An animal model of glomerular light-chain-associated amyloidogenesis depicts the crucial role of lysosomes
2014; Elsevier BV; Volume: 86; Issue: 4 Linguagem: Inglês
10.1038/ki.2014.122
ISSN1523-1755
AutoresJiamin Teng, Elba A. Turbat‐Herrera, Guillermo A. Herrera,
Tópico(s)Ion Transport and Channel Regulation
ResumoIn vitro and ex vivo studies have elucidated the step-by-step process whereby some physicochemically abnormal light chains are processed by mesangial cells to form amyloid fibrils. Although crucial steps in the cascade of events have been determined, these findings have not been reproduced in vivo. This has led to some doubts as to the significance and clinical application of the information that has been deciphered. Here, we developed an animal model which uses mice injected with amyloidogenic light chains purified from the urine of patients with biopsy-proven, light-chain-associated glomerular amyloidosis which validated in vitro/ex vivo findings. This animal model showed internalization of the light chains utilizing caveolae followed by trafficking to the mature lysosomal compartment where fibrils were formed. This model permits evaluation of mesangial amyloidogenesis for prolonged periods of time, is potentially useful to test maneuvers to modulate events that take place, and can be used to design novel therapeutic interventions. In vitro and ex vivo studies have elucidated the step-by-step process whereby some physicochemically abnormal light chains are processed by mesangial cells to form amyloid fibrils. Although crucial steps in the cascade of events have been determined, these findings have not been reproduced in vivo. This has led to some doubts as to the significance and clinical application of the information that has been deciphered. Here, we developed an animal model which uses mice injected with amyloidogenic light chains purified from the urine of patients with biopsy-proven, light-chain-associated glomerular amyloidosis which validated in vitro/ex vivo findings. This animal model showed internalization of the light chains utilizing caveolae followed by trafficking to the mature lysosomal compartment where fibrils were formed. This model permits evaluation of mesangial amyloidogenesis for prolonged periods of time, is potentially useful to test maneuvers to modulate events that take place, and can be used to design novel therapeutic interventions. In 1927, Smetana1.Smetana H. The relation of the reticulo-endothelial system to the formation of amyloid.J Exp Med. 1927; 45: 619-632Crossref PubMed Scopus (17) Google Scholar pointed out the important role of the reticuloendothelial system in amyloidosis. In vitro models were used since the 1960s to elucidate mechanisms involved in the process of amyloidogenesis. In the early 1960s and 70s, Shirahama and Cohen conducted seminal research in the pathogenesis of AA-amyloidosis using macrophages where they indicated that the lysosomes played an important role in amyloidogenesis2.Gueft G. Ghidoni J.J. The site of formation and ultrastructure of amyloid.Am J Pathol. 1963; 43: 837-854PubMed Google Scholar, 3.Shirahama T. Cohen A.S. Lysosomal breakdown of amyloid fibrils by macrophages.Am J Pathol. 1971; 63: 463-486PubMed Google Scholar, 4.Shirahama T. Cohen A.S. An analysis of the close relationship of lysosomes to early deposits of amyloid. Ultrastructural evidence in experimental mouse amyloidosis.Am J Pathol. 1973; 73: 97-114PubMed Google Scholar, 5.Shirahama T. Cohen A.S. Intralysosomal formation of amyloid fibrils.Am J Pathol. 1975; 81: 101-116PubMed Google Scholar and studied glomerular changes in human primary and secondary amyloidosis and experimental AA-amyloidosis (in rabbits).6.Shirahama T. Cohen A.S. Fine structure of the glomerulus in human and experimental amyloidosis.Am J Pathol. 1973; 73: 97-114PubMed Google Scholar Interestingly, the authors noted a distinct population of mesangial cells with a macrophage phenotype that they incorrectly interpreted at the time as representing degenerating mesangial cells. Gueft, at the time a post-doctoral research fellow, and Guidoni in 1963 stated: ''We suggest that the amyloid fibrils are formed at the moment that a histiocyte, etc, delivers the precursor (protein) to the outside''2.Gueft G. Ghidoni J.J. The site of formation and ultrastructure of amyloid.Am J Pathol. 