Volume 84, Issue 5, Pages 902-910 (November 2013) Increased lysosomal proteolysis counteracts protein accumulation in the proximal tubule during focal segmental glomerulosclerosis  Rikke Nielsen, Geraldine Mollet, Ernie L. Esquivel, Kathrin Weyer, Pia K. Nielsen, Corinne Antignac, Erik I. Christensen  Kidney International  Volume 84, Issue 5, Pages 902-910 (November 2013) DOI: 10.1038/ki.2013.218 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Morphology and expression of proinflammatory and profibrotic mediators. (a) Hematoxylin–eosin (HE) staining of a podocin knockout (KO) mouse from group 3 (scale bar=25μm). (b) mRNA levels of transforming growth factor-β (TGF-β), monocyte chemoattractant protein 1 (MCP-1), nuclear factor-κB (NF-κB), and kidney injury molecule 1 (KIM-1) normalized to aquaporin 1, of podocin-knockout (KO) mice as compared with wild type (WT) mice during progression of proteinuria (groups 1–4). Each bar represents the ratio between the mean content of the specific protein in KO and WT mice. Asterisks designate a significant difference between the mean of the WT mice and the mean of the KO mice (P<0.05; n=4 or 5). The dotted line represents the ratio 1 at which the WT and KO mice have equal means. The error bars represent the estimated s.e. of the KO/WT ratio. The estimation is obtained by the Delta method of finding approximations based on the Taylor series expansions to the variance of functions of random variables. Kidney International 2013 84, 902-910DOI: (10.1038/ki.2013.218) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Content of megalin and cubilin ligands. (a) Content of endocytosed retinol-binding protein (RBP) in a wild-type (WT) and a podocin-knockout (KO) mouse from group 3 analyzed by immunohistochemistry (scale bars=25μm). (b) Localization of RBP in lysosomes, visualized by double staining of RBP (green) and cathepsin b (red) in a podocin-KO mouse from group 3 (scale bar=25μm). (c) Accumulation of four different proteins normalized to aquaporin 1 in the renal proximal tubules of podocin-KO mice with progression of proteinuria (groups 1–4). Each bar represents the ratio between the mean content of the specific protein in KO and WT mice. Asterisks designate a significant difference between the mean of the WT mice and the mean of the KO mice (P<0.05; n=4 or 5). The dotted line represents the ratio 1 at which the WT and KO mice have equal means. The error bars represent the estimated s.e. of the KO/WT ratio. The estimation is obtained by the Delta method of finding approximations based on the Taylor series expansions to the variance of functions of random variables. DBP, vitamin D–binding protein. (d) Content of endogenous albumin in a WT and a podocin-KO mouse from group 2 analyzed by immunohistochemistry (scale bars=25μm). Kidney International 2013 84, 902-910DOI: (10.1038/ki.2013.218) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Megalin and cubilin levels. (a) Protein and (b) mRNA levels of megalin and cubilin normalized to aquaporin 1 during progression of proteinuria. Each bar represents the ratio between the mean content of the specific protein or mRNA in knockout (KO) and wild-type (WT) mice. Asterisks designate a significant difference between the mean of the WT mice and the mean of the KO mice (P<0.05; n=4 or 5). The dotted line represents the ratio 1 at which the WT and KO mice have equal means. The error bars represent the estimated s.e. of the KO/WT ratio. The estimation is obtained by the Delta method of finding approximations based on the Taylor series expansions to the variance of functions of random variables. (c) Immunohistochemical analysis for megalin in a podocin-KO and a WT mouse from group 3. Arrows indicate collapsed tubules, also shown in enlargement (scale bars=50μm). (d) Immunohistochemical analysis for cubilin in a podocin-KO and a WT mouse from group 3 (scale bars=50μm). Kidney International 2013 84, 902-910DOI: (10.1038/ki.2013.218) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 Lysosomal enzyme levels. (a) Protein levels of lysosomal enzymes normalized to aquaporin 1 during progression of proteinuria. (b) Immunohistochemical analysis for cathepsin b on a podocin-knockout (KO) and wild-type (WT) mouse (scale bars=25μm). (c) mRNA levels of lysosomal enzymes normalized to aquaporin 1 during progression of proteinuria. (d) mRNA levels of additional eight enzymes normalized to aquaporin 1 analyzed only in group 3. Each bar represents the ratio between the mean content of the specific protein or mRNA in KO and WT mice. Asterisks designate a significant difference between the mean of the WT mice and the mean of the KO mice (P<0.05; n=4 or 5). The dotted line represents the ratio 1 at which the WT and KO mice have equal means. The error bars represent the estimated s.e. of the KO/WT ratio. The estimation is obtained by the Delta method of finding approximations based on the Taylor series expansions to the variance of functions of random variables. Kidney International 2013 84, 902-910DOI: (10.1038/ki.2013.218) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Turnover of endocytosed material. (a) Content of iodinated albumin at 5, 30, and 180min, and 24h after injection of iodinated mouse serum albumin (MSA) in wild-type (WT) and podocin-knockout (KO) mice from group 3 (n≥3; line represents mean in the groups). (b) Dye-quenched bovine serum albumin (DQ-BSA), which is quenched until degraded, in WT and podocin-KO mice from group 3 at 5min, 30min, and 24h after injection (scale bars=20μm). (c) Degraded DQ-BSA remaining in collapsed tubules from a podocin-KO mouse from group 3 at 24h after injection (scale bar=20μm). (d) Oil Red O staining for lipid in a WT and a podocin-KO mouse from group 3 (scale bar=20μm). Kidney International 2013 84, 902-910DOI: (10.1038/ki.2013.218) Copyright © 2013 International Society of Nephrology Terms and Conditions