Hiroaki Miyazaki

Overt nephrogenic diabetes insipidus in mice lacking the CLC-K1 chloride channel

CLC-K1 is a kidney-specific chloride channel that mediates transepithelial chloride transport in the thin ascending limb of Henles loop (tAL) in the inner medulla. Transport of NaCl in the tAL is thought to be a component of urinary concentration in a passive model of the countercurrent multiplication system, but there has been no direct evidence that CLC-K1 is involved in urine concentration. To analyse the physiological function of CLC-K1 in vivo, we generated mice lacking CLC-K1 by targeted gene disruption. Clcnk1–/– mice were physically normal appearance, but produced approximately five times more urine than Clcnk1+/– and Clcnk1+/+ mice. After 24 hours of water deprivation, Clcnk1–/– mice were severely dehydrated and lethargic, with a decrease of approximately 27% in body weight. Intraperitoneal injection of the V2 agonist 1-deamino-8-D-arginine vasopressin (dDAVP) induced a threefold increase in urine osmolarity in Clcnk1+/– and Clcnk1+/+ mice, whereas only a minimal increase was seen in Clcnk1–/– mice, indicating nephrogenic diabetes insipidus. After in vitro perfusion of the tAL, the lumen-to-bath chloride gradient did not produce a diffusion potential in Clcnk1–/– mice in contrast to Clcnk1+/+ and Clcnk1+/– mice. These results establish that CLC-K1 has a role in urine concentration, and that the countercurrent system in the inner medulla is involved in the generation and maintenance of hypertonic medullary interstitium.

Excellent salinity tolerance of Mozambique tilapia (Oreochromis mossambicus): elevated chloride cell activity in the branchial and opercular epithelia of the fish adapted to concentrated seawater.

Abstract Changes in morphology and cellular activity of the chloride cells in branchial and opercular epithelia were examined in tilapia, Oreochromis mossambicus, adapted to fresh water (FW), seawater (SW) and concentrated SW (180% SW). The tilapia are adaptable to a wide range of salinity, maintaining the plasma osmolality within physiological levels. Gill Na+, K+-ATPase activity was remarkably increased in response to elevated environmental salinity. Using immunocytochemical staining with an antiserum specific for α-subunit of Na+, K+-ATPase, chloride cells were detected on the afferent half of the filament epithelia. The size of immunoreactive chloride cells was twice larger in SW and four times larger in 180% SW than in FW. Confocal laser scanning microscopic observations revealed the frequent occurrence of chloride cell complexes under hypersaline conditions. By electron microscopy, a deeply invaginated apical crypt and well-developed tubular network were observed in chloride cells of SW- and 180% SW-adapted fish, indicating enhanced cellular activity. Chloride cells present in the opercular membrane were also developed in response to increased salinity. These findings suggest that highly activated chloride cells in branchial and opercular epithelia may be responsible for salt secretion in hyperosmotic environments. The excellent salinity tolerance of tilapia appears to be attributed to their ability to develop chloride cells in response to increased environmental salinity.

Developmental changes in drinking rate and ion and water permeability during early life stages of euryhaline tilapia, Oreochromis mossambicus, reared in fresh water and seawater

Euryhaline tilapia (Oreochromis mossambicus) can breed either in fresh water (FW) or in seawater, and the developing embryos and larvae withstand direct transfer from FW to SW or vice versa, before the development of osmoregulatory organs. In the study, developmental changes in drinking rate and ion and water permeability were examined after transfer of the embryos from FW to SW. Drinking was measured by accumulation of fluorescent beads in the intestine and also by 14C-dextran accumulation he whole body. The drinking rate increased steadily from 2 to 10 days after hatching, and the larvae in SW consistently imbibed more water than those in FW. The diffusional water permeability remained low during embryonic stages but increased markedly after hatching in both FW and SW; the water permeability was consistently less in SW-adapted embryos and larvae than those in FW. In contrast, the turnover rates of chloride ion in SW were 50 to 100 times greater than those in FW, and increased markedly after hatching. The drinking rate as well as ion and water permeability of the tilapia embryos and larvae in FW and SW were comparable with those reported for stenohaline species. These results clearly indicate that different water and ion regulatory mechanisms are operating in the tilapia embryos and larvae in FW and those in SW to convey their strong euryhalinity.

