Circulating Permeability Factors in Idiopathic Nephrotic Syndrome and Focal Segmental Glomerulosclerosis

Mini-Review

Ellen T. McCarthy,* Mukut Sharma,† and Virginia J. Savin† *Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas; and †Kansas City VA Medical Center, Kansas City, Missouri

Circulating permeability factors may be important in idiopathic nephrotic syndrome (INS) including focal segmental glomerulosclerosis (FSGS) and in recurrence after renal transplantation. Evidence for plasma factors includes posttransplant recurrence of proteinuria and its response to plasmapheresis or immunoadsorption and induction of proteinuria in experimental animals by infusion of patient plasma or its fractions. The authors and other investigators have used proteomic techniques to seek pathogenic molecules. The authors have recently proposed cardiotrophin-like cytokine-1 (CLC-1) as an active factor in FSGS. Other potential permeability factors include hemopexin and vascular permeability factor in minimal change nephrotic syndrome (MCNS) and soluble urokinase receptor in FSGS. In the authors’ studies, in vitro plasma permeability activity is blocked by diverse substances that may decrease levels of active molecules or block the effects of circulating permeability factors. It has been shown that the simple sugar galactose blocks the effect of FSGS serum on albumin permeability in vitro and decreases permeability activity when administered to patients. Because identities of permeability factors and their mechanisms of action are not well defined, therapy of INS/FSGS is empiric. Corticosteroids are the mainstay of initial therapy whereas calcineurin inhibitors such as cyclosporine A (CsA) and immunosuppressive medications provide adjunctive therapy. Nonspecific therapies such as blocking the renin-angiotensin system and controlling blood pressure and plasma lipids may also diminish proteinuria and slow progression. Identification of molecules that initiate proteinuria and application of findings from in vitro studies may lead to development of new treatments to arrest progression and prevent recurrence after transplantation. Clin J Am Soc Nephrol 5: 2115–2121, 2010. doi: 10.2215/CJN.03800609

linical syndromes of idiopathic nephrotic syndrome pathophysiological process. In this review, we have considered (INS) include minimal change nephrotic syndrome reports concerning circulating “factors” in NS and in recurrent C (MCNS) and focal segmental glomerulosclerosis (FSGS): NS after renal transplantation regardless of the terminology INS describes a clinical syndrome that includes heavy proteinuria, used. In the future, it is possible that measurement of such edema, hypoalbuminemia, and hyperlipidemia in the absence of factors may be used to refine various diagnostic categories. evident systemic disease. A kidney biopsy is required to further Evidence supporting the presence of circulating factors in NS is classify INS. Terminology used in the literature may be confus- summarized in Table 1. ing because routine practice regarding biopsy varies among patient age groups, clinical presentation, and geographical Clinical Course and Therapy area. Patient groups that may not routinely undergo biopsy MCNS generally has a fairly benign course and is usually include children, some patients with steroid-resistant nephrotic steroid responsive. Renal disease progresses slowly even in syndrome (NS) and progression to renal failure, and some patients with frequent relapses or steroid dependence. Treat- patients with recurrent NS after transplantation. The most com- ment and clinical responses have been reviewed (1). In contrast, mon histologic diagnoses among patients with INS who un- FSGS has a highly variable course. No randomized controlled dergo biopsy are MCNS and FSGS. Investigators occasionally trials of sufficient numbers of patients are available to provide include membranous nephropathy in reports concerning INS. high-quality information to guide therapy of FSGS in native The question of whether MCNS and FSGS are distinct entities kidneys or renal allografts. Current therapy results in full or or are variants of a single disease has been debated extensively. partial remission in only approximately 50% of patients. Treat- However, as genetic and viral etiologies of NS have been iden- ments that have been used include corticosteroids with or tified, it has become apparent that neither entity reflects a single without cyclophosamide (1,2), cyclosporine (3), mycophenolate mofitil (MMF) (4), and rituximab (5,6). If proteinuria can be diminished by these agents or by nonspecific therapies such as

Published online ahead of print. Publication date available at www.cjasn.org. angiotensin converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs), and/or lipid lowering agents, pro- Correspondence: Dr. Ellen T. McCarthy, Kidney Institute, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160. Phone: 913- gression of renal dysfunction is slowed (7,8). Rapid progression 588-9252; Fax: 913-588-3867; E-mail: [email protected] to ESRD is associated with an increased incidence of posttrans-

