With improvements in short-term organ survival after transplantation, focus has shifted to long-term organ function. It has become evident that increasing 1-year graft survival rates, as the result of improved prevention of acute rejections, have decreased the rate of subsequent attrition of renal allografts (1). Both immune and non-immune factors have been implicated in the pathogenesis of chronic allograft nephropathy (2). There are several non-immunologic factors that substantially affect long-term survival. Approximately half of the kidneys from cadaveric donors develop progressive dysfunction over time and ultimately fail within a decade despite stringent immunosuppressive measures (3). Although attention to potential causes of the condition has focused primarily on events occurring after engraftment, both short- and long-term function of the organ may be influenced by a variety of donor-associated factors that may develop before removal of the organ for transplantation. Indeed, allografts, particularly from less than optimal sources, may not be biologically inert at the time of placement but already programmed to trigger or amplify subsequent host activity by provoking a continuum between the nonspecific inflammatory changes and the onset of alloresponsiveness (4). Several donor-associated factors implicated in long-term graft dysfunction alone or in combination involve donor age, diabetes, hypertension, and the systemic effects of brain death (5–7).
Hypertension has been shown both experimentally and clinically to produce chronic graft changes that include glomerulosclerosis, arteriosclerosis, and interstitial fibrosis. In animal models, recipient hypertension is deleterious to long-term organ structure and function (8). Similarly, kidney allografts from hypertensive donors seem to experience more frequent and severe rejection episodes after transplantation. Organs from genetically hypertensive animals may transfer hypertension to normotensive recipients (9). Observations in patients emphasize these experimental findings. Because the effects of the donor’s condition on the organ after engraftment have not been fully defined, however, the question was asked whether kidneys transplanted from hypertensive donors experience differences in intensity and progression of chronic rejection compared with those from normotensive sources. The influence of this putative donor risk factor was assessed in a well-established rat model of chronic graft dysfunction.
MATERIALS AND METHODS
Model of Donor Hypertension
Inbred adult (200–250 g) male Fisher (F344) rats of the same age (16±2 weeks) and from the same shipment acted as kidney donors in the hypertensive and normotensive donor groups. Lewis rats of the same age (16±2 weeks) were used as recipients (Harlan Sprague-Dawley, Indianapolis, IN). Donor hypertension was induced by partial occlusion of the right renal artery with a silver clip (diameter 0.25 mm) (10). The right kidney of sham controls was mobilized, but the artery was not clipped. Blood pressure values in both donors and recipients were determined weekly in regularly spaced intervals (7 days) using the tail-cuff method. Values were expressed as mean arterial pressure (mm Hg). Three measurements per animal were performed during 60 min, and the average was calculated. Only rats with a permanent increase in blood pressure (>30%) in all measurements 2 weeks after clipping of the renal artery were accepted as donors. After 10 weeks, the left kidney unclipped was removed for transplantation in the hypertensive and control groups. This interval was chosen specifically because hypertension was well established despite serial morphologic assessments showing that irreversible changes had not yet developed in the unclipped kidney.
Operative Technique and Experimental Groups
The left donor kidneys were transplanted 10 weeks after induction of donor hypertension. Normotensive, age-matched rats served as controls. All allografted animals were given low-dose cyclosporine (1.5 mg/kg, Novartis, Basel, Switzerland) for 10 days beginning on the day of transplantation. The nephrectomy of the remaining right kidney of the recipient was performed 10 days after transplantation.
Proteinuria over 24 hr was measured in rats housed in individual metabolic cages (n=15/group) every 4 weeks for 32 weeks. Protein excretion was determined by measuring precipitation after addition of 3% sulfosalicylic acid (Fisher Scientific, New York, NY). Turbidity was assessed by absorbance at a wavelength of 595 nm using a Coleman Junior II spectrophotometer (Shimidzu, Tokyo, Japan). Creatinine levels were measured on serum samples taken at 0, 24, and 32 weeks by the creatinine assay kit (Sigma Chemical Co, St. Louis, MO). Survival was assessed up to 32 weeks in separate groups (n=15/group).
