Prostaglandin E2 affects mitochondrial function in adult mouse cardiomyocytes and hearts.

Prostaglandin E2 (PGE2) signals differently through 4 receptor subtypes (EP1-EP4) to elicit diverse physiologic/pathologic effects. We previously reported that PGE2 via its EP3 receptor reduces cardiac contractility and male mice with cardiomyocyte-specific deletion of the EP4 receptor (EP4 KO) develop dilated cardiomyopathy. The aim of this study was to identify pathways responsible for this phenotype. We performed ingenuity pathway analysis (IPA) and found that genes differentiating WT mice and EP4 KO mice were significantly overrepresented in mitochondrial (adj. p value = 6.28 × 10-26) and oxidative phosphorylation (adj. p value = 1.58 × 10-27) pathways. Electron microscopy from the EP4 KO hearts show substantial mitochondrial disarray and disordered cristae. Not surprisingly, isolated adult mouse cardiomyocytes (AVM) from these mice have reduced ATP levels compared to their WT littermates and reduced expression of key genes involved in the electron transport chain (ETC) in older mice. Moreover, treatment of AVM from C57Bl/6 mice with PGE2 or the EP3 agonist sulprostone resulted in changes of various genes involved in the ETC, measured by the Mitochondrial Energy Metabolism RT2-profiler assay. Lastly, the EP4 KO mice have reduced expression of superoxide dismuatse-2 (SOD2), whereas treatment of AVM with PGE2 or sulprostone increase superoxide production, suggesting increased oxidative stress levels in these EP4 KO mice. Altogether the current study supports the premise that PGE2 acting via its EP4 receptor is protective, while signaling through its other receptors, likely EP3, is deleterious.

Targeted Gene Deletion or Antagonism of the Prostaglandin E2 EP3 Receptor Protects Against Cardiac Injury Postmyocardial Infarction.

BACKGROUND: Prostaglandin E2 acts through 4 G-protein-coupled receptors (EP1-EP4). We previously reported that activation of the EP3 receptor reduces cardiac contractility, and its expression increases after a myocardial infarction (MI), mediating the reduction in cardiac function. In contrast, cardiac overexpression of the EP4 receptor in MI substantially improves cardiac function. Moreover, we recently reported that mice overexpressing EP3 have heart failure under basal conditions and worsened cardiac function after MI. Thus, the deleterious effects of the prostaglandin E2 EP receptors in the heart are mediated via its EP3 receptor. We, therefore, hypothesized that cardiomyocyte-specific knockout (CM-EP3 KO) or antagonism of the EP3 receptor protects the heart after MI. METHODS: To test our hypothesis, we made the novel CM-EP3 KO mouse and subjected CM-EP3 KO or controls to sham or MI surgery for 2 weeks. In separate experiments, C57BL/6 mice were subjected to 2 weeks of MI and treated with either the EP3 antagonist L798 106 or vehicle starting 3 days post-MI. RESULTS: CM-EP3 KO significantly prevented a decline in cardiac function after MI compared with WT animals and prevented an increase in hypertrophy and fibrosis. Excitingly, mice treated with L798 106 3 days after MI had significantly better cardiac function compared with vehicle-treated mice. CONCLUSIONS: Altogether, these data suggest that EP3 may play a direct role in regulating cardiac function, and pharmaceutical targeting of the EP3 receptor may be a therapeutic option in the treatment of heart failure.

Deleterious effects of cardiomyocyte-specific prostaglandin E2 EP3 receptor overexpression on cardiac function after myocardial infarction.

