Evaluation of C1q Status and Titer of De Novo Donor-Specific Antibodies as Predictors of Allograft Survival.

De novo donor-specific antibodies (dnDSAs) that develop after renal transplantation are independent predictors of allograft loss. However, it is unknown if dnDSA C1q status or titer at the time of first detection can independently predict allograft loss. In a consecutive cohort of 508 renal transplant recipients, 70 developed dnDSAs. Histologic and clinical outcomes were correlated with the C1q assay or dnDSA titer. C1q positivity correlated with dnDSA titer (p < 0.01) and mean fluorescence intensity (p < 0.01) and was more common in class II versus class I dnDSAs (p < 0.01). C1q status correlated with tubulitis (p = 0.02) and C4d status (p = 0.03) in biopsies at the time of dnDSA development, but not T cell-mediated rejection (TCMR) or antibody-mediated rejection (ABMR). De novo DSA titer correlated with Banff g, i, t, ptc, C4d scores, TCMR (p < 0.01) and ABMR (p < 0.01). Post-dnDSA graft loss was observed more frequently in recipients with C1q-positve dnDSA (p < 0.01) or dnDSA titer ≥ 1:1024 (p ≤ 0.01). However, after adjustment for clinical phenotype and nonadherence in multivariate models, neither C1q status nor dnDSA titer were independently associated with allograft loss, questioning the utility of these assays at the time of dnDSA development.

Trabecular bone score in kidney transplant recipients.

SUMMARY: It is uncertain whether bone mineral density (BMD) can accurately predict fracture in kidney transplant recipients. Trabecular bone score (TBS) provides information independent of BMD. Kidney transplant recipients had abnormal bone texture as measured by lumbar spine TBS, and a lower TBS was associated with incident fractures in recipients. INTRODUCTION: Trabecular bone score (TBS) is a texture measure derived from dual energy X-ray absorptiometry (DXA) lumbar spine images, providing information independent of bone mineral density. We assessed characteristics associated with TBS and fracture outcomes in kidney transplant recipients. METHODS: We included 327 kidney transplant recipients from Manitoba, Canada, who received a post-transplant DXA (median 106 days post-transplant). We matched each kidney transplant recipient (mean age 45 years, 39% men) to three controls from the general population (matched on age, sex, and DXA date). Lumbar spine (L1-L4) DXA images were used to derive TBS. Non-traumatic incident fracture (excluding hand, foot, and craniofacial) (n = 31) was assessed during a mean follow-up of 6.6 years. We used multivariable linear regression models to test predictors of TBS, and multivariable Cox proportional hazard regression was used to estimate hazard ratios (HRs) per standard deviation decrease in TBS to express the gradient of risk. RESULTS: Compared to the general population, kidney transplant recipients had a significantly lower lumbar spine TBS (1.365 ± 0.129 versus 1.406 ± 0.125, P < 0.001). Multivariable linear regression revealed that receipt of a kidney transplant was associated with a significantly lower mean TBS compared to controls (-0.0369, 95% confidence interval [95% CI] -0.0537 to -0.0202). TBS was associated with fractures independent of the Fracture Risk Assessment score including BMD (adjusted HR per standard deviation decrease in TBS 1.64, 95% CI 1.15-2.36). CONCLUSION: Kidney transplant recipients had abnormal bone texture as assessed by TBS and a lower lumbar spine TBS was associated with fractures in recipients.

Rates and determinants of progression to graft failure in kidney allograft recipients with de novo donor-specific antibody.

Understanding rates and determinants of clinical pathologic progression for recipients with de novo donor-specific antibody (dnDSA), especially subclinical dnDSA, may identify surrogate endpoints and inform clinical trial design. A consecutive cohort of 508 renal transplant recipients (n = 64 with dnDSA) was studied. Recipients (n = 388) without dnDSA or dysfunction had an eGFR decline of -0.65 mL/min/1.73 m(2) /year. In recipients with dnDSA, the rate eGFR decline was significantly increased prior to dnDSA onset (-2.89 vs. -0.65 mL/min/1.73 m(2) /year, p < 0.0001) and accelerated post-dnDSA (-3.63 vs. -2.89 mL/min/1.73 m(2) /year, p < 0.0001), suggesting that dnDSA is both a marker and contributor to ongoing alloimmunity. Time to 50% post-dnDSA graft loss was longer in recipients with subclinical versus a clinical dnDSA phenotype (8.3 vs. 3.3 years, p < 0.0001). Analysis of 1091 allograft biopsies found that dnDSA and time independently predicted chronic glomerulopathy (cg), but not interstitial fibrosis and tubular atrophy (IFTA). Early T cell-mediated rejection, nonadherence, and time were multivariate predictors of IFTA. Independent risk factors for post-dnDSA graft survival available prior to, or at the time of, dnDSA detection were delayed graft function, nonadherence, dnDSA mean fluorescence intensity sum score, tubulitis, and cg. Ultimately, dnDSA is part of a continuum of mixed alloimmune-mediated injury, which requires solutions targeting T and B cells.

