M. Żabińska

The influence of non‐HLA antibodies directed against angiotensin II type 1 receptor (AT1R) on early renal transplant outcomes

Non‐HLA antibodies (Abs) targeting vascular receptors are thought to have an impact on renal transplant injury. Anti‐angiotensin II type 1‐receptor‐activating antibodies (anti‐AT1R) have been mentioned to stimulate a severe vascular rejection, but the pretransplant screening has not been introduced yet. The aim of our study was to assess the incidence and importance of anti‐AT1R antibodies and their influence on renal transplant in the 1st year of observation. We prospectively evaluated the presence of anti‐AT1R antibodies in 117 consecutive renal transplant recipients in pre‐ and post‐transplant screening. Anti‐AT1R antibodies were observed in 27/117 (23%) of the analyzed recipients already before transplantation. The function of renal transplant was considerably worse in anti‐AT1R(+) group. The patients with anti‐AT1R Abs >9 U/ml lost their graft more often. Biopsy‐proven AR was described in 4/27 (15%) pts in the anti‐AT1R(+) group and 13/90 (14.4%) in the anti‐AT1R(−) group, but more severe cases of Banff IIB or antibody‐mediated rejection (AMR) were more often observed in anti‐AT1R (+) 4/27 (15%) vs. 1/90 (1.1%) in anti‐AT1R(+) (P = 0.009). Patients with anti‐AT1R Abs level >9 U/ml run a higher risk of graft failure independently of classical immunological risk factors. The recipients with anti‐AT1R Abs developed more severe acute rejections described as IIB or AMR in Banff classification. More recipients among the anti‐AT1R‐positive ones lost the graft. Our study suggests monitoring of anti‐AT1R Abs before renal transplantation for assessment of immunologic risk profiles and the identification of patients highly susceptible to immunologic events, graft failure, and graft loss.

The Complement Cascade and Renal Disease

Serum complement cascade, a part of innate immunity required for host protection against invading pathogens, is also a mediator of various forms of disease and injury. It is activated by classical, lectin, and alternative pathways that lead to activation of C3 component by C3 convertases, release of C3b opsonin, C5 conversion and eventually membrane attack complex formation. The tightly regulated activation process yields also C3a and C5a anaphylatoxins, which target a broad spectrum of immune and non-immune cells. The review discusses the involvement of the complement cascade in kidney disease pathogenesis and injury. The role of the complement pathways in autoantibody-mediated forms of glomerulonephritis (lupus nephritis, anti-glomerular basement membrane disease, anti-neutrophil cytoplasmic autoantibody-induced or membranoproliferative glomerulonephritis, membranous nephropathy), C3 glomerulopathy, atypical forms of hemolytic uremic syndrome, ischemic-reperfusion injury of transplanted kidney, and antibody-mediated renal allograft rejection are discussed. The disturbances in complement activation and regulation with underlying genetics are presented and related to observed pathology. Also promising strategies targeting the complement system in complement-related disorders are mentioned.

The impact of non-HLA antibodies directed against endothelin-1 type A receptors (ETAR) on early renal transplant outcomes.

BACKGROUND Non-HLA antibodies (Abs) targeting vascular receptors are considered to have an influence on renal transplant injury. Anti-endothelin-1 type A receptor (anti-ETAR) antibodies were associated with cellular and antibody-mediated rejection and early onset of vasculopathy in heart transplant patients but their role in renal transplantation remains unclear. The aim of our study was to assess the incidence and importance of anti-ETAR antibodies and their impact on renal transplant during the first year observation. METHODS We evaluated the presence of anti-ETAR antibodies in 116 consecutive renal transplant recipients in pre- and post-transplant screening (before and in 1st, 3rd, 6th, 12th month after transplantation). Additionally, we assessed the presence of anti-HLA antibodies. Anti-ETAR antibodies were assayed by ELISA. The diagnosis of acute rejection was based on the Banff criteria. RESULTS Anti-ETAR antibodies were observed in 55 (47.4%) of the analyzed recipients before transplantation. The function of renal transplant was significantly worse in the anti-ETAR(+) group compared to the anti-ETAR(-) group during the first post-transplant year. One month after transplantation the serum creatinine in anti-ETAR (+) patients (pts) was 1.86±0.8mg/dl and 1.51±0.5 in anti-ETAR(-) pts (p=0.009). Twelve months after transplantation the difference between the groups was still observed 1.70±0.7 vs. 1.40±0.4 (p=0.04). Biopsy proven acute rejection was recognized in 8/55 (14.5%) in ETAR(+) and 9/61 (14.8%) in ETAR(-) patients but cases with mild to severe intimal arteritis (v1-v3) were more often observed in patients with the presence of anti-ETAR Abs 4/55 (7.2%) comparing with 1/61 (1.6%) in anti-ETAR(-) patients. The anti-ETAR antibody levels varied at different measurement intervals during the one-year follow-up. CONCLUSIONS The presence of anti-ETAR antibodies is associated with a worse renal transplant function during the first 12months after transplantation. Including anti-ETAR antibodies in the diagnostics of renal transplant recipient immune status should be considered to provide comprehensive assessment of humoral alloimmunity.

