Mitochondria-targeted antioxidant MitoQ ameliorates ischaemia-reperfusion injury in kidney transplantation models.

BACKGROUND: Ischaemia-reperfusion (IR) injury makes a major contribution to graft damage during kidney transplantation. Oxidative damage to mitochondria is an early event in IR injury. Therefore, the uptake, safety, and efficacy of the mitochondria-targeted antioxidant MitoQ were investigated in models of transplant IR injury. METHODS: MitoQ uptake by warm and cooled pairs of pig and declined human kidneys was measured when preserved in cold static storage or by hypothermic machine perfusion. Pairs of pigs' kidneys were exposed to defined periods of warm and cold ischaemia, flushed and stored at 4°C with or without MitoQ (50 nmol/l to 250 µmol/l), followed by reperfusion with oxygenated autologous blood in an ex vivo normothermic perfusion (EVNP). Pairs of declined human kidneys were flushed and stored with or without MitoQ (5-100 µmol/l) at 4°C for 6 h and underwent EVNP with ABO group-matched blood. RESULTS: Stable and concentration-dependent uptake of MitoQ was demonstrated for up to 24 h in pig and human kidneys. Total blood flow and urine output were significantly greater in pig kidneys treated with 50 µmol/l MitoQ compared with controls (P = 0.006 and P = 0.007 respectively). In proof-of-concept experiments, blood flow after 1 h of EVNP was significantly greater in human kidneys treated with 50 µmol/l MitoQ than in controls (P ≤ 0.001). Total urine output was numerically higher in the 50-µmol/l MitoQ group compared with the control, but the difference did not reach statistical significance (P = 0.054). CONCLUSION: Mitochondria-targeted antioxidant MitoQ can be administered to ischaemic kidneys simply and effectively during cold storage, and may improve outcomes after transplantation.

Repeat duplex ultrasound scanning in suspected Deep Vein Thrombosis (DVT): putting the onus back on the referring clinician.

Duplex scanning is utilised by many departments in the investigation of suspected DVT. NICE Guideline CG144 recommended repeat scanning for patients in whom the initial Wells score was 'likely' in the presence of a raised D-Dimer, following a normal first scan. Following implementation of this recommendation in our department there was a dramatic rise in the number of repeat scans being undertaken, all of which were negative for DVT. Introduction of an electronic message to the report, placing the onus back on the referring clinician to arrange repeat scan if deemed appropriate resulted in a fall in the number of scans being undertaken without impacting on patient outcome.

Baseline donor chronic renal injury confers the same transplant survival disadvantage for DCD and DBD kidneys.

Histological assessment of baseline chronic kidney injury may discriminate kidneys that are suitable for transplantation, but has not been validated for appraisal of donation after circulatory death (DCD) kidneys. 'Time-zero' biopsies for 371 consecutive, solitary, deceased-donor kidneys transplanted at our center between 2006 and 2010 (65.5% DCD, 34.5% donation after brain death [DBD]) were reviewed and baseline chronic degenerative injury scored using Remuzzi's classification. High scores correlated with donor age and extended criteria donors (42% of donors), but the spectrum of scores was similar for DCD and DBD kidneys. Transplant outcomes for kidneys scoring from 0 to 4 were comparable (1 and 3 year graft survival 95% and 92%), but were much poorer for kidneys scoring ≥5, with 1 year graft survival only 73%, and 12.5% suffering primary nonfunction. Critically, high Remuzzi scores conferred the same survival disadvantage for DCD and DBD kidneys. On multi-variable regression analysis, time-zero biopsy score was the only independent predictor for graft survival, whereas one-year graft estimated glomerular filtration rate (eGFR) correlated with donor age and biopsy score. In conclusion, the relationship between severity of chronic kidney injury and transplant outcome is similar for DCD and DBD kidneys. Kidneys with Remuzzi scores of ≤4 can be implanted singly with acceptable results.

Donor kidney disease and transplant outcome for kidneys donated after cardiac death.

