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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.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.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%.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.

A 33-year-old Japanese woman at 40 weeks gestation visited the maternity hospital after imminent labor had begun. After the delivery, persistent bleeding developed resulting in hemorrhagic shock. Although the hemorrhage was eventually controlled, hepatic and renal dysfunction occurred, leading to acute kidney injury (AKI). The patient's clinical presentation was suggestive of amniotic fluid embolism (AFE). We subsequently initiated continuous renal replacement therapy (RRT) for AKI. The patient's condition improved, she discontinued RRT, and her renal function recovered. We herein report a patient who successfully recovered from AKI caused by AFE.

Pseudohypoaldosteronism type II (PHAII), an autosomal dominant disorder characterized by hypertension, hyperkalemia, and hyperchloremic acidosis, is reportedly due to mutations in WNK1 and WNK4 kinase genes. However, the pathogenesis of the disease remains unknown. Mutations in the WNK1 gene are the deletions in the first intron, which reportedly increases WNK1 mRNA expression. Thus, we generated WNK1 over-expressing stable cell lines using MDCKII cells to model the distal nephron of PHAII patients. Using these cell lines, we investigated whether increased WNK1 expression might affect paracellular chloride permeability and claudin phosphorylation, since we previously observed an increase in both with a disease-causing mutant WNK4. WNK1 expression in MDCKII cells increased chloride permeability two to threefold. Co-expression of wild-type WNK4 did not further increase WNK1-enhanced chloride permeability. WNK1 expression also induced phosphorylation of endogenous claudin-4 in MDCKII cells, as well as over-expressed claudin-4. Combined, these results suggest that increased WNK1 expression has the same effect on chloride permeability and claudin phosphorylation as the mutant WNK4. Thus, increased chloride shunt may be involved in the pathogenesis of PHAII caused by WNK1 mutations.

Frame-shift mutations within the C terminus of aquaporin 2 (AQP2) cause autosomal-dominant nephrogenic diabetes insipidus (AD-NDI). To identify the molecular mechanism(s) of this disease in vivo and to test possible therapeutic strategies, we generated a mutant AQP2 (763–772 del) knockin mouse. Heterozygous knockin mice showed a severely impaired urine-concentrating ability. However, they were able to slightly increase urine osmolality after dehydration. This milder phenotype, when compared with autosomal-recessive NDI, is a feature of AD-NDI in humans, thus suggesting successful establishment of an AD-NDI mouse model. Immunofluorescence of collecting duct cells in the AD-NDI mouse revealed that the mutant AQP2 was missorted to the basolateral instead of apical plasma membrane. Furthermore, the mutant AQP2 formed a heterooligomer with wild-type AQP2 and showed a dominant-negative effect on the normal apical sorting of wild-type AQP2 even under dehydration. Using this knockin mouse, we tested several drugs for treatment of AD-NDI and found that rolipram, a phosphodiesterase 4 inhibitor, was able to increase urine osmolality. Phosphodiesterase inhibitors may thus be useful drugs for the treatment of AD-NDI. This animal model demonstrates that a mutant monomer gains a dominant-negative effect that reverses the normal polarized sorting of multimers.

Background/Aim: Intradialytic hypotension is the most common complication associated with hemodialysis, and its cause is multifactorial. However, the relationship between hypertension and intradialytic hypotension is not clear. We investigated the influence of predialysis blood pressure and antihypertensive drugs on intradialytic hypotension. Methods: Risk factors for intradialytic hypotension were analyzed in 111 patients undergoing regular hemodialysis treatment and had annual echocardiography performed. The correlation between the addition of antihypertensive medications and the incidence of intradialytic hypotensive episodes was studied in 21 hypertensive patients. Results: Based on multivariate logistic regression analysis, diabetes (odds ratio OR 8.18, 95% confidence interval CI 1.47–45.5; p = 0.016), interdialytic weight gain (OR 2.45, 95% CI 1.24–4.82; p = 0.010), ejection fraction (OR 0.88, 95% CI 0.81–0.95; p = 0.001), and left ventricular volume (OR 0.97, 95% CI 0.94–0.99; p = 0.013) were determined as risk factors for intradialytic hypotension. However, there was no association between predialysis blood pressure and intradialytic hypotension. After additional antihypertensive medications, the predialysis blood pressure fell from 175/85 mm Hg to 154/78 mm Hg (p < 0.0001/p < 0.0001). The mean 24-hour interdialytic blood pressure fell from 165/87 mm Hg to 147/80 mm Hg (p < 0.0001/p = 0.006). However, the frequency of hypotensive episodes was not increased (p = 0.77). Conclusions: Diabetes, excessive interdialytic weight gain, low ejection fraction, and low left ventricular volume are independent risk factors for intradialytic hypotension. However, no correlation was found between predialysis blood pressure values or addition of antihypertensive medications and the incidence of intradialytic hypotension. Thus, hypertension may be controlled without aggravating intradialytic hypotension in hemodialysis patients.

