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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

Injection of high doses of recombinant mouse suPAR into uPAR-knockout (Plaur−/−) mouse model induces down-regulation of nephrin expression.(a) Quantification (left panel) of the ratio between urine total protein (mg)/creatinine (mg) concentration of suPAR treated mice with high dose of 20 μg of mouse recombinant for 24 hours vs control mice (Mock) (N = 3 mice for group). Immune-fluorescence in green (right panel) of suPAR (488 Alexa Fluor) deposit into glomerular tissue of suPAR treated Plaur−/− mice. (b) Quantification (left panel) of immunoflourescence staining of nephrin and synaptopodin expression in Mock and suPAR treated mice. (N = 3 mice for group). DAPI staining was used to determine cell numbers. Data are expressed as average of MFI/cell ±SD. Representative immunoflourescence staining (right panel) of nephrin in green (488 Alexa Fluor), synaptopodin in red (594 Alexa Fluor) and nucleus in blue (DAPI) expression in untreated (Mock) and suPAR treated mice (N = 3 mice for group). (c) QPCR analysis of nephrin and WT-1 expression in Mock and suPAR treated mice obtained by using specific mice TaqMan assays and expressed as relative fold change ±SD vs. mock cells. (N = 3 mice for group). Statistical significance (P) is indicated by asterisks and is represented as: *<0.05; **<0.01; ***<0.001.
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f6: Injection of high doses of recombinant mouse suPAR into uPAR-knockout (Plaur−/−) mouse model induces down-regulation of nephrin expression.(a) Quantification (left panel) of the ratio between urine total protein (mg)/creatinine (mg) concentration of suPAR treated mice with high dose of 20 μg of mouse recombinant for 24 hours vs control mice (Mock) (N = 3 mice for group). Immune-fluorescence in green (right panel) of suPAR (488 Alexa Fluor) deposit into glomerular tissue of suPAR treated Plaur−/− mice. (b) Quantification (left panel) of immunoflourescence staining of nephrin and synaptopodin expression in Mock and suPAR treated mice. (N = 3 mice for group). DAPI staining was used to determine cell numbers. Data are expressed as average of MFI/cell ±SD. Representative immunoflourescence staining (right panel) of nephrin in green (488 Alexa Fluor), synaptopodin in red (594 Alexa Fluor) and nucleus in blue (DAPI) expression in untreated (Mock) and suPAR treated mice (N = 3 mice for group). (c) QPCR analysis of nephrin and WT-1 expression in Mock and suPAR treated mice obtained by using specific mice TaqMan assays and expressed as relative fold change ±SD vs. mock cells. (N = 3 mice for group). Statistical significance (P) is indicated by asterisks and is represented as: *<0.05; **<0.01; ***<0.001.

Mentions: We next examined whether exogenous circulating full-length suPAR could cause nephrin down-modulation in uPAR-knockout (Plaur−/−) mice in which we injected i.v. 20 μg (1 mg/Kg) of murine full-length recombinant mouse suPAR. After 24 hours, we observed an increased level of proteinuria and higher amount of suPAR deposit in the glomeruli of Plaur−/− mice infused with full-length recombinant murine suPAR compared to control experiments (Fig. 6a)8. Moreover, we performed experiments using confocal microscopy in the same experimental setting and we observed a significant down-modulation of nephrin expression with no changes in synaptopodin levels in Plaur−/− mice infused with full-length recombinant murine suPAR compared to control experiments (Fig. 6b). Finally, we confirmed our results obtained in vitro in the in vivo model by showing that the decreased expression of nephrin is associated with the down-modulation of WT-1 in suPAR treated Plaur−/− mice (Fig. 6c). These data demonstrate that suPAR is able to activate a specific repressor-signaling pathway that leads to suppression of WT-1 and Nephrin genes.


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)

Injection of high doses of recombinant mouse suPAR into uPAR-knockout (Plaur−/−) mouse model induces down-regulation of nephrin expression.(a) Quantification (left panel) of the ratio between urine total protein (mg)/creatinine (mg) concentration of suPAR treated mice with high dose of 20 μg of mouse recombinant for 24 hours vs control mice (Mock) (N = 3 mice for group). Immune-fluorescence in green (right panel) of suPAR (488 Alexa Fluor) deposit into glomerular tissue of suPAR treated Plaur−/− mice. (b) Quantification (left panel) of immunoflourescence staining of nephrin and synaptopodin expression in Mock and suPAR treated mice. (N = 3 mice for group). DAPI staining was used to determine cell numbers. Data are expressed as average of MFI/cell ±SD. Representative immunoflourescence staining (right panel) of nephrin in green (488 Alexa Fluor), synaptopodin in red (594 Alexa Fluor) and nucleus in blue (DAPI) expression in untreated (Mock) and suPAR treated mice (N = 3 mice for group). (c) QPCR analysis of nephrin and WT-1 expression in Mock and suPAR treated mice obtained by using specific mice TaqMan assays and expressed as relative fold change ±SD vs. mock cells. (N = 3 mice for group). Statistical significance (P) is indicated by asterisks and is represented as: *<0.05; **<0.01; ***<0.001.
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Related In: Results  -  Collection

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f6: Injection of high doses of recombinant mouse suPAR into uPAR-knockout (Plaur−/−) mouse model induces down-regulation of nephrin expression.(a) Quantification (left panel) of the ratio between urine total protein (mg)/creatinine (mg) concentration of suPAR treated mice with high dose of 20 μg of mouse recombinant for 24 hours vs control mice (Mock) (N = 3 mice for group). Immune-fluorescence in green (right panel) of suPAR (488 Alexa Fluor) deposit into glomerular tissue of suPAR treated Plaur−/− mice. (b) Quantification (left panel) of immunoflourescence staining of nephrin and synaptopodin expression in Mock and suPAR treated mice. (N = 3 mice for group). DAPI staining was used to determine cell numbers. Data are expressed as average of MFI/cell ±SD. Representative immunoflourescence staining (right panel) of nephrin in green (488 Alexa Fluor), synaptopodin in red (594 Alexa Fluor) and nucleus in blue (DAPI) expression in untreated (Mock) and suPAR treated mice (N = 3 mice for group). (c) QPCR analysis of nephrin and WT-1 expression in Mock and suPAR treated mice obtained by using specific mice TaqMan assays and expressed as relative fold change ±SD vs. mock cells. (N = 3 mice for group). Statistical significance (P) is indicated by asterisks and is represented as: *<0.05; **<0.01; ***<0.001.
Mentions: We next examined whether exogenous circulating full-length suPAR could cause nephrin down-modulation in uPAR-knockout (Plaur−/−) mice in which we injected i.v. 20 μg (1 mg/Kg) of murine full-length recombinant mouse suPAR. After 24 hours, we observed an increased level of proteinuria and higher amount of suPAR deposit in the glomeruli of Plaur−/− mice infused with full-length recombinant murine suPAR compared to control experiments (Fig. 6a)8. Moreover, we performed experiments using confocal microscopy in the same experimental setting and we observed a significant down-modulation of nephrin expression with no changes in synaptopodin levels in Plaur−/− mice infused with full-length recombinant murine suPAR compared to control experiments (Fig. 6b). Finally, we confirmed our results obtained in vitro in the in vivo model by showing that the decreased expression of nephrin is associated with the down-modulation of WT-1 in suPAR treated Plaur−/− mice (Fig. 6c). These data demonstrate that suPAR is able to activate a specific repressor-signaling pathway that leads to suppression of WT-1 and Nephrin genes.

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