Class II HLA epitope matching-A strategy to minimize de novo donor-specific antibody development and improve outcomes.

De novo donor-specific antibody (dnDSA) develops in 15-25% of renal transplant recipients within 5 years of transplantation and is associated with 40% lower graft survival at 10 years. HLA epitope matching is a novel strategy that may minimize dnDSA development. HLAMatchmaker software was used to characterize epitope mismatches at 395 potential HLA-DR/DQ/DP conformational epitopes for 286 donor-recipient pairs. Epitope specificities were assigned using single antigen HLA bead analysis and correlated with known monoclonal alloantibody epitope targets. Locus-specific epitope mismatches were more numerous in patients who developed HLA-DR dnDSA alone (21.4 vs. 13.2, p < 0.02) or HLA-DQ dnDSA alone (27.5 vs. 17.3, p < 0.001). An optimal threshold for epitope mismatches (10 for HLA-DR, 17 for HLA-DQ) was defined that was associated with minimal development of Class II dnDSA. Applying these thresholds, zero and 2.7% of patients developed dnDSA against HLA-DR and HLA-DQ, respectively, after a median of 6.9 years. Epitope specificity analysis revealed that 3 HLA-DR and 3 HLA-DQ epitopes were independent multivariate predictors of Class II dnDSA. HLA-DR and DQ epitope matching outperforms traditional low-resolution antigen-based matching and has the potential to minimize the risk of de novo Class II DSA development, thereby improving long-term graft outcome.

Evolution and clinical pathologic correlations of de novo donor-specific HLA antibody post kidney transplant.

The natural history for patients with de novo donor-specific antibodies (dnDSA) and the risk factors for its development have not been well defined. Furthermore, clinical and histologic correlation with serologic data is limited. We studied 315 consecutive renal transplants without pretransplant DSA, with a mean follow-up of 6.2 ± 2.9 years. Protocol (n = 215) and for cause (n = 163) biopsies were analyzed. Solid phase assays were used to screen for dnDSA posttransplant. A total of 47 out of 315 (15%) patients developed dnDSA at a mean of 4.6 ± 3.0 years posttransplant. Independent predictors of dnDSA were HLA-DRß1 MM > 0 (OR 5.66, p < 0.006); and nonadherence (OR 8.75, p < 0.001); with a strong trend toward clinical rejection episodes preceding dnDSA (OR 1.57 per rejection episode, p = 0.061). The median 10-year graft survival for those with dnDSA was lower than the No dnDSA group (57% vs. 96%, p < 0.0001). Pathology consistent with antibody-mediated injury can occur and progress in patients with dnDSA in the absence of graft dysfunction and furthermore, nonadherence and cellular rejection contribute to dnDSA development and progression to graft loss.

Protocol biopsies in renal transplantation: insights into patient management and pathogenesis.

A 1-day symposium on the application of protocol biopsies in renal transplantation was held in Boston, 21 July 2006. Representatives from centers with extensive experience in the use of protocol biopsies for routine patient care and research reported results on the pathological findings and their value in patient management. The consensus was that protocol biopsies, in experienced hands, are a safe and valuable means of detecting subclinical disease that can benefit from modification of therapy. Furthermore, molecular studies reveal evidence of activity or progression not readily appreciated by histological techniques. Wider application is expected in multicenter clinical trials to predict and validate outcomes. The principal barrier to wider use of protocol biopsies is knowledge of the benefits of intervention.

Kidney transplantation outcomes in Canadian aboriginals.

