Lymphangiogenesis in the tubulointerstitium correlates with severity of interstitial fibrosis (A), proteinuria (B), plasma creatinine levels (C) and creatinine clearance (D).

<p>Interstitial fibrosis, proteinuria (8 wks post transplantation), plasma creatinine levels (8 wks post transplantation) and creatinine clearance (8 wks post transplantation) were determined as recently described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009095#pone.0009095-Rienstra1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009095#pone.0009095-Waanders1" target="_blank">[15]</a>. LYVE-1 expression was quantified as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009095#s2" target="_blank"><i>Methods</i></a> section. (gray circle isografts, black circle allografts).</p

Proteoglycan expression in arteries, glomeruli and tubulointerstitium of non-transplanted control kidneys.

<p>All proteoglycans were strongly expressed in the intima of arteries (A–D). HS proteoglycans were located in the subendothelial BM while versican was located in the endothelial cell membrane (insert D, confocal image, magnification 3780×). The BMs of vascular SMCs in the media showed a strong expression of collXVIII and a patchy expression of perlecan (A and B). In the glomeruli (E–H), the glomerular BM moderately expressed collXVIII (E) while perlecan was virtually absent (F) whereas agrin was abundantly present (G). All HS proteoglycans were expressed in Bowman's capsule but only minimally in the mesangial matrix (E–G). Dotted staining pattern for versican suggested expression by podocytes. (H). In the tubulointerstitium (I–L), tubular BMs minimally expressed collXVIII and perlecan in the cortex (I.1 and J). Compared with the cortex, collXVIII expression was increased in medullary tubular BMs (I.2). Perlecan was moderately to strongly expressed in peritubular capillaries (J). Agrin was uniformly expressed in tubular BMs (K). Versican was not present in tubular BMs but strongly expressed in the tubulointerstitial matrix (L). Magnifications: A–G, H & J: 640×; I: 320×.</p

Lymphangiogenesis in the tubulointerstitium is associated with perlecan expression.

<p>Expression of perlecan in allografts was significantly increased compared with non-transplanted control kidneys and isografts (A–C). After transplantation (of both iso- and allografts), the area with CD31 expression slightly decreased (D–F) (NS: not significant). In allografts, LYVE-1 expression was significantly increased compared with non-transplanted control kidneys and isografts (G–I). Double staining for perlecan and LYVE-1 revealed that perlecan is expressed in association with lymphatic endothelium in the newly-formed lymphatics (J–L). Arrowheads indicate peritubular capillaries strongly positive for perlecan but negative for LYVE-1. C, F and I represent the quantification of surface area stained for perlecan, CD31 and LYVE-1, respectively. Magnification A, B, D, E, G, H: 320×; J–L: 640×. *p<0.05, **p<0.01</p

Proteoglycan expression in transplant vasculopathy (TV), focal glomerulosclerosis (FGS) and tubulointerstitial fibrosis (IF) in allografts.

<p>In the neointima in TV, collXVIII and perlecan were strongly expressed (A and B). Expression of agrin and versican was less prominent in the neointima but their expression was slightly upregulated in the media (compared with non-transplanted control tissue) (C and D). Dotted lines indicate the internal elastic lamina. In the glomeruli (E–H), expression of collXVIII in the glomerular BM was variable with strong expression in glomerulosclerotic lesions (E). Perlecan was strongly induced in the glomerular BM (F). Agrin expression remained similar to its expression in glomerular BMs in non-transplanted control tissue (G). Versican staining was comparable with non-transplanted control tissue (H). In the tubulointerstitium (I–L), collXVIII (I) and perlecan (J) were minimally present in IF in which agrin expression was absent (K). CollXVIII was clearly expressed by tubular BMs in cortical (I.1) and medullary (I.2) regions at similar levels. Versican was strongly expressed in IF (L). In the cortical tubular BM, collXVIII was strongly expressed with a strong, but slightly interrupted, expression of agrin (I and K). Perlecan was only weakly expressed in the tubular BM but strongly expressed in peritubular capillaries (J). Magnification 640×.</p

Proteoglycan expression in the specific renal compartments in non-transplanted control kidneys, isografts, and allografts.

<p>Semi-quantitative scores (ranging from 0–4) of proteoglycan expression presented as the staining intensity and, between parentheses, the surface area stained (as described in detail in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009095#s2" target="_blank"><i>Methods</i></a> section). Scores given are the group means of the different grafts analyzed. Numbers of grafts analyzed are: non-transplanted control, n = 5; isografts, n = 5; allografts, n = 11. The values represented in <b><i>bold/italic</i></b> indicate differentially expressed proteoglycans in the various groups as discussed in more detail in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009095#s3" target="_blank"><i>Results</i></a> section. Abbreviations: NI, neointima; BC, Bowman's capsule; GBM, glomerular basement membrane; MM, mesangial matrix; TBM, tubular basement membrane; med, medulla.</p

Proteoglycan core proteins expressed in transplant vasculopathy, glomerulosclerosis and interstitial fibrosis in allografts contain functional glycosaminoglycan side chains.