1963; 43: 837-854PubMed Google Scholar supporting the important role of macrophages or facultative macrophages in the process of amyloid formation and suggesting that amyloid fibrils are released at the surface of the cells engaged in their genesis. These concepts at the time were highly innovative and provocative. Studies by other investigators 30–40 years later further highlighted the important role played by lysosomes in macrophages in the pathogenesis of AA-amyloidosis, but none of these studies specifically addressed the pathogenesis of renal amyloidosis.7.Kluve-Beckerman B. Manaloor J.J. Liepnieks J.J. A pulse-chase study tracking the conversion of macrophage-endocytosed serum amyloid A into extracellular amyloid.Arthritis Rheum. 2003; 46: 1905-1913Crossref Scopus (49) Google Scholar,8.Kluve-Beckerman B. Liepnieks J.J. Wang L. et al.A cell culture system for the study of amyloid pathogenesis.Am J Pathol. 1990; 155: 123-133Abstract Full Text Full Text PDF Scopus (65) Google Scholar These studies concluded that amyloid formation occurred intracellularly supporting other early studies on the subject performed using immunofluorescence and electron microscopy.9.Zucker-Franklin D. Franklin E.C. Intracellular localization of human amyloid by fluorescence and electron microscopy.Am J Pathol. 1970; 59: 23-42PubMed Google Scholar Whether amyloid is ever located intracytoplasmically before it is extruded from the cells during amyloidogenesis has been a source of debate. For years, the definition of amyloid indicated that it was an extracellular material; however, such definition has been changed to indicate that it may also be found intracellularly, based on experimental data obtained from studies of macrophages engaged in the formation of fibrils in AA-amyloidosis. In 1987, Cohen and Connors10.Cohen A.S. Connors L.H. The pathogenesis and biochemistry of amyloidosis.J Pathol. 1987; 151: 1-10Crossref PubMed Scopus (110) Google Scholar summarized the knowledge that had been collected on the subject of amyloidogenesis in a comprehensive manuscript which addressed biochemical aspects and pathogenesis. In 1999, Solomon et al.11.Solomon A. Weiss D.T. Schell M. et al.Transgenic mouse model of AA amyloidosis.Am J Pathol. 1999; 154: 1267-1272Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar created a transgenic mouse model of AA-amyloidosis. These animals developed systemic amyloidosis and died of renal failure. However, no detailed studies of renal amyloidogenesis were performed using this experimental platform. Tagouri et al.12.Tagouri Y. Sanders P.W. Picken M.M. et al.In vitro AL-amyloid formation by rat and human mesangial cells.Lab Invest. 1996; 74: 290-302PubMed Google Scholar were the first to demonstrate in vitro amyloid formation by mesangial cells grown in monolayers and on Matrigel incubated with monoclonal light-chains (LCs) obtained from the urine of patients with renal biopsy-proven glomerular light chain derived (AL)-amyloidosis. Additional work performed later addressed the formation of AL-amyloid in glomeruli in an ex vivo experimental platform,13.Herrera G.A. Welbourne T.C. Russell W.J. Isolated perfused rat kidney: a new model to evaluate light chain nephrotoxicity (Abstract).Lab Invest. 2001; 81: 188AGoogle Scholar perfusing monoclonal LCs through the renal artery in mice kidneys maintained physiologically intact. The ex vivo platform could only be utilized up to 72h after the kidney is removed from its normal blood supply, as the kidney could not be maintained physiologically intact for a more extended period of time. LCs purified from the urine of patients with AL-amyloidosis (Figure 1) and renal biopsy-proven involvement injected in the penile vein of mice were successfully delivered to the mesangium (Figure 2). Not all glomeruli received the same amount of LCs at any given time frame, but the injected LCs were demonstrated consistently in the mesangium in the majority of the glomeruli in both kidneys. The amount of LCs in different mesangial areas varied but was similar at 1 and 2 weeks post initial LC injection (Figure 2). This finding was important as the delivery of the LCs to the kidneys