Chronic transplant dysfunction (CTD) is a major risk factor for late graft loss after kidney transplantation (1). Clinically, CTD after renal transplantation is defined as gradual deterioration of graft function, manifesting as a slow progressive decline in glomerular filtration rate, generally along with proteinuria and hypertension. Because these clinical variables are not specific, diagnosis is made after histopathologic evaluation. Histopathologic features of CTD after renal transplantation include transplant glomerulopathy, interstitial fibrosis, and tubular atrophy (2, 3), at times accompanied by neointimal thickening and duplication of the internal elastic lamina (IEL). Although immunosuppressive therapy has come a long way in reducing acute graft loss, it has hardly contributed to reduction in long-term graft loss (4). New strategies with different immune suppressive regiments may improve the long-term survival of renal allografts (5).
The initiating mechanism of CTD is poorly understood although several insults associated with transplantation, such as ischemia-reperfusion, the alloimmune response, and immunosuppressive treatment lead to graft injury (6). This injury triggers vascular remodeling (7) and tubular repair (8). Surviving tubular cells have the capacity to proliferate and regenerate tubules (8). Vascular remodeling is characterized by the accumulation of α-smooth muscle actin (α-SMA)-positive smooth muscle cells (SMCs) between the endothelial lining and the IEL, forming an occlusive neointima. This latter process is referred to as transplant vasculopathy (TV) or chronic allograft arteriopathy. Neointimal SMCs secrete extracellular matrix leading to arterial stiffness, luminal stenosis and, eventually, ischemic graft failure.
Data on the origin of neointimal SMCs in human CTD are contrasting, because both kidney graft (donor) origin and contribution of recipient-derived cells have been reported. Although Grimm et al. (9) reported that 34% of neointimal SMCs are recipient derived, Bittmann et al. (10) found no contribution of recipient-derived cells to neointima formation after transplantation. If recipient-derived SMCs do indeed contribute to neointima formation, these cells or their ancestral progenitors should be demonstrably present in the circulation. Indeed, circulating smooth muscle precursor cells (SMPCs) were identified (11, 12). Deeper insight into the origin of neointimal cells may lead to the development of new therapeutic approaches that may, for example, target the engraftment and function of recipient-derived cells in graft arteries or the recruitment, migration, and function of donor-derived cells to the arterial intimal space.
Because the scarce literature available concerning the origin of neointimal SMCs after renal transplantation in human CTD did not reach a consensus (9, 10), the aim of this study was to revisit the origin of neointimal cells after renal transplantation. We used nephrectomy samples from sex-mismatched renal allograft recipients. As opposed to biopsies used previously (9), nephrectomy samples have the advantage of containing larger arteries with prominent neointima formation.
As previous studies demonstrated the plasticity of circulating precursors to differentiate endothelial and epithelial lineages as well (13–15), we also determined the contribution of recipient-derived cells to endothelial and epithelial regeneration in CTD.
Chimerism was determined using fluorescent in situ hybridization (FISH) for X and Y chromosomes, combined with immunofluorescent staining on grafts transplanted in a sex-mismatched combination.
PATIENTS AND METHODS
To distinguish between recipient- and donor-derived cells, sex-mismatched nephrectomy samples (n=4) of female kidney grafts transplanted into male recipients were investigated (for patient characteristics see Table 1). From 1998 to 2007, 31 nephrectomies were performed within the University Medical Center Groningen. Of these patients, nine male patients received a female allograft. Four of these nine patients had developed TV and were included in the study for origin investigations. As positive and negative controls for FISH, two male grafts transplanted into male recipients and one female graft transplanted into a female recipient were used, respectively. All patients had returned to dialysis before nephrectomy. Although the immunosuppression regimen was tapered after return to dialysis in all patients, it was never discontinued. None of the patients were retransplanted before nephrectomy. Reasons for nephrectomy were chronic renal allograft dysfunction (CTD)/ongoing rejection (n=5), surgical/infectious complications (n=1), and primary nonfunction/rejection (n=1). All procedures and use of tissue were performed according to national ethical guidelines.
