Bisphenol A and Bisphenol S in Hemodialyzers.

Bisphenol A (BPA)-based materials are used in the manufacturing of hemodialyzers, including their polycarbonate (PC) housings and polysulfone (PS) membranes. As concerns for BPA's adverse health effects rise, the regulation on BPA exposure is becoming more rigorous. Therefore, BPA alternatives, such as Bisphenol S (BPS), are increasingly used. It is important to understand the patient risk of BPA and BPS exposure through dialyzer use during hemodialysis. Here, we report the bisphenol levels in extractables and leachables obtained from eight dialyzers currently on the market, including high-flux and medium cut-off membranes. A targeted liquid chromatography-mass spectrometry strategy utilizing stable isotope-labeled internal standards provided reliable data for quantitation with the standard addition method. BPA ranging from 0.43 to 32.82 µg/device and BPS ranging from 0.02 to 2.51 µg/device were detected in dialyzers made with BPA- and BPS-containing materials, except for the novel FX CorAL 120 dialyzer. BPA and BPS were also not detected in bloodline controls and cellulose-based membranes. Based on the currently established tolerable intake (6 µg/kg/day), the resulting margin of safety indicates that adverse effects are unlikely to occur in hemodialysis patients exposed to BPA and BPS quantified herein. With increasing availability of new data and information about the toxicity of BPA and BPS, the patient safety limits of BPA and BPS in those dialyzers may need a re-evaluation in the future.

Steviol glycosides as an alternative osmotic agent for peritoneal dialysis fluid.

Background: Peritoneal dialysis (PD) is a renal replacement technique that requires repeated exposure of the peritoneum to hyperosmolar PD fluids (PDFs). Unfortunately, it promotes alterations of the peritoneal membrane (PM) that affects its functionality, including mesothelial-mesenchymal transition (MMT) of mesothelial cells (MCs), inflammation, angiogenesis, and fibrosis. Glucose is the most used osmotic agent, but it is known to be at least partially responsible, together with its degradation products (GDP), for those changes. Therefore, there is a need for more biocompatible osmotic agents to better maintain the PM. Herein we evaluated the biocompatibility of Steviol glycosides (SG)-based fluids. Methods: The ultrafiltration and transport capacities of SG-containing and glucose-based fluids were analyzed using artificial membranes and an in vivo mouse model, respectively. To investigate the biocompatibility of the fluids, Met-5A and human omental peritoneal MCs (HOMCs) were exposed in vitro to different types of glucose-based PDFs (conventional 4.25% glucose solution with high-GDP level and biocompatible 2.3% glucose solution with low-GDP level), SG-based fluids or treated with TGF-ß1. Mice submitted to surgery of intraperitoneal catheter insertion were treated for 40 days with SG- or glucose-based fluids. Peritoneal tissues were collected to determine thickness, MMT, angiogenesis, as well as peritoneal washings to analyze inflammation. Results: Dialysis membrane experiments demonstrated that SG-based fluids at 1.5%, 1%, and 0.75% had a similar trend in weight gain, based on curve slope, as glucose-based fluids. Analyzing transport capacity in vivo, 1% and 0.75% SG-based fluid-exposed nephrectomized mice extracted a similar amount of urea as the glucose 2.3% group. In vitro, PDF with high-glucose (4.25%) and high-GDP content induced mesenchymal markers and angiogenic factors (Snail1, Fibronectin, VEGF-A, FGF-2) and downregulates the epithelial marker E-Cadherin. In contrast, exposition to low-glucose-based fluids with low-GDP content or SG-based fluids showed higher viability and had less MMT. In vivo, SG-based fluids preserved MC monolayer, induced less PM thickness, angiogenesis, leukocyte infiltration, inflammatory cytokines release, and MMT compared with glucose-based fluids. Conclusion: SG showed better biocompatibility as an osmotic agent than glucose in vitro and in vivo, therefore, it could alternatively substitute glucose in PDF.

A porcine model of hemodialyzer reactions: roles of complement activation and rinsing back of extracorporeal blood.

