Robert T. McCluskey

Glomerulopathy Associated with Cytomegalovirus Viremia in Renal Allografts

Abstract We investigated the relation between cytomegalovirus (CMV) infection and renal-allograft dysfunction in 14 patients. In seven instances (including two successive transplants in one patient), allograft dysfunction occurred during clinically manifest, viremic CMV infection. In five of these, biopsies revealed little or no tubulointerstitial change but a distinctive, diffuse glomerulopathy characterized by enlargement or necrosis of endothelial cells and accumulation of mononuclear cells and fibrillar material in glomerular capillaries. Two of these allografts recovered their function, both with cessation of high-dose immunosuppression. Biopsies in the other 10 patients revealed predominantly tubulointerstitial changes typical of cellular rejection, and most of these patients did not have viremia. One additional patient, studied prospectively, manifested both forms of allograft injury: tubulointerstitial changes occurring two weeks after transplantation and responding to increased immunosuppression,...

Role of Megalin (gp330) in Transcytosis of Thyroglobulin by Thyroid Cells A NOVEL FUNCTION IN THE CONTROL OF THYROID HORMONE RELEASE

When thyroglobulin (Tg) is endocytosed by thyrocytes and transported to lysosomes, thyroid hormones (T4 and T3) are released. However, some internalized Tg is transcytosed intact into the bloodstream, thereby avoiding proteolytic cleavage. Here we show that megalin (gp330), a Tg receptor on thyroid cells, plays a role in Tg transcytosis. Following incubation with exogenous rat Tg at 37 °C, Fisher rat thyroid (FRTL-5) cells, a differentiated thyroid cell line, released T3 into the medium. However, when cells were incubated with Tg plus either of two megalin competitors, T3 release was increased, suggesting that Tg internalized by megalin bypassed the lysosomal pathway, possibly with release of undegraded Tg from cells. To assess this possibility, we performed experiments in which FRTL-5 cells were incubated with either unlabeled or 125I-labeled Tg at 37 °C to allow internalization, treated with heparin to remove cell surface-bound Tg, and further incubated at 37 °C to allow Tg release. Intact 330-kDa Tg was released into the medium, and the amount released was markedly reduced by megalin competitors. To investigate whether Tg release resulted from transcytosis, we studied FRTL-5 cells cultured as polarized layers with tight junctions on permeable filters in the upper chamber of dual chambered devices. Following the addition of Tg to the upper chamber and incubation at 37 °C, intact 330-kDa Tg was found in fluids collected from the lower chamber. The amount recovered was markedly reduced by megalin competitors, indicating that megalin mediates Tg transcytosis. We also studied Tg transcytosis in vivo, using a rat model of goiter induced by aminotriazole, in which increased release of thyrotropin induces massive colloid endocytosis. This was associated with increased megalin expression on thyrocytes and increased serum Tg levels, with reduced serum T3 levels, supporting the conclusion that megalin mediates Tg transcytosis. Tg transcytosis is a novel function of megalin, which usually transports ligands to lysosomes. Megalin-mediated transcytosis may regulate the extent of thyroid hormone release.

Correlation of antineutrophil cytoplasmic antibodies with the extrarenal histopathology of Wegener's (pathergic) granulomatosis and related forms of vasculitis.

We studied the histologic findings from extrarenal biopsies (especially of the lung or upper respiratory tract) or autopsies of 68 patients who were tested for serum antineutrophil cytoplasmic antibodies (ANCAs). We used antigen-specific assays to detect antibodies against proteinase 3 (PR3) and myeloperoxidase (MPO), the two types of ANCAs of proven diagnostic value for the spectrum of diseases that includes Wegeners (pathergic) granulomatosis, microscopic polyarteritis (microscopic polyangiitis), Churg-Strauss syndrome, idiopathic necrotizing and crescentic glomerulonephritis, and their variants. Twenty-eight patients had antibodies to PR3 and 16 had antibodies to MPO; no patient had antibodies to both. All 44 patients with ANCAs had histologic evidence of this spectrum of diseases. Thirteen patients without histologic evidence of this spectrum of diseases had negative tests for ANCAs. There were no pathologic features that reliably identified patients with one or the other type of ANCA. Eighteen of 31 patients with lesions of Wegeners granulomatosis had antibodies to PR3, seven had antibodies to MPO, and six had neither. Three of four patients with necrotizing arteries without granulomas had anti-MPO antibodies, but similar lesions were seen, together with extravascular granulomas, in three patients with anti-PR3 antibodies. Of 16 patients with alveolar hemorrhage, nine had anti-PR3 and five had anti-MPO antibodies. Two patients diagnosed clinically as having Churg-Strauss syndrome had anti-MPO antibodies. In 16 of the 25 patients with ANCAs and a histologic diagnosis of Wegeners granulomatosis the diagnosis was made on the basis of extravascular granulomatous lesions alone, which argues against the requirement for vasculitis. Of six patients with negative tests for ANCAs and histologically diagnosed Wegeners granulomatosis, none had evidence of renal involvement. We conclude that in the appropriate clinical setting the presence of anti-PR3 or anti-MPO antibodies provides reliable evidence of the above spectrum of diseases, but that subclassification (to the extent this is possible) depends on the presence of distinctive clinical or pathologic features. In patients with negative tests for ANCAs, interpretation of clinical and histologic findings remains the only definitive method of diagnosis.

