Factor In Focal Segmental Glomerulosclerosis Research Articles

Anti-nephrin antibodies in recurrence of focal segmental glomerulosclerosis: closer to discovering the Holy Grail?

Recurrent forms of primary focal segmental glomerulosclerosis (FSGS) pose an unmet challenge to nephrologists, both in terms of understanding the underlying pathophysiology and in terms of identifying an effective management strategy of this disease, which frequently leads to kidney graft loss. In the past few decades, experimental observations both in patients and in animal models have led to the hypothesis of the existence of circulating factors driving the loss of integrity of the glomerular filtration barrier in FSGS. Although different circulating factor candidates have been postulated, none has been unequivocally shown to be pathogenic. In the current study, Shirai etal. propose a new candidate for this role by identifying circulating anti-nephrin autoantibodies in a cohort of patients with post-transplant recurrence of primary FSGS. Recent evidence by Watts etal. has also identified anti-nephrin autoantibodies in the circulation and in the kidney biopsies of patients with minimal change disease. If confirmed, the identification of these autoantibodies would both contribute to identifying the elusive circulating factor in FSGS and increase our understanding of the spectrum of proteinuric glomerular lesions, spanning from minimal change disease to FSGS. The quest for the Holy Grail is perhaps closer to completion.

Current understanding of the molecular mechanisms of circulating permeability factor in focal segmental glomerulosclerosis.

The pathogenetic mechanisms underlying the onset and the post-transplant recurrence of primary focal segmental glomerulosclerosis (FSGS) are complex and remain yet to be fully elucidated. However, a growing body of evidence emphasizes the pivotal role of the immune system in both initiating and perpetuating the disease. Extensive investigations, encompassing both experimental models and patient studies, have implicated T cells, B cells, and complement as crucial actors in the pathogenesis of primary FSGS, with various molecules being proposed as potential "circulating factors" contributing to the disease and its recurrence post kidney-transplantation. In this review, we critically assessed the existing literature to identify essential pathways for a comprehensive characterization of the pathogenesis of FSGS. Recent discoveries have shed further light on the intricate interplay between these mechanisms. We present an overview of the current understanding of the engagement of distinct molecules and immune cells in FSGS pathogenesis while highlighting critical knowledge gaps that require attention. A thorough characterization of these intricate immune mechanisms holds the potential to identify noninvasive biomarkers that can accurately identify patients at high risk of post-transplant recurrence. Such knowledge can pave the way for the development of targeted and personalized therapeutic approaches in the management of FSGS.

Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS.

CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy.Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.

Soluble Urokinase Receptors in Focal Segmental Glomerulosclerosis: A Review on the Scientific Point of View.

Focal segmental glomerulosclerosis (FSGS) is one of the primary glomerular disorders in both children and adults which can progress to end-stage renal failure. Although there are genetic and secondary causes, circulating factors have also been regarded as an important factor in the pathogenesis of FSGS, because about 40% of the patients with FSGS have recurrence after renal transplantation. Soluble urokinase-type plasminogen activator receptor (suPAR) is a soluble form of uPAR, which is a membrane-bound protein linked to GPI in various immunologically active cells, including podocytes. It has recently been suggested as a potential circulating factor in FSGS by in vitro podocyte experiments, in vivo mice models, and human studies. However, there have also been controversies on this issue, because subsequent studies showed conflicting results. suPAR levels were also increased in patients with other glomerular diseases and were inversely correlated with estimated glomerular filtration rate. Nevertheless, there has been no balanced review on this issue. In this review, we compare the conflicting data on the involvement of suPAR in the pathogenesis of FSGS and shed light on interpretation by taking into account many points and the potential variables and confounders influencing serum suPAR levels.

