Chronic Allograft Injury Research Articles

Through a glass darkly: seeking clarity in preventing late kidney transplant failure.

A common lament is that long-term kidney transplant outcomes remain the same despite improvements in early graft survival. To be fair, progress has been made-in both our understanding of chronic injury and modestly, graft survival. However, we are still a long way from actually solving this important and difficult problem. In this review, we outline recent data supporting the existence of several causes of renal allograft loss, the incidences of which peak at different time points after transplantation. On the basis of this broadened concept of chronic renal allograft injury, we examine the challenges of clinical trial design in long-term studies, including the use of surrogate end points and biomarkers. Finally, we suggest a path forward that, ultimately, may improve long-term renal allograft survival.

Transcriptional Profiling of Belatacept and Calcineurin Inhibitor Therapy in Renal Allograft Recipients

Calcineurin inhibitor (CNI) use may lead to allograft injury and compromised renal function. Gene expression profiles of 12-month kidney biopsies from a Phase 3 study of belatacept and a CNI comparator, cyclosporine (CsA), were compared with expression profiles of a set of historical, demographically matched, preimplantation control biopsies. Gene set enrichment analysis was used to test each set of differentially expressed genes (DEGs) for the enrichment of an in vitro-derived CNI toxicity (CNIT) gene set and published gene sets associated with chronic allograft injury (CAI), immune modulation and tissue remodeling. The unique set of genes differentially expressed in CNI biopsies compared with preimplantation controls was enriched for genes associated with fibrosis, early tubulointerstitial damage and in vitro CNIT. The DEGs from belatacept biopsies were not enriched for the CNIT genes but, instead, exhibited enrichment for gene sets associated with immune response and tissue remodeling. A combined analysis of DEGs across both treatment groups identified select solute transporter and cellular differentiation genes whose expression at 12 months correlated with renal function at 36 months. These results provide mechanistic insights into the reduced CAI and higher renal function observed in belatacept- versus CsA-treated patients.

Possible Mechanisms and Potential Urine Protein Biomarkers through Quantitative Proteomics and Bioinformatics.

Introduction: Inability to effectively monitor graft injuries leads to graft dysfunction. A non-invasive tool to monitor such injuries is an unmet need. Method: Through a robust and comprehensive proteomics analysis, rigorous bioinformatics, and ELISA on a set of 480 urine samples with matched biopsies, we identified and validated potential biomarkers for acute rejection (AR), chronic allograft injury (CAI), and BK virus nephropathy (BKVN). Result: A panel of 100 statistically significant proteins as potential biomarkers for different transplant injury is identified. Four proteins, FGB, FGG, SUMO2, and HLA-DRB1 were selected based on their relevance in AR. Urinary FBB in AR was significantly higher vs STA (p=0.04), vs CAI (p=0.05), and vs BKV (p=0.03). The increased urine protein level of FBG in AR (p=0.04), vs CAI (p=0.05), and vs BKV (p=0.02). The urine protein level of HLA-DRB1 was significantly higher in AR vs STA (p=0.001), vs CAI (p=0.003), and vs BKV (p=0.04). Urine SUMO2 level was significantly higher in AR vs STA urine (p=0.005), and vs BKVN urine (p=0.04). An ROC analysis to identify AR from the rest of the phenotypes (CAI, BK, and STA) on the data from FBB, FGG, and HLA-DRB1 yielded an AUC of 0.8.Table: No Caption available.Figure: No Caption available.Conclusion: A panel of 3 urinary proteins, mined by high throughput proteomics, and validated by customized ELISA, allows for non-invasive diagnosis of AR, not confounded by BKVN. This urinary assay requires no local sample processing, is robust and can be used as a sensitive assay for out-patient renal transplant injury surveillance.

Activin Inhibition Reduces Early Injury After Kidney Transplantation.