Epithelial renal collecting duct cells express multiple types of aquaporin (AQP) water channels in a polarized fashion. AQP2 is specifically targeted to the apical cell domain, whereas AQP3 and AQP4 are expressed on the basolateral membrane. It is crucial that these AQP variants are sorted to their proper polarized membrane domains, because correct AQP sorting enables efficient water transport. However, the molecular mechanisms involved in the polarized targeting and membrane trafficking of AQPs remain largely unknown. In the present study, we have examined the polarized trafficking and surface expression of AQP3 in Madin-Darby canine kidney type II (MDCKII) cells in an effort to identify the molecular determinants of polarized targeting specificity. When expressed in MDCKII cells, the majority of the exogenous wild-type AQP3 was found to be targeted to the basolateral membrane, consistent with its localization pattern in vivo. A potential sorting signal consisting of tyrosine- and dileucine-based motifs was subsequently identified in the AQP3 NH2 terminus. When mutations were introduced into this signaling region, the basolateral targeting of the resulting mutant AQP3 was disrupted and the mutant protein remained in the cytoplasm. AQP2-AQP3 chimeras were then generated in which the entire NH2 terminus of AQP2 was replaced with the AQP3 NH2 terminus. This chimeric protein was observed to be mislocalized constitutively in the basolateral membrane, and mutations in the AQP3 NH2-terminal sorting signal abolished this effect. On the basis of these results, we conclude that an NH2-terminal sorting signal mediates the basolateral targeting of AQP3.

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.

The C-terminal tail of aquaporin-2 determines apical trafficking . Background Aquaporin-2 (AQP-2) proteins are mainly expressed at the apical region of the collecting duct cells. We previously reported three different mutations in the C-terminus of AQP-2 that all-cause autosomal-dominant nephrogenic diabetes insipidus. When one of these mutant AQP-2s was expressed in Madin-Darby canine kidney (MDCK) cells, it was mistargeted to the basolateral membrane, suggesting a critical role of the C-terminal tail in the apical trafficking of AQP-2. METHODS Portions of the AQP-2 C-terminal tail (residues 226-271) were mutated by the polymerase chain reaction (PCR) technique and inserted into the pcDNA3.1 vector. Constructs were transfected into MDCK cells to examine the localization of mutated AQP-2 proteins by immunofluorescence microscopy. Cell surface expression was detected by biotinylation assay. RESULTS The wild-type AQP-2 was localized at the apical membrane, whereas mutants lacking residues 262-271 (the last 10 amino acids) were predominantly distributed in the endoplasmic reticulum. Deletion mutants of the initial (226-240del) and middle (241-252del) portions of the C-terminal tail were identified at the apical membrane, suggesting that residues 226-252 have no involvement in apical targeting. An AQP-4-AQP-2 chimera in which a portion of the AQP-4 C-terminal tail was replaced by the corresponding site in AQP-2 (residues 256-271) was found at the apical membrane. The sequence of the last 4 amino acids of AQP-2 (G-T-K-A) corresponds to a PDZ-interacting motif. Our investigations identified a mutant of this portion mostly localized to the subapical region. Further, apical expression was found to be significantly decreased in mutants lacking a consensus sequence for cyclic adenosine monophosphate (cAMP)-dependent phosphorylation (residues 253-256). Conclusion The sequence at 256-271 is sufficient for apical trafficking in AQP-2. The putative PDZ-interacting motif (G-T-K-A, residues 268-271) plays a key role in apical membrane expression. In addition, cAMP-dependent phosphorylation was found to be critical for apical targeting.

