Membranous nephropathy and lupus-like syndrome after hematopoietic cell transplantation: a case report

<p>Abstract</p> <p>Introduction</p> <p>The kidney is increasingly recognised as a target organ of chronic graft-versus-host disease after hematopoietic cell transplantation in the context of the development of the nephrotic syndrome. Chronic graft-versus-host disease is associated with autoimmune phenomena similar, but not identical, to those observed in various rheumatologic disorders, implicating autoimmunity as an important component of the disease.</p> <p>Case presentation</p> <p>We report the case of a 57-year-old Caucasian man who developed the nephrotic syndrome due to membranous nephropathy in association with recurrent chronic graft-versus-host disease, along with a lupus-like syndrome manifested with pancytopenia, hair loss, positive anti-DNA antibodies and sub-epithelial and mesangial immune deposits. To the best of our knowledge, this is the first case reported in the literature. The nephrotic syndrome subsided soon after he was treated with a short course of cyclosporin with steroids. Unfortunately he died seven months later due to a relapse of leukemia.</p> <p>Conclusions</p> <p>Our case report confirms the notion that chronic graft-versus-host disease is characterized by the appearance of autoimmune phenomena similar, but not identical, to those seen in autoimmune diseases. The decision for more immunosuppression has to be weighed against the need for preservation of the graft versus leukemia phenomenon.</p

In vitro study of TNFa effect in the cytoskeleton of gromerular epithelial cells (podocytes)

