Aberrantly glycosylated IgA1 induces mesangial cells to produce platelet-activating factor that mediates nephrin loss in cultured podocytes
2009; Elsevier BV; Volume: 77; Issue: 5 Linguagem: Inglês
10.1038/ki.2009.473
ISSN1523-1755
AutoresRosanna Coppo, Valentina Fonsato, Sabrina Balegno, Emanuela Ricotti, Elisa Loiacono, Roberta Camilla, Licia Peruzzi, Alessandro Amore, Benedetta Bussolati, Giovanni Camussi,
Tópico(s)Chronic Kidney Disease and Diabetes
ResumoThe reaction of mesangial cells with aberrantly glycosylated IgA1 has been implicated in the etiology of IgA nephropathy (IgAN). Tumor necrosis factor, which is assumed to mediate the interaction between mesangial cells and podocytes, also induces the expression of platelet-activating factor (PAF). In this study, we determined whether PAF affects the expression of nephrin (an adhesion molecule critical to glomerular permselectivity) and cytoskeletal F-actin organization in podocytes. We treated human mesangial cells with atypically glycosylated IgA1 either prepared in vitro or derived from the sera of patients with IgAN. We then prepared conditioned media from these cells and added them to cultured human podocytes in the presence of PAF receptor antagonists. Podocytes transfected to overexpress acetylhydrolase, the main catabolic enzyme of PAF, served as controls. Downregulation of nephrin expression and F-actin reorganization occurred when podocytes were cultured with mesangial cell-conditioned medium. Preincubation of podocytes with a PAF receptor antagonist prevented the loss and redistribution of nephrin. In control podocytes overexpressing acetylhydrolase, nephrin loss was abrogated. Our results suggest that atypically glycosylated IgA-induced PAF from mesangial cells is a mediator of podocyte changes, which, when more directly tested elsewhere, were found to be associated with proteinuria. Hence, it is possible that these in vitro findings may be relevant to the proteinuria of IgAN. The reaction of mesangial cells with aberrantly glycosylated IgA1 has been implicated in the etiology of IgA nephropathy (IgAN). Tumor necrosis factor, which is assumed to mediate the interaction between mesangial cells and podocytes, also induces the expression of platelet-activating factor (PAF). In this study, we determined whether PAF affects the expression of nephrin (an adhesion molecule critical to glomerular permselectivity) and cytoskeletal F-actin organization in podocytes. We treated human mesangial cells with atypically glycosylated IgA1 either prepared in vitro or derived from the sera of patients with IgAN. We then prepared conditioned media from these cells and added them to cultured human podocytes in the presence of PAF receptor antagonists. Podocytes transfected to overexpress acetylhydrolase, the main catabolic enzyme of PAF, served as controls. Downregulation of nephrin expression and F-actin reorganization occurred when podocytes were cultured with mesangial cell-conditioned medium. Preincubation of podocytes with a PAF receptor antagonist prevented the loss and redistribution of nephrin. In control podocytes overexpressing acetylhydrolase, nephrin loss was abrogated. Our results suggest that atypically glycosylated IgA-induced PAF from mesangial cells is a mediator of podocyte changes, which, when more directly tested elsewhere, were found to be associated with proteinuria. Hence, it is possible that these in vitro findings may be relevant to the proteinuria of IgAN. A clue to the pathogenesis of IgA nephropathy (IgAN) is thought to be the mesangial deposition of circulating aberrantly glycosylated IgA1.1.Coppo R. Amore A. Aberrant glycosylation in IgA nephropathy (IgAN).Kidney Int. 2004; 65: 1544-1547Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar IgA1 is heavily glycosylated, with O-glycosylated carbohydrate chains based on N-acetyl galactosamine (GalNAc) residues, usually extended with galactose (Gal) to form Galβ1,3GalNAc, which may be covered with α-2,6 and/or α-2,3 sialic acid (Sia). Several authors reported the presence of aberrantly glycosylated IgA, with reduced Gal and/or Sia and increased exposure of GalNAc, in sera of patients with IgAN.2.Allen A.C. Harper S.J. Feehally J. Galactosylation of N-and O-linked carbohydrate moieties of IgA1 and IgG in IgA nephropathy.Clin Exp Immunol. 