AIMS: Prostaglandin E2 (PGE2) is a lipid hormone that signals through 4 different G-protein coupled receptor subtypes which act to regulate key physiological processes. Our laboratory has previously reported that PGE2 through its EP3 receptor reduces cardiac contractility at the level of isolated cardiomyocytes and in the isolated working heart preparation. We therefore hypothesized that cardiomyocyte specific overexpression of the PGE2 EP3 receptor further decreases cardiac function in a mouse model of heart failure produced by myocardial infarction. MAIN METHODS: Our study tested this hypothesis using EP3 transgenic mice (EP3 TG), which overexpress the porcine analogue of human EP3 in the cardiomyocytes, and their wildtype (WT) littermates. Mice were analyzed 2 wks after myocardial infarction (MI) or sham operation by echocardiography, RT-PCR, immunohistochemistry, and histology. KEY FINDINGS: We found that the EP3 TG sham controls had a reduced ejection fraction, reduced fractional shortening, and an increased left ventricular dimension at systole and diastole compared to the WT sham controls. Moreover, there was a further reduction in the EP3 TG mice after myocardial infarction. Additionally, single-cell analysis of cardiomyocytes isolated from EP3 TG mice showed reduced contractility under basal conditions. Overexpression of EP3 significantly increased cardiac hypertrophy, interstitial collagen fraction, macrophage, and T-cell infiltration in the sham operated group. Interestingly, after MI, there were no changes in hypertrophy but there were changes in collagen fraction, and inflammatory cell infiltration. SIGNIFICANCE: Overexpression of EP3 reduces cardiac function under basal conditions and this is exacerbated after myocardial infarction.

Inhibition of spleen tyrosine kinase decreases donor specific antibody levels in a rat model of sensitization.

Antibody mediated rejection is a major cause of renal allograft loss. Circulating preformed donor specific antibodies (DSA) can result as a consequence of blood transfusion, pregnancy or prior transplantation. Current treatment strategies are limited due to partial or transient efficacy, adverse side-effects or patient unsuitability. Previous in vivo studies exploring autoimmune diseases have shown that spleen tyrosine kinase (SYK) signalling is involved in the development of pathogenic autoantibody. The role of SYK in allogenic antibody production is unknown, and we investigated this in a rodent model of sensitization, established by the transfusion of F344 whole blood into LEW rats. Two-week treatment of sensitized rats with selective SYK inhibitor fostamatinib strongly blocked circulating DSA production without affecting overall total immunoglobulin levels, and inhibition was sustained up to 5 weeks post-completion of the treatment regimen. Fostamatinib treatment did not affect mature B cell subset or plasma cell levels, which remained similar between non-treated controls, vehicle treated and fostamatinib treated animals. Our data indicate fostamatinib may provide an alternative therapeutic option for patients who are at risk of sensitization following blood transfusion while awaiting renal transplant.

Gastrointestinal Bleeding in a Pancreas Transplant Recipient: A Case to Remember.

BACKGROUND Pancreas transplantation has proven to be the most effective therapeutic option for insulin-dependent diabetes mellitus. However, despite advances in surgical technique and continuously improving outcomes, pancreas transplantation has the highest complication rate among all solid-organ transplants. Vascular complications in particular can be catastrophic, with graft- and life-threatening potential. Ectopic variceal bleeding is less common and is rarely reported in the literature. CASE REPORT A 51-year-old man presented with recurrent intermittent gastrointestinal bleeding (GIB) associated with hepatic dysfunction and portal hypertension 4 years after a successful pancreas-after-kidney transplant. Apart from positive serology for hepatitis E virus, all the other liver disease screening results were negative. He was extensively investigated with 6 computed tomography (CT) scans, 3 esophago-gastro-duodenoscopies (EGD), 3 colonoscopies, and 1 visceral arteriogram before the plausible diagnosis of ectopic trans-anastomotic variceal bleeding involving the pancreas transplant was established. Selective variceal catheterization and embolization were done with 3% sodium tetradecyl sulphate (STD). He remained free of bleeding after embolization. CONCLUSIONS This case report adds to the scanty literature on the management of ectopic variceal bleeding in a pancreas transplant recipient. Diagnosis of ectopic varix is usually challenging and frequently requires a visceral arteriogram. We describe a novel minimally-interventional technique to obtain source control and also discuss the complexity involved in the management, along with future implications.

Transfusion-Dependent Anemia in a Simultaneous Pancreas and Kidney Transplant Recipient.

A case of transfusion-dependent anemia in a simultaneous pancreas and kidney (SPK) transplant recipient that masqueraded as gastrointestinal bleeding (GIB) is described. The anemia was attributed to bleeding from the donor duodenal cuff based on balloon enteroscopy findings. The patient underwent multiple contrast-enhanced computed tomography scans and multiple endoscopies with confounding features until, eventually, the diagnosis was established. We discuss the diagnostic difficulties and the therapeutic dilemma, along with the pitfalls in ascertaining the final diagnosis.

