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NDT Advance Access originally published online on November 8, 2005
Nephrology Dialysis Transplantation 2006 21(1):10-13; doi:10.1093/ndt/gfi250
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© The Author [2005]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Editorial Comment

Possible new perspectives for our understanding of nephrotic syndrome recurrence

Caroline Hervé1 and Jacques Dantal2

1 INSERM U643, ITERT and 2 Service de Néphrologie, Immunologie Clinique, 30 Bd Jean Monnet, CHU Nantes, 44093 Nantes, France

Correspondence and offprint requests to: Jacques Dantal, Service de néphrologie, immunologie clinique, 30 Bd Jean Monnet, 44093 Nantes Cedex, France. Email: jacques.dantal{at}chu-nantes.fr

Keywords: nephrotic syndrome; permeability factors; podocyte; recurrence



   Introduction
Top
 Introduction
Hypothetical plasmatic factors
 Genetic contribution
 Podocyte culture techniques
 The nephrotic stage: a...
 Conclusion
 References
 
The identification and characterization of circulating factors that alter glomerular permeability in idiopathic nephrotic syndrome (INS) remain the subject of intensive research in nephrology. The existence of such factors was initially evoked by Shalhoub in 1974 [1] and, although they are thought to be of T-cell origin, their precise nature remains elusive. INS recurs immediately after kidney transplantation in 30% of patients with corticosteroid- or cyclosporin A-resistant INS [2]. The rapidity of this recurrence and the beneficial effects of plasmapheresis [2] and/or immunoadsorptions [3], together with several cases of materno-fetal transmission of glomerular abnormalities [4], lend credence to the involvement of plasmatic factors. In addition, the beneficial effects of corticosteroids and/or immunosuppressive therapies on the initial disease and the T-cell abnormalities recently reported in steroid-sensitive INS [5] indirectly support a T-cell origin.



   Hypothetical plasmatic factors
Top
 Introduction
 Hypothetical plasmatic factors
Genetic contribution
 Podocyte culture techniques
 The nephrotic stage: a...
 Conclusion
 References
 
Major discrepancies have been revealed in the results of studies on the putative plasmatic factor(s) performed in vitro or in vivo in rodents. These discrepancies could be related to the origin of the biological fraction tested [i.e. cortico-sensitive disease, recurrence after transplantation, genetic variant of focal and segmental glomerulosclerosis (FSGS)] and/or to the sensitivity or specificity of the experiments in question. Studies of patients with steroid-sensitive minimal change disease using stimulated cultured T cells, T-cell hybridomas or serum have described hypothetical plasmatic factors. Nevertheless, the specificity of these factors is poor and no firm conclusions could be drawn. Currently, the best characterized candidate in these steroid-sensitive patients is haemopexin [6]. The activity of this 100 kDa haem-binding protein correlated tightly with INS evolution (remission/relapse). Moreover, haemopexin has been shown to reduce the anionic charges of the filtration barrier [7] and to have protease activity [8].

Other groups have studied the putative FSGS factor following INS recurrence post-kidney transplantation. The first reported approach consisted of injecting different plasmas or plasma fractions into rats (intravenously and/or intraperitoneally). These reports were encouraging as they demonstrated the transfer of (an) albuminuric factor(s) in vivo [3,9]. Nevertheless, the results were difficult to reproduce and a clear lack of specificity was demonstrated [10]. These negative results could have been due to a low final concentration of a putative factor, species specificity and/or the presence of an antagonist/neutralizing factor in normal rat serum. The second approach consisted of using isolated rat glomeruli in vitro. Using this technique, pre-transplantation plasma or fractions from INS patients were shown to increase the albumin permeability of normal glomeruli but did not seem to be specific to the disease (i.e. 30% of membranous glomerulonephritis patients) [11]. Greater risk of recurrence was reported for patients in whom pre-transplant sera induced a high albumin permeability [12], but the predictive value of this particular test was not a consistent finding [11]. This bioassay was used in an attempt to detect and identify the plasmatic factor but, despite the use of various samples, no biochemical characterization was performed.



   Genetic contribution
Top
 Introduction
 Hypothetical plasmatic factors
 Genetic contribution
Podocyte culture techniques
 The nephrotic stage: a...
 Conclusion
 References
 
More recently, major progress in our knowledge concerning the mechanisms of INS has come from the identification of some key proteins of the podocyte slit diaphragm. These proteins, which are inter-related, are critical for congenital and early childhood onset nephrotic syndrome [13,14]. Their role is to maintain the integrity of the slit diaphragm and thus its permselectivity [15]. Late recurrence of INS reported in transplanted children with congenital nephrotic syndrome of the Finnish type was elegantly demonstrated to be related to an immunization against nephrin inherent to normal kidney grafts [16]. However, the difference between the intrinsic ‘genetic’ form and the extrinsic ‘immunological’ form due to an extra-renal factor of nephrotic syndrome is not clear-cut. Recurrence of nephrotic syndrome after kidney transplantation is observed in 35% of patients with the ‘immunological’ form, but also in 8% of patients with podocin mutation [17]. Moreover, the involvement of plasmatic factors in the recurrence of congenital nephrotic syndrome cannot be totally excluded, as an increase in albumin permeability was detected with the serum of these patients, both before and after transplantation [18]. The pathophysiological mechanisms underlying INS and its recurrence after kidney transplantation are more complex than the simple hypothesis of an albuminuric factor. They probably involve disequilibria between permeability-inducing as well as protective factors, rendering their identification more difficult.



