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miR-200b precursor can ameliorate renal tubulointerstitial fibrosis.

Oba S, Kumano S, Suzuki E, Nishimatsu H, Takahashi M, Takamori H, Kasuya M, Ogawa Y, Sato K, Kimura K, Homma Y, Hirata Y, Fujita T - PLoS ONE (2010)

Bottom Line: A single injection of 0.5 nM miR-200b precursor was sufficient to inhibit the increase of collagen types I, III and fibronectin in obstructed kidneys, and amelioration of fibrosis was confirmed by observation of the kidneys with Azan staining. miR-200 family members have been previously shown to inhibit EMT by reducing the expression of ZEB-1 and ZEB-2 which are known repressors of E-cadherin.We demonstrated that expression of ZEB-1 and ZEB-2 was increased after ureter obstruction and that administration of the miR-200b precursor reversed this effect.In summary, these results indicate that miR-200 family is up-regulated after ureter obstruction, miR-200b being strongly induced, and that miR-200b ameliorates tubulointerstitial fibrosis in obstructed kidneys.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology and Endocrinology, University of Tokyo School of Medicine, Tokyo, Japan. OBA-2IM@h.u-tokyo.ac.jp

ABSTRACT
Members of the miR-200 family of micro RNAs (miRNAs) have been shown to inhibit epithelial-mesenchymal transition (EMT). EMT of tubular epithelial cells is the mechanism by which renal fibroblasts are generated. Here we show that miR-200 family members inhibit transforming growth factor-beta (TGF-beta)-induced EMT of tubular cells. Unilateral ureter obstruction (UUO) is a common model of EMT of tubular cells and subsequent tubulointerstitial fibrosis. In order to examine the role of miR-200 family members in tubulointerstitial fibrosis, their expression was investigated in the kidneys of UUO mice. The expression of miR-200 family miRNAs was increased in a time-dependent manner, with induction of miR-200b most pronounced. To clarify the effect of miR-200b on tubulointerstitial fibrosis, we injected miR-200b precursor intravenously. A single injection of 0.5 nM miR-200b precursor was sufficient to inhibit the increase of collagen types I, III and fibronectin in obstructed kidneys, and amelioration of fibrosis was confirmed by observation of the kidneys with Azan staining. miR-200 family members have been previously shown to inhibit EMT by reducing the expression of ZEB-1 and ZEB-2 which are known repressors of E-cadherin. We demonstrated that expression of ZEB-1 and ZEB-2 was increased after ureter obstruction and that administration of the miR-200b precursor reversed this effect. In summary, these results indicate that miR-200 family is up-regulated after ureter obstruction, miR-200b being strongly induced, and that miR-200b ameliorates tubulointerstitial fibrosis in obstructed kidneys. We suggest that members of the miR-200 family, and miR-200b specifically, might constitute novel therapeutic targets in kidney disease.

