Super-swelling behavior of stacked lipid bilayer systems.

Bilayer systems comprising lipid mixtures are the most well-studied model of biological membranes. While the plasma membrane of the cell is a single bilayer, many intra- and extra-cellular biomembranes comprise stacks of bilayers. Most bilayer stacks in nature are periodic, maintaining a precise water layer separation between bilayers. That equilibrium water separation is governed by multiple inter-bilayer forces and is highly responsive. Biomembranes re-configure inter-bilayer spacing in response to temperature, composition, or mass transport cues. In synthetic bilayer systems for applications in cosmetics or topical treatments, control of the hydration level is a critical design handle. Herein we investigate a binary lipid system that leverages key inter-bilayer forces leading to unprecedented levels of aqueous swelling while maintaining a coherent multilamellar form. We found that combining cationic lipids with bicontinuous cubic phase-forming lipids (lipids with positive Gaussian modulus), results in the stabilization of multilamellar phases against repulsive steric forces that typically lead to bilayer delamination at high degrees of swelling. Using ultra-small-angle X-ray scattering alongside confocal laser scanning microscopy, we characterized various super-swelled states of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and glycerol monooleate (GMO) lipids, as well as other analogous systems, at varied concentration and molar ratios. Through these experiments we established swelling profiles of various binary lipid systems that were near-linear with decreasing lipid volume fraction, showing maximum swelling with periodicity well above 200 nanometers. Confocal fluorescence micrograph of super-swelled multilamellar structures in 90GMOD sample at 25 mM concentration. Inset plot shows intensity profile of orange line, with pink triangles indicating maxima.

Pathological cardiolipin-promoted membrane hemifusion stiffens pulmonary surfactant membranes.

Lower tract respiratory diseases such as pneumonia are pervasive, affecting millions of people every year. The stability of the air/water interface in alveoli and the mechanical performance during the breathing cycle are regulated by the structural and elastic properties of pulmonary surfactant membranes (PSMs). Respiratory dysfunctions and pathologies often result in, or are caused by, impairment of the PSMs. However, a gap remains between our knowledge of the etiology of lung diseases and the fundamental properties of PSMs. For example, bacterial pneumonia in humans and mice has been associated with aberrant levels of cardiolipin, a mitochondrial-specific, highly unsaturated 4-tailed anionic phospholipid, in lung fluid, which likely disrupts the structural and mechanical integrity of PSMs. Specifically, cardiolipin is expected to significantly alter PSM elasticity due to its intrinsic molecular properties favoring membrane folding away from a flat configuration. In this paper, we investigate the structural and mechanical properties of the lipidic components of PSMs using lipid-based models as well as bovine extracts affected by the addition of pathological cardiolipin levels. Specifically, using a combination of optical and atomic force microscopy with a surface force apparatus, we demonstrate that cardiolipin strongly promotes hemifusion of PSMs and that these local membrane contacts propagate at larger scales, resulting in global stiffening of lung membranes.

A Randomized Trial of Roxadustat in Anemia of Kidney Failure: SIERRAS Study.

INTRODUCTION: Erythropoiesis-stimulating agents, standard of care for anemia of end-stage kidney disease, are associated with cardiovascular events. We evaluated the efficacy and safety of roxadustat, an oral hypoxia-inducible factor prolyl hydroxylase inhibitor that stimulates erythropoiesis. METHODS: SIERRAS was a phase 3, randomized, open-label, active-controlled study enrolled adults on dialysis for end-stage kidney disease receiving erythropoiesis-stimulating agents for anemia. Patients were randomized (1:1) to thrice-weekly roxadustat or epoetin alfa. Doses were based on previous epoetin alfa dose and adjusted in the roxadustat arm to maintain hemoglobin at ∼11 g/dl during treatment. Epoetin alfa dosing was adjusted per US package insert. Primary efficacy endpoint was mean hemoglobin (g/dl) change from baseline averaged over weeks 28 to 52. Treatment-emergent adverse events were monitored. RESULTS: Enrolled patients (roxadustat, n = 370 and epoetin alfa, n = 371) had similar mean (SD) baseline hemoglobin levels (10.30 [0.66] g/dl). Mean (SD) hemoglobin changes for weeks 28 to 52 were 0.39 (0.93) and -0.09 (0.84) in roxadustat and epoetin alfa, respectively. Roxadustat was noninferior (least squares mean difference: 0.48 [95% confidence interval: 0.37, 0.59]; P < 0.001) to epoetin alfa. Tolerability was comparable between treatments. CONCLUSION: In end-stage kidney disease, roxadustat was noninferior to epoetin alfa in up to 52 weeks of treatment in this erythropoietin-stimulating agent conversion study. Roxadustat had an acceptable tolerability profile.

