The use of vitamin D analogs is independently associated with the favorable renal prognosis in chronic kidney disease stages 4-5: the CKD-ROUTE study.

BACKGROUND: Vitamin D analogs have generally been recommended for treatment of mineral bone disease in chronic kidney disease (CKD). However, the association between this treatment and CKD progression has not yet been established. METHODS: We designed a post hoc propensity score-matched cohort analysis derived from 3-year follow-up data of a prospective cohort. Adult participants with pre-dialysis CKD stages 4-5 who had newly been prescribed active vitamin D analogs during the observation period were eligible as matched cases. Then, matched controls were extracted from participants who had never been prescribed active vitamin D analogs. The primary outcome was a composite of end-stage renal disease or a 50 % reduction in estimated glomerular filtration rate (eGFR). A Cox proportional hazards model evaluated the association between the use of vitamin D analogs and the primary outcome. RESULTS: We enrolled 240 patients (males, 65 %). The number of matched cases and controls was 30 and 210, respectively. The primary outcome was observed in 94 patients, whereas 25 patients died. The mean ± standard deviation age and eGFR were 69 ± 12 years and 17 ± 5.7 ml/min/1.73 m2, respectively. In a Cox proportional hazard model, the use of vitamin D analogs was independently associated with a lower risk of the primary outcome (crude hazard ratio 0.41; 95 % confidence interval 0.19, 0.89; adjusted hazard ratio 0.38; 95 % confidence interval 0.17, 0.88). CONCLUSION: The use of vitamin D analogs is independently associated with the preservation of renal function in patients with pre-dialysis CKD stages 4-5.

Anaemia management and mortality risk in newly visiting patients with chronic kidney disease in Japan: The CKD-ROUTE study.

AIM: To investigate the association between iron deficiency anaemia and mortality risk and assess the changes in anaemia and iron status after primary management by a nephrologist. METHODS: In this prospective cohort study, we stratified 951 non-dialysis chronic kidney disease (CKD) G2-G5 patients newly visiting 16 nephrology centres into four groups according to the presence of anaemia with or without iron deficiency. All-cause mortality, cardiovascular (CV)-related mortality, and a change in anaemia and iron status after specialized primary care were the endpoints evaluated. RESULTS: During a median follow-up time of 19 months, the number of all-cause deaths and CV-related deaths were 56 and 26, respectively. Compared with the control group, the groups with isolated anaemia and iron deficiency anaemia had significantly higher all-cause mortalities (isolated anaemia: hazard ratio (HR), 3.37; 95% confidence intervals (CI), 1.76-6.44; iron deficiency anaemia: HR, 3.11; 95% CI, 1.21-8.01) and CV-related mortalities (isolated anaemia: HR, 3.64; 95% CI, 1.36-9.73; iron deficiency anaemia: HR, 3.86; 95% CI, 1.11-13.41). In the isolated anaemia group, erythropoietin-stimulating agent (ESA) prescriptions significantly increased to approximately 70%. However, in patients with both anaemia and iron deficiency, iron prescriptions only increased to 48.1%. CONCLUSIONS: Iron deficiency anaemia and isolated anaemia were associated with all-cause and CV-related mortality. The absence of relative increase in iron prescriptions suggests that iron deficiency should be accurately assessed and iron supplementation should be appropriately used to manage anaemia in non-dialysis patients with CKD.

Actin directly interacts with different membrane channel proteins and influences channel activities: AQP2 as a model.

The interplay between actin and 10 membrane channel proteins that have been shown to directly bind to actin are reviewed. The 10 membrane channel proteins covered in this review are aquaporin 2 (AQP2), cystic fibrosis transmembrane conductance regulator (CFTR), ClC2, short form of ClC3 (sClC3), chloride intracellular channel 1 (CLIC1), chloride intracellular channel 5 (CLIC5), epithelial sodium channel (ENaC), large-conductance calcium-activated potassium channel (Maxi-K), transient receptor potential vanilloid 4 (TRPV4), and voltage-dependent anion channel (VDAC), with particular attention to AQP2. In regard to AQP2, most reciprocal interactions between actin and AQP2 occur during intracellular trafficking, which are largely mediated through indirect binding. Actin and the actin cytoskeleton work as cables, barriers, stabilizers, and force generators for motility. However, as with ENaC, the effects of actin cytoskeleton on channel gating should be investigated further. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

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.

FAPP2 is required for aquaporin-2 apical sorting at trans-Golgi network in polarized MDCK cells.

