Even though renal transplantation has been a clinical reality for nearly 40 years, chronic rejection remains a primary cause of late graft loss. Although advances in immunosuppressive therapy have increased 1-year graft survival, the percentage of allografts developing chronic rejection has not changed and no treatment, other than retransplantation, is currently available to treat this posttransplant complication. Regardless of the organ affected, chronic rejection manifests itself by progressive vasculo-occlusive disease, resulting in ischemic damage and clinical deterioration of organ function. Chronic rejection in renal allografts also involves the appearance of a more disparate collection of pathologic sequelae, including the processes of characteristic glomerulopathy, tubulointerstitial appearance of inflammatory cells, and interstitial fibrosis accompanied by tubular atrophy and drop-out (1).
Tubulointerstitial lesions were identified in the early clinical experiences of renal transplantation and are known to affect up to 48% of renal allografts after the first year (2). Even though vascular and glomerular lesions contribute to the clinical deterioration of graft function, tubular damage and loss associated with interstitial inflammation and fibrosis may be the most important determinants in chronic renal allograft rejection. Indeed, impairment of creatinine clearance and glomerular filtration correlate well with the degree of tubular damage and interstitial fibrosis in a wide range of interstitial diseases (3-5). The contribution of the inflammatory process in chronic rejection has been described (6, 7) and provides a potential mechanism for targeted delivery of polypeptide growth factors and cytokines capable of inducing, coordinating, and sustaining a wound healing process. After injury, the renal parenchyma undergoes a nephrogenic repair process that is similar to events associated with developmental nephrogenesis (8-10).
Fibroblast growth factor (FGF*) has been implicated as an initiator of fibrogenic mechanisms in a wide range of pathologic conditions (11-13), including renal diseases (14). Several studies have confirmed that acidic FGF (FGF-1) is expressed in the undifferentiated mesenchymal cells of the developing kidney (15-17), an observation that suggests a role for this polypeptide during nephrogenesis. In addition, injury to proximal tubular epithelial cells induces expression of FGF-1, which is competent to coordinate nephrogenic repair in vitro (18). Collectively, these observations predict the involvement of FGF-1 during development of pathophysiologic lesions associated with chronic rejection of renal allografts. Indeed, recent evidence from this laboratory has described the exaggerated appearance of FGF-1 in the glomeruli and vasculature of chronically rejected human renal allografts (19, 20). In these studies, immunoreactive FGF-1 protein and mRNA co-localized with the presence of cellular infiltrate, suggesting inflammatory-mediated delivery of this growth factor.
The present study examined these issues further and was designed to explore the expression of FGF-1 and high-affinity receptors in the tubulointerstitial compartment of both normal and chronically rejected human renal allografts. Several lines of evidence provide a compelling argument that during chronic rejection, the appearance of interstitial FGF-1 includes not only inflammatory-mediated delivery, but also enhanced expression by tubular epithelial cells.
MATERIALS AND METHODS
Tissue specimens. Formalin-fixed, paraffin-embedded renal tissue from 17 patients (19, 20), who underwent 19 nephrectomies after graft loss secondary to chronic rejection, were analyzed in this study. Five control samples included: (i) renal tissue from three separate, heart-beating organ donors, whose kidneys were determined unsuitable for transplantation; (ii) tissue obtained from a normal, functioning renal allograft, which resided in a patient who died 25 years after transplantation from unrelated complications; and (iii) a renal biopsy obtained from a normal donor kidney immediately after transplantation.
In situ hybridization and immunohistochemistry. Detection of endogenous mRNA and protein was performed on routine paraffin sections, using specific riboprobes and antibodies, essentially as previously described (19, 20). The percentage of proliferating cell nuclear antigen (PCNA) staining was determined by dividing 3,3′-diaminobenzidine-stained (brown) nuclei by the total observed (blue plus brown) nuclei in at least 100 tubular epithelial cells. Twenty different intact tubular segments, including proximal and distal nephrons from each specimen, were evaluated and included approximately equal representation of structures both adjacent to and removed from areas of inflammation. Statistical significance was assessed using Student's t test (Abacus Concepts, Inc., Berkeley, CA). Immunohistochemical staining for FGF-1 and high-affinity receptors was assessed by four independent investigators with blinded evaluation. Twenty different intact tubular segments from each specimen were evaluated and assigned a grade, according to the following protocol: 0, no staining identified; 1+, less than 25% of tubular cells positive, with mild to moderate intensity; 2+, 25-50% of tubular cells positive, with moderate intensity; 3+, 50-75% of tubular cells positive, with moderate to marked intensity; and 4+, greater than 75% of tubular cells positive, with marked intensity.
The total cumulative score for each specimen was determined and divided by 20, to obtain an average grade, which was approximated to the nearest whole number.
