| Abstract |
Up until the end of 1990’s idiopathic membranous nethropathy (IMN) was the most frequent nephropathy among male adults. Gradually, due to the extension of diagnostic renal biopsies to the poorer spanish-spoken and black population, the frequency of IMN was reduced from 30% to 15-23% but remained the most common glomerulopathy among adult male Caucasians. IMN is characterized by thickening of the glomerular basement membrane (GBM), subepithelial deposition of immune complexes, eradication of the foot processes and progressive extension of basement membrane all-around the depositions (spikes), in order to isolate and absorb them. Alongside, IgG and C3 deposits are detected by immunofluorescence whereas the cellularity of the glomerular tuft is not substantially increased.
Pathogenetically, IMN is caused by the deposition of autoantibodies in the sybepithelial aspect of the GBM and the subsequent activation of complement. Recent studies have shown that the antigenic target of the circulating antibodies in the majority of cases of IMN (70%) is phospholipase A2 receptor (PLA2R) and to a lesser degree (10%), thrombospontin type I domain containing 7A (THSD7A) which are present on podocytes as is megalin in rat models of MN. Similarly, neutral endopeptidase has been found as the target antigen in newborns’ podocytes with alloimmune neonatal membranous nephropathy, cationic bovine serum albumin as a planted antigen in early childhood MN and circulating auto-antibodies against human male antigens. The immunocomplex depositions activate complement, leading to reorganization of podocytic cytoskeleton, elimination of the slit diaphragm proteins and eventually to proteinuria. Additionally intracellural proteins and cryptic epitopes may be exposed, thus inducing “a second wave of immunisation”.
Heymann Nephritis is a faithful experimental model of the disease that has been extensively studied since first described by Heymann et al. in 1959. The active model of HN is induced by immunization of Lewis rats with preparations of brush-border proteins. The passive model of HN (PHN) is induced by a single i.v. injection of heterologous anti-brush border antiserum (anti-Fx1A) that produces heterologous IgG subepithelial deposits within hours to days. Proteinuria occurs in all animals within five days. This “heterologous phase” is followed, two weeks later, by an “autologous phase” during which rat IgG antibodies are produced against the heterologous IgG.
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The autologous IgG alloantibodies are also deposited at the subepithelial space, inducing a further increase in proteinuria. The second (autologous) phase of PHN mimics idiopathic MN because during the autologous phase there is production of autoantibodies, against a planted exogenous antigen but also against neoantigens that are exposed in the subepithelial space during the initial injury. Therefore it is the autologous phase of passive HN that shares the same pathophysiological mechanisms to those recently identified in idiopathic MN in humans.
The clinical manifestations of the disease include peripheral edema, nephrotic range proteinuria, hypoalbuminemia, dyslipidemia and abnormal urine sediment (oval fat bodies, red blood cells of glomerular origin, and occasionally epithelial cells). In the initial phase, glomerular filtration rate may be maintained at normal levels.
The therapeutic approach depends on the severity of proteinuria, the degree of renal dysfunction and the renal biopsy findings at the time of diagnosis. Usually in patients with proteinouria less than 8gr/24h and normal renal function are treated conservatively with inhibitors of the renin-angiotensin-aldosterone axis, statins and occasionally with antithrombotic agents. If, after 6 months of conservative treatment, nephrotic syndrome and/or kidney function are not improving or even they are deteriorating then the need for immunosuppressive therapy arises. The immunosuppressive treatment of choice remains the monthly alterations of chlorambucil or cyclophosphamide with steroids (classic or alternative Ponticelli scheme). The new discoveries regarding the implication of auto-antibodies in the pathogenesis of IMN and the recent introduction of rituximab (which targets B-lymphocytes) in our armamentarium made the use of this monoclonal antibody an attractive new therapeutic choice that gradually proves to be effective.
Moreover the new discoveries regarding the immunosuppressive actions of the mTOR inhibitors rapamycin and everolimus on the inhibition of humoral immunity make these agents also attractive for the treatment of IMN. Bonegio et al. demonstrated that low dose rapamycin ameliorated proteinuria in experimental PHN and limited tubulointerstitial inflammation and interstitial fibrosis in association with reduced expression of proinflammatory and profibrotic genes. The beneficial effects of rapamycin have also been observed in active HN. Here we tried to investigate more specific effects of rapamycin, beyond the known antifibrotic ones. In particular we
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examined the effect of rapamycin on podocytes architecture and slit diaphragm proteins nephrin and podocin, as well as on the deposition of pathogenic autoantibodies that coincides with the autologous phase of PHN.
Methods:
Passive Heymann Nephritis (PHN) was induced by a single i.v infusion of 0.5ml anti-Fx1 per 100gr of body weight, in 12 Sprague-Dawley male rats. The anti-Fx1 is a generous donation from Dr Kerjascki. One week later, six of these rats commenced daily subcutaneous injections of Rapamycin 0.5 mgr/Kg (PHN-RAPA group).The remaining six rats with PHN were used as the proteinuric control group (PHN group) while six more rats without PHN were used as the healthy control group (Control). Weekly 24-hour urine collections were made over the following 7 weeks and the animals were sacrificed at the end of the 7th week. Renal tissue was processed for photon and electron microscopy studies as well as for PCR and Western Blot analysis of the slit diaphragm proteins podocin and nephrin. Alongside chemical tests for creatinine, metabolic markers and rapamycin blood levels were measured. For the comparison of urine protein levels between groups, repeated measures ANOVA with Bonferroni correction in post hoc analysis was used. Statistically significant differences were considered if p was less than 0,05 and for the statistical analysis SPSS17 was used.
Results:
At the end of the 7th day (when rapamycin was given to the PHN-RAPA group) both PHN and PHN-RAPA groups displayed severe nephrotic range proteinuria which was sustained until the end of the second week. At this time proteinuria started to continuously decline in the PHN-RAPA group while it remained stable in the PHN group. At the end of the seven weeks period, proteinuria was reduced to the 1/3 of that of the comparator PHN group (p=0.007 in post hoc analysis). However, proteinuria in the PHN-RAPA group approached but did not reached the normal levels of the control group (p<0.001). Concerning the metabolic indices total protein and albumin improved in the PHN-RAPA group, although body weight was reduced compared to the PHN group. Histological lesions were markedly improved by rapamycin. Immunofluorescence revealed attenuated deposits of autologous alloantibodies in
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treated rats. Untreated rats showed decreased glomerular content of both nephrin and podocin whereas rapamycin restored their expression.
Electron microscopy showed reduction of the deposits and GBM width closer to normal.
Conclusions:
Rapamycin has shown to reduce proteinuria and to restore histological lesions in experimental models of immune toxicity. B lymphocytes and autoantibodies play a crucial role in the pathogenesis of membranous nephropathy. Rapamycin inhibits the production of both B and T lymphocytes and promotes the expansion of regulatory T cells which inhibit auto-reactive B-clones.
The present study showed that the administration of Rapamycin after the appearance of the nephrotic syndrome reduced proteinuria, restored the histological lesions and the expression of podocyte proteins nephrin and podocin during the autologous phase of PHN. The elimination of immune deposits during the second phase of PHN, implys that rapamycin depressed allo- and auto-reactive B clones, while on the other hand it restored the normal expression of podocytic proteins crucial for the integrity of the glomerular sieve. Therefore rapamycin could be considered as a new therapeutic choice for membranous nephropathy in the future either alone or combined with other immunsuppressive agents.
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Μελέτη της έκφρασης ποδοκυτταρικών πρωτεϊνών στη μεμβρανώδη σπειραματονεφρίτιδα
