Benito A. Yard

Effects of Donor Pretreatment With Dopamine on Graft Function After Kidney Transplantation A Randomized Controlled Trial

CONTEXT Kidney graft function after transplantation can be improved through pharmacological donor pretreatment to limit organ injury from cold preservation. OBJECTIVE To determine whether pretreatment of brain-dead donors with low-dose dopamine improves early graft function in human renal transplant recipients. DESIGN, SETTING, AND PATIENTS Randomized, open-label, multicenter, parallel-group trial of 264 deceased heart-beating donors and 487 subsequent renal transplants performed at 60 European centers between March 2004 and August 2007 (final follow-up, December 31, 2008). Eligible donors were stable under low-dose norepinephrine with a normal serum creatinine concentration on admission. INTERVENTIONS Donors were randomized to receive low-dose dopamine (4 mug/kg/min). MAIN OUTCOME MEASURES Dialysis requirement during first week after transplantation. RESULTS Dopamine was infused for a median of 344 minutes (IQR, 215 minutes). Dialysis was significantly reduced in recipients of a dopamine-treated graft. Fewer recipients in the treatment group needed multiple dialyses (56/227; 24.7%; 95% CI, 19.0%-30.3%; vs 92/260; 35.4%; 95% CI, 29.5%-41.2%; P = .01). The need for multiple dialyses posttransplant was associated with allograft failure after 3 years (HR, 3.61; 95% CI, 2.39-5.45; P < .001), whereas a single dialysis was not (HR, 0.67; 95% CI, 0.21-2.18; P = .51). Besides donor dopamine (OR, 0.54; 95% CI, 0.35-0.83; P = .005), cold ischemic time (OR, 1.07; 95% CI, 1.02-1.11 per hour; P = .001), donor age (OR, 1.03; 95% CI, 1.01-1.05 per year; P < .001), and recipient body weight (OR, 1.02; 95% CI, 1.01-1.04 per kg; P = .009) were independent explanatory variables in a multiple logistic regression model. Dopamine resulted in significant but clinically meaningless increases in the donors systolic blood pressure (3.8 mm Hg; 95% CI, 0.7-6.9 mm Hg; P = .02) and urine production before surgical recovery of the kidneys (29 mL; 95% CI, 7-51 mL; P = .009) but had no influence on outcome. CONCLUSION Donor pretreatment with low-dose dopamine reduces the need for dialysis after kidney transplantation. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00115115.

Increased circulating endothelial progenitor cells in septic patients: Correlation with survival

Objective:Endothelial damage and detachment of endothelial cells are known to occur in septic patients. Thus, recruitment of circulating endothelial progenitor cells (cEPCs) to these lesions might have a beneficial effect on the clinical course in septic patients. Therefore, we were interested in whether EPCs, detected by flow cytometry, are increasingly mobilized during sepsis and if this mobilization is associated with clinical outcome. Design:Prospective, nonrandomized study. Setting:Intensive care unit of a university hospital. Patients:Patients with (n = 32) and without (n = 15) sepsis and healthy volunteers (n = 15). Interventions:Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and cEPCs were characterized by three-color fluorescence flow cytometry using antibodies against CD133, CD34, and vascular endothelial growth factor receptor-2. Serum concentrations of vascular endothelial growth factor, granulocyte macrophage-colony stimulating factor, and erythropoietin were determined by enzyme-linked immunosorbent assay. Severity of sepsis was assessed according to Acute Physiology and Chronic Health Evaluation II scoring. Measurements and Main Results:In septic patients, the number of cEPCs was significantly higher than in nonseptic intensive care unit patients (p < .05) and healthy controls (p < .02). Nonsurvivors (n = 8), defined as death within 28 days after onset of sepsis, had significantly lower numbers of cEPCs than survivors (n = 24) (p < .0001). The number of cEPCs was correlated with survival in septic patients. Serum vascular endothelial growth factor concentrations were significantly higher in septic patients compared with nonseptic intensive care unit patients and healthy controls (p < .01) and correlated with the cEPC numbers (p < .0001). Similar findings were observed for granulocyte macrophage-colony stimulating factor and erythropoietin. Conclusions:Our data suggest that cEPC enumeration in peripheral blood of septic patients might be a valuable marker to assess the clinical outcome in these patients.

Carnosine Prevents Apoptosis of Glomerular Cells and Podocyte Loss in STZ Diabetic Rats

