Dietmar Abendroth

Up-regulation of cell cycle regulatory genes after renal ischemia/reperfusion : Differential expression of p16(INK4a), p21(WAF1/CIP1) and p27(Kip1) cyclin-dependent kinase inhibitor genes depending on reperfusion time

The aim of this study was to evaluate the influence of renal ischemia, cold preservation and reperfusion on the degree of renal kidney senescence. An experimental model of ex vivo renal hemoperfusion was used. Expression of p16(INK4a), p21(WAF1/CIP1) and p27(Kip1) cyclin‐dependent kinase inhibitor genes (CDKIGs) was studied immunohistochemically in kidney biopsy samples at baseline and different time points after reperfusion. All three markers were up‐regulated in kidney tissue after the reperfusion; however, their activation in different renal cells varied according to the reperfusion time. Expression of p16 was significantly increased in tubular cells at 180 min of reperfusion when compared with the baseline. Activation of p27 was detected in glomerular cells at 15 min and was significantly higher at 60, 120 and 180 min of reperfusion. The marker started increasing in tubular cells at 15 min and was elevated at every time point afterwards. p21 was significantly over‐expressed in all renal cells after the reperfusion. It has been shown by the results of the current study that renal ischemia/reperfusion is associated with over‐expression of CDKIGs indicating on substantial DNA damage and/or accelerated tissue senescence. For the first time it has been shown that tissue expression of CDKIGs is positively related with the reperfusion time.

Rapamycin rescue therapy in patients after kidney transplantation : first clinical experience

This study was aimed at analysing rapamycin (RAPA) rescue therapy with calcineurin inhibitor (CNI) withdrawal in renal transplant patients primarily presenting with CNI‐nephrotoxicity (CNI‐Neph), chronic allograft nephropathy (CAN) without [CAN(a)] and with histological changes suggestive of chronic rejection [CAN(b)].

Production of proinflammatory cytokines and adhesion molecules in ex-vivo xenogeneic kidney perfusion

Abstract Xenogeneic transplantation of solid organs is limited due to hyperacute rejection. In concordant systems, the mechanisms of rejection can be studied due to cross‐reactivity of mediators with anti‐human monoclonal antibodies. The aim of this study was to obtain information about the kinetics of proinflammatory cytokines and production of soluble adhesion molecules in the acute phase of reperfusion, eight kidneys from rhesus monkeys were perfused ex‐vivo with human blood (group B/0) for 1 hour in a closed system. Blood levels of IL‐1b, IL‐6, TNFα, soluble ICAM, and E‐electin were measured using an ELISA technique under steady‐tate conditions. Cytokine levels rose significantly within the 60‐min interval (IL‐1b, 6.1 ± 2.6–161.1 + 98.5 pg/ml; IL‐6, 30.2 ± 7.7–274.2 ± 75.8 pg/ml; TNFα, 544.2 ± 363.6–1651.0±25.7 pg/ml; P < 0.05). Immediately after the beginning of reperfusion, soluble ICAM‐1 and selectin levels were abnormally high and rose constantly throughout the observation period, reaching significance at 60 min. High levels of proinflammatory cytokines may lead to an induction of adhesion molecules, thus, upregulating the leukocyte‐endothelial interaction in a complement‐independent mechanism. Specific pretreatment with monoclonal antibodies against ICAM‐1, LFA‐1, or other soluble mediators may be useful in down‐regulating hyperacute rejection in trans‐pecies transplantation.

IMPDH activity in whole blood and isolated blood cell fraction for monitoring of cellcept-mediated immunosuppression

THE DOSAGE regimen of CellCept (mycophenolate mofetil, MMF) is based on the pharmacokinetics of the active metabolite, mycophenolic acid (MPA), and on empirically obtained efficacy data rather than on the kinetics of the immunosuppressive activity. This activity is based on the inhibition of inosine 59-monophosphate dehydrogenase (IMPDH), a pivotal enzyme in the biosynthesis of guanine nucleotides. PK–PD relationships based on monitoring of MPA plasma concentrations and on the inhibition of IMPDH in whole blood cell fractions have been determined in animals following a single dose of MMF. There is no reliable information on the kinetics of enzyme activity after repeated MMF dosing in humans. Therefore, a new assay using HPLC was developed by one of the present investigators (W.A.) to quantify IMPDH activity in whole plasma and isolated lymphocytes of renal transplant patients and healthy controls. This study describes the assay and initial results obtained with this method.

