Volume 88, Issue 5, Pages 990-998 (November 2015) Common histological patterns in glomerular epithelial cells in secondary focal segmental glomerulosclerosis  Christoph Kuppe, Hermann-Josef Gröne, Tammo Ostendorf, Toin H. van Kuppevelt, Peter Boor, Jürgen Floege, Bart Smeets, Marcus J. Moeller  Kidney International  Volume 88, Issue 5, Pages 990-998 (November 2015) DOI: 10.1038/ki.2015.116 Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 1 Small adhesions are formed by cells expressing podocyte and parietal epithelial cell (PEC) markers. (a–d) Synaptopodin (Synpo)-positive (presumptive podocytes, arrows with tails), and annexin A3 (ANXA3)-positive adhesions (presumptive PECs, arrowheads) can be observed within the same glomeruli. Blow ups of marked insets are shown in panels a′–d″. Asterisks mark sclerotic lesions in a′ and a″. In general, synaptopodin-positive areas on Bowman’s capsule (arrows with tails) were negative for PEC matrix (LKIV69). Representative glomeruli quadruple immunofluorescence (IF) staining for LKIV69/synaptopodin/ANXA3/Hoechst. (a) Transplant glomerulopathy (Transpl. glomerulop.), (b) lupus nephritis, (c) fibrillary glomerulonephritis (GN) with focal and segmental glomerulosclerosis (FSGS), and (d) membranoproliferative glomerulonephritis (MPGN). Scale bars=100μm (a–d);25μm (a′-d′);50μm (d″). Kidney International 2015 88, 990-998DOI: (10.1038/ki.2015.116) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 2 Cells expressing parietal epithelial cell (PEC) markers in sclerotic lesions. Sclerotic lesions. Representative periodic acid-Schiff (PAS)-stained sections of (a) small and (b–d) advanced sclerotic focal and segmental glomerulosclerosis (FSGS) lesions (serial sections, principal glomerular disease: (a) membranous nephropathy, (b) transplant glomerulopathy (Transpl. glomerulop.), (c) lupus nephritis, and (d) collapsing FSGS not showing the classical extracapillary proliferations in this particular glomerulus). (a) Only a small area of sclerosis (a, asterisk) and no adhesion is visible on this plane of the section (PAS staining). Cells expressing PEC markers covering the synaptopodin (Synpo)-negative sclerotic region (asterisk) can only be identified by annexin A3 (ANXA3)/LKIV69 staining (arrowheads). (b–d) Advanced sclerotic lesions (asterisks) always showed positive staining for PEC markers LKIV69/ANXA3. (b) Inset highlights the classical double contoured GBM. (c1) Nonspecific staining in erythrocytes (arrow with tails). (d1) Note the parietal podocyte (d1, arrow with tails) and PECs spreading on top of the still detectable synaptopodin-positive capillaries (d1′ arrow with tails). Arrows indicate LKIV69 staining for PEC matrix, arrowheads indicate PECs, and asterisk indicate sclerotic lesion. Scale bars=100μm (a–d);100μm (a1–d1);50μm (a1′–d1′). GBM, glomerular basement membrane. Kidney International 2015 88, 990-998DOI: (10.1038/ki.2015.116) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 3 Cellular lesions. (a) Cellular lesion and subtotal sclerosis (asterisks) of the glomerular tuft. LKIV69/annexin A3 (ANXA3)-positive parietal epithelial cells (PECs;arrowheads) coexpress activation markers CD44/KRT19 (a1–a2). (a3) PEC marker claudin-1 stains the cells within the cellular lesion (a3, arrows). Whereas ANXA3 was expressed by all cells (a1′, arrows), CD44 and keratin 19 (KRT19) were differentially expressed (a2, a2′, arrowheads vs. arrows with tails). (b) This cellular lesion (asterisk) again stains for LKIV69/ANXA3 (presumptive PECs, b1). In addition, infiltrating ANXA3 brightly positive (b1–b1′, arrows with tails) or CD44-positive presumptive leukocytes (b2′, arrows with tails) can be detected because of the nature of the underlying disease lupus nephritis. Again, differential expression of the activation markers CD44 and KRT19 in presumptive PECs can be observed (arrows in b2). (b3) Virtually all cells of the cellular lesion express claudin-1, suggesting that they are PECs. (a, b) Representative serial sections from patients with (a) transplant glomerulopathy and (b) lupus nephritis. Scale bars=100μm (a+b);100μm (a1+b2);50μm (a1′+b1′). Kidney International 2015 88, 990-998DOI: (10.1038/ki.2015.116) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 4 Three histological patterns of parietal epithelial cell (PEC) involvement in focal and segmental glomerulosclerosis (FSGS) lesions in humans. (a) Normal glomerulus with podocytes (blue) and parietal epithelial cells (PECs, green) in the extracapillary compartment separated by the glomerular basement membrane (GBM) from the endocapillary compartment. (b) Glomerular adhesions/synechiae were formed by presumptive podocytes (B1) and/or PECs (B2). PEC matrix could no longer be observed along the Bowman's capsule underneath parietal podocytes (B1+B3). PECs migrating onto the GBM deposited PEC matrix at the site of adhesion to the glomerular tuft (B2, red). It cannot be ruled out that in contrast to mice, podocytes in direct contact with cellular adhesions undergo trans- or de-differentiation in situ to express PEC markers de novo. (c) Sclerotic FSGS lesions. Note that presumptive PEC migration onto the GBM is also associated with the development of sclerosis within the adjacent endocapillary compartment (C1). In some cases, presumptive podocytes were still detectable within adhesions (C2), and cells expressing PEC markers lay on top of synaptopodin-positive podocytes (arrow). Note that only presumptive PEC migration onto affected tuft segments, not podocyte migration, was associated with the development of sclerotic lesions. (d) Advanced sclerosis. Presumptive PECs cap entire sclerotic tuft segments. As a consequence, the tubuloglomerular outlet may become obstructed or lost (D1). (e) Cellular lesions are almost exclusively populated by cells expressing PEC markers, where more presumptive PECs express activation markers compared with sclerotic lesions. Kidney International 2015 88, 990-998DOI: (10.1038/ki.2015.116) Copyright © 2015 International Society of Nephrology Terms and Conditions