Urinary excretion of factor VIII after renal transplantation.

The urinary excretion of factor-VIII-related antigen (VIIIRAg) was measured in 72 patients with kidney transplants and compared with that of two end-products of fibrin-fibrinogen lysis (fragments D and E) to assess their usefulness in monitoring the onset of rejection episodes. Specific and sensitive radioimmunoassays were used to measure the three proteins. Unconcentrated urine samples of 24-hour collections were obtained from 20 healthy subjects, 48 patients with stable transplants, and 24 patients with recent transplants serially followed up from the day of transplantation. Factor VIIIRAg and fragments E and D were not detectable in the urine from healthy subjects but were present in 39%, 60%, and 100% respectively of samples from patients with stable transplants. During 33 acute rejection episodes in 19 patients with recent transplants factor VIIIRAg and fragments E and D were significantly increased above the values observed in patients with stable transplants in 82%, 73%, and 64% of samples respectively; in patients with recent transplants showing no clinical sign of rejection increased excretion of these proteins was observed in 11%, 26%, and 22% of samples respectively. The presence of factor VIIIRAg in urine from patients with kidney allografts suggests that endothelial cell-factor VIII-platelet interactions might pay a key part in the pathogenesis of acute rejection. The results suggest that the assay of factor VIIIRAg in urine is more useful than assays of fragments D and E as a corroborative index of transplant rejection.     

Renal synthesis of leukaemia inhibitory factor (LIF), under normal and inflammatory conditions

Leukaemia inhibitory factor (LIF) is a pleiotropic cytokine that is particularly involved in nephrogenesis and repair of the extracellular matrix. Transgenic mice overexpressing LIF have mesangial proliferative glomerulonephritis. Also, during local inflammatory reactions, such as kidney graft rejection or urinary tract infections, urinary LIF excretion is enhanced. The aim of the study therefore was to study LIF production by normal and inflammatory diseased kidneys (glomerulonephritis or graft rejection), maintained in short cultures. To determine the responsibility of the kidney itself in LIF synthesis, we measured LIF secretion into the culture supernatants of human mesangial or renal tubular epithelial cells. Fragments from diseased kidneys, whether grafts or not, released more LIF than normal human kidney fragments, mesangial or renal tubular epithelial cells. However, LIF production was delayed in renal transplants compared to glomerulonephritic samples taken from untreated patients. In every case, LIF production was enhanced by interleukin 1beta (IL-1beta) and inhibited by IL-4 or dexamethasone, except in two severe rejection episodes. So, LIF appeared to respond to pro- and anti-inflammatory stimuli, in vitro and in vivo. Considering its biological effects, LIF could play a role in inflammatory renal diseases.     

Renal Transplantation: An Analysis of 33 Cases

Thirty-three patients with end-stage renal failure have had transplants over a three-year period, four patients receiving kidneys from siblings and the remainder cadaver organs. Twenty-seven kidneys survived with stable function for periods of six months to three years. Graft survival at one year was 85% and at two years 82%. One patient died and five were returned to dialysis. Complications included rejection episodes, technical problems, respiratory and wound infections, gastrointestinal disorders, and side effects of steroids.     

Urinary prostaglandin E2 excretion in renal allotransplantation in man

The urinary prostaglandin E₂ excretion was measured daily for 28 days in 15 patients (10 men and 5 women) after renal allotransplantation. Patients with acute oliguric renal failure immediately after the transplantation showed high urinary PGE₂ concentrations, but no or minimal increase in the total excretion rates. The median PGE₂ excretion was 211 μg/24 h after establishment of stable renal function, but with great individual variations. Rejection crises were characterized by a two-fold increase in PGE₂ excretion, with a subsequent fall induced by the steroid treatment. The PGE₂ excretion correlated better with urinary sodium excretion than diuresis.The pathophysiological role of the renal prostaglandin ssynthesis remains incompletely defined. The prostaglandin E₂ (PGE₂) appears to act as a modulator of the renal salt and water excretion (1,2) and prostaglandins are important mediators of the immunresponses (3,4). The eraly renal allograft rejection is an event characterized by salt and water retention together with decreasing renal function (5). Antibodies against renal tissue as well as cytotoxic leukocytes (“killer cells”) are active in the process (6,7) and many hormonal systems are involved, among them renin and vasopressin (8). Both hormones are known to stimulate the synthesis of prostaglandin in the kidneys and interact with its effect (9,10,11). The present material was therefore designed to study the urinary excretion of PGE₂ in the kidney allografts before and during rejection crises.     

