Beyond operational tolerance: effect of ischemic injury on development of chronic damage in renal grafts

BACKGROUND: The induction of operational tolerance is the holy grail of clinical transplantation. However, in animal models with operational tolerance, long- term grafts still develop chronic damage. The elucidation of the impact of allogenic versus nonallogeneic factors in such a model is important. This study examined the effect of a clinically relevant combination of warm ischemia and cold preservation in the absence of allogeneic response (isografts) and in the context of operational tolerance. METHODS: Dark Agouti (DA) rat kidneys were transplanted into DA recipients (isografts) or Albino Surgery recipients (allografts) tolerized by two transfusions of DA blood, under cover of cyclosporin A. Grafts were subjected to minimal cold preservation or to 30 mins warm ischemia followed by 24 hrs cold preservation. RESULTS: After an initial peak of renal dysfunction, serum creatinine concentration returned to normal in isografts and nonischemic allografts, but remained significantly elevated in ischemic allografts (P<0.0002) throughout 6 months follow-up. Both allograft groups developed proteinuria. At 6 months, ischemic isografts and nonischemic allografts demonstrated very mild tubular atrophy and interstitial fibrosis. Tubulointerstitial injury was significantly more severe in ischemic allografts (P<0.01 vs. nonischemic allografts) and was associated with increased infiltrating monocyte/macrophages and NK cells (P<0.05). Moderate glomerulosclerosis was a feature of both allograft groups (P<0.05). CONCLUSIONS: The modified allogeneic response in operationally tolerant recipients acts in synergy with ischemia/reperfusion injury in the development of chronic damage. Strategies to limit or modify the initial ischemia/reperfusion injury may ameliorate chronic tubulointerstitial damage. Progressive glomerular damage and proteinuria in allografts may require other pharmacological intervention.     

Early and late injury to renal transplants from non-heart-beating donors

BACKGROUND: The lack of adequate numbers of kidneys for transplantation has stimulated interest in the use of organs from non-heart-beating donors (NHBDs). The short- and long-term effects of this risk factor on kidney isografts and allografts were examined with a rat model. METHODS: NHBDs were killed by ether overdose. Kidney isografts (male Lewis rats [LEW]-->LEW) were transplanted orthotopically into bilaterally nephrectomized recipients 15, 30, 45, and 90 min after asystole to determine short-term survival patterns, which were compared to those of rats bearing kidneys from living donors (LDs, 0 min). Isografts and allografts (Fisher 344 rats-->LEW) from 45-min and 105-min NHBDs and from LD controls were placed in additional recipients in which contralateral native nephrectomy was performed on day 10 to allow the injured graft to recover from its ischemic insult. Serum creatinine, proteinuria, and graft morphology were assessed serially over a 24-week follow-up period. RESULTS: Early survival and renal dysfunction of isografted rats correlated with the interval of donor cardiac arrest before transplantation. Long-term survival of recipients of kidneys from LDs and between 45-min and 105-min NHBDs was also significantly different (100% vs. 87% vs. 37% at 24 weeks, respectively, P<0.03). Proteinuria increased progressively over time, proportionate to the period of donor asystole, and was associated with increasing cellular infiltration, tubular atrophy, and glomerulosclerosis in the grafts. The development of important functional and structural changes was intensified in NHBD allografts. LD allografts showed early changes of chronic rejection. CONCLUSIONS: The results emphasize the continuum between an initial nonspecific, donor-associated renal injury and late functional and morphologic changes associated with the interval of donor cardiac arrest. These events are accelerated in NHBD allografts that experience the added insult of host alloreactivity.     

Long-term kidney isografts develop functional and morphologic changes that mimic those of chronic allograft rejection.

