Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis,”NatureMedicine,

Abstract

Focal segmental glomerulosclerosis (FSGS) is a cause of proteinuric kidney disease, compromising both native and transplanted kidneys. Treatment is limited because of a complex pathogenesis, including unknown serum factors. Here we report that serum soluble urokinase receptor (suPAR) is elevated in two-thirds of subjects with primary FSGS, but not in people with other glomerular diseases. We further find that a higher concentration of suPAR before transplantation underlies an increased risk for recurrence of FSGS after transplantation. Using three mouse models, we explore the effects of suPAR on kidney function and morphology. We show that circulating suPAR activates podocyte b 3 integrin in both native and grafted kidneys, causing foot process effacement, proteinuria and FSGS-like glomerulopathy. Our findings suggest that the renal disease only develops when suPAR sufficiently activates podocyte b 3 integrin. Thus, the disease can be abrogated by lowering serum suPAR concentrations through plasmapheresis, or by interfering with the suPAR-b 3 integrin interaction through antibodies and small molecules targeting either uPAR or b 3 integrin. Our study identifies serum suPAR as a circulating factor that may cause FSGS. A R T I C L E S NATURE MEDICINE VOLUME 17 | NUMBER 8 | AUGUST 2011 953 It can be elevated in some malignant neoplasms (for example, ovarian cancer 21 ) as well as in HIV infection In conclusion, our study suggests circulating suPAR as a previously undescribed cause for both primary and recurrent FSGS. RESULTS suPAR is increased in serum of subjects with FSGS We found that suPAR serum concentrations are significantly elevated in people with FSGS when compared to healthy subjects We then stratified the FSGS cases into three different subpopulations: primary FSGS, recurrent FSGS in the allograft and FSGS without recurrence after transplantation. We found the highest suPAR concentrations in pretransplantation blood from subjects with FSGS who later developed recurrent FSGS after transplantation We also compared suPAR serum concentrations in transplanted FSGS patients 1 year after transplantation and found significantly higher suPAR serum concentrations in patients that developed recurrent FSGS than in FSGS patients who received kidney transplants and then had normal renal function A R T I C L E S 954 VOLUME 17 | NUMBER 8 | AUGUST 2011 NATURE MEDICINE As multiple forms of suPAR have been attributed to domain cleavage or alternative splicing Concentrations of the ligand of uPAR, urokinase (uPA), are often elevated in certain types of cancers that also present with elevated suPAR concentrations in various body fluids 27 . Thus, we measured serum uPA concentrations in the groups within our glomerular disease cohort. Notably, and unlike suPAR, we found no difference in the serum uPA concentrations among the groups ( We hypothesized that suPAR could activate 3 integrin in a similar manner to membrane-bound uPAR in podocytes 18 . The activity of 3 integrin is typically measured using the activation epitope-recognizing antibodies such as the 3 integrin-specific antibody AP5 (refs. 29,30). We used human differentiated podocytes 31 and incubated them either with FSGS serum that contains high concentrations of suPAR or with recombinant suPAR, in the absence or presence of a blocking antibody to uPAR or with the 3 integrin small molecule-inhibitor cycloRGDfV A R T I C L E S NATURE MEDICINE VOLUME 17 | NUMBER 8 | AUGUST 2011 955 adhesions are known to be the location of 3 integrin 32 . We also found that this effect could be blocked by a blocking antibody specific to uPAR or by cycloRGDfV To show that circulating suPAR affects the transplanted kidney by activating podocyte 3 integrin, we used double immunofluorescent staining with synaptopodin, a podocyte marker 33 , to analyze aftertransplantation graft biopsies for the presence of AP5 signal in podocytes. We found that 3 integrin activity is low in graft podocytes before reperfusion, whereas it is markedly increased 2 h after reperfusion in recurrent FSGS, but not in nonrecurrent FSGS suPAR and b 3 integrin activity during plasmapheresis To further define the relationship between suPAR and podocyte 3 integrin activity, we did fluorescence-activated cell sorting (FACS) analysis for 3 integrin activity in cultured human podocytes g h e f Figure 3 suPAR serum concentrations and podocyte 3 integrin activity determine treatment response to plasmapheresis in recurrent FSGS. (a) Human podocytes incubated with different pooled serum samples and assayed for 3 integrin activity. MFI, mean fluorescence intensity. *P < 0.05 for nonrecurrent FSGS versus normal subjects, ***P < 0.001 for recurrent versus nonrecurrent FSGS or versus healthy subjects. The respective suPAR concentration of the pooled sera is marked in red. NS, normal (healthy) subject; NR, nonrecurrent FSGS; REC, recurrent FSGS (representative of three experiments). (b) Pharmacological modulation of 3 integrin activity in podocytes. **P < 0.01 for cylcoRGDfv co-treated cells versus recurrent FSGS serum alone; ***P < 0.001 for uPAR-specific mAb co-treated cells versus recurrent FSGS serum alone. (c) suPAR in serum from subjects with recurrent FSGS (n = 4) before and after a course of plasmapheresis. **P < 0.01. (d) Effect of plasmapheresis on 3 integrin activity