Prolonged renal allograft survival by donor interleukin-6 deficiency: association with decreased alloantibodies and increased intragraft T regulatory cells

Both humoral and cellular immune responses are involved in renal allograft rejection. Interleukin (IL)-6 is a regulatory cytokine for both B and Foxp3 (forkhead box P3)-expressing regulatory T (Treg) cells. This study was designed to investigate the impact of donor IL-6 production on renal allograft survival. Donor kidneys from IL-6 knockout (KO) vs. wild-type (WT) C57BL/6 mice (H-2(b)) were orthotopically transplanted to nephrotomized BALB/c mice (H-2(d)). Alloantibodies and Treg cells were examined by fluorescence-activated cell sorting analysis. Graft survival was determined by the time to graft failure. Here, we showed that a deficiency in IL-6 expression in donor kidneys significantly prolonged renal allograft survival compared with WT controls. IL-6 protein was upregulated in renal tubules and endothelium of renal allografts following rejection, which correlated with an increase in serum IL-6 compared with that in those receiving KO grafts or naive controls. The absence of graft-producing IL-6 or lower levels of serum IL-6 in the recipients receiving IL-6 KO allografts was associated with decreased circulating anti-graft alloantibodies and increased the percentage of intragraft CD4(+)CD25(+)Foxp3(+) Treg cells compared with those with WT allografts. In conclusion, the lack of graft-producing IL-6 significantly prolongs renal allograft survival, which is associated with reduced alloantibody production and/or increased intragraft Treg cell population, implying that targeting donor IL-6 may effectively prevent both humoral and cellular rejection of kidney transplants.     

Short term suppression of hyperacute renal allograft rejection in presensitised dogs with prostacyclin

One of the main features of hyperacute renal allograft rejection in presensitised dogs is platelet aggregation within the kidney as detected by light microscopy and renal arterio-venous platelet counts. Graft failure, as determined by reduction and ultimate cessation of renal blood flow and urine production, can be abrogated in the short term by prostacyclin which is the most potent inhibitor of platelet aggregation yet discovered. After 4 h of extracorporeal perfusion, by which time all control kidneys had been rejected, all prostacyclin treated kidneys had normal or above normal blood flow rates, were producing urine and were similar histologically (light microscopy) to 4-hour autografts.     

Human and porcine early kidney precursors as a new source for transplantation

Kidney transplantation has been one of the major medical advances of the past 30 years. However, tissue availability remains a major obstacle. This can potentially be overcome by the use of undifferentiated or partially developed kidney precursor cells derived from early embryos and fetal tissue. Here, transplantation in mice reveals the earliest gestational time point at which kidney precursor cells, of both human and pig origin, differentiate into functional nephrons and not into other, non-renal professional cell types. Moreover, successful organogenesis is achieved when using the early kidney precursors, but not later-gestation kidneys. The formed, miniature kidneys are functional as evidenced by the dilute urine they produce. In addition, decreased immunogenicity of the transplants of early human and pig kidney precursors compared with adult kidney transplants is demonstrated in vivo. Our data pinpoint a window of human and pig kidney organogenesis that may be optimal for transplantation in humans.     

Activated macrophages require T cells for xenograft rejection under the kidney capsule

Transplantation of tissues from other species has been advocated as a way to overcome the extreme shortage of human donors. Rejection, however, remains a major hurdle for clinical xenotransplantation. Although activation of macrophages by T cells is critical for the cellular rejection of xenografts, what other important interactions between these two types of cells remain less defined. When we activated macrophages of immuno-deficient mice (SCID or Rag-/-) using interferon-gamma and lipopolysacharide, xenogeneic cells were rejected by activated macrophages in the peritoneal cavity (which has an abundance of resident macrophages), but were not rejected under the kidney capsule (which requires the recruitment of effectors). This difference between the two sites implies that activated macrophages are inefficient for self-recruitment to peripheral graft sites and that T cells may still be required for the process. To test this hypothesis further, immunodeficient mice that had received xenogeneic cells were infused with peritoneal exudate cells (containing activated macrophages and activated T cells) from preimmunized immunocompetent mice. Xenogeneic cells at both the kidney capsule and peritoneal sites were rejected soon after cell transfer. However, when the exudate cells were transferred into SCID recipients that first had been injected with T cell depleting antibodies, xenograft rejection was only prominent at the peritoneal site but not kidney capsule site. These results argue that activated macrophages (as the result of T cell activation) still require T cells for xenograft rejection at peripheral sites.     

