonic hedgehog and its Gli1 transcription factor in murine kidney.A) Ethanol-induced accumulation of Sonic hedgehog and Gli1 mRNA is dependent on PTAFR. Kidneys of wild-type BL6 or ptafr-/- mice ingesting ethanol or control diets were harvested and mRNA was extracted for qPCR quantitation and analyzed as in Fig 1. SEM (n = 4); *, p<0.05. B) Shh induction by chronic ethanol ingestion is dependent on PTAFR. Kidneys of wild-type BL6 or ptafr-/- mice fed ethanol or their pair-fed controls were harvested and stained by fluorescent immunohistochemistry for Shh and Fluor 568 secondary antibody. C) Gli1 induction by chronic ethanol ingesting is dependent on PTAFR. Sections of kidneys of mice with the stated genotype and diet were stained with anti-Gli1 and Fluor 488-conjugated secondary antibody as in the preceding panel. D) Shh and Gli1 co-localize in the kidneys of ethanol-fed mice. Image overlay of Panels B and C show Shh and Gli1 co-localize, and that the doubly positive cells are positioned to surround vascular lumens as shown by inclusion of a panel reporting DAPI (fluorescing blue) counterstained nuclei.
Mentions: The hedgehog signaling pathway induces fibrosis in acutely damaged kidney [25,31], and we find mRNA for Sonic hedgehog (Shh) also was significantly induced in the kidney when the insult was chronic ethanol metabolism (Fig 3A). As with inflammation [28], genetic abolition of ptafr abolished the increase in Shh mRNA. Similarly, the transcription factor Gli1, which is induced by hedgehog signaling [22], also was induced in the kidneys of ethanol-fed mice, and loss of PTAFR signaling abolished the increase in Gli1 mRNA. Fate mapping shows kidney pericytes are the precursors of kidney myofibroblasts when injury is acutely induced by unilateral ureteral obstruction [25,31], and that this response is through Shh signaling [32]. Fluorescent immunohistochemistry showed expression of both Shh (Fig 3B) and Gli1 (Fig 3C) proteins were increased in the kidneys of ethanol-fed mice and that the two proteins were specifically associated with cells surrounding microvessels. Overlaying the images of fluorescent anti-Shh and anti-Gli1 showed these two proteins were extensively co-localized (Fig 3D). These data show ethanol induces Shh along with Gli1 in the kidney during chronic ethanol feeding and, like the ethanol-induced inflammatory response, that Gli1 and Shh induction require PTAFR.

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