in acceptable quantities for prolonged periods of time, so that the process of amyloidogenesis could be studied, has been a challenge in previous experimental in vivo platforms. The LCs were more concentrated in the mesangial areas at two weeks post initial injection (Figure 2). There were striking differences in glomeruli from mice injected with myeloma cast nephropathy (MCN), light chain deposition disease (LCDD), and amyloidogenic light chains at 2 weeks post initial injection (Figure 3).Figure 2Monotypical light chains in glomeruli. (a, b) × 500 (a) and × 500 (b), direct immunofluorescence for kappa and lambda light chains, respectively; fluorescein isothiocyanate, marker dye. One (a) and two (b) weeks post initiation of injection of amyloidogenic light chains. Note deposition of light chains in mesangial areas at 1 and 2 weeks. At 2 weeks, the deposited light chains are more concentrated in mesangial areas.View Large Image Figure ViewerDownload (PPT)Figure 3Mice kidneys injected with myeloma cast nephropathy, light chain deposition disease (LCDD) and amyloidogenic light chains: comparison of glomerular morphology at 2 weeks post initial injection. (a–o) Mice kidneys 2 weeks after initial injection with myeloma cast nephropathy light chains (a, b, c). (a) Periodic acid–Schiff (PAS; × 500) stain, (b) × 500, thioflavin T stain, and (c) × 5000, transmission electron microscopy, uranyl acetate, and lead citrate. Mice kidneys two weeks after initial injections with LCDD light chains (d–i). (d) PAS, × 500, (e) × 500, silver methenamine, (f) × 7500, transmission electron microscopy, uranyl acetate, and lead citrate, (g) × 500, Congo red, (h) × 500, thioflavin T stain, and (i) × 500, CD68 immunohistochemical stain with diaminobenzidine as marker. Mice kidneys two weeks after initial injection of amyloidogenic light chains (j–n) (j) × 500, PAS stain; (k) × 750, Congo red stain; (l) × 350, polarized Congo red stain; (m) × 350, Thioflavin T stain, and (n) × 500, CD68 immunohistochemical stain with diaminobenzidine as marker; (o) × 13,500 transmission electron microscopy, uranyl acetate, and lead citrate. Normal glomerulus with unremarkable peripheral capillary walls and mesangium (a). Negative Congo red stain indicating absence of amyloid (b). Normal ultrastructural appearance of glomerulus (c). No mesangial expansion. No matrix accumulation or amyloid deposits in mesangium. In d, note mesangial nodularity as a result of matrix deposition. The increased matrix stains positive with the silver methenamine stain (e). Increased mesangial matrix is confirmed ultrastructurally (f). No transformed mesangial cells are present. Note absence of amyloid in Congo red and thioflavin T stains (g, h). In i, note absence of CD68 immunoreactivity in mesangial cells, supporting lack of phenotypic transformation. In j, amyloid appears as PAS-positive deposits in mesangial areas (circles) and in the wall of arteriole (arrowhead). In k, congophilia is demonstrated in the mesangial deposits and l reveals apple-green birefringence characterizing the deposits as amyloid. m shows typical fluorescence associated with amyloid deposits with thioflavin stain. n reveals strong immunohistochemical staining in the cytoplasm of mesangial cells for CD68 consistent with the macrophage phenotype that they have acquired. o shows abundance of lysosomes in the cytoplasm of mesangial cells.View Large Image Figure ViewerDownload (PPT) In this in vivo model, amyloid deposits were identified in the glomerular mesangium and in arterioles and small-size arteries (Figure 3j–m). The entire process of amyloid formation in the mesangium could be followed sequentially and documented. In contrast, in mice injected with LCDD-LCs, initial mesangial expansion with hypercellularity and later increased matrix was observed (Figure 3d–f), and glomeruli in those injected with MCN-LCs (Figure 3a–c) were normal. No amyloid was seen on Congo red or Thioflavin T stains in the renal parenchyma of mice injected with LCDD (Figure 3g and h) or MCN-LCs (Figure 3b and c). Following internalization, the amyloid-forming (but not the LCDD-LCs which were catabolized in endosomes or the MCN-LCs which did not interact with mesangial cells) LCs reached the mature lysosomal