Stefan G. Tullius

Identification of a microRNA signature of renal ischemia reperfusion injury

Renal ischemia reperfusion injury (IRI) is associated with significant morbidity and mortality. Given the importance of microRNAs (miRNAs) in regulating gene expression, we examined expression profiles of miRNAs following renal IRI. Global miRNA expression profiling on samples prepared from the kidneys of C57BL/6 mice that underwent unilateral warm ischemia revealed nine miRNAs (miR-21, miR-20a, miR-146a, miR-199a-3p, miR-214, miR-192, miR-187, miR-805, and miR-194) that are differentially expressed following IRI when compared with sham controls. These miRNAs were also differently expressed following IRI in immunodeficient RAG-2/common γ-chain double-knockout mice, suggesting that the changes in expression observed are not significantly influenced by lymphocyte infiltration and therefore define a lymphocyte-independent signature of renal IRI. In vitro studies revealed that miR-21 is expressed in proliferating tubular epithelial cells (TEC) and up-regulated by both cell-intrinsic and -extrinsic mechanisms resulting from ischemia and TGF-β signaling, respectively. In vitro, knockdown of miR-21 in TEC resulted in increased cell death, whereas overexpression prevented cell death. However, overexpression of miR-21 alone was not sufficient to prevent TEC death following ischemia. Our findings therefore define a molecular fingerprint of renal injury and suggest miR-21 may play a role in protecting TEC from death.

Cytokines, adhesion molecules, and the pathogenesis of chronic rejection of rat renal allografts

Little is known of the host immune mechanisms responsible for initiation and progression of chronic rejection. We describe immunopathologic features associated with progressively deteriorating function of kidney allografts in the F344-to-Lewis rat strain combination, which differ at MHC and non-MHC loci. Initial rejection in untreated recipients was controlled by a brief course of CsA (5 mg/kg/day, for 10 days), resulting in >80% of recipients surviving up to a year despite declining renal function. In contrast to controls (isografts placed in untreated or CsA-treated Lewis rats), allografts from 12–16 weeks post-Tx showed segmental or global glomerulosclerosis, increasing tubular atrophy, interstitial fibrosis, and intimal proliferation leading ultimately to vascular occlusion. By flow cytometry, IgM and IgG alloantibodies peaked at 2–4 weeks, with a gradual decline to baseline thereafter. Immunohistology showed early and progressive deposition of IgM, IgG, C3, and fibrin in vessel walls and glomeruli. In addition, by 12 weeks, extensive infiltration by activated (IL-2R+) macrophages and CD4+ T cells were noted in glomeruli and blood vessels, in conjunction with staining for the cytokines TNF-α, IL-1, and IL-6. The persistent and dense intraglomerular expression of IL-6 was of particular interest, given its potent mitogenic effects for mesangial cells in vitro, and suggests a role for this cytokine as a mediator of mesangial expansion, advanced glomerular injury, and glomerulosclerosis in chronic rejection. Parallel timing of IL-6 and TNF-α expression was shown in serum samples by ELISA and bioassays. In vitro binding studies showed increased binding of naive host lymphocytes to allograft versus isografts, correlating with upregulation (peaking at week 16) of intercellular adhesion molecule-1 expression by graft endothelium. We conclude that cytokine production and upregulation of adhesion molecules occurring as part of a cellular immune response may be as important, to the etiology of chronic rejection as the hitherto widely emphasized antibody-mediated host responses.

Brain Death Activates Donor Organs and Is Associated with a Worse I/R Injury After Liver Transplantation

The majority of transplants are derived from donors who suffered from brain injury. There is evidence that brain death causes inflammatory changes in the donor. To define the impact of brain death, we evaluated the gene expression of cytokines in human brain dead and ideal living donors and compared these data to organ function following transplantation.

Three Patients with Full Facial Transplantation

Unlike conventional reconstruction, facial transplantation seeks to correct severe deformities in a single operation. We report on three patients who received full-face transplants at our institution in 2011 in operations that aimed for functional restoration by coaptation of all main available motor and sensory nerves. We enumerate the technical challenges and postoperative complications and their management, including single episodes of acute rejection in two patients. At 6 months of follow-up, all facial allografts were surviving, facial appearance and function were improved, and glucocorticoids were successfully withdrawn in all patients.