Glomerular permeability for macromolecules depends partially on proper attachment of the glomerular epithelial cells (GEC) to the glomerular basement membrane (GBM). The latter requires integrity of the actin cytoskeleton, which in turn is regulated by specific actin – associated proteins. Since several glomerulopathies characterized by heavy proteinouria are associated with increased glomerular tumor necrosis factor α (TNF-α) expression, we studied the interaction of TNF-α with the actin cytoskeleton of cultured rat GEC. Incubation of GEC with 10 ng/ml TNF-α for variable time periods runding from 15 min to 24 hr demonstrated a marked accentuation and redistribution of actin microfilaments, as shown by direct fluorescence analysis and confocal laser scanning microscopy. Quantitative biochemical determination of the G/total – actin ratio confirmed the above observations. Indeed, this ratio was significantly reduced, indicating substantial polymerization of G-actin and formation of F-actin. Concurrently, TNF-α rapidly induced tyrosine phosphorylation of both paxillin and focal adhesion kinase, without affecting the expression levels of these two proteins. In addition, tyrosine phosphorylation of vinculin became evident, indicating involvement of this focal adhesion marker in the observed actin reorganization. Inhibition of tyrosine phosphorylation by genistein prevented the reorganization of the actin cytoskeleton by TNF-α. We conclude that TNF-α induces substantial reorganizatoin of actin cytoskeleton and focal adhesion. These effects occur simultaneously, with a promet TNF-α – induced tyrosine phosphorylation of paxillin and focal adhesion kinase, indicating that these proteins, shown to regulate actin polymerization and formation of focal adhesions, may be directly involved in the mechanism controlling the observed actin redistribution. We studied effects of TNFα upon the proliferation, cell volume and apoptosis glomerular epithelial cells. Incubation of GEC evidence with TNF-α (10 ng/ml) for 15 min, 2h, 4h and 24h increased of cell volume, while there is no evidence proliferation and apoptosis. These findings suggest that the observed TNF-α-actin cytoskeleton interactions may relate to the pathogenesis of glomerulopathies with heavy proteinuria, in which increased glomerular expression of TNF-α is associated with disturbances in the attachment of podocytes to the GBM.Η ακεραιότητα του φραγµού διήθησης των σπειραµατικών τριχοειδών, ο οποίος παρεµποδίζει τη διήθηση µακροµορίων, εξαρτάται, σε σηµαντικό βαθµό, από την αρτιότητα της πρόσφυσης των σπειραµατικών επιθηλιακών κυττάρων (ποδοκυττάρων) στη βασική σπειραµατική µεµβράνη. Εξάλλου, η πρόσφυση των ποδοκυττάρων εξαρτάται από την ακεραιότητα του κυτταροσκελετού της ακτίνης, η οποία µε τη σειρά της ρυθµίζεται από ειδικές συνδεόµενες µε την ακτίνη πρωτεΐνες. ∆εδοµένου ότι ορισµένες σπειραµατοπάθειες που χαρακτηρίζονται από βαριά λευκωµατινουρία, συνοδεύονται από αυξηµένη έκφραση του σπειραµατικού TNFα, µελετήσαµε την επίδραση του TNFα στον κυτταροσκελετό της ακτίνης, σε καλλιέργειες κυτταρικής σειράς ποδοκυττάρων αρουραίου. Όπως διαπιστώθηκε µε άµεσο ανοσοφθορισµό και µικροσκοπία συνεστίασης, η επώαση των ποδοκυττάρων µε 10 ng/ml TNFα, για χρονικά διαστήµατα από 5 λεπτά έως 24 ώρες, προκάλεσε εκσηµασµένη αύξηση της πυκνότητας και ανακατανοµή των µικροϊνιδίων της ακτίνης. Η παραπάνω παρατήρηση επιβεβαιώθηκε µε ποσοτικό βιοχηµικό προσδιορισµό του κλάσµατος G/ολικής ακτίνης και της de novo σύνθεσης ακτίνης. Πράγµατι, διαπιστώθηκε µια σηµαντική, ταχείας έναρξης και παρατεταµένη µείωση του λόγου G/ολικής ακτίνης, ενδεικτική πολυµερισµού της G- προς F-ακτίνη, αλλά και de novo σύνθεση ακτίνης. Παράλληλα µε τον πολυµερισµό της ακτίνης, ο TNFα προκάλεσε µια ταχέως επαγόµενη τυροσινική φωσφορυλίωση της παξιλλίνης και της κινάσης των εστιακών προσφύσεων (FAK), χωρίς να µεταβάλλει τα επίπεδα έκφρασης των δύο αυτών πρωτεϊνών. Επίσης, η διαπιστωθείσα τυροσινική φωσφορυλίωση της βινκουλίνης απετέλεσε ένδειξη ότι, η πρωτεΐνη αυτή των εστιακών προσφύσεων, συµµετέχει στην παρατηρούµενη αναδιοργάνωση του κυτταροσκελετού της ακτίνης. Επιπλέον, η αναστολή της τυροσινικής φωσφορυλίωσης µε γενιστεϊνη αναστέλει την αναδιοργάνωση του κυτταροσκελετού της ακτίνης από τον TNFα. Τέλος, πέραν των προαναφερόμενων µεταβολών, η έκθεση των ποδοκυττάρων σε TNFα οδηγεί σε αύξηση του κυτταρικού τους όγκου, χωρίς όµως να επηρεάζει τον κυτταροπολλαπλασιασµό ή την κυτταρική απόπτωση. Συμπεραίνουμε ότι, στο κυτταρικό µας σύστηµα, ο TNFα επάγει µια σηµαντική αναδιοργάνωση του κυτταροσκελετού της ακτίνης και των εστιακών προσφύσεων. Τα δύο αυτά φαινόµενα, τα οποία συµπίπτουν χρονικά, συνοδεύονται από µια άµεση, TNFα-επαγώµενη, τυροσινική φωσφορυλίωση της FAK και της παξιλλίνης. ∆εδοµένου ότι, όπως είναι γνωστό, οι δύο αυτές πρωτεΐνες ρυθµίζουν τον πολυµερισµό της ακτίνης και τη συγκρότηση των εστιακών προσφύσεων, θεωρούµε πολύ πιθανό ότι εµπλέκονται ευθέως στην παρατηρούµενη αναδιάταξη της ακτίνης. Στο σύνολό τους, τα ευρήµατα της παρούσας µελέτης επιτρέπουν το συµπέρασµα ότι οι επαγώµενες από τον TNFα διαταραχές, οι οποίες διαπιστώθηκαν στο κυτταρικό µας σύστηµα, ενδέχεται να αποτελούν το µοριακό υπόβαθρο των διαταραχών της πρόσφυσης των ποδοκυττάρων στη βασική σπειραµατική µεµβράνη, οι οποίες χαρακτηρίζουν τις σπειραµατοπάθειες µε βαριά λευκωµατινουρία. Η υπερέκφραση του σπειραµατικού TNFα, η οποία έχει διαπιστωθεί στις σπειραµατοπάθειες αυτές θα µπορούσε να είναι µέρος του µηχανισµού, που προκαλεί ή/και συντηρεί τη σπειραµατική βλάβη

Rapamycin Ameliorates Proteinuria and Restores Nephrin and Podocin Expression in Experimental Membranous Nephropathy