BACKGROUND: Although outcomes of kidney transplants following donation after cardiac death (DCD) and donation after brainstem death (DBD) are similar, generally only optimal younger DCD donors are considered. This study examined the impact of pre-existing donor kidney disease on the outcome of DCD transplants. METHODS: This retrospective study compared the outcome of all DCD kidney transplants performed during 1996-2006 with contemporaneous kidney transplants from DBD donors. Implantation biopsies were scored for glomerular, tubular, parenchymal and vascular disease (global histology score). There were 104 DCD and 104 DBD kidney transplants. RESULTS: Delayed graft function (DGF) occurred more frequently in DCD than DBD kidneys (64.4 versus 28.8 per cent; P < 0.001). Long-term graft outcome was similar. The only donor factor that influenced outcome was baseline kidney disease, which was similar in both groups, even though DCD donors were younger, with a higher predonation estimated glomerular filtration rate. The global histology score predicted DGF (odds ratio 1.85 per unit; P = 0.006) and graft failure (relative risk 1.55 per unit; P = 0.001), although there was no difference for DCD and DBD kidneys. CONCLUSION: Transplant outcomes for DCD and DBD kidneys are comparable. Baseline donor kidney disease influences DGF and graft survival but the impact is no greater for DCD kidneys.

Expression of tumor necrosis factor receptors in normal kidney and rejecting renal transplants.

Activation of the TNF signal transduction cascade is initiated by the interaction of TNF with either of two cell surface receptors, TNFR-1 and TNFR-2. The levels and regulation of expression of these two receptors has been extensively analyzed in cultured cells, but little is known of TNFR expression in situ. We analyzed the expression of TNFR-1 and -2 in normal human renal kidney and in renal transplants undergoing acute cellular rejection. Immunohistochemistry and immunogold electron microscopy indicated a strong expression of TNFR-1 on the endothelium of glomeruli of normal kidney. Immunogold colocalization for TNFR-1 and a marker of the trans-Golgi network (TGN-46) demonstrated TNFR-1 within the Golgi complex in endothelial cells in normal kidney, confirming our previous studies with cultured cells. TNFR-1 expression was lost in glomeruli from acutely rejecting kidney, but TNFR-1 was detected in abundance on infiltrating leukocytes in the interstitium of allografts with acute rejection. In contrast, TNFR-2 was demonstrated predominantly in epithelial cells of distal convoluted tubule (DCT) in acute rejection kidney near TNF-expressing leukocytes. TNF was absent in normal kidney, but present in rejecting allograft. TNF was found in infiltrating leukocytes and in adjacent tubular epithelial cells. In situ hybridization showed TNFR-1 mRNA within the endothelium of the glomeruli and of a few arterioles in normal kidney, whereas TNFR-2 mRNA was seen in tubular epithelial cells of the DCT in acute transplant rejection. These data reveal that there is both differential expression and regulation of the two TNF receptors in human kidney.

Wegener's granulomatosis presenting as acute suppurative interstitial nephritis.

A 78 year old man presented with acute renal failure following a prolonged respiratory illness. A renal biopsy demonstrated severe suppurative interstitial nephritis with normal glomeruli. After nine weeks of antibiotics he remained anuric and a second biopsy demonstrated pauci-immune, necrotising glomerulonephritis. His subsequent clinical course was consistent with a diagnosis of Wegener's granulomatosis and antineutrophil cytoplasmic antibodies (ANCA) were detected. This is the first reported case of Wegener's granulomatosis presenting with an isolated tubulointerstitial lesion.

Long-term survival of nonhuman primates receiving life-supporting transgenic porcine kidney xenografts.