compartment; this process took ∼2h, with some variation depending on the LC tested. The longest component of the sequence of events was the lysosomal processing of the LCs to form the amyloid fibrils which took 8–10h. The overall speed of amyloid formation and the amount produced varied among LCs utilized in the experiments. The identity of the fibrillary material as amyloid was established based on the staining characteristics of the material (salmon pink staining with Congo red stain (Figure 3k) and subsequent apple-green birefringence upon polarization (Figure 3l), and thioflavin T fluorescence (Figure 3m) and, most importantly, the ultrastructural features of the fibrils seen by transmission electron microscopy supported by the scanning electron microscopic appearance. The fibrils were randomly arranged, measured 7–14nm (mean=11nm) in diameter, and did not show branching. Approximately 60min after incubation with the amyloidogenic LCs (depending on the LC), mesangial cells began to transform from a smooth muscle to a macrophage phenotype (Figure 4a), while no such transformation is seen with LCDD-LC incubation (Figure 4b and d). Concomitantly, mesangial cells began to reveal intracytoplasmic CD68 staining (Figure 4c), with immunoreactivity increasing as they acquired a more differentiated macrophage phenotype. By 18–24h after the initial LC injection, virtually every mesangial cell in mesangial areas had acquired lysosomes and lost intracytoplasmic myofilaments and attachment plaques at their membranes appearing at least partially transformed and expression of CD68 became much more pronounced in transformed cells (Figure 3o and 5a–c). Phenotypic transformation of mesangial cells did not occur in glomeruli from mice injected with LCDD (Figure 3d–i) or with MCD LCs (Figure 3a–c). Progressive loss of smooth-muscle characteristics of the mesangial cells ensued and during this process there were mesangial cells with hybrid—smooth muscle/macrophage—features. As more time passed, the lysosomes in the mesangial cells increased in numbers (Figure 5a and b) and began to show changes in their electron density as they processed the LCs (Figure 5c and d) and fibrils were formed (Figure 5e and f). The lysosomes located toward the periphery of the mesangial cells engaged in the process of delivering the amyloid fibrils to the outside (Figure 6a–d). Lysosomes abutted on the mesangial cell membranes which showed gaps (Figure 5g, circle) and extruded the amyloid fibrils into the extracellular matrix (Figure 5h). Extracellular amyloid deposition was first detected 12±2h after injection depending on the LC tested. Lysosomes and surrounding amyloid fibrils were labeled for either kappa or lambda light chains (never both), supporting the derivation of the fibrils from monotypical light chains (Figure 4e and f).Figure 5Mice injected with amyloid forming light chains at 2 weeks post initial injection. (a–h) Two weeks after initial injection of amyloidogenic light chains. (a) × 7500; (b) × 25,500; (c) × 25,500; (d) × 27,000; (e) × 35,000; (f) × 37,500; (g) × 17,500; (h) × 22,550.Transmission electron microscopy, uranyl acetate, and lead citrate. Phenotypic transformation of mesangial cells from smooth muscle to macrophage phenotype is complete. No remaining myofilaments or attachment plaques. The mesangial cells are full of lysosomes of various sizes and shapes (a, b). In c–f, details of lysosomes in mesangial cells are shown. Note variability in size, shape, and electron density. Lysosomes are identified throughout the cytoplasm of the mesangial cells. Lysosomes at the periphery of mesangial cells are intimately associated with cell membranes as shown. Details of lysosomes processing light chains and forming fibrils (arrow in f). Note many lysosomes are abutting the cell membranes in mesangial cells (g) and there are gaps in the cell membranes highlighted in g (circle). In the extracellular space, there are amyloid fibrils, best seen in g and h. The mean diameter of the fibrils is 11nm (f).View Large Image Figure ViewerDownload (PPT)Figure 6Scanning electron microscopy. (a–f) One week after initial injection of amyloidogenic light chains (a) × 2000; (b) × 1500; (c) × 1300; (d) × 1600; (e) × 12,000; (f) × 17,000. In a, note