Safety of a Novel Dialyzer Containing a Fluorinated Polyurethane Surface-Modifying Macromolecule in Patients with End-Stage Kidney Disease.

BACKGROUND: By inhibiting the adsorption of protein and platelets, surface-modifying macromolecules (SMMs) may improve the hemocompatibility of hemodialyzers. This trial aims to assess the performance and safety of a novel dialyzer with a fluorinated polyurethane SMM, Endexo™. METHODS: This prospective, sequential, multicenter, open-label study (NCT03536663) was designed to meet regulatory requirements for clinical testing of new hemodialyzers, including assessment of the in vivo ultrafiltration coefficient (Kuf). Adults prescribed thrice-weekly hemodialysis were eligible for enrollment. After completing 12 hemodialysis sessions with an Optiflux® F160NR dialyzer, patients received 38 sessions with the dialyzer with Endexo. Evaluated parameters included the in vivo Kuf of the dialyzer with Endexo extent of removal of urea, albumin, and ß2-microglobulin (ß2M), as well as complement activation. RESULTS: Twenty-three patients received 268 hemodialysis treatments during the Optiflux period, and 18 patients received 664 hemodialysis treatments during the Endexo period. Three serious adverse events were reported, and none of them were considered device related. No overt complement activation was observed with either dialyzer. Both dialyzers were associated with comparable mean increases in serum albumin levels from pre- to posthemodialysis (Optiflux: 7.9%; Endexo: 8.0%). These increases can be viewed in the context of a mean increase in hemoglobin of approximately 5% and a mean ultrafiltration volume removed of approximately 2.2 L. The corrected mean ß2M removal rate was 47% higher during the Endexo period (67.73%). Mean treatment times (208 vs. 205 min), blood flow rates (447.7 vs. 447.5 mL/min), dialysate flow rates (698.5 vs. 698.0 mL/min), urea reduction ratio (82 vs. 81%), and spKt/V (2.1 vs. 1.9) were comparable for the Endexo and Optiflux periods, respectively. The mean (SD) Kuf was 15.85 (10.33) mL/h/mm Hg during the first use of the dialyzer with Endexo (primary endpoint) and 16.36 (9.92) mL/h/mm Hg across the Endexo period. CONCLUSIONS: The safety of the novel dialyzer with Endexo was generally comparable to the Optiflux dialyzer, while exhibiting a higher ß2M removal rate.

Structure of Lung-Mimetic Multilamellar Bodies with Lipid Compositions Relevant in Pneumonia.

The hierarchical assembly of lipids, as modulated by composition and environment, plays a significant role in the function of biological membranes and a myriad of diseases. Elevated concentrations of calcium ions and cardiolipin (CL), an anionic tetra-alkyl lipid found in mitochondria and some bacterial cell membranes, have been implicated in pneumonia recently. However, their impact on the physicochemical properties of lipid assemblies in lungs and how it impairs alveoli function is still unknown. We use small- and wide-angle X-ray scattering (S/WAXS) and solid-state nuclear magnetic resonance (ssNMR) to probe the structure and dynamics of lung-mimetic multilamellar bodies (MLBs) in the presence of Ca2+ and CL. We conjecture that CL overexpressed in the hypophase of alveoli strongly affects the structure of lung-lipid bilayers and their stacking in the MLBs. Specifically, S/WAXS data revealed that CL induces significant shrinkage of the water-layer separating the concentric bilayers in multilamellar aggregates. ssNMR measurements indicate that this interbilayer tightening is due to undulation repulsion damping as CL renders the glycerol backbone of the membranes significantly more static. In addition to MLB dehydration, CL promotes intrabilayer phase separation into saturated-rich and unsaturated-rich lipid domains that couple across multiple layers. Expectedly, addition of Ca2+ screens the electrostatic repulsion between negatively charged lung membranes. However, when CL is present, addition of Ca2+ results in an apparent interbilayer expansion likely due to local structural defects. Combining S/WAXS and ssNMR on systems with compositions pertinent to healthy and unhealthy lung membranes, we propose how alteration of the physiochemical properties of MLBs can critically impact the breathing cycle.

Rho kinase acts at separate steps in ureteric bud and metanephric mesenchyme morphogenesis during kidney development.

In this study, five different in vitro assays, which together recapitulate much of kidney development, were used to examine the role of the Rho-associated protein serine/threonine kinase (ROCK) in events central to ureteric bud (UB) and metanephric mesenchyme (MM) morphogenensis, in isolation and together. ROCK activity was found to be critical for (1) cell proliferation, growth, and development of the whole embryonic kidney in organ culture, (2) tip and stalk formation in cultures of isolated UBs, and (3) migration of MM cells (in a novel MM migration assay) during their condensation at UB tips (in a UB/MM recombination assay). Together, the data indicate selective involvement of Rho/ROCK in distinct morphogenetic processes necessary for kidney development and that the coordination of these events by Rho/ROCK provides a potential mechanism to regulate overall branching patterns, nephron formation, and thus, kidney architecture.

Spatiotemporal regulation of morphogenetic molecules during in vitro branching of the isolated ureteric bud: toward a model of branching through budding in the developing kidney.

In search of guiding principles involved in the branching of epithelial tubes in the developing kidney, we analyzed branching of the ureteric bud (UB) in whole kidney culture as well as in isolated UB culture independent of mesenchyme but in the presence of mesenchymally derived soluble factors. Microinjection of the UB lumen (both in the isolated UB and in the whole kidney) with fluorescently labeled dextran sulfate demonstrated that branching occurred via smooth tubular epithelial outpouches with a lumen continuous with that of the original structure. Epithelial cells within these outpouches cells were wedge-shaped with actin, myosin-2 and ezrin localized to the luminal side, raising the possibility of a "purse-string" mechanism. Electron microscopy and decoration of heparan sulfates with biotinylated FGF2 revealed that the basolateral surface of the cells remained intact, without the type of cytoplasmic extensions (invadopodia) that are seen in three-dimensional MDCK, mIMCD, and UB cell culture models of branching tubulogenesis. Several growth factor receptors (i.e., FGFR1, FGFR2, c-Ret) and metalloproteases (i.e., MT1-MMP) were localized toward branching UB tips. A large survey of markers revealed the ER chaperone BiP to be highly expressed at UB tips, which, by electron microscopy, are enriched in rough endoplasmic reticulum and Golgi, supporting high activity in the synthesis of transmembrane and secretory proteins at UB tips. After early diffuse proliferation, proliferating and mitotic cells were mostly found within the branching ampullae, whereas apoptotic cells were mostly found in stalks. Gene array experiments, together with protein expression analysis by immunoblotting, revealed a differential spatiotemporal distribution of several proteins associated with epithelial maturation and polarization, including intercellular junctional proteins (e.g., ZO-1, claudin-3, E-cadherin) and the subapical cytoskeletal/microvillar protein ezrin. In addition, Ksp-cadherin was found at UB ampullary cells next to developing outpouches, suggesting a role in epithelial-mesenchymal interactions. These data from the isolated UB culture system support a model where UB branching occurs through outpouching possibly mediated by wedge-shaped cells created through an apical cytoskeletal purse-string mechanism. Additional potential mechanisms include (1) differential localization of growth factor receptors and metalloproteases at tips relative to stalks; (2) creation of a secretory epithelium, in part manifested by increased expression of the ER chaperone BiP, at tips relative to stalks; (3) after initial diffuse proliferation, coexistence of a balance of proliferation vs. apoptosis favoring tip growth with a very different balance in elongating stalks; and (4) differential maturation of the tight and adherens junctions as the structures develop. Because, without mesenchyme, both lateral and bifid branching occurs (including the ureter), the mesenchyme probably restricts lateral branching and provides guidance cues in vivo for directional branching and elongation as well as functioning to modulate tubular caliber and induce differentiation. Selective cadherin, claudin, and microvillar protein expression as the UB matures likely enables the formation of a tight, polarized differentiated epithelium. Although, in vivo, metanephric mesenchyme development occurs simultaneously with UB branching, these studies shed light on how (mesenchymally derived) soluble factors alone regulate spatial and temporal expression of morphogenetic molecules and processes (proliferation, apoptosis, etc.) postulated to be essential to the UB branching program as it forms an arborized structure with a continuous lumen.

Regulation of ureteric bud branching morphogenesis by sulfated proteoglycans in the developing kidney.

Glycosaminoglycans in the form of heparan sulfate proteoglycans (HSPG) and chondroitin sulfate proteoglycans (CSPG) are required for normal kidney organogenesis. The specific roles of HSPGs and CSPGs on ureteric bud (UB) branching morphogenesis are unclear, and past reports have obtained differing results. Here we employ in vitro systems, including isolated UB culture, to clarify the roles of HSPGs and CSPGs on this process. Microarray analysis revealed that many proteoglycan core proteins change during kidney development (syndecan-1,2,4, glypican-1,2,3, versican, decorin, biglycan). Moreover, syndecan-1, syndecan-4, glypican-3, and versican are differentially expressed during isolated UB culture, while decorin is dynamically regulated in cultured isolated metanephric mesenchyme (MM). Biochemical analysis indicated that while both heparan sulfate (HS) and chondroitin sulfate (CS) are present, CS accounts for approximately 75% of the glycosaminoglycans (GAG) in the embryonic kidney. Selective perturbation of HS in whole kidney rudiments and in the isolated UB resulted in a significant reduction in the number of UB branch tips, while CS perturbation has much less impressive effects on branching morphogenesis. Disruption of endogenous HS sulfation with chlorate resulted in diminished FGF2 binding and proliferation, which markedly altered kidney area but did not have a statistically significant effect on patterning of the ureteric tree. Furthermore, perturbation of GAGs did not have a detectable effect on FGFR2 expression or epithelial marker localization, suggesting the expression of these molecules is largely independent of HS function. Taken together, the data suggests that nonselective perturbation of HSPG function results in a general proliferation defect; selective perturbation of specific core proteins and/or GAG microstructure may result in branching pattern defects. Despite CS being the major GAG synthesized in the whole developing kidney, it appears to play a lesser role in UB branching; however, CS is likely to be integral to other developmental processes during nephrogenesis, possibly involving the MM. A model is presented of how, together with growth factors, heterogeneity of proteoglycan core proteins and glycosaminoglycan sulfation act as a switching mechanism to regulate different stages of the branching process. In this model, specific growth factor-HSPG combinations play key roles in the transitioning between stages and their maintenance.

TGF-beta superfamily members modulate growth, branching, shaping, and patterning of the ureteric bud.

Protein-rich fractions inhibitory for isolated ureteric bud (UB) growth were separated from a conditioned medium secreted by cells derived from the metanephric mesenchyme (MM). Elution profiles and immunoblotting indicated the presence of members of the transforming growth factor-beta (TGF-beta) superfamily. Treatment of cultured whole embryonic kidney with BMP2, BMP4, activin, or TGF-beta1 leads to statistically significant differences in the overall size of the kidney, the number of UB branches, the length and angle of the branches, as well as in the thickness of the UB stalks. Thus, the pattern of the ureteric tree is altered. LIF, however, appeared to have only minimal effect on growth and development of the whole embryonic kidney in organ culture. The factors all directly inhibited, in a concentration-dependent fashion, the growth and branching of the isolated UB, albeit to different extents. Antagonists of some of these factors reduced their inhibitory effect. Detailed examination of TGF-beta1-treated UBs revealed only a slight increase in the amount of apoptosis in tips by TUNEL staining, but diminished proliferation throughout by Ki67 staining. These data suggest an important direct modulatory role for BMP2, BMP4, LIF, TGF-beta1, and activin (as well as their antagonists) on growth and branching of the UB, possibly in shaping the growing UB by playing a role in determining the number of branches, as well as where and how the branches occur. In support of this notion, UBs cultured in the presence of fibroblast growth factor 7 (FGF7), which induces the formation of globular structures with little distinction between the stalk and ampullae [Mech. Dev. 109 (2001) 123], and TGF-beta superfamily members lead to the formation of UBs with clear stalks and ampullae. This indicates that positive (i.e., growth and branch promoting) and negative (i.e., growth and branch inhibiting) modulators of UB morphogenesis can cooperate in the formation of slender arborized UB structures similar to those observed in the intact developing kidney or in whole embryonic kidney organ culture. Finally, purification data also indicate the presence of an as yet unidentified soluble non-heparin-binding activity modulating UB growth and branching. The data suggest how contributions of positive and negative growth factors can together (perhaps as local bipolar morphogenetic gradients existing within the mesenchyme) modulate the vectoral arborization pattern of the UB and shape branches as they develop, thereby regulating both nephron number and tubule/duct caliber. We suggest that TGF-beta-like molecules and other non-heparin-binding inhibitory factors can, in the appropriate matrix context, facilitate "braking" of the branching program as the UB shifts from a rapid branching stage (governed by a feed-forward mechanism) to a stage where branching slows down (negative feedback) and eventually stops.

Developmental approaches to kidney tissue engineering.

Recent advances in our understanding of the developmental biology of the kidney, as well as the establishment of novel in vitro model systems, have potential implications for kidney tissue engineering. These advances include delineation of the roles of a number of growth factors in the developmental programs of branching morphogenesis and mesenchymal differentiation, a new understanding of the roles of the extracellular matrix, identification of potential "renal" stem cells, the ex vivo propagation and subsequent recombination of isolated components of the kidney, and successful transplantation of renal primordia into adult hosts. This review will examine these advances in the context of approaches to tissue engineering. Finally, novel approaches that synthesize advances in both cell-based and organ-based approaches are proposed.

A strategy for in vitro propagation of rat nephrons.

BACKGROUND: Recent advances in the understanding of the molecular biology of rodent renal development have lead to the ability to culture the components of the developing rat kidney-the ureteric bud (UB) and the metanephric mesenchyme (MM)-in isolation from one another. Here we here describe a method for subculturing and propagating either whole rat metanephric rudiments or isolated rat UBs. Exploiting the branching program intrinsic to the UB, propagated rat UBs can be recombined with fresh rat mesenchyme to form a large number of rat "neokidneys" derived from a single progenitor that may be amenable to site-specific modulation of function. METHODS: Whole rat metanephric rudiments or isolated rat UBs were cultured and subdivided through several generations. Both cultured progenitor and subsequent generations of isolated rat UBs were recombined with freshly isolated rat metanephric mesenchyme. The tubules of these rat neokidneys were examined for expression of epithelial markers. RESULTS: Isolated rat UBs and whole rat metanephric rudiments could be propagated through several generations and appeared morphologically identical to their progenitors. Generations of isolated rat UB could be recombined with fresh rat mesenchyme and the resultant neokidney displayed the same morphologic appearance as the whole rat kidney rudiment. The UB-derived and MM-derived portions of the tubules of these rat neokidneys appear contiguous. CONCLUSIONS: The recombination of cultured and propagated rat UB with rat mesenchyme yielded rat neokidneys with tubular structures that appeared morphologically identical to whole rat kidney. In vitro propagation of rat metanephric rudiments and recombination of rat UB and MM suggest the possibility of designing nephrons that possess specific desirable functions that can be propagated in vitro.