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

BACKGROUND: The adherence of calcium oxalate crystals to the renal tubule epithelium is considered a critical event in the pathophysiology of calcium nephrolithiasis. Calcium oxalate monohydrate (COM) crystals cannot adhere to the surface of a functional Madin-Darby canine kidney (MDCK) monolayer, but they bind avidly to the surface of proliferating and migrating cells. METHODS: To identify crystal-binding molecules (CBMs) at the surface of crystal-attracting cells, we applied metabolic labeling protocols in combination with differential enzymatic digestion and gel filtration, which was compared with [14C]COM crystal binding and confirmed by confocal microscopy. RESULTS: The indication that hyaluronan [hyaluronic acid (HA)] might act as a CBM in subconfluent cultures came from studies with glycosaminoglycan (GAG)-degrading enzymes. Subsequently, metabolic-labeling studies revealed that hyaluronidase cleaved significantly more radiolabeled glycoconjugates from crystal-attracting cells than from cells without affinity for crystals. During wound repair, crystal binding could be prevented by pretreating the healing cultures with hyaluronate lyase, an enzyme that specifically hydrolyzes HA. Binding to immobilized HA provided evidence that COM crystals physically can become associated with this polysaccharide. Finally, confocal microscopy demonstrated that fluorescently labeled HA binding protein (HABP) adhered to the surface of proliferating cells in subconfluent cultures as well as to cells involved in closing a wound, but not to cells in confluent monolayers. CONCLUSIONS: These results identify HA as binding molecule for COM crystals at the surface of migrating and proliferating MDCK cells.

Sialic acid and crystal binding.

BACKGROUND: We studied the role of cell surface sialic acid in the adherence of calcium oxalate monohydrate (COM) crystals to Madin-Darby canine kidney (MDCK) cells. METHODS: Studies were performed with undifferentiated (crystal-binding) cells in subconfluent cultures and maturated (noncrystal-binding) cells in confluent cultures. Lectins were used to study the emergence and abundance of oligosaccharides at the cell surface during epithelial development. The effect of neuraminidase treatment on crystal binding was studied with [14C]COM crystals, and the enzyme-induced release of cell surface-associated sialic acid molecules was monitored by labeling the cells metabolically with [3H]glucosamine. RESULTS: Binding studies with lectins derived from Maackia Amurensis II (MALII) and Sambucus Nigra (SNA) demonstrated that the cells expressed terminal sialic acids attached to penultimate galactose through alpha 2,3 and alpha 2,6 bonds at different stages of epithelial development. Neuraminidase treatment strongly reduced the affinity of the cell surface for COM crystals in subconfluent cultures. Nevertheless, neuraminidase cleaved more sialic acids from cells in confluent cultures than from those in subconfluent cultures. Peanut agglutinin (PNA), which binds only to sialylated terminal galactose units, adhered to developing but not to maturated cells, unless the latter were pretreated with neuraminidase. Both results indicate that the surface of maturated MDCK cells is more heavily sialylated than that of undifferentiated cells. Free sialic acid molecules showed little or no affinity for COM crystals and did not affect the adherence of the crystals to undifferentiated cells. CONCLUSIONS: There are at least two models that may explain these results. First, sialic acids are presented at the surface of immature cells in an orientation that specifically matches crystal surface characteristics favoring crystal-cell interactions. Second, sialic acid molecules are not directly associated with the crystals, but may be involved in the exposure of another crystal binding molecule at the cell surface.

Attachment sites for particles in the urinary tract.

The adherence of crystals to the surface of renal tubule epithelial cells is one of the initial events in the development of nephrolithiasis. The accumulation of crystalline material in the kidney will sooner or later result in the formation of a stone. Calcium crystals occasionally are present in the urine of even healthy individuals, and mechanisms responsible for the selective attachment of crystals to the tubular epithelium of stone-forming individuals must exist. Although several types of cell surface molecules, including phosphatidylserine (PS) and sialic acid, have been proposed as receptors for crystals in the tubular system, the exact nature of these crystal-binding sites has not yet been revealed. Previously, it was demonstrated that calcium oxalate monohydrate crystals adhere to subconfluent, but not to confluent, Madin-Darby canine kidney-I cultures. This model was used here to investigate whether the surface of cells with affinity for crystals is enriched with one of the proposed crystal-binding molecules. Annexin V was used for the detection of PS at the cell surface, and Sambucus nigra lectin was used to reveal terminal sialic acid in a (alpha2,6) linkage to galactose units. FITC-annexin V binding studies showed that PS was not exposed at the surface of proliferating or growth-inhibited cells, unless they were pretreated with an apoptosis-inducing cytotoxic agent. Sambucus nigra lectin binding, of which the specificity was confirmed by blocking with N-acetylneuraminyl-lactose, demonstrated the abundant presence of (alpha2,6)-linked sialic acid residues at the cell surface of both subconfluent and confluent cultures. While these results seem to rule out a role for PS in the adherence of calcium oxalate monohydrate crystals to the surface of maturating Madin-Darby canine kidney-I cells, they question the role for cell surface-associated sialylated glycoconjugates in this process.

