Saburo Horikawa

Aquaporin‐2 trafficking is regulated by PDZ‐domain containing protein SPA‐1

Targeted positioning of water channel aquaporin‐2 (AQP2) strictly regulates body water homeostasis. Trafficking of AQP2 to the apical membrane is critical to the reabsorption of water in renal collecting ducts. Controlled apical positioning of AQP2 suggests the existence of proteins that interact with AQP2. A biochemical search for AQP2‐interacting proteins led to the identification of PDZ‐domain containing protein, signal‐induced proliferation‐associated gene‐1 (SPA‐1) which is a GTPase‐activating protein (GAP) for Rap1. The distribution of SPA‐1 coincided with that of AQP2 in renal collecting ducts. The site of colocalization was concomitantly relocated by hydration status. AQP2 trafficking to the apical membrane was inhibited by the SPA‐1 mutant lacking Rap1GAP activity and by the constitutively active mutant of Rap1. AQP2 trafficking was impaired in SPA‐1‐deficient mice. Our results show that SPA‐1 directly binds to AQP2 and regulates at least in part AQP2 trafficking.

Molecular cloning and developmental expression of a human kidney S-adenosylmethionine synthetase.

cDNA clones encoding the human kidney S‐adenosylmethionine synthetase (kidney‐type isozyme) were isolated. The amino acid sequence deduced from the cDNA indicates that this enzyme contains 395 amino acids and has a molecular mass of 43,660 Da. The predicted amino acid sequence of this protein shares 84% similarity with that of human liver S‐adenosylmethionine synthetase (liver‐type isozyme). In addition, the developmental expression of these two isozyme mRNAs has been studied in the human liver using the reverse transcription—polymerase chain reaction (RT‐PCR).

Reciprocal interaction with G-actin and tropomyosin is essential for aquaporin-2 trafficking

Trafficking of water channel aquaporin-2 (AQP2) to the apical membrane and its vasopressin and protein kinase A (PKA)–dependent regulation in renal collecting ducts is critical for body water homeostasis. We previously identified an AQP2 binding protein complex including actin and tropomyosin-5b (TM5b). We show that dynamic interactions between AQP2 and the actin cytoskeleton are critical for initiating AQP2 apical targeting. Specific binding of AQP2 to G-actin in reconstituted liposomes is negatively regulated by PKA phosphorylation. Dual color fluorescence cross-correlation spectroscopy reveals local AQP2 interaction with G-actin in live epithelial cells at single-molecule resolution. Cyclic adenosine monophosphate signaling and AQP2 phosphorylation release AQP2 from G-actin. In turn, AQP2 phosphorylation increases its affinity to TM5b, resulting in reduction of TM5b bound to F-actin, subsequently inducing F-actin destabilization. RNA interference–mediated knockdown and overexpression of TM5b confirm its inhibitory role in apical trafficking of AQP2. These findings indicate a novel mechanism of channel protein trafficking, in which the channel protein itself critically regulates local actin reorganization to initiate its movement.

Pharmacological preconditioning protects lung injury induced by intestinal ischemia/reperfusion in rat

Intestinal ischemia/reperfusion (I/R) is a critical and triggering event in the development of distal organ dysfunction, frequently involving the lungs. Respiratory failure is a common cause of death and complications after intestinal I/R. Stress protein heme oxygenase-1 (HO-1) confers the protection against a variety of oxidant-induced cell and tissue injuries. The aim of this study was to investigate the hypothesis that the induced HO-1 expression by pharmacological preconditioning with anticancer drug doxorubicin (Dox) could protect the lung injury induced by intestinal I/R. Intravenous administration of Dox induced HO-1 expression in the lungs and high levels of the expression were sustained at least to 48 h after the injection. Therefore, as pharmacological preconditioning, a low dose of Dox was injected intravenously into rats at 48 h before the start of intestinal ischemia. Rats underwent intestinal I/R by superior mesenteric artery occlusion for 120 min followed by 120 min of reperfusion. Preconditioning with Dox significantly ameliorated the lung injury induced by the intestinal I/R. Administration of a specific inhibitor of HO activity reduced the efficacy of the preconditioning. Our results suggest that this improvement may be mediated at least in part by the HO-1 induction. These findings may offer interesting perspectives for patient management in intestinal surgical operation and intestine transplantation.

Phosphorylation of Aquaporin-2 Regulates Its Water Permeability

Vasopressin-regulated water reabsorption through the water channel aquaporin-2 (AQP2) in renal collecting ducts maintains body water homeostasis. Vasopressin activates PKA, which phosphorylates AQP2, and this phosphorylation event is required to increase the water permeability and water reabsorption of the collecting duct cells. It has been established that the phosphorylation of AQP2 induces its apical membrane insertion, rendering the cell water-permeable. However, whether this phosphorylation regulates the water permeability of this channel still remains unclear. To clarify the role of AQP2 phosphorylation in water permeability, we expressed recombinant human AQP2 in Escherichia coli, purified it, and reconstituted it into proteoliposomes. AQP2 proteins not reconstituted into liposomes were removed by fractionating on density step gradients. AQP2-reconstituted liposomes were then extruded through polycarbonate filters to obtain unilamellar vesicles. PKA phosphorylation significantly increased the osmotic water permeability of AQP2-reconstituted liposomes. We then examined the roles of AQP2 phosphorylation at Ser-256 and Ser-261 in the regulation of water permeability using phosphorylation mutants reconstituted into proteoliposomes. The water permeability of the non-phosphorylation-mimicking mutant S256A-AQP2 and non-phosphorylated WT-AQP2 was similar, and that of the phosphorylation-mimicking mutant S256D-AQP2 and phosphorylated WT-AQP2 was similar. The water permeability of S261A-AQP2 and S261D-AQP2 was similar to that of non-phosphorylated WT-AQP2. This study shows that PKA phosphorylation of AQP2 at Ser-256 enhances its water permeability.