INTRODUCTION The innate immune response augments adaptive immunity and may also trigger repair processes that lead to uncontrolled fibrosis and atherosclerosis as seen in chronic allograft injury. The activins are members of the transforming growth factor β superfamily. They play critical roles in the onset of acute and chronic inflammatory conditions such as septicemia, asthma, and fibrosis. The role of activins in transplantation is unknown. In this study we investigated how activin inhibition affects early inflammation after kidney transplantation. METHODS A rat renal transplantation model was used. Activin A and B signaling was inhibited with a soluble activin receptor ActRIIB-Fc. In the study group ActRIIB-Fc was given to the transplant recipient i.p. two hours before the transplantation (n=5). In addition transplant recipients were treated daily with cyclosporine A (CsA) 1.5 mg/kg s.c. The control group received CsA only (n=5). Rats were followed for 72 hours. Immunohistochemical stainings were used to detect different inflammatory cell populations in the kidney transplants. RESULTS Activin inhibition significantly decreased the number of ED3+ macrophages (control vs. treatment: 50±13 vs. 16±4 cells/mm2, p<0.05), MPO+ neutrophiles (26±5 vs.10±2 cells/mm2, p<0.05) and OX62+ dendritic cells (24±8 vs. 11±6 cells/mm2, p<0.05) in the kidney transplants 72 hours after transplantation. ActRIIB-Fc did not affect CD4+ or CD8+ T cell infiltration. CONCLUSIONS Activin inhibition efficiently decreses the infiltration of macrophages, dendritic cells and neutrophilic cells to the kidney transplant in rat. These cells are characteristic to innate immunity response. The inhibition of innate immunity could be of use in the prevention of kidney allograft injury.

NK Cells Mediate Chronic Kidney Allograft Injury.

Chronic allograft injury remains the leading cause of late kidney graft loss despite improvements in immunosuppressive drugs and a reduction in acute T cell mediated rejection. We have recently demonstrated that NK cells are cytotoxic to tubular epithelial cells (TECs) and contribute to acute kidney ischemia-reperfusion injury (IRI). The role of NK cells in kidney allograft rejection has not been studied. A parent to F1 transplant model in which T cell tolerance is present, was utilized for these studies. C57BL/6 kidneys were transplanted into fully nephrectomized F1 (C57BL/6 x BALB/c, CB6F1) mice. Serum creatinine levels increased from baseline (18.8+5.0 umol/l to 37.2±5.9 umol/l, P<0.001) at 60 days post transplantation. In further support of this, in B6Rag-/--to- CB6F1Rag-/- (B6Rag-/-xBALB/cRag-/-) F1 kidney transplants which lack T and B cells but retain NK cells, similar levels of kidney dysfunction were observed 65 days post transplant (creatinine: 33.8+7.9 umol/l vs. 17.5+5.1 umol/l in non-transplant Rag-/- mice, P<0.05). Depletion of NK cells in Rag1-/- mice prevented kidney injury (24.6±5.5 umol/l, p<0.05). Interestingly, osteopontin (OPN) was highly expressed in 60 days kidney grafts, and OPN deficiency in kidney graft had reduced chronic injury after transplantation (17.7+3.1 umol/l, P<0.001). OPN activated NK cells can mediate TEC death in vitro. In conclusion, these data demonstrate for the first time a critical role for NK cells in mediating chronic kidney injury in transplantation, which is independent of T and B cells. It may require specific therapeutic strategies to attenuate NK cells' contribution to chronic kidney injury.

Long-Term Outcomes Following a Calcineurin Sparing Protocol After Renal Transplantation.