BACKGROUND: Recently, there has been a resumed interest in clinical xenotransplantation using pig organs. However, no data are available yet regarding the capacity of porcine organs to sustain the life of a primate beyond the first month. We have attempted to obtain long-term survival of nonhuman primates using human decay-accelerating factor (hDAF) transgenic pig organs and an immunosuppressive strategy particularly aimed at neutralizing the humoral component of the immune response. METHODS: hDAF transgenic or control kidneys were transplanted into 14 bilaterally nephrectomized cynomolgus monkeys (Macaca fascicularis) that underwent splenectomy and were immunosuppressed with cyclosporine A, cyclophosphamide, and steroids. All animals also received recombinant erythropoietin. Postoperatively, the primates were monitored daily. Laboratory evaluations included serum biochemistry, hematology, and measurements of hemolytic antipig antibodies. To assess the role of splenectomy in the control of humoral response, historical data were also used from a group of monkeys (n=7) that received the same immunosuppressive regimen and an hDAF transgenic porcine kidney but did not have splenectomy or receive recombinant erythropoietin. RESULTS: This immunosuppressive approach obtained the longest survival time (78 days) described to date of a primate receiving a life-supporting porcine renal xenograft. Furthermore, four of nine animals in this series survived for 50 days or more. Most biochemical measurements in this study (including plasma urea, creatinine, sodium, and potassium concentrations) remained within normal ranges for several weeks in all of the longest-surviving animals. CONCLUSIONS: Normalization of renal function (urea and creatinine) in primate recipients of porcine renal xenografts suggests that pig kidneys may be suitable for future clinical xenotransplantation. Additional immunosuppressive approaches, specifically designed to prevent humorally mediated immunological damage, should be explored to further prolong survival of primates that have received porcine xenografts.

Phase I study of an engineered aglycosylated humanized CD3 antibody in renal transplant rejection.

BACKGROUND: The potential therapeutic benefits of CD3 monoclonal antibodies, such as OKT3, have been limited by their immunogenicity and their propensity to activate a severe cytokine release syndrome. This has constrained the clinical use of OKT3 to the treatment of acute rejection episodes of organ allografts. METHODS: We have humanized a rat CD3 antibody and created a single amino acid substitution in position 297 of the IgG1 heavy chain to prevent glycosylation and, consequently, binding of the therapeutic antibody to Fc receptors and to complement. This antibody has been given as first line antirejection therapy in nine kidney transplant recipients with biopsy-proven acute rejection episodes. RESULTS: None of the patients demonstrated any antiglobulin response nor any significant cytokine release syndrome. Seven of the nine showed evidence of resolution of their rejection, although some patients experienced re-rejection. CONCLUSIONS: These findings suggest that CD3 antibodies can be engineered to lose their toxicity while retaining their potency as immunosuppressants. Nonactivating humanized CD3 monoclonal antibodies now merit further investigation in the management of transplant patients and in therapy of autoimmune diseases.

Factors involved in the pathogenesis of neutrophilic vasculitis in MRL/Mp-lpr/lpr mice: a model for human microscopic angiitis.

Anti-neutrophil cytoplasm antibodies (ANCA) directed against myeloperoxidase (MPO) are detected in patients with microscopic angiitis. Human MPO autoantibodies stimulate neutrophil degranulation in vitro and are thought to be pathogenic. We have previously shown that MRL-lpr mice with MPO autoantibodies have a higher incidence of vasculitis than their seronegative littermates. The aim of the present study is to determine the relationship between MPO autoantibodies and microscopic angiitis. The neutrophil binding properties of anti-MPO monoclonal antibodies (mAbs) from MRL-lpr mice were tested using murine heterophils (neutrophils) present in blood and induced peritoneal exudates. MRL anti-MPO mAbs selectively bind activated neutrophils which express MPO in vitro. The pathogenicity of an IgG2b anti-MPO mAb, C6, was investigated in vivo. Anti-MPO mAb, C6 was administered to young MRL mice which had been primed with exogenous TNF alpha to induce neutrophil activation and expression of MPO. Neutrophilic vasculitis similar to microscopic angiitis occurred in 33% of MRL mice which had been treated with anti-MPO mAb. The lesions were mainly restricted to sites of previous endothelial insult which suggests an active role for injured endothelium in this pathology.

Myeloperoxidase autoantibodies distinguish vasculitis mediated by anti-neutrophil cytoplasm antibodies from immune complex disease in MRL/Mp-lpr/lpr mice: a spontaneous model for human microscopic angiitis.