normal glomerulus (control) and compare it with the glomerulus in b with some amyloid already formed (note fibrils). Early formation of amyloid fibrils by mesangial cells is depicted in c, d. Note in c extrusion of single fibrils by mesangial cells into the extracellular matrix. In d and e note more advanced fibrillogenesis by mesangial cells and in e and f tangled, randomly disposed, non-branching fibrils typical of amyloid. Fibrils measure about 7–14nm in diameter. Reference for size calculation: 40nm in diameter Dynabeads (arrow) embedded in tissue (f); these are used as reference to measure diameter of fibrils (noted to have a mean diameter of about 10nm).View Large Image Figure ViewerDownload (PPT) Figure 7a and b portray whole glomeruli viewed with scanning electron microscopy, highlighting the presence of fibrils (Figure 7b—compare with 7a—saline-injected mouse) at 7 days post initial amyloidogenic LC injection. Amyloid formation by mesangial cells at 7 days post injection was clearly evident (Figure 6c and d). Scanning electron microscopy clearly demonstrated an abundance of extracellular amyloid fibrils at 2 weeks post initial penile injection (Figure 6e and f and Figure 7a and b). Amyloid fibrils deposited in the extracellular space replaced the native mesangial matrix (Figure 7b). Plasma cell dyscrasias are characterized by a clone (or more than one clone) of abnormal/neoplastic plasma cells, albeit small in some instances resulting in overproduction of free physicochemically abnormal LCs (∼95% of all plasma cell dyscrasias produce free LCs). These LCs freely circulate in the body to reach the glomerular capillaries. About 85% of these LCs are nephrotoxic and 30% of these are glomerulopathic (associated with glomerular pathology).14.Herrera G.A. Renal manifestations in plasma cell dyscrasias: an appraisal from the patients' bedside to the research laboratory.Annals Diagn Pathol. 2000; 4: 174-200Abstract Full Text PDF PubMed Scopus (70) Google Scholar Glomerulopathic LCs include those that can produce amyloid (amyloidogenic) and those associated with LCDD. MCN-LCs are freely filtered through the peripheral capillary walls due to their low molecular weight producing their pathogenic effects in distal tubules; these do not interact with the mesangial cells. In 1970, Glenner et al.15.Glenner G.G. Ein D. Eanes E.D. et al.Creation of 'amyloid' fibrils from Bence-Jones proteins in-vitro.Science. 1971; 174: 712-714Crossref PubMed Scopus (276) Google Scholar showed the derivation of a type of amyloid from LCs, developing the concept of AL-amyloidosis. Glomerulopathic (but not tubulopathic LCs) compete for purported receptors present on mesangial cells.16.Teng J. Russell W.J. Gu X. et al.Different types of glomerulopathic light chains interact with mesangial cells using a common receptor but exhibit different intracellular trafficking patterns.Lab Invest. 2004; 84: 440-451Crossref PubMed Scopus (99) Google Scholar Glomerular amyloid formation is typically observed first in the glomerular mesangium, as pointed out initially by Shirahama and Cohen6.Shirahama T. Cohen A.S. Fine structure of the glomerulus in human and experimental amyloidosis.Am J Pathol. 1973; 73: 97-114PubMed Google Scholar and confirmed by Gise17.Gise H. Christ H. Bohle A. Early glomerular lesions in amyloidosis.Virchows Arch (A). 1981; 390: 259-272Crossref PubMed Scopus (28) Google Scholar in 1981. In the early 1970s, Linke et al.18.Linke R.P. Zucker-Franklin D. Franklin E.C. Morphologic, chemical, and immunologic studies of amyloid-like fibrils formed from Bence Jones proteins proteolysis.J Immunol. 