Despite remarkable advances in early kidney allograft survival rates, improvements in long-term outcomes remain limited primarily due to chronic progressive renal injury. One of the many possible causes of chronic allograft injury is calcineurin inhibitor (CNI) toxicity. To avoid CNI toxicity and to improve graft longevity, patients have been converted to non-CNI immunosuppressive regimens. Due to the anti-fibrotic effects of TORC1 inhibitors, these agents were proposed as non-nephrotoxic options to replace CNI in renal transplantation. Since the lasting consequences of TORC1 inhibitor therapy are not well defined, we report our 9 year experience with a CNI sparing protocol. We reviewed the medical records of all 600 patients transplanted in our center from 2001 to 2011. We compared the outcomes of patients who received continuous CNI based therapy (CNI, n=365) to those who were converted to sirolimus and remained on this drug throughout the study (CONV, n=109). The two groups were similar for recipient and donor characteristics as well as immunosuppressive therapy. Diabetes was more common in the CNI group whereas hypertension and re-transplants were more frequent in the CONV group. Graft and patient survival rates were equivalent in both groups (Fig1). There was no difference in the causes of death. The causes of graft loss were also similar except that recurrent disease was more common in the CONV group. The rate of acute rejection (19.1% CNI vs. 15% CONV, p=0.47) was similar. Renal function was also not different between groups. There was no difference in outcomes in patients converted within or after one year. Therefore, long-term outcomes following renal transplantation are no different whether patients are switched to sirolimus or remain on CNI therapy.Figure: No Caption available.Figure: No Caption available.

Proteomic Analysis Identified Urine Proteins Predictive of Chronic Allograft Injury.

Aim: To correlate early urine protein biomarkers with long term kidney transplant histology and function; to assess biomarker potential to predict subsequent allograft dysfunction and chronic interstitial fibrosis. Method: Patient cohort was selected from a single center within the GoCAR study who had urine samples at 3 and 12 months and protocol biopsies at 0, 3, and 12 months post transplant. Patients were grouped by chronic damage scores using Banff and Chronic Allograft Damage Index (CADI) from 12 month biopsies or change in estimated glomerular filtration rate (eGFR). Urine proteins at 3 and 12 months associated with chronic damage at 12 months were identified by two-dimensional differential in gel electrophoresis (2D-DIGE) and tandem mass spectrometry. Differential protein abundance was calculated using DeCyder software and further interrogated using the STRING network program. Results: Delayed graft function occurred in 25% and biopsy proven acute rejection in 17% of patients. Acute and chronic damage scores were low at 3 months. Proteinuria or eGFR were not significantly different between chronic damage analysis groups. From the 3 month urine, 9 proteins including kininogen, MASP2 and cystatin M correlated to fibrosis at 12 months (5 with Banff, 4 with CADI) and 2 proteins correlated with change in eGFR. The top KEGG pathway in the STRING network were coagulation and complement cascades. Follow up 12 month urine analysis based on the same chronic damage groups revealed 11 proteins, 4 identified in the previous 3 month analysis and 7 new proteins, including CD14 and β2 microglobulin. The top KEGG pathway at 12 months was antigen processing and presentation. Conclusion: Using traditional proteomic techniques, we have identified potential non-invasive biomarkers that are predictive of chronic allograft damage. The dominant urine proteome alters between 3 and 12 months expressing a different panel of proteins. While early urine protein signatures predict subsequent damage, validation in larger and more diverse cohorts is required.

