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Epithelial Cell Structure and Polarity
Few cell types more elegantly embody the dictum that “form follows function” than do those of polarized epithelia. It is the unique architecture of renal epithelial cells that permits them to mediate vectorial transport. This transport, in turn, essentially determines the body’s fluid and electrolyte composition. This chapter reviews the structures of renal epithelial cells and explores the mechanisms through which these structures are generated and maintained.
Keywords
epithelial; polarity; membrane; domain; matrix; cell adhesion; junctions
Introduction
Many of the chapters in this volume are devoted to the mechanisms through which the nephron is able to convert the glomerular filtrate into concentrated urine that is responsive to the metabolic status of the organism as a whole. The multifactorial nature of this problem means it needs to be treated at several levels of resolution. A meaningful description of renal tubular functions requires an understanding of the nephron’s properties as an integrated tissue, as well as those of its constituent parts, including the cells and molecules that contribute to its transport functions.
As detailed elsewhere in this volume, the nephron is a remarkably heterogeneous structure. Throughout its length, the renal tubule is notable for the marked variations in the morphologic and physiologic properties of its epithelial cells, reflecting the numerous and diverse responsibilities that neighboring segments are called on to fulfill. At the tissue level, the function of the kidney is critically dependent on the geometry and topography of the nephron. The precise juxtaposition of various epithelial cell types, which manifest distinct fluid and electrolyte transport capabilities, in large measure specifies the course of modifications to which the glomerular filtrate is exposed. This dependence on geometry also extends to renal function at the cellular level.
The Nature and Physiologic Implications of Epithelial Polarity
Despite their variations in form and function, all of the epithelial cells that line the nephron share at least one fundamental characteristic. Like their relatives in other tissues, all epithelial cells are polarized. The plasma membranes of polarized epithelial cells are divided into two morphologically and biochemically distinct domains. In the case of the nephron, the apical surfaces of the epithelial cells face the tubular lumen. The basolateral surface rests on the epithelial basement membrane, and is in contact with the interstitial fluid compartment. The lipid and protein components of these two contiguous plasmalemmal domains are almost entirely dissimilar. It is precisely these differences that account for the epithelial cell’s capacity to mediate the vectorial transport of solutes and fluid against steep concentration gradients. Thus, the subcellular geometry of renal epithelial cells is critical to renal function.
The principal cell of the collecting tubule provides a useful illustration of the importance of biochemical polarity for renal function. As described in other contributions to this volume, the principal cell is required to resorb sodium against a very steep concentration gradient. It accomplishes this task through the carefully controlled placement of ion pumps and channels. The basolateral plasma membrane of the principal cell, like that of most polarized epithelial cells, possesses a large complement of Na + /K + -ATPase. This basolateral sodium pump catalyzes the energetically unfavorable transport of three sodium ions out of the cell in exchange for two potassium ions, through the consumption of the energy embodied in one molecule of ATP. The apical surface of the principal cell lacks sodium pump, but is equipped with a sodium channel, which allows sodium ions to move passively down their concentration gradient. Through the action of the sodium pump the intracellular sodium concentration is kept low and the driving forces across the apical membrane favor the influx of sodium from the tubular fluid through the apical sodium channels. Thus, the combination of a basolateral Na + /K + -ATPase and an apical sodium channel lead to the vectorial movement of sodium from the tubule lumen to the interstitial space against its electrochemical gradient. This elegant mechanism is critically dependent upon the principal cell’s biochemical polarity. If the sodium pump and the sodium channel occupied the same plasmalemmal domain, then the gradients generated by the former could not be profitably exploited by the latter. Thus, the vectorial resorption or secretion of solutes or fluid is predicated upon the asymmetric distribution of transport proteins in polarized epithelial cells.
The fact that epithelial cells manifest biochemical polarity implies that they are endowed with the capacity to generate and maintain differentiated subdomains of their cell surface membranes. Newly synthesized membrane proteins must be targeted to the appropriate cell surface domain, and retained there following their delivery. During tissue development, cell division, and wound healing, plasmalemmal domains must be delimited and their biochemical character established. Clearly, specialized machinery and pathways must exist through which this energetically unfavorable compositional asymmetry can be supported. The nature of these specializations has been the subject of intense study for decades. While firm answers are not yet available, a number of fascinating model systems have been developed, and valuable insights have emerged. This chapter will focus on what is known of the processes through which tubular epithelial cells create their polarized geometry.
Epithelial Cell Structure: Morphology and Physiology
The renal tubular epithelium is composed of a remarkably varied collection of cell types, ranging from the highly specialized glomerular epithelial cells with foot processes that faciliate filtration of the blood through the basement membrane, to the simple squamous epithelium of the loop of Henle. A detailed delineation of its morphologic diversity is beyond the scope of this chapter. However, certain essential features are shared among all cell types in the tubular epithelium and, indeed, most other epithelial cell types found in the body. Among these are a differentiated, microvillar apical surface facing the tubular lumen, a lateral surface specialized for cell–cell interactions and regulation of transepithelial permeability, and a basal surface that adheres to the basement membrane. Furthermore, as described previously, the basolateral plasma membrane is particularly important in ion transport, because it is the location of the Na + /K + -ATPase and the cell is able to modulate its surface area in response to the transport activity of individual cell types. The cell–cell adhesive relationships are responsible for the integrity of the epithelium, and also dictate the permeability of the epithelium to small molecules that, in part, give each segment of the epithelium its physiological identity. Furthermore, adhesion of epithelial cells to each other and to the basement membrane sends spatial signals to the cells essential for the establishment and maintenance of epithelial cell polarity. In the following sections the morphology and functional composition of the apical and basolateral domains of the plasma membrane will be described, after detailing the nature of the junctional complex that mediates cell–cell adhesion.
The Junctional Complex
All epithelial cells, including those of the kidney tubule, are joined together along the lateral surfaces by a series of intercellular junctions first noted by their characteristic ultrastructural appearance and relative locations on the lateral plasma membrane. These include the tight junction or zonula occludens , the adherens junction ( zonula adherens or intermediate junction), desmosomes, and gap junctions. In most mammalian epithelia the tight junction is located at the apical-most edge of the lateral membrane closely followed by the adherens junction. Desmosomes and gap junctions have less specific locations on the lateral membrane. Desomosomes and gap junctions will be described briefly, followed by a more comprehensive description of tight junctions and adherens junctions, because of their essential functions in the organization, physiology, and morphogenesis of epithelia.
