NOVEL THERAPIES FOR FSGS: PRECLINICAL AND CLINICAL STUDIES

Abstract

Focal segmental glomerulosclerosis (FSGS) is a rare but important cause of end stage kidney disease in children and adults. Current therapy, consisting of corticosteroids and calcineurin inhibitors, fails to achieve a sustained remission in the majority of patients. Therefore there is a pressing need to develop new treatments for this glomerulopathy. Traditional approaches have focused on agents that modulate the immune system. In this review, we summarize pre-clinical and clinical data with newer agents that may ameliorate FSGS. We focus on drugs that inhibit immune injury or inflammation such as abatacept, rituximab, adalimumab, and stem cells. The potential of agents that block the glomerular action of circulating permeability factors as soluble urokinase receptor is reviewed. Finally, because fibrosis represents the final common pathway of glomerular damage in FSGS, the experience with a wide range of antifibrotic agents is presented. Despite extensive research on the podocyte dysfunction in the pathogenesis of FSGS, there are few agents that directly target podocyte structure or viability. We conclude that FSGS is probably a heterogeneous disorder and that intensified translational research is vital to improve our understanding of distinct subtypes that have a defined prognosis and predictable response to targeted therapeutic interventions.

I. INTRODUCTION

Focal segmental glomerulosclerosis (FSGS) is one of the most common forms of glomerular disease. The entity is defined based on the finding of segmental glomerular sclerosis and hyalinosis¹. Various histopathological variants including tip lesion and a collapsing form have been identified and they may shed light on response to therapy and prognosis². Disturbances in podocyte structure, number and function are considered pivotal to the development of all forms of FSGS and this glomerular disease is now classified as a podocytopathy³.

FSGS accounts for nearly 5–10% of pediatric and adult patients who progress to end stage kidney disease (ESKD). Among those patients who require renal replacement therapy, 15–30% will develop recurrent disease in a transplanted kidney¹. The etiology of FSGS is divided into three categories – primary or idiopathic, genetic, and secondary disease associated with various medications (e.g., pamidronate), infections (e.g. HIV, parvovirus 19), medical conditions (obesity, reflux nephropathy) or critical reduction in kidney mass (e.g. subtotal nephrectomy for Wilms’ tumor)^1,4.

The goal of treatment in patients with FSGS is normalization of urinary protein excretion and preservation of kidney function. However, even partial reduction in proteinuria is beneficial. Studies in children and adults have demonstrated a direct relationship between the degree of lowering of proteinuria and prolongation of renal survival. The standard of care for patients with primary FSGS includes initial treatment with a course of corticosteroids. Up to 25% of patients will respond to this therapy and their prognosis is more favorable¹. For those who are steroid-resistant, the next option is a calcineurin inhibitor with an expected complete or partial remission rate in 40–50% of patients⁵. If these drugs are ineffective, then there is no proven therapy that can consistently achieve a significant and sustained reduction in proteinuria. See companion articles on treatment of FSGS in children (Sethna et al)

There are a number of possible disease mechanisms that can be targeted by novel therapies for FSGS. These include modulation of immunological pathways, inflammation, podocyte cell growth and survival, actin cytoskeleton, circulating factors and fibrosis. In the following sections, we will review pre-clinical and clinical data that support the potential use of novel therapies that are directed at each of these pathophysiological abnormalities.

II. ANIMAL MODELS OF FSGS

Much of our knowledge on the pathophysiology and potential therapeutic targets for FSGS has come from different kinds of animal models that have been developed to mimic the clinical and pathological features of human FSGS. These models induce damage to podocytes. These animal models include the reduction of renal mass by resecting 5/6 of the kidney tissue (unilateral nephrectomy and removal of 2/3 of the contralateral kidney), injury to the renal parenchyma due to drug-induced FSGS using adriamycin, puromycin or streptozocin, virus-induced FSGS, genetically-induced FSGS such as via Mpv-17 inactivation and α-actinin 4 and podocin knockouts using Cre/lox P recombination^6,7. Unfortunately, almost all of these animal models are based on the induction of secondary forms of FSGS and are consequently limited in the ability to mimic primary idiopathic human FSGS.