FAPP2 is an adaptor protein of phosphatidylinositol-4-phosphate and is involved in the transport of some apical cargos from the trans-Golgi network (TGN). To investigate whether the regulated apical transport of aquaporin-2 (AQP2) is involved in the FAPP2-dependent apical protein-sorting machinery, we measured apical sorting of AQP2 in Madin-Darby canine kidney (MDCK) cells with or without FAPP2 knockdown. We established MDCK cell lines that stably express rat AQP2 without any tag sequence. Then, FAPP2-deficient stable cell lines were established from the AQP2-expressing cell lines by a retrovirus-mediated RNA interference system. In the established cell lines, AQP2 was detected in both apical and basolateral membranes. Forskolin increased only the apical localization of AQP2, which was not affected by basolateral treatment with 0.5% tannic acid, indicating that the forskolin-induced apical transport of AQP2 did not include the transcytotic pathway from basolateral to apical membranes but is a direct transport from TGN to the apical membranes. Using these cell lines, we tested the effect of FAPP2 knockdown on the polarized AQP2 transport to plasma membranes and found that the forskolin-induced apical transport of AQP2 was completely abolished by FAPP2 knockdown. By contrast, the basolateral localization of AQP2 was not affected by FAPP2 knockdown. AQP2 phosphorylation by forskolin was also impaired in FAPP2 knockdown MDCK cells. These results suggest that FAPP2 is necessary to generate AQP2-bearing vesicles at trans-Golgi that will undergo phosphorylation by PKA in subapical regions.

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.

Effect of non-essential amino acid glycine administration on the liver regeneration of partially hepatectomized rats with hepatic ischemia/reperfusion injury.

BACKGROUND & AIMS: Liver regeneration after partial hepatectomy or transplantation is a critical problem to affect prognosis. Ischemia/reperfusion (I/R) is an unavoidable process during liver resection or transplantation. The aim of this study was to investigate the effect of glycine on the regeneration of the remnant liver with I/R injury after partial hepatectomy. METHODS: Partially hepatectomized rat with liver I/R injury was prepared by a two-thirds partial hepatectomy following 30 min of total hepatic ischemia. Glycine (5% in water) was orally administered to rats for 3 days as drinking water before the surgery. RESULTS: Mortality rate in partially hepatectomized rats with severe hepatic I/R injury was so high compared to that in the rats with partial hepatectomy alone. However, when glycine was given to the partially hepatectomized rats with hepatic I/R injury, the survival rate, the recovery rate of the remnant liver weight, and the liver injury were obviously improved. On the other hand, when glycine-treated rats underwent partial hepatectomy without hepatic I/R, the recovery rate of the remnant liver weight was decreased as compared with that of the rats with partial hepatectomy alone. In these settings, glycine administration prevented the elevation of serum TNF-alpha levels and liver TNF-alpha mRNA expression. CONCLUSIONS: Glycine improved the regeneration of the remnant liver with severe I/R injury after partial hepatectomy. This improvement may be at least partly due to the amelioration of the hepatic I/R injury by glycine. Glycine seems to be clinically beneficial to the prognosis of patients with liver resection.

Amelioration of hepatic ischemia/reperfusion injury in the remnant liver after partial hepatectomy in rats.

BACKGROUND AND AIMS: Reactive oxygen species have been implicated in the development of hepatic ischemia/reperfusion (I/R) injury. I/R injury remains an important problem in massive hepatectomy and organ transplantation. The aim of this study was to examine the effect of edaravone, a newly synthesized free radical scavenger, on I/R injury in the remnant liver after partial hepatectomy in rats. METHODS: Partial (70%) hepatic ischemia was induced in rats by occluding the hepatic artery, portal vein, and bile duct to left and median lobes of liver. Total hepatic ischemia (Pringle maneuver) was induced by occluding the hepatoduodenal ligament. Edaravone was intravenously administered to rats just before reperfusion and partial (70%) hepatectomy was performed just after reperfusion. RESULTS: Edaravone significantly reduced the increases in the levels of serum alanine aminotransferase and aspartate aminotransferase in rats with liver injury induced by 90-min of partial ischemia followed by 120-min of reperfusion. Histopathological analysis showed that edaravone prevented inflammatory changes in the livers with I/R injury. Edaravone also decreased the levels of myeloperoxidase activity, which is an index of neutrophil infiltration, and interleukin-6 mRNA, which is a proinflammatory cytokine. Additionally, edaravone improved the survival rate in partial hepatectomy rats with I/R injury induced by the Pringle maneuver. CONCLUSIONS: Edaravone administration prior to reperfusion protected the liver against I/R injury. Edaravone also improved the function of the remnant liver with I/R injury after partial hepatectomy. Therefore, edaravone may have applicability for major hepatectomy and liver transplantation in the clinical setting.

Aquaporin-2 protein dynamics within the cell.

