Walter D. Park

Transplant Glomerulopathy: Subclinical Incidence and Association with Alloantibody

Transplant glomerulopathy (TG) usually has been described as part of a constellation of late chronic histologic abnormalities associated with proteinuria and declining function. The current study used both protocol and clinically‐indicated biopsies to investigate clinical and subclinical TG, their prognosis and possible association with alloantibody. We retrospectively studied 582 renal transplants with a negative pre‐transplant T‐cell complement dependent cytotoxicity crossmatch. TG was diagnosed in 55 patients, 27 (49%) based on protocol biopsy in well‐functioning grafts. The cumulative incidence of TG increased over time to 20% at 5 years. The prognosis of subclinical TG was equally as poor as TG diagnosed with graft dysfunction, with progressive worsening of histopathologic changes and function. Although TG was associated with both acute and chronic histologic abnormalities, 14.5% of TG biopsies showed no interstitial fibrosis or tubular atrophy, while 58% (7/12) of biopsies with severe TG showed only minimal abnormalities. TG was associated with acute rejection, pretransplant hepatitis C antibody positivity and anti‐HLA antibodies (especially anti‐Class II), with the risk increasing if the antibodies were donor specific. We suggest that subclinical TG is an under‐recognized cause of antibody‐mediated, chronic renal allograft injury which may be mechanistically distinct from other causes of nephropathy.

Accommodation in ABO-incompatible kidney allografts, a novel mechanism of self-protection against antibody-mediated injury.

To elucidate the mechanism of self‐protection against anti‐donor blood‐group antibody known as accommodation, we studied 16 human ABO‐incompatible living‐donor kidney transplant recipients at 3 and 12 months post transplantation. Both circulating anti‐blood‐group antibody and the target blood‐group antigen in the graft were demonstrable in all patients after transplantation. Thirteen of 16 grafts had normal renal function and histology, while three grafts with prior humoral rejection demonstrated significant glomerulopathy and thus did not meet the criterion for accommodation. Using microarrays, we compared five 1‐year protocol ABO‐compatible renal graft biopsies to four accommodated ABO‐incompatible graft biopsies. Significant alterations in gene expression in 440 probe sets, including SMADs, protein tyrosine kinases, TNF‐α and Mucin 1 were identified. We verified these changes in gene expression using RT‐PCR and immunohistochemistry. Heme oxygenase‐1, Bcl‐2 and Bcl‐xl were not increased in ABO‐incompatible grafts at any time‐point. We conclude that accommodation is always present in well‐functioning, long‐surviving ABO‐incompatible kidney transplants. This self‐protection against antibody‐mediated damage may involve several novel mechanisms including the disruption of normal signal transduction, attenuation of cellular adhesion and the prevention of apoptosis.

Fibrosis with Inflammation at One Year Predicts Transplant Functional Decline

Lack of knowledge regarding specific causes for late loss of kidney transplants hampers improvements in long-term allograft survival. Kidney transplants with both interstitial fibrosis and subclinical inflammation but not fibrosis alone after 1 year have reduced survival. This study tested whether fibrosis with inflammation at 1 year associates with decline of renal function in a low-risk cohort and characterized the nature of the inflammation. We studied 151 living-donor, tacrolimus/mycophenolate-treated recipients without overt risk factors for reduced graft survival. Transplants with normal histology (n = 86) or fibrosis alone (n = 45) on 1-year protocol biopsy had stable renal function between 1 and 5 years, whereas those with both fibrosis and inflammation (n = 20) exhibited a decline in GFR and reduced graft survival. Immunohistochemistry confirmed increased interstitial T cells and macrophages/dendritic cells in the group with both fibrosis and inflammation, and there was increased expression of transcripts related to innate and cognate immunity. Pathway- and pathologic process-specific analyses of microarray profiles revealed that potentially damaging immunologic activities were enriched among the overexpressed transcripts (e.g., Toll-like receptor signaling, antigen presentation/dendritic cell maturation, IFN-γ-inducible response, cytotoxic T lymphocyte-associated and acute rejection-associated genes). Therefore, the combination of fibrosis and inflammation in 1-year protocol biopsies associates with reduced graft function and survival as well as a rejection-like gene expression signature, even among recipients with no clinical risk factors for poor outcomes. Early interventions aimed at altering rejection-like inflammation may improve long-term survival of kidney allografts.