1995; 100: 470-474Crossref PubMed Scopus (255) Google Scholar, 3.Tomana M. Matousovic K. Julian B.A. et al.Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG.Kidney Int. 1997; 52: 509-516Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 4.Hiki Y. Tanaka A. Kokubo T. et al.Analyses of IgA1 hinge glycopeptides in IgA nephropathy by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.J Am Soc Nephrol. 1998; 9: 577-582Crossref PubMed Google Scholar, 5.Amore A. Conti G. Cirina P. et al.Aberrantly glycosylated IgA molecules downregulate the synthesis and secretion of vascular endothelial growth factor in human mesangial cells.Am J Kidney Dis. 2000; 36: 1242-1252Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar Desialylated and degalactosylated IgA (deSial/deGal IgA) have been proven to react with mesangial cells (MCs) and to trigger the synthesis of mediators of inflammation and sclerosis through modifications of integrin expression and proliferation/apoptosis.5.Amore A. Conti G. Cirina P. et al.Aberrantly glycosylated IgA molecules downregulate the synthesis and secretion of vascular endothelial growth factor in human mesangial cells.Am J Kidney Dis. 2000; 36: 1242-1252Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 6.Novak J. Vu N.L. Novak L. et al.Interactions of human mesangial cells with IgA and IgA-containing immune complexes.Kidney Int. 2002; 62: 465-475Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 7.Peruzzi L. Amore A. Cirina P. et al.Integrin expression and IgA nephropathy: in vitro modulation by IgA with altered glycosylation and macromolecular IgA.Kidney Int. 2000; 8: 2331-2340Abstract Full Text Full Text PDF Scopus (34) Google Scholar, 8.Amore A. Cirina P. Conti G. et al.Glycosylation of circulating IgA in patients with IgA nephropathy modulates proliferation and apoptosis of mesangial cells.J Am Soc Nephrol. 2001; 12: 1862-1871PubMed Google Scholar Hematuria is the most common early clinical manifestation of IgAN. When proteinuria develops, the progression toward renal failure is accelerated, and proteinuria is considered as the most powerful risk factor for progression of IgAN.9.Coppo R. D’Amico G. Factors predicting progression of IgA nephropathies.J Nephrol. 2005; 18: 503-512PubMed Google Scholar Investigations on the mechanisms leading to the development of proteinuria in IgAN have focused on podocytes after finding a loss of podocytes in the urine of these patients, with podocytopenia related to the severity of glomerular dysfunction and secondary to unknown podocyte insults.10.Lemley K.V. Lafayette R.A. Safai M. et al.Podocytopenia and disease severity in IgA nephropathy.Kidney Int. 2002; 61: 1475-1485Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar,11.Hara M. Yanagihara T. Kihara I. Cumulative excretion of urinary podocytes reflects disease progression in IgA nephropathy and Schönlein-Henoch purpura nephritis.Clin J Am Soc Nephrol. 2007; 2: 231-238Crossref PubMed Scopus (92) Google Scholar A downregulation of nephrin, a transmembrane adhesion molecule expressed at the slit diaphragm of podocytes, has been shown to have a critical role in glomerular permselectivity.12.Tryggvason K. Unraveling the mechanisms of glomerular ultrafiltration: nephrin, a key component of the slit diaphragm.J Am Soc Nephrol. 1999; 10: 2440-2445Crossref PubMed Google Scholar Nephrin has a structural function in podocyte architecture, and also has the capacity to transfer signals from the podocyte surface to cytoskeleteton.13.Yuan H. Takeuchi E. Salant D.J. Podocyte slit-diaphragm protein nephrin is linked to the actin cytoskeleton.Am J Physiol Renal Physiol. 2002; 282: F585-F591Crossref PubMed Scopus (97) Google Scholar,14.Aaltonen P. Holthöfer H. The nephrin-based slit diaphragm: new insight into the signalling platform identifies targets for therapy.Nephrol Dial Transplant. 2007; 22: 3408-3410Crossref PubMed Scopus (18) Google Scholar Actin cytoskeleton modification with loss of stress fibers has been reported in monolayers of cultured podocytes with altered permeability to albumin, mimicking proteinuric conditions.15.Lennon R. Singh A. Welsh G.I. et al.Hemopexin induces nephrin-dependent reorganization of the actin cytoskeleton in podocytes.J Am Soc Nephrol. 