Causes and risk factors for acute dialysis initiation among patients with end-stage kidney disease-a large retrospective observational cohort study.

BACKGROUND: Patients who require acute initiation of dialysis have higher mortality rates when compared with patients with planned starts. Our primary objective was to explore the reasons and risk factors for acute initiation of renal replacement therapy (RRT) among patients with end-stage kidney disease (ESKD). Our secondary objective was to determine the difference in glomerular filtration rate (GFR) change in the year preceding RRT between elective and acute dialysis starts. METHODS: We conducted a single-centre retrospective observational study. ESKD patients either started dialysis electively (planned starters) or acutely and were known to renal services for >90 (unplanned starters) or <90 days (urgent starters). RESULTS: In all, 825 consecutive patients initiated dialysis between January 2013 and December 2015. Of these, 410 (49.7%) patients had a planned start. A total of 415 (50.3%) patients had an acute start on dialysis: 244 (58.8%) unplanned and 171 (41.2%) urgent. The reasons for acute dialysis initiation included acute illness (58%) and unexplained decline to ESKD (33%). Cardiovascular disease [n = 30 (22%)] and sepsis [n = 65 (48%)] accounted for the majority of acute systemic illness. Age and premorbid cardiovascular disease were independent risk factors for acute systemic illness among unplanned starts, whereas autoimmune disease accounted for the majority of urgent starts. The rate of decline in GFR was greater in the month preceding RRT among acute dialysis starters compared with planned starters (P < 0.001). CONCLUSIONS: Cardiovascular disease and advancing age were independent risk factors for emergency dialysis initiation among patients known to renal services for >3 months. The rapid and often unpredictable loss of renal function in the context of acute systemic illness poses a challenge to averting emergency dialysis start.

Allosensitization after transplant failure: the role of graft nephrectomy and immunosuppression - a retrospective study.

There are conflicting data about the role of transplant nephrectomy and immunosuppression withdrawal on the development of allosensitization and the impact on re-transplantation. We divided 109 first graft recipients into two groups according to whether they underwent nephrectomy (NX+, n = 61) or their graft was left in situ (NX-, n = 48). Sera were assessed for HLA-A/B/Cw/DR/DQ antibodies at the time of NX/transplant failure and after 3, 6, 12, 24 months. The NX+ group showed a higher rate of donor specific antibody (DSA) and non-DSA human leukocyte antigen (HLA) antibody production at all the time points. Multivariable analysis showed that nephrectomy was a strong, independent risk factor for the development of DSAs after 12 and 24 months (P = 0.005 and 0.008). In the NX- group, low tacrolimus levels correlated with DSA formation (AUC 0.817, P = 0.002; best cut-off level 2.9 ng/ml). Analysis with a standardized pool of UK donors showed a more difficult grade of HLA matchability following nephrectomy compared with the NX- group. Nephrectomy is followed by the long-term production of DSA and non-DSA HLA antibodies and negatively impacts on the chances of finding a HLA-compatible kidney. Tacrolimus levels ≥3 ng/ml are protective against the development of allosensitization and could facilitate re-transplantation in the NX- group.

Successful management of post-transplant focal segmental glomerulosclerosis with therapeutic plasma exchange and rituximab.

BACKGROUND: Post-transplant focal segmental glomerulosclerosis (FSGS) is associated with renal allograft loss. Currently, optimal treatment remains controversial. METHODS: The aim of our study was to examine the efficacy and safety of therapeutic plasma exchange (TPE), and rituximab (RTX), in the management of post-transplant FSGS. The treatment protocol consisted of RTX and monthly cycles of 5 plasma exchanges for 6 months. We treated 10 transplant recipients with biopsy-proven post-transplant FSGS. Lastly, we compared the studied group to a historic control group of nine patients with post-transplant FSGS. RESULTS: 9 out of 10 patients achieved remission after the conclusion of treatment (4 complete and 5 partial), while 1 patient did not respond to treatment. During the follow-up period, there was one graft loss and one patient died while in remission from unrelated complications. There was a significant reduction in mean uPCR between diagnosis (517.4 ± 524.2 mg/mmol) and last follow-up (87 ± 121.6 mg/mmol) in the patients with sustained remission (p = 0.026). There was no significant decline in eGFR in the eight relapse-free responders at the end of follow-up. (54.4 ± 16.7 from 49.8 ± 20.4 ml/min) (p = 0.6) An increased response rate to the combined TPE and RTX treatment was demonstrated, when compared to a historic control group of nine patients with post-transplant FSGS, as only five out of nine patients achieved remission (two complete and three partial) in that group. CONCLUSIONS: In this study, treatment with TPE and RTX appears to be safe, well tolerated and effective in the management of patients with post-transplant FSGS.

