László Rosivall

Elevated Fibroblast Growth Factor 23 is a Risk Factor for Kidney Transplant Loss and Mortality

An increased circulating level of fibroblast growth factor 23 (FGF23) is an independent risk factor for mortality, cardiovascular disease, and progression of chronic kidney disease (CKD), but its role in transplant allograft and patient survival is unknown. We tested the hypothesis that increased FGF23 is an independent risk factor for all-cause mortality and allograft loss in a prospective cohort of 984 stable kidney transplant recipients. At enrollment, estimated GFR (eGFR) was 51 ± 21 ml/min per 1.73 m(2) and median C-terminal FGF23 was 28 RU/ml (interquartile range, 20 to 43 RU/ml). Higher FGF23 levels independently associated with increased risk of the composite outcome of all-cause mortality and allograft loss (full model hazard ratio: 1.46 per SD increase in logFGF23, 95% confidence interval: 1.28 to 1.68, P<0.001). The results were similar for each component of the composite outcome and in all sensitivity analyses, including prespecified analyses of patients with baseline eGFR of 30 to 90 ml/min per 1.73 m(2). In contrast, other measures of phosphorus metabolism, including serum phosphate and parathyroid hormone (PTH) levels, did not consistently associate with outcomes. We conclude that a high (or elevated) FGF23 is an independent risk factor for death and allograft loss in kidney transplant recipients.

Integrity of cell-cell contacts is a critical regulator of TGF-β1-induced epithelial-to-myofibroblast transition: Role for β-catenin

Injury of the tubular epithelium and TGF-β1-induced conversion of epithelial cells to α-smooth muscle actin (SMA)-expressing myofibroblasts are key features of kidney fibrosis. Since injury damages intercellular junctions and promotes fibrosis, we hypothesized that cell contacts are critical regulators of TGF-β1-triggered epithelial-to-mesenchymal transition (EMT). Here we show that TGF-β1 was unable to induce EMT in intact confluent monolayers, but three different models of injury-induced loss of epithelial integrity (subconfluence, wounding, and contact disassembly by Ca2+-removal) restored its EMT-inducing effect. This manifested in loss of E-cadherin, increased fibronectin production and SMA expression. TGF-β1 or contact disassembly alone only modestly stimulated the SMA promoter in confluent layers, but together exhibited strong synergy. Since β-catenin is a component of intact adherens junctions, but when liberated from destabilized contacts may act as a transcriptional co-activator, we investigated its role in TGF-β1-provoked EMT. Contact disassembly alone induced degradation of E-cadherin and β-catenin, but TGF-β1 selectively rescued β-catenin and stimulated the β-catenin-driven reporter TopFLASH. Moreover, chelation of free β-catenin with the N-cadherin cytoplasmic tail suppressed the TGF-β1 plus contact disassembly-induced SMA promoter activation and protein expression. These results suggest a β-catenin-dependent two-hit mechanism in which both an initial epithelial injury and TGF-β1 are required for EMT.

Protective effects of mercaptoethylguanidine, a selective inhibitor of inducible nitric oxide synthase, in ligature-induced periodontitis in the rat

Excessive production of nitric oxide (NO), and the generation of peroxynitrite have been implicated in various proinflammatory conditions. In the present study, using mercaptoethylguanidine (MEG), a selective inhibitor of iNOS and a peroxynitrite scavenger, we investigated the role of iNOS and peroxynitrite in a rat model of periodontitis. Periodontitis was produced in rat by a ligature of 2/0 braided silk placed around the cervix of the lower left 1st molar. Animals were then divided into two groups: one group of rats was treated with MEG (30 mg kg−1, i.p., 4 times per day for 8 days), animals in the other group received vehicle. At day 8, the gingivomucosal tissue encircling the mandibular 1st molars was removed on both sides from ligated and sham operated animals for inducible nitric oxide synthase (iNOS) activity assay and for immunocytochemistry with anti‐iNOS serum. Plasma extravasation was measured with the Evans blue technique. Alveolar bone loss was measured with a videomicroscopy. Ligation caused a significant, more than 3 fold increase in the gingival iNOS activity, whereas it did not affect iNOS activity on the contralateral side, when compared to sham‐operated animals. Immunohistochemical analysis revealed iNOS‐positive macrophages, lymphocytes and PMNs in the connective tissue and immunoreactive basal layers of epithelium on side of the ligature, and only a few iNOS‐reactive connective tissue cells on the contralateral side. Ligation significantly increased Evans blue extravasation in gingivomucosal tissue and alveolar bone destruction compared to the contralateral side. MEG treatment significantly reduced the plasma extravasation and bone destruction. The present results demonstrated that ligature‐induced periodontitis increases local NO production and that MEG treatment protects against the associated extravasation and bone destruction. Based on the present data, we propose that enhanced formation of NO and peroxynitrite plays a significant role in the pathogenesis of periodontitis.

Integrity of cell-cell contacts is a critical regulator of TGF-beta 1-induced epithelial-to-myofibroblast transition: role for beta-catenin.