2008; 19: 2140-2149Crossref PubMed Scopus (89) Google Scholar Besides the inherited nephrotic syndrome sustained by mutations of nephrin,16.Kestila M. Lenkkeri U. Mannikko M. et al.Positionally cloned gene for a novel glomerular protein-nephrin is mutated in congenital nephrotic syndrome.Mol Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1480) Google Scholar a reduction in nephrin expression has also been shown in primary glomerular diseases with nephrotic-range proteinuria.17.Langham R.G. Kelly D.J. Cox A.J. et al.Proteinuria and the expression of podocyte slit diaphragm protein, nephrin, in diabetic nephropathy: effect of angiotensin converting enzyme inhibition.Diabetologia. 2002; 45: 1572-1576Crossref PubMed Scopus (190) Google Scholar, 18.Doublier S. Salvidio G. Lupia E. et al.Nephrin expression is reduced in human diabetic nephropaty: evidence for a distinct role for glycated albumin and angiotensin II.Diabetes. 2003; 52: 1023-1030Crossref PubMed Scopus (297) Google Scholar, 19.Doublier S. Ruotsalainen V. Salvidio G. et al.Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome.Am J Pathol. 2001; 158: 1723-1731Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar Nephrin and other podocyte markers, including ezrin and podocin,20.Lai K.N. Leung J.C. Chan L.Y. et al.Podocyte injury induced by mesangial-derived cytokines in IgA nephropathy.Nephrol Dial Transplant. 2009; 24: 62-72Crossref PubMed Scopus (121) Google Scholar as well as glomerular epithelial protein 1,21.Tian J. Wang H.P. Mao Y.Y. et al.Reduced glomerular epithelial protein 1 expression and podocyte injury in immunoglobulin A nephropathy.Int Med Res. 2007; 35: 338-345Crossref PubMed Scopus (18) Google Scholar have been recently reported to be downregulated in mild forms of IgAN and to correlate with proteinuria. In IgAN, a cross-talk between MCs and podocytes has been postulated as a mechanism relevant for the development of proteinuria. In an in vitro model of incubation of podocytes with supernatants of MCs cultured with deSial/deGal IgA or with IgA isolated from sera of patients with IgAN, a loss of nephrin expression and a cytoskeleton protein downregulation have been shown in human glomerular epithelial cells (GECs) (Coppo R et al. J Am Soc Nephrol 2007; 18: 626A). Lai et al.20.Lai K.N. Leung J.C. Chan L.Y. et al.Podocyte injury induced by mesangial-derived cytokines in IgA nephropathy.Nephrol Dial Transplant. 2009; 24: 62-72Crossref PubMed Scopus (121) Google Scholar,22.Lai K.N. Leung J.C. Chan L.Y. et al.Activation of podocytes by mesangial-derived TNF-alpha: glomerulo-podocytic communication in IgA nephropathy.Am J Physiol Renal Physiol. 2008; 294: F945-F955Crossref PubMed Scopus (96) Google Scholar reported that humoral factors (predominantly tumor necrosis factor-α (TNF-α) and transforming growth factor-β) released from MCs are responsible for nephrin loss from podocytes. Platelet-activating factor (PAF) may act as a secondary mediator of TNF-α.23.Camussi G. Albano E. Tetta C. Bussolino F. The molecular action of tumor necrosis factor-alpha.Eur J Biochem. 1991; 1: 3-14Crossref Scopus (235) Google Scholar Moreover, several studies showed that PAF may enhance glomerular permeability, and that TNF-α induced the synthesis of PAF from MCs.24.Schwertschlag U.S. Dennis V.W. Tucker J.A. Nonimmunological alterations of glomerular filtration by s-PAF in the rat kidney.Kidney Int. 1988; 34: 779-785Abstract Full Text PDF PubMed Scopus (14) Google Scholar, 25.Perico N. Remuzzi A. Dadan J. et al.Platelet-activating factor alters glomerular barrier size selectivity for macromolecules in rats.Am J Physiol. 