Immunohistochemical analysis of rat S‐adenosylmethionine synthetase isozymes in developmental liver

Mammalian S‐adenosylmethionine (AdoMet) synthetase exists as two isozymes, liver‐type and kidney(non‐hepatic)‐type enzymes. The developmental expression of these two isozyme proteins has been investigated in rat liver using immunohistochemical techniques. The liver‐type AdoMet synthetase is expressed only in adult liver, but not in fetal liver. On the other hand, the kidney‐type AdoMet synthetase is predominantly expressed in fetal liver and faintly detected in adult liver. It was also found that both isozymes were localized to the hepatocytes of rat liver. These results clearly show that AdoMet synthetase isozymes are developmentally regulated within hepatocytes. In addition, in rat kidney we have shown that the kidney‐type AdoMet synthetase is predominantly localized to the distal tubule.

Prior induction of heme oxygenase-1 with glutathione depletor ameliorates the renal ischemia and reperfusion injury in the rat.

Heme oxygenase (HO)‐1 catalyzes the rate‐limiting step in heme degradation releasing iron, carbon monoxide, and biliverdin. Induction of HO‐1 occurs as an adaptive and protective response to oxidative stress. Ischemia and reperfusion (IR) injury seems to be mainly caused by the oxidative stress. In this study, we have examined whether prior induction of HO‐1 with buthionine sulfoximine (BSO), a glutathione (GSH) depletor, affects the subsequent renal IR injury. BSO (2 mmol/kg body weight) was administered intraperitoneally into rats, the levels of HO‐1 protein increased within 4 h after the injection. When BSO was administered into rats at 5 h prior to the renal 45 min of ischemia, the renal IR injury was assessed by determining the levels of blood urea nitrogen and serum creatinine, markers for renal injury, after 24 h of reperfusion. The renal injury was significantly improved as compared to the rats treated with IR alone. Administration of zinc‐protoporphyrin IX, an inhibitor of HO activity, reduced the efficacy of BSO pretreatment on the renal IR injury. Our findings suggest that the prior induction of HO‐1 ameliorates the subsequent renal IR injury.

Hemin pretreatment ameliorates aspects of the nephropathy induced by mercuric chloride in the rat

Mercuric chloride (HgCl(2)) is known to be a nephrotoxicant. When HgCl(2) is administered into rats, acute renal failure (ARF) is induced. Heme oxygenase-1 (HO-1) is antioxidative enzyme and is known to play a protective role against the oxidative injury. To elucidate the cytoprotective role of HO-1 against the nephrotoxicant-induced ARF, we examined the effect of hemin, HO-1 inducer, on HgCl(2)-induced ARF. Subcutaneous administration of hemin (30 mg/kg body weight) into rats once a day for two successive days obviously induced HO-1 protein in the kidneys at 24 h after the last injection. Under this situation, when HgCl(2) (1 mg/kg body weight) was intraperitoneally injected into rats at 24 h after the last injection of hemin improved the serum creatinine (SCr) and blood urea nitrogen (BUN) levels, markers for renal injury, at 24 h after the HgCl(2) injection as compared with the control rats without hemin pretreatment (HgCl(2) treatment alone). This result was further confirmed by histopathological analysis. These findings strongly suggest that the preinduction of HO-1 ameliorates the subsequent HgCl(2)-induced acute renal injury.

Expression of c-myc oncogene in rat liver by a dietary manipulation

After rats deprived of protein for several days are fed a meal containing protein, hepatic DNA replication is induced. When nuclear DNA synthesis is stimulated in the normally quiescent rat liver by a dietary manipulation, we examined the changes of the steady-state levels of messenger RNA for c-myc. Levels of c-myc mRNA are gradually elevated approximately 4 to 5-fold above normal in the livers of rats that are fed for several days a diet that lacks protein. After a nutritional shift from a protein-free diet to a diet containing 50% casein, the levels of c-myc mRNA decrease rapidly by 2 h and returned to approximately basal levels after 8 h. Our results suggest that c-myc expression during the prereplicative stage of liver is likely to reflect events associated with entry and progression of hepatocytes into the cell cycle.

Molecular cloning and characterization of a novel truncated form (ClC‐2β) of ClC‐2α (ClC‐2G) in rabbit heart

Two cDNAs encoding rabbit heart Cl− channels (rabClC‐2β and rabClC‐2α) were isolated by a PCR cloning strategy. RabClC‐2β is a novel cDNA consisting of 2998 bp and encoding the 822‐amino acid protein, while rabClC‐2α is identical to previously reported ClC‐2G. RabClC‐2β is 68 amino acids truncated from NH2‐terminus of rabClC‐2α, but all 13 putative hydrophobic domains are conserved in rabClC‐2β. Although rabClC‐2α was suggested to be activated by extracellular hypotonicity, expression of rabClC‐2β in Xenopus oocytes induced large Cl− currents even in the absence of extracellular hypotonicity. Induction of external hypotonicity did not further increase the amplitude of membrane currents. On the other hand, as similar to rabClC‐2α, rabClC‐2β current was augmented by PKA activation. Thus, different RNA processing of the same gene appears to provide two highly homologous PKA‐activated Cl− channels with or without responsiveness to cell swelling in rabbit heart.