Injury of the tubular epithelium and TGF-β1-induced conversion of epithelial cells to α-smooth muscle actin (SMA)-expressing myofibroblasts are key features of kidney fibrosis. Since injury damages intercellular junctions and promotes fibrosis, we hypothesized that cell contacts are critical regulators of TGF-β1-triggered epithelial-to-mesenchymal transition (EMT). Here we show that TGF-β1 was unable to induce EMT in intact confluent monolayers, but three different models of injury-induced loss of epithelial integrity (subconfluence, wounding, and contact disassembly by Ca2+-removal) restored its EMT-inducing effect. This manifested in loss of E-cadherin, increased fibronectin production and SMA expression. TGF-β1 or contact disassembly alone only modestly stimulated the SMA promoter in confluent layers, but together exhibited strong synergy. Since β-catenin is a component of intact adherens junctions, but when liberated from destabilized contacts may act as a transcriptional co-activator, we investigated its role in TGF-β1-provoked EMT. Contact disassembly alone induced degradation of E-cadherin and β-catenin, but TGF-β1 selectively rescued β-catenin and stimulated the β-catenin-driven reporter TopFLASH. Moreover, chelation of free β-catenin with the N-cadherin cytoplasmic tail suppressed the TGF-β1 plus contact disassembly-induced SMA promoter activation and protein expression. These results suggest a β-catenin-dependent two-hit mechanism in which both an initial epithelial injury and TGF-β1 are required for EMT.

Animal Models of Complement-Mediated Hypersensitivity Reactions to Liposomes and Other Lipid-Based Nanoparticles

Intravenous injection of some liposomal drugs, diagnostic agents, micelles and other lipid-based nanoparticles can cause acute hypersensitivity reactions (HSRs) in a high percentage (up to 45%) of patients, with hemodynamic, respiratory and cutaneous manifestations. The phenomenon can be explained with activation of the complement (C) system on the surface of lipid particles, leading to anaphylatoxin (C5a and C3a) liberation and subsequent release reactions of mast cells, basophils and possibly other inflammatory cells in blood. These reactions can be reproduced and studied in pigs, dogs and rats, animal models which differ from each other in sensitivity and spectrum of symptoms. In the most sensitive pig model, a few miligrams of liposome (phospholipid) can cause anaphylactoid shock, characterized by pulmonary hypertension, systemic hypotension, decreased cardiac output and major cardiac arrhythmias. Pigs also display cutaneous symptoms, such as flushing and rash. The sensitivity of dogs to hemodynamic changes is close to that of pigs, but unlike pigs, dogs also react to micellar lipids (such as Cremophor EL) and their response includes pronounced blood cell and vegetative neural changes (e.g., leukopenia followed by leukocytosis, thrombocytopenia, fluid excretions). Rats are relatively insensitive inasmuch as hypotension, their most prominent response to liposomes, is induced only by one or two orders of magnitude higher phospholipid doses (based on body weight) compared to the reactogenic dose in pigs and dogs. It is suggested that the porcine and dog models are applicable for measuring and predicting the (pseudo)allergic activity of particulate “nanodrugs”.

Luminal NaCl delivery regulates basolateral PGE2 release from macula densa cells.

Macula densa (MD) cells express COX-2 and COX-2-derived PGs appear to signal the release of renin from the renal juxtaglomerular apparatus, especially during volume depletion. However, the synthetic machinery and identity of the specific prostanoid released from intact MD cells remains uncertain. In the present studies, a novel biosensor tool was engineered to directly determine whether MD cells release PGE2 in response to low luminal NaCl concentration ([NaCl]L). HEK293 cells were transfected with the Ca2+-coupled E-prostanoid receptor EP1 (HEK/EP1) and loaded with fura-2. HEK/EP1 cells produced a significant elevation in intracellular [Ca2+] ([Ca2+]i) by 29.6 +/- 12.8 nM (n = 6) when positioned at the basolateral surface of isolated perfused MD cells and [NaCl]L was reduced from 150 mM to zero. HEK/EP1 [Ca2+]i responses were observed mainly in preparations from rabbits on a low-salt diet and were completely inhibited by either a selective COX-2 inhibitor or an EP1 antagonist, and also by 100 microM luminal furosemide. Also, 20-mM graduated reductions in [NaCl]L between 80 and 0 mM caused step-by-step increases in HEK/EP1 [Ca2+]i. Low-salt diet greatly increased the expression of both COX-2 and microsome-associated PGE synthase (mPGES) in the MD. These studies provide the first direct evidence that intact MD cells synthesize and release PGE2 during reduced luminal salt content and suggest that this response is important in the control of renin release and renal vascular resistance during salt deprivation.

Body mass index, waist circumference and mortality in kidney transplant recipients.