Circulating Permeability Factors in Primary Focal Segmental Glomerulosclerosis: A Review of Proposed Candidates

Primary focal segmental glomerulosclerosis (FSGS) is a major cause of the nephrotic syndrome and often leads to end-stage renal disease. This review focuses on circulating permeability factors in primary FSGS that have been implicated in the pathogenesis for a long time, partly due to the potential recurrence in renal allografts within hours after transplantation. Recently, three molecules have been proposed as a potential permeability factor by different groups: the soluble urokinase plasminogen activator receptor (suPAR), cardiotrophin-like cytokine factor-1 (CLCF-1), and CD40 antibodies. Both CLCF-1 and CD40 antibodies have not been validated by independent research groups yet. Since the identification of suPAR, different studies have questioned the validity of suPAR as a biomarker to distinguish primary FSGS from other proteinuric kidney diseases as well as suPAR's pathogenic role in podocyte damage. Researchers have suggested that cleaved molecules of suPAR have a pathogenic role in FSGS but further studies are needed to determine this role. In future studies, proposed standards for the research of the permeability factor should be carefully followed. The identification of the permeability factor in primary FSGS would be of great clinical relevance as it could influence potential individual treatment regimen.

Permeability factors in idiopathic nephrotic syndrome: historical perspectives and lessons for the future.

The term idiopathic nephrotic syndrome (iNS) traditionally covers minimal change disease and primary focal segmental glomerulosclerosis (FSGS), now thought to be separate disease entities. Clinical and experimental evidence suggest that circulating permeability factors are involved in their pathogenesis. In the past four decades, many investigators have searched for the responsible factors, thus far with little success. The recent report of the soluble urokinase plasminogen activator receptor as a causative factor in FSGS has received much attention, but again the initially promising findings were not confirmed. We describe the history of the search for permeability factors, discuss the pitfalls that are likely responsible for the lack of success and propose criteria that should be used in future studies when evaluating candidate permeability factors.

Has the circulating permeability factor in primary FSGS been found?

A circulating permeability factor has long been implicated in the pathogenesis of primary focal segmental glomerulosclerosis (FSGS). Recent evidence in animal models, and now in several cohorts of patients with primary FSGS, suggest that the soluble urokinase-type plasminogen-activator receptor (suPAR) might fulfill at least a role as biomarker and perhaps even as contributing factor. Although ongoing studies are needed, confirmation of these findings might lead to new diagnostic and therapeutic strategies for this often resistant glomerular disease, as well as a better understanding of podocyte dysfunction.

Plasma from a case of recurrent idiopathic FSGS perturbs non-muscle myosin IIA (MYH9 protein) in human podocytes

The MYH9 gene encodes a non-muscle myosin IIA heavy chain (NMMHC-IIA) expressed in podocytes. Heterozygous MYH9 mutations cause a set of overlapping syndromes characterized by variable degrees of deafness, morphologic abnormalities of platelets and focal segmental glomerulosclerosis (FSGS) with progressive renal dysfunction. Similar glomerular lesions are seen in a variety of nephropathies, including an idiopathic form of FSGS in children which recurs in renal allografts, implying a circulating factor that affects glomerular podocyte biology. It is unknown whether NMMHC-IIA is perturbed in the idiopathic form of FSGS. We describe a pediatric patient with typical idiopathic FSGS, in whom proteinuria recurred within hours of deceased donor renal transplantation but who responded to plasmapheresis. We demonstrate in vitro that plasmapheresis effluent from our patient rapidly decreased cultured podocyte levels of the phosphorylated myosin light chain (MLC) that mediates NMMHC-IIA binding to actin and induced dispersion of NMMHC-IIA from its usual position along actin stress fibers. FSGS plasma also caused dispersion of slit diaphragm proteins (nephrin and podocin) and vinculin-positive focal adhesion complexes. Our observations suggest that the putative circulating factor in idiopathic FSGS disrupts normal NMMHC-IIA function in podocytes and might contribute to the pathogenesis of recurrent FSGS in other children.

Minimal change disease as a modifiable podocyte paracrine disorder.

Minimal change disease as a modifiable podocyte paracrine disorder.

Circulating Permeability Factors in Idiopathic Nephrotic Syndrome and Focal Segmental Glomerulosclerosis

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.

