Lisa A. Robinson

A Role for Fractalkine and Its Receptor (CX3CR1) in Cardiac Allograft Rejection

The hallmark of acute allograft rejection is infiltration of the inflamed graft by circulating leukocytes. We studied the role of fractalkine (FKN) and its receptor, CX3CR1, in allograft rejection. FKN expression was negligible in nonrejecting cardiac isografts but was significantly enhanced in rejecting allografts. At early time points, FKN expression was particularly prominent on vascular tissues and endothelium. As rejection progressed, FKN expression was further increased, with prominent anti-FKN staining seen around vessels and on cardiac myocytes. To determine the capacity of FKN on endothelial cells to promote leukocyte adhesion, we performed adhesion assays with PBMC and monolayers of TNF-α-activated murine endothelial cells under low-shear conditions. Treatment with either anti-FKN or anti-CX3CR1-blocking Ab significantly inhibited PBMC binding, indicating that a large proportion of leukocyte binding to murine endothelium occurs via the FKN and CX3CR1 adhesion receptors. To determine the functional significance of FKN in rejection, we treated cardiac allograft recipients with daily injections of anti-CX3CR1 Ab. Treatment with the anti-CX3CR1 Ab significantly prolonged allograft survival from 7 ± 1 to 49 ± 30 days (p < 0.0008). These studies identify a critical role for FKN in the pathogenesis of acute rejection and suggest that FKN may be a useful therapeutic target in rejection.

Child and parental perspectives of multidimensional quality of life outcomes after kidney transplantation

Anthony SJ, Hebert D, Todd L, Korus M, Langlois V, Pool R, Robinson LA, Williams A, Pollock‐BarZiv SM. Child and parental perspectives of multidimensional quality of life outcomes after kidney transplantation.
Pediatr Transplantation 2010:14:249–256.

Proinflammatory Actions of Thromboxane Receptors to Enhance Cellular Immune Responses

Metabolism of arachidonic acid by the cyclo-oxygenase (COX) pathway generates a family of prostanoid mediators. Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting COX, thereby reducing prostanoid synthesis. The efficacy of these agents in reducing inflammation suggests a dominant proinflammatory role for the COX pathway. However, the actions of COX metabolites are complex, and certain prostanoids, such as PGE2, in some circumstances actually inhibit immune and inflammatory responses. In these studies, we examine the hypothesis that anti-inflammatory actions of NSAIDs may be due, in part, to inhibition of thromboxane A2 synthesis. To study the immunoregulatory actions of thromboxane A2, we used mice with a targeted disruption of the gene encoding the thromboxane-prostanoid (TP) receptor. Both mitogen-induced responses and cellular responses to alloantigen were substantially reduced in TP−/− spleen cells. Similar attenuation was observed with pharmacological inhibition of TP signaling in wild-type splenocytes, suggesting that reduced responsiveness was not due to subtle developmental abnormalities in the TP-deficient mice. The absence of TP receptors reduced immune-mediated tissue injury following cardiac transplant rejection, an in vivo model of intense inflammation. Taken together, these findings show that thromboxane augments cellular immune responses and inflammatory tissue injury. Specific inhibition of the TP receptor may provide a more precise approach to limit inflammation without some of the untoward effects associated with NSAIDs.

Expression and Targeting of CX3CL1 (Fractalkine) in Renal Tubular Epithelial Cells

The chemokine CX3CL1 plays a key role in glomerulonephritis and can act as both chemoattractant and adhesion molecule. CX3CL1 also is upregulated in tubulointerstitial injury, but little is known about the subcellular distribution and function of CX3CL1 in renal tubular epithelial cells (RTEC). Unexpectedly, it was found that CX3CL1 is expressed predominantly on the apical surface of tubular epithelium in human renal transplant biopsy specimens with acute rejection or acute tubular necrosis. For studying the targeting of CX3CL1 in polarized RTEC, MDCK cells that expressed untagged or green fluorescent protein-tagged CX3CL1 were generated. The chemokine was present on the apical membrane and in subapical vesicles. Apical targeting of CX3CL1 was not due to signals that were conferred by its intracellular domain, to associations with lipid rafts, or to O-glycosylation but, rather, depended on N-linked glycosylation of the protein. With the use of fluorescence recovery after photobleaching, it was found that CX3CL1 is immobile in the apical membrane. However, CX3CL1 partitioned with the triton-soluble rather than -insoluble cellular fraction, indicating that it is not associated directly with the actin cytoskeleton or with lipid rafts. Accordingly, disruption of rafts through cholesterol depletion did not render CX3CL1 mobile. For exploration of potential functions of apical CX3CL1, binding of CX3CR1-expressing leukocytes to polarized RTEC was examined. Leukocyte adhesion to the luminal surface was enhanced significantly when CX3CL1 was present. These data demonstrate that CX3CL1 is expressed preferentially on the apical membrane of RTEC and suggest a novel function for the chemokine in recruitment and retention of leukocytes in tubulointerstitial inflammation.

