AL(light chain)-amyloidogenesis by mesangial cells involves active participation of lysosomes: An ultrastructural study

Abstract

Amyloid formation by cells is a stepwise process that occurs in macrophages and cells capable of transforming into a macrophage phenotype. One such cell is the mesangial cell in the kidney. It has been shown that mesangial cells are engaged in AL (light chain associated)- amyloidogenesis after transforming phenotypically from a smooth muscle to a macrophage phenotype. The actual process of amyloid fibril formation has not been dissected. This ultrastructural study which includes the examination of lysosomal gradient specimens addresses this issue by analyzing the sequence of events that takes place as fibrils are formed in endosomes and lysosomes. The findings indicate that fibrillogenesis begins in endosomes but is completed and most pronounced in the lysosomal compartment. As early as 10 min after incubation of human mesangial cells with AL-LCs, amyloid fibrils are formed in endosomes but mostly occurs in the mature lysosomal compartment. This is the first time that fibril formation is demonstrated experimentally occurring inside human mesangial cells and the entire sequence of events taking place is elucidated.

Keywords: Amyloid; Amyloidogenesis; Endosomes; Gradient centrifugation; Immunoglobulin light chains; Kidney; Lysosomal gradient; Lysosomes; Mesangial cells.

Graphical abstract

Fig. 1

AX10000; BX6000; CX8000 – Transmission electron microscopy, uranyl acetate and lead citrate. Incubation of HMCs with AL-LCs for 30 min. Lysosomal gradient. (Scale measurement-A. 400 nm, B. 600 nm, C. 500 nm). Caveolae marked with arrows (A) are significantly increased in numbers when HMCs are incubated with AL-LC. B- Increased numbers of endosomes and loss of smooth muscle characteristics of HMCs at 30 min after incubation with AL-LC. C- Early formation of amyloid fibrils is noted in lysosomes.

Fig. 2

AX5000; BX25000 - Transmission electron microscopy, uranyl acetate and lead citrate. Incubation of HMCs with AL-LC for 1 h. Lysosomal gradient. (Scale measurement-A. 200 nm, B. 100 nm). Formation of early circular profiles with fibrils in lysosomes. Note alignment with lysosomal membranes.

Fig. 3

AX350-Hematoxylin and eosin stain; BX 500-Congo red stain with insert polarization. Figure A, inset- X200. Incubation of HMCs with AL-LCs for 2 h. Lysosomal gradient. (Scale measurement- No way to accurately measure figure). Abundant eosinophilic, amorphous material in supernatant surrounded a few mesangial cells. There is congophilia demonstrated associated with this material and apple green color was obtained upon polarization of the material (insert), confirming the amyloid nature. Thioflavin T stain shows fluorescence associated with the amyloid aggregates (Figure A, inset).

Fig. 4

AX20000; BX20000. Incubation of HMCs with AL-LC for 1 hour. Lysosomal gradient. (Scale measurement-A. 100 nm, B. 200 nm). Co-localization of amyloid fibrils labeled with lambda light chain (gold particles size 15–16 nm) and for lysosomal membrane associated protein (gold particles size 5–6 nm).

Fig. 5

AX12000; BX15000- Transmission electron microscopy, uranyl acetate and lead citrate stain. HMCs incubated with AL-LC for 1 day (cell culture). (Scale measurement-A. 200 nm, B. 200 nm, C. 100 nm). A and B- A- Disorganized fibrils measuring 6–13 nm in diameter, characteristic of amyloid labeled for lambda light chains (14–16 nm gold particles) in B confirming AL-LC nature, in extracellular space.

Fig. 6

AX15000; BX15000 - Transmission electron microscopy, uranyl acetate and lead citrate stain. HMCs incubated with AL-LC for 4 and 6 h, respectively (A and B). Lysosomal gradient. (Scale measurement- A. 600 nm, B. 200 nm). Amyloid fibrils (7–15 nm in diameter) (A) arranged in circular arrays in the cytoplasm of HMCs. Lysosomal appearance is no longer identifiable in the background.

Fig. 7

AX5000- Transmission electron microscopy, uranyl acetate and lead citrate stain. HMCs incubated with AL-LC for 8 h (cell culture). Cell culture. (Scale measurement-A. 800 nm, B. 200 nm). Amyloid fibrils arranged intracytoplasmically with a parallel disposition.

Fig. 8

AX2500; BX6000-- Transmission electron microscopy; uranyl acetate and lead citrate stain. HMCs incubated with AL-LC for 30 min. Immunogold labeling for EEA-1 with 5–6 nm gold particles. Cell culture. (Scale measurement-A. 1 μm, B. 100 nm). Note empty vacuoles left with marked immune reactivity for EEA-1, after the enzymes have been extruded from the endosomes.

Fig. 9

Diagram- Representation of AL-LC- trafficking in HMC first to endosomes and then to mature lysosomal compartment to engage in amyloid fibril formation.