OBJECTIVE: This study examined antigen-independent factors in the pathogenesis of chronic rejection of organ transplants. SUMMARY BACKGROUND DATA: In addition to alloantigen-dependent events, antigen-independent factors can influence chronic rejection of organ allografts. Initial injury, including early ischemia and acute rejection, may contribute. METHODS: Kidney isografts were transplanted orthotopically into bilaterally nephrectomized rat recipients and studied functionally, morphologically and immunohistologically, at serial intervals up to 72 weeks after transplantation. Controls included chronically rejecting kidney allografts using a well-established model, non-nephrectomized and uninephrectomized animals with a native kidney that had undergone initial ischemia and uninephrectomized rats whose remaining kidney had been manipulated operatively. RESULTS: Allograft recipients developed progressive proteinuria after 12 weeks, with gradual renal failure ultimately leading to death. At the same time, morphologic changes, including progressive arteriosclerosis and glomerulosclerosis, tubular atrophy, and interstitial fibrosis, developed. Immunohistologically, macrophages infiltrated glomeruli during this period and cytokines became upregulated. Comparable changes occurred in isografts, but later, beginning after week 24 and progressing thereafter. The single ischemic kidney in uninephrectomized controls also developed the same lesions; no comparable changes were noted in other control kidneys. CONCLUSIONS: Antigen-independent functional and morphologic changes occur in long-term kidney isografts that resemble those appearing considerably earlier in allografts that reject chronically. Initial injury and extent of functioning renal mass may be important factors for such late changes.     

Late graft dysfunction after prolonged cold ischemia of the donor kidney: inhibition by cyclosporine.

BACKGROUND: The present study was devised to elucidate the influence of prolonged cold ischemia on the development of chronic transplant dysfunction (CTD) in kidney isografts (Brown Norway-->Brown Norway; BN-->BN) and in kidney allografts (BN-->Wistar Agouti/ Rij [WAG]) under temporary cyclosporine (CsA) therapy. METHODS: To induce ischemic injury, BN donor kidneys were preserved for 24 hr in 4 degrees C University of Wisconsin solution before transplantation. Renal function (proteinuria), histomorphology according to the BANFF criteria for CTD, and infiltrating cells were assessed. Grafts were examined both early at days 2, 3, 6, and 10, and late at week 26 (allografts) or at week 52 (isografts). RESULTS: Nonischemic isografts preserved a normal function and morphology. Ischemic isografts developed a progressive proteinuria over time and demonstrated significantly more glomerulopathy with macrophage (Me) infiltration and intimal hyperplasia than nonischemic controls at week 52. During the initial 10 days, there was an increased infiltration of MHC class II+ cells, predominantly CD4+ cells and Mphi, coinciding with up-regulated intercellular adhesion molecule-1 expression. CsA treatment in ischemic isografts inhibited infiltration of MHC II+ cells in the early stage, which was accompanied by significantly less renal damage at week 52 compared with untreated controls (proteinuria: 59+/-8 vs. 134+/-19 mg/24 hr; BANFF score: 2.8+/-0.4 vs. 4.3+/-1.0). Under CsA therapy, 24-hr cold ischemia of the allograft affected neither the onset or progress of proteinuria, nor the histomorphology (BANFF score: 7.8+/-2.4 vs. 7.3+/-1.9). In both ischemic and nonischemic allografts, intercellular adhesion molecule-1 expression and mononuclear cell infiltration (CD4, CD8, Mphi was abundantly present during the first 10 days and function deteriorated rapidly. CONCLUSIONS: Prolonged cold ischemia plays a role in the induction of CTD, but its deleterious effect can be successfully inhibited by CsA. Therefore, the alloantigeneic stimulus is the overriding component in the multifactorial pathogenesis of CTD.     

Cold ischemic injury,aortic allograft vasculopathy,and pro-inflammatory cytokine expression

BACKGROUND: The aim of this study was to understand the role of ischemic preservation injury and pro-inflammatory cytokine expression in the progression of allograft vasculopathy. METHODS: Using the rat aortic transplant model, grafts were stored at 4 degrees C for either 1 or 24 h. Graft vasculopathy was assessed at 4 and 8 weeks after transplantation. Intra-graft cytokine expression was measured at days 1, 3 and, 7 after transplantation. RESULTS: At 4 weeks, intimal hyperplasia of allografts was greater than isografts (P<0.05). At 8 weeks, all groups had an increase in graft vascular disease compared to the 4-week groups (P<0.05). Allografts preserved for 24 h displayed a greater degree of vessel-wall reaction than both isograft groups and allografts stored for 1 h (P<0.05). An increased expression of the cytokines, TNF-alpha, TGF-beta, IL-2, INF-gamma, IL-1, and IL-6 was noted in the allografts stored for 24 h compared to similarly treated isografts (P<0.05). CONCLUSIONS: Prolonged ischemic preservation injury induced vascular disease in both isografts and allografts. The vessel wall reaction increased over time and was greater in allografts than isografts. The enhanced expression of T cell- and macrophage associated cytokines in allografts compared to isografts, suggested that early pro-inflammatory cytokine expression played an important role in progression of allograft vasculopathy.     