Formalin-fixed, paraffin-embedded renal nephrectomy specimens were cut into 1.5-μm-thick sections. A hematoxylin-eosin staining was used for morphologic characterization and was evaluated by a pathologist (M.v.d.H.) (2). Classification according to the Banff scheme categorized all nephrectomy samples (including controls) as grade IIB, except patient 4. The nephrectomy sample of patient 4 was categorized as grade III.
Besides providing sufficient material, nephrectomy samples also give the opportunity to investigate neointima formation in larger arteries (Fig. 1A), as opposed to biopsy samples that often contain few and small arteries. On average, 10 (range 7–14) arteries were investigated per nephrectomy sample. The degree of obliteration in arteries was measured in all patients. Arteries were identified based on the presence of an IEL using Verhoeff-van Gieson stained sections. The total area lying at the luminal side of the IEL, including the lumen, was measured and set at 100%. Subsequently, the luminal area was subtracted from this total area, to obtain the neointimal area. The percentage of the neointimal area from the total area was used as a measure for luminal obliteration. Luminal obliteration was expressed as mean percentage±SD of all arteries measured within one patient. Arteries cut diagonally, that is compressed lumen, were excluded from this measurement.
Fluorescent In Situ Hybridization
FISH probes (Kreatech Diagnostics, Amsterdam) were directed against repetitive sequences in the centromere of the X (DXZ1) or Y chromosome (DYZ3). The probe directed against DXZ1 was directly conjugated with PlatinumBright495 (green) and the probe directed against DYZ3 was conjugated with PlatinumBright550 (red). FISH was performed according to the manufacturer's instructions, with some modifications. After deparaffinization, sections were incubated in 0.2 M HCl for 20 min. To make the DNA accessible for hybridization, sections were incubated in 8% sodium thiocyanate (NaSCN, 80°C, 30 min) followed by 0.025% pepsin in 0.2 M HCl (37°C, 20 min). To reduce autofluorescence, sections were incubated in 100 mM copper sulfate (CuSO4, 37°C, 60 min) followed by 0.2% sodium borohydride (NaBH4, RT, 10 min). Probes were applied on dehydrated sections and simultaneously denatured (80°C, 10 min). Hybridization was performed overnight in a humidified chamber at 37°C. Sections were then rinsed in 0.4×saline sodium citrate/0.3% Nonidet P-40 (NP-40) (72°C, 2 min) followed by 2×saline sodium citrate/0.1% NP-40 (RT, 1 min) to remove excess unbound probe.
Immediately after FISH, immunofluorescent stainings were performed for α-SMA to detect SMCs (mouse IgG2a, clone 1A4), CD45 to detect inflammatory cells (mouse IgG1, clone 2B11+PD7/26), or von Willebrand factor (vWF) to detect endothelial cells (ECs; rabbit polyclonal, all from DAKO, Heverlee, Belgium). Sections were first fixed with 2% paraformaldehyde (10 min). Endogenous biotin was blocked using an avidin/biotin blocking kit (DAKO). Sections were blocked with 10% rabbit serum and subsequently incubated with the primary antibody (60 min) followed by a biotinylated rabbit anti-mouse conjugate (DAKO) for the detection of α-SMA and CD45 (30 min) or donkey anti-rabbit fragment (ab′)2-Cyanin5 (Cy5; Jackson Immunoresearch, Soham, UK) for the detection of vWF. Detection of biotinylated antibody complex was performed by incubation with a streptavidin-Cy5 conjugate (Zymed Laboratories Inc., CA) diluted in phosphate buffer containing 4′,6-diamidino-2-phenylindole (DAPI; Sigma, 20 min). Sections were mounted in Citifluor (Agar Scientific, Stansted, UK).
For α-SMA/CD45/vWF triple stainings without FISH, sections were blocked with 10% goat serum followed by incubation with primary antibodies diluted in phosphate buffer containing DAPI (60 min). Isotype-specific goat anti-mouse conjugates (Southern Biotechnology, Birmingham) or a donkey anti-rabbit conjugate (Jackson Immunoresearch) were used for detection of bound primary antibodies (α-mouse IgG1-fluorescein isothiocyanate for CD45, α-mouse IgG2a-tetramethyl rhodamine isothiocyanate for α-SMA, and α-rabbit fragment [ab′]2-Cy5 for the detection of vWF).
