Plasma “Factors” in Recurrent Nephrotic Syndrome After Kidney Transplantation: Causes or Consequences of Glomerular Injury?

The recurrence of nephrotic syndrome after kidney transplantation is a particularly vexing problem. Steroid-resistant nephrotic syndrome with progression to renal failure occurs most often in the setting of focal segmental glomerulosclerosis (FSGS), a pathological diagnosis that is observed in an increasing number of patients who undergo renal biopsy. In patients with FSGS and those with idiopathic nephrotic syndrome (INS) that progresses to renal failure, recurrence of nephrotic syndrome and eventual loss of renal allograft function occurs in about 30% of patients.1, 2 These findings, coupled with the observations that plasmapheresis3 or immunoadsorption with protein A4 or polyclonal anti-immunoglobulin G5 may lead to remission of proteinuria, and that plasma or certain plasma fractions have the capacity to cause proteinuria in experimental animals6, 7 or to alter the function of glomeruli,8, 9, 10 cultured podocytes,11 or mesangial cells,12 support the hypothesis that recurrence is initiated by 1 or more substances in the plasma. Evidence is listed in Box 1.

In this issue of the American Journal of Kidney Diseases, Bruneau et al21 report a component of plasma in patients with FSGS that appears during the recurrence of proteinuria after transplantation and is logically linked to T-cell activation. This substance, soluble ST2 (sST2), is present in greater concentrations in patients with posttransplantation INS recurrence than in healthy individuals or transplant recipients with other diagnoses. It is important to determine whether sST2 is a cause or a marker of recurrent INS after transplantation.

Understanding of nephrotic syndrome has been advanced by studies that defined the essential role of the podocyte and slit-pore junction in control of the glomerular protein permeability barrier. Since nephrin (encoded by the NPHS1 gene) was identified as the essential component of the podocyte slit-pore junction and nephrin deficiency was identified as the molecular defect in congenital nephrotic syndrome of the Finnish type,24 a number of alterations in podocyte genes have been identified in both familial and sporadic FSGS. They include abnormalities in nephrin itself, podocin (NPHS2), alpha actinin 4 (ACTN4), CD2AP (CD2-associated protein), NEPH1 (KIRREL), the cation channel TRPC6, and others. Studies using transgenic mice and ongoing investigations of humans are identifying defects in the structure of the slit-pore junction, localization of membrane proteins to lipid rafts where signaling may occur, active remodeling of the actin cytoskeleton by gain of function of actin-binding proteins, and by prolonged activation of TRPC6 with a net increase in calcium signaling after stimulation with such hormones as angiotensin. These have been nicely summarized.25

These mutations, taken together, may account for the development of nephrotic syndrome and FSGS in only a minority of affected patients. FSGS has been described in adult patients who are compound heterogygotes for podocin,26 and the contribution of such heterozygous mutations in sporadic FSGS is not fully understood. Perhaps surprisingly, some affected individuals with primary genetic disorders show activity during in vitro testing and experience recurrent disease.27, 28, 29 We speculate that FSGS in these individuals may be related to a response to an environmental stimulus by a genetically susceptible individual. Some environmental causes of podocyte injury and INS/FSGS have been defined. These include such toxins as puromycin aminonucleoside, doxorubicin,30 or bisphosphonates.31 Certain viral infections also may lead to FSGS. The most well documented of these is human immunodeficiency virus type 1 (HIV-1), which leads to severe and rapidly progressive collapsing glomerulopathy.32 Collapsing FSGS also is seen in the absence of HIV disease and has been proposed to be a consequence of parvovirus B19.33

Bruneau et al21 report that sST2, a soluble protein with a molecular weight of 50 to 60 kDa, is a novel component in serum of patients with recurrent INS after renal transplantation. sST2 constitutes the extracellular domain of the protein produced by the ST2 gene. It is secreted by activated T-helper type 2 cells; these cells express ST2L on their surface. A putative c-Maf recognition element in the promoter region of the ST2 gene (IL1RL1) provides a potential mechanism for c-Maf to increase its synthesis. The decision to study this protein was based on observations that atypical T-helper type 2 cell polarization in INS relapse is associated with activation of c-Maf and interleukin 4 downregulation and that sST2 contains 3 immunoglobulin repeats and thus is likely to bind to protein A. The investigators compared sST2 concentrations in patients with and without recurrence with additional patients who had posttransplantation proteinuria for reasons other than INS. They determined that sST2 binds to and can be eluted from protein A, and levels in patients with established recurrence of INS were markedly different from those of patients with INS, but no recurrence. This difference was not evident in pretransplantation specimens. Patients with proteinuria from other causes after transplantation also had somewhat increased sST2 levels. Immunoprecipitation coupled with mass spectrometric analysis failed to show an abnormal sST2 isoform. The investigators also studied effects of patient sera and sST2 on cultured mouse podocytes and proteinuria in rats. Sera from patients with recurrence caused distinctive changes in the podocyte actin skeleton that persisted after removal of sST2 and were not replicated by sST2 itself. Neither injection of sST2 nor increased plasma levels after transient transfection by using an adenoviral vector resulted in proteinuria in rats. Thus, the investigators conclude that sST2 may serve as a marker for recurrence of INS, but it does not appear to either predict or cause recurrent nephrotic syndrome.

