Volume 91, Issue 2, Pages 423-434 (February 2017) Unravelling the immunopathological mechanisms of heavy chain deposition disease with implications for clinical management  Frank Bridoux, Vincent Javaugue, Sébastien Bender, Fannie Leroy, Pierre Aucouturier, Céline Debiais-Delpech, Jean-Michel Goujon, Nathalie Quellard, Amélie Bonaud, Marie Clavel, Patrick Trouillas, Florent Di Meo, Jean-Marc Gombert, Jean-Paul Fermand, Arnaud Jaccard, Michel Cogné, Guy Touchard, Christophe Sirac  Kidney International  Volume 91, Issue 2, Pages 423-434 (February 2017) DOI: 10.1016/j.kint.2016.09.004 Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 1 Kidney biopsy sample from patient 1 with γ1-heavy chain deposition disease. (a) Light microscopy (periodic–acid Schiff staining, original magnification ×200). Section of renal cortex showing nodular glomerulosclerosis and diffuse tubular basement membrane thickening. Bar = 50 μm. (b) Immunofluorescence microscopy (fluorescein isothiocyanate–conjugate antisera to γ1-heavy chain, original magnification ×200). Linear deposits in the mesangium, along the glomerular and tubular basement membranes, and Bowman’s capsule. No significant staining was observed with κ and λ light chains or α and μ heavy chains (not shown). Bar = 50 μm. (c,d) Indirect immunofluorescence (original magnification ×400) showed positive staining with anti-γCH2 antibody (c) but not with anti-γCH1 antibody (d). Bar = 50 μm. (e,f) Electron microscopy. (e) Enlarged multilayered tubular basement membrane with electron-dense powdery deposits (arrows) (original magnification ×2500). Bar = 2 μm. (f) Linear electron-dense deposits that predominate in the inner aspect of the glomerular basement membrane (original magnification ×10,000). Bar = 1 μm. Inset: immunoelectron microscopy (original magnification ×50,000). Presence of anti-γ1 heavy- chain–conjugated gold particles along the inner aspect of glomerular basement membrane. Bar = 0.5 μm. Kidney International 2017 91, 423-434DOI: (10.1016/j.kint.2016.09.004) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 2 Kidney biopsy sample from patient 15 with α heavy chain deposition disease. Light microscopy. (a) Periodic acid-Schiff staining (original magnification ×400). Section of renal cortex showing nodular glomerulosclerosis with diffuse mesangial nodular deposits and moderate mesangial hypercellularity. Bar = 50 μm. (b) Marinozzi silver staining (magnification ×200). Note the presence of double contours of the glomerular basement membranes, mesangiolysis with aneurysmal dilation of the capillary lumens, and tubular basement membrane thickening. Bar = 50 μm. Immunofluorescence microscopy with anti-α (c) and anti-κ (d) conjugates (original magnification ×400). Linear α heavy chain deposits were observed in the mesangium and along the tubular basement membranes (c), whereas no significant staining was seen with κ (c) and λ light chains or γ and μ heavy chains (not shown). Bar = 50 μm. (e) Electron microscopy (original magnification ×10,000). Linear electron-dense deposits (arrows) involving the outer aspect of the tubular basement membrane. Bar = 1 μm. (f) Immunoelectron microscopy (original magnification ×50,000). The presence of anti-α heavy chain–conjugated gold particles along the glomerular basement membrane. Gold particles were predominantly observed along the inner aspect of the glomerular basement membrane. Bar = 0.5 μm. Kidney International 2017 91, 423-434DOI: (10.1016/j.kint.2016.09.004) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 3 Bone marrow biopsy. Immunofluorescence microscopy (original magnification ×600) with fluorescein isothiocyanate–conjugate antisera to γ-heavy chain (a) and phycoerythrin-conjugate antisera to λ-light chain (b). Coexpression of both heavy chain and light chain by a single plasma cell clone (c). Bar = 50 μm. Kidney International 2017 91, 423-434DOI: (10.1016/j.kint.2016.09.004) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 4 Deduced amino-acid sequences of heavy chain variable regions compared with germline sequences according to IMGT numbering. Mutated amino acids are indicated by shaded squares. Kidney International 2017 91, 423-434DOI: (10.1016/j.kint.2016.09.004) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 5 Calculated isoelectric point (pI) of heavy chain deposition disease (HCDD) heavy chain variable regions (HCDD HC) compared with light chain (LC) and heavy chain (HC) germlines. Kidney International 2017 91, 423-434DOI: (10.1016/j.kint.2016.09.004) Copyright © 2016 International Society of Nephrology Terms and Conditions