Randomized Trial of Three Induction Antibodies in Kidney Transplantation

In searching for an optimal induction regimen, we conducted two separate randomized trials of 38 living donor and 90 deceased donor adult, primary kidney transplant recipients comparing antithymocyte globulin (Thymoglobulin) (group A, N=43) versus alemtuzumab (group B, N=43) versus daclizumab (group C, N=42), using exactly the same three treatment arms in each trial. For the purpose of maximizing statistical power, results from the two randomized trials were combined. Groups A and C received standard maintenance dosing with tacrolimus (TAC), mycophenolate mofetil (MMF), and corticosteroids. Because of intense lymphodepletion expected with alemtuzumab use (and hoped-for achievement of a truer immunoregulatory state), group B received lower TAC and MMF dosing and corticosteroid avoidance. Long-term target TAC trough level and MMF dosing were 5 to 7 ng/mL and 1,000 mg b.i.d. in groups A and C; 4 to 6 ng/mL and 500 mg b.i.d. in group B. With median follow-up of 95 months, biopsy-proven cute rejection incidence was similar in the three groups (8/43, 14/43, and 12/42, P=0.34), but biopsy-proven chronic allograft injury incidence was significantly higher in group B (19/43) in comparison with groups A (9/43) and C (7/42) combined (P=0.0008). Mean calculated creatinine clearance was significantly lower in group B versus the average of groups A and C means throughout 60 months posttransplant (62.9±4.2 vs. 83.6±6.9 and 79.8±5.9 at 60 months, P=0.01), and death-censored graft failure was significantly higher in group B (13/43) versus groups A (5/43) and C (5/42) combined (P=0.009). Total infection and new-onset diabetes after transplant rates were not significantly different. Ad hoc analysis suggested that the inferior results in group B were specifically a result of reduced dosing and greater withholding of TAC and MMF occurring in that group. Long-term results clearly indicate inferior clinical outcomes in group B.

Histopathology and biomarkers in prediction of renal function in children after kidney transplantation

BackgroundEarly detection of chronic allograft injury is a major challenge after kidney transplantation (RTx) in adults and children. We correlated the expression of four immunohistochemical biomarkers, P-selectin glycoprotein ligand-1 (PSGL-1), vimentin, α-smooth muscle actin (α-SMA) and collagen IV, to the kidney graft histology and function in pediatric RTx patients. MethodsWe analyzed the histopathology and immunohistochemical stainings of 165 biopsies from 56 patients. Histopathology was scored according to Banff '05 classification and biomarker expression semiquantitatively. Glomerular filtration rate (GFR) was measured annually by 51Cr-EDTA clearance. ResultsIn protocol biopsies, the expression of all four biomarkers correlated with the interstitial fibrosis and tubular atrophy (IF/TA) changes, which increased during the first 36months after RTx. At the time of 18month biopsy, we observed the deterioration of GFR in patients with high (≥2) IF/TA score (50 vs. 68ml/min/1.73m2, p=0.004) or collagen IV expression (45 vs. 65ml/min/1.73m2, p=0.016). Intense stainings of IF/TA, collagen IV and vimentin are also associated with poor GFR at 36 and 48months, however, the biomarker scores revealed no additional predictive value for concomitant or late GFR compared to IF/TA score. Patients with high and low biomarker expressions showed no significant differences in annual deterioration of GFR, which declined on average 2.2ml/min/1.73m2/year over the 7years follow-up. ConclusionsOverall, the results suggest that traditional histopathology is a sufficient predictor for graft function, and the routine use of these histochemical markers as surrogates for graft function deterioration is questioned.

Expression of nestin after renal transplantation in the rat

Chronic allograft injury (CAI) limits the long-term success of renal transplantation. Nestin is a marker of progenitor cells, which probably contribute to its pathogenesis. We hypothesize that nestin is induced by ischemia/reperfusion injury and acute rejection, main risk factors for CAI. Syngeneic renal transplantation was performed in Lewis rats and allogeneic transplantation in the Fischer 344 to Lewis strain combination, which results in reversible acute rejection and in CAI in the long-run. The Dark Agouti to Lewis rat strain combination was used to study fatal acute rejection. In untreated kidneys, nestin immunoreactivity was detected in glomeruli and in very few interstitial or microvascular cells. Syngeneic transplantation induced nestin expression within 4days, which decreased until day 9 and returned to control levels on day 42. Nestin expression was strong during acute rejection and still detected during the pathogenesis of CAI on day 42. Nestin-positive cells were identified as endothelial cells and interstitial fibroblast-like cells co-expressing alpha-smooth muscle actin. A sub-population of them expressed proliferating cell nuclear antigen. In conclusion, nestin is induced in renal grafts by ischemia/reperfusion injury and acute rejection. It is expressed by proliferating myofibroblasts and endothelial cells and probably contributes to the pathogenesis of CAI.

