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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.

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Related in: MedlinePlus

Full length DIDIIDIII suPAR down-regulates nephrin expression in CIHPs.(a) QPCR analysis using specific human nephrin TaqMan assay in Mock and treated human podocytes with different concentration of full lengh suPAR (fl-suPAR) or short suPAR (c-suPAR) variants of suPAR. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and normalized to the expression of GAPDH gene. Results are represent as the average of 3 independent experiments ±SD. (b) Quantification of immunoflourescence staining (left panel) of nephrin expression in control (Mock) and treated human podocytes for 24 hours with 0.4 nM of the full-length (fl-suPAR) or cleaved (c-suPAR) variants of suPAR. Results are expressed as MFI/cell and represent the average of 3 experiments ±SD. Right picture shows one representative immunoflourescence staining out of 3 of nephrin expression (488 Alexa Fluor) in green and nucleus (DAPI) in blue.Statistical significance (P) is indicated by asterisks and is represented as: **<0.01; ***<0.001.
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f3: Full length DIDIIDIII suPAR down-regulates nephrin expression in CIHPs.(a) QPCR analysis using specific human nephrin TaqMan assay in Mock and treated human podocytes with different concentration of full lengh suPAR (fl-suPAR) or short suPAR (c-suPAR) variants of suPAR. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and normalized to the expression of GAPDH gene. Results are represent as the average of 3 independent experiments ±SD. (b) Quantification of immunoflourescence staining (left panel) of nephrin expression in control (Mock) and treated human podocytes for 24 hours with 0.4 nM of the full-length (fl-suPAR) or cleaved (c-suPAR) variants of suPAR. Results are expressed as MFI/cell and represent the average of 3 experiments ±SD. Right picture shows one representative immunoflourescence staining out of 3 of nephrin expression (488 Alexa Fluor) in green and nucleus (DAPI) in blue.Statistical significance (P) is indicated by asterisks and is represented as: **<0.01; ***<0.001.

Mentions: Structurally, uPAR is a GPI-anchored membrane glycoprotein consisting of three homologous domains (DIDIIDIII)35. Cleavage of uPAR from the cell membrane is catalyzed by various enzymes and can occur both at the GPI-anchor and the linker region between DI and DII. The suPAR resulting from cleavage may thus consist of domain DIDIIDIII, DIIDIII or DI. Since the increased level of plasma-associated suPAR in human disorders reflects the increased concentration of both DIDIIDIII (fl-suPAR) and DIIDIII (c-suPAR) variants, we evaluated the contribution of suPAR variants in down-modulation of nephrin in CIHPs46. To compare the dose-dependent effects of fl-suPAR and c-suPAR variants on nephrin inhibition, two suPAR variants were tested in the range of concentration between 0.4–0.02 nM that resulted with the previously observed maximum inhibitory effect of fl-suPAR (Fig. 2b). Interestingly, only fl-suPAR protein was able to significantly reduce nephrin expression at transcription level (Fig. 3a). In addition immunoflourescence analysis showed a statistically significant reduction of nephrin also at protein level only after stimulation with fl-suPAR and not with its short variant c-suPAR (Fig. 3b). These data provide strong evidences of fl-suPAR and not c-suPAR variant capability to induce nephrin down-regulation in human podocytes.


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)

Full length DIDIIDIII suPAR down-regulates nephrin expression in CIHPs.(a) QPCR analysis using specific human nephrin TaqMan assay in Mock and treated human podocytes with different concentration of full lengh suPAR (fl-suPAR) or short suPAR (c-suPAR) variants of suPAR. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and normalized to the expression of GAPDH gene. Results are represent as the average of 3 independent experiments ±SD. (b) Quantification of immunoflourescence staining (left panel) of nephrin expression in control (Mock) and treated human podocytes for 24 hours with 0.4 nM of the full-length (fl-suPAR) or cleaved (c-suPAR) variants of suPAR. Results are expressed as MFI/cell and represent the average of 3 experiments ±SD. Right picture shows one representative immunoflourescence staining out of 3 of nephrin expression (488 Alexa Fluor) in green and nucleus (DAPI) in blue.Statistical significance (P) is indicated by asterisks and is represented as: **<0.01; ***<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Full length DIDIIDIII suPAR down-regulates nephrin expression in CIHPs.(a) QPCR analysis using specific human nephrin TaqMan assay in Mock and treated human podocytes with different concentration of full lengh suPAR (fl-suPAR) or short suPAR (c-suPAR) variants of suPAR. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and normalized to the expression of GAPDH gene. Results are represent as the average of 3 independent experiments ±SD. (b) Quantification of immunoflourescence staining (left panel) of nephrin expression in control (Mock) and treated human podocytes for 24 hours with 0.4 nM of the full-length (fl-suPAR) or cleaved (c-suPAR) variants of suPAR. Results are expressed as MFI/cell and represent the average of 3 experiments ±SD. Right picture shows one representative immunoflourescence staining out of 3 of nephrin expression (488 Alexa Fluor) in green and nucleus (DAPI) in blue.Statistical significance (P) is indicated by asterisks and is represented as: **<0.01; ***<0.001.
Mentions: Structurally, uPAR is a GPI-anchored membrane glycoprotein consisting of three homologous domains (DIDIIDIII)35. Cleavage of uPAR from the cell membrane is catalyzed by various enzymes and can occur both at the GPI-anchor and the linker region between DI and DII. The suPAR resulting from cleavage may thus consist of domain DIDIIDIII, DIIDIII or DI. Since the increased level of plasma-associated suPAR in human disorders reflects the increased concentration of both DIDIIDIII (fl-suPAR) and DIIDIII (c-suPAR) variants, we evaluated the contribution of suPAR variants in down-modulation of nephrin in CIHPs46. To compare the dose-dependent effects of fl-suPAR and c-suPAR variants on nephrin inhibition, two suPAR variants were tested in the range of concentration between 0.4–0.02 nM that resulted with the previously observed maximum inhibitory effect of fl-suPAR (Fig. 2b). Interestingly, only fl-suPAR protein was able to significantly reduce nephrin expression at transcription level (Fig. 3a). In addition immunoflourescence analysis showed a statistically significant reduction of nephrin also at protein level only after stimulation with fl-suPAR and not with its short variant c-suPAR (Fig. 3b). These data provide strong evidences of fl-suPAR and not c-suPAR variant capability to induce nephrin down-regulation in human podocytes.

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