NEWS & VIEWS GLOMERULAR DISEASE
The search goes on: suPAR is not the elusive FSGS factor Jeroen K. Deegens and Jack F. Wetzels
New research indicates that intact soluble urokinase plasminogen activator receptor (suPAR) does not induce albuminuria in mice. These data corroborate the most recent clinical findings, showing that intact suPAR is not the plasma permeability factor responsible for recurrence of focal segmental glomerulosclerosis after renal transplantation. Deegens, J. K. & Wetzels, J. F. Nat. Rev. Nephrol. 10, 431–432 (2014); published online 24 June 2014; doi:10.1038/nrneph.2014.113
Focal segmental glomerulosclerosis (FSGS) is a common cause of nephrotic syndrome. The aetiology of FSGS is diverse and includes genetic, viral and environmental factors. 1 However, primary (idiopathic) FSGS is generally considered to be caused by an unidentified circulating factor. 2 An important study investigating soluble urokinase plasminogen activator receptor (suPAR) in mice reported by Cathelin et al.3 suggests suPAR is not the circulating factor that causes FSGS. The most convincing evidence supporting the presence and pathogenetic function of a circulating factor comes from clinical observations in patients with recurrent FSGS after kidney transplantation. 2 Indeed, FSGS often recurs within hours to days after transplantation of an organ from a donor without the disease. Plasma exchange can induce a remission if instituted early in the course of recurrent disease. Other studies have shown that serum or plasma from patients with post-transplant FSGS induces proteinuria in rats and increases albumin permeability in isolated glomeruli. Case reports have demonstrated remission of proteinuria following implantation of a kidney with FSGS in recipients who did not have FSGS as their indication for transplantation.4 Unfortunately, identification of the factor or factors responsible for these characteristics has proven to be elusive and remains the subject of intensive research.5 In their new paper, Cathelin and coworkers infused two different forms of recombinant suPAR—a monomeric and a commercially available chimeric mouse
‘‘
…there is no clinical role for measuring suPAR in patients with FSGS
’’
suPAR—into wild-type C57BL/6J and 129S2svPas mice. 3 Neither intravenous administration of single-dose recombinant suPAR (20 μg or 100 μg), nor prolonged delivery of suPAR (200 μg), over 1 week induced albuminuria. Also, podocyte foot processes remained normal, arguing against subclinical podocyte injury. The presence of massive suPAR deposits in the glomeruli was confirmed by confocal microscopy. In additional experiments, the effect of suPAR administration in lipopoly saccharide (LPS)-treated mice was tested. It was previously shown that LPS can both increase endogenous suPAR secretion by monocytes and induce podocyte effacement and proteinuria in mice.6,7 Injection of LPS to C57BL/6J mice did indeed induce albuminuria, although co-administration of either monomeric or chimeric suPAR (25 μg) produced no additional effect. The authors concluded that both short-term and prolonged administration of suPAR, and its deposition in glomeruli, is not alone sufficient to cause albuminuria in mice. These results corroborate the conclusions of the clinical research, and argue against a causal role of full-length suPAR in FSGS. But, how should all these seemingly inconsistent data be interpreted? In 2011, suPAR was proposed to be the disease-causing permeability factor.6 suPAR is a 20–50 kDa protein produced by
NATURE REVIEWS | NEPHROLOGY
cleavage and release of membrane bound urokinase plasminogen activator receptor (uPAR).2 suPAR consists of three homologous domains: DI, DII and DIII, which have various subunit configurations.2 In cultured podocytes, uPAR and suPAR were shown to activate integrin β3, one of the main proteins anchoring podocytes to the glomerular basement membrane.2,6 Increased podocyte integrin β3 activity results in reorganization of the actin cytoskeleton of podocytes and seems to be a specific feature of FSGS. Animal studies provided evidence for a role of suPAR in FSGS. High-dose recombi nant mouse suPAR induced proteinuria and foot process effacement in mice lacking the gene for uPAR (Plaur –/–).6 Kidneys from these mice showed prominent deposits of suPAR along the podocytes and increased podocyte integrin β3 activity. 