Anti-neutrophil cytoplasm antibodies (ANCA) with specificity for myeloperoxidase (MPO) occur in the sera of patients with microscopic angiitis, an autoimmune disease characterized by necrotizing vasculitis and crescentic glomerulonephritis. These autoantibodies have been shown to stimulate neutrophil degranulation and are believed to participate in pathogenesis. A neutrophilic vasculitis has been reported in MRL-lpr mice which has histological appearances similar to microscopic angiitis. In the present study we show that 22% of female MRL-lpr mice develop MPO autoantibodies. These animals develop a clinical syndrome of vasculitis and glomerulonephritis that is distinct from immune complex disease. Anti-MPO monoclonal antibodies derived from these mice are polyreactive and react with double-stranded DNA. They bind a conformational epitope on human MPO which is also expressed by activated human neutrophils. The results suggest that a subset of MRL-lpr mice develop ANCA-related vasculitis rather than systemic lupus erythematosus and may be used as a model for human microscopic angiitis.

Life-supporting pig-to-primate renal xenotransplantation using genetically modified donors.

BACKGROUND: In order to circumvent the complement-mediated hyperacute rejection of discordant xenografts, a colony of pigs transgenic for the human regulator of complement activity, human decay-accelerating factor (hDAF), has been produced. METHODS: Seven kidneys from hDAF transgenic pigs and six kidneys from nontransgenic control pigs were transplanted into cynomolgus monkeys; both native kidneys were removed during the same operation. The recipient animals were immunosuppressed with cyclosporine, steroids, and cyclophosphamide. RESULTS: In the transgenic group, the median survival time was 13 days (range, 6-35 days); the median survival time in the control group was 6.5 days (range, 0.3-30 days). There were no cases of hyperacute rejection in the transgenic group, and the two longest-surviving kidneys in this group showed no evidence of rejection on histological examination. In contrast, all control kidneys underwent antibody-mediated rejection, one demonstrating hyperacute rejection and the others acute vascular rejection. CONCLUSION: This study demonstrates that (i) a kidney from an hDAF transgenic pig can support the life of a primate for up to 35 days (and also shows the basic physiological compatibility between the pig and nonhuman primate); (ii) nontransgenic kidneys are not routinely hyperacutely rejected; and (iii) the presence of hDAF on the kidney confers some protection against acute vascular rejection. Improved immunosuppression and immunological monitoring may enable extended survival.

Gold and D-penicillamine induce vasculitis and up-regulate mRNA for IL-4 in the Brown Norway rat: support for a role for Th2 cell activity.

D-penicillamine (DP) and gold salts which are used as immune-modulating agents in the treatment of rheumatoid arthritis are known to be capable of causing autoimmune manifestations. Most autoimmune diseases in man are dominated by Th1-type responses, and one might presume that effective immunotherapy counteracts Th activity, perhaps by causing a shift to a Th2 response. The mechanism of action of gold and DP is not clear, but some clues may be obtained from their effects in animal models. DP, gold salts and mercuric chloride (HgCl2) are known to induce Th2-dominated autoimmune syndromes in genetically susceptible rodent strains, and we have demonstrated recently that HgCl2 up-regulates messenger RNA (mRNA) for IL-4 in the Brown Norway (BN) rat. In the BN rat HgCl2 treatment is also associated with the development of vasculitis, and anti-myeloperoxidase (MPO) antibodies are found in the serum. The present study examined and confirmed the hypothesis that, since gold and DP induce an autoimmune syndrome similar to HgCl2 in the BN rat, they may also induce vasculitis and an up-regulation in mRNA for IL-4. Tissue injury was assessed macroscopically and histologically on day 5 and day 15 after the start of injections with gold, DP or HgCl2, serum titres of IgE and presence of anti-MPO antibodies were determined using ELISA, and a semi-quantitative assay using reverse transcription-polymerase chain reaction was used to assay the level of mRNA for IL-4 in spleen and caecum. The relative degree of tissue injury reflected the potency of induction of IgE by the three agents (HgCl2 being most potent and DP least potent). The lesions were identical histologically, supporting the premise that the vasculitis is a manifestation of the autoimmune syndrome rather than non-specific HgCl2 toxicity. Both gold and DP induced less up-regulation of mRNA for IL-4 than HgCl2. HgCl2 (but not gold or DP) induced anti-MPO antibodies. It would be interesting to examine patients treated with gold and DP to see if there is evidence of a Th2-type response in those developing autoimmune complications, and whether or not the bias to a Th2 environment contributes to efficacy of treatment of the underlying disease.