Preparation of polyacrylamide derivatives showing thermo-reversible coacervate formation and their potential application to two-phase separation processes

Abstract A synthetic polymer forming a thermo-reversible coacervate in aqueous milieu was prepared in this study by radical copolymerization of N,N-dimethylacrylamide (DMAA) with N-phenylacrylamide (PA). Increased content of PA in the copolymer (DPA) led to an abrupt decrease in its cloud point (lower critical solution temperature, LCST) owing to an increase in hydrophobicity. The heat of transition (ΔH) for a 1.0wt% aqueous solution of the copolymer at the LCST was determined to be in the range of 3–4 cal g−1 by differential scanning calorimetry, considerably smaller than that obtained for a neutral amphiphilic polymer, such as poly(N-isopropylacrylamide), undergoing a steep dehydration at the LCST. This small value of ΔH suggests the incomplete dehydration of the polymer chain at the LCST. Indeed, microscopic observation revealed the formation of coacervate droplets in the aqueous solution of the copolymer, which is clear evidence of liquid—liquid phase separation. The concentration of the copolymer in the coacervate phase formed from 1.0wt% aqueous solution of the copolymer at 37°C is approximately 20 wt%. It is of interest that the formation and disappearance of the coacervate are completely thermo-reversible. To estimate the feasibility of applying this thermo-sensitive coacervate system to thermo-modulated aqueous two-phase separation, partitioning of model solutes from aqueous milieu to the coacervate was carried out. By increasing the temperature, preferential partitioning of Trypan Blue (3,3′-[(3,3′-dimethyl[1,1′-biphenyl] 4,4′-diyl)bis(azo)]bis(5-amino-4-hydroxy-2,7-naphthalenedisulfonic acid) tetrasodium salt) from aqueous phase to coacervate phase was observed, whereas no partitioning of vitamin B12 to coacervate phase took place. Consequently, the separation of Trypan Blue from the mixed solution of vitamin B12 and Trypan Blue was achieved solely through the change in the environmental temperature. It may be feasible to apply such copolymers showing liquid—liquid phase separation that responds to temperature as the stationary phase in thermo-regulated liquid chromatography for the separation of water-soluble drugs and dyestuffs.

Localization of mouse CLC-6 and CLC-7 mRNA and their functional complementation of yeast CLC gene mutant

Abstract. CLC-6 and CLC-7 belong to the family of voltage-dependent chloride channels. To learn more about the in vivo roles of CLC-6 and CLC-7, we performed in situ hybridization of these CLC channels in various mouse organs. Mouse CLC-6 (mCLC-6) was expressed in the peripheral region of seminiferous tubules in the testis, tracheal epithelium, epithelium of bronchioles, alveolar cells in the lung, acinar cells in the pancreas, and intestinal epithelium, but we could not detect signals from pancreatic islets. Mouse CLC-7 (mCLC-7) was expressed in neurons in the medulla oblongata, Purkinje cells in the cerebellum, proximal tubules in the kidney, and hepatocytes in the liver. The distribution of mCLC-6 and mCLC-7 were similar in the lung, pancreas, and testis. mCLC-6 functionally complemented the gef1 phenotype of a yeast strain in which a single CLC channel (GEF1) had been disrupted by homologous recombination. In contrast, mCLC-7 did not complement this gef1 phenotype. This study identified the cell types that express mCLC-6 and mCLC-7 in the mouse tissues, and the complementation assay suggested that mCLC-6 functions as an intracellular chloride channel.

Molecular Design of Artificial Lectin; Recognition and Killer Cell Induction of Lymphocytes by a Novel Water Soluble Polymer Having Phenylboronic Acid Moiety

To design of synthetic polymer with mitogenic propertiy, a novel water-soluble polymer was synthesized by radical copolymerization of 3-acrylamidophenylboronic acid with dimethylacrylamide (poly(DMAA-co-PBA)). Since boronic acid moiety has an affinity for vicinal diol compounds, boronic acid was introduced as recognition site of sugar residues existing on the plasma membrane surface of lymphocyte. Interaction of phenylboronic acid with cell was confirmed through 11B-NMR. Then, proliferative response of mouse lymphocytes was evaluated by the incorporation of 3H-thymidine. Bornate-containing polymer with higher molecular weight induced higher proliferation of lymphocytes, and significant proliferation was achieved in the presence of polymers with Mw 300,000(DB30). Moreover, Killer cell activity was estimated by Cr51release assay. In the presence of boronte-containing polymer, Cytolysis of YAC-1 was increased similar to, typical killer cell inducer, PHA lectin. Further, significant accerelation in lymphocyte proliferation was found in the presence of both IL-2 and boronate-containing polymer.