Up-regulation of the Homophilic Adhesion Molecule Sidekick-1 in Podocytes Contributes to Glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a leading cause of nephrotic syndrome and end-stage renal disease worldwide. Although the mechanisms underlying this important disease are poorly understood, the glomerular podocyte clearly plays a central role in disease pathogenesis. In the current work, we demonstrate that the homophilic adhesion molecule sidekick-1 (sdk-1) is up-regulated in podocytes in FSGS both in rodent models and in human kidney biopsy samples. Transgenic mice that have podocyte-specific overexpression of sdk-1 develop gradually progressive heavy proteinuria and severe FSGS. We also show that sdk-1 associates with the slit diaphragm linker protein MAGI-1, which is already known to interact with several critical podocyte proteins including synaptopodin, alpha-actinin-4, nephrin, JAM4, and beta-catenin. This interaction is mediated through a direct interaction between the carboxyl terminus of sdk-1 and specific PDZ domains of MAGI-1. In vitro expression of sdk-1 enables a dramatic recruitment of MAGI-1 to the cell membrane. Furthermore, a truncated version of sdk-1 that is unable to bind to MAGI-1 does not induce podocyte dysfunction when overexpressed. We conclude that the up-regulation of sdk-1 in podocytes is an important pathogenic factor in FSGS and that the mechanism involves disruption of the actin cytoskeleton possibly via alterations in MAGI-1 function.

Focal Segmental Glomerulosclerosis and Renal Transplantation

Primary focal segmental glomerulosclerosis (FSGS) is a major cause of nephrotic syndrome and eventual end-stage renal disease. It is known to be due to an abnormality of the visceral epithelial cells (podocytes) of the glomerulus. The morphological hallmark of primary FSGS is diffuse effacement of podocyte foot processes. The etiology of the podocyte damage is not been clearly established. FSGS can also be a secondary process due to underlying conditions including obesity and heroin use. In the secondary processes, the mechanism appears to be a decreased ratio of podocytes to the glomerular filtration surface area. Familial forms of FSGS also exist due to alterations of several different podocyte proteins. Primary FSGS is an increasing cause of end-stage renal disease. Recurrence of severe FSGS in renal allograft recipients presents a major challenge to transplant physicians. The incidence of recurrence is generally accepted to be between 20% and 30%. Risk factors for and characteristics of recurrence include a rapid progression of the primary disease to end-stage renal failure, early onset of nephrotic range proteinuria after allografting, frequent loss of the allograft, a high frequency of recurrence in subsequent allografts, and children less than 15 years of age. Some investigators have identified a circulating factor called the FSGS factor that appears to be associated with recurrence after transplantation. This factor has been shown to be a protein between 30 and 50 kd molecular weight. Logically, the possibility of a circulating factor associated with recurrence of FSGS led investigators to treat patients with plasmapheresis. Several studies have been reported with varying success. The response of patients to plasmapheresis seems to be completely individual. Other studies have added cyclophosphamide and/or mycophenolate mofetil to the plasmapheresis protocol. Again success in these studies has been variable. However, because some patients show complete recovery with plasmapheresis, individuals who develop recurrent FSGS after transplantation usually are given a trial of plasmapheresis therapy.

Proteinuria after injection of human focal segmental glomerulosclerosis factor.

Recurrent focal segmental glomerulosclerosis (FSGS) is heralded by proteinuria that may remit after treatment with plasmapheresis or immunoadsorption. Study of recurrent FSGS has been hampered by lack of an animal model that exhibits a pattern of proteinuria that mimics human disease. We have obtained a component of FSGS patient plasma (FSGS factor) that increases glomerular albumin permeability (P(alb)) in vitro and causes transient proteinuria in vivo. Plasma fractions containing FSGS factor and comparable plasma fractions from normal donors were injected into normal male Sprague-Dawley rats. Urinary protein, albumin, and creatinine were measured at various time points. Additionally, plasma samples from test animals were collected after injection and tested for FS activity defined by increased P(alb). Finally, glomeruli were isolated from animals after injection and P(alb) of these glomeruli tested. Proteinuria and albuminuria were increased by 24 hr after injection with FSGS factor, and returned to baseline by 48 hr after injection. Injection with the same fraction of normal plasma had no effect on urinary protein. FSGS factor increased urinary protein in a dose-dependent manner. Serum collected from rats 15 or 60 min after injection with FSGS factor increased P(alb) of glomeruli in vitro, whereas serum collected 3 or more hours after injection had no effect. Glomeruli isolated from rats receiving injections with FSGS factor had increased in vitro P(alb) compared with glomeruli from rats injected with a fraction from normal plasma. We have demonstrated that a single injection of FSGS factor increases P(alb) and, causes transient albuminuria and proteinuria in rats. FS activity in the plasma of recipient rats is also transient. This is the first detailed description of the time course and dose-dependence of proteinuria caused by FSGS factor in an animal model.