Further Assessment of Rosette Inhibition Test in Clinical Organ Transplantation

The rosette inhibition test was used in the clinical management of organ allografts to estimate the amount of immunosuppressive drugs necessary to prevent rejection. In patients surviving more than three months renal function appeared to be better than in a similar group of patients managed without the test. It is suggested that this was due to a reduction in the number of clinical or subclinical rejection episodes. On the other hand, the test indicates that in many cases the level of immunosuppression should be much higher, and if this advice is followed the patients become increasingly exposed to the risk of infection. In other words, those patients with good renal function remained well, whereas those who might otherwise have rejected their kidney and survived had in fact died of sepsis.     

Contribution of renal secreted complement C3 to the circulating pool in humans

Complement C3 produced within the kidney may be an important mediator of local inflammatory and immunological injury. The overall level of renal C3 production and consequently its contribution to the total circulating C3 level are, however, unknown. This was investigated by using the conversion of C3 from recipient to donor allotype following renal transplantation. The C3 F and S allotypes of 80 consecutive renal donor-recipient pairs (148 individuals) were determined by amplification refractory mutation system analysis. The extent of allotype conversion in C3 F/S mismatched recipients was quantified at different stages after transplantation, using an enzyme-linked immunosorbent assay specific for the HAV 4-1 polymorphism of C3 that is strongly associated with C3F. Twenty-one of the eighty recipients were potentially informative, i.e., were C3 SS recipients of C3 FF or FS donor kidneys. In the early postoperative period, donor-derived C3 (HAV 4-1-positive) was undetectable, increasing to 9.6% of the total circulating C3 at times of acute allograft rejection. When graft dysfunction occurred from causes other than rejection, donor C3 remained undetectable. After stable graft function was attained (3-13 mo after transplantation), donor C3 made up 4.5% of the total circulating C3 pool. Our findings demonstrate that human transplant kidney in the resting state is a significant source of extrahepatic C3. Its heightened local synthesis during rejection episodes suggests a possible pathogenic role for C3 in this immunological process.     

Detection of early renal transplant rejection by minimally-invasive monitoring of impedance variability

BACKGROUND: Allograft rejection that occurs after renal transplants is identified by surveillance biopsies or by abnormal laboratory and/or hemodynamic data. The latter are insensitive markers of rejection that may not appear until significant histologic damage has already occurred. Therefore, a sensitive and specific non-invasive method of detecting early rejection of transplanted solid organs is needed. METHOD: A single canine renal allograft was implanted followed by bilateral nephrectomy. Bipolar pacing electrodes were implanted at each end of the transplanted kidney. A second set of electrodes was implanted in the liver, which served as a non-rejecting normal organ. Electrodes were connected to an implantable sensor placed in the subcutaneous tissue. Electrical tissue impedance levels were telemetrically downloaded daily. The clinical status of the transplanted organ was monitored by following the blood urea nitrogen and serum creatinine levels, urine output, and clinical appearance. After tissue impedance levels had stabilized, all immunosuppressants were abruptly discontinued. Clinical signs of rejection were then observed after a few days. RESULTS: Rejection was accompanied by changes in electrical impedance of the implanted organ. These changes, when observed, occurred 1-5 days before clinical signs of rejection appeared. CONCLUSION: Analyses of these data suggest that development of a minimally-invasive high-confidence sensor of early rejection of solid organ transplants is feasible.     

Contributions of direct and indirect alloresponses to chronic rejection of kidney allografts in nonhuman primates

The relative contribution of direct and indirect allorecognition pathways to chronic rejection of allogeneic organ transplants in primates remains unclear. In this study, we evaluated T and B cell alloresponses in cynomolgus monkeys that had received combined kidney/bone marrow allografts and myeloablative immunosuppressive treatments. We measured donor-specific direct and indirect T cell responses and alloantibody production in monkeys (n = 5) that did not reject their transplant acutely but developed chronic humoral rejection (CHR) and in tolerant recipients (n = 4) that never displayed signs of CHR. All CHR recipients exhibited high levels of anti-donor Abs and mounted potent direct T cell alloresponses in vitro. Such direct alloreactivity could be detected for more than 1 y after transplantation. In contrast, only two of five monkeys with CHR had a detectable indirect alloresponse. No indirect alloresponse by T cells and no alloantibody responses were found in any of the tolerant monkeys. Only one of four tolerant monkeys displayed a direct T cell alloresponse. These observations indicate that direct T cell alloresponses can be sustained for prolonged periods posttransplantation and result in alloantibody production and chronic rejection of kidney transplants, even in the absence of detectable indirect alloreactivity.     