Fluorescence microscopy was performed using a Leica DMLB microscope (Leica Microsystems, Rijswijk, The Netherlands) equipped with a Leica DC300F camera and Leica QWin 2.8 software. To confirm the presence of FISH signals inside the nucleus, confocal laser scanning microscopy (LEICA TCS SP2) was performed. Ten consecutive optical sections were scanned at the excitation wavelengths of 488 nm (fluorescein isothiocyanate) and 543 nm (tetramethyl rhodamine isothiocyanate), generating a sequential Z series of 10 optical sections. Computerized stacking of these 10 optical sections generated a three-dimensional image of cells of interest (Leica confocal 2.5 software), revealing the presence of two FISH signals within one nucleus.
Quantification of Neointimal, Endothelial, and Tubular Chimerism
Nephrectomy samples of female grafts transplanted into male recipients were used to study the origin of cells contributing to kidney remodeling after transplantation. In these samples, donor-derived cells contain two X chromosomes, whereas recipient-derived cells contain one X and one Y chromosome. For chimerism investigations, all cells containing a Y chromosome (with or without an X chromosome) were included.
Arteries with a clear visible IEL were selected for counting neointimal Y-chromosome-positive cells in sections where FISH was combined with staining for α-SMA. Photomicrographs of these arteries were taken using a 40× objective. Arteries with a diameter smaller than 150 μm were excluded. In the case of large arteries, several photomicrographs were taken to cover the entire arterial area. These photomicrographs were electronically assembled using Hugin software (http://hugin.sourceforge.net/) to obtain an overview of the entire artery. In this process, duplicate photographic areas are overlaid, thus ensuring that no cells are counted twice. Using these assembled photographs, Y-chromosome-positive and α-SMA-positive cells were counted using Adobe Photoshop software, which has a module for keeping track of counted events. First, all α-SMA-positive cells between the lumen and IEL were counted. Subsequently, all Y+/α-SMA double- positive cells within this area were counted. In sporadic cases, where a duplication of the elastic lamina was observed, recipient-derived cells were never quantified in the tissue between the laminae. The percentage of Y+/α-SMA double-positive cells of the α-SMA-positive cell population was calculated. To determine interobserver variations, multiple arteries were sampled at random and recounted by an independent investigator. Percentages of recipient-derived α-SMA-positive cells of all recounted arteries were within the 95% confidence interval, when compared with the initial percentages.
To quantify the number of recipient-derived ECs, FISH was combined with a staining for vWF. Recipient-derived (Y-chromosome-positive) cells co-expressing vWF were quantified and expressed as a percentage of the total endothelial (vWF+) cell population, respectively.
To quantify the number of recipient-derived tubular epithelial cells, FISH was combined with a staining for CD45 to exclude inflammatory cells. Tubuli and tubular cells were identified based on morphology and localization. Y+/CD45− cells were counted and expressed as a percentage of the total number of CD45− tubular cells.
In this study, we used nephrectomy samples from seven patients (four female to male, two male to male, and one female to female; Table 1) with CTD after renal transplantation. The degree of luminal obliteration varied to some extent between arteries within one nephrectomy sample (Table 1). Mean luminal obliteration in the 67 arteries analyzed in seven nephrectomy samples was 83%±17% (mean±SD). The neointima consisted of deposited extracellular matrix (Fig. 1C) and of α-smooth muscle actin-positive (α-SMA+) cells, intermingled with inflammatory CD45+ cells (Fig. 1B,D). Cells co-expressing α-SMA and CD45 were never found (Fig. 1D). Tubuli were identified by morphology. To distinguish between tubular and inflammatory cells within tubuli, staining with CD45 was performed (Fig. 1E).
A punctuate FISH signal was considered a genuine sex chromosome if it was located within the nucleus. The location of FISH signals was examined by confocal microscopy (Fig. 2A). Z planes were made (Fig. 2A) and FISH signals were detected within the DAPI signal, confirming their presence inside the nucleus.