Polycystin: in vitro synthesis, in vivo tissue expression, and subcellular localization identifies a large membrane-associated protein.

The primary structure of polycystin predicts a large integral membrane protein with multiple cell recognition motifs, but its function remains unknown. Insight into polycystin's normal function and its role in the development of autosomal dominant polycystic kidney disease (PKD1) requires the assembly of an extensive collection of molecular reagents to examine its expression and create model systems for functional studies. Development of these crucial reagents has been complicated due to the presence of transcriptionally active homologous loci. We have assembled the authentic full-length PKD1 cDNA and demonstrated expression of polycystin in vitro. Polyclonal antibodies directed against distinct extra- and intracellular domains specifically immunoprecipitated in vitro translated polycystin. The panel of antibodies was used to determine localization of polycystin in renal epithelial and endothelial cell lines and tissues of fetal, adult, and cystic origins. In normal adult kidney and maturing fetal nephrons, polycystin expression was confined to epithelial cells of the distal nephron and vascular endothelial cells. Expression in the proximal nephron was only observed after injury-induced cell proliferation. Polycystin expression was confined to ductal epithelium in liver, pancreas, and breast, and restricted to astrocytes in normal brain. We report clear evidence for the membrane localization of polycystin by both tissue sections and by confocal microscopy in cultured renal and endothelial cells. Interestingly, when cultured cells made cell-cell contact, polycystin was localized to the lateral membranes of cells in contact. These data suggest that polycystin is likely to have a widespread role in epithelial cell differentiation and maturation and in cell-cell interactions.

Treatment of refractory Wegener's granulomatosis with humanized monoclonal antibodies.

Conventional immunosuppression for systemic vasculitides is limited by substantial side-effects, cumulative drug toxicity and refractoriness in some patients. Six Wegener's granulomatosis patients who had been refractory to conventional therapy for at least 6 months, were treated with humanized monoclonal antibodies specific to lymphocyte CD52 or CD4 antigens. Diagnosis was on clinicopathological grounds, supported by the presence of autoantibodies to Proteinase 3. Histological evidence of persistent disease activity was obtained for each patient. Humanized monoclonal anti-CD52, with or without anti-CD4, was given intravenously up to 40 mg/day for up to 10 days. Remission, (programmed withdrawal of drug therapy without return of refractory disease) was achieved in all patients. Cytotoxic drugs were discontinued at the time of monoclonal antibody treatment and not used again; steroids were withdrawn gradually. Four patients relapsed at 1.5, 5, 10 and 18 months, and were treated successfully with further monoclonal antibody therapy alone. Three years after the study began, five patients are well; one patient died at surgery whilst in remission. Humanized monoclonal antilymphocyte antibodies may provide an effective treatment in patients with systemic vasculitis which is refractory to steroids or cytotoxic agents, or who are intolerant of these drugs.

The treatment of systemic lupus erythematosus (SLE) in NZB/W F1 hybrid mice; studies with recombinant murine DNase and with dexamethasone.

The effects of recombinant mouse DNase on a well established murine model of spontaneous SLE have been evaluated. Daily intraperitoneal injections of DNase were given to female NZB/NZW F1 mice during the period of disease development from 4 to 7 months of age or at the height of disease activity from the age of 7 months for 3 weeks. This treatment was compared with the injections of diluent and with an immunosuppressive dose of dexamethasone. The effects of treatment were evaluated using the immunological parameters of disease activity (antinucleoprotein antibody, immune complexes, serum immunoglobulins, anti-cardiolipin antibodies), protein-uria, serum creatinine and renal histopathology (light microscopy, immunofluorescence and electron microscopy). The dose of dexamethasone used (1 mg/kg per day from the age of 4 months) was sufficient to suppress the development of lupus entirely. Treatment with DNase starting at the age of 4 months postponed the development of the disease by about 1 months and extended the period from the onset of disease to death by about 30%. Mice treated for 3 weeks during the most active phase of the disease at 7 months of age showed more dramatic effects. Proteinuria and serum creatinine were significantly reduced and renal histopathology was strikingly less severe than in the control group. Immune complexes involving DNA-containing antigens are believed to play a crucial role in the pathogenesis of SLE. DNA-nucleoprotein, even in immune complexes, can be destroyed by DNase. This enzyme therefore provides a rational way to interfere with the disease process. The results reported here encourage a trial of recombinant human DNase in human SLE and lupus nephritis.