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

ObjectiveThis study examined antigen-independent factors in the pathogenesis of chronic rejection of organ transplants. Summary Background DataIn 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. MethodsKidney 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. ResultsAllograft 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. ConclusionsAntigen-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.

Methylprednisolone therapy in deceased donors reduces inflammation in the donor liver and improves outcome after liver transplantation: a prospective randomized controlled trial.

Objective:To investigate potential beneficial effects of donor treatment with methylprednisolone on organ function and outcome after liver transplantation. Summary Background Data:It is proven experimentally and clinically that the brain death of the donor leads to increased levels of inflammatory cytokines and is followed by an intensified ischemia/reperfusion injury after organ transplantation. In experiments, donor treatment with steroids successfully diminished these effects and led to better organ function after transplantation. Methods:To investigate whether methylprednisolone treatment of the deceased donor is applicable to attenuate brain death-associated damage in clinical liver transplantation we conducted a prospective randomized treatment-versus-control study in 100 deceased donors. Donor treatment (n = 50) consisted of 250 mg methylprednisolone at the time of consent for organ donation and a subsequent infusion of 100 mg/h until recovery of organs. A liver biopsy was taken immediately after laparotomy and blood samples were obtained after brain death diagnosis and before organ recovery. Cytokines were assessed by real-time reverse transcriptase-polymerase chain reaction. Soluble serum cytokines were measured by cytometric bead array system. Results:After methylprednisolone treatment, steroid plasma levels were significantly higher (P < 0.05), and a significant decrease in soluble interleukins, monocyte chemotactic protein-1, interleukin-2, interleukin-6, tumor necrosis factor-&agr;, and inducible protein-10 was observed. Methylprednisolone treatment resulted in a significant downregulation of intercellular adhesion molecule-1, tumor necrosis factor-&agr;, major histocompatibility complex class II, Fas-ligand, inducible protein-10, and CD68 intragraft mRNA expression. Significantly ameliorated ischemia/reperfusion injury in the posttransplant course was accompanied by a decreased incidence of acute rejection. Conclusions:Our present study verifies the protective effect of methylprednisolone treatment in deceased donor liver transplantation, suggesting it as a potential therapeutical approach.

Enzyme-linked immunosorbent spot assay for donor-reactive interferon-gamma-producing cells identifies T-cell presensitization and correlates with graft function at 6 and 12 months in renal-transplant recipients.

Background. A major goal in clinical transplantation is the individualization of immunosuppression. This requires a definition of markers that identify patients at heightened risk of acute rejection and immune-mediated chronic allograft nephropathy. Methods. Frequencies of interferon–&ggr;-producing donor-reactive cells were serially determined in unselected renal-transplant patients in an enzyme-linked immunosorbent spot assay (ELISPOT) before transplantation (n=42) and up to 10 (mean 5.0) times during the first 6 months posttransplantation (n=48) to determine detailed kinetics and analyze for correlation with acute rejection and graft function at 6 and 12 months posttransplantation. Results. Pretransplant ELISPOT frequencies were significantly higher in patients with acute rejection (16/42) versus nonrejecters (26/42). Highly elevated pretransplant frequencies (>200 spots/300,000 peripheral blood mononuclear cells [PBMCs], n=5/42) were associated with a risk of severe acute rejection episodes but were independent of risk factors such as high panel reactive antibodies. Early graft failure exclusively occurred in this group. Importantly, mean ELISPOT frequencies at weeks 2 and 3 but not at month 6 posttransplant correlated inversely with 6 and 12 months glomerular filtration rate. The correlation between ELISPOT frequencies and renal function showed the highest significance in patients without acute rejection. Conclusions. The pretransplant ELISPOT assay might be useful to identify T-cell presensitized patients, who are at heightened risk for severe early acute rejection. An analysis of ELISPOT donor-reactive cells during the early posttransplant period might allow an identification of patients at risk for immune-mediated graft deterioration.