1991; 261: F85-F90PubMed Google Scholar, 26.Sharma R. Sharma M. Li J.Z. et al.Direct effects of platelet-activating factor on glomerular capillary permeability.Kidney Blood Press Res. 1997; 20: 25-30Crossref PubMed Scopus (12) Google Scholar, 27.Camussi G. Turello E. Tetta C. et al.Tumor necrosis factor induces contraction of mesangial cells and alters their cytoskeletons.Kidney Int. 1990; 38: 795-802Abstract Full Text PDF PubMed Scopus (35) Google Scholar The aim of this study was to investigate whether supernatants of MCs incubated with aberrantly glycosylated IgA or with sera from IgAN patients induced nephrin loss from cultured human podocytes and whether this effect was mediated by PAF. Desialylated and degalactosylated polymeric IgA (pIgA) and aberrantly glycosylated IgA1 isolated from sera of patients were removed after 24 h of incubation with MCs, substituted with fresh medium, which was recovered after an additional 12 or 24 h, and incubated with GECs to avoid direct contact between IgA and GECs. The expression of nephrin on nonpermeabilized GECs was evaluated by indirect immunofluorescence using a monoclonal antibody (mAb) specific for its extracellular domain. The staining for nephrin showed a fine punctuated pattern on the surface of GECs (Figure 1a). Aberrantly glycosylated IgA did not directly induce loss of nephrin by GECs (not shown). In contrast, when GECs were stimulated for 1 h with supernatants of MCs previously challenged with deSial/deGal pIgA (Figure 1d and f) or with aberrantly glycosylated IgA1 isolated from patients with IgAN (Figure 2d, e and f), a loss of nephrin was observed, as indicated by the decrease in immunofluorescence staining intensity. The supernatants recovered from MCs incubated with native pIgA or IgA1 isolated from healthy controls did not induce a downregulation of nephrin expression (Figures 1b, c, e, and g). The effect of supernatants from MCs incubated with IgA1 from patients with IgAN was significantly greater than when incubated with IgA1 from healthy controls (Table 1 and Figure 2h). No correlation was found with individual levels of proteinuria. The loss of nephrin induced by aberrantly glycosylated IgA was confirmed by western blot (Figure 1 and 2h) and fluorescence-activated cell sorting analysis (Figure 3).Figure 2Effect of aberrantly glycosylated IgA1 isolated from sera of patients with IgA nephropathy (IgAN) on nephrin expression by glomerular epithelial cells (GECs). (a–f) Representative micrographs of nephrin expression detected by immunofluorescence in control GECs (a), or in GECs treated for 1 h with conditioned medium from mesangial cells (MCs) stimulated for 24 h with native polymeric IgA (pIgA) (b), degalactosylated pIgA (deSial/deGal pIgA) (c) or with aberrantly glycosylated IgA1 isolated from sera of three representative patients with IgAN (d–f). Original magnification × 630. (g) Semiquantitative analysis of nephrin expression evaluated as relative fluorescence intensity after podocyte stimulation with conditioned medium recovered from MCs treated with vehicle alone (Ctr), native pIgA (pIgA), deSial/deGal pIgA or with aberrantly glycosylated IgA1 isolated from sera of three representative IgAN patients (p1, p2, p3). Data are expressed as mean±s.d. of five different experiments. ANOVA (analysis of variance) with Newmann–Keuls multicomparison test was performed: *P<0.05 deSial/deGal pIgA and sera from IgAN patients versus control podocytes. (h) Densitometric analysis and representative western blot of nephrin protein levels in lysates of control podocytes (lane 1, Ctr) or in those treated with conditioned medium from MCs stimulated for 24 h with aberrantly glycosylated IgA1 isolated from sera of p1, p2, p3 IgAN patients (lanes 2, 3 and 4), or from sera of a healthy subject (lane 5), in those treated with conditioned medium from MCs stimulated with deSial/deGal pIgA (lane 6) and with native pIgA (lane 7). Data are expressed as mean±s.d. of three different experiments. ANOVA with Newmann–Keuls multicomparison test was performed: *P<0.05 sera from IgAN patients versus healthy controls or deSial/deGal pIgA versus control podocytes.View Large Image Figure ViewerDownload (PPT)Table 1Nephrin expression on cultured podocytes (GECs) after stimulation with conditioned medium recovered from mesangial cells treated with vehicle alone or with IgA1 isolated from healthy controls or from patients with IgANMesangial cell conditioningNephrin: percentage of positive GECsNephrin: mean relative fluorescence intensityVehicle9840IgA1 from healthy subjects97.1±1.737.0±16.1IgA1 from IgAN patients90.0±0.53**P<0.04 and *P<0.05: significance of the difference of the effect obtained with IgA1 from healthy subjects.27.1±0.88*P<0.04 and *P<0.05: significance of the difference of the effect obtained with IgA1 from healthy subjects.Abbreviations: GEC, glomerular epithelial cell; IgAN, IgA nephropathy.Results of flow cytometry analysis are expressed as percentage of positive cells and as mean relative fluorescence intensity. Data are expressed as mean±standard deviation.