Table 1. Evidence for circulating factor(s) in nephrotic syndrome caused by MCNS and FSGS

Clinical Observations/Identified Mediators Experimental Findings References

MCNS vascular permeability activity of Generation of permeability factor 12 serum of INS patients by T lymphocytes (VPF) increased urinary protease, Proteinuria after hemopexin 14 to 16 hemopexin, in active INS injection into rats. Altered nephrin expression and glomerular endothelial cell monolayer permeability by hemopexin FSGS immediate recurrence of proteinuria Induction of proteinuria in rat 2, 21, 22, 24, 28, 30 to 35, 38 to 40 after transplantation; efficacy of after injection of FSGS serum plasmapheresis, immunoadsorption or plasma; increased in reducing proteinuria glomerular permeability after incubation with plasma/ fraction suPAR increased in FSGS plasma suPAR activates uPAR 17, 18 CLC-1 in active fraction of CLC-1 increases glomerular 52 plasmapheresis fluid from permeability, decreases nephrin recurrent FSGS patients expression, FSGS permeability activity blocked by anti-CLC-1

plant recurrence of proteinuria and FSGS. Therapy for MCNS and disappears during remission (16). Studies of VPR and in children and adults and of FSGS has been recently reviewed hemopexin in patients with FSGS have not been reported. (9–11). Regardless of debates regarding the etiology of NS, current and proposed therapies include general strategies such as (1) Permeability Factors in FSGS identifying and reversing the primary cause of renal injury, (2) We have been studying permeability activity in serum and decreasing proteinuria by interventions relating to hemody- plasma of FSGS patients for more than 20 years. Details of our namic and/or glomerular cellular responses, and (3) slowing observations are given below. Investigators have recently iden- renal fibrosis by the action of nonspecific agents. Current re- tified soluble urokinase receptor (suPAR) in the plasma of search using animal models and clinical trials is aimed at patients with recurrent FSGS. They postulate that it may be a identifying novel therapies that will address each of these cause of proteinuria in FSGS (17). Induction of urokinase re- strategies. ceptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that in- ␣ ␤ Permeability Factors in MCNS cludes lipid-dependent activation of 5 3 integrin (18). The It has long been postulated that a T cell-derived humoral relationship between these factors and the FSGS factor that we factor damages the glomerular permeability barrier in MCNS. have been studying is not known. Candidates include a vascular permeability factor (VPF) and hemopexin. VPF is a lymphokine that is elaborated by con- canavalin A-stimulated T lymphocytes from patients with INS. Differentiation between MCNS and FSGS VPF acts on systemic capillaries and on the glomerular perme- Early in the course of INS, histologic changes may not be ability barrier (12). Its secretion is enhanced by IL-2, IL-15, present, making the distinction between MCNS and FSGS prob- IL-12, and IL-18 and is inhibited by TGF␤1 (13). Hemopexin is lematic. None of the permeability factors proposed for MCNS a protease that activates protein kinase B and Rho A and or FSGS are currently measured in clinical laboratories. In the induces nephrin-dependent reorganization of the actin cy- future, it may be possible to examine patient specimens for a toskeleton in cultured podocytes (14). It reduces endothelial panel of circulating substances. Similarly, diagnostic tests of glycocalyx and increases albumin diffusion across glomerular urine may be developed. Urinary CD80 has been proposed as a endothelial cell monolayers (14). Injection into rats causes pro- diagnostic test to distinguish between MCNS and FSGS (19,20). teinuria and glomerular alterations characteristic of MCNS (15). CD80 appears to arise from podocytes and is found in urine, It is found in the urine of children with steroid-responsive NS but it has not been shown in circulation. Clin J Am Soc Nephrol 5: 2115–2121, 2010 Circulating Permeability Factors in Nephrotic Syndrome 2117

