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1.  Macrophages: Contributors to Allograft Dysfunction, Repair or Innocent Bystanders? 
Purpose of this review
Macrophages are members of the innate immune response. However, their role in the adaptive immune response is not known. The purpose of this review is to highlight our current understanding of macrophage structure and function and how they may participate in allograft injury.
Recent Findings
Studies in acute kidney injury models identify macrophages as key mediators of inflammatory injury while more recent studies indicate that they may play a reparative role, depending on phenotype—M1 or M2 type macrophages. Mregs, generated in vitro, appear to have immune suppressive abilities and a unique phenotype. In solid organ transplant, the emphasis of studies has been on acute or chronic injury. These data are derived from animal models using depletion of macrophages or antagonizing their activation and inflammatory responses. The relative contribution of macrophage phenotype in transplantation has not been explored.
These studies suggest that macrophages play an injurious role in acute cellular allograft rejection, as well as in chronic injury. Infiltration of an allograft with macrophages is also associated with worse graft function and poor prognosis. Further studies are needed to understand the mechanisms of macrophage mediated injury, explore their potential reparative role and determine if they or their functional products are biomarkers of poor graft outcomes.
PMCID: PMC3319132  PMID: 22157320
macrophage; kidney transplant; rejection; antibody; inflammation
2.  Optimal Cut-off Point for Immunoperoxidase Detection of C4d in the Renal Allograft: Results from a Multicenter Study 
Transplantation  2010;90(10):1099-1105.
Although C4d deposition in peritubular capillaries has been identified as a strong risk factor for subsequent renal allograft loss, the optimal cut-off for the fraction of peritubular capillaries needed to establish a positive stain in formalin-fixed paraffin-embedded material has not been systematically defined. The objective of this study was to establish the threshold for positive staining that best predicts renal outcome in renal biopsies in a multicenter study in which local and central pathology were compared.
Unstained renal biopsy slides were obtained from 296 patients. The percentage of peritubular capillaries staining positively for C4d was detected by immunoperoxidase staining.
The percentage C4d deposition ranged from 0% to 90% with 44% (129/296) having a positive percentage of C4d staining. The median for positive cases was 25%. Local C4d+ results were reported qualitatively, with 28% recorded as positive for C4d. Using a centrally-determined cut-off of 10%, tests for agreement of local and central C4d staining were fair (Kappa 0.40, 95% CI 0.29-0.51). Raising the centrally-determined cut-off to 25% or 50% did not change the Kappa values (0.44 and 0.41, respectively). By Cox proportional hazards model, C4d positivity (centrally-determined assessment) using a cut-off of 10% was the strongest predictor of time to graft loss (HR 2.66, 95% CI [1.68, 4.21]). Centrally-determined C4d positivity correlated with Banff scores indicative of acute inflammation, but not with scores indicative of fibrosis/atrophy or transplant glomerulopathy.
Our findings indicate that C4d positivity, defined as ≥10% by immunoperoxidase, is a strong predictor of graft loss.
PMCID: PMC3171966  PMID: 21430605
C4d; graft survival; immunoperoxidase
3.  Inflammation in Areas of Tubular Atrophy in Kidney Allograft Biopsies: A Potent Predictor of Allograft Failure 
The Banff scoring schema provides a common ground to analyze kidney transplant biopsies. Interstitial inflammation (i) and tubulitis (t) in areas of viable tissue are features in scoring acute rejection, but are excluded in areas of tubular atrophy. We studied inflammation and tubulitis in a cohort of kidney transplant recipients undergoing allograft biopsy for new-onset late graft dysfunction (N=337). We found inflammation (“iatr”) and tubulitis (“tatr”) in regions of fibrosis and atrophy to be strongly correlated with each other (p<0.0001). Moreover, iatr was strongly associated with death-censored graft failure when compared to recipients whose biopsies had no inflammation, even after adjusting for the presence of interstitial fibrosis (Hazard Ratio=2.31, [1.10-4.83]; p=0.0262) or tubular atrophy (Hazard Ratio=2.42, [1.16-5.08]; p=0.191), serum creatinine at the time of biopsy, time to biopsy, and i score. Further, these results did not qualitatively change after additional adjustments for C4d staining or donor specific antibody. Stepwise regression identified the most significant markers of graft failure which include iatr score. We propose that a more global assessment of inflammation in kidney allograft biopsies to include inflammation in atrophic areas may provide better prognostic information. Phenotypic characterization of these inflammatory cells and appropriate treatment may ameliorate late allograft failure.