Urinary fibrin-fibrinogen degradation products in nephrotic syndrome.

The urinary concentration of fibrin-fibrinogen degradation products (F.D.P.) was measured in 90 patients with proteinuria above 2 g/1 and correlated with proteinuria, differential protein clearances, serum urea and creatinine, and renal biopsy findings. There was a linear correlation (r equals 0-7; P less than 0-001) between the urinary F.D.P. excretion and the selectivity of the proteinuria such that patients with highly selective proteinuria excreted only small amounts of F.D.P. whereas those with non-selective proteinuria excreted much higher levels. There was a significant correlation between the urinary F.D.P. excretion and the urine:serum (U:S) ratio of IgG excretion but not with the U:S ratio or urinary excretion of albumin or transferrin. Sephadex G200 column chromatography of the concentrated urine in 26 cases showed that patients with highly selective proteinuria excreted predominantly F.D.P. of low molecular weight in the urine whereas those with non-selective proteinuria excreted mainly fibrinogen and products of high molecular weight. Hence the type and quantity of F.D.P. in the urine are determined primarily by the differential filtration of fibrinogen and the various degradation products from the plasma through the glomerular basement membrane, which in turn is determined by the "pore size" of the basement membrane. In clinical nephrology measurement of the urinary F.D.P. level provides a rapid and convenient means of estimating the differential protein clearance.     

Antibody-dependent cell cytotoxicity and spontaneous cytotoxicity of peripheral blood lymphocytes of organ recipients and cells infiltrating the kidney allogeneic transplant

It is supposed that determination of antibody-dependent cellular-mediated cytotoxicity and spontaneous cellular-mediated cytotoxicity in the peripheral blood is non-informative for early diagnosis of acute rejection crisis. Activity of K- and NK-cells in peripheral blood is observed to increase during a peak of the rejection crisis. While studying cellular infiltrate obtained from the transplanted kidney, activity of K- and NK-cells is shown to considerably increase in the rejected transplants, which can testify that they contribute much in the loss of the allotransplant function.     

Antiproteinuric treatment reduces urinary loss of vitamin D-binding protein but does not affect vitamin D status in patients with chronic kidney disease

Vitamin D deficiency is common in chronic kidney disease (CKD). Increased urinary loss of vitamin D binding protein (VDBP), the main transporter of 25-hydroxyvitamin D(3) in the circulation, has been postulated to contribute to vitamin D deficiency in proteinuria. To test this hypothesis we analyzed urinary and plasma levels of VDBP, 25-hydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(3) from proteinuric patients, before and after antiproteinuric interventions. We performed a post-hoc analysis of a clinical trial in CKD patients (n=13, creatinine clearance median 60 (range 25-177) ml/min) subjected to the following study periods: washout (no antiproteinuric treatment, 4 weeks), lisinopril 40mg QD (ACEi, 6 weeks), or indomethacin 75mg BID (NSAID, 4 weeks) in randomized sequence. Healthy subjects screened for donation (n=10) served as controls. Plasma and urine VDBP levels were measured by ELISA, 25-hydroxyvitamin D(3) levels by LC-MS and 1,25-dihydroxyvitamin D(3) levels by radioimmunoassay. In CKD patients urinary VDBP excretion was strongly increased (median (range) 5413 (155-211,027) μg/24h) as compared to healthy controls (64 (23-111) μg/24h, p<0.001). Both NSAID and ACEi significantly decreased urinary VDBP excretion, in proportion to proteinuria reduction. Plasma VDBP, 25-hydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(3) levels, however, were similar between patients and controls and not affected by antiproteinuric intervention. Urinary VDBP excretion is markedly increased in proteinuria and responds to antiproteinuric treatment. Urinary VDBP loss is not associated with plasma VDBP or vitamin D(3) levels, suggesting that urinary loss of VDBP does not affect vitamin D status.     