Effects of Donor Age and Cell Senescence on Kidney Allograft Survival

The biological processes responsible for somatic cell senescence contribute to organ aging and progression of chronic diseases, and this may contribute to kidney transplant outcomes. We examined the effect of pre-existing donor aging on the performance of kidney transplants, comparing mouse kidney isografts and allografts from old versus young donors. Before transplantation, old kidneys were histologically normal, but displayed an increased expression of senescence marker p16 ^INK4a . Old allografts at day 7 showed a more rapid emergence of epithelial changes and a further increase in the expression of p16 ^INK4a . Similar but much milder changes occurred in old isografts. These changes were absent in young allografts at day 7, but emerged by day 21. The expression of p16 ^INK4a remained low in young kidney allografts at day 7, but increased with severe rejection at day 21. Isografts from young donors showed no epithelial changes and no increase in p16 ^INK4a . The measurements of the alloimmune response—infiltrate, cytology, expression of perforin, granzyme B, IFN-γ and MHC—were not increased in old allografts. Thus, old donor kidneys display abnormal parenchymal susceptibility to transplant stresses and enhanced induction of senescence marker p16 ^INK4a , but were not more immunogenic. These data are compatible with a key role of somatic cell senescence mechanisms in kidney transplant outcomes by contributing to donor aging, being accelerated by transplant stresses, and imposing limits on the capacity of the tissue to proliferate.     

Cold ischemia augments allogeneic-mediated injury in rat kidney allografts

BACKGROUND: Some clinical studies demonstrate that kidney grafts with prolonged cold ischemia experience early acute rejection more often than those with minimal ischemia. The mechanism, however, is putative. Therefore, the aim of this study was to unravel the impact of ischemia on the immune response in rat kidney allografts compared with that in isografts. METHODS: To induce ischemic injury, donor kidneys were preserved for 24 hours in 4 degrees C University of Wisconsin solution before transplantation. No immunosuppression was administered. The histomorphology according to the BANFF criteria for acute rejection and infiltrating cells were assessed at days 1, 2, 3, 4, 6, and 8 post-transplantation. RESULTS: In allografts, exposure of the kidney to ischemia led to a significantly earlier onset of interstitial cell infiltration and tubulitis compared with nonischemic allografts. The BANFF score of interstitial cell infiltration was 1 +/- 0 vs. 0.25 +/- 0.29 at day 3 and 2 +/- 0 vs. 1.25 +/- 0.25 at day 4. In contrast, in isografts, the effect of ischemia on the histology was not significant. From day 6, the histologic differences between ischemic and nonischemic grafts disappeared. Ischemia led to a more intense expression of P-selectin (day 1), intercellular adhesion molecule-1 (ICAM-1; day 2), and major histocompatibility complex (MHC) class II on endothelium and proximal tubular cells (day 2) in both allografts and isografts. Concurrently with the up-regulated ICAM-1 and MHC expression, significantly more CD4(+) cells and macrophages infiltrated the ischemic allografts at days 2 and 3 and the ischemic isografts at day 4. Importantly, the influx of these cells after ischemia was significantly greater in allografts than in isografts. CONCLUSIONS: Cold ischemia augments allogeneic-mediated cell infiltration in rat kidney allografts. The earlier onset of acute rejection in 24-hour cold preserved allografts may be prevented by better preservation or treatment using tailored immunosuppression.     