Long-term Follow-up of Non-HLA and Anti-HLA Antibodies: Incidence and Importance in Renal Transplantation

BACKGROUND Detection of antibody-mediated injury is becoming increasingly important in post-transplant patient care. The role of donor-specific anti-human leukocyte antigen (HLA) antibodies in kidney transplant damage is known, whereas the significance of non-HLA antibodies remains an unresolved concern. The aim of the study was to determine the presence and influence on renal function of non-HLA and anti-HLA antibodies in stable patients at 5 years after kidney transplantation. METHODS We evaluated the antibodies in 35 consecutive patients with stable renal function at 5 years after transplantation. RESULTS Pretransplant screening for donor-specific antibodies by CDC cross-matches was negative in all patients. Anti-endothelial cell antibodies (AECA), anti-angiotensin II type 1 receptor antibodies (anti-AT1R), and anti-endothelin receptor antibodies (anti-ETAR) were assayed as non-HLA antibodies. Non-HLA antibodies were observed in 12 (34%) patients, including AECA (n = 5; 14%), anti- AT1R (n = 6; 17%), anti-ETAR (n = 4; 11%), and both anti-AT1R and anti-ETAR (n = 3). Among 13 (37%) patients with anti-HLA antibodies, 7 also had both non-HLA antibodies: AECA (n = 1), anti-AT1R (n = 3), and anti-ETAR (n = 3). The antibody-negative group (n = 13) showed significantly better renal function than the antibody-positive group (non-HLA and/or anti-HLA; n = 22). Biopsy-proven acute rejection had occurred in 2 of 13 (15%) antibody-negative versus 8 of 22 (36%) antibody-positive patients. These preliminary data revealed an high prevalence of autoantibody and alloantibody production among stable patients at 5 years after kidney transplantation. CONCLUSION Simultaneous production of these antibodies and their association with reduced renal function suggests that active humoral immune responses are poorly controlled by immunosuppression.

Non-HLA antibodies: angiotensin II type 1 receptor (anti-AT1R) and endothelin-1 type A receptor (anti-ETAR) are associated with renal allograft injury and graft loss.

INTRODUCTION Non-HLA antibodies specific for angiotensin II type 1 receptor (anti-AT1R) and endothelin-1 type A receptor (anti-ETAR) of vascular cells activate signaling pathways leading to cell proliferation and vascular injury. The aim of this study was to evaluate the impact of non-HLA antibodies on kidney allograft morphology and function in patients who underwent a kidney biopsy due to renal function impairment. PATIENTS AND METHODS The study included 65 consecutive renal transplant patients who were evaluated for the presence of non-HLA and anti-HLA antibodies at the time of transplant biopsy. Results of pre-transplant CDC cross-match were negative. A kidney allograft biopsy was performed between 6 days and 13 years (42 ± 49 months) after transplantation, and the diagnosis was made on the basis of the Banff criteria. The level >9 U/L of anti-AT1R and anti-ETAR antibodies was considered high. RESULTS A high level of non-HLA antibodies (anti-AT1R and/or anti-ETAR) was found in 7 (10.7%) of 65 patients at the time of biopsy. Graft loss in the non-HLA-positive patients was significantly higher (71% in non-HLA-positive cases after 7.8 ± 2.6 months vs 11% after 6 months in non-HLA-negative cases [P = .00099]). In these non-HLA-positive patients, the mean anti-AT1R level was 15.3 ± 9.4 U/L and the mean anti-ETAR level was 13.8 ± 8.6 U/L. In only 2 of these patients were anti-HLA antibodies additionally detected: anti-class I in 1 and anti-class II in both patients. The mean serum creatinine level was 2.34 ± 0.6 mg/dL at the time of biopsy. Results of an early biopsy revealed acute vascular rejection (Banff grade IIB). Chronic allograft injury was found (grading cg1-3, cv1-2, ci1-2, ct1-2) in the remaining 6 patients. C4d was present in 3 of 7 patients. CONCLUSIONS High levels of anti-AT1R and/or anti-ETAR antibodies were associated with morphological and functional allograft injury and graft loss in these study patients. Non-HLA antibodies can be helpful in assessing the risk of graft failure.

