Arjang Djamali

Diagnosis and management of antibody-mediated rejection: Current status and novel approaches

Advances in multimodal immunotherapy have significantly reduced acute rejection rates and substantially improved 1‐year graft survival following renal transplantation. However, long‐term (10‐year) survival rates have stagnated over the past decade. Recent studies indicate that antibody‐mediated rejection (ABMR) is among the most important barriers to improving long‐term outcomes. Improved understanding of the roles of acute and chronic ABMR has evolved in recent years following major progress in the technical ability to detect and quantify recipient anti‐HLA antibody production. Additionally, new knowledge of the immunobiology of B cells and plasma cells that pertains to allograft rejection and tolerance has emerged. Still, questions regarding the classification of ABMR, the precision of diagnostic approaches, and the efficacy of various strategies for managing affected patients abound. This review article provides an overview of current thinking and research surrounding the pathophysiology and diagnosis of ABMR, ABMR‐related outcomes, ABMR prevention and treatment, as well as possible future directions in treatment.

Heat shock protein 27 (HSP27): biomarker of disease and therapeutic target

Heat shock protein 27 (HSP27) is a multidimensional protein which acts as a protein chaperone and an antioxidant and plays a role in the inhibition of apoptosis and actin cytoskeletal remodeling. In each of these capacities, HSP27 has been implicated in different disease states playing both protective and counter-protective roles. The current review presents HSP27 in multiple disease contexts: renal injury and fibrosis, cancer, neuro-degenerative and cardiovascular disease, highlighting its role as a potential biomarker and therapeutic target.

Epithelial-to-mesenchymal transition and oxidative stress in chronic allograft nephropathy.

Epithelial‐to‐mesenchymal transition (EMT) and oxidative stress contribute to kidney tissue fibrosis in various forms of native kidney disease. However, their role in chronic allograft nephropathy (CAN) remains somewhat uncertain. To address this question, kidney transplants were performed in 3‐month‐old rats, using the Fisher 344 → Lewis model of CAN. Six‐month posttransplant, kidney allografts displayed significant tubular atrophy, interstitial fibrosis and vascular wall thickening. Allograft recipients had significantly higher levels of serum creatinine (4.7 ± 1.3 versus 0.59 ± 0.08 mg/dL, p = 0.03) and proteinuria (380 ± 102 versus 30.2 ± 8 mg/dL, p = 0.04) compared to syngeneic grafts. Semiquantitative PCR, immunoblot and immunohistochemical analyses demonstrated increased α‐smooth muscle actin (α‐SMA) mRNA and protein levels coupled with reduced E‐cadherin mRNA and protein immunoreactivity, confirming the presence of CAN‐associated EMT. Allograft α‐SMA levels were increased as early as 1–2 weeks posttransplant. Immunohistochemical studies for collagen type I and III, superoxide anion (O2−), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) confirmed that tubular O2−, eNOS and iNOS, and interstitial collagen I, III and O2− levels were significantly increased in CAN‐associated EMT. In conclusion, these observations suggest that CAN‐associated EMT may be a link between oxidative stress and allograft fibrosis.

Medical Care of Kidney Transplant Recipients after the First Posttransplant Year

Kidney transplantation is the treatment of choice for patients with ESRD. Despite improvements in short-term patient and graft outcomes, there has been no major improvement in long-term outcomes. The use of kidney allografts from expanded-criteria donors, polyoma virus nephropathy, underimmunosuppression, and incomplete functional recovery after rejection episodes may play a role in the lack of improvement in long-term outcomes. Other factors, including cardiovascular disease, infections, and malignancies, also shorten patient survival and therefore reduce the functional life of an allograft. There is a need for interventions that improve long-term outcomes in kidney transplant recipients. These patients are a unique subset of patients with chronic kidney disease. Therefore, interventions need to address disease progression, comorbid conditions, and patient mortality through a multifaceted approach. The Kidney Disease Outcomes Quality Initiative from the National Kidney Foundation, the European Best Practice Guidelines, and the forthcoming Kidney Disease: Improving Global Outcomes clinical practice guidelines can serve as a cornerstone of this approach. The unique aspects of chronic kidney disease in the transplant recipient require the integration of specific transplant-oriented problems into this care schema and a concrete partnership among transplant centers, community nephrologists, and primary care physicians. This article reviews the contemporary aspects of care for these patients.

Epithelial-to-mesenchymal transition and chronic allograft tubulointerstitial fibrosis.