III. MODULATION OF IMMUNOLOGICAL PATHWAYS AND INFLAMMATION

A. Rituximab

Rituximab, a monoclonal antibody against CD20 on B-cells, was first demonstrated to induce remission of proteinuria in a single patient with a transplant-related lymphoma and recurrent FSGS after kidney transplantation⁸. Subsequent reports have evaluated the effect of rituximab in case series of patients with primary FSGS. Overall, the response has been low, in the range of 20–30%, suggesting that this therapy may have a role in select patients with primary FSGS⁹. There is evidence that rituximab may have off-target effects on lipid metabolism in podocytes by binding to sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b) protein and regulating acid sphingomyelinase activity. This action may contribute to the efficacy of rituximab in post-transplant FSGS¹⁰. Further research is required in order to place rituximab into a rational framework for the treatment of FSGS

B. ACTH

Injections with Adrenocorticotropic hormone (ACTH), a pituitary neuroimmunoendocrine polypeptide, were one of the first therapies used for childhood nephrotic syndrome^11,12. Broad clinical and experimental evidence had long suggested that ACTH has antiproteinuric, lipid lowering and renoprotective properties¹³, and the drug was reintroduced as a treatment alternative for nephrotic syndrome, initially in Europe with a synthetic ACTH depot and then in the United States with natural ACTH gel. Hogan and colleagues treated 24 adult patients with steroid resistant or steroid-dependent FSGS with ACTH and achieved remission in 7 (29%), indicating that this drug may represent an alternative in patients who do not respond to steroids and other common second-line agents¹⁴. It is suggested that ACTH may have actions beyond those attributable to corticosteroids, possibly acting via anti-inflammatory mechanisms or directly on podocytes via the melanocortin 1 receptor¹⁵.

C. Abatacept

Abatacept (CTLA-4–Ig) is a costimulatory inhibitor that targets B7-1 and is currently approved for the treatment of rheumatoid arthritis and juvenile idiopathic arthritis. Reiser et al have shown that induction of the T-cell costimulatory molecule B7-1 in podocytes is associated with nephrotic syndrome¹⁶. Chih-Chuan et al randomly selected biopsy specimens of native human kidneys and identified a subpopulation of patients with minimal-change disease or primary FSGS who had B7-1 immunostaining of podocytes. The authors selected five patients with FSGS and B7-1 staining of podocytes in kidney-biopsy for management with Abatacept. Four of these patients had rituximab-resistant recurrent FSGS after transplantation and one patient had glucocorticoid-resistant primary FSGS. Clinical remission, more specifically, nephrotic-range proteinuria resolved in all the patients over a period of weeks to months¹⁷ If validated, abatacept may be a new therapeutic tool for the subgroup of patients with FSGS who exhibit B7-1 immunostaining in the kidney biopsy specimens. This drug may stabilize β1-integrin activation in podocytes and reduce proteinuria in patients with B7-1–positive glomerular disease.

D. Adalimumab

Monoclonal antibodies are increasingly being used in the treatment of steroid-resistant and steroid-dependent FSGS. Adalimumab is a human monoclonal antibody directed against tumor necrosis factor α (TNF-α). Since TNF-α is upregulated in both human and experimental models of FSGS, attempts to lower proteinuria by inhibiting TNF-α have been made. In the phase 1 trial conducted by the “Novel Therapies for Resistant FSGS” (FONT) study group in children and adults, adalimumab was well tolerated and after sixteen months of follow up, 4 patients of the adalimumab-treated group (n= 10) showed stabilization of kidney function and reduced proteinuria^18,19. This observation suggests that adalimumab may have a role in slowing the progression of FSGS in a select subgroup of patients, but further studies are needed to confirm this.

IV. CIRCULATING FACTORS

The rapid recurrence of FSGS after kidney transplantation, the ability of plasma from patients with FSGS to induce proteinuria after infusion into animals, and the remission of post-transplant FSGS achieved by plasmapheresis have fostered the notion that proteinuria in FSGS is caused by excessive levels of circulating factors that cause dysfunction of the glomerular filtration barrier in some patients²⁰. Over the years, a number of molecules have proposed a variety of circulating permeability factors including hemopexin and vascular endothelial growth factor. Savin and colleagues have searched for these molecules by using a volumetric assay of changes in glomerular size following imposition of an oncotic gradient. They have linked high levels of a circulating FSGS permeability factor with recurrence of disease after transplant and investigated the effect of various therapeutic interventions on the glomerular permeability to albumin²⁰.