Histologic Phenotype on 1-Year Posttransplantation Biopsy and Allograft Survival in HLA-Incompatible Kidney Transplants

The correlation between histopathologic phenotypes and allograft outcomes among patients desensitized for donor-specific antibody (HLA-incompatible) is unknown. We analyzed 1-year biopsies from desensitized recipients transplanted between 1999 and 2010 and estimated graft survival for each histologic phenotype identified. Median time posttransplant for the 1-year biopsy was 367 days (interquartile range 357-388 days) and median follow-up of all patients post-1-year biopsy was 42 months (interquartile range 19.5-65 months). Transplant glomerulopathy was present in 25.0% of biopsies and resulted in worse graft survival (66.7% vs. 96.7%, P<0.001). C4d positivity and transplant glomerulopathy together portended exceptionally poor graft survival (33.3% vs. 97.2%, P<0.001). Microcirculation inflammation was prevalent, with glomerulitis and peritubular capillaritis found in 60.0% and 47.6% of 1-year biopsies, respectively. Glomerulitis was associated with worse graft survival (82.1% vs. 98.1%, P=0.004), whereas capillaritis was not (88.1% vs. 97.7% respectively, P=0.091). Among C4d-negative HLA-incompatible recipients (82.6% of biopsies), no difference in graft survival was observed between patients with or without microcirculation inflammation in contrast to previous reports by other investigators. Patients who had no C4d deposition, transplant glomerulopathy, or microcirculation inflammation had a 100.0% graft survival. On Cox regression analysis, no independent histopathological parameter was associated with graft survival. We have identified several histopathologic phenotypes in HLA-incompatible kidney recipients that correlate with allograft outcomes. Characterization of these phenotypes is the first step towards better understanding the pathophysiologic basis of chronic antibody-mediated allograft injury and individualizing therapeutic intervention.

Protocol Biopsy Findings in Living Donor Kidney Transplant Patients Treated With Once-daily or Twice-daily Tacrolimus Formulation

BackgroundOnce-daily extended-release tacrolimus (Tac-QD) has been shown to have equivalent efficacy and safety to the twice-daily formulation (Tac-BID) in kidney transplant patients. However, detailed comparison of allograft pathology found on a protocol biopsy (PB) in Tac-QD– versus Tac-BID–based regimens has not been described. MethodsWe retrospectively investigated 119 de novo living donor kidney transplant patients treated with Tac-QD (n = 90) or Tac-BID (n = 29) and their 3- and 12-month PB results. Other immunosuppressive drugs administered included basiliximab, mycophenolate mofetil, and methylprednisolone. We evaluated daily doses and trough levels of Tac and serum creatinine levels, and compared pathologic findings. ResultsDaily doses were higher in the Tac-QD group, but trough levels and serum creatinine levels were comparable. On 3- and 12-month PB, the frequency of subclinical rejection was similar between the groups, whereas interstitial fibrosis and tubular atrophy (IF/TA) were less common in the Tac-QD group at 12 months (42.2% vs 20.6%, P = .04). Univariate and multivariate logistic regression analyses revealed that allograft rejection (borderline changes or higher) was associated with IF/TA (odds ratio 4.09, 95% confidence interval 1.76–10.10, P = .001). The Tac-QD–based regimen showed a trend toward the absence of IF/TA but it did not reach statistical significance. Tubular vacuolization and arteriolar hyaline changes were also comparable in the two groups. ConclusionsWe found a trend toward milder IF/TA, but no significant differences in kidney allograft pathology in patients who were administered Tac-QD– versus Tac-BID–based regimens at 12 months. The effects of Tac-QD on chronic allograft injury must be studied by longer observation.