Shared alloimmune responses against blood and transplant donors result in adverse clinical outcomes following blood transfusion post-renal transplantation.

De novo HLA donor-specific antibodies (DSA) following transplantation are associated with alloimmune injury and allograft failure. Blood transfusions are allogeneic, and when given posttransplant (PTBT) they may independently increase the risk of HLA antibody development. This study aims to analyze the development of HLA transfusion-specific antibodies (TSA) to blood donors of transfusions given posttransplant and examine the impact on clinical outcomes. A total of 244 blood donors of transfusions received by 86 transplant patients (46 who developed a DSA post transfusion and 40 who remained DSA negative) were HLA typed. De novo TSA developed against 150/244 (61.5%) blood donors. In 70/150 (46.7%) cases the TSA was of shared HLA antibody specificity with a DSA response in the recipient (DSA+ = TSA+). This occurred when there was a greater overall HLA match between the blood and transplant donor. DSA+ = TSA+ patients had increased risk of allograft failure (P = .0025) and AMR (P = .02) compared with the DSA+ ≠ TSA+ patients. To conclude, PTBT may elicit de novo HLA antibodies. Enhanced HLA matching between the blood and transplant donor is more likely to result in a DSA and TSA of shared antibody specificities. Transfusion avoidance or the use of HLA matched or selected blood may reduce this risk and improve outcomes.

Neurological complications of renal dialysis and transplantation.

Neurological complications from renal replacement therapy contribute significantly to morbidity and mortality in patients with renal failure. Such complications can affect either the central or peripheral nervous systems. Most neurological disturbances associated with the uraemic state do not respond fully to renal replacement therapy. There are also complications specifically associated with dialysis and transplantation. A multidisciplinary approach, involving both nephrologists and neurologists, is critical for the diagnosis and effective management of these disorders.

Terasaki Epitope Mismatch Burden Predicts the Development of De Novo DQ Donor-Specific Antibodies and are Associated With Adverse Allograft Outcomes.

BACKGROUND: De novo DQ donor-specific antibody (DSA) are associated with antibody-mediated rejection and allograft loss. Given the lack of effective treatment of de novo DQ DSA, their prevention is vital if there is to be an improvement of long-term allograft survival. Using the HLA Matchmaker program, DQ epitope matching has been shown to be superior to HLA antigen mismatching in predicting de novo DQ DSA development. Whether DQ epitopes determined by Terasaki may more accurately predict de novo DQ development over HLA antigen matching is not known. METHODS: We retrospectively analyzed the immunogenicity of the different HLA antigens, DQB1 alleles and DQB1 Teraskai epitopes (TerEp) in a large cohort of renal transplant recipients, by comparing patient mismatches with de novo DSA development. RESULTS: Patients mismatched at a DQB1 allele were at significantly higher risk of developing de novo DSA compared with other mismatched HLA antigens. Patients mismatched at the DQ7 allele appear to be at specific risk. For patients mismatched at a single DQB1 allele, the risk of de novo DQ DSA development increases with the number of TerEp mismatches. However, the immunogenicity of the different DQ TerEps does not appear to be equal. Patients who develop antibodies against TerEps are at increased risk of adverse allograft outcomes, specifically antibody-mediated rejection. CONCLUSIONS: Epitope mismatch burden, determined by TerEps, helps predict risk of de novo DQ DSA development and offers an alternative approach to predict an alloimmune response.

Dual-balloon assisted super-selective embolisation of high flow arterial venous fistula within a transplant kidney.

