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Full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes.

Alfano M, Cinque P, Giusti G, Proietti S, Nebuloni M, Danese S, D'Alessio S, Genua M, Portale F, Lo Porto M, Singhal PC, Rastaldi MP, Saleem MA, Mavilio D, Mikulak J - Sci Rep (2015)

Bottom Line: This phenomenon is mediated only by full-length suPAR, is time-and dose-dependent and is associated with the suppression of Wilms' tumor 1 (WT-1) transcription factor expression.Moreover, an antagonist of αvβ3 integrin RGDfv blocked suPAR-induced suppression of nephrin.This study unveiled that interaction of full-length suPAR with αvβ3 integrin expressed on podocytes results in down-modulation of nephrin that may affect kidney functionality in different human pathologies characterized by increased concentration of suPAR.

View Article: PubMed Central - PubMed

Affiliation: Division of Oncology, Urological Research Institute URI; IRCCS Ospedale San Raffaele, Milan, Italy.

ABSTRACT
Increased plasma level of soluble urokinase-type plasminogen activator receptor (suPAR) was associated recently with focal segmental glomerulosclerosis (FSGS). In addition, different clinical studies observed increased concentration of suPAR in various glomerular diseases and in other human pathologies with nephrotic syndromes such as HIV and Hantavirus infection, diabetes and cardiovascular disorders. Here, we show that suPAR induces nephrin down-modulation in human podocytes. This phenomenon is mediated only by full-length suPAR, is time-and dose-dependent and is associated with the suppression of Wilms' tumor 1 (WT-1) transcription factor expression. Moreover, an antagonist of αvβ3 integrin RGDfv blocked suPAR-induced suppression of nephrin. These in vitro data were confirmed in an in vivo uPAR knock out Plaur(-/-) mice model by demonstrating that the infusion of suPAR inhibits expression of nephrin and WT-1 in podocytes and induces proteinuria. This study unveiled that interaction of full-length suPAR with αvβ3 integrin expressed on podocytes results in down-modulation of nephrin that may affect kidney functionality in different human pathologies characterized by increased concentration of suPAR.

No MeSH data available.


Related in: MedlinePlus

SuPAR induces lower WT-1 binding to the Nephrin gene promoter.(a) Chromatin immunoprecipitation (ChIP) analysis of WT-1 binding to the cis region of Nphs1 gene promoter in CIHPs. Results are represented as fold change in the enrichment of precipitated chromatin fragments of Nphs1 gene promoter with either anti-WT-1 Ab or Rabbit Normal IgG in non treated (Mock) or treated with suPAR cells. Precipitated products were amplified by qPCR by using SYBR Green assay and normalized to DNA lacking any WT-1 site, located in the promoter of GAPDH gene of Input. (b) Binding of the specific anti-RNA polymerase II (CTD4H8) Ab to the DNA fragment of GAPDH gene was used as the positive control (Ctrl). Amplification of GAPDH gene promoter in the IgG and WT-1 chromatin immunoprecipitated of Mock and su-PAR treated samples were used as a negative Ctrl. ChIP samples were analyzed in triplicate and represented the average of 3 independent experiments ±SD. (c) QPCR analysis of WT-1 gene expression in Jurkat and K562 and CIHPs cell lines. TaqMan assays of 3 independent experiments ±SD. Values were normalized to the expression of GAPDH gene. (d) Detection of WT-1 protein by Western blotting assay (WB) after the chromatin immunoprecipitation with IgG control or anti-WT-1 antibodies in Jurkat and K562 cells. Analysis of β-actin of Inputs were used for normalization. One representative cropped blot out of two is shown. Statistical significance (P) is indicated by asterisks and is represented as: *<0.05; **<0.01; ***<0.001.
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f5: SuPAR induces lower WT-1 binding to the Nephrin gene promoter.(a) Chromatin immunoprecipitation (ChIP) analysis of WT-1 binding to the cis region of Nphs1 gene promoter in CIHPs. Results are represented as fold change in the enrichment of precipitated chromatin fragments of Nphs1 gene promoter with either anti-WT-1 Ab or Rabbit Normal IgG in non treated (Mock) or treated with suPAR cells. Precipitated products were amplified by qPCR by using SYBR Green assay and normalized to DNA lacking any WT-1 site, located in the promoter of GAPDH gene of Input. (b) Binding of the specific anti-RNA polymerase II (CTD4H8) Ab to the DNA fragment of GAPDH gene was used as the positive control (Ctrl). Amplification of GAPDH gene promoter in the IgG and WT-1 chromatin immunoprecipitated of Mock and su-PAR treated samples were used as a negative Ctrl. ChIP samples were analyzed in triplicate and represented the average of 3 independent experiments ±SD. (c) QPCR analysis of WT-1 gene expression in Jurkat and K562 and CIHPs cell lines. TaqMan assays of 3 independent experiments ±SD. Values were normalized to the expression of GAPDH gene. (d) Detection of WT-1 protein by Western blotting assay (WB) after the chromatin immunoprecipitation with IgG control or anti-WT-1 antibodies in Jurkat and K562 cells. Analysis of β-actin of Inputs were used for normalization. One representative cropped blot out of two is shown. Statistical significance (P) is indicated by asterisks and is represented as: *<0.05; **<0.01; ***<0.001.