Constitutive Endocytosis of the Chemokine CX3CL1 Prevents Its Degradation by Cell Surface Metalloproteases

CX3CL1, a chemokine with transmembrane and soluble species, plays a key role in inflammation by acting as both chemoattractant and adhesion molecule. CX3CL1 is the only chemokine known to undergo constitutive internalization, raising the possibility that dynamic equilibrium between the endocytic compartment and the plasma membrane critically regulates the availability and processing of CX3CL1 at the cell surface. We therefore investigated how transmembrane CX3CL1 is internalized. Inhibition of dynamin using a nonfunctional allele or of clathrin using specific small interfering RNA prevented endocytosis of the chemokine in CX3CL1-expressing human ECV-304 cells. Perusal of the cytoplasmic domain of CX3CL1 revealed two putative adaptor protein-2 (AP-2)-binding motifs. Accordingly, CX3CL1 co-localized with AP-2 at the plasma membrane. We generated a mutant allele of CX3CL1 lacking the cytoplasmic tail. Deletion of the cytosolic tail precluded internalization of the chemokine. We used site-directed mutagenesis to disrupt AP-2-binding motifs, singly or in combination, which resulted in diminished internalization of CX3CL1. Although CX3CL1 was present in both superficial and endomembrane compartments, ADAM10 (a disintegrin and metalloprotease 10) and tumor necrosis factor-converting enzyme, the two metalloproteases that cleave CX3CL1, localized predominantly to the plasmalemma. Inhibition of endocytosis using the dynamin inhibitor, Dynasore, promoted rapid metalloprotease-dependent shedding of CX3CL1 from the cell surface into the surrounding medium. These findings indicate that the cytoplasmic tail of CX3CL1 facilitates its constitutive clathrin-mediated endocytosis. Such regulation enables intracellular storage of a sizable pool of presynthesized CX3CL1 that protects the chemokine from degradation by metalloproteases at the plasma membrane.

The prevalence of BK viremia and urinary viral shedding in a pediatric renal transplant population: A single‐center retrospective analysis

Abstract:  Polyomavirus‐induced nephropathy has emerged as an important cause of renal graft dysfunction. Limited pediatric data are available for this disease. We therefore reviewed the results of the first year of polyomavirus screening in our pediatric renal transplant recipients to determine the prevalence of polyomavirus viremia and urinary shedding. Screening included detection of polyomavirus in plasma by polymerase chain reaction (PCR) and in urine by electron microscopy (EM). In patients with a positive screening test, an assessment of graft dysfunction was made. Fifty‐two patients met the inclusion criteria. Urinary EM was performed in 205 samples and polyomavirus was detected in 10 patients, representing 19% of the study population. PCR was performed on 222 samples and was positive for the BK virus in plasma from seven patients or 13.4% of the study population. Eight patients had a positive screening test and increased creatinine. All these patients underwent renal transplant biopsy. This revealed evidence of polyomavirus nephropathy in four patients. Our findings reveal a high prevalence of polyomavirus in both urine and plasma that is frequently associated with graft dysfunction. These findings support the routine screening of pediatric post‐renal transplant patients for polyomavirus replication.

Eight-Hour Continuous Normothermic Ex Vivo Kidney Perfusion Is a Safe Preservation Technique for Kidney Transplantation: A New Opportunity for the Storage, Assessment, and Repair of Kidney Grafts.

Background Hypothermic kidney storage causes preservation injury and is poorly tolerated by renal grafts. We investigated whether static cold storage (SCS) can be safely replaced with a novel technique of pressure-controlled normothermic ex vivo kidney perfusion (NEVKP) in heart-beating donor kidney transplantation. Methods Right kidneys were removed from 30 kg Yorkshire pigs in a model of heart-beating donation and either preserved in cold histidine-tryptophan-ketoglutarate solution for 8 hours (n = 5), or subjected to 8 hours of pressure-controlled NEVKP (n = 5) followed by renal heterotopic autotransplantation. Results During NEVKP, physiologic perfusion conditions were maintained with low intrarenal resistance and normal electrolyte and pH parameters. Aspartate aminotransferase and lactate dehydrogenase as injury markers were below the detectable analyzer range (<4 and <100 U/L, respectively). Perfusate lactate concentration decreased from baseline until the end of perfusion (10.38 ± 0.76 mmol/L vs 1.22 ± 0.26 mmol/L; P < 0.001). Posttransplantation, animals transplanted with NEVKP versus SCS grafts demonstrated similar serum creatinine peak levels (NEVKP, 2.0 ± 0.5 vs SCS 2.7 ± 0.7 mg/dL; P = 0.11) and creatinine clearance on day 10 (NEVKP, 65.9 ± 18.8 mL/min vs SCS 61.2 ± 15.6 mL/min; P = 0.74). After 10 days of follow-up, animals transplanted with NEVKP grafts had serum creatinine and blood urea nitrogen values comparable to their basal levels (P = 0.49 and P = 0.59), whereas animals transplanted with SCS grafts had persistently elevated serum creatinine and blood urea nitrogen when compared with basal levels (P = 0.01 and P = 0.03). Conclusions Continuous pressure-controlled NEVKP is feasible and safe in good quality heart-beating donor kidney grafts. It maintains a physiologic environment and excellent graft function ex vivo during preservation without causing graft injury.