This retrospective study included analysis of five specific controls and 19 transplant nephrectomies, from 17 recipients whose medical history has been reported previously (19, 20). Routine histologic analysis of both a nontransplant control specimen (Fig. 1A) and tissue obtained from an allograft that had maintained normal renal function for 25 years after transplantation (Fig. 1B) were unremarkable, except for mild focal vascular sclerosis. Tubulointerstitial elements were grossly intact and showed no pathologic abnormalities. Immunohistochemical analysis of these controls demonstrated minimal staining for FGF-1 in the tubular epithelium in both cortical and medullary locations (Fig. 1C) and no staining in the interstitium (Fig. 1D).
In contrast to observations in normal human tubulointerstitium, histochemical analysis of chronically rejected allografts demonstrated varying degrees of tubular atrophy and loss, tubular dilatation, and interstitial fibrosis (Fig. 2A). In addition, almost all of the tissues examined exhibited areas of inflammatory infiltration within the tubulointerstitium (Fig. 2B). Exaggerated staining for FGF-1 was observed in the tubular epithelium, associated with chronic rejection (Fig. 2C). No significant differences in FGF-1 staining were noted between proximal and distal tubular structures. In the interstitium, FGF-1 staining was localized to areas of inflammatory infiltration and to small vascular structures within the stromal matrix (Fig. 2D).
In situ hybridization analysis of control tubular elements (Fig. 3A) failed to demonstrate expression of FGF-1 mRNA. In contrast, increased expression of FGF-1 mRNA in tubular epithelial cells of chronically rejected kidneys (Fig. 3B) was readily apparent. This pattern of staining was consistent throughout the tubular compartments and did not appear localized to any particular tubular region. Enhanced immunolocalization of FGF-1 mRNA also was detectable in the interstitial inflammatory cells (Fig. 3C). Exaggerated staining for FGF-1 mRNA in both tubular epithelial and inflammatory cells was consistent with immunohistochemical analyses of FGF-1 protein.
Inflammatory cells within the tubulointerstitium associated with chronic rejection included CD3+ T cells (Fig. 4A) and macrophages (Fig. 4B), distributed throughout the interstitium and frequently localized to perivascular areas. Representative staining for the procoagulant, von Willebrand factor, demonstrated increased number and size of vascular structures within the interstitium of chronically rejected kidneys (Fig. 4C). Analysis of chronically rejected kidneys with the antibody directed against von Willebrand factor frequently identified dense vascular plexus-like structures within the interstitial compartment (Fig. 4D). The average number of separate von Willebrand factor-positive vascular structures in chronically rejected renal tissue (58.8±6.5) was significantly (P<0.01) higher than that found in control samples (6.9±2.4). Routine histochemical analysis further demonstrated the appearance of red blood cells and hemosiderin deposits localized in areas near vascular structures, an observation that suggests active hemorrhage from leaky neocapillary formation.
Positive staining for PCNA was observed in the tubulointerstitial compartment of chronically rejected renal allografts, associated with not only the tubular epithelium (Fig. 5A), but also the recruited inflammatory infiltrate (Fig. 5B) and dense areas of von Willebrand-positive vascular structures (Fig. 5D). Total levels of PCNA staining in the tubular epithelium (55.2%) were increased significantly (P<0.001) when compared with controls (1.3%), including the 25-year-old normal renal allograft (Fig. 5C). However, the percentage of PCNA staining varied, depending on the involvement of inflammatory processes. Tubular epithelial cells distant to inflammatory cells (55.2%) demonstrated a significant (P<0.001) increase in PCNA staining, compared with those residing within areas of active cellular infiltrate (40.8%).
Of the four FGF receptor (FGFR) isoforms analyzed (Fig. 6) in control samples, each demonstrated moderate staining of the tubular epithelium. Similar immunostaining for each FGFR was noted in the tubulointerstitial compartment of chronically rejected renal allografts (Fig. 6), and included immunoreactivity against not only the tubular epithelium, but also inflammatory cells and vascular structures within the interstitium. The localization pattern of the immunoreactive FGFR was consistent in both control and chronically rejected tubular structures. A composite summary of immunohistochemical tubular staining intensity for both FGF-1 and its high-affinity receptors is presented in Table 1. Differential levels of immunostaining for FGF-1 and FGFR could not be attributed significantly to any aspect of the donor or recipient data previously presented (19, 20). The main consistent observation derived from these immunohistochemical analyses demonstrated that the tubulointerstitial compartment of kidneys undergoing chronic allograft rejection harbor the exaggerated appearance of FGF-1 protein, levels of which seem related to activated transcriptional and translational processes in both tubular epithelial cells and infiltrating inflammatory cells.