For histologic and molecular biologic assessments, kidneys were harvested at serial intervals (0, 2, 12, 24, and 32 weeks, n=6/time point) and snap-frozen; additional samples were fixed in 10% buffered formalin and paraffin-embedded; and sections were stained with hematoxylin-eosin, periodic acid Schiff, trichrome, and elastin stains. Morphologic and morphometric analysis of leukocyte infiltration, interstitial fibrosis, tubular atrophy, and glomerulosclerosis were performed. Briefly, glomerulosclerosis in periodic acid Schiff-stained sections was scored 0 to 3 in more than 40 glomeruli per kidney. Fibrosis (trichrome) and leukocyte infiltration (hematoxylin-eosin) were evaluated by image analysis (proportion of cortical area) (IPLab software, Scananalytics, Richmond, VA) in six kidneys per group per time point.
Competitive Reverse Transcriptase-Polymerase Chain Reaction
Expression of interleukin (IL)-2, IL-4, interferon-γ, perforin, Fas-ligand, tumor necrosis factor-α, and macrophage inflammatory protein (MIP)-1α was determined using real-time polymerase chain reaction (PCR) on the ABI PRISM 7700 Sequence Detection System (TaqMan, Perkin Elmer, Applied Biosystems, Weiterstadt, Germany). This method uses the 5′ nuclease activity of Taq polymerase to cleave a non-extendable hybridization probe during the extension phase of the PCR. The approach uses dual-labeled fluorogenic hybridization probes. One fluorescent dye serves as a reporter (FAM, 6-carboxyfluorescein), and its emission spectrum is quenched by the second fluorescent dye (TAMRA, 6-carboxy-tetramethyl-rhodamine). The reactions are monitored in real time during the log-phase of product accumulation. The increase in the reporter dye fluorescence intensity during PCR is proportional to the amplification of the target sequence. The cycle number at which the amplification plot crosses a fixed threshold above baseline is defined as the threshold cycle (Ct). To control for variation in DNA content across different preparations, all results were related to the concentration of a housekeeping gene, in our case rat aldolase. Relative quantitation was performed according to the comparative ΔCt method. The difference between the mean Ctcytokine and the mean Ctaldolase was calculated to normalize for different amounts of DNA (ΔCt). The result for the cytokine gene expression was given by a unit-less value through the formula 2–ΔCt.
PCR reaction was performed in a final volume of 25 μL containing 1 μL cDNA, 12.5 μL Master Mix (TaqMan Universal PCR Master Mix), 200 nM hybridization probe, 50 to 900 nM of each primer, and 5.5 μL distilled water. After an initial step of 2 min at 50°C involving activation of uracil-N-glycosylase and degradation of any preexisting contaminating RNA sequences, a denaturation and a hot start for AmpliTaq Gold DNA polymerase were performed for 10 min at 95°C. The amplification took place in a two-step PCR (40 cycles; 15-sec denaturation step (95°C) and a 1-min annealing and extension step (60°C). The mean Ct values for aldolase and the cytokines were calculated from double determinations.
Statistical significance was ascertained using the Student t test, log-rank sum test, and Mann-Whitney test; results were expressed as mean±standard deviation and considered significant if the P value was less than 0.05. Functional, histologic, and molecular assessments were performed in a blinded fashion by the investigators.
Physiologic Changes After Induction of Donor Hypertension
Mean arterial blood pressures of donor animals increased within 2 weeks (150±10 mm Hg, P <0.05 vs. baseline, n=45) after renal artery clipping, stabilizing thereafter (170±11 mm Hg) at 4 weeks and (179±13 mm Hg, n=45) 10 weeks (Fig. 1). Control animals remained consistently normotensive (108±8 mm Hg at 10 weeks, not significant [NS], n=45). After transplantation of the contralateral kidney from the hypertensive donors, the systemic blood pressure of the recipients increased slightly (112±8 mm Hg, n=39, P <0.05 vs. controls at 24 weeks) (Fig. 2). Controls remained normotensive (102±3 mm Hg at 24 weeks, n=39) (Fig. 2).
No significant differences in survival of the animals undergoing transplantation were observed (log-rank sum P =NS, n=15).