Aboriginal populations experience a very high rate of end-stage renal disease (ESRD); however, little is known about the outcomes of transplantation in this population. We performed a retrospective database review to determine the short- and long-term outcomes of kidney transplantation in Aboriginals. Adult Aboriginal (AB) and Caucasian (C) individuals receiving primary kidney transplants between 1969 and 2003 in Manitoba, Canada were examined. A total of 705 recipients were included (126 AB and 579 C). AB recipients were younger, had different etiologies of ESRD, longer cold-ischemic times for deceased donor transplants, and higher peak panel reactive antibody levels. At 1 year post-transplant, there was no difference in serum creatinine, acute rejection or graft survival between AB and C recipients. However, AB recipients experienced greater weight gains early post-transplant and were more likely to develop post-transplant diabetes mellitus. AB recipients exhibited inferior 10-year graft (AB 26% vs. C 47%, p < 0.01) and patient survival (AB 50% vs. 75%, p < 0.01). When graft survival was censored for death with a functioning graft, there was no difference between the two groups. Multivariate analysis revealed AB race to be an independent predictor of premature graft failure and patient death. In conclusion, kidney transplant outcomes have historically been inferior in the Manitoba population of Canadian Aboriginals.

The urine protein to creatinine ratio (P/C) as a predictor of 24-hour urine protein excretion in renal transplant patients.

BACKGROUND: The purpose of this study was to examine the utility of the random urine protein to creatinine ratio (P/C) in evaluation and longitudinal management of proteinuria in adult renal transplant recipients with or without overt nephropathy in an outpatient clinic. METHODS: A total of 289 adult renal transplant recipients provided 24-hr urine collections for total protein and creatinine, followed by a random urine for protein and creatinine. For longitudinal analysis, 192 of these patients provided two 24-hr urine collections with concomitant random urine specimens separated on average by 6.8 months. As well, 134 patients provided a total of 851 multiple-paired spot and 24-hr urine samples (range 2 to 12) over a 2-year period. RESULTS: The log random urine P/C ratio correlated significantly to the log 24 UP (r=0.749, P<0.0001) with or without nephrotic range proteinuria. High sensitivity (74.4-90%) and specificity values (93-98%) were found for estimating proteinuria from 0.5 to 2 g/day. However, the precision of estimation decreased as the level of urinary protein excretion increased to >3 g/day. The positive predictive value decreased as proteinuria became >3 g/day, perhaps because of the low prevalence of patients with high level proteinuria in our sample. The direction of change in P/C ratio longitudinally was accompanied by a similar direction of change in 24 UP, which was highly significant (r=0.7555, P<0.0001). CONCLUSION: We conclude that the urine P/C ratio is a useful and convenient screening and longitudinal test for proteinuria.

Neointimal and tubulointerstitial infiltration by recipient mesenchymal cells in chronic renal-allograft rejection.

BACKGROUND: Tissue remodeling depends on mesenchymal cells (fibroblasts and myofibroblasts) and is a prominent feature of chronic renal-transplant rejection. It is not known whether the mesenchymal cells that participate in remodeling originate locally or from circulating precursor cells. METHODS: We obtained biopsy specimens of renal allografts from six male recipients of an allograft from a female donor, four female recipients of an allograft from a male donor, two male recipients of an allograft from a male donor, and two female recipients of an allograft from a female donor. All the allografts were undergoing chronic rejection. All but two specimens were obtained within six months after transplantation. We used immunohistochemical methods to identify mesenchymal cells with smooth-muscle alpha-actin and in situ hybridization to identify mesenchymal cells with Y-chromosome DNA. RESULTS: No Y-chromosome bodies were identified in the case of the two renal-allograft specimens in which both the donor and the recipient were female. In the case of the two renal-allograft specimens in which both the donor and the recipient were male, approximately 40 percent of mesenchymal cells contained a Y-chromosome body. In the case of the six specimens in which the donor was female and the recipient was male, a mean (+/-SD) of 34+/-16 percent of mesenchymal cells in the neointima, 38+/-12 percent of such cells in the adventitia, and 30+/-7 percent of such cells in the interstitium contained the Y-chromosomal marker, indicating that they originated from the recipient rather than the donor. In the case of the four renal-allograft specimens in which the donor was male and the recipient was female, the respective values were 24+/-15 percent, 33+/-9 percent, and 23+/-8 percent, indicating a persistent population of donor mesenchymal cells. CONCLUSIONS: The presence of mesenchymal cells of host origin in the vascular and interstitial compartments of renal allografts undergoing chronic rejection provides evidence that a circulating mesenchymal precursor cell has the potential to migrate to areas of inflammation.

Increased levels of GALbeta1-4GLCNACalpha2-6 sialyltransferase pretransplant predict delayed graft function in kidney transplant recipients.