Time course and cellular localization of inducible nitric oxide synthases expression during cardiac allograft rejection

BACKGROUND: We have demonstrated that inhibition of inducible nitric oxide synthase (NOS) ameliorated acute cardiac allograft rejection. This study determined the time course and cellular localization of inducible NOS expression during the histologic progression of unmodified acute rat cardiac allograft rejection. METHODS: Tissue from syngeneic (ACI to ACI) and allogeneic (Lewis to ACI) transplants were harvested on postoperative days 3 through 10 and analyzed for inducible NOS mRNA expression (ribonuclease protection assay), inducible NOS enzyme activity (conversion of L-[3H]arginine to nitric oxide and L-[3H]citrulline), and nitric oxide production (serum nitrite/nitrate levels). Inducible NOS mRNA and protein expression were localized using in situ hybridization and immunohistochemistry. RESULTS: Inducible NOS mRNA and enzyme activity were expressed in allografts during mild, moderate, and severe acute rejection (postoperative days 4 through 10), but were not detected in normals, isografts, or allografts before histologic changes of mild acute rejection (postoperative day 3). Inducible NOS expression resulted in increased serum nitrite/nitrate levels during mild and moderate rejection (postoperative days 4 through 6). Inducible NOS mRNA and protein expression localized to infiltrating mononuclear inflammatory cells in allograft tissue sections during all stages of rejection but were not detected in allograft parenchymal cells or in normals or isografts. CONCLUSIONS: Inducible NOS expression and increased nitric oxide production occurred during the early stages of acute rejection, persisted throughout the unmodified rejection process, and localized to infiltrating inflammatory cells but not allograft parenchymal cells during all stages of acute rejection.     

Respiratory epithelial expression of integrin alphaVbeta6 in chronic progressive allograft rejection.

BACKGROUND: Obliterative bronchiolitis (OB) is the major cause of morbidity and mortality after lung transplantation. One initiating event in the development of obliteration of the airway lumen is epithelial injury. In our model of chronic rejection, initial ischemic injury and denudation of the epithelium occurs in the isografts, with eventual re-epithelialization and partial patency of the airway lumen. In contrast, allografts do not recover epithelium, and the airway lumen becomes obliterated. We hypothesized that because integrin alphaVbeta6 is expressed in healing epithelium, integrin alphaVbeta6 expression would be greatly increased in isografts, but not in allografts. METHODS: Using a rat tracheal allograft rejection model as a source of 4- to 5-microm tissue sections, we compared integrin staining in allografts vs isografts from animals at post-transplant Days 7, 14, 28, and 60. We analyzed the sections using immunohistochemistry after incubation with a specific monoclonal antibody E7P6 against integrin alphaVbeta6. Negative control slides were processed identically, except that primary antibody was omitted. RESULTS: The sections from healing, re-epithelializing isografts showed intense staining when using the antibody recognizing integrin alphaVbeta6, compared with the allografts studied. Days 7 and 14 isografts had increased epithelial expression of alphaVbeta6. As the isograft epithelium recovered, the intensity diminished at Days 28 and 60. In allografts, at Days 7 to 60, we detected only a comparatively low-level of expression in injured epithelium. CONCLUSIONS: Integrin alphaVbeta6 is readily detectable in healing isografts. Integrin alphaVbeta6 may be crucial in maintaining a viable epithelial cell layer, which is related to slowed progression of airway obliteration in OB.     

Discovering novel injury features in kidney transplant biopsies associated with TCMR and donor aging

We previously characterized the molecular changes in acute kidney injury (AKI) and chronic kidney disease (CKD) in kidney transplant biopsies, but parenchymal changes selective for specific types of injury could be missed by such analyses. The present study searched for injury changes beyond AKI and CKD related to specific scenarios, including correlations with donor age. We defined injury using previously defined gene sets and classifiers and used principal component analysis to discover new injury dimensions. As expected, Dimension 1 distinguished normal vs. injury, and Dimension 2 separated early AKI from late CKD, correlating with time posttransplant. However, Dimension 3 was novel, distinguishing a set of genes related to epithelial polarity (e.g., PARD3) that were increased in early AKI and decreased in T cell–mediated rejection (TCMR) but not in antibody-mediated rejection. Dimension 3 was increased in kidneys from older donors and was particularly important in survival of early kidneys. Thus high Dimension 3 scores emerge as a previously unknown element in the kidney response-to-injury that affects epithelial polarity genes and is increased in AKI but depressed in TCMR, indicating that in addition to general injury elements, certain injury elements are selective for specific pathologic mechanisms. (ClinicalTrials.gov NCT01299168).