Posttransplant Recurrence of NS/FSGS We and others have studied plasma from patients with re- Proteinuria recurs after initial renal transplantation in ap- current FSGS in an attempt to identify the injurious substance. proximately 30% of patients whose underlying diagnosis is Our efforts have been aided by the use of an in vitro functional FSGS (2,21). Recurrence may exceed 85% in patients with a assay of glomerular permeability (41). Isolated rat glomeruli are history of allograft loss because of recurrence. Risk factors for briefly incubated with patient serum, plasma, or plasma frac- recurrence include young age, rapid course of primary disease tion. An albumin oncotic gradient across the glomerular wall is (i.e., renal failure within 3 years of diagnosis of primary dis- established by replacing the isolation medium with one of ease), previous recurrence in an allograft, and elevated perme- lower oncotic pressure. When the protein permeability barrier ability activity in an in vitro assay (22). Renal allografts are often is intact and uninjured, water flows into the capillaries, causing biopsied to establish a diagnosis in the setting of recurrent the glomeruli to increase in size. Incubation with injurious proteinuria. However, recurrent FSGS is the presumptive diag- substances such as sera from patients with recurrent FSGS nosis in patients with primary FSGS and proteinuria in the causes a decrease in the albumin reflection coefficient of the ␴ early posttransplant period. Biopsies in the first hours after membrane ( alb) and a decrease in the effective oncotic gradi- proteinuria have no morphologic changes. Subsequent biopsies ent. The increase in glomerular volume that occurs in response may show only foot process effacement, but glomerulosclerosis to an albumin oncotic gradient is then diminished compared to eventually appears unless a remission can be induced. Prema- the response of control glomeruli. Because the concept of re- ture allograft loss after recurrence is common (23,24). Therapy flection coefficient has no intuitive relationship to clinical dis- of recurrent FSGS includes early plasmapheresis intended to ease, we have calculated the convectional albumin permeabil- Ϫ ␴ remove injurious substances(s). Possible alternative interpreta- ity, Palb,as(1 alb), a dimensionless parameter that ranges tions of the observed benefit include the addition of a salutary from 0 in normal glomeruli to 1.0 with maximal injury. The

substance or immunomodulation. High doses of calcineurin reproducibility of the Palb assay is high with a correlation of inhibitors often improve proteinuria and stabilize renal func- 0.72 (P Ͻ 0.001) (33), and an agreement of repeated values tion. These agents likely have multiple targets (25–27), but they within 0.3 in Ͼ83% of patients in whom repeated samples have do not decrease circulating permeability activity (27). We spec- been tested (37). The assay reflects injury to the glomerular ulate that long-term remissions after relatively short courses of protein permeability barrier and is not specific to FSGS serum/ plasmapheresis may also be related to protective effects of plasma. For example, agents as diverse as TNF␣, superoxide, or calcineurin inhibitors or other agents. It is also possible that antibodies to protein tyrosine phosphatase receptor and ␤1

susceptibility to injurious agents is enhanced by ischemic or integrin increase Palb (42–46). immunological injury at the time of transplant, and recovery Sera from some but not all patients with FSGS cause in- Ͼ confers some degree of resistance. creased Palb in isolated glomeruli. In our initial report, Palb 0.5 was found in approximately 30% of FSGS samples tested (33). It should be noted that this was a sample of convenience Prevention of Recurrence made up of sera from patients with a wide spectrum of disease Pretransplant plasmapheresis appears to prevent or delay collected at multiple centers. In that study, sera from nine recurrence in high-risk patients (28). Encouragement regarding patients with steroid-sensitive MCNS were negative. We sub- pretransplant immunotherapy comes from a trial in which sequently reported that 42% (11 of 26) of children at presenta- transplant patients received hematopoietic donor cells after a tion with NS had P Ͼ 0.5. P did not discriminate between nonablative preconditioning regimen. Early recurrence of FSGS alb alb steroid-responsive and steroid-resistant patients (47). A pro- was significantly reduced, although only minimal donor-de- spective study of a larger number of children will be needed to rived engraftment occurred (29). The precise mechanism for interpret these results. In patients whose specimens have been this protection is not well defined but might include suppres- submitted to our laboratory for evaluation, P Ͼ 0.5 was sion of generation of a permeability factor by conditioning alb associated with more rapid progression to ESRD (48). In pa- regimen or by the generation of a chimeric state. tients at high risk for recurrence, defined by rapid progression

or previous graft loss to recurrence, Palb was uniformly high.