Extracorporeal photopheresis as an antirejection prophylaxis in kidney transplant recipients: preliminary results.

Extracorporeal photopheresis (ECP) is considered a promising immunomodulatory therapy of acute allograft rejection in organ transplantation and graft-versus-host disease. Our aim was to investigate the biological responses of 10 patients who underwent kidney transplantation with ECP as prophylactic treatment. They received conventional immunosuppressive therapy plus ECP immediately after transplantation: 12 to 16 applications over the course of 2.5 months. ECP procedures were performed using an automated system for leukocyte separation and photoactivation with methoxsalen. All recipients were followed by estimated glomerular filtration rate (eGFR) and peripheral T, B, natural killer, T-regulatory (Treg) and dendritic cells (DC) counts and phenotypes. An acute rejection episode appeared in one control group recipient. The ECP group showed a positive trend to an higher GFR at months 3 (53±11 vs 47.1±9; P=.17) and 6 (67.5±10 vs 53.6±3; P=.03, Wilcoxon test). An increased percentage of Treg (CD3+ CD4+ CD25+) among the total CD3 cell count (4.9%±1% to 9.4%±15%) as well as inducible Treg (CD3+ CD8+ CD28-) was observed among CD3 cells (3.3%±3% to 11.8%±8%, P=.025) within 3 months of ECP treatment. A significant difference in the percentage of Treg was noted at month 3 (completed ECP) between the ECP and the control groups (9.4%±15% vs 3%±1%; P=.01). Addition of ECP to standard immunosuppression was associated with a significantly higher GFR at 6 months and with a significant increase in natural Treg among CD3 cells.

Imbalance of Metallaproteinase/Tissue Inhibitors of Metalloproteinase System in Renal Transplant Recipients With Chronic Allograft Injury

INTRODUCTION Nowadays, renal allografts continue to be lost at the rate of 2% to 4% per year beyond the first year after transplantation due to chronic allograft injury. Excessive accumulation of extracellular matrix results from overproduction and/or defective degradation by proteolytic enzymes, among which metalloproteinases (MMPs) play a major role. The aim of this study was to assess the role of MMPs in renal transplant recipients (RTR) in the context of allograft injury or proteinuria. MATERIALS AND METHODS Plasma and urine MMP-2 and MMP-9 and tissue inhibitors of metalloproteinases (TIMPs) were assessed by enzyme-linked immunoassay in 150 RTR including 66% males with an overall mean age of 49.2±11.5 years. The subjects were examined at a mean of 73.4±41.2 months (range=12-240) after kidney transplantation. Thirty-seven healthy volunteers including 54% male with an overall mean age of 48.4±14.1 years served as a control group. RESULTS Renal transplant recipients displayed significantly decreased plasma MMP-2 activity compared with healthy controls (P<.000) probably due to increased inhibitory plasma (p) TIMP-2 activity (P=.0029), and lower plasma MMP-2:TIMP-2 index (P<.0001). Plasma MMP-9 and pTIMP-1 activities were twofold increased in RTR compared with controls (P=.0015 and P<.000) but with a nearly stable plasma MMP-9:TIMP-1 index (P=NS). There was no difference between RTR and controls according to urine (u) MMP-2 activity, but uMMP-9 was increased in RTR compared with healthy controls (P=.0032). Urine MMP-9 potential was probably diminished by increased uTIMPs (uTIMP-2, P=.017; uTIMP-1, P=.000), which contributed to graft impairment or proteinuria. CONCLUSION Our study revealed profibrotic MMP/TIMP constellations in RTR that show an imbalance in plasma MMP-2 and MMP-9 with increased plasma and urinary TIMPs. The net proteolytic potential of increased plasma and urinary MMP-9 may be diminished significantly by enhanced plasma and urine TIMP activities.

Advanced age of renal transplant recipients correlates with increased plasma concentrations of interleukin-6, chemokine ligand 2 (CCL2), and matrix metalloproteinase 2, and urine concentrations of CCL2 and tissue inhibitor of metalloproteinase 1.