Background: Cell cycle progression and arrest of renal tubular cells after acute injury is a reactive process of renal regeneration. The p16INK4a/p19ARF (alternative reading frame) locus encodes two proteins involved in cell cycle regulation. We investigated the transcriptional regulation and tissue distribution of p19ARF in ischemic acute renal failure (ARF). Methods: We examined the time course and immunohistochemistry of p19ARF in rat kidneys following the induction of ischemic ARF. We also examined the effect of p19ARF overexpression on p53 levels and cell cycle progression in MDCK cells. Results: The protein expression of p19ARF strongly increased 72 h after the ischemia. Immunohistochemical studies showed that the renal tubular cells in the outer medulla expressed p19ARF protein 72 h after ischemic injury. The time course of E2F1 induction was observed at 6–24 h, and it was found to precede p19ARF expression. In MDCK cells, the overexpression of E2F1 increased promoter activity and the protein level of p19ARF and induced apoptosis. Transfection of the p19ARF expression vector caused an increase in p53 protein, cell cycle arrest and apoptosis. Conclusions: These data support the hypothesis that the E2F1-p19ARF-p53 pathway forms a negative feedback loop to regulate the cell cycle of renal tubular cells in the ischemic ARF.

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.

Urea transporter UT-A2, the major urea transporter of the thin descending limb of the loop of Henle in short loop nephrons, has been implicated in urea recycling in the medulla, thereby producing concentrated urine. To investigate the physiological role of UT-A2 in vivo, we generated UT-A2-selective knockout mice by deleting the UT-A2 promoter. Western analysis, immunohistochemistry, and quantitative reverse transcription-PCR were used to confirm the specific deletion of UT-A2 with preservation of other UT-A transporters. Compared to wild-type mice, differences in the urine outputs of UT-A2(-/-) mice consuming a normal protein diet (20% protein) were not observed under normal conditions or with dehydration. Likewise, impairment of urea accumulation in the inner medulla of UT-A2(-/-) mice was not observed. On a low-protein diet (4% protein), however, significantly reduced maximal urine osmolality was observed in dehydrated UT-A2(-/-) mice compared to wild-type littermates (2,500 mosmol versus 3,450 mosmol, respectively). A significant reduction in urea accumulation in the inner medulla was also observed in UT-A2(-/-) mice; however, differences in Na(+) and Cl(-) accumulation were not observed. Thus, UT-A2 is important for maintaining a high concentration of urea in the inner medulla when urea supply to the kidney is limited.

Recently, attention has been focused on the role of aldosterone in the pathophysiology of hypertension and cardiovascular disease. Several clinical and experimental data support the hypothesis that aldosterone contributes to the progression of renal injury. However, the molecular mechanisms of the effects of aldosterone in signal transduction and the cell-cycle progression of mesangial cells are not well known. For determining the signaling pathway of aldosterone in cultured mesangial cells, the effects of aldosterone on the mitogen-activated protein kinase 1/2 (MAPK1/2) pathway and the promoter activities of cyclin D1, cyclin A, and cyclin E were investigated. First, it was shown that the mineralocorticoid receptor (MR) was expressed in rat mesangial cells and glomeruli and that aldosterone stimulated the proliferation of mesangial cells via the MR and MAPK1/2 pathway. Next, it was demonstrated that aldosterone stimulated Ki-RasA, c-Raf kinase, MEK1/2, and MAPK1/2 in rat mesangial cells. Aldosterone induced cyclin D1 and cyclin A promoter activities and protein expressions, as well as the increments of CDK2 and CDK4 kinase activities. The presence of CYP11B2 and 11beta-HSD2 mRNA in rat mesangial cells also was shown. In conclusion, aldosterone seems to exert mainly MR-induced effects that stimulate c-Raf, MEK1/2, MAPK1/2, the activities of CDK2 and CDK4, and the cell-cycle progression in mesangial cells. MR antagonists may serve as a potential therapeutic approach to mesangial proliferative disease.

Vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct cells is critical to regulate the urine concentration. To better understand the mechanism of subcellular trafficking of AQP2, we examined MDCK cells expressing AQP2 as a model. We first performed double-immunolabeling of AQP2 with endosomal marker proteins, and showed that AQP2 is stored at a Rab11-positive subapical compartment. After the translocation to the plasma membrane, AQP2 was endocytosed to EEA1-positive early endosomes, and then transferred back to the original Rab11-positive compartment. When Rab11 was depleted by RNA interference, retention of AQP2 at the subapical storage compartment was impaired. We next examined the role of cytoskeleton in the AQP2 trafficking and localization. By the treatment with microtubule-disrupting agent such as nocodazole or colcemid, the distribution of AQP2 storage compartment was altered. The disruption of actin filaments with cytochalasin D or latrunculin B induced the accumulation of AQP2 in EEA1-positive early endosomes. Altogether, our data suggest that Rab11 and microtubules maintain the proper distribution of the subapical AQP2 storage compartment, and actin filaments regulate the trafficking of AQP2 from early endosomes to the storage compartment.

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. Recently, we have identified for the first time proteins which directly bind to AQP2: SPA-1, a GTPase-activating protein for Rap1, and cytoskeletal protein actin. Based on these findings, we have speculated the existence of a multiprotein complex which includes AQP2, SPA-1, and actin, for providing the mechanism which generates force and motion in AQP2 trafficking. To clarify the proteins comprising the complex, a large amount of AQP2-associated protein complex was isolated from the extract of rat kidney papilla using immunoaffinity column coupled with anti-AQP2 antibody and was analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition to SPA-1 and actin, 11 proteins were identified using this method: ionized calcium binding adapter molecule 2, myosin regulatory light chain smooth muscle isoforms 2-A and 2-B, alpha-tropomyosin 5b, annexin A2 and A6, scinderin, gelsolin, alpha-actinin 4, alpha-II spectrin, and myosin heavy chain nonmuscle type A. Our findings show for the first time an AQP2-binding multiprotein "force generator" complex. This multiprotein complex may provide the machinery of driving AQP2 movement.

Aquaporin-7 (AQP7) is a water/glycerol transporting protein expressed in adipocyte plasma membranes. We report here remarkable age-dependent hypertrophy in adipocytes in AQP7-deficient mice. Wild type and AQP7 null mice had similar growth at 0–16 weeks as assessed by body weight; however, by 16 weeks AQP7 null mice had 3.7-fold increased body fat mass. Adipocytes from AQP7 null mice of age 16 weeks were greatly enlarged (diameter 118 μm) compared with wild type mice (39 μm). Adipocytes from AQP7 null mice also accumulated excess glycerol (251 versus 86 nmol/mg of protein) and triglycerides (3.4 versus 1.7 μmol/mg of protein). In contrast, at age 4 weeks, adipocyte volume and body fat mass were comparable in wild type and AQP7 null mice. To investigate the mechanism(s) responsible for the progressive adipocyte hypertrophy, glycerol permeability and fat metabolism were studied in adipocytes isolated from the younger mice. Plasma membrane glycerol permeability measured by [14C]glycerol uptake was 3-fold reduced in AQP7-deficient adipocytes. However, adipocyte lipolysis, measured by free fatty acid release and hormone-sensitive lipase activity, and lipogenesis, measured by [14C]glucose incorporation into triglycerides, were not affected by AQP7 deletion. These data suggest that adipocyte hypertrophy in AQP7 deficiency results from defective glycerol exit and consequent accumulation of glycerol and triglycerides. Increasing AQP7 expression/function in adipocytes may reduce adipocyte volume and fat mass in obesity.

Background. Aquaporin-2 (AQP-2) is an arginine vasopressin (AVP)-regulated water channel in kidney collecting duct cells. The present study was undertaken to determine whether a change in tonicity could directly regulate the AQP-2 gene in an in vitro experiment. Methods. Various fragments of the 5'-flanking region of the murine AQP-2 gene up to -9.5 kb were cloned into a luciferase (Luc) reporter plasmid, and they were transiently transfected into Madin-Darby canine kidney cells. Results. Hypertonicity significantly increased the Luc activity of the constructs containing >6.1 kb of the 5'-flanking region of the AQP-2 gene (-6.1AQP2). However, promoter regions