Five‐Year Outcomes in Living Donor Kidney Transplants With a Positive Crossmatch

Renal transplant candidates with high levels of donor‐specific anti‐HLA antibodies have low transplantation rates and high mortality rates on dialysis. Using desensitization protocols, good short‐term outcomes are possible in “positive crossmatch kidney transplants (+XMKTx)”, but long‐term outcome data are lacking. The aim of the current study was to determine actual 5‐year graft outcomes of +XMKTx. We compared graft survival and the functional and histologic status of 102 +XMKTx to 204 −XMKTx matched for age and sex. Actual 5‐year death‐censored graft survival was lower in the +XMKTx group (70.7% vs. 88.0%, p < 0.01) and chronic injury (glomerulopathy) was present in 54.5% of surviving grafts. Graft survival was higher in recipients with antibody against donor class I only compared with antibody against class II (either alone or in combination with class I) (85.3% vs. 62.6%, p = 0.05) and was similar to −XMKTx (85.3 vs. 88.0%, p = 0.64). Renal function and proteinuria ranged across a wide spectrum in all groups reflecting the different histological findings at 5 years. We conclude that when compared to −XMKTx, +XMKTx have inferior outcomes at 5 years, however, almost half of the surviving grafts do not have glomerulopathy and avoiding antibodies against donor class II may improve outcomes.

The Banff 2015 Kidney Meeting Report: Current Challenges in Rejection Classification and Prospects for Adopting Molecular Pathology

The XIII Banff meeting, held in conjunction the Canadian Society of Transplantation in Vancouver, Canada, reviewed the clinical impact of updates of C4d‐negative antibody‐mediated rejection (ABMR) from the 2013 meeting, reports from active Banff Working Groups, the relationships of donor‐specific antibody tests (anti‐HLA and non‐HLA) with transplant histopathology, and questions of molecular transplant diagnostics. The use of transcriptome gene sets, their resultant diagnostic classifiers, or common key genes to supplement the diagnosis and classification of rejection requires further consensus agreement and validation in biopsies. Newly introduced concepts include the i‐IFTA score, comprising inflammation within areas of fibrosis and atrophy and acceptance of transplant arteriolopathy within the descriptions of chronic active T cell–mediated rejection (TCMR) or chronic ABMR. The pattern of mixed TCMR and ABMR was increasingly recognized. This report also includes improved definitions of TCMR and ABMR in pancreas transplants with specification of vascular lesions and prospects for defining a vascularized composite allograft rejection classification. The goal of the Banff process is ongoing integration of advances in histologic, serologic, and molecular diagnostic techniques to produce a consensus‐based reporting system that offers precise composite scores, accurate routine diagnostics, and applicability to next‐generation clinical trials.

The histology of solitary renal allografts at 1 and 5 years after transplantation

Previous studies suggest that the majority of renal allografts are affected by progressive, severe chronic histologic injury, yet studies using current protocols are lacking. The goal of this study was to examine the prevalence and progression of histologic changes using protocol allograft biopsies at 1 and 5 years after solitary kidney transplantation in patients transplanted between 1998 and 2004. Chronic histologic changes generally were mild at both 1 and 5 years and were similar in deceased and living donor kidneys. The overall prevalence of moderate or severe fibrosis was 13% (60/447) at 1 year and 17% (60/343) at 5 years. In a subgroup of 296 patients who underwent both 1‐ and 5‐year biopsies, mild fibrosis present at 1 year progressed to more severe forms at 5 years in 23% of allografts. The prevalence of moderate or severe arteriolar hyalinosis was similar in tacrolimus and calcineurin inhibitor‐free immunosuppression. These results in the recent era of transplantation demonstrate fewer, less severe and less progressive chronic histologic changes in the first 5 years after transplantation than previously reported.

Kidney Transplantation in Patients with Antibodies against Donor HLA Class II

The immunologic risk associated with donor‐specific antibodies (DSA) against Class II human leukocyte antigens (HLA) in kidney transplant (KTx) recipients is unclear. The aim of this study was to determine the outcome of KTx when DSA was detected only against HLA Class II. To isolate the impact of anti‐Class II DSA, we retrospectively analyzed 12 KTx recipients who at baseline had a positive B‐cell flow cytometric crossmatch (FXM) and a negative T‐cell FXM. Using alloantibody specification analysis, 58.3% (7/12) had DSA against donor Class II and 41.7% had no demonstrable DSA. Biopsy‐proven AMR occurred in 57% (4/7) in the Class II+ group and 0% in the Class II− group (p > 0.05). Peritubular capillaries stained positive for C4d in 86% (6/7) of the Class II+ patients and in 40% (2/5) of the Class II− patients (p > 0.05). One patient in the Class II+ group lost their graft at 3 months to accelerated transplant glomerulopathy, while all other grafts were functioning 3–37 months posttransplant despite the persistence of anti‐Class II DSA. We conclude that KTx recipients with clearly defined anti‐Class II DSA are at risk for humoral rejection suggesting that desensitization and/or close posttransplant monitoring may be needed to prevent AMR.

