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Increased circulating miR-21 levels are associated with kidney fibrosis.

Glowacki F, Savary G, Gnemmi V, Buob D, Van der Hauwaert C, Lo-Guidice JM, Bouyé S, Hazzan M, Pottier N, Perrais M, Aubert S, Cauffiez C - PLoS ONE (2013)

Bottom Line: While there is evidence that miRNAs deregulation plays a causative role in various complex disorders, their role in fibrotic kidney diseases is largely unexplored.Circulating miR-21 levels are significantly increased in patients with severe IF/TA grade (IF/TA grade 3: 3.0±1.0 vs lower grade of fibrosis: 1.5±1.2; p = 0.001).In a multivariate linear regression model including IF/TA grade and estimated GFR, independent associations were found between circulating miR-21 levels and IF/TA score (ß = 0.307, p = 0.03), and between miR-21 levels and aMDRD (ß = -0.398, p = 0.006).

View Article: PubMed Central - PubMed

Affiliation: EA4483, Département de Biochimie et Biologie Moléculaire, Faculté de Médecine H Warembourg, Pôle Recherche, Lille, France. francois.glowacki@chru-lille.fr

ABSTRACT
MicroRNAs (miRNAs) are a class of noncoding RNA acting at a post-transcriptional level to control the expression of large sets of target mRNAs. While there is evidence that miRNAs deregulation plays a causative role in various complex disorders, their role in fibrotic kidney diseases is largely unexplored. Here, we found a strong up-regulation of miR-21 in the kidneys of mice with unilateral ureteral obstruction and also in the kidneys of patients with severe kidney fibrosis. In addition, mouse primary fibroblasts derived from fibrotic kidneys exhibited higher miR-21 expression level compared to those derived from normal kidneys. Expression of miR-21 in normal primary kidney fibroblasts was induced upon TGFβ exposure, a key growth factor involved in fibrogenesis. Finally, ectopic expression of miR-21 in primary kidney fibroblasts was sufficient to promote myofibroblast differentiation. As circulating miRNAs have been suggested as promising non-invasive biomarkers, we further assess whether circulating miR-21 levels are associated with renal fibrosis using sera from 42 renal transplant recipients, categorized according to their renal fibrosis severity, evaluated on allograft biopsies (Interstitial Fibrosis/Tubular Atrophy (IF/TA). Circulating miR-21 levels are significantly increased in patients with severe IF/TA grade (IF/TA grade 3: 3.0±1.0 vs lower grade of fibrosis: 1.5±1.2; p = 0.001). By contrast, circulating miR-21 levels were not correlated with other renal histological lesions. In a multivariate linear regression model including IF/TA grade and estimated GFR, independent associations were found between circulating miR-21 levels and IF/TA score (ß = 0.307, p = 0.03), and between miR-21 levels and aMDRD (ß = -0.398, p = 0.006). Altogether, these data suggest miR-21 has a key pathogenic role in kidney fibrosis and may represent a novel, predictive and reliable blood marker of kidney fibrosis.

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miR-21 is expressed in activated renal fibroblasts and upregulated in response to TGFβ stimulation. miR-21 and ACTA2 expression levels obtained by real time PCR in (A–B) control primary kidney fibroblasts exposed or not to TGF-β1 (10 ng/mL), and in (C–D) primary fibroblasts derived from fibrotic kidneys (UUO mice) and primary fibroblasts derived from normal kidneys (sham mice). (E–F) ACTA2 and COL1A1 expression levels obtained by real time PCR in control primary kidney fibroblasts transfected with either scrambled miRNA (neg) or premiR-21 (10 nM). Data are expressed as mean ± SEM. * p<0.05.
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pone-0058014-g003: miR-21 is expressed in activated renal fibroblasts and upregulated in response to TGFβ stimulation. miR-21 and ACTA2 expression levels obtained by real time PCR in (A–B) control primary kidney fibroblasts exposed or not to TGF-β1 (10 ng/mL), and in (C–D) primary fibroblasts derived from fibrotic kidneys (UUO mice) and primary fibroblasts derived from normal kidneys (sham mice). (E–F) ACTA2 and COL1A1 expression levels obtained by real time PCR in control primary kidney fibroblasts transfected with either scrambled miRNA (neg) or premiR-21 (10 nM). Data are expressed as mean ± SEM. * p<0.05.

Mentions: As depicted in Figure 2A, mice with UUO exhibited a time dependent increased expression of miR-21 during disease progression. In situ hybridization experiments performed in the injured kidneys 28 days after UUO revealed a selective expression of miR-21 in area consistent with fibrotic lesions (Figure 2B). In addition, miR-21 levels were correlated with the expression of two major extracellular matrix proteins (fibronectin and type I collagen, COL1A1) released by activated kidney fibroblasts, as well as TGFβ, a central pro-fibrotic growth factor, and α Smooth Muscle Actin (ACTA2), a marker of myofibroblast differentiation (Figure 2C–F). Furthermore, expression of miR-21 in normal primary kidney fibroblasts was significantly induced upon TGFβ exposure (Figure 3A and 3B). Fibroblasts derived from fibrotic kidneys (28 days UUO) exhibited higher miR-21 expression level (Figure 3C) and higher ACTA2 expression (Figure 3D) compared to control fibroblasts. Finally, we further showed that ectopic expression of miR-21 in primary kidney fibroblasts was sufficient to promote myofibroblast differentiation, as determined by an increased in both ACTA2 (Figure 3E) and COL1A1 (Figure 3F) expression levels. Altogether, these findings strongly suggest that miR-21 is likely to be involved in the pathogenic activation of kidney fibroblasts during fibrosis.


