Detlef Lang

Cisplatin Nephrotoxicity Is Critically Mediated via the Human Organic Cation Transporter 2

Cis-platin is an effective anti-neoplastic agent, but it is also highly nephrotoxic. Here, we clearly identify the human organic cation transporter 2 (hOCT2) as the critical transporter for cis-platin nephrotoxicity in isolated human proximal tubules and offer a potential mechanism for reducing nephrotoxicity in clinical practice. Interaction of cis-platin with hOCT2 in kidney or hOCT1 in liver was investigated with the fluorescent cation 4-[4-(dimethyl-amino)styril]-methylpyridinium in stably transfected HEK293 cells and for the first time in tissues physiologically expressing these transporters, human proximal tubules, and human hepatocyte couplets. Cis-platin (100 micromol/L) inhibited transport via hOCT2-HEK293 but not hOCT1-HEK293. In human proximal tubules cis-platin competed with basolateral organic cation transport, whereas it had no effect in tubules from a diabetic kidney or in hepatocytes. In hOCT2-HEK293 cells treated for 15 hours, incubation with cis-platin induced apoptosis, which was completely suppressed by contemporaneous incubation with the hOCT2 substrate cimetidine (100 micromol/L). These findings demonstrate that uptake of cis-platin is mediated by hOCT2 in renal proximal tubules, explaining its organ-specific toxicity. A combination of cis-platin with other substrates that compete for hOCT2 offers an effective option to decrease nephrotoxicity in the clinical setting.

Increased inorganic phosphate induces human endothelial cell apoptosis in vitro

Chronic kidney disease with hyperphosphatemia is associated with accelerated atherosclerosis and endothelial dysfunction. However, the contribution of high serum phosphate levels to endothelial injury is incompletely understood. The aim of this work was to evaluate the responses of endothelial cells to elevated levels of extracellular phosphate in vitro. High phosphate in concentrations similar to those observed in uremia-associated hyperphosphatemia (>2.5 mM) induced apoptosis in two endothelial cell lines (EAhy926 cells and GM-7373 cells). This effect was enhanced when cells were incubated for 24 h in the presence of 2.8 mM calcium instead of 1.8 mM. By treating cells with 0.5 or 1.0 mM phosphonoformic acid, an inhibitor of the phosphate transporter, death was completely prevented. The process of phosphate-induced apoptosis was further characterized by increased oxidative stress, as detected by increased ROS generation and disruption of the mitochondrial membrane potential at approximately 2 h after treatment, followed by caspase activation. These findings show that hyperphosphatemia causes endothelial cell apoptosis, a process that impairs endothelial integrity. Endothelial cell injury induced by high phosphate concentrations may be an initial event leading to vascular complications in patients with chronic kidney disease.

Individual PKC-Phosphorylation Sites in Organic Cation Transporter 1 Determine Substrate Selectivity and Transport Regulation

To elucidate the molecular mechanisms underlying stimulation of rat organic cation transporter type 1 (rOCT1) by protein kinase C (PKC) activation, functional properties and regulation of rOCT1 stably expressed in HEK293 cells after site-directed mutagenesis of putative PKC phosphorylation-sites were compared with wild-type (WT) rOCT1 using microfluorometric measurements with the fluorescence organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)). Either substitutions of single (S286A, S292A, T296A, S328A, and T550A) or of all five PKC-sites (5x-PKC) with alanine suppressed PKC-induced stimulation of ASP(+) uptake, whereas regulation by p56(lck) tyrosine kinase was conserved in all mutants. Remarkably, the apparent affinities for TEA(+), TPA(+), and quinine were changed differently in each mutant (EC(50) in WT, S286A, S292A, T296A, S328A, T550A, and 5x-PKC in mumol: TEA(+): 105, 153, 56, 1135, 484, 498, 518; TPA(+): 0.1, 2.1, 0.3, 1.0, 43, 0.3, 2.2; quinine: 1.5, 3.0, 2.5, 4.8, 81, 7.6, 8.9, respectively). After mutations, no effects of PKC activation on apparent affinity of rOCT1 for these substrates could be detected, in contrast to what was observed in WT. PKC activation had no significant effect on rOCT1 trafficking from intracellular pools to the cell membrane. Substitution of all PKC sites suppressed PKC-induced phosphorylation of rOCT1. In conclusion, it was found that the presence of all five potential PKC phosphorylation sites is necessary for the PKC-induced stimulation of rOCT1. The different effects on the EC(50) values by the different mutations suggest that the large intracellular loop participates in building the substrate binding pocket of rOCT1 or specifically modulates its structure.