A. Galactose

Savin and colleagues have conducted extensive studies on permeability factors in patients with FSGS. They have demonstrated that the molecule(s) has a high affinity for galactose²¹. This has led to the use of oral galactose supplementation in single patients or small case series. There have been several published case reports showing some success with oral galactose in reducing the plasma activity of the FSGS soluble factor^22–24, but these results were difficult to attribute only to galactose since the patients received concomitant treatments including plasmapheresis. Galactose was included as a test treatment in the FONT study and results of this trial are pending. Recent evidence suggests that cardiotrophin-like cytokine factor 1 may be another candidate for the unidentified permeability factor in FSGS that mediates podocyte injury.^20,24a

B. suPAR

A recently identified circulating factor that may contribute to podocyte injury and proteinuria in FSGS is soluble urokinase receptor (suPAR). It is detected in the serum of patients with primary FSGS and activates β3 integrin in podocytes leading to foot process effacement and proteinuria²⁵. Subsequent studies confirmed that suPAR was elevated in 55–85% of patients with primary steroid-resistant FSGS in two large clinical cohorts²⁶. In patients with recurrent FSGS post-transplant, the degree of podocyte effacement correlates with the suPAR concentration and successful therapy is associated with reduction in suPAR levels and restoration of podocyte integrity²⁷. Recent reports have disputed these findings and questioned whether elevated suPAR levels are indicative of higher likelihood of having primary FSGS^28,29. Studies to clarify the role of suPAR in the pathogenesis of primary FSGS in the native kidney and allograft will be key to justifying efforts to develop novel therapies with suPAR as a target.

V. ANTIFIBROTIC AGENTS

Renal fibrosis, characterized by glomerulosclerosis and tubulointerstitial fibrosis, is the final common manifestation leading to ESKD of a wide variety of chronic kidney diseases including FSGS. Characterized by an excessive deposition and net accumulation of extracellular matrix (ECM) components, renal fibrosis represents a complex process involving various intricate intracellular signaling pathways. These include fibroblast and mesangial activation, tubular epithelial to mesenchymal transition, infiltration by inflammatory cells and apoptosis³⁰. Antagonizing the fibrogenic action of these different signaling pathways could therefore be a potential therapeutic strategy in FSGS.

It is important to provide a justification for such a heavy focus on antifibrotic strategies in a review of novel therapies for FSGS. Despite intensive research and advances in the role of the podocyte in this disease, none of the findings have identified a target that is shared by a large percentage of patients with FSGS and proteinuria. Some of the rare genetic mutations in podocyte proteins have pointed towards a specific intervention such as dietary supplementation with coenzyme Q10³¹, yet this appears to be a key target for a very rare subset of the Mendelian causes of nephrotic syndrome¹. Glomerulosclerosis is the only feature that is shared by all forms of FSGS, primary or secondary, and prevention of glomerular fibrosis has been successfully applied in animal models of FSGS and demonstrated to slow the rate of progression of kidney disease. Fibrosis represents the final common pathway of tissue damage in other organs such as hepatic cirrhosis and cardiac fibrosis. Thus, this strategy should benefit from utilization of basic science findings in a wide range of specialties. It is hoped that ongoing prospective studies of patients with primary glomerular disease that include genomics and proteomics will yield more meaningful pathophysiological mechanisms of disease that can be targeted by rational drug design.

A.TGF-β receptor I inhibitors

It is widely accepted that transforming growth factor (TGF)-β and its downstream Smad cascade is a key mediator in the pathogenesis of renal fibrosis both in experimental models and in human kidney diseases. Upregulation of TGF-β is a universal finding in CKD in humans and animal models. TGF-β mediates progressive renal fibrosis by stimulating extracellular matrix production, while inhibiting its degradation. Expression of TGF- β is increased in patients with primary FSGS, particularly in podocytes of sclerotic segments³² and intrarenal transcription of TGF-β is increased in children with FSGS compared to those with minimal change disease³³ suggesting that TGF-β gene transcription is indicative of progressive renal damage typical of FSGS. Therefore, tremendous efforts have been made to develop strategies to reduce the expression or antagonize the effects of TFG-β action in an attempt to hamper the progression of renal fibrosis in FSGS.

1.Direct TGF-β1 inhibitors

Li and colleagues³⁴ reported that a combined therapy using the p38 MAPK pathway inhibitor (SB203580) and a TGF-β receptor I inhibitor (ALK5I) can attenuate renal injury and reduce the progression of adriamycin (ADR)-induced nephropathy in vivo. In a similar study, low-dose of GTW, a natural medicinal plant from Chinese traditional medicine was used to ameliorate the glomerulosclerosis in the same model of ADR-induced FSGS through TGF-β receptor I inhibition³⁵.

a. Resveratrol

Resveratrol, a polyphenol with well documented antifibrotic and anti-inflammatory properties, attenuated the expression of extracellular matrix proteins in both the 5/6th remnant kidney model in rats and cultured mesangial cells exposed to TGF-β1. This effect was at least partially associated with Sirt1, an antifibrotic factor that inhibits TGF-β1 signaling by deacetylating Smad3³⁶.