Increased circulating miR-21 levels are associated with kidney fibrosis.

Glowacki F, Savary G, Gnemmi V, Buob D, Van der Hauwaert C, Lo-Guidice JM, Bouyé S, Hazzan M, Pottier N, Perrais M, Aubert S, Cauffiez C - PLoS ONE (2013)

miR-21 is expressed in activated renal fibroblasts and upregulated in response to TGFβ stimulation. miR-21 and ACTA2 expression levels obtained by real time PCR in (A–B) control primary kidney fibroblasts exposed or not to TGF-β1 (10 ng/mL), and in (C–D) primary fibroblasts derived from fibrotic kidneys (UUO mice) and primary fibroblasts derived from normal kidneys (sham mice). (E–F) ACTA2 and COL1A1 expression levels obtained by real time PCR in control primary kidney fibroblasts transfected with either scrambled miRNA (neg) or premiR-21 (10 nM). Data are expressed as mean ± SEM. * p<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585177&req=5

pone-0058014-g003: miR-21 is expressed in activated renal fibroblasts and upregulated in response to TGFβ stimulation. miR-21 and ACTA2 expression levels obtained by real time PCR in (A–B) control primary kidney fibroblasts exposed or not to TGF-β1 (10 ng/mL), and in (C–D) primary fibroblasts derived from fibrotic kidneys (UUO mice) and primary fibroblasts derived from normal kidneys (sham mice). (E–F) ACTA2 and COL1A1 expression levels obtained by real time PCR in control primary kidney fibroblasts transfected with either scrambled miRNA (neg) or premiR-21 (10 nM). Data are expressed as mean ± SEM. * p<0.05.
Mentions: As depicted in Figure 2A, mice with UUO exhibited a time dependent increased expression of miR-21 during disease progression. In situ hybridization experiments performed in the injured kidneys 28 days after UUO revealed a selective expression of miR-21 in area consistent with fibrotic lesions (Figure 2B). In addition, miR-21 levels were correlated with the expression of two major extracellular matrix proteins (fibronectin and type I collagen, COL1A1) released by activated kidney fibroblasts, as well as TGFβ, a central pro-fibrotic growth factor, and α Smooth Muscle Actin (ACTA2), a marker of myofibroblast differentiation (Figure 2C–F). Furthermore, expression of miR-21 in normal primary kidney fibroblasts was significantly induced upon TGFβ exposure (Figure 3A and 3B). Fibroblasts derived from fibrotic kidneys (28 days UUO) exhibited higher miR-21 expression level (Figure 3C) and higher ACTA2 expression (Figure 3D) compared to control fibroblasts. Finally, we further showed that ectopic expression of miR-21 in primary kidney fibroblasts was sufficient to promote myofibroblast differentiation, as determined by an increased in both ACTA2 (Figure 3E) and COL1A1 (Figure 3F) expression levels. Altogether, these findings strongly suggest that miR-21 is likely to be involved in the pathogenic activation of kidney fibroblasts during fibrosis.

Bottom Line: While there is evidence that miRNAs deregulation plays a causative role in various complex disorders, their role in fibrotic kidney diseases is largely unexplored.Circulating miR-21 levels are significantly increased in patients with severe IF/TA grade (IF/TA grade 3: 3.0±1.0 vs lower grade of fibrosis: 1.5±1.2; p = 0.001).In a multivariate linear regression model including IF/TA grade and estimated GFR, independent associations were found between circulating miR-21 levels and IF/TA score (ß = 0.307, p = 0.03), and between miR-21 levels and aMDRD (ß = -0.398, p = 0.006).

View Article: PubMed Central - PubMed

Affiliation: EA4483, Département de Biochimie et Biologie Moléculaire, Faculté de Médecine H Warembourg, Pôle Recherche, Lille, France. francois.glowacki@chru-lille.fr

ABSTRACT
MicroRNAs (miRNAs) are a class of noncoding RNA acting at a post-transcriptional level to control the expression of large sets of target mRNAs. While there is evidence that miRNAs deregulation plays a causative role in various complex disorders, their role in fibrotic kidney diseases is largely unexplored. Here, we found a strong up-regulation of miR-21 in the kidneys of mice with unilateral ureteral obstruction and also in the kidneys of patients with severe kidney fibrosis. In addition, mouse primary fibroblasts derived from fibrotic kidneys exhibited higher miR-21 expression level compared to those derived from normal kidneys. Expression of miR-21 in normal primary kidney fibroblasts was induced upon TGFβ exposure, a key growth factor involved in fibrogenesis. Finally, ectopic expression of miR-21 in primary kidney fibroblasts was sufficient to promote myofibroblast differentiation. As circulating miRNAs have been suggested as promising non-invasive biomarkers, we further assess whether circulating miR-21 levels are associated with renal fibrosis using sera from 42 renal transplant recipients, categorized according to their renal fibrosis severity, evaluated on allograft biopsies (Interstitial Fibrosis/Tubular Atrophy (IF/TA). Circulating miR-21 levels are significantly increased in patients with severe IF/TA grade (IF/TA grade 3: 3.0±1.0 vs lower grade of fibrosis: 1.5±1.2; p = 0.001). By contrast, circulating miR-21 levels were not correlated with other renal histological lesions. In a multivariate linear regression model including IF/TA grade and estimated GFR, independent associations were found between circulating miR-21 levels and IF/TA score (ß = 0.307, p = 0.03), and between miR-21 levels and aMDRD (ß = -0.398, p = 0.006). Altogether, these data suggest miR-21 has a key pathogenic role in kidney fibrosis and may represent a novel, predictive and reliable blood marker of kidney fibrosis.

Show MeSH
Related in: MedlinePlus