Mentions: Different activating and suppressing transcription factors have been identified as being involved in the transcriptional regulation of Nephrin gene expression363738. The most documented transcription factor involved in the regulation of Nphs1 gene expression is WT-1. Knockout, transgenic, and siRNA analyses have demonstrated the importance of WT-1 at several stages of kidney development394041. Moreover, expression of WT-1 continues in podocytes of adult kidneys and is required for physiological levels of nephrin expression. Consistent with this observation, it was demonstrated that nephrin expression in podocytes is lower in the kidneys of mice with reduced expression of WT-13742. In addition, both in vitro and in vivo functional approaches have shown that WT-1 can bind and activate the nephrin promoter and that this binding is essential for nephrin-specific expression in vivo3743. Since our results showed suPAR-dependent down-regulation of nephrin at transcription level, we assessed whether WT-1 is involved in this process. QPCR analysis of WT-1 transcription showed a statistically significant decrease of WT-1 expression in CIHPs after treatment with fl-suPAR (Fig. 4b), thus indicating that WT-1 is a possible target of an activated suPAR-αvβ3 signaling down-stream pathway. In line with the kinetic observed for the down-modulation of nephrin (Fig. 4a), experiments performed with different concentrations of cycloRGDfv inhibitor revealed a full restoration of WT-1 transcripts after pre-treatment with 10 μM of cycloRGDfv (Fig. 4b), while we still could observe a lower but significant inhibition of nephrin incubating CIHPs with 5 μM of the αvβ3 inhibitor. To assess the specific suPAR-dependent attenuating role of WT-1 transcription factor in nephrin gene expression we evaluated the binding of WT-1 in the promoter region of Nphs1 by chromatin immunoprecipitation assay (ChIP). We detected a significantly lower binding of WT-1 protein in the regulatory region of Nphs1 gene in fl-suPAR treated cells compared to the non-stimulated podocytes (Fig. 5a). Amplification of GAPDH promoter was used as a positive control in CIHPs by immunoprecipitation of chromatin with anti-RNA polymerase II antibody (Fig. 5b). GAPDH promoter is lacking any WT-1 site thus as a negative control we amplified GAPDH promoter after chromatin immunoprecipitation with IgG or anti-WT-1 antibody in Mock and suPAR treated samples, however, we did not observe any significant changes (Fig. 5b). Specificity of the anti-WT-1 antibody used in ChIP assay was tested in the Jurkat and in K562 cell lines, known to be respectively negative and positive cells for WT-1 expression (Fig. 5c–d)4445. These data strongly indicate that the suPAR-dependent down regulation of nephrin might occur through decreased transcription levels of WT-1 factor resulting in the attenuated binding to Nphs1 gene promoter and thus lower transcription of nephrin. In addition, we analyzed the expression of the transcription regulator Snail that has recently been proposed as a repressor of Nephrin gene expression38. However, although we found detectable levels of Snail in CIHPs, we did not measure any increased amount of Snail after suPAR treatment (data not shown).