The in situ hybridization and immunohistochemical studies reported here demonstrated minimal expression of FGF-1 mRNA and protein in the tubulointerstitium of normal human kidney transplant controls. The near absence of detectable FGF-1 mRNA and protein predicts that normal tubular epithelium contains a quiescent cell population, which is consistent with the absence of significant PCNA staining. This observation is consistent with the relatively low cellular turnover occurring in the tubular compartment under normal physiologic conditions (21, 22). No change in staining pattern for FGF-1 mRNA and protein was observed in specific control samples. In contrast, tubulointerstitial lesions in transplant kidney allografts that experience chronic rejection demonstrated the exaggerated appearance of FGF-1 mRNA and protein. Immunostaining for FGF-1 protein correlated well with the increased appearance of FGF-1 mRNA, both of which were associated with resident inflammatory cells. The tubulointerstitial appearance of both macrophages and T lymphocytes is consistent with the pathologic features of chronic rejection in the human renal allograft (6, 7). Furthermore, both cell types are a rich source of FGF-1 (23-25), suggesting local delivery of the polypeptide mitogen by circulating and resident inflammatory cells. A similar mechanism has been proposed for the involvement of FGF-1 in both vasculopathy and glomerular lesions associated with chronic rejection of human renal allografts (19, 20).
The appearance of both PCNA staining and increased von Willebrand factor-positive vascular structures within areas of active inflammation further supports the potential significance of the increased appearance of FGF-1 protein and mRNA in interstitial lesions associated with chronic rejection. Increased von Willebrand factor staining may represent unmasking of preexisting peritubular vascular structures after tubular atrophy and loss. However, the appearance of positive PCNA staining in some endothelial cells suggests a role for angiogenesis during chronic rejection that mimics induction of neovascularization during renal development (16). The appearance of potentially leaky neovascular structures is consistent with a normal repair response to FGF-1 (26-28). However, this physiologic response may perpetuate and amplify processes associated with tubulointerstitial lesions, including increased recruitment of inflammatory cells, delivery of additional FGF-1, activation of T cells (29), enhanced angiogenesis, and induction of interstitial fibrogenesis. During renal interstitial scar formation, increased synthesis of heparan sulfate proteoglycan containing specific FGF binding domains has been identified (30). This observation predicts the involvement of extracellular matrix remodeling activity during fibrogenesis, associated with chronic rejection (19).
In addition to inflammatory-mediated synthesis and delivery of FGF-1, results provided here also demonstrate the exaggerated appearance of FGF-1 mRNA and protein in the tubular epithelium, associated with chronic rejection. This observation is consistent with a response-to-injury hypothesis (31), wherein damaged tubular epithelial cells induce expression of FGF-1 mRNA and protein competent to both promote nephrogenic repair (18) and function as a survival factor (32). However, intracellularly sequestered FGF-1, which lacks a classical signal sequence for secretion, is not available to mediate its biological effects on cell growth (33). Previous in vitro studies have demonstrated that FGF-1 is released from cells in response to heat shock (34) and oxidative stress (33, 35), by an endoplasmic reticulum-independent pathway. The secretion of FGF-1 in response to biologic stress may be responsible for inducing an extracellular autocrine/paracrine transforming loop in chronically rejecting tubular epithelium because of the following: (i) chronic rejection involves inflammatory-mediated processes (6, 7); (ii) inflammation in vivo is usually accompanied by localized hyperthermia (36) and production of reactive oxygen species (37); and (iii) pathophysiologic responses in chronic rejection include oxidative stress initiated by ischemia/reperfusion injury (38) and amplified by inactivation of antioxidant defenses (39). Consequently, the appearance of increased PCNA staining in tubular epithelial cells from chronically rejected renal allografts is consistent with an extracellular FGF-1 pathway and represents a normal regenerative response of these structures. The decrease in PCNA staining of tubular epithelial cells within areas of active cellular infiltrate may relate to the ability of FGF-1 to modulate oxidant-mediated apoptosis (37, 40), particularly because these structures exhibited a more damaged phenotype.
Mitogenic response to extracellular FGF-1 demands expression of specific high-affinity receptors to complete signal transduction processes. In the studies described here, immunostaining for FGFR-1, FGFR-2, FGFR-3 and FGFR-4 was detected readily in the tubular epithelium of both control and chronically rejected samples. The immuno-appearance of the FGFR in chronically rejected tissue also included enhanced focal staining of the inflammatory infiltrate and microvasculature residing within the tubulointerstitial compartment. Consequently, a local growth response is anticipated by receptor-positive cells exposed to the presence of a competent mitogen, a prediction consistent with PCNA staining in tubular epithelium, interstitium, and neovascular structures. Although the enhanced appearance of FGF-1 in the tubulointerstitial compartment argues in support of a transforming role for this polypeptide mitogen during development of renal lesions associated with chronic rejection, additional efforts will be required to establish a direct cause-and-effect relationship beyond that suggested in a limited retrospective analysis.
This work was supported by NIH grants HL45990 (J.A.T.), HL48491 (J.A.T.), and HL09270 (J.D.K.)
Abbreviations: FGF-1, acidic fibroblast growth factor; FGFR, fibroblast growth factor receptor; PCNA, proliferating cell nuclear antigen.
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