BACKGROUND: In this case report, we describe a novel application of the technique of 'dual-balloon assisted' cannulation and embolisation of a high flow arterial venous fistula (AVF) in transplant kidney, where attempts at standard and previously described embolisation techniques were proving difficult to achieve. CASE PRESENTATION: Seventy year old gentleman with renal transplant presenting with high output cardiac failure and deteriorating renal function. Angiography demonstrated high flow traumatic AV fistula within transplanted kidney, secondary to multiple biopsies. Attempts at guidewire and guiding sheath placement and stability for occlusion plug deployment were failing due to combination of very high back-flow pressures within the AVF and challenging vascular anatomy; with an aneurysmal, tortuous iliac artery as well as intra renal transplanted vessels. A combination of angioplasty and remodeling aortic balloons in the transplant artery and the host external iliac vein respectively, facilitated stabilization of guiding sheath and hence controlled delivery of an occlusion plug from the venous side of the fistula. The fistula was successfully embolised, leading to complete resolution of patient symptoms and improvement of renal function beyond his previous baseline. CONCLUSIONS: Percutaneous embolisation is an established technique to treat iatrogenic AVF in transplant kidneys. High flow pressure through an AVF, as demonstrated in this case, can cause difficulty and raise safety issues in accessing and embolising the AVF using previously described techniques. This case report describes an effective and novel application of the technique of using a second balloon in the host common iliac vein to; lower flow pressure, stabilise the guidewires during plug deployment and prevent displacement of wires and/or plug into the common iliac vein.

High Intrapatient Variability of Tacrolimus Levels and Outpatient Clinic Nonattendance Are Associated With Inferior Outcomes in Renal Transplant Patients.

BACKGROUND: Nonadherence to immunosuppressants is associated with rejection and allograft loss. Intrapatient variability (IPV) of immunosuppression levels is a marker of nonadherence. This study describes the impact of IPV of tacrolimus levels in patients receiving a tacrolimus monotherapy immunosuppression protocol. METHODS: We retrospectively analyzed the outpatient tacrolimus levels of kidney-only transplant patients taken between 6 and 12 months posttransplant. IPV was determined using the coefficient of variance. RESULTS: Six hundred twenty-eight patients with a mean number of 8.98 ± 3.81 tacrolimus levels and a mean follow-up of 4.72 ± 2.19 years were included. Multivariate analysis showed death was associated with increasing age (1.04 [1.01-1.07], P = 0.0055), diabetes at time of transplant (2.79 [1.44-5.41], P = 0.0024), and rejection (2.34 [1.06-5.19], P = 0.036). Variables associated with graft loss included the highest variability group (2.51 [1.01-6.27], P = 0.048), mean tacrolimus level less than 5 ng/mL (4.32 [1.94-9.63], P = 0.0003), a high clinic nonattendance rate (1.10 [1.01-1.20], P = 0.03), and rejection (9.83 [4.62-20.94], P < 0.0001). Independent risk factors for rejection were de novo donor-specific antibody (3.15 [1.84-5.39], P < 0.0001), mean tacrolimus level less than 5 ng/mL (2.57 [1.27-5.19], P = 0.00860, and a high clinic nonattendance rate (1.11 [1.05-1.18], P = 0.0005). CONCLUSIONS: This study shows that high tacrolimus IPV and clinic nonattendance are associated with inferior allograft survival. Interventions to minimize the causes of high variability, particularly nonadherence are essential to improve long-term allograft outcomes.

Alemtuzumab dose adjusted for body weight is associated with earlier lymphocyte repletion and less infective episodes in the first year post renal transplantation - a retrospective study.

The optimal dose of alemtuzumab for renal transplant induction is not known, and the doses reported in the literature vary. This study compares two separate dosing regimens of alemtuzumab in renal transplantation. The first is a standard fixed dose of 30 mg (SD), and the second is a dose adjusted for body weight at 0.4 mg/kg (AD). In this first year post-transplant, there was no difference in patient [HR 0.64 (0.22-1.86), P = 0.39] or allograft survival [HR 1.18 (0.48-2.90), P = 0.72] between the two groups. There was also no difference in overall rejection-free survival [HR 1.12 (0.79-1.58), P = 0.53]. However, absolute lymphocyte count was significantly higher at all measured time points in the first year in the AD group. There were also less episodes of urosepsis [HR 1.38 (1.03-1.85), P = 0.037] and fungal infection [HR 5.15 (2.00-13.28), P = 0.015] in the AD group compared with the SD group. This study shows that AD alemtuzumab is associated with earlier lymphocyte repletion and less infective episodes in the first year postrenal transplant, without increasing the risk of rejection. This work highlights the need for studies into the optimal dosing of monoclonal antibodies used in transplantation.