BACKGROUND: Galbeta1-4GlcNAcalpha2-6 sialyltransferase (ST6GalI) is an acute phase reactant whose release from cells can be induced by proinflammatory cytokines. Because patients with chronic renal failure have high circulating levels of proinflammatory cytokines, we hypothesized that patients on the renal transplant waiting list would have high circulating levels of ST6GalI, which might adversely affect post-transplant events. METHODS: Levels of ST6GalI were measured in the serum of 70 patients immediately before renal transplant; these were correlated with posttransplant events, such as delayed graft function and rejection. RESULTS: The mean serum level of ST6GalI was significantly higher in the patients (3162+/-97 U) than in 19 controls (2569 +/- 125 U; P<0.003). Patients who required dialysis posttransplant for treatment of delayed graft function (n=20) had significantly higher levels of ST6GalI pretransplant (3735+/-228 U) than patients (n=50) who did not require dialysis (2933+/-83 U; P<0.0001). In a multivariate analysis the ST6GalI level and cold ischemic time were found to be independent risk factors for the development of delayed graft function. CONCLUSIONS: ST6GalI levels are high in renal failure patients awaiting a renal transplant and may be a risk factor for the development of delayed graft function. The assessment and perhaps modulation of a potential transplant recipient's ST6GalI systemic level may be beneficial.

Quantitation of allograft fibrosis and chronic allograft nephropathy.

Despite improvements in the prevention and treatment of acute renal allograft rejection, the long-term survival of renal transplants has not increased. Immunologic and non-immunologic factors contribute to the gradual deterioration of graft function and to the histologic lesion characterized by vascular and interstitial fibrosis ('chronic rejection'). Quantitation of this process has been attempted using various invasive and non-invasive methods. These methods, performed at different times post-transplant, are reviewed in this article. In particular, pathology scoring systems and the potential of using computerized image analysis of biopsy material are discussed.

Clinical rejection is distinguished from subclinical rejection by increased infiltration by a population of activated macrophages.

It has been reported previously that one-third of protocol renal biopsies in asymptomatic, biochemically stable renal transplant recipients in the first 6 mo show unsuspected subclinical graft rejection (both infiltrate and tubulitis) and that subclinical rejection is a risk factor for chronic renal dysfunction. This study was performed to determine whether differences in phenotype or activation status of graft-infiltrating cells underlie these different manifestations of acute rejection. Biopsies with normal histology (n = 10), subclinical rejection (n = 13), and clinical rejection (n = 9) were studied using immunohistochemistry and computerized image analysis. Subclinical and clinical rejections had similar histologic Banff scores. Univariate analysis showed a trend for a higher infiltration with CD8+ (P = 0.053) and CD68+(P = 0.06) cells in clinical rejection. Of the activation markers studied (CD25, perforin, tumor necrosis factor-alpha), only allograft inflammatory factor-1+-activated macrophages were significantly (P = 0.014) increased in the infiltrate of clinical rejection biopsies. These data suggest that activated macrophages or their products are responsible for acute renal dysfunction associated with clinical rejection episodes.

Does subclinical rejection contribute to chronic rejection in renal transplant patients?

Renal allograft biopsies have traditionally been performed in the setting of acute graft dysfunction. However, several groups have performed graft biopsies at times of stable graft function, and more recently, after treatment of rejection episodes. Surprisingly, unequivocal histologic criteria for acute rejection have been demonstrated in a high proportion of these protocol biopsies. The Winnipeg Transplant Group has documented the high prevalence of clinically silent inflammatory infiltrates in early protocol biopsies, and demonstrated their inflammatory and cytotoxic potential by immunohistochemical and molecular biological techniques. Furthermore, in a randomized trial, our group has demonstrated that subclinical rejection, if untreated, is associated with the development of early chronic pathology and late graft dysfunction. In this overview, we will summarize the early data on subclinical allograft inflammation, present the experience of the Winnipeg Transplant Group, and discuss the possible implications of subclinical rejection on the development of chronic rejection.

Protocol biopsies in renal transplantation: research tool or clinically useful?

Early protocol biopsies of stable, well functioning renal allografts reveal a high prevalence of clinically unsuspected acute and chronic pathology. It is becoming increasingly apparent that these histopathological findings are both pathogenic and predictive of long-term allograft outcome. Therefore, protocol biopsies may be required for optimal post-transplant surveillance until non-invasive methods to detect allograft inflammation are developed.

Matching for private or public HLA epitopes reduces acute rejection episodes and improves two-year renal allograft function.