Indomethacin protects permeability barrier from focal segmental glomerulosclerosis serum

Eicosanoids are believed to play a role in the pathophysiology of several models of glomerular disease. The cyclooxygenase inhibitor indomethacin reduces proteinuria in patients with focal segmental glomerulosclerosis (FSGS) or other glomerular diseases. We have shown that sera of some patients with FSGS significantly increase glomerular albumin permeability (Palb) in an in vitro assay. To determine the role of eicosanoids in the increased Palb caused by the FSGS factor, glomeruli were isolated from normal rats, preincubated with indomethacin, then incubated with FSGS serum or normal serum and Palb was calculated. To study the direct effect of individual eicosanoids on Palb, glomeruli were incubated with prostaglandin E2, prostaglandin F2alpha or a thromboxane A2 mimetic, and Palb was calculated. In the final set of experiments, normal glomeruli were preincubated with the thromboxane synthase inhibitor furegrelate, incubated with FSGS serum, and Palb was calculated. Preincubation of isolated glomeruli with either the cyclooxygenase inhibitor indomethacin or the thromboxane synthase inhibitor furegrelate protected glomeruli from the increase in Palb caused by FSGS serum. Each of the three principal glomerular eicosanoids significantly increased Palb of isolated glomeruli. These studies implicate a product of the cyclooxygenase pathway of arachidonic acid metabolism as mediating the increased Palb caused by FSGS serum in our in vitro assay and possibly the proteinuria seen in patients with FSGS.

Consideration of vaccination against the FSGS factor

Focal segmental glomerulosclerosis (FSGS) is a common cause of nephrotic disease in adults, and it is thought to be increasing in incidence. FSGS is often difficult to treat and even has a high propensity to relapse after transplant. Indeed, this suggests that there might be some circulating factor responsible for proteinuria (‘FSGS factor’). Here I suggest that if and when the FSGS factor is found, vaccination against this factor might be a useful treatment modality for FSGS.

COMPOUND FROM TRIPTERYGIUM WILFORDII PROTECTS ISOLATED GLOMERULI FROM INCREASED ALBUMIN PERMEABILITY CAUSED BY FSGS SERUM

276 Extracts from the plant Tripterygium wilfordii have been used extensively by physicians in China to treat proteinuric renal disease. We have previously shown that sera from patients with focal segmental glomerulosclerosis (FSGS) increase albumin permeability of isolated rat glomeruli in a unique in vitro bioassay, and that crude extract from T. wilfordii prevents the increase in albumin permeability caused by FSGS serum (Nephrol Dial Transplant 12:2064-2068, 1997). The active compound(s) responsible for this protective effect have yet to be identified. In the present studies, we tested four compounds purified from T. wilfordii to determine which may confer protection against the FSGS factor. All compounds were similar in chemical structure with minor differences and included celastrol, tingenone, salaspermic acid and oleanolic acid. Glomeruli were isolated from normal male Sprague-Dawley rats in medium containing 5% bovine serum albumin (BSA). Glomeruli were preincubated with test compound (100 μg/ml) for 10 minutes at 37°C, then washed with fresh isolation medium. Glomeruli were then incubated with FSGS serum for 10 minutes at 37°C. An oncotic gradient was generated by changing the bathing medium to 1% BSA, and volumetric change of glomeruli recorded using videomicroscopy. This change in glomerular volume due to the oncotic gradient was used to calculate convectional albumin permeability (Palbumin). FSGS serum alone and pooled normal serum were used as positive and negative controls, respectively. Palbumin of <0.5 indicated protection from the effect of FSGS serum on glomeruli. Our results show that preincubation with celastrol protected glomeruli from the increase in Palbumin caused by FSGS serum: 0.05 ± 0.12 (celastrol, n=10 glomeruli) vs 0.02 ± 0.02 (normal serum, n=10) vs 0.90 ± 0.13 (FSGS serum, n=15). The other compounds tested did not protect glomeruli from FSGS serum, with Palbumin values significantly higher than normal serum: 0.54 ± 0.13 (tingenone, n=5), 0.63 ± 0.22 (salaspermic acid, n=5), 0.68 ± 0.18 (oleanolic acid, n=5). We conclude that celastrol, a compound purified from extract of T. wilfordii, protects the protein permeability barrier of isolated glomeruli from the effect of FSGS serum. Whether this is the only protective compound from T. wilfordii remains to be determined by further testing.