b. Tranilast (n-[3,4-dimethoxycinnamoyl] anthranilic acid)

Several pre-clinical studies have reported beneficial effects of tranilast, an anti-allergic compound used in Japan for the treatment of hypertrophic scars, as an inhibitor of TGF-β1 induced fibrosis in a range of diseases³⁷. No studies of this agent in human FSGS have been published to date.

c. Decorin

Proteoglycans represent a major component of the ECM. Decorin, also known as PGII or PG-40, plays an important role in collagen fibrillogenesis and fibrocyte differentiation. Decorin can bind TGF-β and neutralize its biological activity and is considered a natural regulator of TGF-β1. Since the discovery that administration of decorin inhibited the increased production of ECM and attenuated the manifestations of renal fibrosis³⁸, similar studies have been performed in animal models of FSGS. However, this therapeutic approach to targeting the adverse effects of TGF-β has not been successfully applied in patients.

2.Neutralizing TFG-β antibodies - Fresolimumab

The administration of neutralizing antibodies against TGF-β has been studied intensively, and has been successful and well tolerated in several types of animal models, mostly in the setting of diabetic nephropathy^39,40. Trachtman and colleagues conducted a phase 1, single-dose study of fresolimumab, a polyclonal anti-TGF-β antibody, for the treatment of resistant primary FSGS. In this study, 3 out of 16 patients had at least a 50% reduction in proteinuria⁴¹. In addition, because of the important anti-inflammatory effect of TGF-β, it may be necessary to develop molecules that can selectively inhibit the profibrotic effects of TGF-β.

3. Pirfenidone [5-methyl-1-phenyl-2(1H)-pyridone]

Pirfenidone is an orally active small molecule known for its anti-fibrotic action. Pirfenidone inhibits TGF-β via multiple pathways: it reduces TGF-β promoter activity, TGF-β protein secretion, TGF-β-induced Smad2 phosphorylation and generation of reactive oxygen species. The beneficial effect of pirfenidone has also been proved not only in vitro but also in patients with FSGS⁴². However, despite the efficacy of pirfenidone in reducing proteinuria, there was no demonstrable decrease in the rate of decline in GFR. It is unknown whether earlier implementation of pirfenidone could achieve a lowering of proteinuria and stabilization in kidney function.

4. microRNAs

MicroRNAs (miRNAs), short non-coding RNAs (about 22 nucleotides), have emerged as participants in the pathogenesis of human diseases, including renal fibrosis^43–45. miRNAs bind to the 3′-untranslated region of target genes to regulate gene expression by post-translational repression or induction of target mRNA degradation.

The kidney is an attractive organ for antifibrotic miRNA therapy due to the observed accumulation of miRNA inhibitors within it after systemic administration. These inhibitors, however, must be delivered selectively to the kidney to avoid deleterious effects of modulating miRNAs in other organs.

MicroRNA (miR)-214 and miR-21 are upregulated in models of renal injury⁴⁶. Denby et al recently showed that miR-214 functions by promoting fibrosis in renal injury independent of TGF-β signaling in vivo. Genetic deletion of miR-214 in mice significantly attenuated interstitial fibrosis induced by unilateral ureteral obstruction (UUO), and antagonism of miR-214 by treatment of wild-type mice with an anti-miR directed against miR-214 (anti-miR-214) before UUO resulted in similar antifibrotic effects. The authors also showed how TGF-β blockade combined with miR-214 deletion granted additional renal protection⁴⁷. Using a similar model, Li and colleagues had previously identified microRNA miR-433 overexpression as an important component of the TGF-β/Smad3-driven renal fibrosis. Delivering a miR-433 knockdown plasmid to the kidney by ultrasound microbubble–mediated gene transfer was able to suppress the induction and progression of fibrosis in the UUO model⁴⁸.

Sun and collaborators used low-dose paclitaxel to ameliorate fibrosis in the remnant kidney model by down-regulating miR-192⁴⁹. Although paclitaxel may have some therapeutic value in ameliorating fibrosis in patients with FSGS, it has not been tested.

The miR-30 family is down-regulated in the podocytes of patients with FSGS⁵⁰. In cultured podocytes, treatment with TGF-β or PAN leads to cytoskeletal damage and apoptosis. In this particular study, the damage was ameliorated by exogenous miR-30 expression and aggravated by miR-30 knockdown. The authors nicely showed how the protective role of miR-30 is due to direct inhibition of Notch1 and p53 (a pathway independent of TGF- β signaling), and how glucocorticoid treatment maintains miR-30 expression in vitro. A potential avenue for development of miRNA therapies and defining a novel mechanism underlying the therapeutic effect of glucocorticoids in the treatment of podocytopathies will be two areas for future research.