Desmosomes are large, multiprotein complexes primarily responsible for the mechanical strength of cell–cell interactions. They are formed after the assembly of adherens and tight junctions. By transmission electron microscopy they appear as discrete, focal concentrations of dense material in the cytoplasm of adjacent cells, as well as in the intercellular space. In contrast to adherens and tight junctions, desmosomes do not form an adhesive belt around the entire epithelial cell, but are a kind of “spot weld” at various points on adjacent lateral membranes. They are composed of both integral membrane proteins of the cadherin family called desmogleins and desmocollins, and peripheral membrane proteins known as desmoplakins, as well as a variety of other protein constituents. Adjacent cells adhere to each other through cadherin-mediated interactions. The peripheral components then provide mechanical stability to this interaction, via keratin intermediate filaments in the cytoplasm of each cell. Ultrastructurally, these appear as a mass of hair-like protrusions interacting in parallel with each plaque and then splaying out into the cytoplasm. In this manner, desmosomes link all cells in the epithelium. While there is evidence that desmosomal components may play an active role in regulating some aspects of cell–cell adhesion and even gene expression, in general their function is considered to be relatively passive.
Gap junctions are so named because of the characteristic 3 nm gap between adjacent cells that is evident using transmission electron microscopy. Examination of freeze-fracture specimens, which permits visualization of the internal topography of membranes, reveals the gap junction as a discrete array of intramembranous particles or connexons. Each connexon is composed of five identical connexins, a family of transmembrane proteins. Connexons on adjacent cells interact through their extracytoplasmic domains to form a series of low-resistance channels. These permit the passage of small molecules of less than 1 kDa, linking neighboring cells in the epithelium both electrically and metabolically. In the kidney, it is likely that gap junctions play important roles during morphogenesis and repair, although their precise functions have not been investigated in detail.
Among the numerous functions subserved by epithelia, perhaps the most important is that of a barrier between the intra- and extracorporeal spaces. In the case of the kidney, the extracorporeal space is defined by the lumen of the renal tubule. That the chemical composition of urine differs substantially from that of the interstitial extracellular fluid bathing the epithelial basement membranes is evidence that the barrier provided by the tubular epithelium is tight to both small and large molecules. There are two components to this barrier, arranged in parallel. The first is comprised of the apical and basolateral membranes of the epithelial cells, which together serve as a pair of series resistances to the flow of solutes across the epithelia. The second barrier is provided by the tight junction or zonula occludens that controls movement of molecules between the cells along the so-called paracellular pathway.
The tight junction defines a border between the apical and basolateral plasma membranes. In columnar and cuboidal cells of the renal epithelium, it is found at the apical extremity of the lateral membrane and in the plane of the apical surface. Analysis by transmission electron microscopy originally suggested that the tight junction is a zone of partial fusion between the plasma membranes of adjacent cells. Although this is no longer believed to be the case (see below), the ultrastructure of the junctions is consistent with this interpretation. When cells that have been treated with osmium are examined at high magnification, their membranes are distinguished by a characteristic pattern. The two leaflets of the lipid bilayer appear as a “unit membrane,” defined by a pair of darkly stained parallel lines separated from one another by 5–10 nm. In areas corresponding to the tight junction, the four parallel lines representing the two unit membrane of adjacent cells are replaced by three lines, which led to the suggestion that the two outer leaflets contributed by the neighboring cells have in some way merged to form a new trilaminar membrane structure.
The putative outer leaflet fusion suggested by morphologic studies received some support from examination of lipid mobility in polarized epithelial cells. The mobility of outer leaflet lipids is restricted by the tight junction. Labeled lipid probes inserted into the outer leaflets of epithelial apical or basolateral plasma membranes have unimpeded mobility within their respective domains, but cannot cross the tight junction. Furthermore, outer leaflet lipids are unable to diffuse between neighboring epithelial cells through the tight junction. In contrast, inner leaflet lipids can apparently move freely between the two plasma membrane domains, suggesting that the tight junction presents no barrier to their diffusion. These observations are consistent with a model of the tight junction, in which the outer leaflets of the lipid bilayer participate in the formation of some junctional structure, while the inner leaflet remains unperturbed. These results also suggest that the lipid composition of the apical inner leaflet is necessarily identical to that of the basolateral one, because any differences would quickly be randomized by diffusion. Thus, the differences in lipid compositions of the apical and basolateral surfaces alluded to in the introduction to this chapter must be entirely contributed by the constituents of the outer leaflet.
Electron microscopy has provided further insights into the structure of the tight junction. Examination of freeze-fracture replicas of epithelial cells reveals the tight junction to be composed of continuous branching and interwoven strands that surround the entire perimeter of the cell. These strands appear as elevations in the P or cytoplasmic fracture face, and are matched by grooves in the E or external face. In some cell types the strands have a fibrillar appearance, and no discrete subunit structure can be resolved. In other cell types, and in samples not fixed with glutaraldehyde, the strands can appear more as a series of particles. Although some early models postulated that the strands were composed of unusually structured lipids, it is now certain that they are primarily composed of integral membrane proteins (see below). Observations of a number of cell types with different amounts of transepithelial electrical resistance revealed a rough correlation between the number and complexity of the anastomosing strands and the degree of transepithelial electrical resistance. While this correlation certainly exists in at least some epithelia, the amount of resistance is now known to be a function of the specific complement of proteins making up tight junctions in different cells.
The first tight junction protein identified was, appropriately, ZO-1 ( zonula occludens -1). ZO-1, however, turned out to be a cytoplasmic peripheral membrane protein, suggesting that other, integral transmembrane proteins capable of mediating cell–cell contact and the intermembrane permeability barrier must exist. Shortly thereafter, occludin, a multispanning membrane protein, was identified, followed by many other proteins. It is now clear that the tight junction is an extremely complex structure composed of at least three different families of transmembrane proteins including: multiple claudins; occludin and other members of the MARVEL family; and the junctional adhesion molecules or JAMS. Additional peripheral membrane proteins are also part of the tight junction, including ZO-1, -2, and -3, and cingulins. It is also evident that the functions of these protein complexes extend beyond regulating solute permeability to participation in epithelial cell polarization.
Claudins are the most important tight junctional proteins controlling paracellular permeability of small molecules. They are the major protein constituent of the tight junctional strands seen in freeze-fracture; expression of claudins in fibroblasts produces characteristic strands and promotes cell–cell adhesion. The claudin family consists of at least 24 members in mammals. All are tetraspanning transmembrane proteins of 20–27 kD, with two extracellular loops. With one exception, the cytoplasmic C-terminal sequence of claudins interacts with ZO-1, -2, and -3. Interacting claudins on neighboring epithelial cells create charge selective channels, with the overall permeability of the tight junction to ions dependent on the particular mix of claudins expressed in the cell. This was illustrated dramatically in the renal epithelial cell line MDCK (Madin–Darby canine kidney) when expression of claudin 8, in addition to other endogenous claudins, reduced the paracellular movement of mono- and divalent cations without affecting the permeability of anions or uncharged solute molecules. In the kidney tubular epithelium, cells of the proximal tubule, which has a transepithelial electrical resistance of 6–10 Ωcm 2 , express claudins 2, 6, 9, 10, and 11, while cells of the collecting duct, with a much higher resistance of 1000 Ωcm 2 , express claudins 3, 4, 6, 7, 8, 10, and 14 ( Figure 1.1 ). Other cell types along the nephron express other combinations, yielding a range of increasing resistances in the proximal-to-distal direction ( Figure 1.1 ).