Outpatient pediatric renal transplant biopsy – Is it safe?

Abstract:  The safe observation time following pediatric renal transplant biopsy is unknown. To predict the safety of day‐care pediatric renal transplant biopsy, we retrospectively evaluated the timing, incidence and severity of post‐biopsy complications in children observed overnight. Biopsies were performed under real time ultrasound guidance using an 18‐gauge Bard Biopty needle. Coagulation screen and platelet counts were measured preprocedure. Hemoglobin (Hb) was measured preprocedure, at 6 h and at 1‐day post‐procedure. Twenty‐eight of 45 children transplanted between January 2002 and May 2004 underwent 65 biopsies. There was gross hematuria following 8 (12%) biopsies; 2/8 occurred after 6 h. Hb fell by >15 g/L in six cases (9%) – three had Hb drop within 6 h post‐procedure and three had a steady decline over 24 h. No patient required blood transfusion. Oral analgesia post‐procedure was required in seven cases (11%). One of these had gross hematuria. No patient required surgical intervention or transfusion. Three complications were recorded >6 h post‐biopsy but none required intervention. Daycare renal transplant biopsy appears to be safe in selected patients.

Continuous Normothermic Ex Vivo Kidney Perfusion Improves Graft Function in Donation After Circulatory Death Pig Kidney Transplantation.

Background Donation after circulatory death (DCD) is current clinical practice to increase the donor pool. Deleterious effects on renal graft function are described for hypothermic preservation. Therefore, current research focuses on investigating alternative preservation techniques, such as normothermic perfusion. Methods We compared continuous pressure-controlled normothermic ex vivo kidney perfusion (NEVKP) with static cold storage (SCS) in a porcine model of DCD autotransplantation. After 30 minutes of warm ischemia, right kidneys were removed from 30-kg Yorkshire pigs and preserved with 8-hour NEVKP or in 4°C histidine-tryptophan-ketoglutarate solution (SCS), followed by kidney autotransplantation. Results Throughout NEVKP, electrolytes and pH values were maintained. Intrarenal resistance decreased over the course of perfusion (0 hour, 1.6 ± 0.51 mm per minute vs 7 hours, 0.34 ± 0.05 mm Hg/mL per minute, P = 0.005). Perfusate lactate concentration also decreased (0 hour, 10.5 ± 0.8 vs 7 hours, 1.4 ± 0.3 mmol/L, P < 0.001). Cellular injury markers lactate dehydrogenase and aspartate aminotransferase were persistently low (lactate dehydrogenase < 100 U/L, below analyzer range; aspartate aminotransferase 0 hour, 15.6 ± 9.3 U/L vs 7 hours, 24.8 ± 14.6 U/L, P = 0.298). After autotransplantation, renal grafts preserved with NEVKP demonstrated lower serum creatinine on days 1 to 7 (P < 0.05) and lower peak values (NEVKP, 5.5 ± 1.7 mg/dL vs SCS, 11.1 ± 2.1 mg/dL, P = 0.002). The creatinine clearance on day 4 was increased in NEVKP-preserved kidneys (NEVKP, 39 ± 6.4 vs SCS, 18 ± 10.6 mL/min; P = 0.012). Serum neutrophil gelatinase-associated lipocalin at day 3 was lower in the NEVKP group (1267 ± 372 vs 2697 ± 1145 ng/mL, P = 0.029). Conclusions Continuous pressure-controlled NEVKP improves renal function in DCD kidney transplantation. Normothermic ex vivo kidney perfusion might help to decrease posttransplant delayed graft function rates and to increase the donor pool.

Role of thromboxane A2 in the induction of apoptosis of immature thymocytes by lipopolysaccharide.

ABSTRACT Lipopolysaccharide (LPS) causes apoptotic deletion of CD4+ CD8+ thymocytes, a phenomenon that has been linked to immune dysfunction and poor survival during sepsis. Given the abundance of thromboxane-prostanoid (TP) receptors in CD4+ CD8+ thymocytes and in vitro evidence that thromboxane A2 (TXA2) causes apoptosis of these cells, we tested whether enhanced generation of TXA2 plays a role in LPS-induced thymocyte apoptosis. Mice injected with 50 μg of LPS intraperitoneally displayed a marked increase in generation of TXA2 and prostaglandin E2 in the thymus as well as apoptotic deletion of CD4+ CD8+ thymocytes. Administration of indomethacin or rofecoxib inhibited prostanoid synthesis but did not affect thymocyte death. In contrast, thymocyte apoptosis in response to LPS was significantly attenuated in TP-deficient mice. These studies indicate that TXA2 mediates a portion of apoptotic thymocyte death caused by LPS. The absence of an effect of global inhibition of prostanoid synthesis suggests a complex role for prostanoids in this model.