PMCID: PMC2951299  PMID: 20883541
biopsy; inflammation; fibrosis; injury; graft failure; Banff schema
4.  Multi-Center Evaluation of a Standardized Protocol for Non-Invasive Gene Expression Profiling 
Gene expression profiling of transplant recipient blood and urine can potentially be used to monitor graft function, but the multitude of protocols in use make sharing data and comparing results from different laboratories difficult. The goal of this study was to evaluate the performance of current methods of RNA isolation, reverse transcription, and quantitative polymerase chain reaction (qPCR) and to test whether multiple centers using a standardized protocol can obtain the same results. Samples, reagents, and detailed instructions were distributed to six participating sites that performed RNA isolation, reverse transcription and qPCR for 18S, PRF, GZB, IL8, CXCL9 and CXCL10 as instructed. All data were analyzed at a single site. All sites demonstrated proficiency in RNA isolation and qPCR analysis. Gene expression measurements for all targets and samples had correlations >0.938. The coefficient of variation of fold-changes between pairs of samples was less than 40%. All sites were able to accurately quantify a control sample of known concentration within a factor of 1.5. Collectively, we have formulated and validated detailed methods for measuring gene expression in blood and urine that can yield consistent results in multiple laboratories.
PMCID: PMC3781926  PMID: 23802725
Transplantation  2012;94(9):971-977.
Hyperlipidemia is a common adverse effect of sirolimus (SRL). We previously showed significant associations of ABCB1 3435C>T and IL-10 -1082G>A with log-transformed SRL dose-adjusted, weighted-normalized trough. We further examined to see whether these polymorphisms were also associated with SRL-induced dyslipidemia.
Genotyping was performed for ABCB1 1236C>T, 2677 G>T/A, and 3435C>T, CYP3A4 -392A>G, CYP3A5 6986A>G and 14690G>A, IL-10 -1082G>A, TNF - 308G>A, and ApoEε2,ε3, and ε4 alleles. The longitudinal changes of total cholesterol (TC), low-density lipoprotein (LDL)-C, and triglyceride (TG) levels after SRL treatment prior to statin therapy were analyzed by a linear mixed-effects model, with adjustments for selected covariates for each lipid.
Under the dominant genetic model, ABCB1 3435C>T was associated with TC (p=0.0001) and LDL-C (p<0.0001) values after SRL administration. Mean TC and LDL-C levels were 26.9 and 24.9 mg/dL higher, respectively, in ABCB1 3435T carriers than 3435CC homozygotes at an average SRL trough concentration of 4 ng/mL without concomitant medication. ABCB1 1236C>T under the recessive model and IL-10 -1082G>A under the dominant model were associated with log-transformed TG values (p=0.0051 and 0.0436, respectively). Mean TG value was 25.1% higher in ABCB1 1236TT homozygotes compared to ABCB1 1236C carriers and was 12.4% higher in IL-10-1082AA homozygotes than -1082G carriers.
ABCB1 polymorphisms were found to be associated with lipid responses to SRL treatment, confirming the role of ABCB1 gene in SRL pharmacokinetics and pharmacodynamics. Further studies are necessary to define the role of ABCB1 and IL-10 polymorphisms on SRL-induced dyslipidemia in renal transplantation.
PMCID: PMC3491093  PMID: 23073467
Sirolimus; ABCB1; total and LDL cholesterol; triglyceride; pharmacogenetics
6.  A reproducible mouse model of chronic allograft nephropathy with vasculopathy 
Kidney international  2012;82(11):1231-1235.
