Differential expression of proteoglycans in tissue remodeling and lymphangiogenesis after experimental renal transplantation in rats

Abstract

Background: Chronic transplant dysfunction explains the majority of late renal allograft loss and is accompanied by extensive tissue remodeling leading to transplant vasculopathy, glomerulosclerosis and interstitial fibrosis. Matrix proteoglycans mediate cell-cell and cell-matrix interactions and play key roles in tissue remodeling. The aim of this study was to characterize differential heparan sulfate proteoglycan and chondroitin sulfate proteoglycan expression in transplant vasculopathy, glomerulosclerosis and interstitial fibrosis in renal allografts with chronic transplant dysfunction.

Methods: Renal allografts were transplanted in the Dark Agouti-to-Wistar Furth rat strain combination. Dark Agouti-to-Dark Agouti isografts and non-transplanted Dark Agouti kidneys served as controls. Allograft and isograft recipients were sacrificed 66 and 81 days (mean) after transplantation, respectively. Heparan sulfate proteoglycan (collXVIII, perlecan and agrin) and chondroitin sulfate proteoglycan (versican) expression, as well as CD31 and LYVE-1 (vascular and lymphatic endothelium, respectively) expression were (semi-) quantitatively analyzed using immunofluorescence.

Findings: Arteries with transplant vasculopathy and sclerotic glomeruli in allografts displayed pronounced neo-expression of collXVIII and perlecan. In contrast, in interstitial fibrosis expression of the chondroitin sulfate proteoglycan versican dominated. In the cortical tubular basement membranes in both iso- and allografts, induction of collXVIII was detected. Allografts presented extensive lymphangiogenesis (p<0.01 compared to isografts and non-transplanted controls), which was associated with induced perlecan expression underneath the lymphatic endothelium (p<0.05 and p<0.01 compared to isografts and non-transplanted controls, respectively). Both the magnitude of lymphangiogenesis and perlecan expression correlated with severity of interstitial fibrosis and impaired graft function.

Interpretation: Our results reveal that changes in the extent of expression and the type of proteoglycans being expressed are tightly associated with tissue remodeling after renal transplantation. Therefore, proteoglycans might be potential targets for clinical intervention in renal chronic transplant dysfunction.

Figure 1. Allografts present severe development of transplant vasculopathy (A), focal glomerulosclerosis (B) and interstitial fibrosis (C).

(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×.

Figure 2. Proteoglycan expression in arteries, glomeruli and tubulointerstitium of non-transplanted control kidneys.

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×.

Figure 3. Proteoglycan expression in transplant vasculopathy (TV), focal glomerulosclerosis (FGS) and tubulointerstitial fibrosis (IF) in allografts.

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×.

Figure 4. Proteoglycan core proteins expressed in transplant vasculopathy, glomerulosclerosis and interstitial fibrosis in allografts contain functional glycosaminoglycan side chains.

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.

Figure 5. Lymphangiogenesis in the tubulointerstitium is associated with perlecan expression.

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

Figure 6. Lymphangiogenesis in the tubulointerstitium correlates with severity of interstitial fibrosis (A), proteinuria (B), plasma creatinine levels (C) and creatinine clearance (D).

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 , . LYVE-1 expression was quantified as described in the Methods section. (gray circle isografts, black circle allografts).