Outcome of Kidney Transplantation in Patients with Inherited Thrombophilia: Data of a Prospective Study

Inherited prothrombotic risk factors predispose patients to thromboembolic events. In kidney transplant recipients, thrombophilia may manifest itself with venous thrombosis, microvascular occlusion, or acute rejection with major consequences for allograft survival. This is a prospective study on 165 renal allograft recipients to evaluate the contribution of genetic thrombophilic risk factors to transplant outcome. Besides antithrombin, protein C, and protein S deficiencies, none of which was found in our patient group, factor V G1691A (FV G1691A), prothrombin G20210A (PT G20210A) mutations, and methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphisms were studied. The primary endpoint of the study was occurrence of an acute rejection within the first 90 d and transplant loss within 1 yr. Heterozygous FV G1691A and PT G20210A mutations and the MTHFR T677T variant were significantly associated with acute rejections with rejection rates of 68%, 67%, and 71%, respectively, as compared with 35% in patients not carrying these genotypes. Many rejections that were histologically proven were acute vascular ones. Transplant loss was significantly associated exclusively with the PT G20210A group (50% 1-yr graft survival; odds ratio, 10.0; 95% confidence interval, 1.8 to 56.1). PT G20210A patients exerted the highest prothrombotic activity pretransplant, as determined by prothrombin 1.2 fragments (PT F1.2), which may be the background for minor outcome. In conclusion, common prothrombotic mutations are significantly associated with acute rejections, especially vascular rejections, and for PT G20210A also with early transplant failure. Screening for hypercoagulable states pretransplant is recommended to intensify anticoagulatory treatment posttransplant.

High phosphate directly affects endothelial function by downregulating annexin II

Hyperphosphatemia is associated with increased cardiovascular risk in patients with renal disease and in healthy individuals. Here we tested whether high phosphate has a role in the pathophysiology of cardiovascular events by interfering with endothelial function, thereby impairing microvascular function and angiogenesis. Protein expression analysis found downregulation of annexin II in human coronary artery endothelial cells, an effect associated with exacerbated shedding of annexin II-positive microparticles by the cells exposed to high phosphate media. EAhy926 endothelial cells exposed to sera from hyperphosphatemic patients also display decreased annexin II, suggesting a negative correlation between serum phosphate and annexin II expression. By using endothelial cell-based assays in vitro and the chicken chorioallantoic membrane assay in vivo, we found that angiogenesis, vessel wall morphology, endothelial cell migration, capillary tube formation, and endothelial survival were impaired in a hyperphosphatemic milieu. Blockade of membrane-bound extracellular annexin II with a specific antibody mimicked the effects of high phosphate. In addition, high phosphate stiffened endothelial cells in vitro and in rats in vivo. Thus, our results link phosphate and adverse clinical outcomes involving the endothelium in both healthy individuals and patients with renal disease.

Down-Regulation of Monocyte Apoptosis by Phagocytosis of Platelets: Involvement of a Caspase-9, Caspase-3, and Heat Shock Protein 70-Dependent Pathway

Monocytes interact and cross-talk with platelets in many settings including inflammation, hemostasis, or vascular disorders. During inflammatory diseases, there is a rapid targeting of monocytes and platelets to points of inflammation and endothelial injury, where they lie side-by-side. In this in vitro study, we investigated different interactions between monocytes and platelets and elucidated whether platelets might affect monocyte apoptosis. Freshly isolated human monocytes were rendered apoptotic by serum deprivation or CD95 ligation and cocultured with platelets. Monocyte apoptosis was determined by flow cytometry, TUNEL staining, DNA electrophoresis, and transmission electron microscopy imaging. We could show that monocyte apoptosis was highly suppressed when platelets were added to the cultures. Transmission electron microscopy depicted that monocytes completely ingested thrombocytes by phagocytosis. Blocking thrombocyte uptake by the phagocytosis inhibitor cytochalasin D abrogated the enhanced monocyte survival and led to high apoptosis levels. Monocyte survival was paralleled by down-regulation of caspase-9 and -3 and up-regulation of heat shock protein 70 during uptake of platelets. Platelet supernatants and contents of platelet granules were ineffective in altering monocyte senescence. Also, ingestion of latex beads or zymosan by monocytes was ineffective to mimic platelet-dependent rescue from apoptosis. In conclusion, this study shows that platelets can suppress apoptosis of monocytes by a specific phagocytosis-dependent process with further consequences for atherosclerotic or inflammatory conditions.

Monocyte activation for enhanced tumour necrosis factor-α and interleukin 6 production during chronic renal allograft rejection

To evaluate the contribution of immune mechanisms in the initiation and progression of chronic renal allograft rejection we investigated monocyte-derived cytokine synthesis in vitro in 16 patients with histologically proven chronic rejection; 22 transplant patients with stable function served as controls. Basal tumour necrosis factor-alpha (TNF-alpha) production, measured by L 929 bioassay, was low and not significantly different in both groups. By triggering TNF-alpha formation in vitro by lipopolysaccharide (LPS), high concentrations of TNF-alpha (155.1 +/- 243.0 ng/ml) were measured in monocyte cultures from chronic rejection patients which greatly exceeded the TNF-alpha levels of 11.5 +/- 14.5 ng/ml in the control group. Measurement of interleukin 6 (IL-6) levels by enzyme immunoassay gave similar results, with significantly higher IL-6 concentrations in LPS-triggered monocyte cultures from chronic rejection compared with stable function patients. Additional stimulation of LPS-treated monocytes with interferon-gamma (IFN-gamma) as a priming agent enhanced TNF-alpha formation in stable function patients and, in contrast, slightly reduced monokine formation in chronic rejection patients, which suggests that in this group a high activation level of monocytes has already been reached in vivo by T cell factors such as IFN-gamma. Treatment of monocyte cultures in vitro with prednisolone reduced TNF-alpha formation differently in most but not all cultures from chronic rejection and stable function patients; thus this in vitro test system might be helpful in predicting the benefit of an intensified immunosuppressive regimen for chronic rejection patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood pressure, antihypertensive treatment, and graft survival in kidney transplant patients.