<p>Neointimal cells in TV (A), glomerular BMs in non-sclerotic areas (B) and tubular BMs in IF (C) express heparan sulfate domains with N-unsubstituted glucosamine residues as recognized by antibody JM-403. Following heparitinase treatment presence of heparan sulfate stub regions was identified in medial and neointimal cells in TV (D), in glomerular BMs (E) and in tubular BMs (F) using antibody F69-3G10. Dotted line in panel A and D represents the internal elastic lamina. Abbreviations: M: media; NI: neointima. L-selectin-IgM chimeric protein binding in the tubulointerstitium in no pre-treated sections (G), sections pre-treated with heparitinase I [hep I] (H), sections pre-treated with chondroitinase ABC [chonABC] (I) and sections pre-treated with both heparitinase I and chondroitinase ABC (J). Insets show high power magnifications of the framed areas. Arrows: tubular BMs, asterisks: interstitium.</p

Allografts present severe development of transplant vasculopathy (A), focal glomerulosclerosis (B) and interstitial fibrosis (C).

<p>(A) Intrarenal artery with a neointima (Verhoeff staining [elastic laminae: black; collagen: red; smooth muscle cells: yellow]). (B) Glomerulus with a sclerotic lesion (periodic acid-Schiff staining [glycans in connective tissue: purple-magenta]). (C) Part of the tubulointerstitium with a fibrotic area (Masson's trichrome staining [collagen: blue]). Stainings were performed on 2 µm formalin-fixed paraffin sections. Abbreviations: IEL: internal elastic lamina; IF: interstitial fibrosis; M: media; NI: neointima. Magnification 400×.</p

Reduced macrophage/monocyte influx after renal I/R in double mutant mice for collagen XV and XVIII.

<p><i>A:</i> Immunofluorescent staining for collagen IV, XV and XVIII (green) and macrophages (red) in WT and double mutant mice at day 5 after I/R showed presence of collagen IV, XV and XVIII in peritubular capillaries (white arrows) in WT mice and accumulation of macrophage/monocytes around these capillaries. Less macrophage influx (in red) was observed in double mutant mice, also lacking collagen XV and XVIII signal (in green). The nuclei are stained in blue. Scale bars 20 µm. <i>B:</i> Immunofluorescent staining for macrophages (red) and nuclei (blue) in WT and double mutant sham operated mice at day 5 after I/R showed no macrophage/monocyte in renal tissues. Scale bars 20 µm. <i>C:</i> Number of macrophage/monocytes per HPF (High Power Field) at different timepoints after I/R. At day 5 significantly less macrophage/monocytes were observed in kidneys of <i>Col15a1^−/−×Col18a1^−/−</i> mice compared to WT (p<0.05). Data is presented as mean ± SEM. *: p<0.05.</p

Increased expression of TNF-α and MCP-1 in double mutant mice, lacking both collagen XV and XVIII compared to WT.

<p><i>A–B:</i> Immunofluorescent staining of MCP-1 (red) in WT (<i>A</i>) and double mutant mice (<i>B</i>) at day 5. Double mutant mice showed an increased expression of MCP-1 in peri-tubular capillaries. Scale bars 20 µm. <i>C:</i> mRNA expression of MCP-1 in renal tissue of <i>Col15a1^−/−</i>, <i>Col18a1^−/− and Col15a1^−/−×Col18a1^−/−</i> double compound compared to WT mice at day 5 after I/R (***: p<0.001 double mutant mice compared to WT). <i>D:</i> mRNA expression of TNF-α in renal tissue of <i>Col15a1^−/−</i>, <i>Col18a1^−/− and Col15a1^−/−×Col18a1^−/−</i> double compound compared to WT mice at day 5 after I/R (**: p<0.01 double mutant mice compared to WT).</p

MCP-1-induced monocyte migration is increased in the presence of immobilized heparin-albumin and glycosylated collagen XVIII.

<p><i>A:</i> MCP-1 dose dependently increased the migration of monocytes over a porous membrane. Immobilization of heparin-albumin, mimicking an artificial BM HSPG, promotes monocyte transmigration. Spontaneous migration over albumin-coated membrane in the absence of MCP-1 was set as 1 and the other values were calculated accordingly. The error bars represent SEM. <i>B:</i> Transmigration of monocytes towards MCP-1 (10 ng/ml) was increased in the presence of heparin-albumin and collagen XVIII with long GAG chains. Heparin-albumin increased the monocyte migration significantly compared to albumin coated membrane (p<0.01). N-terminal fragment of short collagen XVIII with long GAG chains promoted transmigration significantly compared to albumin and N-terminal fragment without GAG chain (both p<0.05). Relative to albumin, also the full-length short collagen XVIII promoted MCP-1-induced monocyte transmigration to some extent (not significant). Data is calculated relative to migration over albumin-coated membrane towards 10 ng/ml MCP-1. The error bars represent SEM. *: p<0.05, **: p<0.01.</p