Chronic allograft tubular atrophy/interstitial fibrosis (TA/IF) is a major cause of late allograft loss. A major challenge to the future of kidney transplantation is to dissect the identifiable causes of chronic allograft TA/IF and to develop cause-specific treatment strategies. Emerging evidence suggests that epithelial-to-mesenchymal transition (EMT) is an important event in native and transplant kidney injury, including chronic allograft TA/IF. During EMT, tubular epithelial cells are transformed into myofibroblasts through a stepwise process including loss of cell-cell adhesion and E-cadherin expression, de novo alpha-smooth muscle actin expression, actin reorganization, tubular basement membrane disruption, cell migration, and fibroblast invasion with production of profibrotic molecules such as collagen types I and III and fibronectin. We examined in this review the molecular and cellular pathways of EMT and their involvement in chronic allograft tubulointerstitial fibrosis. We examined the role of alloimmune T cells and oxidative stress in this context and evaluated EMT as a marker of disease progression. Potential therapeutic options are discussed. In conclusion, there is enough evidence demonstrating that EMT is involved in the pathogenesis of chronic allograft tubulointerstitial fibrosis. However, the extent of its contribution to allograft fibrogenesis remains unknown, and only interventional trials will enable us to clarify this question. Furthermore, additional data are required to determine whether EMT may be used as a surrogate marker of disease progression in kidney transplant recipients.

C1q Binding Activity of De Novo Donor-specific HLA Antibodies in Renal Transplant Recipients With and Without Antibody-mediated Rejection.

Background Complement fixation by donor-specific HLA antibodies (DSA) is a primary mechanism for antibody-mediated damage of organ allografts. Using a recently developed kit that measures C1q binding to distinguish complement fixing and nonfixing antibodies, studies showed that C1q + DSAs have a higher risk of rejection and graft loss compared to C1q-DSA. The objective of this study was to assess the ability of the C1q-binding assay to identify clinically significant de novo DSA in renal transplant recipients and to define the properties of DSA that confer C1q binding ability. Methods The DSA-positive sera from 34 kidney recipients, 19 with biopsy-proven antibody-mediated rejection (AMR) + and 15 who were AMR−, were assayed in C1q-binding assays (C1q Screen; One Lambda, Inc. Canoga Park, CA). The correlation between C1q-binding activity, presence of AMR, DSA mean fluorescence intensity (MFI) values, and immunoglobulin G isotype was determined. Results Fifty-three percent (10/19) of sera from AMR+ patients had C1q + DSA, whereas only 13% (2/15) of sera from AMR− patients contained C1q + DSA. C1q + DSA exhibited significantly higher MFI values regardless of whether they were from AMR+ or AMR− patients (16,118 ± 6698 vs 6429 ± 4003; P < 0.0001). C1q + DSA converted to C1q − when diluted to a comparable MFI level as the C1q − DSA from AMR− patients, and some C1q − antibodies converted to C1q + when concentrated to MFI levels comparable to those observed for AMR+/C1q + sera. Conclusions The C1q binding activity by de novo DSA in patients with AMR largely reflects differences in antibody strength. The C1q assay does not appear to distinguish functionally distinct DSA with clinical significance.

Pretransplant donor-specific antibodies detected by single-antigen bead flow cytometry are associated with inferior kidney transplant outcomes.

Background. The clinical significance of pretransplant donor-specific antibodies (pre-Tx DSAs) detected by single-antigen bead flow cytometry (SAB-FC) remains unclear. Methods. To investigate the impact that pre-Tx DSAs detected by SAB-FC have on early clinical outcomes, we tested pre-Tx sera from all consecutive deceased-donor kidney transplants performed between January 2005 and July 2006 (n=237). Results. In the study population of which 66% had a high-immunologic risk, mean fluorescence intensity (MFI) more than or equal to 100 for class I and more than or equal to 200 for class II were the lowest DSA thresholds associated with inferior antibody-mediated rejection-free graft survival (75% vs. 90%, P=0.004 and 76% vs. 87%, P=0.017, respectively). The hazard ratio for antibody-mediated rejection increased linearly with higher class II DSA from MFI 100 to 800 (1.7[0.8–3.2], P=0.1 for MFI≥100 vs. 4.7[2.4–8.8], P<0.001 for MFI ≥800). Differences in graft function were only evident in patients with class II MFI more than or equal to 500 (estimated glomerular filtration rate: 47.6 vs. 54.3, P=0.02 and proteinuria: 0.6±0.6 vs. 0.4±0.3, P=0.03). A difference in death-censored graft survival was detected in patients with class II MFI more than or equal to 1000 (75% vs. 91.9%, P=0.055). Conclusion. High-pre-Tx DSAs detected by SAB-FC are associated with incrementally poor graft outcomes in deceased-donor kidney transplant with high-immunologic risk.