The selective barrier created primarily by claudins is sometimes referred to as the “pore pathway,” because it permits movement only of small ions and other uncharged small solute molecules. However, in at least some epithelia, there is also a kind of “leak pathway” that allows passage of larger molecules, including macromolecules. The nature of the leak pathway and its regulation is poorly understood. Occludin, which is also a tetraspanning membrane protein unrelated to claudins, may play a role in the leak pathway, together with ZO-1 and the actin cytoskeleton. Even though it is counterintuitive, an electrically tight pore pathway can co-exist with an active leak pathway, although the molecular and structural basis of this has not been fully clarified.
The tight junction is a structure whose function is highly dependent on interactions between integral and peripheral components and the actin cytoskeleton. ZO-1 and its family members are perhaps the most important class of proteins linking the various tight junctional proteins together. ZO-1 contains multiple PDZ (PSD95-Dlg-ZO-1) protein interaction domains. These bind to both claudins and JAMs, while other regions of the molecule bind to occludin and actin. ZO-1 is also capable of binding to components usually identified with adherens junctions, and to a wide variety of signaling molecules. While it is still valid to view the regulation of paracellular permeability as the primary function of the tight junction, it is more appropriate to think of the overall structure as a component of a larger apical junctional complex responsible for a multiplicity of adhesive, signaling, and membrane trafficking functions.
Originally, the tight junction was looked at as a stable, static structure in intact epithelia. Recent results using, among other approaches, expression of fluorescent tight junction proteins in cultured and intact epithelia, indicate that, in fact, the tight junction is highly dynamic. In the intestine, the epithelial leak pathway will open to permit uptake of glucose beyond the capacity of the Na + -glucose transporter in the apical membrane. This process is controlled by the actomyosin cytoskeleton, since drug-induced actin depolymerization, as well as activation of myosin light chain kinase (MLCK), compromises the epithelial barrier. Breakdown in the barrier is accompanied by simultaneous endocytosis of occludin, both implicating occludin in the regulation of the leak pathway and further demonstrating the cell’s capacity to reshape the junction. Tumor necrosis factor (TNF), which is involved in the pathogenesis of Crohn’s disease, will cause barrier breakdown through a mechanism also dependent on MLCK. Although these studies of tight junction plasticity have concentrated on the intestine, it would be surprising if similar mechanisms were not operable in the renal tubular epithelium, especially in the proximal tubule which morphologically resembles intestinal absorptive cells, and where uptake of a variety of filtered materials occurs.
The adherens junction, or zonula adherens , forms a belt just below the tight junction in most epithelial cells, connecting them via extracellular interactions and cytoplasmic linkages to the actin cytoskeleton ( Figure 1.2 ). In the electron microscope, adherens junctions appear as a dense, somewhat amorphous concentration of submembranous staining, with a mass of impinging actin filaments. The major adhesive component of the adherens junction is E-cadherin. E-cadherin is a single-pass transmembrane protein that consists of a series of calcium-binding extracellular or EC repeat domains, and a cytoplasmic tail that interacts with members of the catenin family. In adherent cells E-cadherin is concentrated in the adherens junction, but can also be more diffusely distributed over the lateral plasma membrane. Adhesion between cells occurs through trans interactions between the EC1 domains contributed by different cells in the presence of calcium, which maintains the proper conformation of the extracellular part of E-cadherin. Interactions occur initially through individual molecules, but are then consolidated and strengthened through lateral interactions of individual units.
The stability of E-cadherin-mediated adhesion is dependent on the binding of catenins to the cytoplasmic tail of E-cadherin. P120-catenin binds to a specific octapeptide located in the cytoplasmic juxtamembranous part of the cytoplasmic tail, and appears to be responsible for maintaining the stability of E-cadherin in the membrane, preventing its endocytosis and degradation. It is also involved in signaling related to cell motility, and is a substrate for the Src-receptor tyrosine kinase. The second catenin that associates with E-cadherin is β-catenin, which binds to the carboxy terminus of the cytoplasmic tail in a phosphorylation-dependent manner. Certain serine phosphorylations of E-cadherin increase the affinity of the β-catenin–E-cadherin interaction, while phosphorylation of serines on β-catenin disrupt the interaction with E-cadherin, and with α-catenin. In addition to its role in cell–cell adhesion, β-catenin is itself an important signaling molecule that is capable of entering the nucleus and regulating transcription of genes related to cell proliferation and differentiation. Its function in transcription is carefully regulated by the Wnt signaling pathway by keeping the cytoplasmic concentration of β-catenin low, either through its interaction with E-cadherin or through its degradation by a mechanism dependent on a cytoplasmic “destruction complex” and ubiquitination.
In the adherens junction, β-catenin serves as a bridge between E-cadherin and α-catenin that, in turn, interacts with the actin cytoskeleton. In this manner, cell–cell adhesion through the adherens junction is given both a degree of mechanical stability and mobility through actomyosin contraction. Originally the β-catenin–α-catenin–actin interaction was believed to be somewhat static, but recent evidence indicates that it is very dynamic. Alpha-catenin can exist as either a monomer or dimer, with the monomer able to bind β-catenin, but not actin, and the dimer able to bind actin, but not β-catenin. Three pools then exist in the cell: a monomer pool bound to β-catenin; a free cytoplasmic monomer pool; and a dimer pool bound to actin. As the adherens junction forms and consolidates, a high concentration of monomers is transported to a localized site on the membrane through β-catenin interactions. This then drives dimer formation and a dynamic linkage to the actin cytoskeleton. Localized concentration of α-catenin dimers can also inhibit Arp2/3, a mediator of actin branching essential for cell migration, and thus facilitate the transformation from a migrating cell to an adherent polarized cell during processes such as injury repair. The recognition that the adherens junction is dynamic and plays a role in cell motility has helped to transform our view of epithelia from that of a static sheet of cells to one of interlocking cells capable of constant motion and remodeling, all the while maintaining a precise permeability barrier between the inside and outside compartments of the body.