Serum Endocan Correlated With Stage of Chronic Kidney Disease and Deterioration in Renal Transplant Recipients

Earlier detection and intervention for chronic renal allograft injury (CRAI) remain major challenges for transplantation physicians. Endocan plays a key role in the regulation of cell adhesion, inflammatory disorders, and tumor progression. We conducted this cross-sectional study of 97 renal transplant (RT) recipients with mean RT duration of 7.0 ± 5.7 years to determine whether Endocan could be a diagnostic and prognostic marker. The patients' mean age was 43.6 ± 13.2 years, and 55.7% (54/97) were male. Higher Endocan levels were found in more advanced chronic kidney disease (CKD) stages in a dose-dependent manner. Interestingly, the Endocan ≥643.19 pg/mL group had higher creatinine (Cr; 1.2 ± 0.4 vs 1.6 ± 1.1 mg/dL; P = .029) and lower estimated glomerular filtration rate (eGFR; 67.8 ± 23.8 mL/min vs 54.4 ± 22.0; P = .006) than the Endocan <643.19 pg/mL group after 3 months of follow-up, respectively. Linear regression analysis found tumor necrosis factor (TNF)-α correlated well with Endocan. To elucidate the response of endothelium activation, we stimulated human umbilical vein endothelial cells (HUVECs) with TNF-α in vitro, and found the levels of Endocan (P = .022) and transforming growth factor (TGF)-β1 (P = .034) increased with time, but interleukin (IL)-10 decreased (P = .013). In summary, Endocan may reflect the degree of endothelial cell injury in renal allografts, and showed a trend of elevation in late-stage CKD. An in vitro study demonstrated TNF-α–activated HUVECs secreted high levels of Endocan and TGF-β1, which could lead to a better understanding of the role of endothelium in immune balance. In conclusion, Endocan may have potential as a useful long-term indicator of CRAI in RT recipients, but further study is needed to verify our findings.

T helper subsets &amp; regulatory T cells: rethinking the paradigm in the clinical context of solid organ transplantation

Recent years have witnessed remarkable expansion in the knowledge of how various immune/inflammatory cells and T helper (Th) cell subsets, including Th1, Th2, Th9, Th17, Th22, follicular T helper (Tfh) and Treg subpopulations, reciprocally regulate each other. This review highlights current understanding of the Th subsets paradigm, who are the old school players, who are the new kids on the block and how does each come to play in different clinical contexts in solid organ transplantation. The article commences with a brief overview of the development and characteristic cytokine profiles of individual members of the paradigm. However, the main focus of this review is on the current understanding of the Th subset paradigm, and how these unique subpopulations impact host responses towards solid organ allografts. More specifically, it will highlight the recent findings that implicate the paradigm in transplantation. The interplay among different subsets is discussed collectively in the clinical context of pretransplant immunological risk factors such as alloimmunization as well as post-transplant immunological consequences such as rejection. Accumulating evidence suggests that Th17 cells play a role in the development of chronic allograft injury in transplantation of various organs. In vitro, tacrolimus suppressed Th1 and Th2 cells but not Th17 cells. Animal studies suggest that regulatory T cells (Treg)-based therapies could be effective as mechanisms of long-term drug-free transplant tolerance in humans. Indeed, a dual role for TGF-β and Foxp3 in induced tolerance has been proposed, in which TGF-β stimulates Foxp3 expression and is associated with the induction of Treg-facilitating acquisition of tolerance. Exploiting Th subsets' regulatory functions could potentially offer opportunities for immunological interventions in solid organ transplantation.