PTA Plus Stent Implantation Versus PTA Alone for Central Venous Stenoses

From 1990 through 1992, 17 severely stenosed central venous segments were diagnosed in 13 patients. In 9 patients, 11 stenoses were detected during follow-up after trans- femoral venous thrombectomy. The other 6 stenoses occurred in the venous outflow tract of 4 hemodialysis shunts. Six stenoses were treated by percutaneous transvenous angio plasty (PTA) alone, and 11 stenoses by PTA plus stent implantation (PTA+S). For PTA+S, a flexible, self-expanding stent was used. Acute procedure-related complications were not observed. After a median follow-up time of eighteen months (three to thirty-six months), all patients were examined clinically, duplex-sonographically, and angiographically. Following PTA alone (n = 6), there was only 1 excellent result, and 5 patients developed high-grade restenoses. Two of these patients had to be reoperated on, and 1 patient underwent another PTA with stent placement. PTA+S (n = 11) for initial treatment of central venous stenoses gave excellent results in 8 cases, a good result in 1 case, and 2 insufficient results caused by intimal hyperplasia within the stent. These restenoses were successfully treated by another PTA.

Na-K/2Cl transporter inhibition for reduction of postis-chemic kidney failure tested in autologous reperfusion

Abstract  Postischemic kidney function may be influenced by donor conditioning. The sulfamoyl‐benzoate “piretanide” (P) is a diuretic agent with an inhibitory effect on the luminal Na‐K‐2CL‐transporter system in the ascending part of the loop of Henle. A clinical pilot study demonstrated a lower rate of organ dysfunction following transplantation in humans when the donor organs were pretreated with piretanide. In an experimental ex vivo model the effect of piretanide on immediate organ function following long or short cold ischemia was studied. Porcine kidneys (n = 36) were removed after in situ transaortal hypothermic flushing with 21 Eurocollins solution. Following short storage (1 h, n= 18) or long storage (24 h, n= 18) the kidneys were reperfused with intraoperatively drawn heparinized autologous blood diluted with Ringers lactate to a hematocrit of 25 %. Urine flow was higher in the piretanide‐pretreated group (p), especially after long storage. The electrolyte loss was comparable in both groups. Postischemic endogenous creatinine clearance was significantly elevated in the treatment group (4.45 ± 0.6 ml/min per 100 mg in P vs 1.91 ± 0.4 ml/min per 100 mg, in control, P < 0.05 Mann‐Whitney test). Renal hemodynamics were improved by piretanide, resulting in significantly lower resistance and allowing higher flow during pressure‐controlled perfusion. O2 consumption, representing general metabolic activity, was higher after long storage, indicating an earlier recovery from cold ischemia. In this ex vivo model, autologous reperfusion of porcine kidneys could be improved by piretanide pretreatment. Auto‐regulation of kidney vasculature was maintained as well as functional parameters such as creatinine clearance or gluconeogenesis. Therefore, piretanide may be used in larger clinical trials to further improve organ quality in times of donor shortage.

The function of transgenic human DAF‐expressing porcine livers during hemoperfusion with human blood

Abstract  Extracorporal pig liver perfusion could bridge the deadly problem of acute human liver failure. However, preformed natural antibodies and complement activation (CA) are the predominant mechanisms of hyperacute xenoge‐neic rejection. The blockade of both pathways of CA in the xenograft, using transgenic livers expressing human decay accelerating factor on the endothelial surface results in prolonged graft survival and lower release of mediators.

Expression of human decay accelerating factor (hDAF) in transgenic pigs regulates complement activation during ex vivo liver perfusion — immunopathological findings

Abstract  Ex vivo perfusions of human decay accelerating factor‐ex pressing transgenic (n = 3), and nontransgenic (n = 6) porcine livers with human blood revealed a higher degree of organ damage in non transgenic pig livers. Transgenic livers were protected from immuno‐histologically detectable complement deposition, despite corresponding IgM and IgG deposits in both groups. Complement activation and consumption of C3 and C4 turned out to be lower in transgenic pig livers. In contrast to livers of normal landrace pigs, livers from genetically manipulated pigs showed no morphological alterations after perfusion.