The Apical Microvillar Surface
The apical brush border membrane is perhaps best epitomized by the one that graces the epithelial cells of the proximal tubule. Named for its appearance, the proximal tubular brush border is comprised of densely packed parallel microvilli which rise like the bristles of a brush from the level of the tight junctions to a height of 1 to 1.3 μm. The proximal tubular brush border is by far the most luxuriant to be found in the nephron; although the apical membranes of other renal epithelial cell types are endowed with small collections of microvillus-like structures, much less is known about the structural specializations characteristic of the apical membranes of more distal renal epithelial cells.
The functions subserved by apical microvilli are not entirely clear. Certainly their most dramatic and obvious effect upon the properties of the apical membrane is manifest as a tremendous amplification of the apical membrane surface area. For the proximal tubule this amplification is in the order of 20-fold. As is the case for the epithelia of the small intestine, it is through this redundancy that the proximal tubular epithelial cells markedly increase the efficiency of both their absorptive and degradative functions.
Physiologically, the proximal tubule is responsible for the resorption of ~60% of the filtered load of fluid and solutes. Furthermore, it mediates the digestion of essentially all of the polysaccharides and peptides present in the glomerular filtrate, and transports the resultant sugars and amino acids from the lumen to the interstitial fluid space. It is apparent, therefore, that the epithelial cells of the proximal tubule must be specially equipped, in order to cope efficiently with the comparatively enormous quantities of fluid and substrates that rapidly transit this nephron segment. The presence of an extravagant brush border greatly increases the fraction of the tubular fluid that comes into close contact with the enzymatic and transport systems arrayed on the microvillar surfaces prior to its passage from this tubule segment into the descending loop of Henle. Concomitantly, it proportionally multiplies the number of enzymatic and transport systems available to modify the substrates dissolved in the tubular fluid. Thus, the brush border membrane provides the scaffolding for the relatively massive arsenal of enzymatic and transport machinery required to accomplish the proximal tubule’s function as a high-capacity and high-throughput absorptive system.
Ultrastructurally, a microvillus is composed of a bundle of ~19 parallel thin filaments that are linked to one another and to the overlying surface membrane by protein cross bridges. The thin filaments extend well beyond the base of the microvillus, and are anchored in a dense matrix of fibers oriented parallel to the plane of the membrane. This meshwork, referred to as the terminal web, underlies the entire apical surface and anastomses with the filaments that radiate from the lateral desmosomes and zonulae adherens ( Figure 1.2 ). The functional implications of these structural arrangements have become clearer as their components have been biochemically identified.
The thin filaments that form the microvillar core are composed of actin ( Figure 1.2 ). Ultrastructural studies employing heavy meromyosin reveal that all of the filaments in the bundle share a single polarity, and are oriented with their nucleating end towards the microvillar tip. At their termination in the microvillar tip the filaments are received by an electron-dense cap whose molecular identity has yet to be established. As they emerge from the base of the microvillus, the actin filaments are caught up in the fibers of the terminal web ( Figure 1.2 ). Fodrin, or non-erythroid spectrin, comprises one of the major components of this network. It appears to function beneath the brush border as an actin fiber cross-linker. Another of the chief constituents of this fibrillar matrix is a non-muscle form of myosin II that belongs to the same myosin subfamily as its skeletal muscle counterpart. Bipolar myosin thick filaments appear to interact with the actin filaments as they sweep out of the microvillar sheath to join the terminal web. Paired anti-parallel myosin filaments cross-link the actin filaments of neighboring microvilli to one another, forming a connection which bears close comparison to the actin–myosin arrangement characteristic of the striated muscle sarcomere. The analogy is strengthened by the presence in the microvillar rootlet of tropomyosin, a protein that functions in skeletal muscle to regulate the interaction between actin and myosin.
This marked molecular similarity between the terminal web and the skeletal muscle contractile unit prompted speculation that this arrangement might also be functionally homologous. A number of investigators have postulated that activation of myosin-based contraction at the microvillar base might lead to microvillar shortening. Repetitive activation of such a mechanism would lead to a piston-like extension and retraction of these membranous processes, which in turn might stir the surrounding tubular fluid. Such a mixing motion is certainly teleologically appealing, in that it would help to ensure that the tubular fluid is uniformly exposed to the enzymatic and transport systems of the proximal tubular apical membrane surface. No evidence for any such concerted and dynamic properties of microvilli has yet been gathered.
Biochemical studies have shed light on the identities and functional properties of some of the proteins which contribute to the interfibrillar cross bridges observed in transmission electron micrographic profiles of microvilli. Howe and Mooseker identified a protein of molecular weight 110 kDa that participates in cross-linking the filaments of intestinal microvilli to the plasma membrane. This protein exhibits a high affinity for the calcium-binding protein calmodulin, which participates in the transduction of a number of calcium-regulated phenomena. Of further interest was the fact that the 110 kDa protein manifests a myosin-like Mg-ATPase activity. Addition of ATP to intact microvilli results in solubilization of the 110 kDa protein, and disruption of the cross-links between the actin filaments and the microvillar membrane. Thus, attachment of the plasma membrane to the thin filaments may be regulated by ATP and calcium. The degree to which this putative capacity for structural modulation plays a role in microvillar function has yet to be clarified. Subsequent molecular analysis revealed that the brush border 110 kDa protein belongs to the myosin I family of unconventional myosin molecules. Unlike skeletal muscle myosin (which is assigned to the myosin II classification), brush border myosin I molecules possess a single globular head group, and do not form bipolar filaments. Members of the myosin I family, including brush border myosin (myosin Ia), have been found to associate with the membranes of intracellular vesicles, prompting the hypothesis that these motor proteins serve to propel vesicles through the cytoplasm along actin filament tracks. Co-localization studies have demonstrated that brush border myosin I and the microtubule-dependent motor protein dynein can be found together on the membranes of post-Golgi vesicles. This observation has inspired the hypothesis that apically-directed vesicles depart the Golgi along microtubule tracks powered by the action of dynein. Upon their arrival at the actin-rich terminal web, they switch engines and are carried the rest of the way to the brush border by myosin I. While brush border myosin I is abundantly expressed in intestinal epithelial cells, it may be present at lower levels in the renal proximal tubule. Since the myosin I family is large and diverse, however, it is extremely likely that an as-yet-unidentified member of this class subserves similar structural and mechanical functions in the epithelial cells of the kidney.