Higher body mass index (BMI) appears paradoxically associated with better outcomes in patients with chronic kidney disease. Whereas higher BMI reflects both increased visceral and subcutaneous fat and/or muscle mass, a combined assessment of BMI and waist circumference may enable differentiation of visceral adiposity from muscle and/or nonvisceral fat mass. We examined the association of BMI and waist circumference with all‐cause mortality in a prospective cohort of 993 kidney transplant recipients. Associations were examined in Cox models with adjustment for demographic and comorbid conditions and for inflammatory markers. Unadjusted death hazard ratios (95%CI) associated with one standard deviation higher BMI and waist circumference were 0.94 (0.78, 1.13), p = 0.5 and 1.20 (1.00, 1.45), p = 0.05, respectively. Higher BMI was associated with lower mortality after adjustment for waist circumference (0.48 [0.34, 0.69], p < 0.001), and higher waist circumference was more strongly associated with higher mortality after adjustment for BMI (2.18 [1.55–3.08], p < 0.001). The associations of waist circumference with mortality remained significant after additional multivariable adjustments. Higher BMI and waist circumference display opposite associations with mortality in kidney transplant recipients. Waist circumference appears to be a better prognostic marker for obesity than BMI.

Complement activation by polyethoxylated pharmaceutical surfactants: Cremophor-EL, tween-80 and tween-20

Immunosafety analysis of pharmaceutical surfactants is an important step in understanding the complex mechanisms by which they induce side effects in susceptible patients. This paper provides experimental evidences that polyethoxylated surfactants, Cremophor-EL and Tween-80, also known as Polysorbate-80, activate the complement system in vitro, in normal human serum and plasma. They appeared to be more efficient reactogens than their structural homolog, Tween-20. Cremophor-EL and Tween-80 promoted the generation of biologically active complement products, C3a, C5a and C5b-9. Consistently, Paclitaxel and Taxotere (Docetaxel), pharmaceuticals formulated in Cremophor-EL and Tween-80, activated the complement system in similar extent. Moreover, comparison of serum reactivity against the drug-loaded and drug-free formulations exhibited a significant linear correlation. Taken together, these results are consistent with the hypothesis that therapeutic side effects, such as acute hypersensitivity and systemic immunostimulation, caused by intravenous nanomedicines containing polyethoxylated detergents such as Cremophor-EL and Tween-80, can be attributed to complement activation-derived inflammatory mediators.

Liposome-induced complement activation and related cardiopulmonary distress in pigs: factors promoting reactogenicity of Doxil and AmBisome

UNLABELLED Hypersensitivity reactions to liposomal drugs, often observed with Doxil and AmBisome, can arise from activation of the complement (C) system by phospholipid bilayers. To understand the mechanism of this adverse immune reaction called C activation-related pseudoallergy (CARPA), we analyzed the relationship among liposome features, C activation in human serum in vitro, and liposome-induced cardiovascular distress in pigs, a model for human CARPA. Among the structural variables (surface charge, presence of saturated, unsaturated, and PEGylated phospholipids, and cisplatin vs. doxorubicin inside liposomes), high negative surface charge and the presence of doxorubicin were significant contributors to reactogenicity both in vitro and in vivo. Morphological analysis suggested that the effect of doxorubicin might be indirect, via distorting the sphericity of liposomes and, if leaked, causing aggregation. The parallelism among C activation, cardiopulmonary reactions in pigs, and high rate of hypersensitivity reactions to Doxil and AmBisome in humans strengthens the utility of the applied tests in predicting the risk of CARPA. FROM THE CLINICAL EDITOR The authors studied complement activation-related pseudoallergy (CARPA) in a porcine model and demonstrate that high negative surface charge and drug effects leading to distortion of liposome sphericity might be the most critical factors leading to CARPA. The applied tests might be used to predict CARPA in humans.

In vitro and in vivo complement activation and related anaphylactic effects associated with polyethylenimine and polyethylenimine-graft-poly(ethylene glycol) block copolymers

Complement activation by polymeric gene and drug delivery systems has been overlooked in the past. As more reports appear in the literature concerning immunogenicity of polymers and their impact on gene expression patterns, it is important to address possible immune side effects of polymers, namely complement activation. Therefore, in this study the activity of low and high molecular weight poly(ethylene imine) and two PEGylated derivatives to induce complement activation were investigated in human serum. These in vitro results revealed that PEI 25 kDa caused significant and concentration dependent complement activation, whereas none of the other polymers induced such effects at their IC(50) concentrations determined by MTT-assays. To verify these in vitro results, additionally, studies were carried out in a swine model after intravenous administration, showing complement activation-related pseudoallergy (CARPA), reflected in symptoms of transient cardiopulmonary distress. Injections of PEI 25 kDa or PEI(25k)-PEG(2k)(10) at a dose of 0.05 and 0.1 mg/kg caused strong reactivity, while PEI 5 kDa and with PEI(25k)-PEG(20k)(1) were also reactogenic at 0.1 mg/kg. It was found that PEI 25 kDa caused both self- and cross-tolerance, whereas the PEG-PEIs were neither self- nor cross-reactively tachyphylactic. As a result of this study, it was shown that PEGylation of polycations with PEG of 20 kDa or higher molecular weight may be favorable. However, potential safety concerns in the development of PEI-based polymeric carriers for drugs and nucleic acids and their translation from bench to bedside need to be taken into consideration for human application.