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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 mediated down-regulation of nephrin depends on αvβ3 integrin interaction and is associated with reduced activity of WT-1.(a–b) Quantification of qPCR analysis of nephrin (a) and WT-1 (b) expression in Mock and suPAR treated (20 ng/mL) for 24 hours in CIHPs pre-incubated with different concentration (1, 5 and 10 μM) of αvβ3 antagonist (RGDfv). Results obtained by using the specify human TaqMan assays are expressed as relative fold change in treated cells vs. mock cells (ΔΔCt) and represent the average of 3 independent experiments ±SD. Values were normalized to the expression of GAPDH gene. Right upper picture of panel A shows one representative immunoflourescence staining out of 3 of αvβ3 (594 Alexa Fluor) integrin expression in red and nucleus in blue (DAPI) in CIHPs.
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f4: SuPAR mediated down-regulation of nephrin depends on αvβ3 integrin interaction and is associated with reduced activity of WT-1.(a–b) Quantification of qPCR analysis of nephrin (a) and WT-1 (b) expression in Mock and suPAR treated (20 ng/mL) for 24 hours in CIHPs pre-incubated with different concentration (1, 5 and 10 μM) of αvβ3 antagonist (RGDfv). Results obtained by using the specify human TaqMan assays are expressed as relative fold change in treated cells vs. mock cells (ΔΔCt) and represent the average of 3 independent experiments ±SD. Values were normalized to the expression of GAPDH gene. Right upper picture of panel A shows one representative immunoflourescence staining out of 3 of αvβ3 (594 Alexa Fluor) integrin expression in red and nucleus in blue (DAPI) in CIHPs.

Mentions: It has been demonstrated that suPAR binds and activates αvβ3 integrin in human podocytes8. In order to understand whether the αvβ3 integrin molecule is involved in suPAR dependent down-regulation of nephrin we used the αvβ3 small molecule-inhibitor cycloRGDfv8. Pre-treatment of CIHPs constitutively expressing αvβ3 integrin with cycloRGDfv prior stimulation with fl-suPAR resulted in a significant inhibition of suPAR-dependent downmodulation of nephrin at the transcription level (Fig. 4a). Moreover, qPCR analysis revealed that the effect of cycloRGDfv on nephrin expression on CIHPs incubated with suPAR is dose dependent, since incubation with 5 μM and 10 μM of this αvβ3 inhibitor, respectively, partially or fully restored the amount of nephrin transcripts compared to control experiments. These findings suggest that suPAR is able to induce the down-modulation of nephrin in CIHPs via αvβ3 interaction


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 mediated down-regulation of nephrin depends on αvβ3 integrin interaction and is associated with reduced activity of WT-1.(a–b) Quantification of qPCR analysis of nephrin (a) and WT-1 (b) expression in Mock and suPAR treated (20 ng/mL) for 24 hours in CIHPs pre-incubated with different concentration (1, 5 and 10 μM) of αvβ3 antagonist (RGDfv). Results obtained by using the specify human TaqMan assays are expressed as relative fold change in treated cells vs. mock cells (ΔΔCt) and represent the average of 3 independent experiments ±SD. Values were normalized to the expression of GAPDH gene. Right upper picture of panel A shows one representative immunoflourescence staining out of 3 of αvβ3 (594 Alexa Fluor) integrin expression in red and nucleus in blue (DAPI) in CIHPs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585377&req=5

f4: SuPAR mediated down-regulation of nephrin depends on αvβ3 integrin interaction and is associated with reduced activity of WT-1.(a–b) Quantification of qPCR analysis of nephrin (a) and WT-1 (b) expression in Mock and suPAR treated (20 ng/mL) for 24 hours in CIHPs pre-incubated with different concentration (1, 5 and 10 μM) of αvβ3 antagonist (RGDfv). Results obtained by using the specify human TaqMan assays are expressed as relative fold change in treated cells vs. mock cells (ΔΔCt) and represent the average of 3 independent experiments ±SD. Values were normalized to the expression of GAPDH gene. Right upper picture of panel A shows one representative immunoflourescence staining out of 3 of αvβ3 (594 Alexa Fluor) integrin expression in red and nucleus in blue (DAPI) in CIHPs.
Mentions: It has been demonstrated that suPAR binds and activates αvβ3 integrin in human podocytes8. In order to understand whether the αvβ3 integrin molecule is involved in suPAR dependent down-regulation of nephrin we used the αvβ3 small molecule-inhibitor cycloRGDfv8. Pre-treatment of CIHPs constitutively expressing αvβ3 integrin with cycloRGDfv prior stimulation with fl-suPAR resulted in a significant inhibition of suPAR-dependent downmodulation of nephrin at the transcription level (Fig. 4a). Moreover, qPCR analysis revealed that the effect of cycloRGDfv on nephrin expression on CIHPs incubated with suPAR is dose dependent, since incubation with 5 μM and 10 μM of this αvβ3 inhibitor, respectively, partially or fully restored the amount of nephrin transcripts compared to control experiments. These findings suggest that suPAR is able to induce the down-modulation of nephrin in CIHPs via αvβ3 interaction

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