Another protein that apparently participates in the organization of the microvillus has a molecular weight of 95 kDa, and has been dubbed villin. Villin belongs to a large family of actin-binding proteins. Prominent in its structure is a pair of sequence domains that appear to be involved in associations with f-actin. The presence of this tandem repeat justifies the contention that villin mediates the bundling of actin fibers. It is interesting to note that villin is a calcium-binding protein, and that interaction with calcium alters its behavior in the presence of actin filaments. In experiments carried out with purified villin in solution, it has been found that this protein bundles actin filaments when the free calcium concentration is less than 1 μM. When the calcium concentration rises to 10 μM, villin severs actin filaments into short protofilaments. At intermediate calcium concentrations, villin binds to actin filaments at their growing ends, forming a cap that prevents further elongation. Due to the dynamic nature of the microfilament polymer, this capping results in the formation of shortened filaments. It is not known whether these calcium-dependent activities of villin are manifest in vivo. If villin does indeed sever or shorten actin filaments within the living cell, it would seem likely that perturbations which produce elevations of intracellular calcium concentrations may lead to structurally significant alterations in the organization of the microvillar scaffolding. During embryonic development, villin is expressed throughout the cytoplasm of epithelial cells prior to the elevation of a brush border. At later stages, villin becomes localized to the cytosolic surface of the apical membrane, and is subsequently incorporated into forming microvilli. This behavior has led to the suggestion that the localization of villin to the apical surface is a watershed event in the biogenesis of microvilli. Thus, the formation of inter-filamentous bridges, presumably mediated by villin, may be a critical first step in the organization of the microvillar infrastructure. Supporting this model are the results of experiments in which Caco-2 intestinal epithelial cells were stably transfected with a vector encoding antisense villin mRNA. The consequent reduction in villin expression resulted in a loss of the brush border and mis-sorting of a subset of apical microvillar proteins. It must be noted, however, that results from gene knockout experiments argue against an obligate role for villin in microvillus formation. Mice whose villin genes have been disrupted, and which produce no villin protein, are able nonetheless to generate morphologically and apparently physiologically normal brush borders. Presumably, other components of the microvillar infrastructure can shoulder the cross-linking and organizational duties normally performed by villin. Such functional redundancy is typical of biological systems endowed with architecture as esthetically elegant and complex as that which graces the microvillus.
While villin is limited in its distribution to those cell types endowed with brush borders, another actin-bundling component of the microvillus is present in numerous structures. Plastin-1, which is also known as fimbrin, is a 68 kDa polypeptide associated with the interfilamentous cross bridges that can also be detected in hair cell stereocilia and in ruffled borders. Plastin-1 is clearly a multivalent actin-binding protein, and participates in the cross-linking of the microvillar actin filament array. Structural studies suggest that the cross-linking activity of plastin-1 constitutes the principal means through which the parallel actin filaments are interconnected in microvilli, and the length and organization of brush borders are abnormal in plastin-1 knockout mice. A third bundling protein, known as espin, also participates in the organization of the microvillar actin filaments. While microvilli appear to form normally in the absence of espin, espin overexpression leads to microvillar lengthening by exerting subtle effects on the relative rates of actin filament polymerization and depolymerization. Simultaneous knockout of plastin-1, villin, and espin produces animals whose brush borders are short, and characterized by reduced numbers of disorganized actin filaments and mislocalized myosin. Interestingly, localization of enzymes that are normally concentrated in microvilli is markedly compromised in epithelial cells from these animals, suggesting that the organization of the overlying plasma membrane is dependent upon the structural integrity of the microvillar actin bundle.
Several other polypeptides, associated with the microvillus core and the terminal web, have also been identified. Among the most interesting and important of these is ezrin, a member of the ezrin–radixin–moesin family of proteins. The C-terminal tails of these polypeptides bind to actin filaments, while their N-termini interact with proteins in the membrane. A number of proteins involved in the generation or regulation of intracellular second messengers associate in macromolecular complexes with ezrin–radixin–moesin family members, suggesting that in addition to functioning as linkers these proteins may also act as scaffolding for the assembly of components involved in signal transduction. Knockout of ezrin expression results in shortened and poorly formed brush border microvilli, and perturbations in the organization of the terminal web. In addition, ezrin participates in forming complex molecular scaffolds that regulate and stabilize the expression of solute transport proteins in the apical membranes of renal epithelial cells.
The terminal web mentioned above consists of three morphologically distinguishable domains. In addition to the cytoskeletal fibers that receive the rootlets of the microvilli, fibers that arise from desmosomes and the zonula adherens contribute to this meshwork. The desmosomal fibers consist primarily of 10 nm intermediate filaments composed of keratins. At the level of the zonula adherens , the cell is ringed by a complex of randomly polarized actin filaments which also contains myosin and tropomyosin ( Figure 1.2 ). In vitro experiments have demonstrated that this ring has the capacity to contract circumferentially. This capacity has led to the idea that contraction of the zonula adherens ring might contribute to the alterations in tight junctional permeability which have been observed in several epithelial systems in response to certain second messengers and osmotic stress, as described earlier. Thus, activation of sodium-coupled glucose uptake in cultured intestinal epithelial cells has been shown to induce a decrease in transepithelial resistance. This effect is dependent upon the activity of myosin light chain kinase. It is thought that by shortening in a “purse-string” fashion, these filaments might actually draw neighboring cells away from one another, and thus modify the structure and permeability of the occluding junctions. The relevance of this model to the functioning of renal epithelia has yet to be established.
The anisotropy and structural complexity that characterize the filamentous core of the microvillus apparently also extend to its overlying plasma membrane. The proteins embedded in, and associated with, the plasmalemma of the proximal tubule brush border are not uniformly distributed over its surface, but rather are restricted to specific subdomains. This lateral segregation is epitomized by the behavior of two transmembrane polypeptides, maltase and gp330. The 300 kDa enzyme maltase is distributed over the entire surface of the microvilli themselves, but is absent from the intermicrovillar membrane regions. In contrast, the heavily glycosylated gp330 (also known as megalin) is restricted in its distribution to these intermicrovillar regions. The restriction of megalin to the intermicrovillar regions appears to be mediated by its interactions with protein components of the endocytic machinery. Ultrastructural examination of the intermicrovillar regions reveals the presence of coated pits. The cytosolic surface of the plasma membrane in these domains is coated with an electron dense material that biochemical and immunoelectron microscopic studies have demonstrated to be clathrin. The presence in these intermicrovillar pits of morphologic and compositional features associated with the process of endocytosis has led investigators to believe that this domain mediates the retrieval of large peptides and proteins from the proximal tubular fluid. The proximal tubular epithelial cells are responsible for capturing and degrading any proteins that pass through the glomerular filtration barrier. This function is apparently served by the profusion of coated pits and vesicles that decorate the surfaces of membranes at the microvillar base. Megalin is a member of the LDL receptor family and, together with cubulin, serves as a receptor that binds to and mediates the uptake of filtered proteins and peptides. Megalin knockout mice exhibit low molecular weight proteinuria, establishing the critical role for megalin as the proximal tubule’s pre-eminent scavenger.