While short-term outcomes in kidney transplantation have improved dramatically, long-term survival remains a major challenge. A key component of long-term, chronic allograft injury in solid organ transplants is arteriosclerosis characterized by vascular neointimal hyperplasia and inflammation. Establishing a model of this disorder would provide a unique tool, not only to identify mechanisms of disease, but also test potential therapeutics for late graft injury. To this end, we utilized a mouse orthotopic renal transplant model in which C57BL/6J (H-2b) recipients were given either a kidney allograft from a completely mismatched Balb/cJ mouse (H-2d), or an isograft from a littermate. A unilateral nephrectomy was performed at the time of transplant followed by a contralateral nephrectomy on post-transplant day seven. Recipients were treated with daily cyclosporine subcutaneously for 14 days and then studied 8 and 12 weeks post transplantation. Renal function was significantly worse in allograft compared to isograft recipients. Moreover, the allografts had significantly more advanced tubulointerstitial fibrosis and profound vascular disease characterized by perivascular leukocytic infiltration and neointimal hyperplasia affecting the intrarenal blood vessels. Thus, we describe a feasible and reproducible murine model of intrarenal transplant arteriosclerosis useful to study allograft vasculopathy.
PMCID: PMC3495090  PMID: 22874842
Transplantation  2011;92(12):1342-1347.
SRL absorption and metabolism are affected by Pgp-mediated transport and CYP3A enzyme activity, which are further under the influences of cytokine concentrations. This retrospective study determined the associations of ABCB1 1236C>T, 2677 G>T/A, and 3435C>T, CYP3A4 -392A>G, CYP3A5 6986A>G and 14690G>A, IL-10 -1082G>A, and TNF -308G>A polymorphisms with SRL dose-adjusted, weight-normalized trough concentrations (C/D) at 7 days, and at 1, 3, 6, and 12 months post initiation of SRL.
Genotypes for 86 renal transplant patients who received SRL-based maintenance immunosuppressive therapy were determined using polymerase chain reaction followed by chip-based mass spectrometry. The changes of log-transformed C/D over the days post transplantation were analyzed using a linear mixed-effects model, with adjustments for body mass index and weight-normalized doses of tacrolimus, prednisone, clotrimazole, and statins.
ABCB1 3435C>T and IL-10 -1082G>A were significantly associated with log C/D (p=0.0016 and 0.0394, respectively). Mean SRL C/D was 48% higher in patients with ABCB1 3435CT/TT genotype than those with 3435CC genotype, and was 24% higher in IL-10 -1082GG compared to -1082AG/AA.
ABCB1 3435C>T and IL-10 -1082G>A were significantly associated with long-term SRL dose requirements. Genetics can play a significant role in SRL dosing and may be useful in therapeutic monitoring of SRL in renal transplantation. Future replication studies are needed to confirm these associations.
PMCID: PMC3237821  PMID: 22094953
Sirolimus; ABCB1; CYP3A5; pharmacogenetics; pharmacokinetics
8.  Elevated Expression Levels of ANXA11, Integrins β3 and α3, and TNFα Contribute to a Candidate Proteomic Signature in Urine for Kidney Allograft Rejection 
Proteomics. Clinical applications  2011;5(5-6):311-321.
Kidney transplantation is the treatment of choice for end stage renal disease, with long term allograft loss being the major obstacle, and for which potential treatments are based on a histological diagnosis. The problem is that markers for predicting graft rejection are limited in number and invasive and quite non-specific. We have hypothesized that protein biomarkers might be discovered in the urine of patients when acute or chronic rejection might be occurring.
Experimental design
We have established a workflow in which initial screening for candidate biomarkers is first performed using urine samples on large scale antibody microarrays. This approach generated several dozen candidates. The next step is to qualify some of the strongest signals using the high throughput Reverse Capture Protein Microarray platform.
Four top candidates including ANXA11, Integrin α3 and Integrin β3, and TNFα initially identified by the antibody microarray platform were all qualified using Reverse Capture Protein Microarrays. We also used Receiver Operating Condition (ROC) curves to independently quantify the specificity and sensitivity of these four analytes.
Conclusions and clinical relevance
The present data suggest that these novel four analytes in the urine, together or independently, may contribute to a robust and quantitative urine proteomic signature for diagnosing acute or chronic rejection of renal allografts.
PMCID: PMC3444813  PMID: 21591265
graft rejection; urine; proteomics; protein arrays
9.  Chemokines and their Receptors in Human Renal Allotransplantation 
Transplantation  2011;91(1):70-77.
Chemokines and their receptors play a critical role in leukocyte trafficking, and inhibition of select chemokines has been shown to attenuate kidney disease and allograft rejection in animal models. We therefore evaluated chemokine and chemokine receptor transcripts in human renal allograft biopsies, correlating transcript levels with clinical course and immunohistochemical analysis to relate chemokine expression to relevant clinical human disease phenotypes.