Role of neutrophils in the pathogenesis of experimental vasculitis.

In the Brown-Norway rat, mercuric chloride (HgCl2) induces an autoimmune syndrome characterized by high IgE levels. There is widespread necrotizing leukocytoclastic vasculitis involving lung, skin, mucous membranes, pancreas, liver, and gut, with tissue injury being most marked in the cecum. As in systemic vasculitis in man, there are neutrophils at the site of tissue injury and the animals develop anti-neutrophil cytoplasmic antibodies, which in the Brown-Norway rat are directed against myeloperoxidase. To determine whether neutrophils are involved in the pathogenesis of the vasculitis, we have used a monoclonal antibody that was reported to deplete neutrophils in other rat strains. Rats treated with HgCl2 received antibody by intravenous injection at various time points. Serial blood samples were taken for neutrophil counts and to assay for anti-myeloperoxidase and IgE antibodies. The guts of animals killed after antibody therapy were scored for vasculitic changes and neutrophils infiltrate. RP3 (but not the control antibody MAC6) was shown to bind to Brown-Norway rat neutrophils and to block glycogen-induced influx of neutrophils into the peritoneum. When given at peak disease, RP3 caused a dose-dependent reduction in tissue injury with a marked reduction in circulating blood neutrophil numbers and in tissue neutrophil infiltrate. RP3 treatment did not affect the rise in titer of IgE and anti-myeloperoxidase antibodies. The data presented demonstrate that in this model neutrophils are necessary for the induction of vasculitis and that the degree of vasculitis correlates with neutrophil number. To our knowledge, this study is the first to provide direct evidence for a role for neutrophils in vasculitis. We suggest that antibodies directed against neutrophils, especially if they deplete neutrophils, may be useful in the therapy of vasculitis in man.

Nitric oxide contributes to tissue injury in mercuric chloride-induced autoimmunity.

Recent data has suggested a role for nitric oxide (NO) both in the induction of immunity and as an effector of tissue injury in experimental models of inflammation. In this study, we have tested the efficacy of two inhibitors of NO synthase, NG-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG), to modify the autoimmune leucocytoclastic necrotizing vasculitis which develops following the administration of mercuric chloride (HgCl2) to the Brown Norway rat. Neither agent affected the induction of autoimmunity as judged by plasma IgE titres or the degree of tissue neutrophil infiltration; however, L-NMMA did significantly attenuate tissue injury scores. We conclude that inhibition of NO synthase does not influence the induction of autoimmunity by HgCl2, but that NO does contribute to the development of tissue injury in this experimental model.

Hydrogen peroxide-induced endothelial retraction is accompanied by a loss of the normal spatial organization of endothelial cell adhesion molecules.

Treatment of confluent monolayers of human umbilical vein endothelial cells with sublethal concentrations of hydrogen peroxide (H2O2) produces reversible cell retraction that opens gaps between adjacent cells. Despite the retraction, adjacent cells remain in contact through a network of dendrite-like processes. Retraction depends on cellular metabolism but not new protein synthesis or protein kinase C. Shape changes induced by H2O2 are accompanied by partial redistribution of actin filaments from the cell periphery in resting endothelial cells to a tangled network of centrally located filaments in H2O2-treated endothelial cells. This change in actin organization is associated with a loss of the normal distribution pattern of surface protein expression. Specifically, beta 1 and beta 3 integrins partly escape from focal adhesion plaques and migrate to the lateral and apical surface of the cell; PECAM-1 redistributes from the lateral borders to the basal surface; and ICAM-1 and ICAM-2 spread from apical caps to the basal surface and to the dendrite-like processes. The likely consequence of endothelial retraction accompanied by abnormal membrane protein distribution is a loss of normal endothelial cell functions. These changes are best considered manifestations of H2O2-induced sublethal injury that may cause endothelial dysfunction.