Toshiyuki Matsuzaki

Disruption of Aquaporin-11 Produces Polycystic Kidneys following Vacuolization of the Proximal Tubule

ABSTRACT Aquaporin-11 (AQP11) has been identified with unusual pore-forming NPA (asparagine-proline-alanine) boxes, but its function is unknown. We investigated its potential contribution to the kidney. Immunohistochemistry revealed that AQP11 was localized intracellularly in the proximal tubule. When AQP11 was transfected in CHO-K1 cells, it was localized in intracellular organelles. AQP11-null mice were generated; these mice exhibited vacuolization and cyst formation of the proximal tubule. AQP11-null mice were born normally but died before weaning due to advanced renal failure with polycystic kidneys, in which cysts occupied the whole cortex. Remarkably, cyst epithelia contained vacuoles. These vacuoles were present in the proximal tubules of newborn mice. In 3-week-old mice, these tubules contained multiple cysts. Primary cultured cells of the proximal tubule revealed an endosomal acidification defect in AQP11-null mice. These data demonstrate that AQP11 is essential for the proximal tubular function. AQP11-null mice are a novel model for polycystic kidney diseases and will provide a new mechanism for cystogenesis.

Aquaporin-5 (AQP5), a water channel protein, in the rat salivary and lacrimal glands: immunolocalization and effect of secretory stimulation.

Abstract Aquaporin-5 (AQP5) is a water channel protein and is considered to play an important role in water movement across the plasma membrane. We raised anti-AQP5 antibody and examined the localization of AQP5 protein in rat salivary and lacrimal glands by immunofluorescence microscopy. AQP5 was found in secretory acinar cells of submandibular, parotid, and sublingual glands, where it was restricted to apical membranes including intercellular secretory canaliculi. In the submandibular gland, abundant AQP5 was also found additionally at the apical membrane of intercalated duct cells. Upon stimulation by isoproterenol, apical staining for AQP5 in parotid acinar cells tended to appear as clusters of dots. These results suggest that AQP5 is one of the candidate molecules responsible for the water movement in the salivary glands.

Water Channel Protein AQP3 Is Present in Epithelia Exposed to the Environment of Possible Water Loss

Aquaporins (AQPs) are membrane water channel proteins expressed in various tissues in the body. We surveyed the immunolocalization of AQP3, an isoform of the AQP family, in rat epithelial tissues. AQP3 was localized to many epithelial cells in the urinary, digestive, and respiratory tracts and in the skin. In the urinary tract, AQP3 was present at transitional epithelia. In the digestive tract, abundant AQP3 was found in the stratified epithelia in the upper part, from the oral cavity to the forestomach, and in the simple and stratified epithelia in the lower part, from the distal colon to the anal canal. In the respiratory tract, AQP3 was present in the pseudostratified ciliated epithelia from the nasal cavity to the intrapulmonary bronchi. In the skin, AQP3 was present in the epidermis. Interestingly, AQP3 was present at the basal aspects of the epithelia: in the basolateral membranes in the simple epithelia and in the multilayered epithelia at plasma membranes of the basal to intermediate cells. During development of the skin, AQP3 expression commenced late in fetal life. Because these AQP3-positive epithelia have a common feature, i.e., they are exposed to an environment of possible water loss, we propose that AQP3 could serve as a water channel to provide these epithelial cells with water from the subepithelial side to protect them against dehydration.

Aquaporins in the digestive system

Fluid transfer such as secretion and absorption is one of the major functions of the digestive system. Aquaporins are water channel proteins providing water transfer across the cellular membrane. At least six aquaporin isoforms are expressed in the digestive system. Aquaporin-1 (AQP1) is widely distributed in endothelial cells of capillaries and small vessels as well as in the central lacteals in the small intestine. AQP1 is also present in the duct system in the pancreas, liver, and bile duct. AQP3 is mainly expressed in the epithelia of the upper digestive tract from the oral cavity to the stomach and of the lower digestive tract from the distal colon to the anus. AQP4 is present in the parietal cells of the stomach and in the intestinal epithelia. AQP5 is expressed in acinar cells of the salivary, pyloric, and duodenal glands. AQP8 is expressed in the intestinal epithelia, salivary glands, pancreas, and liver. AQP9 is present in the liver and intestinal goblet cells. Aquaporins have important roles in the digestive system, such as AQP5 in saliva secretion, as shown by the studies on AQP5-null mice. In addition, water transfer across the digestive epithelia seems to occur not only via aquaporins but also via other transporter or channel systems.