Finally, it is worth noting that most or all of the epithelial cells of the nephron are endowed with a single primary cilium ( Figure 1.2 ). This non-motile cilium possesses a ring of nine microtubules, but lacks the central pair of microtubules found in motile cilia. This primary cilium appears to serve sensory functions. Bending the primary cilium, in response to flow or mechanical stimuli, induces calcium signaling in renal epithelial cells. Furthermore, the functional integrity of the primary cilium appears to be a prerequisite for the maintenance of normal renal tubular architecture. A number of cystic diseases of the kidney are attributable to mutations in genes encoding proteins found in cilia. Similarly, mice in which expression of ciliary proteins has been disrupted develop cysts. The mechanisms through which loss of the cilium’s mechanosensory functions leads to cystic transformation remain to be established.
The Basolateral Plasma Membrane
The rigid subservience of structure to function so elegantly exemplified by the apical microvillar membrane also extends to the basolateral surface of the epithelial plasma membrane. As was mentioned above, the basolateral membrane possesses the ion pumps that power the transepithelial resorption of solutes and water. The resorptive capacity of a given cell type is thus largely dependent on the quantity of ion pumps embedded within its basolateral membrane. This parameter appears, in turn, to be roughly proportional to the surface area encompassed by this membrane domain. Consequently, renal epithelial cells that participate in resorption of large quantities of ions and fluid (such as those of the proximal tubule), as well as cells that carry out resorption of ions against steep concentration gradients (such as those of the thick ascending limb of the loop of Henle), are endowed with basolateral plasma membranes whose surface areas are amplified through massively redundant infoldings.
As was detailed in the discussion of the apical membrane, the lateral distribution of proteins within the plane of the basolateral membrane is not uniform. This fact is most dramatically illustrated by epithelial cell types that lack the deeply invaginated basolateral infoldings discussed above. Studies have demonstrated that the Na + /K + -ATPase is concentrated in subdomains of the basolateral membranes of small intestinal epithelial cells. The sodium pump is essentially restricted to the lateral membranes of these cells, and is absent from the basal surfaces that rest on the basement membrane. Dislodging these cells from the underlying basement membrane produces a redistribution of the sodium pump throughout the entire basolateral surface. These results suggest that the sodium pump is either actively or passively prevented from entering the basal domain of the plasma membrane, in some manner that is dependent on an intact interaction with the basement membrane. The meshwork of cytoskeletal elements associated with those sites at which the epithelial cell is anchored to the basement membrane may be too dense to allow membrane proteins such as the sodium pump to penetrate. Conversely, cytoskeletal restraints whose integrity requires cell attachment to the basement membrane might retain the sodium pump within the lateral subdomains. In each of these scenarios, the cytoskeleton plays an important role in determining the subcellular distribution of a transmembrane protein. Research over the years has made it quite clear that the cytoskeleton plays a critical role in defining polarized domains, and in determining aspects of their protein compositions.
The Basement Membrane
The basement membrane, while not strictly part of the epithelium, is such an essential contributor to epithelial function that it cannot be excluded from any comprehensive description of the renal epithelium. The basement membrane is a thin layer of secreted and assembled extracellular matrix that underlies all epithelia and endothelia in the body, and also surrounds skeletal muscle fibers and peripheral nerves. In the past, the terms basement membrane and basal lamina were used inconsistently to describe morphological features of this layer, but there is no longer sufficient reason to distinguish these terms from each other, and they may be used interchangeably. In the kidney, the tubular basement membrane is comparable to that found under other epithelia in the body, while that of the glomerulus is more complex and unusual. In the glomerulus, the basement membrane is synthesized from proteins secreted by both podocytes and the closely apposed endothelium, resulting in a double-thick layer of matrix proteins that is an essential part of the glomerular blood filter. Diseases affecting the glomerular basement membrane often lead to compromise of the filter and proteinuria. Detailed discussion of this barrier and its specialized and distinctive composition is beyond the scope of our overall discussion of the biology of the renal epithelium, and will not be pursued in this chapter.
Basement membranes are visible by transmission electron microscopy of glutaraldehyde-fixed and heavy metal stained thin sections of epithelia, and classically appear as an electron dense layer ( lamina densa ) separated from an electron lucid ( lamina lucida ) layer adjacent to the basal epithelial surface. While these morphological features were originally believed to have a structural basis, there is now evidence that they may be fixation artifacts. All basement membranes are composed of a common set of protein components which include laminins, type IV collagen, heparan sulfate proteoglycans, and nidogen, although the specific types of these can vary depending on both developmental stage and tissue, as well as accompanying pathology. The most important component is probably laminin, because of its role in both assembly of the basement membrane and signaling. Laminins consist of large (~400–800 kDa) heterotrimeric secreted glycoproteins. In mammals, five α-, three β-, and three γ-subunits have been identified in at least 15 different heterotrimeric complexes. Prototypical laminins are cross-shaped molecules in which the short arms of the cross are contributed by the amino-termini of each subunit, and the stem by a coiled-coil made up of the carboxy-terminal halves of each subunit. Typically, the amino-termini of each subunit consist of a globular LN or polymer domain that is involved in basement membrane assembly. The carboxy-terminus of the α-subunit is folded into a series of five globular domains (G1–5) that are essential for binding to the cell surface. Laminins are named according to their subunit composition, such that LM-511, the most common laminin in the kidney, is composed of the α5-, β1-, and γ1-subunits. Like all collagens, collagen IV is a trimeric molecule composed of combinations of various type IV α-subunits that fold into an elongated triple helix. In contrast with fibrillar collagens such as collagen I, type IV collagen does not form bundles, because of the persistence of carboxy-terminal noncollagenous domains (NC1) and interruptions in the collagen repeats within the triple-helix forming regions that render the molecule more flexible. The most common types of proteoglycans found in the basement membrane are perlecan and agrin. Each is a complex molecule that contains a variety of structural motifs resembling those found in laminins, in addition to substantial negatively-charged heparan sulfate polysaccharides. Nidogen (also called entactin) is a relatively small basement membrane protein that acts primarily to link laminin and collagen IV in the assembled structure. In addition to the core components of laminin, collagen IV, and proteoglycans, a variety of other minor components may also be present under particular, poorly-defined conditions, including extracellular matrix proteins normally considered to be primarily components of the interstitial matrix, such as fibronectin.
Basement membranes initially form during embryogenesis, and are then remodeled during development. In addition, de novo basement membrane assembly may occur in adults following injuries that interrupt basement membrane continuity. Assembly is believed to occur through a mass-action process driven primarily by laminin polymerization. Laminin molecules secreted by epithelial cells bind to receptors on the basal cell surface until the density of bound molecules permits formation of heterotrimeric complexes of α, β, and γ amino-terminal LN domains contributed by three different laminin molecules. The resulting structure is a polymerized network of molecules closely associated with the basal surface. Subsequently, collagen IV intercalates into this primary network to form a secondary network created by head-to-tail interactions between collagen IV molecules. The two networks are then linked through nidogen interactions between laminin and collagen, and other molecules, including notably proteoglycans, fill the spaces within the interlocked laminin and collagen networks.