6 Similar results were seen in Plaur–/– mouse kidneys transplanted into wild-type mice after LPSinduced suPAR production.6 Furthermore, proteinuria and FSGS-like lesions developed in wild-type mice after injection of plasmids producing only suPAR D I and DII domains. By contrast, a plasmid with a point mutation in the DII domain induced synthesis of suPAR in wild-type mice that was unable to bind to integrin β3, and did not induce proteinuria. Taken together, these results provide evidence that wholedomain suPARI–III, as well as shorter fragments, can cause proteinuria in mice through a ctivation of integrin β3. The pathogenetic role of suPAR in FSGS was further supported by human data, demonstrating significantly increased serum suPAR levels in patients with FSGS compared with healthy and disease controls. 6 However, these clinical data were criticized because the study did not adjust for reduced renal function in patients with FSGS.2 Following the initial report in 2011,6 subs equent reports have demonstrated an inverse relationship between elevated suPAR levels and estimated glomerular filtration rate. 8 When adjusted for renal function, suPAR levels did not differentiate between FSGS and other renal diseases.9,10 Also, serum suPAR did not correlate with the degree of proteinuria. Moreover, higher serum or plasma suPAR concentrations have VOLUME 10 | AUGUST 2014
© 2014 Macmillan Publishers Limited. All rights reserved
NEWS & VIEWS also been reported in patients with cancer, sepsis, atherosclerosis and paroxysmal nocturnal haemoglobinuria.2,3 Despite higher suPAR levels, patients in these studies did not develop FSGS. 2,3 Of note, the ELISA tests used in all clinical studies cannot discern full-length glycosylated suPARI–III from fragments. The latest investigation has some limitations. Although the authors confirmed presence of suPAR in the glomeruli, they did not evaluate whether administration of suPAR increased integrin β3 activation in podocytes. Still, in view of the overwhelming recent clinical data, we feel confident in the conclusion that intact, full-length suPAR is not the cause of FSGS. It is notable that administration of recombinant suPAR induced albuminuria in Plaur –/– mice, but not in their wild-type counterparts. As suggested by the authors, this finding might reflect that genetic ablation of Plaur renders these kidneys more susceptible to the effect of suPAR. However, not all the data can be explained by this mechanism. Unglycosylated and/or suPAR fragments, but not the full-length suPAR, might cause FSGS. Obviously, if confirmed, this would
AUGUST 2014 | VOLUME 10
explain the negative ELISA findings in the clinical studies, as the assay used mainly reflects the full-length receptor. Moreover, it would explain why prolonged delivery of recombinant full-length suPAR in the study by Cathelin et al. 3 did not induce albuminuria in wild-type mice, whereas genetically engineered wild-type mice producing suPAR DI and DII domains became albuminuric. Although fragments of suPAR remain important candidates for the circulating permeability factor, further studies are needed to provide proof of concept. Meanwhile, there is no clinical role for measuring suPAR in patients with FSGS. Department of Nephrology, Radboud University Medical Centre, PO Box 9101, 6500 HB Nijmegen, Netherlands (J.K.D., J.F.W.). Correspondence to: J.F.W. [email protected] Competing interests The authors declare no competing interests. 1.
2.
Deegens, J. K. & Wetzels, J. F. Immunosuppressive treatment of focal segmental glomerulosclerosis: lessons from a randomized controlled trial. Kidney Int. 80, 798–801 (2011). Maas, R. J., Deegens, J. K. & Wetzels, J. F. Serum suPAR in patients with FSGS: trash
or treasure? Paediatr. Nephrol. 28, 1041–1048 (2013). 3. Cathelin, D. et al. Administration of recombinant soluble urokinase receptor per se is not sufficient to induce podocyte alterations and proteinuria in mice. J. Am. Soc. Nephrol. http:// dx.doi.org/10.1681/ASN.2013040425. 4. Rea, R., Smith, C., Sandhu, K., Kwan, J. & Tomson, C. Successful transplant of a kidney with focal segmental glomerulosclerosis. Nephrol. Dial. Transplant. 16, 416–417 (2001). 5. McCarthy, E. T., Sharma, M. & Savin, V. J. Circulating permeability factors in idiopathic nephrotic syndrome and focal segmental glomerulosclerosis. Clin. J. Am. Soc. Nephrol. 5, 2115–2121 (2010). 6. Wei, C. et al. Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis. Nat. Med. 17, 952–960 (2011). 7. Wei, C. et al. Modification of kidney barrier function by the urokinase receptor. Nat. Med. 14, 55–63 (2008). 8. Schlondorff, D. Are serum suPAR determinations by current ELISA methodology reliable diagnostic biomarkers for FSGS? Kidney Int. 85, 499–501 (2014). 9. Meijers, B. et al. The soluble urokinase receptor is not a clinical marker for focal segmental glomerulosclerosis. Kidney Int. 85, 636–640 (2014). 10. Bock, M. E., Price, H. E., Gallon, L. & Langman, C. B. Serum soluble urokinase-type plasminogen activator receptor levels and idiopathic FSGS in children: a single-centre report. Clin. J. Am. Soc. Nephrol. 8, 1304–1311 (2013).