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miR-200 family members can ameliorate EMT and tubulointerstitial fibrosis in UUO model mouse kidneys.a, Western blotting analysis confirms down-regulation of E-cadherin and up-regulation of N-cadherin during EMT. b,c, Western blotting of E-cadherin and N-cadherin in HK-2 cells treated with TGF-beta and transfected with control miR or miR-200 members precursors individually. In western blotting, each band has been quantitated and subjected to densitometry to determine if statistically significant difference exists between groups d, Changes in miR-200 family levels in UUO model mice kidneys, as measured by TaqMan qRT-PCR and normalized to U6 expression. The data are means from a representative time course experiment measured in triplicate and are presented as mean±S.E. e, Azan staining of intact Balbc mouse kidney, UUO day-6 kidneys after transfection of negative control pre-miR or miR-200b precursor. Fibrosis score; The tubulointerstitial fibrosis score in kidneys of miR-200b precursor injected group was significantly lower than of control group: P<0.05, The data are means from experiment measured in triplicate and are presented as mean±S.E (n = 8) f, Immunofluorescence studies of vimentin and cytokeratin 18 in the kidneys of UUO day6 mice and UUO day6 mice injected by miR-200b precursor. miR-200b ameliorates up-regulation of vimentin and down-regulation of cytokeratin 18. g, real-time PCR confirms that miR-200b precursor reverses up-regulation of type I,III collagen and fibronectin of UUO day-6 kidneys. The data are normalized to beta-actin mRNA and shown as mean±S.E.(n = 8).
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pone-0013614-g001: miR-200 family members can ameliorate EMT and tubulointerstitial fibrosis in UUO model mouse kidneys.a, Western blotting analysis confirms down-regulation of E-cadherin and up-regulation of N-cadherin during EMT. b,c, Western blotting of E-cadherin and N-cadherin in HK-2 cells treated with TGF-beta and transfected with control miR or miR-200 members precursors individually. In western blotting, each band has been quantitated and subjected to densitometry to determine if statistically significant difference exists between groups d, Changes in miR-200 family levels in UUO model mice kidneys, as measured by TaqMan qRT-PCR and normalized to U6 expression. The data are means from a representative time course experiment measured in triplicate and are presented as mean±S.E. e, Azan staining of intact Balbc mouse kidney, UUO day-6 kidneys after transfection of negative control pre-miR or miR-200b precursor. Fibrosis score; The tubulointerstitial fibrosis score in kidneys of miR-200b precursor injected group was significantly lower than of control group: P<0.05, The data are means from experiment measured in triplicate and are presented as mean±S.E (n = 8) f, Immunofluorescence studies of vimentin and cytokeratin 18 in the kidneys of UUO day6 mice and UUO day6 mice injected by miR-200b precursor. miR-200b ameliorates up-regulation of vimentin and down-regulation of cytokeratin 18. g, real-time PCR confirms that miR-200b precursor reverses up-regulation of type I,III collagen and fibronectin of UUO day-6 kidneys. The data are normalized to beta-actin mRNA and shown as mean±S.E.(n = 8).

Mentions: Initial western blotting analysis demonstrated TGF-beta-mediated repression of E-cadherin, and induction of N-cadherin, in HK-2 kidney tubular cells (Fig. 1a). All miR-200 family precursors tested were capable of inhibiting the reduction of E-cadherin and up-regulation of N-cadherin affected by TGF-beta in HK-2 cells (Fig. 1b, c).


miR-200b precursor can ameliorate renal tubulointerstitial fibrosis.

Oba S, Kumano S, Suzuki E, Nishimatsu H, Takahashi M, Takamori H, Kasuya M, Ogawa Y, Sato K, Kimura K, Homma Y, Hirata Y, Fujita T - PLoS ONE (2010)

miR-200 family members can ameliorate EMT and tubulointerstitial fibrosis in UUO model mouse kidneys.a, Western blotting analysis confirms down-regulation of E-cadherin and up-regulation of N-cadherin during EMT. b,c, Western blotting of E-cadherin and N-cadherin in HK-2 cells treated with TGF-beta and transfected with control miR or miR-200 members precursors individually. In western blotting, each band has been quantitated and subjected to densitometry to determine if statistically significant difference exists between groups d, Changes in miR-200 family levels in UUO model mice kidneys, as measured by TaqMan qRT-PCR and normalized to U6 expression. The data are means from a representative time course experiment measured in triplicate and are presented as mean±S.E. e, Azan staining of intact Balbc mouse kidney, UUO day-6 kidneys after transfection of negative control pre-miR or miR-200b precursor. Fibrosis score; The tubulointerstitial fibrosis score in kidneys of miR-200b precursor injected group was significantly lower than of control group: P<0.05, The data are means from experiment measured in triplicate and are presented as mean±S.E (n = 8) f, Immunofluorescence studies of vimentin and cytokeratin 18 in the kidneys of UUO day6 mice and UUO day6 mice injected by miR-200b precursor. miR-200b ameliorates up-regulation of vimentin and down-regulation of cytokeratin 18. g, real-time PCR confirms that miR-200b precursor reverses up-regulation of type I,III collagen and fibronectin of UUO day-6 kidneys. The data are normalized to beta-actin mRNA and shown as mean±S.E.(n = 8).
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pone-0013614-g001: miR-200 family members can ameliorate EMT and tubulointerstitial fibrosis in UUO model mouse kidneys.a, Western blotting analysis confirms down-regulation of E-cadherin and up-regulation of N-cadherin during EMT. b,c, Western blotting of E-cadherin and N-cadherin in HK-2 cells treated with TGF-beta and transfected with control miR or miR-200 members precursors individually. In western blotting, each band has been quantitated and subjected to densitometry to determine if statistically significant difference exists between groups d, Changes in miR-200 family levels in UUO model mice kidneys, as measured by TaqMan qRT-PCR and normalized to U6 expression. The data are means from a representative time course experiment measured in triplicate and are presented as mean±S.E. e, Azan staining of intact Balbc mouse kidney, UUO day-6 kidneys after transfection of negative control pre-miR or miR-200b precursor. Fibrosis score; The tubulointerstitial fibrosis score in kidneys of miR-200b precursor injected group was significantly lower than of control group: P<0.05, The data are means from experiment measured in triplicate and are presented as mean±S.E (n = 8) f, Immunofluorescence studies of vimentin and cytokeratin 18 in the kidneys of UUO day6 mice and UUO day6 mice injected by miR-200b precursor. miR-200b ameliorates up-regulation of vimentin and down-regulation of cytokeratin 18. g, real-time PCR confirms that miR-200b precursor reverses up-regulation of type I,III collagen and fibronectin of UUO day-6 kidneys. The data are normalized to beta-actin mRNA and shown as mean±S.E.(n = 8).
Mentions: Initial western blotting analysis demonstrated TGF-beta-mediated repression of E-cadherin, and induction of N-cadherin, in HK-2 kidney tubular cells (Fig. 1a). All miR-200 family precursors tested were capable of inhibiting the reduction of E-cadherin and up-regulation of N-cadherin affected by TGF-beta in HK-2 cells (Fig. 1b, c).