During pathological processes such as renal cyst formation and recovery from ischemic injury to the tubular epithelium, there is evidence that the atypical laminin isoform LM-332 is expressed. This laminin consists of the α3-, β3-, and γ2-subunits, with both the α3- and γ2-subunits lacking amino-terminal LN domains, precluding the molecule from participating in typical network formation. The specific function of LM-332 in these pathological situations is unknown, but one hypothesis is that it interacts with prototypical laminins, such as LM-511, to terminate or even disrupt normal basement membrane assembly, facilitating remodeling of the basement membrane, and possibly signaling the epithelium to differentiate into a more plastic state suitable for injury repair.
The basement membrane interacts with epithelial cells primarily by binding to the integrin family of extracellular matrix receptors. Integrins are a superfamily of cell adhesion receptors found in nearly all cells. Each integrin consists of a heterodimer of α- and β-subunits, both of which are transmembrane glycoproteins. A total of 18 α- and 8 β-subunits are known in mammals, resulting in at least 24 heterodimers. Although integrins are known primarily as receptors for extracellular matrix proteins, they may also participate in cell–cell adhesion. Epithelial cells of the kidney and other organs typically express an array of integrins, including multiple forms with the β1-subunit, such as α2β1 and α3β1, as well as integrins with the β3-, β5-, and β6-subunits in combination with αV (A. Manninen, personal communication). Many, if not all, epithelial cells also express integrin α6β4. The β4-subunit is uniquely found in epithelial cells and, unlike most other epithelial integrins, interacts on the cytoplasmic side with cytokeratins, rather than the actin cytoskeleton. Integrin α2β1 is a collagen receptor, while α3β1 and α6β4 are receptors for multiple isoforms of laminin. The various αV-containing integrins are receptors for ligands containing the binding sequence arginine–glycine–aspartate (RGD), such as fibronectin and vitronectin. They may also play a role in activation of transforming growth factor β (TGFβ), which is important in epithelial repair and other processes. The MDCK cell line, for example, expresses α2β1, α3β1, α6β4, and several αV-containing integrins, with α3β1 and α6β4 mediating adhesion to different laminins, and with αVβ3 (and possibly other αV integrins) activating TGFβ to turn on specific laminin expression during wound-healing. In the kidney tubule, the complement of integrins expressed varies along the nephron, as does the expression of their extracellular matrix ligands, underlining their involvement not only in cell adhesion, but also in differentiation.
In adherent cells, most integrins facilitate adhesion through dynamic interactions with the actin cytoskeleton. Linkage to actin is mediated by adapter protein complexes that bind to integrin cytoplasmic tails and then to actin. Proteins found in these complexes include talin, which binds directly to integrins and activates their adhesive properties, paxillin, α-actinin, and vinculin. Studies of migrating cells suggest that initial adhesive interactions occur through small “focal complexes” that form on leading lamellipodia and are linked to branched actin through the action of the Rac1, a small GTPase of the rho family. As the cell moves over these contacts, they mature into larger “focal adhesions” that associate with robust actin stress fibers (at least in culture) controlled by another GTPase RhoA and its effectors. While the general elements of this model have been somewhat validated in epithelial cells during wound healing, the status of focal complexes and focal adhesions in mature polarized epithelia of the kidney and elsewhere remain, for the most part, unexplored. As mentioned previously, α6β4 is a novel integrin, in that it is epithelial-specific and is capable of assembling adhesion complexes that interact with the cytokeratin cytoskeleton. In normal skin, α6β4 is an essential part of hemidesmosomes. These are large protein complexes containing a second transmembrane protein BP180 in addition to α6β4, as well as the cytoplasmic proteins BP230 and plectin that interact with cytokeratin filaments. The type of hemidesmosome found in the skin (type I) is visible as a dense plaque on the cytoplasmic surface of the basal plasma membrane under the electron microscope. In the kidney such structures have not been reported. However, it is possible that a less developed type of hemidesmosome that is not apparent ultrastructurally (type II) is present in the kidney, although this has not been examined.
In addition to their role in mechanical adhesion, focal complexes and focal adhesions are also platforms for signaling. A variety of kinases including, notably, focal adhesion kinase (FAK) and members of the src family of tyrosine kinases, associate with integrin adhesion complexes and are activated by binding to the extracellular matrix. Subsequent signals then activate downstream serine/threonine kinases, such as integrin-linked kinase (ILK), and MAP kinases, such as ERK, to regulate a diverse range of processes including proliferation, migration, and apoptosis. Indeed, at least 180 different cytoskeletal, adapter, and signaling proteins are known to be associated with integrin adhesion complexes, depending on the cell type and circumstances.
In addition to integrins, other membrane proteins are involved in epithelial cell adhesion to the extracellular matrix, including dystroglycan, a laminin receptor, and possibly a membrane-bound form of the Lutheran antigen. There is evidence that glycolipids may also serve as transient laminin receptors. While it is not proven that any of these receptors play a direct role in epithelial polarization, they may act indirectly by affecting assembly of the basal lamina.
Biogenesis of Epithelial Polarity
In the kidney, polarization of epithelial cells occur under two different circumstances: early development of the tubular epithelium; and repair of an existing epithelium following injury. In mammals, formation of the renal tubular epithelium is initiated by induction of the metanephric mesenchyme by the invading ureteric bud. Following induction, cells of the mesenchyme form aggregates known as condensates, and these subsequently differentiate into polarized epithelial cells facing a central lumen. Extension of this lumen and further, more specialized, differentiation of epithelial cells eventually forms the nephron. In the case of injury by, for example, nephrotoxic substances or ischemia, the existing polarized tubular epithelium is disrupted in spots. Cells at the periphery of these damaged areas then convert from relatively sessile cells polarized along an apical-to-basal axis to flatter, more migratory cells that now have front–rear rather than apical–basal polarity and the capacity to proliferate, enabling them to fill gaps in the epithelium. Once continuity has been achieved, apical–basal polarity is restored. While front–rear and apical–basal polarization would seem to be quite distinctive, there is evidence that many of the important molecules and signals are shared. Indeed, as we are now beginning to understand, even front–rear polarization during injury repair is a close mechanistic cousin of apical–basal polarization.
In the following sections, our current understanding of the mechanism of epithelial polarization will be described, after a brief introduction to the predominant experimental system used to study these processes.