   Podocyte culture techniques
Top
 Introduction
 Hypothetical plasmatic factors
 Genetic contribution
 Podocyte culture techniques
The nephrotic stage: a...
 Conclusion
 References
 
The successful transfection of primary cultures of human or mouse podocytes with a temperature-sensitive gene construct represents a new and practical tool to study podocyte biology [19,20]. Differentiated podocytes express the typical markers of in vivo podocytes, including key proteins of the slit diaphragm such as synaptopodyn, P-cadherin, nephrin, podocin and CD2AP. This cell line can thus be used as a new tool to examine the effect of nephrotic plasma directly, with a particular focus on the organization of the slit diaphragm complex.

First, it has been reported that incubation of this immortalized podocyte cell line with normal or non-nephrotic plasma is associated with the development of long foot processes and the localization of nephrin, podocin and CD2AP at the cell surface [19]. Recently, Coward et al. [21] published significant data using these cells. They showed an abnormal distribution of slit diaphragm proteins after 48 h exposure to nephrotic plasma, with nephrin, podocin and CD2AP being retained within the cytosol. Previously, two key findings have come from human and animal models of nephrosis: it was shown that nephrin and podocin are localized away from the slit diaphragm in nephrotic patients and that there was a change in slit diaphragm proteins from a linear capillary loop pattern in normal glomerulus to a granular intracellular translocation in conditions of heavy proteinuria [22–24]. The experiments on podocyte culture are therefore able to reproduce in vitro what is thought to occur in vivo during nephrotic syndrome.

Immortalized podocytes are also unique tools for the analysis of intracellular mechanisms of podocyte differentiation and dedifferentiation. The maintenance of the differentiated form, observed with sera from patients in remission, is mediated within minutes through the suppression of calcium flow, via a kinase pathway. Moreover, recent studies showed that blockade of integrin-linked kinase prevented podocyte detachment [25] and that activation of a mitogen-activated protein kinase (MAPK) such as p38 was necessary for podocyte injury [26]. Thus, podocyte activation by kinases seems to play a crucial role in the dedifferentiation process. Another interesting observation made by Coward and colleagues [21] was that normal serum could reverse the delocalization of nephrin, strongly supporting the possibility of a defective or missing protective factor in nephrotic plasma.



   The nephrotic stage: a critical balance
Top
 Introduction
 Hypothetical plasmatic factors
 Genetic contribution
 Podocyte culture techniques
 The nephrotic stage: a...
Conclusion
 References
 
The hypothesis that nephrotic patients lack plasmatic factors essential for the maintenance of glomerular permeability was initially evoked by studies based on isolated glomerular experiments as well as the results obtained using immortalized podocyte cell lines. This hypothesis agrees with the failure to transfer albuminuria to animals using sera, plasma or immunoadsorption eluates and with the inability to reproduce these experiments due to the presence of natural factors that could reverse the effect of the transferred material. Further evidence for the loss of factor(s) necessary for the maintenance of glomerular permeability comes from the description of increased glomerular permeability in the isolated glomerular test using sera from FSGS patients bearing the NPHS2 gene mutation [18]. In the latter case, urine from nephrotic patients, but not from normal controls, blocks in vitro the induction of glomerular abnormalities, strongly suggesting the loss of a protective factor in the urine of nephrotic patients.

Finally, is this hypothesis plausible with clinical observations? The mechanisms of action of plasma treatment are not clear: to our knowledge, ~60% of adult patients with FSGS recurrence present an initial complete or partial remission after such treatment, but no differences can be found with respect to the substitution fluids used (albumin vs fresh frozen plasma). Moreover, immunoadsorption onto protein A or Ig-Therasorb columns can give equivalent results without any protein substitution [2,28]. In addition, this effect is not specific for FSGS recurrence and was also reported in nephrotic syndrome of other origins [29]. Thus, these observations do not support a decrease of a protective factor and suggest that if these treatments could modify the balance between circulating factors, they act mainly by the removal of proteinuric factors. In the recurrent patients, the only widely accepted concept is the immediate recurrence of albuminuria, suggesting that the presence of a pre-formed factor is able to affect glomerular permeability of the kidney graft. The discrepancies concerning the predictive value of the effect of pre-transplantation sera on isolated glomeruli also need to be reconsidered. The defect of a protective factor is difficult to explain by a urinary loss process as these pre-transplanted patients are generally anuric and non-nephrotic. In addition, the high doses of cyclosporin delivered as the first-line therapy for patients with immediate FSGS recurrence could also protect the glomerulus from an extra-renal aggression [27] rather than only block the production of an albuminuric factor of T-cell origin. The non-specific finding reported recently by the loss of a protective factor(s) seems to be more related to the consequences of FSGS that could lead to an amplification loop of the glomerulonephritis, rather than to the cause of the initial disease. In addition, circulating factors have to be considered as well as factors that could be of autocrine origin.



   Conclusion
Top
 Introduction
 Hypothetical plasmatic factors
 Genetic contribution
 Podocyte culture techniques
 The nephrotic stage: a...
 Conclusion
References
 
The balance between different factors able to alter or protect the glomerulus leads us to a new and stimulating field of research certainly more complex than the simple search for a proteinuric factor. The immortalized podocyte model will provide an additional in vitro test for a more precise analysis of the factors involved in the maintenance of podocyte differentiation and will provide new insights into the puzzling mechanisms of nephrotic syndrome.



   Acknowledgments
 
We thank Joanna Ashton for editing the manuscript and D. Sahali (Créteil, France) for critical reading of this manuscript.

Conflict of interest statement. None declared.



   References
Top
 Introduction
 Hypothetical plasmatic factors
 Genetic contribution
 Podocyte culture techniques
 The nephrotic stage: a...
 Conclusion
 References
 

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Received for publication: 5. 9.05
Accepted in revised form: 7.10.05


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