2134 provides an electrical driving force for reabsorption of Ca2+ and Mg2+ paracellularly. The distal tubule and collecting duct fine-tune urinary composition at the end of the nephron. Active, transcellular Na+ reabsorption and K+ and H+ secretion generate steep transtubular concentration gradients for these ions. In these segments, the paracellular pathway is relatively imperme- able and acts as a barrier to solutes, so that these ion gradients are not dissipated. Tight junctions regulate paracellular permeability of epithelia Epithelial cells are connected via multiple junctional com- plexes. The tight junction separates the apical and basolateral membrane domains and acts as the paracellular barrier while remaining selectively permeable to ions and water. By electron microscopy, the tight junction appears as a band of parallel fibril strands in the subapical compartment. The tight junction has been shown to be the site of paracellular permeability in epithelia. Ussing and Windhager [6] incubated frog skin cells apically with Ba2+ and basolaterally with SO42- and showed that, under the condition of hyperosmotic solution on the apical side, BaSO4 precipitated in junctions. Machen [7] demonstrated that La3+ permeates the junctional complex in rabbit gallbladder and ileum epithelium and proposed the concept that some tight junctions are permeable to ions and small molecules like water and mannitol. Studies on the biochemical composition of the tight junction have helped to elucidate the regulation of tight junction permeability. The tight junction is constituted of several groups of proteins (Fig. 1). The first group consists of membrane-spanning proteins (claudin, occudin, and junctional adhesion molecules), which are of particular interest because their extracellular domains face the paracellular space and are thought to regulate paracellular transport directly. The second group consists of scaffolding proteins (e.g., zona occludens family), which link the membrane-spanning proteins to the actin cytoskeleton. The third group consists of signaling molecules, including transcription factors and kinases/phos- phatases that regulate tight junction protein transcription and expression (for a more detailed review, see [8]). Claudins are tight junction proteins that constitute the paracellular barrier and pore Pediatr Nephrol (2011) 26:2133–2142 proteins that have two extracellular loops facing the para- cellular space. Among the membrane-spanning proteins at the tight junction, claudins are by far the most variable in terms of number of genes/isoforms. These isoforms are expressed variably in epithelia of different organs in a tissue-specific manner, which probably correlates with the variability in permeability of these epithelia. The initial discovery of the pathogenic linkage of claudin-16 to familial hypomagnesemia hypercalciuria nephrocalcinosis (FHHNC) first suggested that the claudins might be general regulators of paracellular permeability at the tight junction. At the tight junction, claudins work in concert with the other components of the tight junction, which makes it difficult to control for and define the specific roles in permeability of any individual claudin. Nevertheless, a variety of experimental approaches have been used to answer three key questions: what are the permeability properties of a specific claudin? What part of the claudin determines the permeability? What amino acids are the active sites of paracellular transport? These studies are summarized in the following section. Note that we focus here on renal epithelial physiology, but claudins have a broader role in many other aspects of biology, e.g., barrier function in gastrointestinal epithelia, retina and cochlea, cancer, etc. Expression studies in epithelial cell lines and knockout studies in mice The function of claudins has been addressed by numerous overexpression studies in epithelial cell lines (for a detailed review, see [9]). To briefly summarize, claudin-1 [10], 4 [11], 5 [12], 8 [13], 9 [14], 11 [11], 15 [11], and 19 [15] are considered to be barrier claudins because they increase transepithelial resistance (TER) when overexpressed in leaky cell lines. Claudin-2 [16] and 16 [17] are considered to be pore claudins because they decrease the TER in most cell lines. Because epithelial cells express endogenous claudins, the change of TER and ion selectivity reflects the overall effect of exogenous and endogenous claudins interaction rather than the properties of the expressed claudin alone. The function of claudins has also been addressed by knockout studies in mice. This study design minimizes the interaction effects and may better reveal the function of the target claudin of interest. Knockout of claudins-1 and -5 increases paracellular permeability in the skin and blood– brain barrier, respectively, while knockout of claudin-2 decreases permeability in the proximal renal tubule [18]. Taken together, these findings confirm that claudins regulate paracellular permeability at the tight junction. General properties of claudins Chimera and mutagenesis studies Twenty-four claudin genes have so far been identified in mammalian cells. Claudins are 4-transmembrane domain While the previous studies showed that claudins regulate the paracellular permeability, evidence that claudins form