An animal model of glomerular light-chain-associated amyloidogenesis depicts the crucial role of lysosomes  Jiamin Teng, Elba A Turbat-Herrera, Guillermo A Herrera  Kidney International  Volume 86, Issue 4, Pages 738-746 (October 2014) DOI: 10.1038/ki.2014.122 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 SDS-polyacrylamide gel electrophoresis (PAGE) of purified light chains. Two of the amyloidogenic and two light chain deposition disease (LCDD) light chains injected in mice are shown as single bands at about 20–25Kd. Kidney International 2014 86, 738-746DOI: (10.1038/ki.2014.122) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Monotypical 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. Kidney International 2014 86, 738-746DOI: (10.1038/ki.2014.122) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Mice 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. Kidney International 2014 86, 738-746DOI: (10.1038/ki.2014.122) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Comparison of early changes (6 hours post initial injection) in glomeruli of mice injected with light chain deposition disease (LCDD) and amyloidogenic light chains. (a–f) Comparison of mice injected with amyloidogenic (a, e, f) and LCDD (b) light chains 6h post initial injections. (a) × 6500 and (b) × 6750. Transmission electron microscopy, uranyl acetate, and lead citrate. (c) × 500 and (d) × 500, immunohistochemical stain for CD68. (e) × 25,000 and (f) × 35,000, immunogold labeling for lambda light chains, 10nm gold particles. Note in a, a couple of mesangial cells with aggregates of lysosomes in their cytoplasm, circle indicating early transformation to a macrophage phenotype. Most mesangial cells are not yet transformed. In b, none of the mesangial cells exhibit lysosomes in their cytoplasm as no macrophage transformation of mesangial cells has occurred. Few cells with intracytoplasmic staining for CD68 indicating early transformation to macrophage phenotype (c) in amyloidogenic light chain-injected mouse and no staining in d, indicating lack of mesangial cell transformation in LCDD-light chain-injected mouse. In e, the lysosomes are decorated with gold particles indicating the presence of monotypical light chains (no labeling for kappa light chains) and in f, the adjacent fibrillary material is also labeled with gold particles indicating that the amyloid is amyloid light chain (AL) type, lambda light chain-related in mouse injected with amyloid-forming light chain. Kidney International 2014 86, 738-746DOI: (10.1038/ki.2014.122) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Mice 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). Kidney International 2014 86, 738-746DOI: (10.1038/ki.2014.122) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 Scanning 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). Kidney International 2014 86, 738-746DOI: (10.1038/ki.2014.122) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 7 Scanning electron microscopy. (a, b); (a) × 1700; (b) × 7500. Two weeks after initial injection of amyloidogenic light chains. Whole glomerulus with abundant amyloid at 2 weeks post initial injection. Glomerular architecture entirely disrupted by tangled, haphazardly disposed fibrils (a). Details of amyloid fibrils in b. Kidney International 2014 86, 738-746DOI: (10.1038/ki.2014.122) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 8 Schematic representation of process of amyloid formation in mesangial cells and contrast with processing of light chain deposition disease (LCDD) light chains by mesangial cells. Glomerulopathic light chains (amyloid-forming and light chain deposition light chains) compete for receptors on the surface of mesangial cells. Amyloidogenic light chains (blue in the diagram) are internalized and initially processed for endosomes. The endosomes recruit mature lysosomes and transfer the light chains to them. Eventually the light chains reach the mature lysosomal system where they are processes and fibrils are formed. The lysosomes extrude the fibrils into the extracellular compartment. They abut on the cell membranes and expel their fibrils. In contrast, light chain deposition disease light chains (pink in the diagram) are catabolized by endosomes and are not transported to mature lysosomes. These light chains, though glomerulopathic, do not engage in the formation of amyloid. Kidney International 2014 86, 738-746DOI: (10.1038/ki.2014.122) Copyright © 2014 International Society of Nephrology Terms and Conditions