Hemodialysis reactions (HDRs) resemble complement-activation-related pseudoallergy (CARPA) to certain i.v. drugs, for which pigs provide a sensitive model. On this basis, to better understand the mechanism of human HDRs, we subjected pigs to hemodialysis using polysulfone (FX CorDiax 40, Fresenius) or cellulose triacetate (SureFlux-15UX, Nipro) dialyzers, or Dialysis exchange-set without membranes, as control. Experimental endpoints included typical biomarkers of porcine CARPA; pulmonary arterial pressure (PAP), blood cell counts, plasma sC5b-9 and thromboxane-B2 levels. Hemodialysis (60 min) was followed by reinfusion of extracorporeal blood into the circulation, and finally, an intravenous bolus injection of the complement activator zymosan. The data indicated low-extent steady rise of sC5b-9 along with transient leukopenia, secondary leukocytosis and thrombocytopenia in the two dialyzer groups, consistent with moderate complement activation. Surprisingly, small changes in baseline PAP and plasma thromboxane-B2 levels during hemodialysis switched into 30%-70% sharp rises in all three groups resulting in synchronous spikes within minutes after blood reinfusion. These observations suggest limited complement activation by dialyzer membranes, on which a membrane-independent second immune stimulus was superimposed, and caused pathophysiological changes also characteristic of HDRs. Thus, the porcine CARPA model raises the hypothesis that a second "hit" on anaphylatoxin-sensitized immune cells may be a key contributor to HDRs.

Genomic reprograming analysis of the Mesothelial to Mesenchymal Transition identifies biomarkers in peritoneal dialysis patients.

Peritoneal dialysis (PD) is an effective renal replacement therapy, but a significant proportion of patients suffer PD-related complications, which limit the treatment duration. Mesothelial-to-mesenchymal transition (MMT) contributes to the PD-related peritoneal dysfunction. We analyzed the genetic reprograming of MMT to identify new biomarkers that may be tested in PD-patients. Microarray analysis revealed a partial overlapping between MMT induced in vitro and ex vivo in effluent-derived mesothelial cells, and that MMT is mainly a repression process being higher the number of genes that are down-regulated than those that are induced. Cellular morphology and number of altered genes showed that MMT ex vivo could be subdivided into two stages: early/epithelioid and advanced/non-epithelioid. RT-PCR array analysis demonstrated that a number of genes differentially expressed in effluent-derived non-epithelioid cells also showed significant differential expression when comparing standard versus low-GDP PD fluids. Thrombospondin-1 (TSP1), collagen-13 (COL13), vascular endothelial growth factor A (VEGFA), and gremlin-1 (GREM1) were measured in PD effluents, and except GREM1, showed significant differences between early and advanced stages of MMT, and their expression was associated with a high peritoneal transport status. The results establish a proof of concept about the feasibility of measuring MMT-associated secreted protein levels as potential biomarkers in PD.

Nalfurafine suppresses pruritogen- and touch-evoked scratching behavior in models of acute and chronic itch in mice.

The kappa-opioid agonist, nalfurafine, has been approved in Japan for treatment of itch in patients with chronic kidney disease. We presently investigated if systemic administration of nalfurafine inhibited ongoing or touch-evoked scratching behavior (alloknesis) following acute intradermal injection of histamine or the non-histaminergic itch mediator, chloroquine, in mice. We also investigated if nalfurafine suppressed spontaneous or touch-evoked scratching in an experimental model of chronic dry skin itch. Nalfurafine reduced scratching evoked by histamine and chloroquine. Following acute histamine, but not chloroquine, low-threshold mechanical stimuli reliably elicited directed hindlimb scratching behavior, which was significantly attenuated by nalfurafine. In mice with experimental dry skin, nalfurafine abolished spontaneous scratching but had no effect on alloknesis. Nalfurafine thus appears to be a promising treatment for acute itch as well as ongoing itch of dry skin.

Novel selective MMP-13 inhibitors reduce collagen degradation in bovine articular and human osteoarthritis cartilage explants.