www.nature.com/nrneph © 2014 Macmillan Publishers Limited. All rights reserved
The search goes on: suPAR is not the elusive FSGS factor Jeroen K. Deegens and Jack F. Wetzels
New research indicates that intact soluble urokinase plasminogen activator receptor (suPAR) does not induce albuminuria in mice. These data corroborate the most recent clinical findings, showing that intact suPAR is not the plasma permeability factor responsible for recurrence of focal segmental glomerulosclerosis after renal transplantation. Deegens, J. K. & Wetzels, J. F. Nat. Rev. Nephrol. 10, 431–432 (2014); published online 24 June 2014; doi:10.1038/nrneph.2014.113
Focal segmental glomerulosclerosis (FSGS) is a common cause of nephrotic syndrome. The aetiology of FSGS is diverse and includes genetic, viral and environmental factors. 1 However, primary (idiopathic) FSGS is generally considered to be caused by an unidentified circulating factor. 2 An important study investigating soluble urokinase plasminogen activator receptor (suPAR) in mice reported by Cathelin et al.3 suggests suPAR is not the circulating factor that causes FSGS. The most convincing evidence supporting the presence and pathogenetic function of a circulating factor comes from clinical observations in patients with recurrent FSGS after kidney transplantation. 2 Indeed, FSGS often recurs within hours to days after transplantation of an organ from a donor without the disease. Plasma exchange can induce a remission if instituted early in the course of recurrent disease. Other studies have shown that serum or plasma from patients with post-transplant FSGS induces proteinuria in rats and increases albumin permeability in isolated glomeruli. Case reports have demonstrated remission of proteinuria following implantation of a kidney with FSGS in recipients who did not have FSGS as their indication for transplantation.4 Unfortunately, identification of the factor or factors responsible for these characteristics has proven to be elusive and remains the subject of intensive research.5 In their new paper, Cathelin and coworkers infused two different forms of recombinant suPAR—a monomeric and a commercially available chimeric mouse
‘‘
…there is no clinical role for measuring suPAR in patients with FSGS
’’
suPAR—into wild-type C57BL/6J and 129S2svPas mice. 3 Neither intravenous administration of single-dose recombinant suPAR (20 μg or 100 μg), nor prolonged delivery of suPAR (200 μg), over 1 week induced albuminuria. Also, podocyte foot processes remained normal, arguing against subclinical podocyte injury. The presence of massive suPAR deposits in the glomeruli was confirmed by confocal microscopy. In additional experiments, the effect of suPAR administration in lipopoly saccharide (LPS)-treated mice was tested. It was previously shown that LPS can both increase endogenous suPAR secretion by monocytes and induce podocyte effacement and proteinuria in mice.6,7 Injection of LPS to C57BL/6J mice did indeed induce albuminuria, although co-administration of either monomeric or chimeric suPAR (25 μg) produced no additional effect. The authors concluded that both short-term and prolonged administration of suPAR, and its deposition in glomeruli, is not alone sufficient to cause albuminuria in mice. These results corroborate the conclusions of the clinical research, and argue against a causal role of full-length suPAR in FSGS. But, how should all these seemingly inconsistent data be interpreted? In 2011, suPAR was proposed to be the disease-causing permeability factor.6 suPAR is a 20–50 kDa protein produced by
NATURE REVIEWS | NEPHROLOGY
cleavage and release of membrane bound urokinase plasminogen activator receptor (uPAR).2 suPAR consists of three homologous domains: DI, DII and DIII, which have various subunit configurations.2 In cultured podocytes, uPAR and suPAR were shown to activate integrin β3, one of the main proteins anchoring podocytes to the glomerular basement membrane.2,6 Increased podocyte integrin β3 activity results in reorganization of the actin cytoskeleton of podocytes and seems to be a specific feature of FSGS. Animal studies provided evidence for a role of suPAR in FSGS. High-dose recombi nant mouse suPAR induced proteinuria and foot process effacement in mice lacking the gene for uPAR (Plaur –/–).6 Kidneys from these mice showed prominent deposits of suPAR along the podocytes and increased podocyte integrin β3 activity. 