BACKGROUND: The current role of HLA matching in renal transplantation is controversial. Public HLA epitope matching has been suggested to be as advantageous as private HLA matching, with the added benefit of increasing recipients' access to well-matched grafts. METHODS: In this single-center study of 105 renal transplant recipients, we examined the association of HLA matching with early (0-3 months) and late (4-6 months) rejection episodes (RE), as well as renal allograft function up to 2 years after transplant. RESULTS: Poor HLA-DR, but not HLA-A or -B, matching was associated with early RE (0 DR matches, RE=2.7+/-0.19, 1 DR match, RE=2.37+/-0.18, vs. 2 DR matches, RE=1.5+/-0.38; P < 0.01). In contrast, poor HLA-B, but not HLA-A or -DR, matching was associated with late rejections (0 HLA-B matches, RE=1.1+/-0.51 vs. 1-2 HLA-B matches, RE=0.51+/-0.1; P < 0.004). HLA-B matching was also associated with a significantly lower serum creatinine (SCr) level at 24 months (0 HLA-B matches, SCr=178+/-20 micromol/L vs. SCr=132+/-6 micromol/L for 1-2 HLA-B matches; P < 0.025). Matching for 10 supertypic HLA-A and -B cross-reactive groups was associated with reduced late graft rejection (0-2 residue matches, RE=1.15+/-0.18 vs. RE=0.62+/-0.12 for 3 to 7 residue matches; P < 0.013) as well as a significantly lower SCr level at 24 months (0-2 residue matches, SCr=205+/-29 micromol/L vs SCr=131+/-6 micromol/L for 3 to 7 residue matches; P < 0.001) after transplantation. CONCLUSIONS: HLA-DR matching was associated with a reduced frequency of early rejection episodes, whereas HLA-B or residue/cross-reactive group matching was associated with a reduced frequency of late rejection episodes and improved graft function at 2 years.

Immune-activation gene expression in clinically stable renal allograft biopsies: molecular evidence for subclinical rejection.

BACKGROUND: A significant percentage of biopsies from stable, well-functioning renal allografts have histologic findings consistent with acute rejection or borderline rejection. The implication of this finding is not yet fully understood. We analyzed immune-activation gene transcripts in stable protocol biopsies to determine the extent of immunologic activity of graft-infiltrating cells in this setting. Histologic classification of the biopsies was based on the Banff criteria. To emphasize that the tissue samples were procured from grafts with no clinical evidence of impaired function, we interjected the term "subclinical" into the Banff terminology. This produced three histologic categories: normal, borderline subclinical rejection, and acute subclinical rejection. METHODS: We used competitive template polymerase chain reaction techniques to quantify transcript amounts for the constant region of the T-cell receptor beta chain; the cytokines, tumor necrosis factor alpha, interleukin (IL)-1beta, transforming growth factor beta, interferon gamma, IL-2, IL-4, IL-10, and IL-15; and the cytotoxic T lymphocyte effector molecules, granzyme B, perforin, and Fas ligand. RESULTS: We found that histologically normal biopsies were typically devoid of gene transcripts or had very low amounts. Conversely, biopsies with acute subclinical rejection by histologic examination had heightened amounts of transcripts for many of the genes assayed. Borderline subclinical rejection samples showed an intermediate amount of expression. CONCLUSIONS: These results demonstrate that histologic features of rejection are often accompanied by enhanced expression of pro-inflammatory gene transcripts, despite the absence of clinically overt graft dysfunction. As this state of subclinical rejection could prove detrimental to long-term graft function, a role for surveillance biopsies of stable grafts with intent to treat subclinical rejection should be considered.

Sequential protocol biopsies in renal transplant patients. Clinico-pathological correlations using the Banff schema.

Twenty-five renal transplant patients on a triple immunosuppressive regimen of cyclosporine, azathioprine, and prednisone underwent protocol graft biopsies at 1, 2, 3, 6, and 12 months after transplant regardless of renal function. The histological diagnosis was made with the Banff schema. As reported previously, protocol biopsies revealed a high prevalence of subclinical rejection, as well as "borderline" inflammation, despite levels of CsA considered to be in the therapeutic range. Every biopsy was given a score for the severity of the histological changes (the Banff Score for Inflammatory Changes [BSI]), which permitted the generation of a cumulative BSI over the year of follow-up for each patient. At the end of 1 year, normal histology and excellent renal function (mean serum creatinine < 110 mumol/L) were seen only in transplant patients with the lowest cumulative BSI (P < 0.001). These results suggest that repeated inflammation in the renal allograft, even if subclinical, can lead to its dysfunction. Moreover, it would appear that, at least for the present, protocol biopsies may be required to assess adequately the effectiveness of current immunosuppressive therapies in renal transplant patients.