Having described the generalities of epithelial junctional complexes, I will now focus on the relevance of TJs in the healthy and diseased kidney. While TJs are present all along the renal tubule their composition and morphology vary considerably between different tubule segments. This contributes to a more than a 100-fold difference in permeability that steadily increases from the proximal tubule to the collecting duct (Fig 3-12). As previously stated, TJs along the tubule first form during kidney development and can reassemble following tubular injury. The key role of TJs in kidney health and disease is illustrated by hereditary diseases, by recent animal models and by in vitro studies [217,218].
Figure 3-12 Schematic of the nephron and collecting duct system depicting segment-specific expression of major claudins (CLDN) and ZO-1. Transepithelial resistance (TER) varies inversely with paracellular flux and estimates of ETR in each tubule segment are shown at top (From Bradley M.
Clau d
As previously mentioned, TJs restrict diffusion of small molecules across the intercellular space. Besides its fence function, TJs also function as gates that regulate paracellular transport, a crucial component of normal renal physiology. TJs are differently distributed along the renal tubule, where each segment is endowed with distinct characteristics [41,101]. Proximal tubule segments have “leaky” TJs, with fewer strands that are more discontinuous. This correlates with low TER and substantial movement of isosmotic fluid. By contrast, more distal segments have“tight” TJs, with more strands and more continuity. This correlates with high TER [219]. Renal tubules with “tight” TJs and high TER can maintain high electrochemical gradients produced by active transcellular transport. This configuration is essential for the production of either highly concentrated or diluted urine with concentrations of solutes several fold higher or lower than that of plasma [41]. It should be mentioned that other epithelia, such as those of bladder and skin, display an even higher TER than renal collecting duct.
It is clear that claudins are major determinants of paracellular permeability and play a large role in differential solute and water selectivity between tubule segments [220]. Most claudins are differently expressed along the renal tubule (Fig 3-12). For example, claudin-2, a
“leaky” claudin, is highly expressed in the proximal tubule and early segment of the thin descending limb. On the other hand, claudin-4 and -8 are restricted to tight CD [41].
Expression of exogenous claudin-2 in high-resistance MDCK I cells generated a low-resistance phenotype [221]. In vitro studies additionally showed that claudin-2 is responsible for paracellular sodium and water transport [222,223]. Claudin-2-deficient mice displayed a significant decrease in net sodium and water reabsorption in the PT and loss of
sodium selectivity [224]. Claudin-4 and -8 were shown to interact with each other and function as a Cl- channel in CD cells [225]. Claudin-4-deficient mice exhibit a small but significant increase in fractional excretion of Cl- and Ca2+, a concomitant decrease of claudin-8 expression, and increased mortality due to urothelial hyperplasia and hydronephrosis as animals age [226]. In CD, Na+ is reabsorbed via the transcellular pathway through apical expressed epithelial Na+ channel (ENaC) and basolateral expressed Na+-K+-ATPase in principal cells. The paracellular pathway acts as a barrier to prevent backleak of reabsorbed Na+ while allowing Cl- to diffuse down its electrical gradient. This process is likely regulated by claudin-4 and claudin-8.
Tight junctions also regulate the permeability of uncharged macromolecules more than 4 Å in diameter [41], which might depend on occludin and ZO-1 but not claudins [144,227-229]. Occludin, ZO-1 and ZO-2 expression increase along isolated rabbit renal tubules along with junction complexity, and expression levels of these three proteins are significantly higher in distal segments than in more proximal regions [158]. Other roles of TJ proteins in the kidney, besides their role in regulating paracellular permeability, remain to be explored.
Tight junction pathologies are highly heterogeneous and difficult to classify [230]. As described in “Chapter 1.3: Effects of injury on tubular cells and on their recovery”, TJs are essential for renal tubule recovery after ischemic injury. However, the detailed mechanisms remain unclear. Genetic studies also revealed several TJ-related kidney diseases known to be caused by mutations of genes encoding TJ proteins.
Mutations in claudin-16 and claudin-19 cause FHHNC (familial hypomagnesemia with hypercalciuria and nephrocalcinosis) [231,232]. Claudin-16 is highly expressed in the thick ascending limb of Henle's loop (TAL) and regulates paracellular transport of Mg2+ and Ca2+
[233,234]. Patients with mutations in claudin-16 display increased urinary loss of Mg2+ and Ca2+ and reduced plasma Mg2+ levels, leading to fatigue and seizures. Claudin-19 is highly expressed in the retinal epithelium and in the TAL [235]. Patients with mutations in claudin-19 display ocular abnormalities, more severe renal impairment than patients with claudin-16 mutations and have a high risk of progression to chronic renal disease [236,237].
Mutations in claudin-14 cause nonsyndromic deafness [238], whereas carriers of common sequence variants in claudin-14 have been implicated in the pathogenesis of kidney stones and reduced bone mineral density [239]. Recently, a novel deletion/rearrangement of the occludin gene was identified, and this mutation causes brain calcification and renal dysfunction, such as asymmetry of kidney size and chronic kidney disease [240].
IV Aim of the study
Early in my study, I investigated how chronic hypertonic challenge induces hypertrophy in renal CD principal cells. During this investigation, I found that expression of all ZO proteins was significantly increased in CD cells in response to chronic hypertonic stress and that this is correlated with a decrease of cell proliferation. Unexpectedly, I found that depletion of either ZO-1 or ZO-2 under isotonic conditions decreased cell proliferation and generated a severe hypertrophic phenotype. ZO-1 and ZO-2 were previously considered as negative regulators of cell proliferation. However, ZO-1 and ZO-2-deficient mice are embryonic lethal and exhibit delayed growth at early embryonic days (Ⅲ 2.1.2). Along with my preliminary data, I suspect that the proliferating role of ZO proteins in renal CD principal cells is different from that previously described. This led me to investigate the putative roles of ZO proteins in regulating renal CD principal cell proliferation and to focus my study on the roles they play under isotonic conditions, i.e. conditions that prevail in the renal cortex. This is an important issue since the functional properties of the CD rely on the scaffolding function of ZO proteins and their interactions with both transmembrane junctional proteins and multiple signaling molecules. Because low cell proliferation likely benefits intercellular adhesion, and thus diffusion barrier efficiency, it stands to reason that ZO proteins influence CD permeability not only as part of a diffusion barrier but also by helping to maintain the intercellular seal by restricting cell proliferation. The aim of this study was to establish the influence of ZO proteins on the proliferative potential of renal CD principal cells. This study would lend invaluable insight on how TJs help control cell proliferation, and thus the kidney's ability to regulate extracellular homeostasis, in both healthy and diseased kidney.
V Materials and methods
1. Cell culture and transfection