Background/Aims: We identified carnosinase-1 (CN-1) as risk-factor for diabetic nephropathy (DN). Carnosine, the substrate for CN-1, supposedly is a protective factor regarding diabetic complications. In this study, we hypothesized that carnosine administration to diabetic rats might protect the kidneys from glomerular apoptosis and podocyte loss. Methods: We examined the effect of oral L-carnosine administration (1g/kg BW per day) on apoptosis, podocyte loss, oxidative stress, AGEs and hexosamine pathway in kidneys of streptozotocin-induced diabetic Wistar rats after 3 months of diabetes and treatment. Results: Hyperglycemia significantly reduced endogenous kidney carnosine levels. In parallel, podocyte numbers significantly decreased (-21% compared to non-diabetics, p<0.05), apoptotic glomerular cells numbers increased (32%, compared to non-diabetic, p<0.05) and protein levels of bax and cytochrome c increased (175% and 117%). Carnosine treatment restored carnosine kidney levels, prevented podocytes loss (+23% compared to diabetic, p<0.05), restrained glomerular apoptosis (-34% compared to diabetic; p<0.05) and reduced expression of bax and cytochrome c (-63% and -54% compared to diabetics, both p<0.05). In kidneys of all diabetic animals, levels of ROS, AGEs and GlcNAc-modified proteins were increased. Conclusion: By inhibition of pro-apoptotic signaling and independent of biochemical abnormalities, carnosine protects diabetic rat kidneys from apoptosis and podocyte loss.

Oral Carnosine Supplementation Prevents Vascular Damage in Experimental Diabetic Retinopathy

Backgrounds/Aims: Pericyte loss, vasoregression and neuroglial activation are characteristic changes in incipient diabetic retinopathy. In this study, the effect of the antioxidant and antiglycating dipeptide carnosine was studied on the development of experimental diabetic retinopathy. Materials/Methods: STZ-induced diabetic Wistar rats were orally treated with carnosine (1g/kg body weight/day). Retinal vascular damage was assessed by quantitative morphometry. Retinal protein extracts were analyzed for markers of oxidative stress, AGE-formation, activation of the hexosamine pathway and changes in the expression of Ang-2, VEGF and heat shock proteins Hsp27 and HO-1. Glial cell activation was analyzed using Western blot analysis and immunofluorescence of GFAP expression and retinal neuronal damage was histologically examined. Results: Oral carnosine treatment prevented retinal vascular damage after 6 months of experimental hyperglycemia. The protection was not caused by ROS- or AGE-inhibition, but associated with a significant induction of Hsp27 in activated glial cells and normalization of increased Ang-2 levels in diabetic retinas. A significant reduction of photoreceptors in retinas of carnosine treated animals was noted. Conclusion: Oral carnosine treatment protects retinal capillary cells in experimental diabetic retinopathy, independent of its biochemical function. The vasoprotective effect of carnosine might be mediated by the induction of protective Hsp27 in activated glial cells and normalization of hyperglycemia-induced Ang-2.

Prevention of Cold‐Preservation Injury of Cultured Endothelial Cells by Catecholamines and Related Compounds

The present study was conducted to dissect the underlying mechanisms by which catecholamines protect cells against preservation injury.

Abnormalities of CD4+ T cell subpopulations in ANCA-associated vasculitis

In patients with ANCA‐associated vasculitis (AAV), CD25 expression is increased on circulating T cells. Although in animal experiments the role of CD4+ CD25+ T‐regulatory‐cells (Treg) in protection against autoimmunity is well established, the role of these cells in AAV is unknown. To investigate the hypothesis that an increased expression of CD25 on T cells is related to persistent T cell activation and not to disturbances in Treg cells in AAV (34 patients, six of them after renal transplantation), we investigated CD25 expression in different subpopulations of CD4+ cells and FOXP3 mRNA expression by reverse transcription‐polymerase chain reaction (RT‐PCR). In addition, T cell proliferation and cytokine secretion after stimulation with anti‐CD3 and anti‐CD28 and intracellular cytokine production after stimulation with phorbol myristate acetate (PMA)‐ionomycin was determined. Controls were non‐vasculitic renal transplant patients (n = 9) and healthy controls (HC) (n = 13). In AAV the total number of lymphocytes, CD4+ lymphocytes and the percentage of naive T cells are lower than in HC and RTX. An increased percentage of CD25+ cells was found in AAV and AAV/RTX, irrespective of disease activity, but not in HC or RTX. This was confined to the naive (CD4+ CD45RBhigh) population only. FOXP3 mRNA expression in CD4+ T cells did not differ between AAV patients and healthy controls. In vitro T cell proliferation was enhanced in AAV patients compared to HC (P < 0·01). PBMC of AAV patients produced significantly less interleukin (IL)‐10 and interferon (IFN)‐γ after anti‐CD3/CD28 stimulation. The percentage of IL‐10 and IL‐12, but not IFN‐γ, IL‐4 or tumour necrosis factor (TNF)‐α‐producing cells was significantly higher in patients compared to HC. These findings were confined to the memory population of CD4+ cells. We conclude that AAV patients are lymphopenic and have low numbers of CD4+ T cells, which seem to be in a persistent state of activation.

Hypothermic injury: the mitochondrial calcium, ATP and ROS love-hate triangle out of balance.