Whether the use of angiotensin‐converting enzyme inhibitor or angiotensin receptor blocker inhibitor (ACEI/ARB) is beneficial in renal transplant recipients remains controversial. In this retrospective study on 505 renal transplant recipients, we analyzed blood pressure and graft survival according to antihypertensive treatment with ACE‐I/ARB and/or calcium channel blockers (CCB) over a period of 10 years. Patients were stratified according to their blood pressure 1 year after transplantation [controlled (≤130/80 mmHg; CTR, 181 patients) and noncontrolled (>130/80 mmHg; non‐CTR, 324 patients)] and according to antihypertensive treatment (ACE‐I/ARB and/or CCB taken for at least 2 years). One year after transplantation, 88.4% of CTR and 96.6% of non‐CTR received antihypertensive treatment (P < 0.05). Graft survival was longer in CTR than in non‐CTR (P < 0.05). Importantly, graft survival was longer in patients who received long‐term treatment with ACEI/ARB, CCB, or a combination of ACEI/ARB and CCB (P < 0.001). The beneficial effect of ACEI/ARB therapy was more pronounced in non‐CTR compared with that of CTR. We conclude that blood pressure control is a key target for long‐term graft survival in renal transplant patients. Long‐term ACEI/ARB and CCB therapy is beneficial for graft survival, especially in patients with diabetes and/or albuminuria.

A randomized, double-blind study of valsartan versus metoprolol on arterial distensibility and endothelial function in essential hypertension

BACKGROUND Antihypertensive drugs may have differential, pressure-independent effects on hypertension-associated alterations of arterial function. We compared the effects of a 12-week therapy with the AT(1)-receptor antagonist valsartan (Val) versus the beta-blocker metoprolol (Met) on arterial stiffness and endothelial function in mildly hypertensive patients at rest and during generalized sympathetic stimulation. METHODS Sixty-eight patients (37 male, 31 female, 46 +/- 6 years) were randomized to Val (80-160 mg/d) or Met (50-100 mg/d). Effects of therapy on endothelial function, brachial and carotid artery distensibility coefficients, pulse wave velocity, carotid intima-media thickness and elastic modulus were assessed at rest and during the cold pressor test. RESULTS Fifty-two patients were available for per protocol analysis. Blood pressure was comparably reduced in both treatment groups. Effects on endothelial function and large artery elastic wall properties did not differ significantly between the two antihypertensive treatment regimens. Trends did not differ significantly between groups for any parameter including carotid intima-media thickness and elastic modulus. CONCLUSION Short-term treatment with Val and Met had similar effects on large artery functional vessel wall properties in a population of mildly hypertensive patients.

INFECTION BY CANDIDA ALBICANS INHIBITS APOPTOSIS OF HUMAN MONOCYTES AND MONOCYTIC U937 CELLS

Infectious microorganisms can differently induce or Inhibit apoptosis of immunocompetent effector and host cells. In this study we examined the influence of an infection by Candida albicans (C. albicans) on programmed cell death of monocytic U937 cells and human monocytes. Basal and tumor necrosis factor α (TNF‐α)‐induced DNA fragmentation of U937 cells was significantly inhibited by an infection with C. albicans. Enhanced apoptosis of U937 cells, induced by TNF‐α, caused a diminished candidacidal activity of the effector cells, whereas inhibition of apoptosis by granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) was paralleled by an intensified host defense. Pretreatment of U937 cells or monocytes with the cyclooxygenase blocker indomethacin completely abolished the reduction of DNA fragmentation induced by the yeast. Studying the underlying mechanisms we found that C. albicans induced formation of prostaglandin E2 (PGE2) by U937. Exogenous administration of PGE2 down‐regulated apoptosis of U937 or human monocytes to a similar extent as did fungal infection. Activation of protein kinase A by the cAMP analogue 8‐bromo‐cAMP inhibited U937 apoptosis, as did PGE2 On the other hand, rp‐cAMP, a blocker of the cAMP‐dependent signal transduction, restored and elevated DNA fragmentation levels down‐regulated by C. albicans. U937 cells expressed the bcl‐2 protein but the infection with fungi or PGE2 treatment did not increase proto‐oncogene expression. Monocytic effector cells may therefore strengthen the defense against C. albicans by an autocrine feedback regulation via a PGE2‐dependent, cAMP‐transduced inhibition of apoptosis. J. Leukoc. Biol. 60: 737–743; 1996.