Two general approaches have been used to attempt to identify the permeability factor in FSGS. The first is a biochemical and analytic approach, augmented by modern proteomic analyses. These studies are based on standard methods of biochemical purification and analyses of molecular characteristics followed by gel electrophoresis and mass spectrometry. We and others have used a functional assay of permeability activity that shows changes in the glomerular capillary albumin reflection coefficient (σalbumin) and albumin permeability (Palb) after incubation with the patient plasma or serum.8, 9, 10, 34 This assay has made it possible to perform sequential purification steps and select fraction(s) with enhanced activity. We recently have used galactose as an effective affinity material to enrich activity of FSGS plasma16 and reported that cardiotrophin-like cytokine factor 1 (CLC-1; encoded by CLCF1), a member of the interleukin 6 family, is present in the enriched fraction of FSGS plasma, and that CLC-1 increases glomerular Palb, and its injection causes proteinuria in rats.22 Other investigators have used similar assays of Palb and have shown activity in plasma before transplantation and during recurrence and found that Palb activity is a strong predictor of recurrence.35 Mass spectrometry has shown the presence of other components in FSGS sera that include apolipoprotein E (APOE) and oxidized albumin.18, 19 However, as with sST2, they do not appear to act on the podocyte or filtration barrier. Urine markers in proteinuric renal diseases include distinctive patterns of 11 proteins, including orosomucoid, transferrin, α1-microglobulin, zinc α2-glycoprotein, α1-antitrypsin, complement factor B, haptoglobin, transthyretin, plasma retinol-binding protein, albumin, and hemopexin.36, 37

An alternative strategy for discovering the permeability factor in nephrotic syndrome has been to identify candidate molecules based on proposed mechanisms of action or associations with abnormalities in regulation of the immune system. This approach was used to identify sST2 and its association with posttransplantation recurrence of INS in the present article. A similar approach led to identification of soluble urokinase receptor (suPAR), another candidate molecule proposed as a mediator of podocyte injury, in FSGS serum.23 CD80 has been found in urine of patients with active INS,37 but no effect on glomerular function has been shown. Successful identification of the molecules that cause recurrence of nephrotic syndrome in renal allografts may require combining proteomic analyses with the search for candidates associated with proposed mechanisms.

The precise sequence of events that lead to INS/FSGS with steroid resistance, rapid progression of kidney injury, and posttransplantation recurrence is not clear. T cells have long been implicated, and recent reports of remission after treatment with rituximab suggest additional involvement of B cells. Several naturally occurring animal models of FSGS, including those in the Buffalo/Mna rat38 and soft-coated Wheaton terrier,39 are characterized by immune disorders, as well as renal disease. The problem of whether minimal change nephrotic syndrome, steroid-resistant INS, and FSGS are encompassed within the spectrum of a single disease or are distinct entities is unresolved. In the absence of clear definition of disease process, it is not surprising that management of recurrent disease is empirical, expensive, and of limited efficacy.

Additional investigations of humans with these disorders and the effects of soluble proteins, cytokines, and other injurious agents on the glomerulus are required. Identification of the molecule(s) that mediate glomerular injury in FSGS/INS and its recurrent phenotype may provide a platform for the design of specific therapies based on molecular interactions. In addition, animal models based on the purported etiologic agents will be essential to test potential therapies. Identification of the unique components of plasma from patients with INS/FSGS and the biological activity of these components may reveal essential clues to the mechanisms of FSGS. Our continued hope is that identifying permeability factors and their actions will lead to treatment to reverse functional changes in the glomerular capillary wall, arrest disease progression, and prevent posttransplantation recurrence.