Permeability Factors in FSGS Palb was also very high in nearly every patient with collapsing and Posttransplant Recurrence glomerulopathy in two independent samples (49,50). Investigators have hypothesized the presence of one or more In vitro assays offer the unique opportunity to study inte- circulating substances injurious to the glomerulus. This hypoth- grated glomerular function in the absence of the influence of esis is supported by observations that proteinuria may begin systemic humoral, hemodynamic, or rheologic forces. The im- immediately after transplantation and that injection of patient mediate responses to various mediators can be measured and plasma or its fractions into rats causes proteinuria (30–32). agents that may have a multitude of systemic effects can be Additionally, plasmapheresis may improve proteinuria and readily studied. In addition, more chronic responses can be lessen the risk of development of progressive FSGS. Our work studied using glomeruli isolated from animal models of disease (31,33–37) and that of several other groups (38–40) has con- or after in vivo manipulations. Glomerular cells maintain struc- firmed that serum or plasma from patients with recurrent FSGS ture and interaction with other glomerular cells and the extra- contains one or more substances that injure the filtration barrier cellular matrix, features that are increasingly recognized as and/or cause proteinuria. being crucial. However, like other technically demanding as- 2118 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 5: 2115–2121, 2010 says, appropriate effort and time are required to standardize patients with genetic mutations in podocyte proteins (54,55). the assay. Several other laboratories have successfully estab- We can only speculate about the relationship between podocyte lished this technique. There is potential for selection bias, es- mutations and circulating permeability factors. It may be that pecially when glomeruli are isolated from animals with estab- the co-occurrence is happenstance (i.e., two relatively common lished disease. It is thus complementary to other assays of conditions coexist). On the other hand, it may be that podocyte glomeruli function. abnormalities predispose to more severe injury by circulating We have used various techniques for protein isolation and substance(s) and result in severe and recurrent renal disease. A described characteristics of the substance(s) in FSGS serum/ study of many podocyte genes and serum activity in patients plasma that exhibit activity in the Palb assay. We have shown with renal disease will be required to understand the relation- that the active substance in FSGS serum or plasma is a small ship between these mechanisms for glomerular injury. protein with one or more glycation sites. It has high affinity for protein A and also has some affinity for cationic materials. It Observations with Implications for Future has a hydrophobic domain as evidenced by affinity in a hydrophobic interaction column. During initial fractionation studies, Therapy We have identified several experimental agents that inhibit we used ammonium sulfate precipitation and found that P alb P activity (36,56). Inclusion in incubation medium of normal activity was present in a fraction with an apparent molecular alb plasma (53), eicosanoids including 20-hydroxyeicosatetraenoic weight of approximately 50 kD. All of the activity of the orig- acid (57) or 8,9-epoxyeicosatrieneoic acid (58), nitric oxide (59), inal specimen was retained in this fraction. P activity per alb certain glycosides of Trypterigium wilfordii (60), or cyclosporine milligram of protein was enriched by more than 10,000 fold (61) prevents the FSGS-induced increase in P . Inhibition of compared with the activity of the initial plasma specimen (51). alb cyclooxygenase (62), phosphatases, or kinases also prevents the More recent studies (unpublished data) using affinity chroma- increase in P (35). These observations may lead to future tography and fractionation by membrane sieving indicate that alb specific therapeutic interventions in FSGS. the Palb activity is present in a fraction with an apparent molecular weight of Յ30 kD. With progressive purification it is likely that noncovalently or otherwise loosely associated pro- Standard Therapy and Potential New teins are removed, accounting for changes in apparent molec- Therapies ular size of the active substance we are studying. Recently, we Steroid therapy is a reasonable first-line therapy and is likely have used galactose affinity purification to enrich specimens to be effective in the early stages of disease, especially in and have used mass spectrometry to identify the proteins in the children. Cyclosporine or MMF may induce remissions in ad- active fraction (37). Galactose affinity chromatography has al- ditional patients. The relative effectiveness of these agents is lowed us to achieve a high degree of enrichment of activity in not yet known, but clinical experience suggests that they may a single step. have approximately the same likelihood of benefit. Control of We have found cardiotrophin-like cytokine-1 (CLC-1) in the blood pressure and use of ACEIs and/or ARBs and control of active fraction from galactose affinity chromatography. CLC-1, lipid levels with statins or fibrates provide useful adjunctive a member of the IL-6 family, is the only cytokine present. measures to decrease proteinuria and slow progression. New Further studies suggest that CLC-1 may be the permeability agents that block effects of aldosterone, renin, vasopressin, or factor in recurrent FSGS. It is present in active patient plasma, endothelin may be useful in limiting podocyte activation as it mimics the effects of FSGS plasma on Palb, and it decreases well as in decreasing proteinuria and controlling blood pres- nephrin expression by glomeruli and cultured podocytes. Strik- sure, but these agents have not been formally tested. ingly, a monoclonal antibody to CLC-1 blocks the Palb effect of New therapies for patients with steroid-resistant or recurrent active FSGS sera. The concentration of CLC-1 in the circulation NS/FSGS are sorely needed, and multicenter trials will be of patients with recurrent FSGS may be up to 100 times higher required. Design of these trials will depend on understanding than in normal subjects (52). Studies to confirm and expand of mechanisms of glomerular injury and or progression of renal these observations are ongoing. damage. The FSGS Clinical Trial (FSGS-CT NCT00135811) is an example of such a trial. Entry into this multicenter randomized Functional Properties of FSGS Permeability controlled trial comparing cyclosporine to MMF plus pulse Factor and Clinical Associations dexamethasone has recently been completed. The study is on- The permeability factor in FSGS plasma not only has a strong going and analysis has not yet been reported. A multicenter affinity for galactose as noted above, but its activity in the Palb phase I trial of rosiglitazone (63,64) and of adalimumab has also Ϫ assay is blocked by galactose in concentrations of 10 12 M (37). been completed (64,65). The salutary responses in some pa-