Spontaneous acceptance of liver transplants in rodents: evidence that liver leucocytes induce recipient T-cell death by neglect

In many animal models transplanted livers are not rejected, even when there is a complete MHC mismatch between the donor and recipient and the recipient is not immunosuppressed. This distinguishes liver transplants from other organs, such as kidneys and hearts, which are rapidly rejected in mismatched individuals. Acceptance of transplanted livers in a rat model is not due to the absence of an immune response to the liver and there is a rapid, abortive response that is ultimately exhausted. Donor leucocytes transferred with the liver appear to be responsible for both liver acceptance and the abortive activation of the recipient's T cells. The immune mechanism of liver transplant acceptance appears to be due to 'death by neglect' in which T cells are activated to express IL-2 and IFN-gamma mRNA in the recipient lymphoid tissues, but not at adequate levels within the graft. Subsequently the activated T cells die leading to specific clonal deletion of liver donor-reactive T cells. These findings have important implications for liver transplant patients as immunosuppressive drugs that are given to prevent rejection can also interfere with this form of tolerance. In addition, it might be possible to modify the immunosuppressive drug treatment of transplant patients to promote the process of death by neglect of recipient alloreactive T cells.     

Antibody immunosuppressive therapy in solid-organ transplant: Part I

Currently, a wide variety of both polyclonal and monoclonal antibodies are being routinely utilized to prevent and treat solid organ rejection. More commonly, these agents are also administered in order to delay introduction of calcineurin inhibitors, especially in patients with already compromised renal function. While these antibody therapies dramatically reduced the incidence of acute rejection episodes and improved both short and long-term graft survival, they are also associated with an increased incidence of opportunistic infections and neoplastic complications. Therefore, effective patient management must necessarily balance these risks against the potential benefits of the therapy.Key words: monoclonal, polyclonal, induction, transplants, kidney, lung, liver, heart, rejection, complications     

Development of either split tolerance or robust tolerance along with humoral tolerance to donor and third-party alloantigens in nonmyeloablative mixed chimeras

Hematopoietic chimerism is considered to generate robust allogeneic tolerance; however, tissue rejection by chimeras can occur. This "split tolerance" can result from immunity toward tissue-specific Ags not expressed by hematopoietic cells. Known to occur in chimeric recipients of skin grafts, it has not often been reported for other donor tissues. Because chimerism is viewed as a potential approach to induce islet transplantation tolerance, we generated mixed bone marrow chimerism in the tolerance-resistant NOD mouse and tested for split tolerance. An unusual multilevel split tolerance developed in NOD chimeras, but not chimeric B6 controls. NOD chimeras demonstrated persistent T cell chimerism but rejected other donor hematopoietic cells, including B cells. NOD chimeras also showed partial donor alloreactivity. Furthermore, NOD chimeras were split tolerant to donor skin transplants and even donor islet transplants, unlike control B6 chimeras. Surprisingly, islet rejection was not a result of autoimmunity, since NOD chimeras did not reject syngeneic islets. Split tolerance was linked to non-MHC genes of the NOD genetic background and was manifested recessively in F(1) studies. Also, NOD chimeras but not B6 chimeras could generate serum alloantibodies, although at greatly reduced levels compared with nonchimeric controls. Surprisingly, the alloantibody response was sufficiently cross-reactive that chimerism-induced humoral tolerance extended to third-party cells. These data identify split tolerance, generated by a tolerance-resistant genetic background, as an important new limitation to the chimerism approach. In contrast, the possibility of humoral tolerance to multiple donors is potentially beneficial.     

The activating immunoreceptor NKG2D and its ligands are involved in allograft transplant rejection

Although the linkage between innate and adaptive immunity in transplantation has been recognized, the mechanisms underlying this cooperation remain to be fully elucidated. In this study, we show that early "danger" signals associated with transplantation lead to rapid up-regulation of NKG2D ligands. A second wave of NKG2D ligand up-regulation is mediated by the adaptive immune response to allografts. Treatment with an Ab to NKG2D was highly effective in preventing CD28-independent rejection of cardiac allografts. Notably, NKG2D blockade did not deplete CD8(+) T cells or NK1.1(+) cells nor affect their migration to the allografts. These results establish a functional role of NKG2D and its ligands in the rejection of solid organ transplants.