1973; 111: 10-23PubMed Google Scholar reported the formation of amyloid fibrils by digesting LCs with proteolytic enzymes and the following year, Epstein19.Epstein W.C. Tran M. Wood I.S. Formation of 'amyloid' fibrils in vitro by action of human kidney lysosomal enzymes on Bence Jones proteins.J Lab Clin Med. 1974; 84: 107-110PubMed Google Scholar digested human LCs with kidney lysosomal enzymes also demonstrating the formation of amyloid fibrils, further advancing the idea that lysosomes are integral elements in the sequence of events that lead to the genesis of amyloid fibrils and that lysosomal enzymes are operative in the formation of the fibrillary material in the kidney. Although AA-amyloidosis occurs spontaneously in a number of animals, no studies addressing the process of amyloid formation in the kidney have been conducted. There have been a number of previous attempts to create an animal model of renal AL-amyloidosis. Solomon et al.20.Solomon A. Weiss D.T. Kattine A.A. Nephrotoxic potential of Bence Jones proteins.N Engl J Med. 1991; 324: 1845-1851Crossref PubMed Scopus (256) Google Scholar, in seminal work reported in 1991, delivered monoclonal LCs intraperitoneally to mice and killed them 48h later demonstrating the presence of amyloid deposits in the renal vessels but not in glomeruli. This seminal work cemented the concept that circulating LCs are responsible for producing renal AL-amyloidosis. In our laboratory, several previous attempts to deliver LCs to the kidneys failed, including the use of an osmotic pump placed in the jugular vein of mice delivering large concentrations LCs at prescribed time frames for several days. Only very small amounts of LCs could be demonstrated in the kidneys of these animals, making it impossible to carry out detailed studies. When mesangial cells are incubated with immunoglobulin LCs extracted and purified from the urine of patients with renal biopsy-proven AL-amyloidosis, they engage in fibrillogenesis-forming amyloid. From our previous studies, rat and human mesangial cells incubated with LCs from the urine of patients with renal biopsy-proven AL-amyloidosis formed amyloid, which eventually went to the extracellular domain (supernatant).12.Tagouri Y. Sanders P.W. Picken M.M. et al.In vitro AL-amyloid formation by rat and human mesangial cells.Lab Invest. 1996; 74: 290-302PubMed Google Scholar Key to the ability of mesangial cells to engage in amyloidogenesis is that their plasticity allows them to transform from a smooth muscle to a macrophage phenotype, endowing them with the machinery necessary to engage in LC and processing.21.Teng J. Turbat-Herrera E.A. Herrera G.A. The role of translational research in advancing the understanding of the pathologenesis of light chain (LC)-mediated glomerulopathies.Pathol Int. 2007; 57: 398-412Crossref PubMed Scopus (20) Google Scholar, 22.Keeling J. Teng J. Herrera G.A. AL-amyloidosis and light chain deposition disease light chains induce divergent phenotypic transformations of human mesangial cells.Lab Invest. 2004; 84: 1322-1338Crossref PubMed Scopus (102) Google Scholar, 23.Herrera G.A. Plasticity of mesangial cells: a basis for understanding pathological alterations.Ultrastruct Pathol. 2006; 30: 471-479Crossref PubMed Scopus (33) Google Scholar, 24.Russell W.J. Cardelli J. Harris E. et al.Monoclonal light chain-mesangial cells interactions: early signaling events and subsequent pathologic effects.Lab Invest. 2001; 81: 689-703Crossref PubMed Scopus (43) Google Scholar Previously conducted in vitro and ex vivo studies have dissected the sequence of events responsible for the eventual formation of amyloid by mesangial cells. The process of amyloidogenesis begins with the interaction of LCs with purported receptors on mesangial cells followed by endocytotic uptake of the LCs. The endocytotic process is mediated by caveolae.25.Herrera G.A. Teng J. Turbat-Herrera E.A. Renal amyloidosis: current views on pathogenesis and impact on diagnosis.in: Ronco C. Experimental Models for Renal Diseases: Pathogenesis and Diagnosis Herrera GA ed. Contributions to Nephrology Serieswith some changes. Vol. 169. Karger Pub, Basel, Switzerland2011: 232-244Crossref Scopus (8) Google Scholar, 26.Galbiati F. Razani B. Lisanti M.P. Emerging themes in lipid and rafts and caveolae.Cell. 2001; 106: 403-411Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar, 27.Cohen A.W. Hnasko R. Schubert W. et al.Role of caveolae and caveolins in health and disease.Physiol Rev. 2004; 84: 1341-1379Crossref PubMed Scopus (736) Google Scholar Caveolae are specialized lipid rafts that were originally identified as 'flask-shaped' invaginations of the plasma membrane that are about 50–100nm in size which Palade28.Palade G.E. Fine structure in blood capillaries.J Appl Phys. 1953; 24: 1424Google Scholar referred to plasmalemmal vesicles and a couple of years later, Yamada29.Yamada E. The fine structure of the gall bladder epithelium of the mouse.J Biophys Biochem Cytol. 