Background/Aims: Catecholamines prevent hypothermic cell death which accounts for severe tissue damage and impaired allograft function after prolonged organ preservation. Here, we identified cellular processes which govern hypothermia-mediated cell death in endothelial cells and how they are influenced by dopamine. Methods: Lactate dehydrogenase assay, intracellular ATP, reactive oxygen species and reduced thio-group measurement, intracellular calcium measurement and mitochondrial calcium staining were performed in the study. Results: Intracellular ATP was almost completely depleted within 12 hrs of hypothermic preservation in untreated human umbilical vein endothelial cells (HUVEC), while dopamine pre-treatment significantly delayed ATP depletion. 4 hrs after hypothermia a redox imbalance was observed in untreated cells, which increased with the duration of hypothermia. The redox imbalance was primarily caused by depletion of SH reduction equivalents and was significantly inhibited by dopamine. In addition, hypothermia-induced Ca2+ influx and mitochondrial Ca2+ accumulation were both prevented by dopamine. The protective effect of dopamine was abrogated by ionomycin and sodium azide and partly by oligomycin and CCCP. Conclusions: Our data demonstrated that loss of intracellular ATP, generation of a redox imbalance and accumulation of intracellular Ca2+ underlie cold preservation injury. Dopamine improves the redox balance, prevents intracellular Ca2+ accumulation and delays ATP depletion.

A CTG Polymorphism in the CNDP1 Gene Determines the Secretion of Serum Carnosinase in Cos-7–Transfected Cells

Recently, we demonstrated that a polymorphism in exon 2 of the serum carnosinase (CNDP1) gene is associated with susceptibility to developing diabetic nephropathy. Based on the number of CTG repeats in the signal peptide, five different alleles coding for 4, 5, 6, 7, or 8 leucines (4L–8L) are known. Diabetic patients without nephropathy are homozygous for the 5L allele more frequently than those with nephropathy. Since serum carnosinase activity correlates with CNDP1 genotype, we hypothesized in the present study that secretion of serum carnosinase is determined by the CNDP1 genotype. To test this hypothesis, we transfected Cos-7 cells with different CNDP1 constructs varying in CTG repeats and assessed the expression of CNDP1 protein in cell extracts and supernatants. Our results demonstrate that CNDP1 secretion is significantly higher in cells expressing variants with more than five leucines in the signal peptide. Hence, our data might explain why individuals homozygous for the 5L allele have low serum carnosinase activity. Because carnosine, the natural substrate for carnosinase, exerts antioxidative effects and inhibits ACE activity and advanced glycation end product formation, our results support the finding that diabetic patients homozygous for CNDP1 5L are protected against diabetic nephropathy.

Modulation of chemokine production and expression of adhesion molecules in renal tubular epithelial and endothelial cells by catecholamines.

Background. The present study was conducted to investigate whether catecholamines influence the production of chemokines and adhesion molecules in proximal tubular epithelial cells (PTECs) and endothelial cells. Methods. PTECs and human umbilical vein endothelial cells (HUVECs) were stimulated with various concentrations of dopamine (DA), adrenaline (AD), or noradrenaline (NA), and the production of interleukin (IL)-8, ENA-78, and Gro-&agr; was assessed by ELISA. The influence of catecholamine pretreatment on tumor necrosis factor (TNF)-&agr;–mediated production of these chemokines and the expression of adhesion molecules was also tested. Results. In PTECs, DA inhibited the production of all three chemokines in a dose-dependent fashion. Although inhibition in ENA-78 and Gro-&agr; production was also found in HUVECs, IL-8 production was up-regulated in these cells. Increased IL-8 secretion was predominantly observed at the apical site of the cells. In AD or NA stimulated cells, the production of Gro-&agr; was increased in PTECs and decreased in HUVECs. Down-regulation in IL-8 production was also observed after AD but not after NA stimulation of both cell types. Interestingly, TNF-&agr;–mediated up-regulation in intercellular adhesion molecule 1, vascular cell adhesion molecule (VCAM), and E-selectin was delayed in DA-pretreated HUVECs but not in PTECs. The influence of DA, but not AD or NA, on chemokine production was completely prevented by the addition of N-acetylcysteine. Conclusions. This study demonstrates that catecholamines differentially influence chemokine production and indicates that DA may have anti-inflammatory properties because it delays the expression of adhesion molecules and inhibits the production of chemokines in PTECs and endothelial cells under basal and inflammatory conditions.

Targeting complement activation in brain-dead donors improves renal function after transplantation.

Kidneys recovered from brain-dead donors have inferior outcomes after transplantation compared to kidneys from living donors. Since complement activation plays an important role in renal transplant related injury, targeting complement activation in brain-dead donors might improve renal function after transplantation. Brain death (BD) was induced in Fisher rats by inflation of an epidurally placed balloon catheter and ventilated for 6h. BD animals were treated with soluble complement receptor 1 (sCR1) 1h before or 1h after BD. Kidney transplantation was performed and 7 days after transplantation animals were sacrificed. Plasma creatinine and urea were measured at days 0, 1, 3, 5 and 7 after transplantation. Renal function was significantly better at day 1 after transplantation in recipients receiving a sCR1 pre-treated donor kidney compared to recipients of a non-treated donor graft. Also treatment with sCR1, 1h after the diagnosis of BD, resulted in a better renal function after transplantation. Gene expression of IL-6, IL-1beta and TGF-beta were significantly lower in renal allografts recovered from treated donors. This study shows that targeting complement activation, during BD in the donor, leads to an improved renal function after transplantation in the recipient.