To determine the efficiency of FISH, the technique was first performed on control nephrectomy samples (i.e., male to male and female to female transplants). As an internal control for FISH, a probe directed against the X chromosomes was included, because all nucleated cells at least contain one X chromosome. No Y chromosomes were detected in sections of female grafts transplanted into female recipients (Fig. 2B). The Y chromosome was clearly visible (with or without an identifiable X chromosome) in 31% (XY=11%±4%, Y=20%±2%; mean±SD) of in total 7076 nuclei analyzed in the two male to male transplants (Fig. 2C, D). In 17%±1% of the nuclei only the X chromosome was detected, whereas in 52%±4% of the nuclei no sex-chromosomes were detected (Fig. 2D). FISH efficiency was similar on serial sections of one sample and between the two control samples, demonstrating equal detection sensitivity for sex chromosomes within and between nephrectomy specimens (data not shown).
Origin of Neointimal Cells
To discriminate between SMCs and infiltrating, recipient- derived, inflammatory cells in the neointima, FISH was combined with an α-SMA staining. A neointimal cell was considered an SMC if the nucleus was directly surrounded by cytoplasmic α-SMA staining and was considered recipient derived if a clear Y chromosome was visibly located within the nucleus (Fig. 3A). In total, 2906 neointimal α-SMA+ SMCs were investigated for chimerism. A mean of 6% (range 3%–11% between patients) of the neointimal α-SMA+/CD45− cells were recipient derived (Table 2). Percentages of chimerism varied between arteries within one nephrectomy sample (Table 2). No correlation between artery size and the degree of SMC chimerism within a graft was detected.
Endothelial and Tubular Chimerism
To investigate EC chimerism, FISH was combined with a vWF staining. An EC was considered to be recipient derived if a punctuate Y chromosome was detected inside the nucleus of a cell that was surrounded by cytoplasmic staining for vWF (Fig. 3B, C). In total, 1404 arterial ECs were investigated for chimerism. A mean of 14% (range 4%–32% between patients) of neointimal ECs were recipient derived (Y-chromosome positive; Table 2). Endothelial chimerism could not be investigated in patient 4 because vWF was present in the extracellular space than intracellularly. ECs in patient 4 were negative for CD31 and α-SMA (data not shown). In total, 660 glomerular ECs were investigated for chimerism. A mean of 19% (range 7%–31% between patients) of glomerular ECs were recipient derived (Y-chromosome positive; Table 2). Again, glomerular endothelial chimerism could not be investigated in patients 2 and 4 because of atypical expression of vWF.
Tubules and tubular epithelial cells were identified based on morphology. Furthermore, FISH was combined with CD45 staining to exclude infiltrating recipient-derived inflammatory cells (Fig. 3D). In total, 1532 CD45− tubular cells were investigated for chimerism. A mean of 3% (range 2%–5% between patients) of CD45− tubular cells were recipient derived (Y+) (Table 2).
In this study, we report neointimal smooth muscle-, endothelial-, and tubular epithelial recipient chimerism in nephrectomy samples of renal allografts with CTD. We show that chimerism is consistent within these different compartments and also between the different patients studied.
Information on the origin (donor vs. recipient) of neointimal cells may contribute to the development of new therapies to treat or prevent TV. The possible contribution of recipient-derived cells to neointima formation has received particular attention, because these cells may form an easily accessible therapeutic target. However, this contribution is still under debate, because currently only two contrasting studies regarding the origin of neointimal cells in human renal allografts are reported (9, 10). Although Grimm et al. (9) reported that approximately one third of neointimal cells were of recipient origin, Bittmann et al. (10) exclusively detected donor-derived neointimal cells. The cause of these diverging results is difficult to pinpoint and is likely due to technical aspects inherent to the complex FISH assay. In agreement with the study of Grimm et al., our study confirms the presence of recipient-derived neointimal cells, albeit at a lower frequency (∼6%). Nevertheless, both our study and the study of Grimm et al. probably underscore the percentage of recipient-derived neointimal cells, because the efficiency of FISH is never 100% (∼38% in 4 μm sections in the study of Grimm et al., ∼31% in 1.5 μm sections in our study). As a result, the actual percentage of recipient- derived cells is expected to be higher in both studies.
