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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

Down-modulation of nephrin expression both at protein and transcription level in CIHPs.(a) Quantification (left panel) of immunoflourescence staining (right upper panel) of nephrin expression in control (Mock) and CIHPs treated with different human recombinant suPAR for 24 hours (suPAR). Results are expressed as MFI/cell and represent the average of 6 experiments ±SD. DAPI staining was used to determine nuclei number. Results are expressed as MFI/cell and represent the average of 4 experiments ±SD. Right picture shows one representative immunoflourescence staining out of 4 of nephrin expression (488 Alexa Fluor) in green and nucleus (DAPI) in blue. (b) Dose-dependent qPCR analysis of nephrin expression in Mock and suPAR treated human podocytes by using specific human TaqMan assays. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and represent the average of 6 experiments ±SD. Values were normalized to the expression of GAPDH gene. (c) Time course qPCR analysis of nephrin and synaptopodin expression in Mock and suPAR treated human podocytes by using specific human TaqMan assays. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and represent the average of 6 experiments ±SD. Values were normalized to the expression of GAPDH gene. Statistical significance (P) is indicated by asterisks and is represented as: ***<0.001.
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f2: Down-modulation of nephrin expression both at protein and transcription level in CIHPs.(a) Quantification (left panel) of immunoflourescence staining (right upper panel) of nephrin expression in control (Mock) and CIHPs treated with different human recombinant suPAR for 24 hours (suPAR). Results are expressed as MFI/cell and represent the average of 6 experiments ±SD. DAPI staining was used to determine nuclei number. Results are expressed as MFI/cell and represent the average of 4 experiments ±SD. Right picture shows one representative immunoflourescence staining out of 4 of nephrin expression (488 Alexa Fluor) in green and nucleus (DAPI) in blue. (b) Dose-dependent qPCR analysis of nephrin expression in Mock and suPAR treated human podocytes by using specific human TaqMan assays. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and represent the average of 6 experiments ±SD. Values were normalized to the expression of GAPDH gene. (c) Time course qPCR analysis of nephrin and synaptopodin expression in Mock and suPAR treated human podocytes by using specific human TaqMan assays. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and represent the average of 6 experiments ±SD. Values were normalized to the expression of GAPDH gene. Statistical significance (P) is indicated by asterisks and is represented as: ***<0.001.

Mentions: Various, genetic and functional, nephrin defects lead to nephritic syndromes and proteinuria222324252627. To verify the effect of suPAR stimulation on nephrin expression, we used human primary podocytes obtained from renal tissue of patients affected by renal adenocarcinoma who underwent a radical nephrectomy. All tissue specimens used for this study were collected from the distal part of the pathological tissue and were free from any disease as was verified by haematoxylin-eosin-staining (Fig. 1a). We then cultured freshly isolated glomerula in order to obtain human podocytes (Fig. 1a), whose phenotype was verified by qPCR analysis detecting gene expression of the following specific podocytes markers: Wilms’ tumor 1 (WT-1), synaptopodin, nephrin and podocin (data not shown). Mean serum and plasma concentration of suPAR in healthy adults have been reported to be 2 ng/mL18, whereas it reaches 20 ng/mL in patients with various diseases, such as cancer, sepsis, liver disease, HIV infection and FSGS81418192021. We then proceeded to incubate human primary podocytes with 20 ng/mL of human recombinant fl-suPAR. After 24 hours of stimulation we detected by fluorescence microscopy a significant down-modulation of nephrin at the protein level (Fig. 1b). Immunoflourescence staining of frozen tissue from human normal kidney served to confirm the specificity of anti-nephrin rabbit clonal antibody (Ab) used for this study (Fig. 1c). The decreased amount of nephrin following incubation of primary podocytes with suPAR was also confirmed by qPCR, thus suggesting a transcriptional control of nephrin expression by suPAR (Fig. 1d). To both confirm the modulation of nephrin exerted by suPAR and to disclose the related mechanism in a post-mitotic podocyte, we repeated the same experimental approach in conditional immortalized human podocytes (CIHPs) in vitro34. By performing assessments of protein expression with immune-flourescence in CIHPs incubated with different concentrations of suPAR for 24 hours, we detected a significant reduction of nephrin flourescence intensity and thus a lower level of nephrin protein in suPAR stimulated CIHPs comparing to control experiments, with the maximum inhibition between 10–20 ng/mL of suPAR (Fig. 2a). To determine whether suPAR stimulation affects nephrin expression at the transcriptional level, we performed qPCR experiments. As shown in Fig. 2b, quantification of nephrin mRNA in CIHPs incubated with suPAR showed a rapid and progressive decrease of Nephrin gene expression (Fig. 2a–c). The reduction of nephrin transcripts was already detectable after 3 hours and became significant after 6 hours of treatment, when it also reached a plateau that was maintained even after 24 hours of stimulation. However, we did not observe any significant suPAR-mediated down-modulation of the expression of synaptopodin, another specific podocyte marker, thus indicating the specificity of suPAR in suppressing nephrin expression in CIHPs (Fig. 2c).