in podocytes incubated with recurrent FSGS serum (n = 6), collected before and after serial treatment with plasmapheresis. ***P < 0.001. (e-h) Clinical cases of recurrent FSGS. Top graphs show serum suPAR, urine protein/creatinine ratio (g/g) and individual plasmapheresis treatment as indicated by arrows and plotted over time (d) from before (−1) to after transplantation. Bottom graphs and images show podocyte 3 integrin activity measured by FACS (left) and immunofluorescence (right) as a result of incubation with pretransplantation serum, or with the after-transplantation serum collected after repetitive plasmapheresis treatments. As a reference, the mean concentration of AP5 from a is marked as a dashed line. (e,f) Patients who obtained full remission after pheresis. (g,h) Patients who did not achieve remission after pheresis. Scale bars, 30 m. Whiskers in plots of AP5 activity and serum suPAR show minimum to maximum. A R T I C L E S 956 VOLUME 17 | NUMBER 8 | AUGUST 2011 NATURE MEDICINE incubated with serum from healthy subjects (n = 5) or with pretransplantation serum from subjects with nonrecurrent (n = 10) and recurrent FSGS (n = 15). We found that incubation with recurrent FSGS pretransplantation serum significantly elevated 3 integrin activity compared to serum from subjects with nonrecurrent FSGS or from healthy subjects Mouse models showing that suPAR causes proteinuria and FSGS To determine whether suPAR is a cause or a consequence of FSGS, we established three different mouse models: (i) uPAR-knockout (Plaur −/− ) mice injected with recombinant suPAR, (ii) hybrid-transplant mice modeling endogenous suPAR release and (iii) genetically engineered wild-type mice that drive expression of a suPAR plasmid in the skin, leading to increased serum suPAR concentrations. First, we examined whether exogenous circulating suPAR could deposit into kidneys and cause albuminuria. We used Plaur −/− mice and injected escalating doses of recombinant mouse suPAR protein intravenously into Plaur −/− mice. We found that low-dose injection at 2 and 10 g did not cause albuminuria, which is consistent with the physiological low concentrations of suPAR we observed in the blood of healthy subjects A R T I C L E S NATURE MEDICINE VOLUME 17 | NUMBER 8 | AUGUST 2011 957 that this deposition was associated with an increase in 3 integrin activity in podocytes, as shown by increased AP5 labeling that, again, is suPAR dose dependent Third, to explore whether prolonged elevation of suPAR in the serum of mice causes a progressive glomerulopathy, we engineered wild-type mice that drive expression of suPAR in the skin. We generated a mouse plasmid (sPlaur WT ) based on a known coding sequence for secreted suPAR 26 that contains the D I and D II domains. We delivered this plasmid into mice by in vivo electroporation into the skin. As a control, we generated a 3 integrin binding-deficient suPAR mutant, sPlaur E134A . This mutant has a point mutation (E134A) in the D II domain We next studied the ultrastructure of podocytes after 4 weeks and noted prominent foot process effacement consistent with glomerular disease; however, we only observed this in mice that expressed suPAR capable of binding 3 integrin A R T I C L E S 958 VOLUME 17 | NUMBER 8 | AUGUST 2011 NATURE MEDICINE To further study the disease-causing effects of suPAR, we also carried out experiments that blocked suPAR action. We administered an uPAR-specific monoclonal antibody to mice expressing sPlaur WT and found protection of proteinuria whereas proteinuria was high when using an IgG isotype control DISCUSSION The present study identifies suPAR as a circulating, causative FSGS factor that is elevated in the serum of approximately two-thirds of primary FSGS patients. suPAR-mediated activation of 3 integrin on podocyte foot processes is the mechanism of injury caused by high suPAR blood concentrations. Since the first clinical description of nephrotic syndrome recurrence after kidney transplantation The amount of podocyte 3 integrin activity that is driven by circulating systemic suPAR depends on the amount of individual serum suPAR and, possibly, also on suPAR post-translational modifications (such as glycosylation status). In addition, podocyte 3 integrin activity can also be driven by augmented podocyte uPAR expression, which is sufficient to initiate podocyte foot process effacement and proteinuria 18 . Podocyte 3 integrin activity seems to be independent of total serum uPA concentrations; this is in contrast to the suPARuPA associations in some forms of cancer Several modes of interference can protect from suPAR-mediated podocyte injury: (i) blockade of suPAR using a blocking antibody specific to suPAR; (ii) protecting 3 integrin from increased activation by cycloRGDfV or 3 integrin-specific antibody 18 ; (iii) blocking suPAR-3 integrin interaction by modulating the suPAR-3 integrin binding site (E134A) and (iv) removing suPAR by plasmapheresis to levels that decrease podocyte 3 integrin activity. Using assays that measure all suPAR forms, we noted that ~70% of subjects with primary FSGS presented with significantly elevated concentrations of serum suPAR before transplantation when compared to other primary glomerulopathies. In addition, we found that total suPAR concentrations remained significantly elevated after kidney transplantation in people who have developed recurrent FSGS compared to those with