We have previously performed studies on the origin of neointimal SMCs in experimental transplant models (aorta and heart) in which severe TV develops after allografting (16, 17). In these models, virtually all neointimal SMCs were found to be of recipient origin. However, in a rat transplant model for long-term renal allograft dysfunction, we found that all neointimal SMCs were of donor origin (18). These studies suggest that the development of TV with regard to the origin of contributing cells is organ dependent, but also varies between species. Because TV can develop to some extent in the absence of alloimmune responses (19), most likely any form of vascular injury can initiate or enhance the development of TV. In this respect, the degree of injury should be taken into account, because the frequency of recipient- derived neointimal cells may vary with the severity of vascular injury (20, 21). In this study, it is conceivable that some degree of injury may have occurred because of tapering of the immune suppressive regimen after graft failure, and thus may have facilitated neointimal engraftment of recipient-derived SMCs. However, we previously found similar percentages of chimerism in biopsy samples (unpublished observations), indicating that recipient cell engraftment precedes graft failure.
The most likely source for recipient-derived neointimal cells is the pool of recipient peripheral blood mononuclear cells, as corroborated by studies that provide evidence for an SMPC subset residing within the myeloid lineage (12, 22). The role of recipient-derived neointimal cells is unclear. Although their number in this study is relatively limited, these cells may have a constructive role in neointimal formation, as suggested by their expression of α-SMA. However, a regulatory role for recipient-derived cells cannot be excluded.
A conventionally accepted source of donor neointimal SMCs is the arterial media from which SMCs migrate across the IEL in response to mechanical arterial injury (the so-called response to injury paradigm) (23). Another possible source for donor neointimal cells are migrating adventitial myofibroblasts. During the response to vascular injury and inflammation, adventitial fibroblasts can be reprogrammed to differentiate into myofibroblasts (24). These myofibroblasts can be attracted to the subendothelial space by chemokines secreted by SMPCs or activated ECs. An alternative source for donor-derived neointimal cells may be ECs by a mechanism termed endothelial to mesenchymal transition (25). In three separate animal models for kidney fibrosis, ECs were shown to differentiate into α-SMA-positive cells, thereby contributing to interstitial fibrosis (26). Whether this mechanism is also applicable to the development of TV is not known.
Next to neointimal chimerism, we addressed arterial and glomerular endothelial chimerism and tubular chimerism, as well, to gain insight into the plasticity of recipient-derived cells. Conflicting evidence exists on the replacement of donor ECs by recipient-derived ECs (10, 14, 27). Most studies find, to a certain extent, replacement of donor ECs with recipient-derived cells. Because Asahara et al. (28) described circulating EPCs, more research has focused on these cells and their therapeutic potential (29). These EPCs are mobilized from the bone marrow into the circulation and home to sites of injury (30). It is suggested that replacement of donor- by recipient-derived ECs may be important for transplantation tolerance in long-term allograft survivors (31), a concept that was however challenged by a study from Lagaaij et al. (14) who found a positive correlation between the extent of EC chimerism and graft rejection. Their data indicate that severe vascular injury might trigger repopulation of severely injured graft endothelium by recipient-derived ECs.
In this study, we found a minor contribution of recipient-derived cells to tubular regeneration in nephrectomy samples after renal transplantation, confirming other studies performed in human biopsies (9, 10, 13, 15, 32, 33). Because of the few studies that are able to correlate chimerism with graft outcome and acknowledging the capacity of renal tubular cells to regenerate the tubules (34), it is more likely that engraftment of recipient-derived tubular cells also depends on the severity of renal tubular damage after transplantation (8).
We showed that both donor- and recipient-derived cells contribute to vascular remodeling and tubular repair after human renal transplantation. The presence of α-SMA in donor- and recipient-derived cells supports a constructive role in neointimal formation. Because donor-derived SMCs predominated in the neointima, we propose that they form an important therapeutic target.
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Keywords:© 2009 Lippincott Williams & Wilkins, Inc.
Transplant vasculopathy; Chimerism; Chronic transplant dysfunction; Clinical renal transplantation