Causes and prognostic value of pre-transplant elevated kynurenine level in kidney allograft recipients

BACKGROUND The activation of the tryptophane catabolizing enzyme Indoleamine 2,3-dioxygenase leads to the formation of kynurenine and other metabolites that counter-regulate immune activation resulting in restoration of immune homeostasis. But in chronic immune activation, as in hemodialysed patients, the immunosuppressive feedback mechanisms continue as indicated by elevated kynurenine concentrations. However, its relevance is still a matter of debate. MATERIAL/METHODS This retrospective analysis presents the pre-transplant kynurenine levels (quantified photometrically) of 307 kidney graft recipients in connection with some pre- and post-transplant variables and the type of immunosuppression (cyclosporine-based triple drug therapy without/with ATG-Fresenius-induction). Statistical analyses performed were analysis of variance, Scheffés test for pairwise comparisons, Cox regression, Spearmans rank correlation, and extended segmentation analysis. RESULTS The pre-transplant kynurenine level was significantly elevated as compared to healthy adults (14.1±5.9 vs. 2.7±0.6 nmol/ml, p<0.0001), significantly higher in PRA positive than in PRA negative patients (16.1 vs. 12.9 nmol/ml, p<0.001) and, supporting this observation, also higher (p<0.0001) in a cohort with predominant (89.7%) pre-sensitized patients (16.4±6.4 nmol/ml) having the longest time on the waiting list (median 39 months) as compared to cohorts with fewer (16.8-22%) pre-sensitized patients (12.7±4.4 resp. 13.4±5.8 nmol/ml) having shorter times on the waiting list (16-24 months). Patients with immediately functioning grafts showed a lower pre-transplant kynurenine level than patients with non-immediately functioning grafts (13.5±6.0 vs. 14.9±5.7 nmol/ml, p=0.053). No associations were found with basic diseases, rejections, or graft survival. CONCLUSIONS The pre-transplant elevated serum kynurenine levels were highly associated with the patients pre-sensitization status and their longer time on hemodialysis treatments, but did not allow prognostic assessments.

Long-term follow-up of lipid metabolism and rheologic properties after successful pancreas and kidney transplantation.

Abstract The long‐term effect of pancreatic and kidney transplantation (spkt) on blood viscosity, lipid metabolism and skin microcirculation in insulin‐dependent diabetes mellitus (IDDM) was studied because impaired rheological properties of blood may play a role in the development of diabetic micro‐ and macroangiopathy. 46 IDDM‐patients (16 f/30 m; 23 ± 34 y mean duration of diabetes; 60 ± 14 mos mean follow up period) underwent spkt (Gr. II n= 28) or solitary kidney (Gr. II: n= 18) transplantation, and were compared with healthy controls (C). Rheological measurements were performed with Mooney‐Ewart rotation‐viscosimeter determining whole blood viscosity (WBV), at shear rates 1, 5, 10, 20, 50, 100, 200 sec‐1 Triglycerides, total and HDL‐, LDL‐ and VLDL cholesterol and fibrinogen were measured. Microcirculation was estimated by transcutaneous oxygen tension measurement (tcpO2) and laser speckle method, in the forefoot area. Hemoglobin al was normalized only in group I (I: 7.2±0.2%; II: 8.3 ± 0.3%; C: < 8%). WBV at low shear (1, 5, 10) was increased in both groups, when compared to healthy controls (I: 12.4 ± 2; 12.5 5 1; 6.8 ± 0.5 mpas; II: 18.7 ± 2; 13.4 ± 15; 9.4±1 mpas; C: 7.5 ±f 0.5; 6.7 ± 0.3; 5.4 ± 0.2 mpas; P<0.05). Plasma fibrinogen was elevated in both groups compared to normals: (I: 384 19; II: 448 ± 20; C: 250±50mg/dl; P<0.05). There was a positive influence of spkt on skin microcirculation: tcpO2prior tx: I: 44 ± 3; II: 49f.6mmHg; post tx: I: 59 ±4; II: 42±3mmHg. Laser speckle prior tx I: 3.3 ± 0.3; II: 4.7±0.2rel. U.; post tx: 3.8 ± 0.2; II: 4.3 ± 0.2 rel. U. Patients with progression of angiopathy showed still higher fibrinogen and shear rates (P < 0.05). There was no significant difference for total HDL‐, LDL‐and VLDL cholesterol. Despite normalization of glucose metabolism and significant improvement of microcirculation in spkt patients, fibrinogen and the shear rates are increased indicating a persisting “individual” vascular risk. It is suggested that an additional hemorheological approach in the treatment posttrransplant might prevent the progression of vascular complications.