OBJECTIVE: MMP-13 is highly upregulated in arthritis and therefore strongly implicated in the pathogenesis of osteoarthritis (OA). Selective inhibition of MMP-13 may provide the desired cartilage degradation protection, while overcoming the musculoskeletal toxicity seen with nonselective inhibition of MMPs. METHODS: Activity and selectivity of novel MMP-13 inhibitors were determined in enzymatic and collagenase assays. Inhibition kinetics and competitive binding experiments were performed. The inhibition of collagen degradation was studied in cartilage explants from OA patients and in bovine and human articular cartilage systems. RESULTS: We have identified a new class of very potent and highly selective non-zinc-binding MMP-13 inhibitors. Selective MMP-13 inhibitors completely blocked type II collagen degradation in bovine explants and showed up to 80% inhibition in human OA cartilage. CONCLUSIONS: These results indicate MMP-13 as the primary collagenase in the human OA cartilage and in the IL-1/OSM-induced cartilage degradation process and suggest that selective MMP-13 inhibitors may be a potential treatment of OA.

A new class of potent matrix metalloproteinase 13 inhibitors for potential treatment of osteoarthritis: Evidence of histologic and clinical efficacy without musculoskeletal toxicity in rat models.

OBJECTIVE: Matrix metalloproteinases (MMPs) have long been considered excellent targets for osteoarthritis (OA) treatment. However, clinical utility of broad-spectrum MMP inhibitors developed for this purpose has been restricted by dose-limiting musculoskeletal side effects observed in humans. This study was undertaken to identify a new class of potent and selective MMP-13 inhibitors that would provide histologic and clinical efficacy without musculoskeletal toxicity. METHODS: Selectivity assays were developed using catalytic domains of human MMPs. Freshly isolated bovine articular cartilage or human OA cartilage was used in in vitro cartilage degradation assays. The rat model of monoiodoacetate (MIA)-induced OA was implemented for assessing the effects of MMP-13 inhibitors on cartilage degradation and joint pain. The surgical medial meniscus tear model in rats was used to evaluate the chondroprotective ability of MMP-13 inhibitors in a chronic disease model of OA. The rat model of musculoskeletal side effects (MSS) was used to assess whether selective MMP-13 inhibitors have the joint toxicity associated with broad-spectrum MMP inhibitors. RESULTS: A number of non-hydroxamic acid-containing compounds that showed a high degree of potency for MMP-13 and selectivity against other MMPs were designed and synthesized. Steady-state kinetics experiments and Lineweaver-Burk plot analysis of rate versus substrate concentration with one such compound, ALS 1-0635, indicated linear, noncompetitive inhibition, and Dixon plot analysis from competition studies with a zinc chelator (acetoxyhydroxamic acid) and ALS 1-0635 demonstrated nonexclusive binding. ALS 1-0635 inhibited bovine articular cartilage degradation in a dose-dependent manner (48.7% and 87.1% at 500 nM and 5,000 nM, respectively) and was effective in inhibiting interleukin-1alpha- and oncostatin M-induced C1,C2 release in human OA cartilage cultures. ALS 1-0635 modulated cartilage damage in the rat MIA model (mean +/- SEM damage score 1.3 +/- 0.3, versus 2.2 +/- 0.4 in vehicle-treated animals). Most significantly, when treated twice daily with oral ALS 1-0635, rats with surgically induced medial meniscus tear exhibited histologic evidence of chondroprotection and reduced cartilage degeneration, without observable musculoskeletal toxicity. CONCLUSION: The compounds investigated in this study represent a novel class of MMP-13 inhibitors. They are mechanistically distinct from previously reported broad-spectrum MMP inhibitors and do not exhibit the problems previously associated with these inhibitors, including selectivity, poor pharmacokinetics, and MSS liability. MMP-13 inhibitors exert chondroprotective effects and can potentially modulate joint pain, and are, therefore, uniquely suited as potential disease-modifying osteoarthritis drugs.

Matrix metalloprotease inhibitors suppress initiation and progression of chondrogenic differentiation of mesenchymal stromal cells in vitro.