Bottom Line: A single injection of 0.5 nM miR-200b precursor was sufficient to inhibit the increase of collagen types I, III and fibronectin in obstructed kidneys, and amelioration of fibrosis was confirmed by observation of the kidneys with Azan staining. miR-200 family members have been previously shown to inhibit EMT by reducing the expression of ZEB-1 and ZEB-2 which are known repressors of E-cadherin.We demonstrated that expression of ZEB-1 and ZEB-2 was increased after ureter obstruction and that administration of the miR-200b precursor reversed this effect.In summary, these results indicate that miR-200 family is up-regulated after ureter obstruction, miR-200b being strongly induced, and that miR-200b ameliorates tubulointerstitial fibrosis in obstructed kidneys.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology and Endocrinology, University of Tokyo School of Medicine, Tokyo, Japan. OBA-2IM@h.u-tokyo.ac.jp

ABSTRACT
Members of the miR-200 family of micro RNAs (miRNAs) have been shown to inhibit epithelial-mesenchymal transition (EMT). EMT of tubular epithelial cells is the mechanism by which renal fibroblasts are generated. Here we show that miR-200 family members inhibit transforming growth factor-beta (TGF-beta)-induced EMT of tubular cells. Unilateral ureter obstruction (UUO) is a common model of EMT of tubular cells and subsequent tubulointerstitial fibrosis. In order to examine the role of miR-200 family members in tubulointerstitial fibrosis, their expression was investigated in the kidneys of UUO mice. The expression of miR-200 family miRNAs was increased in a time-dependent manner, with induction of miR-200b most pronounced. To clarify the effect of miR-200b on tubulointerstitial fibrosis, we injected miR-200b precursor intravenously. A single injection of 0.5 nM miR-200b precursor was sufficient to inhibit the increase of collagen types I, III and fibronectin in obstructed kidneys, and amelioration of fibrosis was confirmed by observation of the kidneys with Azan staining. miR-200 family members have been previously shown to inhibit EMT by reducing the expression of ZEB-1 and ZEB-2 which are known repressors of E-cadherin. We demonstrated that expression of ZEB-1 and ZEB-2 was increased after ureter obstruction and that administration of the miR-200b precursor reversed this effect. In summary, these results indicate that miR-200 family is up-regulated after ureter obstruction, miR-200b being strongly induced, and that miR-200b ameliorates tubulointerstitial fibrosis in obstructed kidneys. We suggest that members of the miR-200 family, and miR-200b specifically, might constitute novel therapeutic targets in kidney disease.

Show MeSH
Related in: MedlinePlus