proper renal function. On the basis of these clinical observations, we created mouse models that could explore the cause or effect nature of suPAR and demonstrate the kidney pathogenicity of elevated systemic suPAR. Notably, we found different forms of suPAR that correspond to different domain fragments in the serum of subjects with FSGS, with molecular weights ranging from 22 to 45 kDa. This is close to the molecular range (30 to 50 kDa) of the factor predicted by others Our study provides the rationale for a more measurable prediction of FSGS risk in subjects with FSGS before and after transplantation. Approximately 70% of subjects with FSGS have elevated concentrations of suPAR compared to other glomerular diseases such as membranous nephropathy, MCD or preeclampsia. This further separates FSGS from other glomerulopathies involving phospholipase A2 receptorspecific antibodies in membranous nephropathy 39 and factors such as angiopoietin-like 40 or c-mip in MCD 41 . Because suPAR is detectable both in healthy human subjects and normal mice, physiological suPAR concentrations or physiological suPAR domain combinations do not seem to be harmful. It is also important to note that there might be species differences with respect to the pathogenic strength of various suPAR domain combinations. Future studies with new and more A R T I C L E S NATURE MEDICINE VOLUME 17 | NUMBER 8 | AUGUST 2011 959 specific suPAR domain-specific antibodies should clarify this question and focus more on the role of suPAR glycosylation in FSGS. Another interesting question is why a few FSGS patients without elevated suPAR still develop FSGS as well as recurrent FSGS. An obvious answer would be that suPAR can act in concert with podocyte uPAR 18 and this might drive FSGS even in the absence of high suPAR concentrations. Another reason might be that native FSGS is caused by a mutation in a podocyte gene 6 . Also, the current ELISA assay for serum suPAR is likely to measure all suPAR domains, and thus it might be possible that FSGS subjects with low total suPAR do have a higher portion of pathological suPAR fragments that current tests cannot readily detect. Once new reagents are developed, even more subjects with FSGS might test positive for pathological suPAR, thereby further increasing the clinical prediction of the test. Alternatively, there is the possibility of the presence of yet-to-be-identified additional permeability factor candidates 17 or the absence of protective podocyte factors Podocyte 3 integrin expression and activation responses must also be evaluated further. Future studies will have to focus more on the expression of the 3 integrin-encoding gene (ITGB3) in the graft In conclusion, we show that suPAR is a circulating factor that can cause FSGS before and after transplantation. Our studies will allow better risk stratification of patients with FSGS by measuring serum and urine concentrations of suPAR, and they will provide the conceptual framework for refined treatment options that remove or neutralize suPAR to a level insufficient to activate podocyte 3 integrin. Regardless of the source of the stimulant (podocyte or systemic), a pathological activation of podocyte 3 integrin is emerging as a key event for the initiation of proteinuric glomerular disease; it is likely to be important in some forms of secondary FSGS, such as diabetic nephropathy 18 , as well. Accordingly, pharmacological modulation of excessive podocyte 3 integrin activation is a promising target for achieving protection from renal disease. METHODS Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturemedicine/. Note: Supplementary information is available on the Nature Medicine website. ACKNOWLEDGMENTS We thank N. Sidenius (Foundation FIRC Institute of Molecular Oncology, Italy) for help with the suPAR assay in mouse samples. We are grateful to L.H. Beck, Jr. and D. Salant (Boston University Medical Center) for providing the membranous nephropathy patient cohort. We thank S. Hsiesh for help with sample collection. We thank G. Høyer-Hansen (the Finsen Laboratory, Denmark) for additional suPAR assays and discussions. The authors are grateful to P.J. Goldschmidt for helpful scientific discussions regarding the manuscript and to M.J. Tracy for critical reading of the manuscript. This work was supported in part by the US National Institutes of Health (grants DK073495 and DK089394 to J.R., DK-82636 to A.F., DK070011 to G.B.), the Halpin Foundation-American Society of Nephrology Research Grant (to C.W.), a grant from the American Diabetes Association (7-09-JF-23 to A.F.), and a grant from the Diabetes Research Institute Foundation (to A.F.). The authors also wish to acknowledge the generous support of the Katz Family Fund. AUTHOR CONTRIBUTIONS J.R. conceived the study. J.R. and C.W. designed the experiments, coordinated the study, analyzed the data and wrote the manuscript. C.W., S.E.H., J.L., D.M., Q.Z., B.N., P.D., V.G. performed the experiments. A.F., N.G., G.B., J.S., S.A.K., H.-K.Y., M.Saleem, A.C., E.S., A.T., M.Salifu, M.M.S., F.S., C.M., V.S., M.Z., D.R., M.P.R., P.R., J.R. contributed to clinical samples and clinical information. M.P.R. and P.R. provided pathology service. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemedicine/. Published online at http://www.nature.com/naturemedicine/. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. A R T I C L E S NATURE MEDICIN