Although proteinuria increased modestly over time in recipients of chronically rejecting allografts from normotensive donors, it became more obviously elevated in animals bearing grafts from hypertensive donors (Fig. 3, n=15). Levels of serum creatinine also increased progressively to higher values in recipients of allografts from hypertensive versus normotensive donors (at 24 weeks, creatinine=1.7±0.3 mg/dL vs. 1.2±0.2 mg/dL; P <0.05, n=27/group; at 32 weeks 2.4±0.4 mg/dL vs. 1.6±0.5 mg/dL, P <0.05, n=21/group).
At the time of transplantation, kidneys from donors that had been hypertensive for 10 weeks showed only mild hypertrophy and minimal endothelial vacuolization (Fig. 4, n=6). By 2 weeks after engraftment, however, injury to the grafts from hypertensive donors was increased in all compartments, with greater numbers of leukocytes (P <0.01), more intense perivascular and periglomerular fibrosis (P <0.01), focal and segmental proliferation, and glomerulosclerosis (P <0.01). Tubular inflammation with severe endothelial ballooning and medial vacuolization with focal intimal proliferation of arteries were obvious (n=6).
By 12 weeks, the infiltrates in the control allografts had largely resolved, leaving focal periglomerular collections. In contrast, cellular infiltration remained in the hypertensive donor allografts. Moderate glomerulosclerosis and interstitial fibrosis persisted (P <0.01). Medial fibrosis and intimal proliferation were evident in most muscular arteries (n=6). After 24 weeks, morphologic changes had progressed in control allografts to mild focal and segmental proliferation and glomerulosclerosis and focal mononuclear cell infiltrates. Comparable hypertensive donor kidneys showed more pronounced proliferation and glomerulosclerosis (P <0.01), moderate transplant arteriosclerosis, and marked interstitial fibrosis (P >0.01, n=6). By 32 weeks, control allografts showed patchy tubular atrophy, minor cell infiltrates, and mild focal and segmental glomerulosclerosis and fibrosis. In contrast, dense cortical infiltrates, interstitial fibrosis tubular atrophy, end-stage glomerulosclerosis (P <0.01), and severe transplant arteriosclerosis had developed in hypertensive donor kidneys (morphometry, analysis of variance, P <0.01, n=6).
Reverse Transcriptase-Polymerase Chain Reaction.
mRNA of representative proinflammatory cell products were present in hypertensive donor kidneys at the time of transplantation despite relatively minimal morphologic changes. The mediators remained significantly (P <0.05) up-regulated throughout the observation period (Fig. 5). Tumor necrosis factor-α and MIP-1α in particular were highly expressed around the time of transplantation and increased progressively over time. Kidneys from normotensive donors showed less activity (n=6/group/time point). IL-4 expression was not detectable before transplantation in kidneys of normotensive or hypertensive donors. The transcription for IL-2 was low in both hypertensive and normotensive donors, although it was expressed slightly higher in hypertensive kidneys (2.44−05±2.3−05 vs. 2.5−06±2.2−06-dCT, P =NS).
As the dichotomy between demand for transplantable organs and organ supply becomes ever greater, pressures to expand the donor pool acceptability grows; suboptimal donors are accepted and used increasingly despite less satisfactory early and late graft function and survival. It is widely accepted that chronic deterioration occurring in the months and years after transplantation is a result of interactions between antigen-dependent and antigen-independent events (2,11). Hypertension is both a donor- and recipient-associated risk factor for chronic renal graft deterioration as shown clinically and experimentally (12–15). The condition is common in chronic renal disease and occurs in more than 80% of patients with end-stage renal failure. In contrast with hypertension in transplant recipients, donor hypertension has received relatively little attention. The practical question posed in the present study is whether attendant sequelae of the donor condition substantially influence the quality of the donor organ, the ensuing response of the host to it, and its ultimate outcome.