LLC-PK1 cells as a model system to study proximal tubule transport of water and other compounds relevant for renal stone disease.

LLC-PK1 cells were cultured on a permeable support in a two-compartment culture system. Confluent monolayers received an ultrafiltrate-like solution at the apical side and a plasma-like solution at the basolateral side. The distribution of various solutes, including phosphate, calcium, and oxalate over both compartments was measured in time. The transport of water was monitored by alterations in fluid concentrations of radiolabeled inulin. Bicarbonate, glucose, and phosphate were transported rapidly from the apical to basolateral side of the monolayer. Sodium and chloride were reabsorbed without major consequences for the osmolality in the apical and basal fluid. Calcium and potassium were also reabsorbed, but to a smaller extent than sodium. The luminal concentration of oxalate gradually increased to values that were at least three times higher (12.0+/-0.4 micromol/l) than those in the contraluminal fluid (3.8+/-0.1 micromol/l). However, since the luminal rise of oxalate completely matched the rise of inulin in the apical fluid this appeared to be the passive consequence of active water reabsorption rather than of net directed oxalate transport. The LLC-PK1 model could prove useful to study the regulation of proximal tubule water transport and its effect on luminal stone salt concentrations under different physiological conditions.

Cell type-specific acquired protection from crystal adherence by renal tubule cells in culture.

BACKGROUND: Adherence of crystals to the surface of renal tubule epithelial cells is considered an important step in the development of nephrolithiasis. Previously, we demonstrated that functional monolayers formed by the renal tubule cell line, Madin-Darby canine kidney (MDCK), acquire protection against the adherence of calcium oxalate monohydrate crystals. We now examined whether this property is cell type specific. The susceptibility of the cells to crystal binding was further studied under different culture conditions. METHODS: Cell-type specificity and the influence of the growth substrate was tested by comparing calcium oxalate monohydrate crystal binding to LLC-PK1 cells and to two MDCK strains cultured on either permeable or impermeable supports. These cell lines are representative for the renal proximal tubule (LLC-PK1) and distal tubule/collecting duct (MDCK) segments of the nephron, in which crystals are expected to be absent and present, respectively. RESULTS: Whereas relatively large amounts of crystals adhered to subconfluent MDCK cultures, the level of crystal binding to confluent monolayers was reduced for both MDCK strains. On permeable supports, MDCK cells not only obtained a higher level of morphological differentiation, but also acquired a higher degree of protection than on impermeable surfaces. Crystals avidly adhered to LLC-PK1 cells, irrespective of their developmental stage or growth substrate used. CONCLUSIONS: These results show that the prevention of crystal binding is cell type specific and expressed only by differentiated MDCK cells. The anti-adherence properties acquired by MDCK cells may mirror a specific functional characteristic of its in situ equivalent, the renal distal tubule/collecting ducts.

Increased calcium oxalate monohydrate crystal binding to injured renal tubular epithelial cells in culture.

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 binding was observed 2 days postinjury when the wounds were already morphologically closed but TER was still low. Confocal images showed that during the repair process, crystals selectively adhered to migrating cells at the wound border and to stacked cells at sites were the wounds were closed. After the barrier integrity was restored, crystal binding decreased again to the same low levels as in undamaged controls. These results indicate that, whereas functional MDCK monolayers are largely protected against COM crystal adherence, epithelial injury and the subsequent process of wound healing lead to increased crystal binding.

Cell cultures and nephrolithiasis.

While the physical chemistry of stone formation has been intensively studied during the last decade, it has become clear that the pathophysiology of renal stone disease cannot be explained by crystallization processes only. In recent years, evidence has emerged that the cells lining the renal tubules can have an active role in creating the conditions under which stones may develop. Since it is difficult to study these mechanisms in vivo, cultured renal tubular cells have become increasingly popular for the study of physiological and cell biological processes that are possibly linked to stone disease. In this paper, we discuss the possible contribution of cellular processes such as transepithelial oxalate transport and crystal--cell interaction to the formation of renal stones. Experimental studies that have been performed with cultured renal cells to elucidate the mechanisms involved in these processes will be summarized.