** P<0.04 and *P<0.05: significance of the difference of the effect obtained with IgA1 from healthy subjects. Open table in a new tab Figure 3Flow cytometric analysis of downmodulation of nephrin expression in glomerular epithelial cells (GECs). (a–f) Representative flow cytometric analysis of GECs (black histograms; white histograms represent isotypic controls) incubated for 1 h with medium alone (a), or with conditioned medium recovered from mesangial cells (MCs) treated with native polymeric IgA (pIgA) (b), degalactosylated pIgA (deSial/deGal pIgA) (c) or with aberrantly glycosylated IgA1 isolated from sera of three representative IgA nephropathy (IgAN) patients (d–f). (g) Semiquantitative analysis of nephrin expression detected as percentage mean fluorescence in control podocytes and in podocytes treated for 1 h with conditioned medium recovered from MCs treated with native pIgA, deSial/deGal pIgA or with aberrantly glycosylated IgA1 isolated from sera of three representative IgAN patients (p1, p2, p3). Data are expressed as mean±s.d. of seven different experiments. ANOVA (analysis of variance) with Newmann–Keuls multicomparison test was performed: *P<0.05 deSial/deGal pIgA and sera from IgAN patients versus control podocytes.View Large Image Figure ViewerDownload (PPT) Abbreviations: GEC, glomerular epithelial cell; IgAN, IgA nephropathy. Results of flow cytometry analysis are expressed as percentage of positive cells and as mean relative fluorescence intensity. Data are expressed as mean±standard deviation. As the loss of nephrin was previously shown to be associated with cytoskeleton redistribution,15.Lennon R. Singh A. Welsh G.I. et al.Hemopexin induces nephrin-dependent reorganization of the actin cytoskeleton in podocytes.J Am Soc Nephrol. 2008; 19: 2140-2149Crossref PubMed Scopus (89) Google Scholar,28.Doublier S. Zennaro C. Spatola T. et al.HIV-1 Tat reduces nephrin in human podocytes: a potential mechanism for enhanced glomerular permeability in HIV-associated nephropathy.AIDS. 2007; 21: 423-432Crossref PubMed Scopus (36) Google Scholar we studied the effect of supernatants from MCs incubated with deSial/deGal pIgA, as described above, on the organization of F-actin. The supernatants of conditioned MCs induced cytoskeleton reorganization, with loss of stress fibers and the cortical accumulation of F-actin associated with cell retraction (Figure 4). Supernatants of MCs stimulated with native pIgA had no detectable effect on F-actin staining. Tumor necrosis factor-α has been recently suggested to be involved in mediating the cross-talk between MCs and GECs induced by abnormally glycosylated IgA.20.Lai K.N. Leung J.C. Chan L.Y. et al.Podocyte injury induced by mesangial-derived cytokines in IgA nephropathy.Nephrol Dial Transplant. 2009; 24: 62-72Crossref PubMed Scopus (121) Google Scholar,22.Lai K.N. Leung J.C. Chan L.Y. et al.Activation of podocytes by mesangial-derived TNF-alpha: glomerulo-podocytic communication in IgA nephropathy.Am J Physiol Renal Physiol. 2008; 294: F945-F955Crossref PubMed Scopus (96) Google Scholar As PAF is known to mediate several of the effects of TNF-α,23.Camussi G. Albano E. Tetta C. Bussolino F. The molecular action of tumor necrosis factor-alpha.Eur J Biochem. 1991; 1: 3-14Crossref Scopus (235) Google Scholar we investigated whether the effect of the supernatant of MCs incubated with aberrantly glycosylated IgA was mediated by PAF. TNF-α, but not deSial/deGal pIgA, induced a time-dependent and dose-dependent synthesis of PAF by GECs (Figures 5a and b). In turn, GECs expressed a PAF receptor (Figure 5g), and when incubated with synthetic PAF, showed a downregulation in the expression of nephrin (Figure 5c, d and h), suggesting a paracrine effect of PAF. This downregulation was abrogated by the PAF receptor antagonist, WEB2170 (Figure 6). When GECs were stimulated with the supernatant of MCs stimulated