Full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes.

Alfano M, Cinque P, Giusti G, Proietti S, Nebuloni M, Danese S, D'Alessio S, Genua M, Portale F, Lo Porto M, Singhal PC, Rastaldi MP, Saleem MA, Mavilio D, Mikulak J - Sci Rep (2015)

SuPAR induces lower WT-1 binding to the Nephrin gene promoter.(a) Chromatin immunoprecipitation (ChIP) analysis of WT-1 binding to the cis region of Nphs1 gene promoter in CIHPs. Results are represented as fold change in the enrichment of precipitated chromatin fragments of Nphs1 gene promoter with either anti-WT-1 Ab or Rabbit Normal IgG in non treated (Mock) or treated with suPAR cells. Precipitated products were amplified by qPCR by using SYBR Green assay and normalized to DNA lacking any WT-1 site, located in the promoter of GAPDH gene of Input. (b) Binding of the specific anti-RNA polymerase II (CTD4H8) Ab to the DNA fragment of GAPDH gene was used as the positive control (Ctrl). Amplification of GAPDH gene promoter in the IgG and WT-1 chromatin immunoprecipitated of Mock and su-PAR treated samples were used as a negative Ctrl. ChIP samples were analyzed in triplicate and represented the average of 3 independent experiments ±SD. (c) QPCR analysis of WT-1 gene expression in Jurkat and K562 and CIHPs cell lines. TaqMan assays of 3 independent experiments ±SD. Values were normalized to the expression of GAPDH gene. (d) Detection of WT-1 protein by Western blotting assay (WB) after the chromatin immunoprecipitation with IgG control or anti-WT-1 antibodies in Jurkat and K562 cells. Analysis of β-actin of Inputs were used for normalization. One representative cropped blot out of two is shown. Statistical significance (P) is indicated by asterisks and is represented as: *<0.05; **<0.01; ***<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4585377&req=5