In Vitro Systems
The kidney’s complicated architecture and cellular heterogeneity renders it a poor substrate for studies designed to examine dynamic cell biological processes. Over the past four decades, the vast majority of research into the mechanisms through which epithelia generate and maintain their polarized phenotype has made use of several continuous lines of cultured epithelial cells. These cell lines retain many of the differentiated properties of their respective parent tissues in vitro . Thus, LLC-PK1 cells resemble the proximal tubule (although their precise origin is uncertain). Similarly, Caco-2, HT-29, and T84 cells behave like their progenitors, the colonocytes of the large intestine. Most importantly for this discussion, they manifest in culture the biochemical and morphologic features of the polarized state. Perhaps the best characterized and most heavily used of these culture models is the Madin–Darby canine kidney (MDCK) cell line ( Figure 1.3 ). MDCK cells were originally derived from normal dog kidney in 1959, and grown in culture as a partially transformed line; that is, MDCK cells grow immortally as a monolayer and will not form tumors in nude mice. Although their precise point of origin along the nephron is not entirely clear, their physiological and morphological properties suggest that they derive from cells of the thick ascending limb, distal tubule or collecting tubule.
The first clues to the polarized nature of the MDCK cell line came from the direct observation of these cells’ capacity for vectorial transport. When grown on impermeable substrata, MDCK cells form domes (also called blisters or hemicysts). Physiological studies have demonstrated that domes develop as a result of the transepithelial transport of solutes from the apical media to the basolateral surface. Water that passively follows these solutes results in the generation of the fluid-filled blisters. It is fair to say that domes arise in regions where the cells have literally pumped themselves up off the dish. In keeping with this dramatic propensity for unidirectional solute movement, each MDCK cell manifests a polarized distribution of ion transport proteins, including several routes for sodium entry into its apical membrane, and approximately one million molecules of the Na + /K + -ATPase in its basolateral plasmalemma. The popularity of MDCK cells for polarity research developed out of the seminal observations of Rodriguez-Boulan and Sabatini in 1978. In studies of enveloped virus budding from infected MDCK cells, these investigators found that influenza virus assembles at, and buds from, the apical cell surface ( Figure 1.3 ). Of even greater significance was the demonstration that the spike glycoproteins which populate the membranes of these viruses accumulate preferentially at the cell surface from which budding is to occur. Thus, the influenza hemagglutinin (HA) protein is predominantly on the apical surface early in infection. Similarly, the G protein of vesicular stomatitis virus (VSV) is almost exclusively basolateral in infected cells. The viral proteins provided investigators with the first experimentally manipulatable system for the study of membrane protein sorting. A large number of studies have subsequently elucidated the sorting of many endogenous MDCK cell proteins, as well as exogenous proteins expressed from vectors. This system remains the most thoroughly investigated paradigm and, as will be detailed below, has yielded important insights into the nature of pathways and signals that participate in membrane protein targeting and the overall biogenesis of epithelial polarity.
More recently, investigators have endeavored to develop new cell lines to study particular aspects of renal cell biology. For example, immortalization genes from human papillomavirus or a hybrid between adenovirus and SV40 have been used to create permanent cell lines from human proximal tubule cells. These lines are of particular interest because of the proclivity of the proximal tubule to suffer injury following ischemic insult. The cell lines retain some differentiated characteristics of the proximal tubule, including expression of brush border markers and sodium dependent/phlorizin-sensitive sugar transport. Cultures of cell lines derived in this fashion are not, however, always able to stably maintain the uniform morphology of a simple epithelium, limiting their usefulness for studies of epithelial polarity. More promising results have been obtained using cell lines derived from mice constitutively expressing immortalization factors as transgenes. Among these is the BUMPT-306 line derived from the mouse proximal tubule. While certainly not perfect, this line does grow as a simple epithelium, and has the added advantage of providing an in vitro correlate to in vivo mouse experiments.
The study of epithelial polarization using cell lines has been facilitated by culturing cells in configurations that more closely resemble in vivo conditions ( Figure 1.4 and 1.5 ). For example, many varieties of epithelial cells can be grown on permeable filter supports. Originally, these were designed to mimic the Ussing chamber used for physiological studies, but later turned out to also be very useful for biochemical and morphological experiments. In their most common commercially-available configuration, these supports are composed of polycarbonate filters with pore sizes typically in the range of 0.4 μm that form the bottom cup. The cup is then suspended in a plastic well containing culture medium, and medium is also added to the inner compartment of the cup ( Figure 1.4 ). Cells are plated on the upper surface of the filter. When a confluent monolayer is formed, it effectively creates a barrier between media compartments. The medium in the interior of the cup bathes the epithelial apical surface, whereas the basolateral surface communicates with the exterior media compartment through the pores of the filter ( Figure 1.4 ). As epithelial cells in the kidney and other organs would normally receive most of their nutrition from the basolateral (serosal) surface, permeable supports are, in a sense, a more natural growth environment than impermeable tissue culture plastic or glass. Indeed, there is some evidence that epithelial cells are more polarized in filter cultures than on solid substrata. Furthermore, the use of filters for the culture of epithelial cells permits investigators simultaneous and independent access to the apical and basolateral plasmalemma surfaces. This useful capacity has been extensively exploited in the experiments described in the protein sorting section of this chapter.
In addition to permeable supports, a number of investigators also culture renal and other epithelial cell lines embedded in a gel of collagen type I or other extracellular matrix molecules ( Figure 1.5 ). These are called three-dimensional (3D) cultures, to distinguish them from more common two-dimensional (2D) cultures on either solid or permeable surfaces. As with permeable supports, the idea behind 3D cultures is that placing the epithelial cell in an environment in which it is surrounded by extracellular matrix similar to that of the interstitium more closely resembles the in vivo environment. While that conclusion is subject to debate, there is no doubt that certain epithelial phenotypes are more readily expressed in 3D than in 2D cultures. Nevertheless, these phenotypes are often slow to develop, frequently taking 7–10 days, and may occur asynchronously; this, and the inaccessibility of the cultures, somewhat limits their usefulness for biochemical studies. With the advent of high resolution confocal fluorescence microscopy and the wide array of fluorescent proteins and probes, the impact of this limitation is lessened. In the case of MDCK cells, individual suspended cells develop into polarized cysts or, when stimulated with certain growth factors, tubules. As will be described below, use of 3D cultures has led to important fundamental observations about epithelial cell polarization. As a final note on the experimental use of 2D and 3D culture modalities, it is important to point out that formation of polarized cysts in 3D may be most closely analogous to the formation of the primordial kidney epithelium from condensed metanephric mesenchyme during development. In contrast, in vitro polarization of kidney epithelial cells in 2D cultures is more akin to the repair of existing kidney tubular epithelia following injury, a scenario that requires the spreading, migration, and proliferation of cells on a pre-existing surface to re-establish a contiguous epithelium.
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