with deSial/deGal pIgA, in the presence of WEB2170, the loss of nephrin was abrogated (Figure 6b and e). Similar results were obtained when GECs were incubated with supernatants derived from incubation of MCs with aberrantly glycosylated IgA1 isolated from sera of IgAN patients (Figure 6h, k and n).Figure 6Abrogation of nephrin downregulation induced by degalactosylated polymeric IgA (deSial/deGal pIgA) and by sera from IgA nephropathy (IgAN) patients in glomerular epithelial cells (GECs) treated with platelet-activating factor (PAF) receptor antagonist or WEB2170, and in PAF acetylhydrolase (PAF-AH) GECs. (a, d, g, j, m) Representative micrographs showing nephrin expression in control GECs (a) or in GECs treated for 1 h with conditioned medium recovered from mesangial cells (MCs) stimulated for 24 h with deSial/deGal pIgA (d) or with aberrantly glycosylated IgA1 isolated from sera of three representative IgAN patients (g, j, m); (b, e, h, k, n) representative micrographs showing nephrin expression in GECs previously treated with PAF receptor antagonist WEB2170 and stimulated with vehicle (b) or treated for 1 h with conditioned medium recovered from MCs stimulated for 24 h with deSial/deGal pIgA (e) or treated with aberrantly glycosylated IgA1 isolated from sera from IgAN patients (h, k, n); (c, f, i, l, o) representative micrographs showing nephrin expression in control PAF-AH-transfected podocytes (c) or in PAF-AH-transfected podocytes treated for 1 h with conditioned medium recovered from MCs stimulated for 24 h with deSial/deGal pIgA (f) or with aberrantly glycosylated IgA1 isolated from sera of IgAN patients (i, l, o). Original magnification × 630. (p) Semiquantitative analysis of nephrin expression detected as relative fluorescence intensity in normal GECs, in GECs after treatment with PAF receptor antagonist WEB2170 and in PAF-AH-transfected podocytes treated for 1 h with conditioned medium recovered from MCs stimulated for 24 h with deSial/deGal pIgA or with aberrantly glycosylated IgA1 isolated from sera of three representative IgAN patients. Data are expressed as mean±s.d. of five different experiments. ANOVA (analysis of variance) with Newmann–Keuls multicomparison test was performed: *P<0.05 deSial/deGal pIgA and sera from IgAN patients versus control podocytes.View Large Image Figure ViewerDownload (PPT)Figure 6Abrogation of nephrin downregulation induced by degalactosylated polymeric IgA (deSial/deGal pIgA) and by sera from IgA nephropathy (IgAN) patients in glomerular epithelial cells (GECs) treated with platelet-activating factor (PAF) receptor antagonist or WEB2170, and in PAF acetylhydrolase (PAF-AH) GECs. (a, d, g, j, m) Representative micrographs showing nephrin expression in control GECs (a) or in GECs treated for 1 h with conditioned medium recovered from mesangial cells (MCs) stimulated for 24 h with deSial/deGal pIgA (d) or with aberrantly glycosylated IgA1 isolated from sera of three representative IgAN patients (g, j, m); (b, e, h, k, n) representative micrographs showing nephrin expression in GECs previously treated with PAF receptor antagonist WEB2170 and stimulated with vehicle (b) or treated for 1 h with conditioned medium recovered from MCs stimulated for 24 h with deSial/deGal pIgA (e) or treated with aberrantly glycosylated IgA1 isolated from sera from IgAN patients (h, k, n); (c, f, i, l, o) representative micrographs showing nephrin expression in control PAF-AH-transfected podocytes (c) or in PAF-AH-transfected podocytes treated for 1 h with conditioned medium recovered from MCs stimulated for 24 h with deSial/deGal pIgA (f) or with aberrantly glycosylated IgA1 isolated from sera of IgAN patients (i, l, o). Original magnification × 630. (p) Semiquantitative analysis of nephrin expression detected as relative fluorescence intensity in normal GECs, in GECs after treatment with PAF receptor antagonist WEB2170 and in PAF-AH-transfected podocytes treated for 1 h with conditioned medium recovered from MCs stimulated for 24 h with deSial/deGal pIgA or with aberrantly glycosylated IgA1 isolated from sera of three representative IgAN patients. Data are expressed as mean±s.d. of five different experiments. ANOVA (analysis of variance) with Newmann–Keuls