The phosphorylation state of serine 256 is dominant over that of serine 261 in the regulation of AQP2 trafficking in renal epithelial cells

Phosphorylation of serine 256 (S256) plays a critical role in vasopressin (VP)-mediated membrane accumulation of aquaporin-2 (AQP2). Recently, phosphorylation of serine 261 was also reported, raising the possibility that it has a role in AQP2 trafficking. We addressed this issue using transfected LLC-PK(1) cells that express point mutations of AQP2 S261 and S256, mimicking the phosphorylated (S to D) or dephosphorylated (S to A) states of these residues. Both AQP2 (S261A) and AQP2 (S261D) were located in the perinuclear cytoplasm without stimulation but, like wild-type AQP2, they both accumulated on the plasma membrane after 20-min exposure to VP or forskolin. Following membrane accumulation, S261A, S261D, and wild-type AQP2 reinternalization was complete over a similar time frame, between 30 and 60 min after VP washout. Using various combinations of point mutations, we showed that the phosphorylation state of S256 is dominant with respect to AQP2 behavior; AQP2 membrane accumulation and internalization were not detectably affected by the phosphorylation state of S261. Finally, blocking AQP2 endocytosis by methyl-beta-cyclodextrin caused membrane accumulation of AQP2 in cells expressing either a single S-A mutation or double mutations of S256 and S261, although as previously reported, the S256D mutation was always present at the cell surface. This suggests that constitutive recycling of AQP2 was not modified by the phosphorylation state of S261. Together, our data indicate that the phosphorylation state of AQP2 at S261 does not detectably affect regulated or constitutive trafficking of AQP2. The potential role of S261 phosphorylation/dephosphorylation in vasopressin action remains to be determined.

Recent advances in fluorescent labeling techniques for fluorescence microscopy.

Tremendous progress in recent computer-controlled systems for fluorescence and laser-confocal microscopy has provided us with powerful tools to visualize and analyze molecular events in the cells. Various fluorescent staining and labeling techniques have also been developed to be used with these powerful instruments. Fluorescent proteins such as green fluorescent protein (GFP) allow us to directly label particular proteins of interest in living cells. This technique has been extended over a large area of cell biology, and a variety of fluorescent protein-derived techniques have been developed to visualize the functions and conditions of the molecules within living cells. In this review, we summarize the techniques for fluorescent staining and labeling for recent fluorescence microscopy.

The NPC Motif of Aquaporin-11, Unlike the NPA Motif of Known Aquaporins, Is Essential for Full Expression of Molecular Function

The recently identified molecule aquaporin-11 (AQP11) has a unique amino acid sequence pattern that includes an Asn-Pro-Cys (NPC) motif, corresponding to the N-terminal Asn-Pro-Ala (NPA) signature motif of conventional AQPs. In this study, we examined the effect of the mutation of the NPC motif on the subcellular localization, oligomerization, and water permeability of AQP11 in transfected mammalian cells. Furthermore, the effect was also assessed using zebrafish. Site-directed mutation at the NPC motif did not affect the subcellular localization of AQP11 but reduced its oligomerization. A cell swelling assay revealed that cells expressing AQP11 with a mutated NPC motif had significantly lower osmotic water permeability than cells expressing wild-type AQP11. Zebrafish deficient in endogenous AQP11 showed a deformity in the tail region at an early stage of development. This phenotype was dramatically rescued by injection of human wild-type AQP11 mRNA, whereas the effect of mRNA for AQP11 with a mutated NPC motif was less marked. Although the NPA motif is known to be important for formation of water-permeable pores by conventional AQPs, our observations suggest that the corresponding NPC motif of AQP11 is essential for full expression of molecular function.

Acute hypertonicity alters aquaporin-2 trafficking and induces a MAPK-dependent accumulation at the plasma membrane of renal epithelial cells.