Activity is also blocked by normal serum or plasma (53). Palb tients and the low incidence of adverse effects support the activity is higher in patients with severe and rapidly progres- potential clinical utility of such agents. A phase II trial is sive disease than in those with slower progression (48). Its underway (IND#103,147) to compare standard conservative removal by plasmapheresis or immunoadsorption is often as- therapy (lisinopril, losartan, and atorvastatin) alone versus stan- sociated with remission of NS (34,38), and pretransplant plas- dard therapy plus rosiglitazone, adalimumab, or galactose. The mapheresis may diminish recurrence (28). Interestingly, activ- rationale for using adalimumab and rosiglitizone is that their ity has been found during posttransplant recurrence in some potential antifibrotic activity may slow or prevent progression Clin J Am Soc Nephrol 5: 2115–2121, 2010 Circulating Permeability Factors in Nephrotic Syndrome 2119 of renal disease independent of their effects on proteinuria. The to define the nature, prevalence of circulating injurious factors, potential efficacy of galactose therapy is supported by a case and mechanisms by which they alter the permeability barrier report of remission of nephrotic syndrome after oral galactose and result in kidney injury must be supported by the renal therapy (66) or complete loss of FSGS permeability activity in a community and by funding agencies. Large multicenter coop- single patient with posttransplant recurrence (37). Other agents erative studies will be required to test efficacy of novel thera- under consideration or in small pilot trials include anti-TGF pies. Meanwhile, physicians must continue to follow the avail- antibodies and rituximab, a monoclonal antibody to the B cell ability of clinical trials (www.clinicaltrials.gov and other sites) surface marker CD20 that depletes B cells. and refer patients to these trials. Broad participation in clinical Strategies for therapy of NS are summarized in Figure 1. trials is essential for significant advances in the care of patients Targets for intervention include circulating substances, the with NS. podocyte itself, the glomerular endothelium, and profibrotic processes. Acknowledgments This work was supported by National Institutes of Health grants Summary DK064969 (McCarthy) and DK 0292588 (Savin) and by support from the There is evidence that circulating factors may play a role in FSGR Foundation. We thank the many patients whose cooperation has MCNS and FSGS in native kidneys and recurrent disease in permitted us to gain understanding of the permeability factors in NS renal allografts. The precise nature of these factors and the and FSGS. mechanisms by which they cause renal injury is the subject of intense investigation. We strongly believe that ongoing studies Disclosures None.

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