MEGALIN (GP330) : A PUTATIVE ENDOCYTIC RECEPTOR FOR THYROGLOBULIN (TG)

Megalin (gp330) is a large glycoprotein receptor found mainly on a group of absorptive epithelial cells, including renal proximal tubule, epididymal and thyroid cells. Megalin has been shown to bind multiple, unrelated ligands, mainly in vitro, and to mediate endocytosis of ligandsin cultured cells. However, physiologic ligands of megalin are largely unknown. In the present study we have demonstrated that purified rat megalin binds rat thyroglobulin (Tg) in solid phase assays, with anestimated Kd of 9.2+/-0.6 nM. Binding was calcium dependent and was almost completely inhibited by excess Tg, by three megalin ligands - lactoferrin, lipoprotein lipase and apolipoprotein J- and by the receptor associated protein (RAP), which inhibits binding of all megalin ligands. Three anti-megalin antibodies partially inhibited Tg binding to megalin. 125I labeled Tg bound to megalin was released by EDTA and heparin; the released product was shown by SDS-PAGE and autoradiography to be 660 kD (dimeric) Tg. However, an immunoblotting experiment showed binding of megalin both to monomeric (330 kD) and dimeric Tg. We propose that megalin, which is known to mediate ligand endocytosis and is found on the apical surface of thyrocytes, may participate in the endocytosis of Tg from the colloid, a process that is required for hormone release from Tg.

Megalin (gp330) Is an Endocytic Receptor for Thyroglobulin on Cultured Fisher Rat Thyroid Cells

We recently reported that megalin (gp330), an endocytic receptor found on the apical surface of thyroid cells, binds thyroglobulin (Tg) with high affinity in solid phase assays. Megalin-bound Tg was releasable by heparin. Here we show that Fisher rat thyroid (FRTL-5) cells, a differentiated rat thyroid cell line, can bind and endocytose Tg via megalin. We first demonstrated that FRTL-5 cells express megalin in a thyroid-stimulating hormone-dependent manner. Evidence of Tg binding to megalin on FRTL-5 cells and on an immortalized rat renal proximal tubule cell line (IRPT cells), was obtained by incubating the cells with125I-Tg, followed by chemical cross-linking and immunoprecipitation of 125I-Tg with antibodies against megalin. To investigate cell binding further, we developed an assay in which cells were incubated with unlabeled Tg at 4 °C, followed by incubation with heparin, which released almost all of the cell-bound Tg into the medium. In solid phase experiments designed to illuminate the mechanism of heparin release, we demonstrated that Tg is a heparin-binding protein, as are several megalin ligands. The amount of Tg released by heparin from FRTL-5 and IRPT cells, measured by enzyme-linked immunosorbent assay (ELISA), was markedly reduced by two megalin competitors, receptor-associated protein (RAP) and 1H2 (monoclonal antibody against megalin), indicating that much of the Tg released by heparin had been bound to megalin (∼60–80%). The amount inhibited by RAP was considered to represent specific binding to megalin, which was saturable and of high affinity (K d ∼11.2 nm). Tg endocytosis by FRTL-5 and IRPT cells was demonstrated in experiments in which cells were incubated with unlabeled Tg at 37 °C, followed by heparin to remove cell-bound Tg. The amount of Tg internalized (measured by ELISA in the cell lysates) was reduced by RAP and 1H2, indicating that Tg endocytosis is partially mediated by megalin.

Alternating antineutrophil cytoplasmic antibody specificity - Drug-induced vasculitis in a patient with Wegener's granulomatosis

We describe a patient who presented with Wegeners granulomatosis associated with antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) with a cytoplasmic immunofluorescence pattern (cANCA), whose ANCA type changed to antimyeloperoxidase antibodies with a perinuclear immunofluorescence pattern (pANCA) when treated with propylthiouracil, and changed back to anti-PR3 antibodies with cANCA after the medication was discontinued. The patient developed flares of vasculitis symptoms associated with rises in either type of ANCA. Tests for antimyeloperoxidase ANCA were repeatedly negative before the drug was started, strongly implicating the drug as the cause of the episode. This case demonstrates that patients with idiopathic ANCA-positive vasculitis may quickly develop a superimposed drug-associated ANCA-positive vasculitis. Iatrogenic vasculitis should be suspected when a patient with idiopathic vasculitis with one type of ANCA develops the other type of ANCA.