The unique phenotype of renal medullary cells allows them to survive and functionally adapt to changes of interstitial osmolality/tonicity. We investigated the effects of acute hypertonic challenge on AQP2 (aquaporin-2) water channel trafficking. In the absence of vasopressin, hypertonicity alone induced rapid (<10 min) plasma membrane accumulation of AQP2 in rat kidney collecting duct principal cells in situ, and in several kidney epithelial lines. Confocal microscopy revealed that AQP2 also accumulated in the trans-Golgi network (TGN) following hypertonic challenge. AQP2 mutants that mimic the Ser256-phosphorylated and -nonphosphorylated state accumulated at the cell surface and TGN, respectively. Hypertonicity did not induce a change in cytosolic cAMP concentration, but inhibition of either calmodulin or cAMP-dependent protein kinase A activity blunted the hypertonicity-induced increase of AQP2 cell surface expression. Hypertonicity increased p38, ERK1/2, and JNK MAPK activity. Inhibiting MAPK activity abolished hypertonicity-induced accumulation of AQP2 at the cell surface but did not affect either vasopressin-dependent AQP2 trafficking or hypertonicity-induced AQP2 accumulation in the TGN. Finally, increased AQP2 cell surface expression induced by hypertonicity largely resulted from a reduction in endocytosis but not from an increase in exocytosis. These data indicate that acute hypertonicity profoundly alters AQP2 trafficking and that hypertonicity-induced AQP2 accumulation at the cell surface depends on MAP kinase activity. This may have important implications on adaptational processes governing transcellular water flux and/or cell survival under extreme conditions of hypertonicity.

Successful gene transfer using adeno-associated virus vectors into the kidney: comparison among adeno-associated virus serotype 1-5 vectors in vitro and in vivo.

Background/Aim: Gene transfer into the kidney has great potential as a novel therapeutic approach. However, an efficient method of gene transfer into the kidney has not been established. We explored the transduction efficiency of renal cells in vitro and in vivo using adeno-associated virus (AAV) serotype 1–5 vectors encoding the β-galactosidase gene. Methods: In the in vitro study, rat kidney epithelial cell line NRK52E cells were transfected with AAV serotype derived vectors. In the in vivo study, AAV serotype derived vectors were selectively injected into the kidney using a catheter-based gene delivery system in rats and mice mimicking the clinical procedure. The efficiency of gene expression was histologically evaluated on the basis of the β-galactosidase expression. Results: AAV serotype 1, 2, and 5 vectors transduced in rat kidney epithelial cell line NRK52E cells in vitro, whereas AAV serotype 3 or 4 vectors showed no transduction. In addition, the kidney-specific injection of AAV serotype 2 vectors successfully transduced in tubular epithelial cells, but not in glomerular, blood vessel, or interstitial cells in vivo, whereas the rest of the serotypes showed no transduction. Conclusion: Since kidney-specific gene delivery via the renal artery by catheterization is highly feasible in humans, these findings provide useful information for promising strategies in renal gene therapy.

Immunolocalization of GLUT1 and connexin 26 in the rat placenta

Abstract.Interhemal membrane in the rat placenta is composed of three trophoblastic layers and endothelial cells. GLUT1, an isoform of the facilitated-diffusion glucose transporter, is abundant in the cells of the placental barrier, i.e., syncytiotrophoblastic layers I and II. GLUT1 is localized at the plasma membranes of the maternal-blood side of syncytiotrophoblastic layer I, and of the fetal-blood side of syncytiotrophoblastic layer II. Double-immunofluorescence microscopy has shown that connexin 26 is present between these GLUT1-positive sites, i.e., between syncytiotrophoblastic layers I and II. Immunogold electron microscopy has revealed that connexin 26 is localized in the gap junctions connecting the two layers. Connexin 26 in these layers therefore makes them functionally a single syncytial layer for the transfer of small molecules such as glucose in the rat placental barrier. These results suggest that glucose transfer in the rat placental barrier is carried out as follows: GLUT1 is used for the entry of glucose into the cytoplasm of syncytiotrophoblastic layer I, connexin 26 for the transfer of glucose from syncytiotrophoblastic layer I to syncytiotrophoblastic layer II, and GLUT1 for the exit of glucose to the fetal circulation.