B. Miscellaneous antifibrotic drugs

1. Paclitaxel

Paclitaxel (taxol) is an anticancer agent which acts by stabilizing polymerized microtubules, enhancing microtubule assembly, arresting the cell cycle in the G0/G1 and G2/M phases, and ultimately leading to cell death. Zhang and colleagues found that low-dose paclitaxel (taxol) ameliorates renal fibrosis in animals with obstructive uropathy by inhibition of TGF-beta/Smad activity⁵¹. Sun L et al used low-dose paclitaxel to ameliorate fibrosis in the remnant kidney model by down-regulating miR-192⁴⁹. Although paclitaxel may have some therapeutic value ameliorating fibrosis in patients with FSGS, it has not yet been tested in patients.

2. Rosiglitazone

Rosiglitazone and pioglitazone are oral peroxisome proliferator-activated receptor-γ agonists that increase insulin sensitivity. They are used as hypoglycemic agents in patients with type 2 diabetes mellitus and have been shown to have antifibrotic effects in the kidney⁵². The FONT phase 1 trial showed that rosiglitazone was well tolerated in children with drug-resistant FSGS and after 16 months of follow up, 71% of participants had stable GFR and reduced proteinuria^19,53. However, testing of the drug was halted after the FDA warning about the potential cardiovascular side effects of rosiglitazone in older patients with type 2 diabetes. No specific serious safety concerns were identified in patients with FSGS who received rosiglitazone in the FONT trial. Future testing of peroxisome proliferator-activated receptor-γ agonists may be warranted.

VI. STEM CELL THERAPIES

Future therapies for FSGS may rely on stem cells. These cells have an unlimited potential to differentiate into multiple distinct lineages, and can potentially lead to structural remodeling and functional regeneration of renal tissue. Studies have reported the use of stem cells in treatment of selected kidney diseases^54,55. Recently, Ma and colleagues investigated the role of human umbilical mesenchymal stem cells (HuMSCs) on the progression of FSGS using a model of adriamycin-induced nephropathy. Their study found that repeated infusions of HuMSCs improved kidney fibrosis and modulated the inflammatory response in these animals⁵⁶. In a similar approach, Ruan and collaborators used autologous stem cell transplantation for the treatment of renal interstitial fibrosis in rabbits with some success⁵⁷.

Belingheri and colleagues recently reported that administration of three human allogeneic bone marrow mesenchymal stem cells (MSC) infusions in a 13-year-old patient with recurrent FSGS after renal transplantation not responding to conventional therapy resulted in improvement of the proteinuria and stabilization of the renal function leading to discontinuation of the plasmapheresis therapy⁵⁸.

VII. CONCLUSION

FSGS continues to be an important cause of CKD and ESKD in pediatric patients. Treatments that can consistently achieve a durable remission in proteinuria and preservation of kidney function are sorely lacking. Despite decades of intensive basic science and clinical research, no therapeutic target has been identified that is applicable to all patients. This suggests that FSGS is a heterogeneous disease like cancer and that multiple approaches will be needed to achieve a “cure” for affected patients. It is hoped that the ongoing NEPTUNE systems biology approach to disease mechanism discovery will identify biomarkers and mechanism-derived therapeutic targets that allow discrimination of patients into clinically relevant subcategories that can guide delineation of prognosis and formulation of a treatment plan⁵⁹. Because of the rarity of FSGS, multicenter collaborative efforts will be required to translate these experimental findings into clinical practice. This will represent the long-awaited arrival of personalized, evidence-based medicine for patients with this serious glomerulopathy.

TABLE 1.

Novel therapies for FSGS

Mechanism of action	Agents
Modulation of immune system/inflammation	Rituximab ACTH Abatacept Adalimumab
Antagonism of circulating factors	Galactose Inhibitors of suPAR
Antifibrotic	TGF-β inhibitors   Direct: fresolimumab   Indirect: microRNAs
Stem cells	Human umbilical mesenchymal stem cells

CLINICAL SUMMARY.

• Current therapy of FSGS is generally ineffective and cannot prevent progression to end stage kidney disease in the majority of patients.

• Novel therapies are under development that target immune-mediated inflammation and glomerular damage, inhibit the action of circulating permeability factors, or prevent glomerular fibrosis.

• None of the novel therapies have been successfully applied to the treatment of patients with FSGS. Intensified translational research is vital to develop targeted therapeutic interventions for patients with this serious glomerulopathy.