6 Similar results were seen in Plaur–/– mouse kidneys transplanted into wild-type mice after LPSinduced suPAR production.6 Furthermore, proteinuria and FSGS-like lesions developed in wild-type mice after injection of plasmids producing only suPAR D I and DII domains. By contrast, a plasmid with a point mutation in the DII domain induced synthesis of suPAR in wild-type mice that was unable to bind to integrin β3, and did not induce proteinuria. Taken together, these results provide evidence that wholedomain suPARI–III, as well as shorter fragments, can cause proteinuria in mice through a ctivation of integrin β3. The pathogenetic role of suPAR in FSGS was further supported by human data, demonstrating significantly increased serum suPAR levels in patients with FSGS compared with healthy and disease controls. 6 However, these clinical data were criticized because the study did not adjust for reduced renal function in patients with FSGS.2 Following the initial report in 2011,6 subs equent reports have demonstrated an inverse relationship between elevated suPAR levels and estimated glomerular filtration rate. 8 When adjusted for renal function, suPAR levels did not differentiate between FSGS and other renal diseases.9,10 Also, serum suPAR did not correlate with the degree of proteinuria. Moreover, higher serum or plasma suPAR concentrations have VOLUME 10 | AUGUST 2014
© 2014 Macmillan Publishers Limited. All rights reserved
NEWS & VIEWS also been reported in patients with cancer, sepsis, atherosclerosis and paroxysmal nocturnal haemoglobinuria.2,3 Despite higher suPAR levels, patients in these studies did not develop FSGS. 2,3 Of note, the ELISA tests used in all clinical studies cannot discern full-length glycosylated suPARI–III from fragments. The latest investigation has some limitations. Although the authors confirmed presence of suPAR in the glomeruli, they did not evaluate whether administration of suPAR increased integrin β3 activation in podocytes. Still, in view of the overwhelming recent clinical data, we feel confident in the conclusion that intact, full-length suPAR is not the cause of FSGS. It is notable that administration of recombinant suPAR induced albuminuria in Plaur –/– mice, but not in their wild-type counterparts. As suggested by the authors, this finding might reflect that genetic ablation of Plaur renders these kidneys more susceptible to the effect of suPAR. However, not all the data can be explained by this mechanism. Unglycosylated and/or suPAR fragments, but not the full-length suPAR, might cause FSGS. Obviously, if confirmed, this would
AUGUST 2014 | VOLUME 10
explain the negative ELISA findings in the clinical studies, as the assay used mainly reflects the full-length receptor. Moreover, it would explain why prolonged delivery of recombinant full-length suPAR in the study by Cathelin et al. 3 did not induce albuminuria in wild-type mice, whereas genetically engineered wild-type mice producing suPAR DI and DII domains became albuminuric. Although fragments of suPAR remain important candidates for the circulating permeability factor, further studies are needed to provide proof of concept. Meanwhile, there is no clinical role for measuring suPAR in patients with FSGS. Department of Nephrology, Radboud University Medical Centre, PO Box 9101, 6500 HB Nijmegen, Netherlands (J.K.D., J.F.W.). Correspondence to: J.F.W. [email protected] Competing interests The authors declare no competing interests. 1.
2.
Deegens, J. K. & Wetzels, J. F. Immunosuppressive treatment of focal segmental glomerulosclerosis: lessons from a randomized controlled trial. Kidney Int. 80, 798–801 (2011). Maas, R. J., Deegens, J. K. & Wetzels, J. F. Serum suPAR in patients with FSGS: trash
or treasure? Paediatr. Nephrol. 28, 1041–1048 (2013). 3. Cathelin, D. et al. Administration of recombinant soluble urokinase receptor per se is not sufficient to induce podocyte alterations and proteinuria in mice. J. Am. Soc. Nephrol. http:// dx.doi.org/10.1681/ASN.2013040425. 4. Rea, R., Smith, C., Sandhu, K., Kwan, J. & Tomson, C. Successful transplant of a kidney with focal segmental glomerulosclerosis. Nephrol. Dial. Transplant. 16, 416–417 (2001). 5. McCarthy, E. T., Sharma, M. & Savin, V. J. Circulating permeability factors in idiopathic nephrotic syndrome and focal segmental glomerulosclerosis. Clin. J. Am. Soc. Nephrol. 5, 2115–2121 (2010). 6. Wei, C. et al. Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis. Nat. Med. 17, 952–960 (2011). 7. Wei, C. et al. Modification of kidney barrier function by the urokinase receptor. Nat. Med. 14, 55–63 (2008). 8. Schlondorff, D. Are serum suPAR determinations by current ELISA methodology reliable diagnostic biomarkers for FSGS? Kidney Int. 85, 499–501 (2014). 9. Meijers, B. et al. The soluble urokinase receptor is not a clinical marker for focal segmental glomerulosclerosis. Kidney Int. 85, 636–640 (2014). 10. Bock, M. E., Price, H. E., Gallon, L. & Langman, C. B. Serum soluble urokinase-type plasminogen activator receptor levels and idiopathic FSGS in children: a single-centre report. Clin. J. Am. Soc. Nephrol. 8, 1304–1311 (2013).
www.nature.com/nrneph © 2014 Macmillan Publishers Limited. All rights reserved