Long-term outcome in biopsy-proven acute interstitial nephritis treated with steroids.

Background: There are no prospective randomized controlled trials describing the outcome of acute interstitial nephritis (AIN) treated with steroids, and retrospective studies are limited. Methods: We identified adult patients with a diagnosis of AIN without glomerular pathology over a 14-year period. Treated patients all received oral prednisolone and three also recieved IV methylprednisolone. Data were collected retrospectively on estimated glomerular filtration rate (eGFR), change in eGFR from time of biopsy, dependence on renal replacement therapy (RRT) and mortality, and outcomes were analysed according to the treatment prescribed. Results: A total of 187 eligible patients with AIN were identified and 158 were treated with steroids. There was no difference in median eGFR or dependence on RRT at the time of biopsy. Steroid-treated patients had significantly higher eGFR at all time points post-biopsy up to 24 months, when median eGFR was 43 mL/min in the steroid-treated group and 24 mL/min in the untreated group (P = 0.01). Fewer patients in the steroid-treated group were dialysis dependent by 6 months (3.2% versus 20.6%, P = 0.0022) and 24 months (5.1% versus 24.1%, P = 0.0019). Conclusions: This large retrospective study suggests a benefit of steroids in treatment of AIN with greater improvement in eGFR and fewer patients progressing to end-stage renal disease.

Risk factors and outcomes of delayed graft function in renal transplant recipients receiving a steroid sparing immunosuppression protocol.

AIM: To analyse the risk factors and outcomes of delayed graft function (DGF) in patients receiving a steroid sparing protocol. METHODS: Four hundred and twenty-seven recipients of deceased donor kidney transplants were studied of which 135 (31.6%) experienced DGF. All patients received monoclonal antibody induction with a tacrolimus based, steroid sparing immunosuppression protocol. RESULTS: Five year patient survival was 87.2% and 94.9% in the DGF and primary graft function (PGF) group respectively, P = 0.047. Allograft survival was 77.9% and 90.2% in the DGF and PGF group respectively, P < 0.001. Overall rejection free survival was no different between the DGF and PGF groups with a 1 and 5 year rejection free survival in the DGF group of 77.7% and 67.8% compared with 81.3% and 75.3% in the PGF group, P = 0.19. Patients with DGF who received IL2 receptor antibody induction were at significantly higher risk of rejection in the early post-transplant period than the group with DGF who received alemtuzumab induction. On multivariate analysis, risk factors for DGF were male recipients, recipients of black ethnicity, circulatory death donation, preformed DSA, increasing cold ischaemic time, older donor age and dialysis vintage. CONCLUSION: Alemtuzumab induction may be of benefit in preventing early rejection episodes associated with DGF. Prospective trials are required to determine optimal immunotherapy protocols for patients at high risk of DGF.

Tubuloreticular Inclusions in Renal Allografts Associate with Viral Infections and Donor-Specific Antibodies.

The presence of tubuloreticular inclusions (TRIs) in native glomerular endothelial cells associates with viral infections and lupus nephritis. However, the associations of TRIs in renal transplant biopsy specimens are not known. We analyzed data from 316 patients who had a transplant biopsy with electron microscopy examination; 41 of 316 (13.0%) patients had TRIs. Patients with TRIs had significantly lower allograft survival rates (50.9%) than patients without TRIs (74.3%; P=0.03). Transplant glomerulopathy-free survival was also inferior in the TRI-positive group (57.5%) compared with the TRI-negative group (87.3%; P=0.002). Serologically, hepatitis C associated with the presence of TRIs (P=0.04) along with donor-specific antibodies (P=0.01). Furthermore, patients who were TRI positive were more likely than patients who were TRI negative to have had a previous rejection episode (P=0.02). On multivariate analysis, TRIs associated with prior rejection, viral infections, and class 1 HLA donor-specific antibodies. These results show that the presence of TRIs in renal allograft biopsy specimens associates with poor allograft outcomes and serologic evidence of viral infections and alloimmunity. The association with alloimmunity is a novel finding that warrants additional investigation.