multicomparison test was performed: *P<0.05 deSial/deGal pIgA and sera from IgAN patients versus control podocytes.View Large Image Figure ViewerDownload (PPT) To further demonstrate the role of PAF, we generated a PAF-deficient GEC line by transfecting cells with a vector carrying the cDNA for human plasmatic PAF acetylhydrolase (PAF-AH), the main catabolic enzyme of PAF (PAF-AH GECs). In PAF-AH GECs, no significant loss of nephrin occurred on stimulation with supernatants of MCs previously conditioned by aberrantly glycosylated IgA, either prepared in vitro (Figure 6f) or isolated from patients with IgAN (Figure 6i, l and o), as well as with synthetic PAF (Figure 5e, f and h). In control GECs transfected with an empty vector, the loss of nephrin occurred as in wild-type GECs. As shown in Figure 7a, we confirmed the expression of the PAF receptor in podocytes in vivo in biopsy samples of patients with IgAN. To evaluate whether the mechanism described in vitro also occurs in IgAN patients, we studied nephrin expression and serum PAF-AH activity, which is considered to be a marker of enhanced PAF synthesis.29.Iatrou C. Moustakas G. Antonopoulou S. et al.Platelet-activating factor levels and PAF acetylhydrolase activities in patients with primary glomerulonephritis.Nephron. 1996; 72: 611-616Crossref PubMed Scopus (26) Google Scholar We found that nephrin expression in renal biopsy samples was significantly reduced in patients with IgAN developing proteinuria (seven cases), but not in nonproteinuric patients (six cases) (Figure 7b–d). Moreover, we found that PAF-AH activity was increased in sera of IgAN patients developing proteinuria, compared with 10 healthy controls (Figure 7e). In this study, we report a downregulation of nephrin expression associated with a modification in cytoskeletal organization in human podocytes cultured with supernatants of MCs stimulated with aberrantly glycosylated IgA generated in vitro or isolated from sera of patients with IgAN. Nephrin has a crucial role in the filtrating barrier and in protein glomerular permselectivity. The redistribution and loss of nephrin induced by genetic mutation or immune-mediated stimuli have been correlated with the development of proteinuria and nephrotic syndrome in humans and experimental models;16.Kestila M. Lenkkeri U. Mannikko M. et al.Positionally cloned gene for a novel glomerular protein-nephrin is mutated in congenital nephrotic syndrome.Mol Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1480) Google Scholar, 30.Topham P.S. Kawachi H. Haydar S.A. et al.Nephritogenic mAb 5-1-6 is direct at the extracellular domain of rat nephrin.J Clin Invest. 1999; 104: 1559-1566Crossref PubMed Scopus (134) Google Scholar, 31.Luimula P. Ahola H. Wang S.X. et al.Nephrin in experimental glomerular disease.Kidney Int. 2000; 58: 1461-1468Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 32.Collino F. Bussolati B. Gerbaudo E. et al.Preclamptic sera induce nephrin shedding from podocytes through endothelin-1 release by endothelial glomerular cells.Am J Physiol Renal Physiol. 2008; 294: F1185-F1194Crossref PubMed Scopus (104) Google Scholar, 33.Savin V.J. Sharma R. Lowell H.B. Welling D.J. Measurement of albumin reflection coefficient with isolated rat glomeruli.J Am Soc Nephrol. 1992; 3: 1260-1269PubMed Google Scholar a reduction of nephrin expression has also been shown in primary acquired nephrotic syndromes.17.Langham R.G. Kelly D.J. Cox A.J. et al.Proteinuria and the expression of podocyte slit diaphragm protein, nephrin, in diabetic nephropathy: effect of angiotensin converting enzyme inhibition.Diabetologia. 2002; 45: 1572-1576Crossref PubMed Scopus (190) Google Scholar, 18.Doublier S. Salvidio G. Lupia E. et al.Nephrin expression is reduced in human diabetic nephropaty: evidence for a distinct role for glycated albumin and angiotensin II.Diabetes. 2003; 52: 1023-1030Crossref PubMed Scopus (297) Google Scholar, 19.Doublier S. Ruotsalainen V. Salvidio G. et al.Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome.Am J Pathol. 2001; 158: 1723-1731Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar Here, we show that nephrin was significantly downregula
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