Acute Nephrotoxicity of Tacrolimus and Sirolimus in Renal Isografts: Differential Intragraft Expression of Transforming Growth Factor-β1 and α-smooth Muscle Actin

Background. Renal dysfunction early after kidney transplantation has multiple causes including ischemia-reperfusion (I/R) injury and drug-induced nephrotoxicity. This study assesses the acute nephrotoxicity of tacrolimus (Tac) and sirolimus (Sir) in a rat renal isograft model. Methods. Lewis renal isografts and uninephrectomized rats that did not undergo transplantation were treated with various doses of Tac (0.5–5.0 mg/kg/d) or Sir (0.5–6.5 mg/kg/d). Kidneys were examined on day 14 by routine histology and immunohistochemistry for transforming growth factor (TGF)-&bgr;1 and &agr;-smooth muscle actin (SMA). Results. Both Tac and Sir demonstrated evidence of nephrotoxicity in the early posttransplant period including increased serum creatinine and morphologic changes in the graft including interstitial inflammation, fibrosis, and tubular vacuolization. Nephrotoxicity was most prominent in the high-dose treatment groups for both drugs and was more severe in transplanted kidneys than in uninephrectomized animals that did not undergo transplantation, suggesting an additive effect of I/R injury and drug nephrotoxicity. Both Tac and Sir increased intragraft TGF-&bgr;1 and &agr;-SMA, but there were distinct differences in the patterns of TGF-&bgr;1 expression. Both demonstrated TGF-&bgr;1 in tubular epithelial cells, but Sir was associated with proximal tubular TGF-&bgr;1 localization in a bright granular pattern, whereas Tac was associated with diffuse distal tubular staining. Conclusions. Both Tac and Sir may be nephrotoxic in the early posttransplant period, especially at high doses and when combined with I/R injury. Immunohistochemical localization of TGF-&bgr;1 in the tubular cells was distinctly different with each drug, suggesting possible differences in the mechanism(s) of nephrotoxicity requiring further study.

Gene expression during acute allograft rejection: novel statistical analysis of microarray data.

High‐throughput microarrays promise a comprehensive analysis of complex biological processes, yet their applicability is hampered by problems of reproducibility and data management. The current study examines some of the major questions of microarray use in a well‐described model of allograft rejection. Using the Brown Norway to Lewis heterotopic heart transplant model, highly purified RNA was isolated from cardiac tissue at postoperative days (POD) 3, 5 and 7 and hybridized onto Affymetrix U34A microarrays. Using the log average ratio (LAR), changes in gene expression were monitored at each timepoint and p‐values generated through statistical analysis. Microarray data were verified for 13 significant transcripts using RT‐PCR. Of the 8800 transcripts studied, 2864 were increased on POD 3, 1418 on POD 5 and 2745 on POD 7. Verifying previous studies, many up‐regulated genes appeared to be associated with the inflammatory process and graft infiltrating cells. Down‐regulated transcripts included many novel molecules such as SC1 and decorin. LAR analysis provides a useful approach to analyze microarray data. Results were reproducible and correlated well with both RT‐PCR and prior studies. Most importantly, these results provide new insights into the pathogenesis of acute rejection and suggest new molecules for future studies.

Intragraft gene expression in positive crossmatch kidney allografts: ongoing inflammation mediates chronic antibody-mediated injury.

We studied intragraft gene expression profiles of positive crossmatch (+XM) kidney transplant recipients who develop transplant glomerulopathy (TG) and those who do not. Whole genome microarray analysis and quantitative rt‐PCR were performed on RNA from protocol renal allograft biopsies in three groups: (1) +XM/TG+ biopsies before and after TG; (2) +XM/NoTG; and (3) negative crossmatch kidney transplants (control). Microarray comparisons showed few differentially expressed genes between paired biopsies from +XM/TG+ recipients before and after the diagnosis of TG. Comparing +XM/TG+ and control groups, significantly altered expression was seen for 2447 genes (18%) and 3200 genes (24%) at early and late time points, respectively. Canonical pathway analyses of differentially expressed genes showed inflammatory genes associated with innate and adaptive immune responses. Comparing +XM/TG+ and +XM/NoTG groups, 3718 probe sets were differentially expressed but these were over‐represented in only four pathways. A classic accommodation phenotype was not identified. Using rt‐PCR, the expression of inflammatory genes was significantly increased in +XM/TG+ recipients compared to the +XM/NoTG and control groups. In conclusion, pretransplant donor‐specific anti‐HLA antibodies results in a gene expression profile characterized by inflammation and cellular infiltration and the majority of +XM grafts are exposed to chronic injury.