f5: SuPAR induces lower WT-1 binding to the Nephrin gene promoter.(a) Chromatin immunoprecipitation (ChIP) analysis of WT-1 binding to the cis region of Nphs1 gene promoter in CIHPs. Results are represented as fold change in the enrichment of precipitated chromatin fragments of Nphs1 gene promoter with either anti-WT-1 Ab or Rabbit Normal IgG in non treated (Mock) or treated with suPAR cells. Precipitated products were amplified by qPCR by using SYBR Green assay and normalized to DNA lacking any WT-1 site, located in the promoter of GAPDH gene of Input. (b) Binding of the specific anti-RNA polymerase II (CTD4H8) Ab to the DNA fragment of GAPDH gene was used as the positive control (Ctrl). Amplification of GAPDH gene promoter in the IgG and WT-1 chromatin immunoprecipitated of Mock and su-PAR treated samples were used as a negative Ctrl. ChIP samples were analyzed in triplicate and represented the average of 3 independent experiments ±SD. (c) QPCR analysis of WT-1 gene expression in Jurkat and K562 and CIHPs cell lines. TaqMan assays of 3 independent experiments ±SD. Values were normalized to the expression of GAPDH gene. (d) Detection of WT-1 protein by Western blotting assay (WB) after the chromatin immunoprecipitation with IgG control or anti-WT-1 antibodies in Jurkat and K562 cells. Analysis of β-actin of Inputs were used for normalization. One representative cropped blot out of two is shown. Statistical significance (P) is indicated by asterisks and is represented as: *<0.05; **<0.01; ***<0.001.
Mentions: Different activating and suppressing transcription factors have been identified as being involved in the transcriptional regulation of Nephrin gene expression363738. The most documented transcription factor involved in the regulation of Nphs1 gene expression is WT-1. Knockout, transgenic, and siRNA analyses have demonstrated the importance of WT-1 at several stages of kidney development394041. Moreover, expression of WT-1 continues in podocytes of adult kidneys and is required for physiological levels of nephrin expression. Consistent with this observation, it was demonstrated that nephrin expression in podocytes is lower in the kidneys of mice with reduced expression of WT-13742. In addition, both in vitro and in vivo functional approaches have shown that WT-1 can bind and activate the nephrin promoter and that this binding is essential for nephrin-specific expression in vivo3743. Since our results showed suPAR-dependent down-regulation of nephrin at transcription level, we assessed whether WT-1 is involved in this process. QPCR analysis of WT-1 transcription showed a statistically significant decrease of WT-1 expression in CIHPs after treatment with fl-suPAR (Fig. 4b), thus indicating that WT-1 is a possible target of an activated suPAR-αvβ3 signaling down-stream pathway. In line with the kinetic observed for the down-modulation of nephrin (Fig. 4a), experiments performed with different concentrations of cycloRGDfv inhibitor revealed a full restoration of WT-1 transcripts after pre-treatment with 10 μM of cycloRGDfv (Fig. 4b), while we still could observe a lower but significant inhibition of nephrin incubating CIHPs with 5 μM of the αvβ3 inhibitor. To assess the specific suPAR-dependent attenuating role of WT-1 transcription factor in nephrin gene expression we evaluated the binding of WT-1 in the promoter region of Nphs1 by chromatin immunoprecipitation assay (ChIP). We detected a significantly lower binding of WT-1 protein in the regulatory region of Nphs1 gene in fl-suPAR treated cells compared to the non-stimulated podocytes (Fig. 5a). Amplification of GAPDH promoter was used as a positive control in CIHPs by immunoprecipitation of chromatin with anti-RNA polymerase II antibody (Fig. 5b). GAPDH promoter is lacking any WT-1 site thus as a negative control we amplified GAPDH promoter after chromatin immunoprecipitation with IgG or anti-WT-1 antibody in Mock and suPAR treated samples, however, we did not observe any significant changes (Fig. 5b). Specificity of the anti-WT-1 antibody used in ChIP assay was tested in the Jurkat and in K562 cell lines, known to be respectively negative and positive cells for WT-1 expression (Fig. 5c–d)4445. These data strongly indicate that the suPAR-dependent down regulation of nephrin might occur through decreased transcription levels of WT-1 factor resulting in the attenuated binding to Nphs1 gene promoter and thus lower transcription of nephrin. In addition, we analyzed the expression of the transcription regulator Snail that has recently been proposed as a repressor of Nephrin gene expression38. However, although we found detectable levels of Snail in CIHPs, we did not measure any increased amount of Snail after suPAR treatment (data not shown).

Bottom Line: This phenomenon is mediated only by full-length suPAR, is time-and dose-dependent and is associated with the suppression of Wilms' tumor 1 (WT-1) transcription factor expression.Moreover, an antagonist of αvβ3 integrin RGDfv blocked suPAR-induced suppression of nephrin.This study unveiled that interaction of full-length suPAR with αvβ3 integrin expressed on podocytes results in down-modulation of nephrin that may affect kidney functionality in different human pathologies characterized by increased concentration of suPAR.

View Article: PubMed Central - PubMed

Affiliation: Division of Oncology, Urological Research Institute URI; IRCCS Ospedale San Raffaele, Milan, Italy.

ABSTRACT
Increased plasma level of soluble urokinase-type plasminogen activator receptor (suPAR) was associated recently with focal segmental glomerulosclerosis (FSGS). In addition, different clinical studies observed increased concentration of suPAR in various glomerular diseases and in other human pathologies with nephrotic syndromes such as HIV and Hantavirus infection, diabetes and cardiovascular disorders. Here, we show that suPAR induces nephrin down-modulation in human podocytes. This phenomenon is mediated only by full-length suPAR, is time-and dose-dependent and is associated with the suppression of Wilms' tumor 1 (WT-1) transcription factor expression. Moreover, an antagonist of αvβ3 integrin RGDfv blocked suPAR-induced suppression of nephrin. These in vitro data were confirmed in an in vivo uPAR knock out Plaur(-/-) mice model by demonstrating that the infusion of suPAR inhibits expression of nephrin and WT-1 in podocytes and induces proteinuria. This study unveiled that interaction of full-length suPAR with αvβ3 integrin expressed on podocytes results in down-modulation of nephrin that may affect kidney functionality in different human pathologies characterized by increased concentration of suPAR.

No MeSH data available.


Related in: MedlinePlus