Renal biopsies were grouped as post-reperfusion (n=10), stable function (n=10), subclinical (n=10) or acute rejection (n=17), or calcineurin inhibitor nephrotoxicity (n=9) based on clinical presentation and histopathological assessment. Using quantitative real-time polymerase chain reaction analysis, chemokine transcripts were assessed relative to transcript levels in pre-procurement biopsies from live donor kidneys (n=15).
Transcripts from several inflammatory chemokines (CCL3, CCL5, CXCL9, CXCL10 and CXCL11) and chemokine receptors (CCR5, CCR7 and CXCR3) were significantly elevated in allografts with subclinical and clinical acute rejection, indicating a strong polarization toward a TH1 effector phenotype during rejection. These transcripts also distinguished acutely rejecting allografts from allografts with non-rejection causes of renal dysfunction. Biopsies from patients with stable function without histological evidence of rejection had increased chemokine transcript levels that were qualitatively similar but quantitatively reduced compared to rejecting allografts.
This comprehensive evaluation of chemokines and their receptors in human renal transplantation defines associations between chemokine expression and clinical phenotypes, may have diagnostic utility, and highlights relevant pathways for therapeutic intervention.
PMCID: PMC3311125  PMID: 21441854
Chemokines; Renal Transplantation; RT-PCR
10.  Low-density array PCR analysis of reperfusion biopsies: an adjunct to histological analysis 
Nephrology Dialysis Transplantation  2010;25(12):4077-4086.
Background. Histologic evaluation of baseline kidney biopsies is an inconsistent tool to predict graft outcomes, which might be assisted by gene expression analysis.
Methods. We evaluated 49 consecutive kidney graft biopsies obtained post-reperfusion in 18 deceased donors (DD) and 31 living donors (LD) at our center. Biopsies were evaluated and scored using Banff criteria. Low-density real-time polymerase chain reaction arrays were used to measure intragraft expression of 95 genes associated with programmed cell death, fibrosis, innate and adaptive immunity and oxidative stress signaling. A pool of 25 normal kidney biopsies was used as control. We applied a stepwise forward selection procedure to build a multiple regression model predicting estimated glomerular filtration rate (eGFR) at 1 year after transplant using baseline clinical characteristics and gene expression levels.
Results. DD grafts displayed a pattern of gene expression remarkably different from LD, including an increased expression of complement protein C3, and chemokines, CXCL1 and CXCL2, consistent with the proinflammatory setting of ischaemia–reperfusion injury. There was no association between any of the reperfusion biopsy histological features and either renal function at 1 year post-transplant or risk of acute rejection. Conversely, older donor age (R2 = 0.17, P < 0.001) and higher integrin β2 gene expression levels (incremental R2 versus Donor Age-only model = 0.23, P < 0.001) jointly predicted lower eGFR at 1 year after transplant (multiple regression R2 = 0.40). Patients with higher ITGβ2 expression levels in baseline biopsies showed lower eGFR, higher levels of proteinuria and more transplant glomerulopathy on the 1-year per-protocol biopsies.
Conclusion. ITGβ2 gene expression in reperfusion biopsies may represent a prognostic marker for kidney transplant recipients, potentially helpful in shaping patients’ treatment. Further studies are needed to confirm our findings.
PMCID: PMC3108365  PMID: 20504838
gene expression; integrin β2; kidney transplant; predictor; reperfusion biopsy
Clinical transplantation  2010;24(4):557-563.
In a cohort of 32 renal transplant patients who are potentially at risk for adverse events, we compared tacrolimus (TAC) abbreviated AUC values calculated by a method developed in Asians (AUCw) with those derived for Caucasians (AUCa). The relationships between TAC trough (C0), abbreviated AUC, and biopsy results were also assessed. Forty-eight AUCs and 15 associated biopsies were evaluated. For AUCs obtained from Caucasian patients only, median AUCw value was lower than that of AUCa (104 vs. 115 ng*h/mL, n=29, p<0.0001). AUCs obtained from both methods for all patients correlated with C0 (rs>0.72, n=48, p<0.0001). Median AUCw (72.9 vs. 174 ng*h/mL, p=0.043) and AUCa (81.0 vs. 203 ng*h/mL, p=0.043) were lower in patients experiencing biopsy-proven acute rejection (AR) than those with normal histology. C0 tended to be lower in biopsies showing AR > 6 months post transplant (5.80 vs. 11.0 ng/mL, p=0.110). Thus, lower abbreviated AUCs were obtained for Caucasians using a method developed in Asians. C0 correlated well with abbreviated AUCs. Lower C0 and AUC appeared to be associated with biopsy-proven AR > 6 months post transplant. Further prospective evaluation of TAC AUC and C0 monitoring in a larger cohort of patients is warranted.