Our experiments were designed to answer the question, if donor hypertension does influence long-term changes in kidney allografts by up-regulation of proinflammatory cytokines before transplantation, the extent and duration of donor hypertension was not considered in this study. Renal allografts from hypertensive donors experience accelerated functional and morphologic changes compared with organs from normotensive sources (8,10,16,17). In addition, donor hypertension “transferred” with the kidney induced slightly elevated blood pressure values in formerly normotensive recipients, a phenomenon also described in experimental models in which the kidney may carry a genetic defect (18). The sequelae of high renal perfusion pressure in the unprotected contralateral organ (as occurs in the clip model used in these studies) may be relevant in the initiation of slightly elevated blood pressure values in the recipient, which are elevated compared with the normotensive control groups but still within physiologic ranges of the examined rat strain. In our model, the minimum endothelial cell swelling and vacuolization in combination with the up-regulation of proinflammatory cytokines may be responsible for the observed change in recipients’ blood pressure because they may affect endothelial function that is necessary to sustain normotension. Because the increase in recipient blood pressure is moderate, it is likely that the resulting acceleration of chronic rejection is mainly caused by the altered immunologic organ quality before transplantation. It also seems obvious that after the occurrence of morphologic changes, as they are observed after long-lasting donor hypertension, the transfer of the hypertensive circulatory condition is a serious problem for the long-term function of the transplanted organ. Despite these clues, however, the precise mechanisms underlying induction of hypertension by renal transplantation from a hypertensive donor remains poorly understood. Most likely, angiotensin II-mediated mechanisms are responsible for the phenomenon.
We have demonstrated that chronic changes developing in kidney grafts from hypertensive donors are intensified and accelerated compared with those from normotensive controls. Functional deterioration correlated with progressive glomerulosclerosis and interstitial fibrosis. The inflammatory activation of the graft before transplantation, as ascertained by intrarenal cytokine mRNA expression, seems to have facilitated a more rapid and intense initial immune response by the recipient. The continuum between nonspecific injury in the donor grafts and subsequent increased specific alloresponsiveness has been shown in various experimental models (4,19,20). Renal damage induced by donor hypertension and then followed by a more intense immunologic response in the recipient supports this concept. However, the activation of the graft before transplantation may also increase its susceptibility to ischemia (21,22). The additional reperfusion injury can enhance acute rejection episodes with significant loss of functional graft tissue (21).
Donor organs experiencing early injury, such as after brain death or ischemia-reperfusion injury, show a common pattern of inflammation mediated by adhesion molecules, cytokines, and chemokines (23,24). These events are similar to those occurring in the model of donor hypertension used in these studies. The nonspecific injuries, regardless of cause, include expression of histocompatibility antigens within a few days. Polymorphonuclear leukocytes enter the graft after interaction with surface molecules on the vascular endothelium. Resultant cellular and molecular inflammatory changes persist in the grafts throughout the entire follow-up period, particularly those associated with macrophage activity such as transforming growth factor-β or MIP-1 α. These fibrosis-inducing factors are important during the evolution of chronic allograft rejection and emphasize the substantial role played by early antigen-independent injuries in the chronic process. This observation is supported by the presence of similar, albeit less intense, changes occurring in long-surviving isografts in which no host immunity is involved (25,26).
The importance of an initial, nonspecific insult on later organ dysfunction and fibrosis has also been associated with ischemia-reperfusion injury alone (21). This insult stimulates the rapid infiltration of host leukocyte populations and production of inflammatory mediators comparable to that in hypertensive donor organs before transplantation. Moreover, when such injured organs are followed over the long-term, their patterns of inflammation consistently resemble those in hypertensive donor organs, particularly the persistence of macrophages and fibrosis-inducing macrophage-associated products. The resultant increased immunogenicity of the organ caused by donor hypertension evokes a more powerful host immune response, increasing both acute and chronic injury. This assumption would explain the apparent correlation between donor risk factors and graft outcome after transplantation and may explain the more severe long-term deterioration of allografts from hypertensive donors. Because of the increased immunogenicity in morphologically normal-appearing organs from donors with borderline hypertension, these organs should be regarded as more susceptible to rejection and long-term changes.
Thus, it seems that the status of the donor organ should be viewed as a dynamic process that directly influences organ function after engraftment rather than as a static condition. The current studies emphasize that transplantable organs may be damaged by donor hypertension with consequences for the allograft recipient, comparable to events occurring in the context of donor brain death-induced graft dysfunction.
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