The Identification of Novel Potential Injury Mechanisms and Candidate Biomarkers in Renal Allograft Rejection by Quantitative Proteomics

Early transplant dysfunction and failure because of immunological and nonimmunological factors still presents a significant clinical problem for transplant recipients. A critical unmet need is the noninvasive detection and prediction of immune injury such that acute injury can be reversed by proactive immunosuppression titration. In this study, we used iTRAQ -based proteomic discovery and targeted ELISA validation to discover and validate candidate urine protein biomarkers from 262 renal allograft recipients with biopsy-confirmed allograft injury. Urine samples were randomly split into a training set of 108 patients and an independent validation set of 154 patients, which comprised the clinical biopsy-confirmed phenotypes of acute rejection (AR) (n = 74), stable graft (STA) (n = 74), chronic allograft injury (CAI) (n = 58), BK virus nephritis (BKVN) (n = 38), nephrotic syndrome (NS) (n = 8), and healthy, normal control (HC) (n = 10). A total of 389 proteins were measured that displayed differential abundances across urine specimens of the injury types (p < 0.05) with a significant finding that SUMO2 (small ubiquitin-related modifier 2) was identified as a "hub" protein for graft injury irrespective of causation. Sixty-nine urine proteins had differences in abundance (p < 0.01) in AR compared with stable graft, of which 12 proteins were up-regulated in AR with a mean fold increase of 2.8. Nine urine proteins were highly specific for AR because of their significant differences (p < 0.01; fold increase >1.5) from all other transplant categories (HLA class II protein HLA-DRB1, KRT14, HIST1H4B, FGG, ACTB, FGB, FGA, KRT7, DPP4). Increased levels of three of these proteins, fibrinogen beta (FGB; p = 0.04), fibrinogen gamma (FGG; p = 0.03), and HLA DRB1 (p = 0.003) were validated by ELISA in AR using an independent sample set. The fibrinogen proteins further segregated AR from BK virus nephritis (FGB p = 0.03, FGG p = 0.02), a finding that supports the utility of monitoring these urinary proteins for the specific and sensitive noninvasive diagnosis of acute renal allograft rejection.

Expression of acetylcholine receptors by experimental rat renal allografts.

Chronic allograft injury (CAI) is a major cause for renal allograft dysfunction and characterized by vasculopathies, tubular atrophy, and fibrosis. We demonstrated that numerous leukocytes interact with vascular endothelial cells of allografts and produce acetylcholine, which contributes to vascular remodeling. The cholinergic system might be a promising target for the development of novel therapies. However, neither the cellular mechanisms nor the acetylcholine receptors involved in CAI are known. Kidney transplantation was performed in the Lewis to Lewis and in the Fischer-334 to Lewis rat strain combination, which is an established experimental model for CAI. Expression of nicotinic and muscarinic acetylcholine receptors mRNA was quantified in renal tissue by real-time RT-PCR on days 9 and 42 after surgery. We detected CHRNA2–7, CHRNA10, CHRNB2, CHRNB4, and CHRM1–3 mRNA in normal kidneys and in renal transplants. In contrast, CHRNA9, CHRM4, and CHRM5 mRNA remained below the threshold of detection. In renal allografts, CHRNA3 and CHRNB4 mRNA expression were dramatically reduced compared to isografts. In conclusion, we demonstrated that most acetylcholine receptor subtypes are expressed by normal and transplanted kidneys. Allograft rejection downmodulates CHRNA3 and CHRNB4 mRNA. The role of different acetylcholine receptor subtypes in the development of CAI remains to be established.

Current Issues in the Treatment of Chronic Antibody-Mediated Rejection in Kidney Transplantation