Reversibility of chronic renal allograft rejection. Critical effect of time after transplantation suggests both host immune dependent and independent phases of progressive injury.

The characteristic and progressive morphological changes of glomerulosclerosis, interstitial fibrosis, and vascular obliteration that occur in renal allografts experiencing chronic rejection correlate directly with declining function and eventual graft loss. Using an established rat transplant model of chronic rejection where such changes occur in predictable sequence, allografts were retransplanted back into the donor strain at serial intervals after the initial engraftment to determine at what stage of development the lesions could still be reversed by removing the continuing immunological drive of the host. Morphological changes were compared with those in retransplanted isografts and nonretransplanted allografts at comparable time intervals. Histological and immunohistological characteristics of chronic rejection were reversible by retransplantation of the kidneys into the donor strain within 12 weeks after their original placement. Retransplantation at or later than week 12 could not reverse the intense humoral and cellular immune responses, or the structural changes (particularly fibrosis) that developed after that period. However, the sparse but inevitably progressing cellular infiltration and cytokine expression in retransplanted and nonretransplanted isograft controls suggest the persistent influence of alloantigen-independent factors in addition to those of host immunity. Thus, the early stages of chronic rejection are alloantigen dependent and reversible, whereas the later changes were irreversible and alloantigen-independent factors appeared increasingly important.

Effects of RS61443 on functional and morphological changes in chronically rejecting rat kidney allografts

No immunosuppression agent is as yet available that prevents the process of chronic allograft rejection, the most critical cause of late organ allograft loss. RS61443 (mycophenolate mofetil) inhibits de novo DNA synthesis as well as diminishes expression of cell surface molecules and antibody production. As these factors seem important in the pathophysiology of the chronic phenomenon, we investigated the effects of the agent in an established model of chronic rejection of kidney allografts in a F344-to-Lewis rat strain combination. All recipients were treated for the first 10 days after engraftment with low-dose cyclosporine (1.5 mg/kg/day) to reverse an initial acute rejection episode. Since functional and morphological changes do not become manifest in this model until after 12 wk, treatment with RS61443 (15 mg/kg/day, p.o.) was either initiated at the day of grafting (Gp 1) or 8 wks thereafter (Gp 2), and continued throughout the follow-up period. Non-RS61443-treated allografted rats receiving vehicle only (Gp 3) developed progressive proteinuria after 12 wk. Peak cellular infiltration (particularly macrophages in glomeruli and perivascular areas) at 16 wk was associated with densely expressed adhesion molecules (ICAM-1 on endothelium), cytokines and growth factors (TNF-alpha and TGF-beta in glomeruli and PDGF on arterial smooth muscle cells). Interstitial fibrosis, with tubular atrophy, glomerulosclerosis, and varying degrees of intimal proliferation and luminal obliteration of vessels, progressed thereafter. In vitro binding of MNC from naive animals to chronically rejecting allografted kidneys generally confirmed the immunohistological observations, peaking at 12 wk; this binding was significantly inhibited by mAbs against specific adhesion molecules (CD11a, CD18, and ICAM-1). Serum-allospecific IgG and IgM peaked at 1-2 wk after engraftment in the control recipients, decreasing thereafter. Although IgM declined to baseline after 12 wk, low levels of allospecific IgG persisted throughout the follow-up period. In contrast, recipient treatment with RS61443 (both Gp 1 and Gp 2) allowed the allografts to function normally throughout follow-up period. Proteinuria was virtually absent, and morphological and immunohistological manifestations of the chronic process were markedly diminished. In addition, treated recipients developed no significant side effects, including leukopenia, anemia, thrombopenia, nephrotoxicity, and hepatotoxicity. It appears that this agent can safely prevent the changes of chronic rejection of kidney allografts in this rat model.

Old-for-Old Kidney Allocation Allows Successful Expansion of the Donor and Recipient Pool

Allocation of kidneys from donors older than 64 years to recipients older than 64 years was started in 1999 to improve use of older donor kidneys. Kidneys are allocated locally without HLA‐matching to keep cold ischemia short.