PURPOSE OF REVIEW: Aquaporin-2 is an aquaporin water channel protein present at the apical membrane of kidney collecting duct cells and plays a key role in urine concentrating ability. Like other membrane proteins, aquaporin-2 undergoes dynamic processes within the cells: synthesized, targeted to the subapical region, exocytosed to the apical membrane, endocytosed, recycled and finally degraded. The understanding of the molecular and cellular mechanisms of these events is advancing rapidly, and recent new findings characterizing such processes are reviewed. RECENT FINDINGS: Hypertonicity itself stimulates aquaporin-2 expression through the tonicity-responsive enhancer and its transcription factors. Gene targeted mouse models for human nephrogenic diabetes insipidus show the importance of the C-terminus of aquaporin-2 in apical sorting and provide mechanistic insights. Evidence for the importance of actin cytoskelton in exocytosis of aquaporin-2 to the apical membrane is accumulating. Actin and other proteins bind to aquaporin-2 and make a multiprotein complex. New proteomic analyses indicate the involvement of a large series of proteins in aquaporin-2 dynamics. SUMMARY: The protein-level understanding of aquaporin-2 dynamics has advanced considerably over the past few years, and continuing studies will open a new way in developing new manoeuvres or drugs to manipulate kidney water homeostasis.

Regulation of aquaporin-2 trafficking and its binding protein complex.

Trafficking of water channel aquaporin-2 (AQP2) to the apical membrane is critical to water reabsorption in renal collecting ducts and its regulation maintains body water homeostasis. However, exact molecular mechanisms which recruit AQP2 are unknown. Recent studies highlighted a key role for spatial and temporal regulation of actin dynamics in AQP2 trafficking. We have recently identified AQP2-binding proteins which directly regulate this trafficking: SPA-1, a GTPase-activating protein (GAP) for Rap1, and cytoskeletal protein actin. In addition, a multiprotein "force generator" complex which directly binds to AQP2 has been discovered. This review summarizes recent advances related to the mechanism for AQP2 trafficking.

Splenic artery ligation ameliorates hepatic ischemia and reperfusion injury in rats.

BACKGROUND/AIMS: Hepatic injury caused by ischemia/reperfusion (I/R) is a key clinical problem associated with liver transplantation and liver surgery. The spleen is involved in hepatic I/R injury. In this study, we examined the effects of splenic artery ligation on hepatic I/R injury. METHODS: Splenic artery ligation was performed 7 days, 3 days, or just before the hepatic ischemia. Hepatic ischemia was conducted by occluding the blood vessels to the median and left lateral lobes with an atraumatic vascular clamp. Hepatic I/R injury was induced by 45 min of ischemia followed by 120 min of reperfusion. RESULTS: When splenic artery ligation was performed at 3 days or just before the ischemia, serum aspartate transaminase and alanine transaminase activities, as markers for hepatic injury, decreased as compared with the rats with I/R alone. Splenic artery ligation also reduced the myeloperoxidase activity, an enzyme present in neutrophils, and the expression of interleukin-6 mRNA, a proinflammatory cytokine, in rat livers with I/R. Efficacy of splenic artery ligation on hepatic I/R injury was also confirmed by histology. On the other hand, when splenic artery ligation was conducted 7 days before the ischemia, efficacy of splenic artery ligation was disappeared. CONCLUSIONS: Splenic artery ligation ameliorates hepatic I/R injury in rats. These results strongly suggest the clinical usefulness of this surgical procedure to protect the liver against I/R injury.

Trafficking mechanism of water channel aquaporin-2.

Targeted positioning of the water channel AQP2 (aquaporin-2) strictly regulates body water homoeostasis. Trafficking of AQP2 to the apical membrane is critical for the reabsorption of water in renal collecting ducts. In addition to the cAMP-mediated effect of vasopressin on AQP2 trafficking to the apical membrane, other signalling cascades can also induce this sorting. Recently, AQP2-binding proteins which could regulate this trafficking have been discovered; SPA-1 (signal-induced proliferation-associated gene-1), a GAP (GTPase-activating protein) for Rap1, and the cytoskeletal protein actin. This review summarizes recent advances related to the trafficking mechanisms of AQP2.

Actions of orexins on individual myenteric neurons of the guinea-pig ileum: orexin A or B?

The actions of orexins (orexin A and B, 10-300 nM) on individual myenteric neurons of the guinea-pig ileum in vitro were compared using intracellular recording methods. Both orexins caused membrane depolarizations associated with an increase in input neuronal resistance in S and AH neurons via a direct action. Orexin depolarizations reversed at about -90 mV, indicating they were due to an inactivation of K+ channels. Orexins facilitated fast excitatory postsynaptic potentials without affecting postsynaptic sensitivity to acetylcholine and adenosine 5'-triphosphate, indicating that the peptides may facilitate ganglionic transmission by increasing presynaptic release of neurotransmitters. Orexin B was sometimes more effective than orexin A and vice versa. It is concluded that orexin B increased neuronal activity via mechanisms similar to orexin A in the guinea-pig myenteric plexus.