Cell biology and genetics of minimal change disease

Introduction

Minimal change disease (MCD) is characterized by massive proteinuria without histological evidence of immune-mediated damage in the glomeruli. The glomerular podocyte plays a key role in filtration and its loss of function results in loss of protein, mainly albumin or smaller proteins, into the urine with high selectivity¹. Proteinuria in MCD is typically reversible with corticosteroid therapy². T cell dysfunction and circulating factors have long been implicated as a cause of the podocyte dysfunction in MCD³, but their nature still remains to be elucidated.

Recent progress in genetics and cell biology has revealed the molecular mechanisms of dysfunction in podocytes⁴. These findings give us clues to focus on target molecules on the podocyte to deduce what those circulating factors may be. At the same time, we can utilize those molecules as biomarkers not only as a diagnostic tool but also in predicting the disease activity or prognosis. This allows us to administer more accurate and precise treatment to patients with MCD while minimizing side effects caused by drugs.

Alongside MCD as one circulating factor disease is a subset of patients with the histological finding of focal segmental glomerulosclerosis (FSGS). These patients are mostly steroid resistant, and therefore the term steroid-resistant nephrotic syndrome (SRNS) is also used here. These patients often have a rapid relapse after transplantation, indicating another circulating factor disease. It is likely that at least a subset of patients with MCD progress to FSGS/SRNS, with a consistent circulating factor in both. The most compelling evidence for this is the observation that patients with initial steroid sensitivity (assumed to be MCD at that stage) who over subsequent years develop steroid resistance/FSGS, and renal failure, have a 90% chance of post-transplant disease recurrence – the archetypal manifestation of circulating factor disease⁵. In this article, known pathogenesis and mechanisms underlying MCD are reviewed.

Clinical features of MCD

MCD is the most common cause of nephrotic syndrome in children⁶ and around 15–20% of cases in adults⁷, and is characterized by massive proteinuria and hypoalbuminemia, resulting in edema and hypercholesterolemia. Histological findings of the disease in glomeruli are typically normal by light microscopy and only electron microscopy shows effacement of podocyte foot processes without electron-dense immune deposits⁸. These manifestations are typically reversible with the use of corticosteroid therapy in steroid-sensitive nephrotic syndrome (SSNS), so that progressive loss of renal function is rare.

The incidence of MCD in childhood is twofold higher in boys, with a prevalence that is inversely proportional to age. Relapse occurs in 50–80% of patients, and recurrent relapse tends to lessen after adolescence⁹. A genetic defect cannot explain these phenomena in MCD.

Genetics in MCD

T cell dysfunction in MCD

T cell dysfunction has long been postulated and many types of cytokines have been investigated. One of the difficulties in examining a hypothesis that immunological disruption underlies MCD in the laboratory is the lack of an animal model that reflects the pathophysiological mechanism. Haddad et al. employed unique methods and established a nephrotic syndrome model by injecting CD34+ peripheral stem cells obtained from FSGS and MCD patients¹⁸ rather than injecting the supernatant of T cells or peripheral blood mononuclear cells (PBMCs) obtained from the patients¹⁹. The injected cells successfully induced the engraftment of human CD45 leukocytes in the thymus, and only the injection of CD34+ stem cells from patients induced albuminuria. Interestingly, stem-cell-injected mice did not have CD3+ mature T cells, suggesting that the cells responsible for the pathogenesis of idiopathic nephrotic syndrome are more likely to be immature differentiating cells rather than mature peripheral T cells. Naïve T cells (Th0s) have been focused on to investigate the difference in DNA methylation in MCD patients²⁰. The change in DNA methylation patterns from remission to relapse occurs predominantly in Th0s. Epigenetic involvement in the pathogenesis of minimal change nephrotic syndrome in T cells has also been suggested in a report showing that nuclear factor related to kappaB binding protein (NFRKB) was highly expressed in the nuclear compartment in T lymphocytes of MCD patients during relapse and that NFRKB promotes hypomethylation of genomic DNA in HEK cells transfected with NFRKB expression plasmid²¹.

Another T cell dysfunction is a Th17 skew in MCD^22,23 (Figure 1). Patients with SSNS demonstrated after corticosteroid treatment that the Th17/regulatory T cell (Treg) balance returned to normal²⁴. More recently, it was reported that Th17 cells are strong candidate drivers for steroid resistance in immune diseases and have selective attenuation by cyclosporine A²⁵. This could be utilized to predict steroid response in early stages of nephrotic syndrome onset by testing peripheral Th17 levels.

Figure 1. Scheme of lymphocyte dysfunction and circulating factors in minimal change disease (MCD).

By immune trigger such as viral infection, vaccination, and exposure to allergen, antigen-presenting cells and memory B cells present antigen to T lymphocyte. These cells are stimulated to secrete circulating factors in MCD. Rituximab depletes B cells and induces remission; on the other hand, rituximab has an effect on cytoskeleton stability of podocytes and blocks albumin permeability. Th17 skew in MCD may cause steroid resistance and cyclosporine A selectively attenuates Th17. Abbreviations: APCs, antigen presenting cells; CysA, cyclosporine A; CF, circulating factor; IL17, interleukin 17; Th17, helper T subset 17; TNF-a, tumor necrosis factor alpha.