Mesenchymal stromal cells (MSC) are an attractive source for cell therapy and tissue engineering of joint cartilage. Common chondrogenic in vitro protocols, however, induce hypertrophic markers like COL10A1, matrix metalloproteinase 13 (MMP13), and alkaline phosphatase (ALP) reminiscent of endochondral bone formation. To direct MSC toward articular chondrocytes more specifically, a better understanding of the regulatory steps is desirable. Proteases are important players in matrix remodeling, display inhibitory effects on growth plate development and MMP13 inhibition prevented hypertrophy of bovine chondrocytes. The aim of this study was to evaluate whether the activity of proteases and MMPs, especially MMP13, is crucial for the transition of MSC toward mature chondrocytes and could allow to selectively influence aspects of early and late chondrogenic differentiation. Protease inhibitors were added during MSC chondrogenesis and stage-specific markers were assessed by histology, qPCR, and ALP quantification. Chondrogenesis was little affected by leupeptin, pepstatin, or aprotinin. In contrast, broad spectrum pan-MMP inhibitors dose dependently suppressed proteoglycan deposition, collagen type II and type X staining, ALP activity, and reduced SOX9 and COL2A1 expression. A selective MMP13 inhibitor allowed chondrogenesis and showed only weak effects on ALP activity. In conclusion, transition of MSC toward mature chondrocytes in vitro depended on molecules suppressed by pan-MMP inhibitors identifying chondrogenic differentiation of MSC as a sophistically regulated process in which catabolic enzymes are capable to directly influence cellular fate. In future therapeutic applications of diseased joints, the tested MMP13-specific inhibitor promises suppression of collagen type II degradation without imposing a risk to impair MSC-driven regeneration processes.

Ketones as building blocks for dynamic combinatorial libraries: highly active neuraminidase inhibitors generated via selection pressure of the biological target.

New and potent inhibitors of neuraminidase, a key enzyme in the influenza virus activity, have been discovered in dynamic combinatorial libraries based on ketones and amines as building blocks. Selective synthesis of a number of inhibitors among multiple theoretically possible combinations of building blocks is driven by the presence of the target enzyme.

Matrilin-2 interacts with itself and with other extracellular matrix proteins.

Matrilin-2 is a component of extracellular filamentous networks. To study the interactions by which it can be integrated into such assemblies, full-length and truncated forms of matrilin-2 were recombinantly expressed in HEK-293 cells and purified from conditioned medium. The recombinant proteins, when used in interaction assays, showed affinity to matrilin-2 itself, but also to other collagenous and non-collagenous extracellular matrix proteins. The interaction between matrilin-2 and collagen I was studied in greater detail and could be shown to occur at distinct sites on the collagen I molecule and to have a K (D) of about 3 x 10(-8) M. Interactions with some non-collagenous protein ligands were even stronger, with matrilin-2 binding to fibrillin-2, fibronectin and laminin-1-nidogen-1 complexes, with K (D) values in the range of 10(-8)-10(-11) M. Co-localization of matrilin-2 with these ligands in the dermal-epidermal basement membrane, in the microfibrils extending from the basement membrane into the dermis, and in the dermal extracellular matrix, indicates a physiological relevance of the interactions in the assembly of supramolecular extracellular matrix structures.

Expression of matrilin-2 in human skin.

The extracellular matrix is composed of a large number of different modular proteins. Matrilin-2 is a newly described member of the protein superfamily with von Willebrand factor A-like modules. To examine the expression of matrilin-2 in human skin, the distribution of protein and mRNA was studied by immunohistochemistry and in situ hybridization. In addition, immunoblotting and real-time reverse transcription polymerase chain reaction were used to investigate the expression of matrilin-2 in keratinocyte and fibroblast cultures. In vivo, keratinocytes and fibroblasts were both found to express matrilin-2 mRNA and deposit the protein at the basal side of the dermal-epidermal basement membrane. Matrilin-2 molecules synthesized by the two cell types in vitro appeared to be processed differently by cell-associated proteases. Transcription of matrilin-2 mRNA in keratinocytes was enhanced by a diffusible factor produced by fibroblasts, suggesting a regulatory mechanism for the production of extracellular matrix at the dermal-epidermal junction. These findings demonstrate that matrilin-2 is expressed in normal skin by keratinocytes and fibroblasts and may thus contribute to cutaneous homeostasis.

Target-induced formation of neuraminidase inhibitors from in vitro virtual combinatorial libraries.

Neuraminidase, a key enzyme responsible for influenza virus propagation, has been used as a template for selective synthesis of small subsets of its own inhibitors from theoretically highly diverse dynamic combinatorial libraries. We show that the library building blocks, aldehydes and amines, form significant amounts of the library components resulting from their coupling by reductive amination only in the presence of the enzyme. The target amplifies the best hits at least 120-fold. The dynamic libraries synthesized and screened in such an in vitro virtual mode form the components that possess high inhibitory activity, as confirmed by enzyme assays with independently synthesized individual compounds.