Hyperosmolality in the renal medullary interstitium is generated by the renal countercurrent multiplication system, in which the medullary thick ascending limb (MAL) and the outer medullary collecting duct (OMCD) primarily participate. Since arginine vasopressin (AVP) regulates Na-K-ATPase activity directly via protein kinase A and indirectly via hyperosmolality, we investigated the acute and chronic effects of hyperosmolality on Na-K-ATPase and AVP-dependent cAMP generation in the MAL and OMCD. Microdissected MAL and OMCD from control and dehydrated rats were used for the measurement of Na-K-ATPase activity, mRNA expression of alpha-1, beta-1, and beta-2 subunits of Na-K-ATPase, and AVP-dependent cAMP generation. Na-K-ATPase activity in the MAL from dehydrated rats, as measured in isotonic medium, was higher than that of control rats. Moreover, incubation of samples in hypertonic medium (490 mOsm/kg H2O) further increased Na-K-ATPase activity. Dehydration increased alpha-1, beta-1, and beta-2 mRNA expression in the MAL without changing that in the OMCD. Western blot analysis revealed that in the outer medulla, the expression of beta-1, but not that of alpha-1 or beta-2, was stimulated by dehydration. Incubation of MAL or OMCD in hypertonic medium increased AVP-dependent cAMP generation. Higher levels of AVP-dependent cAMP were generated in the MAL from dehydrated rats than that of controls, although incubation in hypertonic medium did not lead to additional increases in AVP-dependent cAMP accumulation. In contrast, AVP-dependent cAMP generation in the OMCD was stimulated by dehydration, and was further stimulated by incubation in hypertonic medium. These findings demonstrate that Na-K-ATPase is upregulated short- and long-term hyperosmolality in the MAL, but not in OMCD.

It has been shown that Cl–/HCO3– exchangers and Cl– channels, both of which are sensitive to stilbene derivatives, have essential roles in the mechanism of apoptosis induction. Staurosporine-induced apoptosis in neonatal mouse cardiomyocytes was prevented by a stilbene derivative, DIDS. To clarify whether Cl–/HCO3– exchangers or Cl– channels are targets of DIDS and whether ClC-3 is involved in the apoptotic process, staurosporine-induced reduction of cell viability, DNA laddering and caspase-3 activation were examined in cultured mouse ventricular myocytes derived from wild-type and ClC-3-deficient mice. Staurosporine-induced apoptosis and its DIDS sensitivity in ambient HCO3–-free conditions in which operation of Cl–/HCO3– exchangers is minimized were indistinguishable from when HCO3– was present. Apoptosis was also prevented by application of a non-stilbene-derivative Cl– channel blocker, NPPB, which cannot block Cl–/HCO3– exchangers. Cardiomyocytes derived from ClC-3-deficient mice similarly underwent apoptosis after exposure to staurosporine; moreover, apoptosis was prevented by application of DIDS or NPPB. Thus, we conclude that in cardiomyocytes, apoptosis is critically dependent on operation not of Cl–/HCO3– exchangers but of Cl– channels which are distinct from ClC-3.

ClC chloride channels (ClCs) can be classified into two groups in terms of their cellular localizations: ClCs present in the plasma membranes and those residing in intracellular organelles. Members of the latter group, including ClC-3, ClC-4, ClC-5, ClC-6, and ClC-7, are often co-expressed in a variety of cell types in many organs. Although the localization of individual channels within cells has been investigated, the degree of overlap between the locations of different ClCs in the same cell has not been clarified. To address this question, different combinations of ClCs, engineered to encode specific epitope tags (FLAG or HA), were either transiently or stably transfected into HEK293 cells, and we then compared the intracellular localization of the expressed channel proteins by immunofluorescence microscopy. Immunofluorescence images of the alternatively labeled channels clearly showed significant co-localization between all pair-wise combinations of ClCs. In particular, ClC-3, ClC-4, and ClC-5 showed a high degree of co-localization. As a significant degree of co-localization between ClCs was observed, we used co-immunoprecipitation to evaluate oligomer formation, and found that each ClC tested could form homo-oligomers, and that any pair-wise combination of ClC-3, ClC-4, and ClC-5 could also form hetero-oligomers. Neither ClC-6 nor ClC-7 was co-precipitated with any other channel protein. These results suggest that within cells ClC-3, ClC-4, and ClC-5 may have combinatorial functions, whereas ClC-6 and ClC-7 are more likely to function as homo-oligomers.

We identified the AQP-2 gene mutation (R254Q) in a family with dominant NDI. The patient studied here has NDI with partial response to the anti-diuretic effect of AVP and dDAVP. Hereditary NDI seems to have the uniform clinical manifestations, but this might only reflect the information on screened patients with clear clinical presentations. It may be that a milder form of NDI has been overlooked due to a lack of genetic identification. Gene mutation analysis should be considered even in patients with mild NDI symptoms. Fortunately, both V2R and AQP2 genes are small and can be easily analyzed.