Objective. Recent studies have shown a beneficial effect of rapamycin in passive and active Heymann Nephritis (HN). However, the mechanisms underlying this beneficial effect have not been elucidated. Methods. Passive Heymann Nephritis (PHN) was induced by a single intravenous infusion of anti-Fx1 in 12 Sprague-Dawley male rats. One week later, six of these rats were commenced on daily treatment with subcutaneous rapamycin 0.5 mgr/kg (PHN-Rapa). The remaining six rats were used as the proteinuric control group (PHN) while six more rats without PHN were given the rapamycin solvent and served as the healthy control group (HC). All rats were sacrificed at the end of the 7th week. Results. Rapamycin significantly reduced proteinuria during the autologous phase of PHN. Histological lesions were markedly improved by rapamycin. Immunofluorescence revealed attenuated deposits of autologous alloantibodies in treated rats. Untreated rats showed decreased glomerular content of both nephrin and podocin whereas rapamycin restored their expression. Conclusions. Rapamycin monotherapy significantly improves proteinuria and histological lesions in experimental membranous nephropathy. This beneficial effect may be mediated by inhibition of the alloimmune response during the autologous phase of PHN and by restoration of the normal expression of the podocyte proteins nephrin and podocin

Human TTRV30M localization within podocytes in a transgenic mouse model of transthyretin related amyloidosis: does the environment play a role?

Transthyretin related amyloidosis is a nosological entity that leads to disability, diminished quality of life, all stages of chronic kidney disease and eventually death. Podocytes are polarized, highly differentiated epithelial cells important for proper nephron function. In the present study we investigated whether deposited TTRVal30Met (TTRV30M) molecules could be localized within podocytes in situ under the effect of different housing conditions (i.e. specific pathogen free [SPF] vs. non-SPF). Murine renal glomeruli from human TTRV30M (hTTRV30M) transgenic mice were examined via direct and indirect immunofluorescence techniques for the presence of hTTRV30M, murine serum amyloid P, activated caspase-3 and NPHS1. Association strength and amount of colocalization for NPHS1-hTTRV30M, NPHS1-activated caspase-3, hTTRV30M-murine serum amyloid P were estimated. Localization of hTTRV30M in podocytes was demonstrated by immuno-electron microscopy. Renal hTTRV30M gene and NPHS1 gene expression levels were estimated. Non-SPF transgenic mice showed increased glomerular hTTRV30M deposition compared to their SPF counterparts. Furthermore increased podocytic localization of hTTRV30M was noticed in non-SPF mice. Glomerular caspase-3 activation was increased only in the non SPF housing conditions. Podocytic caspase-3 activation was increased in SPF and in non-SPF transgenic mice when compared to non transgenic controls. Environmental conditions influence glomerular deposition and podocytic localization of hTTRV30M. In this context increased caspase-3 activation occurred

Hsf-1 affects podocyte markers NPHS1, NPHS2 and WT1 in a transgenic mouse model of TTRVal30Met-related amyloidosis

Introduction: Familial amyloid polyneuropathy is characterized by transthyretin (TTR) deposition in various tissues, including the kidneys. While deposition induces organ dysfunction, renal involvement in TTR-related amyloidosis could manifest from proteinuria to end-stage kidney failure. As proteinuria is considered result of glomerular filtration barrier injury we investigated whether TTR deposition affects either glomerular basement membrane (GBM) or podocytes. Materials and methods: Immunohistochemistry, immunoblot and gene expression studies for nephrin, podocin and WT1 were run on renal tissue from human-TTRV30M transgenic mice hemizygous or homozygous for heat shock factor one (Hsf-1). Transmission electron microscopy was used for evaluation of podocyte foot process width (PFW) and GBM thickness in Hsf-1 hemizygous mice with or without TTRV30M or amyloid deposition. Results: Glomeruli of hsf-1 hemizygous transgenic mice showed lower nephrin and podocin protein levels but an increased podocyte number when compared to Hsf-1 homozygous transgenic mice. Nephrin, podocin and WT1 gene expression levels were unaffected by the Hsf-1 carrier status. TTRV30M deposition was associated with increased PFW and GBM thickness. Conclusions: Under the effect of Hsf-1 hemizygosity, TTRV30M deposition has deleterious effects on GBM thickness, PFW and slit diaphragm composition, without affecting nephrin and podocin gene expression