Rituximab and B cell dysfunction

A potential close pathophysiological relationship between MCD and chronic lymphoid neoplasms such as Hodgkin and non-Hodgkin lymphoma has been known since the 1950s, supporting a potential role for B cells in the pathogenesis of MCD (Figure 1). A significant association of HLA-DQA1 (a major histocompatibility complex [MHC] class II) missense coding variants with SSNS recently suggested the possible role of an immune response and the implication of B cells in the pathogenesis of MCD²⁶.

Though the accurate mechanism by which rituximab, a monoclonal antibody against CD20, induces remission in MCD patients remains uncertain, recent observations of the effect of rituximab on complicated refractory SSNS^27–29 suggests a pathophysiological role for B cells in MCD^30,31 (Figure 1). B cell depletion by rituximab resets and suppresses B cell and T cell interactions and keeps the Th17/Treg balance normal, which may lead to sustainable remission^32,33. On the other hand, a direct role for rituximab on podocyte cytoskeleton stabilization was suggested: rituximab prevents disruption of the actin cytoskeleton in cultured normal human podocytes that have been exposed to FSGS patient sera in a sphingomyelin phosphodiesterase acid-like 3b-dependent manner³⁴.

Circulating factors and podocyte cell biology in MCD

A direct test of “circulating factor” activity is to expose human podocytes in culture to active human disease plasma and examine the direct cellular effects on this target cell. It has been shown using this method that nephrotic plasma alters slit diaphragm-dependent signaling and translocates nephrin, podocin, and CD2-associated protein in cultured human podocytes³⁵. This indicated that there is a certain factor increasing or missing in MCD disease plasma.

Hemopexin

Hemopexin (Hpx) is a circulating plasma protease that is synthesized in the liver. The active isoform of Hpx is increased in children with MCD³⁶. In vitro, podocytes showed dramatic reorganization of actin with loss of stress fibers after Hpx treatment³⁷. The Hpx effect on actin is dependent on nephrin followed by RhoA activation and protein kinase B phosphorylation in the downstream intracellular signaling pathway. The effects were reversible and were inhibited by pre-incubation with healthy human plasma or serine protease inhibitors. Though the mechanisms of Hpx activation in the disease are unclear, LPS and tumor necrosis factor (TNF)-α are indicated as possible triggers to activate Hpx in MCD³⁸.

PAR1 signaling axis and VASPp, or suPAR

Because it has serine protease activity³⁹, Hpx may act via the family of protease-activated receptors. There are also matrix metalloproteinases among those proteins that have Hpx homology domains. Recent studies investigated the possibility of a matrix metalloproteinase–protease-activated receptor 1 (PAR1) signaling axis⁴⁰. It was recently reported that proteases present in nephrotic plasma obtained from patients with FSGS can activate PAR1, leading to the podocin-dependent phosphorylation of the actin-associated protein vasodilator-stimulated phosphoprotein (VASP) in human podocytes and increased cell migration, suggesting a novel role for proteases and PARs in the pathogenesis of FSGS^41,42. Although the exact component(s) of FSGS plasma that causes this response remains unknown, the soluble urokinase plasminogen activator receptor (suPAR) has been identified as a potential circulating factor in FSGS via activation of β3 integrin in podocytes and promotes cell motility^43–45. However, correlation of disease activity with suPAR levels has been inconsistent in subsequent reports^46,47. Urinary suPAR was increased in MCD relapse, but it is thought it may simply be a surrogate for proteinuria⁴⁸. These factors are found in FSGS but are potentially also relevant to MCD; this needs experimental verification.

Conclusion

Pathogenic gene mutation analysis in familial MCD has started to reveal insights into underlying mechanisms of pathophysiology in the podocyte, such as endocytosis or Rho GTPase, related to permeability.

We have less evidence of circulating factor activity or from genetic disease in MCD compared to FSGS, perhaps because of less disease severity and lower availability of patient samples in MCD. In terms of circulating factor diseases, findings in FSGS can be examined in relation to MCD. A humanized mouse model might give us a good tool to investigate T cell dysfunction directly related to podocytes.

There are several candidates for biomarkers to predict disease activity or steroid response that allow us to choose precise and acceptable treatment for each individual patient while reducing the side effects of long-term treatment.

New components might be inducible targeting of a specific molecule that is involved in the pathogenesis of MCD both for screening and for treatment.

Abbreviations

MCD, minimal change disease; SSNS, steroid-sensitive nephrotic syndrome; FSGS, focal segmental glomerulosclerosis; SRNS, steroid-resistant nephrotic syndrome; EMP2, epithelial membrane protein 2; LPS, lipopolysaccharide; KANK, kidney ankyrin repeat-containing protein; ARHGDIA, Rho GDP dissociation inhibitor (GDI) alpha; Th0s, naïve T cells; NFRKB, nuclear factor related to kappaB binding protein; Th17, helper T subset 17; Treg, regulatory T cell; PBMC, peripheral mononuclear cell; PAR1, protease activated receptor 1; VASP, vasodilator stimulated phosphoprotein; suPAR, soluble urokinase plasminogen activator receptor; TLR, Toll-like receptor; TNF-α, tumor necrosis factor alpha.