64 Interestingly, FHHNC patients do not exhibit the salt wasting or metabolic alkalosis that is found in Bartter’s syndrome (Wong and Goodenough 1999). Based on these Wndings, it is tempting to hypothesize that PCLN-1 might form a Mg2+- and Ca2+ -conducting pore. It is therefore likely that PCLN-1 selectively mediates the barrier to diva- lent but not monovalent cations. Thus, not only can the paracellular barrier discriminate between charged and uncharged solutes, but it may also exhibit an even higher level of speciWcity distinguishing charge and ionic radius and size of the hydrate shell. As to molecular investigations based on the use of diverse cell culture models for kidney epithelial cells, there is some discrepancy between results obtained upon over- expression of PLCN-1 in two diVerent kidney epithelial cell lines used. In the canine MDCK cell line, another group found PCLN-1 to decrease Na+ permeability, to decrease TER and to enhance transepithelial Mg2+ transport (Ikari et al. 2006). Another recent study performed in the porcine proximal tubule LLC-PK1 cells suggests that paracellin-1 functions to modulate paracellular conductance and not transcellular transport. They hypothesise that PLCN-1 mediates mostly paracellular Na+ permeation to build up the positive lumen potential and that this lumen-positive potential would be the driving force for the reabsorption of Mg2+ (Hou et al. 2005). As described for other molecules relevant to Mg2+ homeostasis such as ACDP2, (ancient conserved domain protein), a divalent metal transporter, or the Mg2+ trans- porter MagT1 (Wong and Goodenough 1999), the human PLCN-1 promoter appears to be responsive to external Mg2+ concentrations, since the renal epithelial cell speciWc PLCN-1 gene expression was shown to positively correlate with the amount of Mg2+ oVered in the growth medium (Efrati et al. 2005). Further, mutations in PLCN-1 can aVect claudin-16 pro- tein targeting at the TJs (Kausalya et al. 2006). The muta- tion T233R for example prevents interaction of claudin-16 with ZO-1 and results in lysosomal targeting of claudin-16 instead. Protein kinase A (PKA) mediates phosphorylation of claudin-16 at Ser217, failure of which equally leads to dissociation of claudin-16 from ZO-1 and its translocation into the lysosomes (Ikari et al. 2006). In conclusion, although the mechanism of PLCN-1/claudin-16-mediated Mg2+ and Ca2+ reabsorbtion is not yet fully understood, it is clear that these claudins play a crucial role in this process. Recently, Konrad et al. (2006) additionally unravelled a role for claudin-19 in the development of FHHNC. They had clinically characterized one Swiss and eight Spanish/ Hispanic families aVected with severe hypomagnesemia due to renal wasting, nephrocalcinosis, and progressive renal failure but had to recognize that the individuals did not have any mutations in CLDN16. They showed that the 123 Histochem Cell Biol (2008) 130:55–70 renal phenotype of the aVected patients was virtually undis- tinguishable from that of patients who presented with FHHNC with proven CLDN16 mutations. However, the aVected individuals in these families also have severe visual impairment, characterized by macular colobomata, signiWcant myopia, and horizontal nystagmus (Konrad et al. 2006). Konrad et al. were able to map a new locus for recessive renal magnesium loss on chromosome 1p34.2 and identiWed mutations in CLDN19, yet another member of the claudin family in patients aVected with FHHNC and addi- tionally, with severe ocular abnormalities. CLDN19, which encodes the tight-junction protein claudin-19 was demon- strated to be highy expressed in renal tubules and the retina. In an eVort to understand the mechanisms underlying the roles of the two claudins in mediating paracellular ion reab- sorption in the kidney, Goodenough and co workers (Hou et al. 2008) showed using pig kidney epithelial cells, that CLDN19 functioned as a Cl(¡) blocker, whereas CLDN16 functioned as a Na(+) channel. Mutant forms of CLDN19 were shown to be associated with FHHNC since they were unable to block Cl(¡) permeation. Coexpression experi- ments further elucidated that CLDN16 interacts with CLDN19 and that their association confers cation selectiv- ity, suggesting a role for mutant forms of CLDN16 and CLDN19 in the development of FHHNC. Vascular system Diabetic retinopathy The barrier between the vascular lumen and neural layers in the retina and the brain parenchyme maintains a charac- teristic microenvironment and is essential for proper neuronal function. This blood-retinal barrier (BRB) consists of two anatomical entities: an inner BRB formed by junctions between endothelial cells of the retinal capillaries, and an outer BRB composed of TJs between retinal pigment epi- thelial cells. Clinical evidence from Xuorescein angiogra- phy has demonstrated that the inner BRB appears to be the primary site of vascular leakage leading to macular edema. The BRB is built up and maintained by TJ complexes that make up the blood vessels of the microvasculature (Mitic and Anderson 1998). The principal proteins found in the retinal endothelial TJs are occludin and claudin-5 (Dej- ana et al. 2001). Loss of the BRB integrity and vascular permeability leading to macular edema (Vitale et al. 1995) characterise diabetic retinopathy. Macular edema in turn is thought to be directly responsible for vision loss in diabetic retinopathy (Felinski and Antonetti 2005). Vascular endo- thelial growth factor (VEGF) is thought to be chieXy responsible for this increase in BRB permeability, albeit other permeabilizing factors like histamin or the cytokines IL-1 and TNF have also been implicated (Felinski and