Circulating alloantibodies are found in a substantial number of renal allograft recipients, and can induce chronic allograft injury, which is represented microscopically as transplant glomerulopathy and diffuse C4d deposition in peritubular capillaries (PTCs). Development of these injuries is significantly correlated with late allograft loss, and in this regard, it was included as a new disease entity named chronic antibody-mediated rejection (cAMR) in the updated Banff 05 classification. Usually, the prognosis of cAMR is poor and conventional immunosuppressants mainly targeting T cell-mediated immunity cannot prevent or reverse it. Therefore, some researchers have suggested that therapies directed at the humoral response may be required for the treatment of cAMR. Recently, some reports have suggested that the combined use of rituximab and intravenous immunoglobulin (IVIg) therapy may be useful for the treatment of cAMR. Our previous study also showed that rituximab and IVIg combination therapy effectively delayed the progression of cAMR. We administered rituximab and IVIg combination therapy to 18 biopsy-proven cAMR patients and found that it significantly slowed the decline of the estimated glomerular filtration rate. However, this effect was limited in patients with heavy proteinuria, and dissipated in all patients by 1 year post-treatment. Recently, new drugs targeting the humoral immune system, such as bortezomib and eculizumab, have been tested for the treatment of cAMR. However, the studies still lack definitive data in terms of successful treatment of cAMR. We speculate that those therapies will compensate for the limitation of previous anti-humoral therapies for cAMR.

A Randomized Pilot Study of Donor Stem Cell Infusion in Living-Related Kidney Transplant Recipients Receiving Alemtuzumab

Transplant tolerance would remove the need for maintenance immunosuppression while improving survival and quality of life. A prospective, randomized pilot study was undertaken to assess the safety and efficacy of donor stem cell infusion (DSCI) in living-related kidney transplant recipients treated with alemtuzumab (C1H) induction and tacrolimus and mycophenolate maintenance with switch to sirolimus and weaning over 2 years. Four patients received DSCI; five patients were controls. Graft failure occurred in two patients in the DSCI arm. Recurrence of glomerular disease occurred in two DSCI recipients, leading to graft loss in one. Biopsy-proven acute rejection episodes occurred in three patients (two in the DSCI vs. one in the control). One DSCI patient, with recurrence, subsequently developed antibody-mediated rejection leading to graft failure. In the remaining two DSCI patients, weaning was attempted but was not successful. All (4 of 4) DSCI patients had biopsy-proven chronic allograft injury and/or recurrence. DSCI with C1H induction and a steroid-free maintenance regimen in a small group of patients failed to induce tolerance, with suboptimal patient and graft survival. The results do not justify extension of this particular trial and underscore the importance of patient selection, specifically avoidance of patients with glomerulopathies whose recurrence may obscure potential benefit.

High-Throughput Proteomic Approaches to the Elucidation of Potential Biomarkers of Chronic Allograft Injury (CAI).

This review focuses on the role of OMICs technologies, concentrating in particular on proteomics, in biomarker discovery in chronic allograft injury (CAI). CAI is the second most prevalent cause of allograft dysfunction and loss in the first decade post-transplantation, after death with functioning graft (DWFG). The term CAI, sometimes referred to as chronic allograft nephropathy (CAN), describes the deterioration of renal allograft function and structure as a result of immunological processes (chronic antibody-mediated rejection), and other non-immunological factors such as calcineurin inhibitor (CNI) induced nephrotoxicity, hypertension and infection. Current methods for assessing allograft function are costly, insensitive and invasive; traditional kidney function measurements such as serum creatinine and glomerular filtration rate (GFR) display poor predictive abilities, while the current “gold-standard” involving histological diagnosis with a renal biopsy presents its own inherent risks to the overall health of the allograft. As early as two years post-transplantation, protocol biopsies have shown more than 50% of allograft recipients have mild CAN; ten years post-transplantation more than 50% of the allograft recipients have progressed to severe CAN which is associated with diminishing graft function. Thus, there is a growing medical requirement for minimally invasive biomarkers capable of identifying the early stages of the disease which would allow for timely intervention. Proteomics involves the study of the expression, localization, function and interaction of the proteome. Proteomic technologies may be powerful tools used to identify novel biomarkers which would predict CAI in susceptible individuals. In this paper we will review the use of proteomics in the elucidation of novel predictive biomarkers of CAI in clinical, animal and in vitro studies.

Renal Heparan Sulfate Proteoglycans Modulate Fibroblast Growth Factor 2 Signaling in Experimental Chronic Transplant Dysfunction

Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non-FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction.