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Research paper thumbnail of Identification of hyaluronan as a crystal-binding molecule at the surface of migrating and proliferating MDCK cells

Kidney international, 2000

at their surface. The purpose of this study is to reveal the identity of these cell surface-assoc... more at their surface. The purpose of this study is to reveal the identity of these cell surface-associated CBMs.

Research paper thumbnail of Identification of hyaluronan as a crystal-binding molecule at the surface of migrating and proliferating MDCK cells

Kidney international, 2000

at their surface. The purpose of this study is to reveal the identity of these cell surface-assoc... more at their surface. The purpose of this study is to reveal the identity of these cell surface-associated CBMs.

Research paper thumbnail of to injured renal tubular epithelial cells in culture Increased calcium oxalate monohydrate crystal binding

Research paper thumbnail of LLC-PK 1 cells as a model system to study proximal tubule transport of water and other compounds relevant for renal stone disease

Research paper thumbnail of Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

Research paper thumbnail of Increased calcium oxalate monohydrate crystal binding to injured renal tubular epithelial cells in culture

The American journal of physiology, 1998

The retention of crystals in the kidney is considered to be a crucial step in the development of ... more The retention of crystals in the kidney is considered to be a crucial step in the development of a renal stone. This study demonstrates the time-dependent alterations in the extent of calcium oxalate (CaOx) monohydrate (COM) crystal binding to Madin-Darby canine kidney (MDCK) cells during their growth to confluence and during the healing of wounds made in confluent monolayers. As determined by radiolabeled COM crystal binding studies and confirmed by confocal-scanning laser microscopy, relatively large amounts of crystals (10.4 +/- 0.4 micrograms/cm2) bound to subconfluent cultures that still exhibited a low transepithelial electrical resistance (TER < 400 omega.cm2). The development of junctional integrity, indicated by a high resistance (TER > 1,500 omega.cm2), was followed by a decrease of the crystal binding capacity to almost undetectable low levels (0.13 +/- 0.03 microgram/cm2). Epithelial injury resulted in increased crystal adherence. The highest level of crystal bindi...

Research paper thumbnail of Cell cultures and nephrolithiasis

World Journal of Urology, 1997

Research paper thumbnail of Cell type-specific acquired protection from crystal adherence by renal tubule cells in culture

Kidney International, 1999

Cell type-specific acquired protection from crystal adherence to concentrate the tubular fluid [1... more Cell type-specific acquired protection from crystal adherence to concentrate the tubular fluid [1]. The selective reabby renal tubule cells in culture. sorption of water and solutes takes place in distinct seg-Background. Adherence of crystals to the surface of renal ments of the nephron [2]. The distribution of these reabtubule epithelial cells is considered an important step in the sorptive capacities is reflected in different risks for development of nephrolithiasis. Previously, we demonstrated crystallization at the various sites in the nephron [3-7]. that functional monolayers formed by the renal tubule cell line, Madin-Darby canine kidney (MDCK), acquire protection The first nucleation risk, for example, is encountered in against the adherence of calcium oxalate monohydrate crystals. the loop of Henle, which disappears as the fluid becomes We now examined whether this property is cell type specific. diluted in the ascending limb and distal tubule [3-7]. The susceptibility of the cells to crystal binding was further During antidiuresis, extensive net water reabsorption studied under different culture conditions. leads to an increased risk for crystal formation in the Methods. Cell-type specificity and the influence of the growth substrate was tested by comparing calcium oxalate monohycollecting duct toward the papillary tip [4-8]. Crystals drate crystal binding to LLC-PK 1 cells and to two MDCK strains formed in the tubular fluid are not necessarily harmful cultured on either permeable or impermeable supports. These as long as they are eliminated with the urine. Crystal cell lines are representative for the renal proximal tubule (LLCgrowth and agglomeration, however, could create parti-PK 1) and distal tubule/collecting duct (MDCK) segments of the nephron, in which crystals are expected to be absent and cles that are too large to freely pass the renal tubules present, respectively. [9-11]. In addition, cell culture studies [12-14] have indi-Results. Whereas relatively large amounts of crystals adcated that crystals can bind to the surface of the renal hered to subconfluent MDCK cultures, the level of crystal bindtubule epithelium. Indeed, crystal deposits with a size ing to confluent monolayers was reduced for both MDCK smaller than the diameter of the tubules have been found strains. On permeable supports, MDCK cells not only obtained a higher level of morphological differentiation, but also acquired attached to the luminal surface of renal tubular cells in a higher degree of protection than on impermeable surfaces. hyperoxaluric patients [15, 16] and rats [17]. Crystals avidly adhered to LLC-PK 1 cells, irrespective of their Previously, we showed that the affinity of the luminal developmental stage or growth substrate used. surface of MDCK-I cells for calcium oxalate monohy-Conclusions. These results show that the prevention of crysdrate (COM) crystals highly depends on the developtal binding is cell type specific and expressed only by differentimental stage of the cultures. Relatively large amounts of ated MDCK cells. The anti-adherence properties acquired by MDCK cells may mirror a specific functional characteristic of crystals could adhere to subconfluent cultures, whereas its in situ equivalent, the renal distal tubule/collecting ducts. intact and functional monolayers were largely protected from crystal binding [18]. It was speculated that the antiadherence capacity of functional MDCK cultures may Renal stones are largely composed of calcium salts reflect a physiologically relevant property. If so, we exthat are precipitated from the tubular fluid. The crystallipect anti-adherence properties to be present in cell types zation of stone salts results from the ability of the kidney that are frequently confronted with crystals and absent in cells that normally do not encounter crystals. Indeed, calcium oxalate crystals did not adhere to the surface of

Research paper thumbnail of Sialic acid and crystal binding

Kidney International, 2000

Research paper thumbnail of Urinary Crystallization Inhibitors Do Not Prevent Crystal Binding

The Journal of Urology, 2002

Research paper thumbnail of Glutathione PEGylated liposomes: pharmacokinetics and delivery of cargo across the blood–brain barrier in rats

Journal of Drug Targeting, 2014

Research paper thumbnail of Glutathione conjugation dose-dependently increases brain-specific liposomal drug delivery in vitro and in vivo

Drug Discovery Today: Technologies, 2016

The blood-brain barrier (BBB) represents a major obstacle for the delivery and development of dru... more The blood-brain barrier (BBB) represents a major obstacle for the delivery and development of drugs curing brain pathologies. However, this biological barrier presents numerous endogenous specialized transport systems that can be exploited by engineered nanoparticles to enable drug delivery to the brain. In particular, conjugation of glutathione (GSH) onto PEGylated liposomes (G-Technology(®)) showed to safely enhance delivery of encapsulated drugs to the brain. Yet, understanding of the mechanism of action remains limited and full mechanistic understanding will aid in the further optimization of the technology. In order to elucidate the mechanism of brain targeting by GSH-PEG liposomes, we here demonstrate that the in vivo delivery of liposomal ribavirin is increased in brain extracellular fluid according to the extent of GSH conjugation onto the liposomes. In vitro, using the hCMEC/D3 human cerebral microvascular endothelial (CMEC) cell line, as well as primary bovine and porcine CMEC (and in contrast to non-brain derived endothelial and epithelial cells), we show that liposomal uptake occurs through the process of endocytosis and that the brain-specific uptake is also glutathione conjugation-dependent. Interestingly, the uptake mechanism is an active process that is temperature-, time- and dose-dependent. Finally, early endocytosis events rely on cytoskeleton remodeling, as well as dynamin- and clathrin-dependent endocytosis pathways. Overall, our data demonstrate that the glutathione-dependent uptake mechanism of the G-Technology involves a specific endocytosis pathway indicative of a receptor-mediated mechanism, and supports the benefit of this drug delivery technology for the treatment of devastating brain diseases.

Research paper thumbnail of Identification of hyaluronan as a crystal-binding molecule at the surface of migrating and proliferating MDCK cells

Kidney international, 2000

at their surface. The purpose of this study is to reveal the identity of these cell surface-assoc... more at their surface. The purpose of this study is to reveal the identity of these cell surface-associated CBMs.

Research paper thumbnail of Identification of hyaluronan as a crystal-binding molecule at the surface of migrating and proliferating MDCK cells

Kidney international, 2000

at their surface. The purpose of this study is to reveal the identity of these cell surface-assoc... more at their surface. The purpose of this study is to reveal the identity of these cell surface-associated CBMs.

Research paper thumbnail of to injured renal tubular epithelial cells in culture Increased calcium oxalate monohydrate crystal binding

Research paper thumbnail of LLC-PK 1 cells as a model system to study proximal tubule transport of water and other compounds relevant for renal stone disease

Research paper thumbnail of Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

Research paper thumbnail of Increased calcium oxalate monohydrate crystal binding to injured renal tubular epithelial cells in culture

The American journal of physiology, 1998

The retention of crystals in the kidney is considered to be a crucial step in the development of ... more The retention of crystals in the kidney is considered to be a crucial step in the development of a renal stone. This study demonstrates the time-dependent alterations in the extent of calcium oxalate (CaOx) monohydrate (COM) crystal binding to Madin-Darby canine kidney (MDCK) cells during their growth to confluence and during the healing of wounds made in confluent monolayers. As determined by radiolabeled COM crystal binding studies and confirmed by confocal-scanning laser microscopy, relatively large amounts of crystals (10.4 +/- 0.4 micrograms/cm2) bound to subconfluent cultures that still exhibited a low transepithelial electrical resistance (TER < 400 omega.cm2). The development of junctional integrity, indicated by a high resistance (TER > 1,500 omega.cm2), was followed by a decrease of the crystal binding capacity to almost undetectable low levels (0.13 +/- 0.03 microgram/cm2). Epithelial injury resulted in increased crystal adherence. The highest level of crystal bindi...

Research paper thumbnail of Cell cultures and nephrolithiasis

World Journal of Urology, 1997

Research paper thumbnail of Cell type-specific acquired protection from crystal adherence by renal tubule cells in culture

Kidney International, 1999

Cell type-specific acquired protection from crystal adherence to concentrate the tubular fluid [1... more Cell type-specific acquired protection from crystal adherence to concentrate the tubular fluid [1]. The selective reabby renal tubule cells in culture. sorption of water and solutes takes place in distinct seg-Background. Adherence of crystals to the surface of renal ments of the nephron [2]. The distribution of these reabtubule epithelial cells is considered an important step in the sorptive capacities is reflected in different risks for development of nephrolithiasis. Previously, we demonstrated crystallization at the various sites in the nephron [3-7]. that functional monolayers formed by the renal tubule cell line, Madin-Darby canine kidney (MDCK), acquire protection The first nucleation risk, for example, is encountered in against the adherence of calcium oxalate monohydrate crystals. the loop of Henle, which disappears as the fluid becomes We now examined whether this property is cell type specific. diluted in the ascending limb and distal tubule [3-7]. The susceptibility of the cells to crystal binding was further During antidiuresis, extensive net water reabsorption studied under different culture conditions. leads to an increased risk for crystal formation in the Methods. Cell-type specificity and the influence of the growth substrate was tested by comparing calcium oxalate monohycollecting duct toward the papillary tip [4-8]. Crystals drate crystal binding to LLC-PK 1 cells and to two MDCK strains formed in the tubular fluid are not necessarily harmful cultured on either permeable or impermeable supports. These as long as they are eliminated with the urine. Crystal cell lines are representative for the renal proximal tubule (LLCgrowth and agglomeration, however, could create parti-PK 1) and distal tubule/collecting duct (MDCK) segments of the nephron, in which crystals are expected to be absent and cles that are too large to freely pass the renal tubules present, respectively. [9-11]. In addition, cell culture studies [12-14] have indi-Results. Whereas relatively large amounts of crystals adcated that crystals can bind to the surface of the renal hered to subconfluent MDCK cultures, the level of crystal bindtubule epithelium. Indeed, crystal deposits with a size ing to confluent monolayers was reduced for both MDCK smaller than the diameter of the tubules have been found strains. On permeable supports, MDCK cells not only obtained a higher level of morphological differentiation, but also acquired attached to the luminal surface of renal tubular cells in a higher degree of protection than on impermeable surfaces. hyperoxaluric patients [15, 16] and rats [17]. Crystals avidly adhered to LLC-PK 1 cells, irrespective of their Previously, we showed that the affinity of the luminal developmental stage or growth substrate used. surface of MDCK-I cells for calcium oxalate monohy-Conclusions. These results show that the prevention of crysdrate (COM) crystals highly depends on the developtal binding is cell type specific and expressed only by differentimental stage of the cultures. Relatively large amounts of ated MDCK cells. The anti-adherence properties acquired by MDCK cells may mirror a specific functional characteristic of crystals could adhere to subconfluent cultures, whereas its in situ equivalent, the renal distal tubule/collecting ducts. intact and functional monolayers were largely protected from crystal binding [18]. It was speculated that the antiadherence capacity of functional MDCK cultures may Renal stones are largely composed of calcium salts reflect a physiologically relevant property. If so, we exthat are precipitated from the tubular fluid. The crystallipect anti-adherence properties to be present in cell types zation of stone salts results from the ability of the kidney that are frequently confronted with crystals and absent in cells that normally do not encounter crystals. Indeed, calcium oxalate crystals did not adhere to the surface of

Research paper thumbnail of Sialic acid and crystal binding

Kidney International, 2000

Research paper thumbnail of Urinary Crystallization Inhibitors Do Not Prevent Crystal Binding

The Journal of Urology, 2002

Research paper thumbnail of Glutathione PEGylated liposomes: pharmacokinetics and delivery of cargo across the blood–brain barrier in rats

Journal of Drug Targeting, 2014

Research paper thumbnail of Glutathione conjugation dose-dependently increases brain-specific liposomal drug delivery in vitro and in vivo

Drug Discovery Today: Technologies, 2016

The blood-brain barrier (BBB) represents a major obstacle for the delivery and development of dru... more The blood-brain barrier (BBB) represents a major obstacle for the delivery and development of drugs curing brain pathologies. However, this biological barrier presents numerous endogenous specialized transport systems that can be exploited by engineered nanoparticles to enable drug delivery to the brain. In particular, conjugation of glutathione (GSH) onto PEGylated liposomes (G-Technology(®)) showed to safely enhance delivery of encapsulated drugs to the brain. Yet, understanding of the mechanism of action remains limited and full mechanistic understanding will aid in the further optimization of the technology. In order to elucidate the mechanism of brain targeting by GSH-PEG liposomes, we here demonstrate that the in vivo delivery of liposomal ribavirin is increased in brain extracellular fluid according to the extent of GSH conjugation onto the liposomes. In vitro, using the hCMEC/D3 human cerebral microvascular endothelial (CMEC) cell line, as well as primary bovine and porcine CMEC (and in contrast to non-brain derived endothelial and epithelial cells), we show that liposomal uptake occurs through the process of endocytosis and that the brain-specific uptake is also glutathione conjugation-dependent. Interestingly, the uptake mechanism is an active process that is temperature-, time- and dose-dependent. Finally, early endocytosis events rely on cytoskeleton remodeling, as well as dynamin- and clathrin-dependent endocytosis pathways. Overall, our data demonstrate that the glutathione-dependent uptake mechanism of the G-Technology involves a specific endocytosis pathway indicative of a receptor-mediated mechanism, and supports the benefit of this drug delivery technology for the treatment of devastating brain diseases.