PMCID: PMC2889034  PMID: 19925470
tacrolimus immunosuppression; trough concentration; abbreviated AUC; biopsy-proven acute rejection; therapeutic drug monitoring
12.  Noninvasive methods to assess the risk of kidney transplant rejection 
In current clinical practice, immune reactivity of kidney transplant recipients is estimated by monitoring the levels of immunosuppressive drugs, and by functional and/or histological evaluation of the allograft. The availability of assays that could directly quantify the extent of the recipient’s immune response towards the allograft would help clinicians to customize the prescription of immunosuppressive drugs to individual patients. Importantly, these assays might provide a more in-depth understanding of the complex mechanisms of acute rejection, chronic injury, and tolerance in organ transplantation, allowing the design of new and potentially more effective strategies for the minimization of immunosuppression, or even for the induction of immunological tolerance. The purpose of this review is to summarize results from recent studies in this field.
PMCID: PMC2756773  PMID: 20161000
biomarker; CD30; Cylex™; ELISPOT; immune monitoring; proteomic; transplantation
13.  Platelet-derived or soluble CD154 induces vascularized allograft rejection independent of cell-bound CD154 
Journal of Clinical Investigation  2006;116(3):769-774.
CD154 is a cell surface molecule expressed on activated T cells that binds to CD40, an activating molecule on APCs. Its blockade has been shown to prevent allograft rejection, presumably by interrupting interactions between T cells and APCs. It is known that activated human platelets express and shed CD154 and can induce APC activation and other immune processes in vitro. Here we show that platelet-derived CD154 is sufficient to initiate cardiac allograft rejection independent of any cellular source of this molecule. CD154-KO mice reject cardiac allografts after receiving CD154-expressing human platelets or recombinant CD154 (rCD154) trimers. Treatment with the human CD154-specific mAb 5c8 specifically prevents this induced rejection. Soluble trimers, but not platelets, induce rejection when infused temporally remote from the surgical procedure, suggesting that surgically induced platelet activation is required for CD154 release. Allograft rejection can thus be instigated by activated platelets through CD154. These data implicate platelets as a proximal component of acquired alloimmunity, providing insight into the mechanisms of allograft rejection and the physiological response to trauma in general.
PMCID: PMC1378189  PMID: 16498500
14.  Angiotensin II regulates cellular immune responses through a calcineurin-dependent pathway 
Journal of Clinical Investigation  1999;104(12):1693-1701.
The renin-angiotensin system (RAS) is a key regulator of vascular tone and blood pressure. In addition, angiotensin II also has a number of cellular effects that may contribute to disease pathogenesis. Using Agtr1a–/– mice, which lack AT1A receptors for angiotensin II, we have identified a novel function of the RAS to modulate the immune system. We find that angiotensin II, acting through type 1 (AT1) receptors on immune cells, triggers the proliferation of splenic lymphocytes. These actions contribute to the vigor of cellular alloimmune responses. Within lymphoid organs, sufficient components of the RAS are present to activate AT1 receptors during an immune response, promoting cell growth. These actions require activation of calcineurin phosphatase. In an in vivo model of cardiac transplantation, the absence of AT1 signaling accentuates the immunosuppressive effects of the calcineurin inhibitor cyclosporine. We conclude that inhibition of AT1 receptor signaling should be useful as an anti-inflammatory and immunosuppressive therapy. Furthermore, the actions of the RAS to promote lymphocyte activation may contribute to inflammation that characterizes a number of diseases of the heart and the vascular system.
J. Clin. Invest. 104:1693–1701 (1999).
PMCID: PMC409880  PMID: 10606623

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