"The FSGS factor:" enrichment and in vivo effect of activity from focal segmental glomerulosclerosis plasma.

A circulating causative factor has been postulated in focal segmental glomerulosclerosis (FSGS). It has been shown that serum or plasma from some FSGS increases glomerular albumin permeability (Palb) in vitro. Palb greater than 0.5 (i.e., FS activity) is associated with recurrence after transplantation. Specimens from 15 FSGS patients were studied to document the presence of a permeability factor, to isolate this factor, to characterize its biochemical properties, and to show its effect in vivo. Total lipids were extracted by chloroform/methanol (2: 1); FS activity was absent from total lipid extract. Chylomicrons and lipoproteins were removed from the plasma with dextran sulfate, followed by sequential precipitation of proteins at 50 and 70% ammonium sulfate saturation. FS activity was retained in the 70% ammonium sulfate supernatant and exhibited a 100-fold purification. FS activity was lost after heating at 100 degrees C for 10 min or after protease digestion. Under nondenaturing conditions, electrophoresis of the FSGS 70% supernatant showed a prominent low molecular weight band that was not evident in the 70% supernatant from normal plasma. Dialysis and centrifugation-based membrane ultrafiltration of the FSGS factor indicated a molecular size between 30 and 50 kD. Injection of the 70% FSGS supernatant into rats caused a threefold increase in urine protein in collections from 6 to 24 h after injection. No increase in proteinuria occurred in rats injected with 70% supernatant from normal individuals. It is concluded that the FSGS factor is a low molecular weight protein with the potential to increase Palb in vitro and to cause proteinuria in vivo.

INJECTION OF PLASMA PROTEIN FRACTION FROM PATIENTS WITH FSGS CAUSES TRANSIENT PROTEINURIA IN RATS

156 Focal segmental glomerulosclerosis (FSGS) is a glomerular disease characterized by heavy proteinuria, progression to end-stage renal disease, and frequent recurrence in renal allografts. Early recurrence of proteinuria following transplantation and therapeutic effect of plasmapheresis suggest the presence of a causative factor in the circulation of some patients with FSGS. We have documented the presence of permeability activity in sera and plasma of patients with FSGS. Through a series of biochemical manipulations we have succeeded in partially purifying a substance from the plasma of patients with recurrent FSGS which markedly increases albumin permeability in our in vitro assay. The current studies were performed to document that injection of partially purified FSGS factor can cause proteinuria in rats, and that this effect is seen with plasma from different patients. The active fraction of FSGS plasma from Patient 1 (Fpf), 12 mg protein in 1 mL saline, or 12 mg of a comparable fraction of pooled normal human plasma fraction (Npf) was injected intravenously into male Sprague Dawley rats (250-350 g). This amount corresponds to the active protein fraction from about 40 mL of plasma. Urine was collected prior to injection and during 6-12 hour intervals for 2 days. Fpf caused an transient increase in urinary protein in every rat while Npf had no effect. Proteinuria was maximal at 12 hr while serum creatinine was unchanged. Proteinuria, expressed as mg protein/mg creatinine, is shown.TablePlasma from each of three additional patients was subjected to identical biochemical manipulations, and injected into rats in separate experiments. The active fraction from each of these patients caused proteinuria in rats at 12 hours. TableMean arterial pressure in rats injected with Fpf dropped significantly within 3 minutes and returned to normal by 10-15 minutes. Injection with a similar fraction of normal plasma had no effect on mean arterial pressure. In summary, Fpf from each of 4 patients with recurrent FSGS caused transient proteinuria in rats. This finding supports an etiologic role for a circulating factor in human FSGS. The model will be useful for studies of mechanisms of proteinuria and for trials of potential therapeutic agents in FSGS.