1995; 1: 445-457Crossref Scopus (525) Google Scholar coined the term caveolae for designation of the same structures. These perform a number of signaling functions30.Anderson R.G.W. The caveolae membrane system.Ann Rev Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1724) Google Scholar and have been identified in a variety of cells. Many proteins have been found in higher quantity (enriched) in caveolae, including caveolin. Caveolin is a transmembrane protein found in caveolae and the principal marker for this structure. According to evidence published by Thomsen et al.31.Thomsen P. Roepstorff K. Stahlhut M. et al.Caveolae are highly immobile plasma membrane microdomains which are not involved in constitutive endocytic trafficking.Mol Biol Cell. 2002; 13: 238-250Crossref PubMed Scopus (373) Google Scholar, caveolae are static structures which are not involved in endocytosis as previously thought. Caveolae play a crucial role in signaling mechanisms that regulate the fate of these LCs once they are endocytosed in the mesangial cells. This animal model clearly confirmed that internalization of the monoclonal LCs utilizing caveolae is followed by trafficking to the mature lysosomal compartment where fibrils are formed.16.Teng J. Russell W.J. Gu X. et al.Different types of glomerulopathic light chains interact with mesangial cells using a common receptor but exhibit different intracellular trafficking patterns.Lab Invest. 2004; 84: 440-451Crossref PubMed Scopus (99) Google Scholar,22.Keeling J. Teng J. Herrera G.A. AL-amyloidosis and light chain deposition disease light chains induce divergent phenotypic transformations of human mesangial cells.Lab Invest. 2004; 84: 1322-1338Crossref PubMed Scopus (102) Google Scholar A striking increase in intracytoplasmic lysosomes was noticeable in transformed mesangial cells exhibiting a macrophage phenotype. Using immunogold labeling at the ultrastructural level, these lysosomes were shown to be processing light chains. Lysosomes containing monotypical light chains were intimately associated with newly formed amyloid fibrils, also labeled for the immunogold monotypical light chains (Figure 4e and f). A significant number of these lysosomes was noted to interact with the membranes of the mesangial cells engaging in the process of delivery of the amyloid fibrils to the extracellular space. Blebbing of the cytoplasm of the mesangial cells with the cytoplasmic protrusions harboring the lysosomes and small cell membrane gaps were observed in the areas where extracellular amyloid was present. This process was best viewed using scanning electron microscopy32.Teng J. Turbat-Herrera E.A. Herrera G.A. Extrusion of amyloid fibrils to the extracellular space in experimental mesangial AL-amyloidosis: transmission and scanning electron microscopy studies and correlation with renal biopsy observations.Ultrast Pathol. 2014; 38: 104-115Crossref PubMed Scopus (15) Google Scholar (Figure 6c and d). The effects of amyloid on the extracellular matrix once it has reached the extracellular space have also been studied in the research laboratory.33.Keeling J. Dempsey S. Joseph L. et al.The role of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in mesangial matrix replacement in AL-amyloidosis: An in vivo and in-vitro correlative study.in: Grateau G. Kyle R.A. Skinner M. Amyloid and Amyloidosis. CRC Press, Boca Raton, FL, USA2005: 136-138Google Scholar, 34.Keeling J. Herrera G.A. The mesangium as a target for glomerulopathic light and heavy chains: Pathogenic considerations in light and heavy chain-mediated glomerular drainage.in: Herrera G.A. The Kidney in Plasma Cell Dyscrasias. Karger, Basel, Switzerland2007: 116-134Google Scholar, 35.Keeling J. Herrera G.A. Matrix metalloproteinases and mesangial remodeling in light chain-related glomerular damage.Kidney Int. 2005; 68: 1590-1603Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar Extracellular amyloid deposition activates metalloproteinases potentiating matrix destruction. In addition, amyloid inhibits transforming growth factor-β making it difficult and eventually virtually impos
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