Autoimmune cell-mediated tubulointerstitial nephritis induced in lewis rats by renal antigens

Abstract Lewis rats were injected with homogenates of renal or liver tissue or saline in complete Freunds adjuvant, plus pertussis vaccine, and were sacrificed at intervals ranging from 4 to 56 days. No renal lesions were observed in liver-or saline-injected animals. However, in rats injected with kidney preparations, severe tubulointerstitial nephritis was observed at 10–14 days; the lesions were characterized by irregular mononuclear cell infiltrates and tubular cell damage. At later intervals, milder more focal lesions were found. By immunofluorescence no tubulointerstitial deposits of immunoglobulin or C3 were detected. Transfer of lymph node, spleen, or peritoneal exudate cells from kidney-injected donors to normal Lewis rats resulted in focal interstitial infiltrates and tubular cell damage at 24 to 48 hr. Transfer of serum from kidney-injected donors or cells or serum from liver- or saline-injected donors produced no renal lesions. The migration of peritoneal macrophages from kidney-injected animals was inhibited by kidney, but not liver or spleen antigens. MIF activity was found in culture supernatants when lymph node or spleen cells from kidney-injected animals were cultured with kidney, but not with liver antigen preparations. The findings are interpreted as indicating that the tubulointerstitial lesions resulted from cell-mediated reactivity against kidney-specific antigens.

Identification of a Heparin-binding Region of Rat Thyroglobulin Involved in Megalin Binding

We recently showed that thyroglobulin (Tg) is a heparin-binding protein and that heparin inhibits binding of Tg to its endocytic receptor megalin (gp330). Here we have identified a heparin-binding region in the carboxyl-terminal portion of rat Tg and have studied its involvement in megalin binding. Rat thyroid extracts, obtained by ammonium sulfate precipitation, were separated by column fractionation into four Tg polypeptides, with apparent masses of 660, 330, 210, and 50 kDa. As assessed by enzyme-linked immunoadsorbent assays and ligand blot binding assays, megalin bound to intact Tg (660 and 330 kDa) and, to a even greater extent, to the 210-kDa Tg polypeptide. Furthermore, the 210-kDa Tg polypeptide inhibited megalin binding to intact Tg by ∼70%. Solid phase assays showed binding of biotin-labeled heparin to intact Tg and to the 210-kDa Tg polypeptide. We characterized the 210-kDa Tg polypeptide by matrix-assisted laser desorption/ionization mass spectrometry analysis and found that it corresponds to the carboxyl-terminal portion of rat Tg. We developed a synthetic peptide corresponding to a 15-amino acid sequence in the carboxyl-terminal portion of rat Tg (Arg689–Lys703), containing a heparin-binding consensus sequence (SRRLKRP) and demonstrated heparin binding to this peptide. A rabbit antibody raised against the peptide recognized intact Tg in its native conformation and under denaturing conditions. This antibody markedly reduced heparin-binding to intact Tg, indicating that the region of native Tg corresponding to the peptide is involved in heparin binding. Furthermore, the anti-Tg peptide antibody almost completely inhibited binding of megalin to Tg, suggesting that the Tg region containing the peptide sequence is required for megalin binding. Physiologically, Tg binding to megalin on thyroid cells may be facilitated by Tg interaction with heparin-like molecules (heparan sulfate proteoglycans) via adjacent binding sites.

Megalin in Thyroid Physiology and Pathology

Megalin, a member of the low density lipoprotein endocytic receptor family, is expressed on the apical surface of thyroid epithelial cells, directly facing the follicle lumen, where colloid is stored in high concentrations. Studies in vivo and with cultured thyroid cells have provided evidence that megalin expression on thyroid cells is TSH-dependent. Thyroglobulin (Tg), the major protein component of the colloid and the precursor of thyroid hormones, binds to megalin with high affinity and megalin mediates in part its uptake by thyrocytes. Tg internalized by megalin avoids the lysosomal pathway and is delivered by transepithelial transport (transcytosis) to the basolateral membrane of thyrocytes, from which it is released into the bloodstream. This process competes with pathways leading to thyroid hormone release from Tg molecules, which occurs following internalization of Tg molecules from the colloid by other means of uptake (fluid phase endocytosis or endocytosis mediated by low affinity receptors) that result in proteolytic cleavage in the lyosomes. During transcytosis of Tg, a portion of megalin (secretory component) remains complexed with Tg and enters the circulation, where its detection may serve as a tool to identify the origin of serum Tg in patients with thyroid diseases. Tg endocytosis via megalin is facilitated by the interaction of Tg with cell surface heparan sulfate proteoglycans, which occurs via a carboxyl terminal heparin binding site of Tg functionally related with a major megalin binding site. Although autoantibodies against megalin can be found in the serum of approximately 50% of patients with autoimmune thyroiditis, a role of megalin in this and other thyroid diseases remains to be established.

Necrotizing arteritis in acute poststreptococcal glomerulonephritis: report of a recovered case.

A patient with biopsy documented acute poststreptococcal glomerulonephritis and arteritis recovered completely with supportive therapy. Illness was preceded by group A streptococcal pharyngitis. At the time of presentation, serum creatinine concentration was 11.5 mg/dl. Serum cryoglobulins containing IgG and C3 were present. The first biopsy, performed during the acute illness, contained glomeruli with typical features of acute PSGN. Medium-sized arteries had extensive necrosis and leukocytic infiltration, and contained IgG, C3, and fibrin. Glomerular filtration rate returned to normal within three weeks; proteinuria cleared by three months, and microscopic hematuria by 11 months. Renal biopsy one year later showed minimal mesangial hypercellularity and no arteritis.