Blood oxygen level-dependent and perfusion magnetic resonance imaging: detecting differences in oxygen bioavailability and blood flow in transplanted kidneys

Functional magnetic resonance imaging (fMRI) is a powerful tool for examining kidney function, including organ blood flow and oxygen bioavailability. We have used contrast enhanced perfusion and blood oxygen level-dependent (BOLD) MRI to assess kidney transplants with normal function, acute tubular necrosis (ATN) and acute rejection. BOLD and MR-perfusion imaging were performed on 17 subjects with recently transplanted kidneys. There was a significant difference between medullary R2 values in the group with acute rejection (R2=16.2/s) compared to allografts with ATN (R2=19.8/s; P=.047) and normal-functioning allografts (R2=24.3/s;P=.0003). There was a significant difference between medullary perfusion measurements in the group with acute rejection (124.4+/-41.1 ml/100 g per minute) compared to those in patients with ATN (246.9+/-123.5 ml/100 g per minute; P=.02) and normal-functioning allografts (220.8+/-95.8 ml/100 g per minute; P=.02). This study highlights the utility of combining perfusion and BOLD MRI to assess renal function. We have demonstrated a decrease in medullary R2 (decrease deoxyhemoglobin) on BOLD MRI and a decrease in medullary blood flow by MR perfusion imaging in those allografts with acute rejection, which indicates an increase in medullary oxygen bioavailability in allografts with rejection, despite a decrease in blood flow.

Nox-2 Is a Modulator of Fibrogenesis in Kidney Allografts

We studied the role of classical phagocytic NADPH oxidase (Nox) in the pathogenesis of kidney allograft tubulointerstitial fibrosis. Immunofluorescence studies showed that Nox‐2 and p22phox (electron transfer subunits of Nox) colocalized in the tubulointerstitium of human kidney allografts. Tubular Nox‐2 also colocalized with α‐SMA in areas of injury, suggestive of epithelial‐to‐mesenchymal transition (EMT). Interstitial macrophages (CD68+) and myofibroblasts (α‐SMA+) expressed Nox‐2 while graft infiltrating T cells (CD3+) and mature fibroblasts (S100A4+) were Nox‐2−. These results were confirmed in the Fisher‐to‐Lewis rat kidney transplant model. Areas of tubulitis were associated with Nox‐2 and α‐SMA, suggestive of EMT. Immunoblot analyses showed that Nox‐2 upregulation was associated with oxidative stress (nitrotyrosine) and fibrogenesis (α‐SMA and phospho‐Smad2) at 3 weeks and 6 months. Allografts treated with Nox inhibitors (DPI or apocynin) for 1 week showed reduced fibronectin and phospho‐Smad2 and increased E‐cadherin levels. Cyclosporine A, TGF‐β1 and angiotensin II increased Nox‐2 mRNA levels 2‐ to 7‐fold in vitro (NRK52E cells). Treatment with specific Nox inhibitors (DPI or apocynin) prevented the downregulation of E‐cadherin and upregulation of fibronectin transcripts. In aggregate, these studies suggest that Nox‐2 is involved in the pathogenesis of allograft tubulointerstitial fibrosis via activation transcription factor Smad2, EMT and myofibroblasts.

Low dose antithymocyte globulins in renal transplantation: daily versus intermittent administration based on T-cell monitoring.

BACKGROUND Despite the long history of use of antithymocyte globulins (ATG) in renal transplantation, ideal doses and duration of ATG administration based on the monitoring of T lymphocytes have yet to be defined. METHODS Two immunosuppressive regimens based on low-dose rabbit ATG (Thymoglobuline; Imtix-Sang-stat, Lyon, France) were assessed during the first year after transplantation: daily ATG (DATG; n=23) where 50 mg of ATG was given every day and intermittent ATG (IATG; n=16) where similar doses of ATG were given for the first 3 days and then intermittently only if CD3+ T lymphocytes (measured by flow cytometry) were > 10/mm3. Both groups received steroids, azathioprine, and cyclosporine. RESULTS ATG-induced depletion was similar for peripheral blood lymphocytes and T cells in both groups: it began at day 1 after transplantation, was submaximal at day 3, and reached maximum intensity between days 6 and 8, from which time cell counts progressively increased. However, T-cell depletion was still present at day 20. The total ATG dose per patient (381.5+/-121 vs. 564+/-135 mg/patient) and the mean cumulative daily dose of ATG (0.60+/-0.17 vs. 0.80+/-0.14 mg/kg/day) were significantly lower in the IATG group (P=0.0001 and 0.0006, respectively). The overlap of ATG and cyclosporine treatment was 6.7+/-3 vs. 7.4+/-4.3 days (P=NS), and the mean duration of ATG therapy was 11.3+/-3.2 vs. 11.6+/-2.7 days in the IATG and DATG groups, respectively (P=NS). ATG was given in an average of one dose every 1.6 days in the IATG group compared with one dose daily in the DATG group (P=7 x 10(-7)). There was no significant difference in renal graft function, the number of acute graft rejections, or ATG-related side effects and complications. Despite the daily immunological follow-up, there was a net saving of