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)

Down-modulation of nephrin expression both at protein and transcription level in CIHPs.(a) Quantification (left panel) of immunoflourescence staining (right upper panel) of nephrin expression in control (Mock) and CIHPs treated with different human recombinant suPAR for 24 hours (suPAR). Results are expressed as MFI/cell and represent the average of 6 experiments ±SD. DAPI staining was used to determine nuclei number. Results are expressed as MFI/cell and represent the average of 4 experiments ±SD. Right picture shows one representative immunoflourescence staining out of 4 of nephrin expression (488 Alexa Fluor) in green and nucleus (DAPI) in blue. (b) Dose-dependent qPCR analysis of nephrin expression in Mock and suPAR treated human podocytes by using specific human TaqMan assays. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and represent the average of 6 experiments ±SD. Values were normalized to the expression of GAPDH gene. (c) Time course qPCR analysis of nephrin and synaptopodin expression in Mock and suPAR treated human podocytes by using specific human TaqMan assays. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and represent the average of 6 experiments ±SD. Values were normalized to the expression of GAPDH gene. Statistical significance (P) is indicated by asterisks and is represented as: ***<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f2: Down-modulation of nephrin expression both at protein and transcription level in CIHPs.(a) Quantification (left panel) of immunoflourescence staining (right upper panel) of nephrin expression in control (Mock) and CIHPs treated with different human recombinant suPAR for 24 hours (suPAR). Results are expressed as MFI/cell and represent the average of 6 experiments ±SD. DAPI staining was used to determine nuclei number. Results are expressed as MFI/cell and represent the average of 4 experiments ±SD. Right picture shows one representative immunoflourescence staining out of 4 of nephrin expression (488 Alexa Fluor) in green and nucleus (DAPI) in blue. (b) Dose-dependent qPCR analysis of nephrin expression in Mock and suPAR treated human podocytes by using specific human TaqMan assays. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and represent the average of 6 experiments ±SD. Values were normalized to the expression of GAPDH gene. (c) Time course qPCR analysis of nephrin and synaptopodin expression in Mock and suPAR treated human podocytes by using specific human TaqMan assays. Results are expressed as relative fold change in suPAR treated cells vs Mock cells (ΔΔCt) and represent the average of 6 experiments ±SD. Values were normalized to the expression of GAPDH gene. Statistical significance (P) is indicated by asterisks and is represented as: ***<0.001.
Mentions: Various, genetic and functional, nephrin defects lead to nephritic syndromes and proteinuria222324252627. To verify the effect of suPAR stimulation on nephrin expression, we used human primary podocytes obtained from renal tissue of patients affected by renal adenocarcinoma who underwent a radical nephrectomy. All tissue specimens used for this study were collected from the distal part of the pathological tissue and were free from any disease as was verified by haematoxylin-eosin-staining (Fig. 1a). We then cultured freshly isolated glomerula in order to obtain human podocytes (Fig. 1a), whose phenotype was verified by qPCR analysis detecting gene expression of the following specific podocytes markers: Wilms’ tumor 1 (WT-1), synaptopodin, nephrin and podocin (data not shown). Mean serum and plasma concentration of suPAR in healthy adults have been reported to be 2 ng/mL18, whereas it reaches 20 ng/mL in patients with various diseases, such as cancer, sepsis, liver disease, HIV infection and FSGS81418192021. We then proceeded to incubate human primary podocytes with 20 ng/mL of human recombinant fl-suPAR. After 24 hours of stimulation we detected by fluorescence microscopy a significant down-modulation of nephrin at the protein level (Fig. 1b). Immunoflourescence staining of frozen tissue from human normal kidney served to confirm the specificity of anti-nephrin rabbit clonal antibody (Ab) used for this study (Fig. 1c). The decreased amount of nephrin following incubation of primary podocytes with suPAR was also confirmed by qPCR, thus suggesting a transcriptional control of nephrin expression by suPAR (Fig. 1d). To both confirm the modulation of nephrin exerted by suPAR and to disclose the related mechanism in a post-mitotic podocyte, we repeated the same experimental approach in conditional immortalized human podocytes (CIHPs) in vitro34. By performing assessments of protein expression with immune-flourescence in CIHPs incubated with different concentrations of suPAR for 24 hours, we detected a significant reduction of nephrin flourescence intensity and thus a lower level of nephrin protein in suPAR stimulated CIHPs comparing to control experiments, with the maximum inhibition between 10–20 ng/mL of suPAR (Fig. 2a). To determine whether suPAR stimulation affects nephrin expression at the transcriptional level, we performed qPCR experiments. As shown in Fig. 2b, quantification of nephrin mRNA in CIHPs incubated with suPAR showed a rapid and progressive decrease of Nephrin gene expression (Fig. 2a–c). The reduction of nephrin transcripts was already detectable after 3 hours and became significant after 6 hours of treatment, when it also reached a plateau that was maintained even after 24 hours of stimulation. However, we did not observe any significant suPAR-mediated down-modulation of the expression of synaptopodin, another specific podocyte marker, thus indicating the specificity of suPAR in suppressing nephrin expression in CIHPs (Fig. 2c).

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