BACKGROUND Advanced age of renal transplant recipients (RTRs) has a negative impact on kidney allograft survival through impaired extracellular matrix degradation by the matrix metalloproteinases/tissue inhibitors of metalloproteinases (MMPs/TIMPs) system. Moreover, older RTRs are at risk of smoldering inflammation, known as inflammaging. AIM The aim of the study was to assess the impact of a RTRs age on plasma and urine concentrations of interleukin 6 (IL-6), chemokine ligand 2 (CCL2), and the MMPs/TIMPs system. MATERIAL AND METHODS One hundred fifty adult RTRs (8.7% ≥ 65 years) and 37 adult healthy volunteers (10.8% ≥ 65 years) were enrolled in the study. The studied factors (IL-6, CCL2, MMP-2, MMP-9, TIMP-1 and TIMP-2) were quantified in plasma and urine with enzyme-linked immunosorbent assay. The Mann-Whitney U test and Spearmans (rs) rank correlation were applied, and differences with a P < .05 were considered statistically significant. RESULTS There was a weak but significant positive correlation between increasing RTRs age and plasma IL-6 (rs = 0.18, P = .028), CCL2 (rs = 0.27, P = .001), and MMP-2 (rs = 0.20, P = .017), as well as urine CCL2 (rs = 0.16, P = 0.050) and TIMP-1 (rs = 0.20, P = .014) concentrations. CONCLUSIONS Advancing age of RTRs correlates with increasing plasma IL-6 and CCL2 concentrations, reflecting smoldering inflammation (known as inflammaging) and alterations in MMPs/TIMPs profiles, especially with increased plasma MMP-2 and urine TIMP-1 concentrations.

Increased Plasma Tissue Inhibitors of Metalloproteinase Concentrations as Negative Predictors Associated With Deterioration of Kidney Allograft Function Upon Long-Term Observation

Chronic allograft injury (CAI) is the most frequent cause of progressive kidney allograft impairment and eventual loss, which is due to interstitial fibrosis and tubular atrophy (IF/TA). Mechanisms of CAI are not fully understood. Chemokines, cytokines, metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) play roles in fibrosis development. The aims of this study were to evaluate plasma and urine TIMPs (TIMP-1 and TIMP-2), MMPs (MMP-2 and MMP-9), proinflammatory interleukin-6 (IL-6), chemokine (C-C motif) ligand 2 (CCL2 chemokines previously known as monocyte chemoattractant protein-1 [MCP-1]) among 150 recipients beyond 1 year post-renal transplantations and to explore the usefulness of these potential biomarkers of ongoing allograft injury. Renal transplant recipients compared with healthy volunteers (control group) showed significantly increased plasma and urine IL-6, MMP-9, TIMP-1, and TIMP-2, as well as lower plasma MMP-2 and urine CCL2 concentrations. Compared with recipients showing good function those with impairments displayed higher plasma TIMP-1 (P < .001) and TIMP-2 (P = .003) concentrations. The recipient estimated glomerular filtration rate (eGFR) values negatively correlated with plasma TIMP-1 and TIMP-2 levels (r = -0.43; P < .0001 and rs = -0.42; P < .0001, respectively) and with urine IL-6 excretion (rs = -0.33; P < .0001). Multivariate and receiver operating characteristic (ROC) analyses showed TIMP-1 plasma level assessments to be useful estimates of allograft injury.

CD3+CD8+CD28− T Lymphocytes in Patients with Lupus Nephritis

The results of studies on the CD3+CD8+CD28− cells in SLE are inconsistent since several analyses describe CD3+CD8+CD28− as either immunosuppressive or cytotoxic. The aim of this study is to inquire whether the quantitative changes of CD3+CD8+CD28− T lymphocytes subpopulation are related to the clinical status of patients with lupus nephritis. Evaluation of Foxp3 expression on CD3+CD8+CD28− cells may shed some light on functional properties of these cells. 54 adult SLE patients and 19 sex and age matched healthy volunteers were enrolled in the study. There were 15 patients in inactive (SLEDAI ≤ 5) and 39 in active (SLEDAI > 5) phase of disease. We determined absolute count of CD3+CD8+CD28− and CD3+CD8+CD28−Foxp3+ subpopulations by flow cytometry. We observed a statistically significant increase in absolute count and percentage of CD3+CD8+CD28− in SLE patients compared to HC (p < 0.001). Moreover there was significant positive correlation between increasing absolute count of CD3+CD8+CD28− cells and disease activity measured by SLEDAI (rs = 0.281, p = 0.038). Active LN patients had increased absolute count of CD3+CD8+CD28− cells compared to HC. Positive correlation of CD3+CD8+CD28− number with disease activity, and lack of Foxp3 expression on these cells, suggests that CD3+CD8+CD28− lymphocytes might be responsible for an increased proinflammatory response in the exacerbation of SLE.