Hemodialysis is rarely terminated by events associated with anticoagulants. A 69-year-old woman on hemodialysis due to chronic renal failure (CRF) developed an anaphylactoid reaction with hypereosinophilia. On the basis of clinical and laboratory findings, we concluded that the causes of this anaphylactoid reaction were low-molecular-weight heparin and heparin. Our patient also showed high levels of soluble interleukin-2 receptor (sIL-2R) and eosinophil cationic protein (ECP), which decreased after the cessation of hemo-dialysis. To date, there has been no report of high levels of cytokines induced by hemodialysis-associated anaphylactoid reaction. In this report, we discuss the mechanism underlying drug-induced anaphylactoid reaction, particularly that involving eosinophilia and cytokines.

We have previously reported the expression of aquaporin-7 (AQP7) in the rat testis and cloned human AQP7 from the testicular cDNA library. However, to our knowledge the spatial expression pattern and biological roles of AQP7 remain to be elucidated in humans.We investigated AQP7 expression in the human testis and ejaculated sperm from fertile men and from infertile patients.AQP7 was expressed at the tail of spermatids and spermatozoa in the human testis. AQP7 protein was also detected at the middle piece and the anterior tale portion of ejaculated sperm. However, some infertile patients lacked AQP7 expression in ejaculated sperm, although all fertile men expressed AQP7 protein. The motility rate of AQP7 negative sperm was significantly lower than that of AQP7 positive sperm, while the sperm concentration was not different between AQP7 positive and negative subjects.AQP7 shows a spatial expression pattern in the human testis. AQP7 may be involved in the maintenance of sperm motility. Furthermore, a lack of AQP7 expression in sperm may be an underlying mechanism of male infertility.

Both mammals and birds can concentrate urine hyperosmotic to plasma via a countercurrent multiplier mechanism, although evolutionary lines leading to mammals and birds diverged at an early stage of tetrapod evolution. We reported earlier (Nishimura H, Koseki C, and Patel TB. Am J Physiol Regul Integr Comp Physiol 271: R1535–R1543, 1996) that arginine vasotocin (AVT; avian antidiuretic hormone) increases diffusional water permeability in the isolated, perfused medullary collecting duct (CD) of the quail kidney. In the present study, we have identified an aquaporin (AQP) 2 homolog water channel in the medullary cones of Japanese quail, Coturnix coturnix (qAQP2), by RT-PCR-based cloning techniques. A full-length cDNA contains an 822-bp open reading frame that encodes a 274-amino acid sequence with 75.5% identity to rat AQP2. The qAQP2 has six transmembrane domains, two asparagine-proline-alanine (NPA) sequences, and putative N-glycosylation (asparagine-124) and phosphorylation sites (serine-257) for cAMP-dependent protein kinase. qAQP2 is expressed in the membrane of Xenopus laevis oocytes and significantly increased its osmotic water permeability (Pf), inhibitable ( P < 0.01) by mercury chloride. qAQP2 mRNA (RT-PCR) was detected in the kidney; medullary mRNA levels were higher than cortical levels. qAQP2 protein that binds to rabbit anti-rat AQP2 antibody is present in the apical/subapical regions of both cortical and medullary CDs from normally hydrated quail, and the intensity of staining increased only in the medullary CDs after water deprivation or AVT treatment. The relative density of the ∼29-kDa protein band detected by immunoblot from the medullary cones was modestly higher in water-deprived/AVT-treated quail. The results suggest that 1) medullary CDs of quail kidneys express a mercury-sensitive functioning qAQP2 water channel, and 2) qAQP2 is at least partly regulated by an AVT-dependent mechanism. This is the first clear identification of AQP2 homolog in nonmammalian vertebrates.

Water channel aquaporin-2 (AQP2) strictly regulates body water homeostasis in mammals. 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 interaction of AQP2 with other proteins. To isolate AQP2-binding proteins, immunoaffinity chromatography of extracts from rat kidney papilla was performed using a column covalently coupled with anti-AQP2 antibody. Using this method 42-kDa protein was purified and subsequently identified as beta- and gamma-isoforms of actin by two-dimensional gel analysis and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. AQP2 was indeed coimmunoprecipitated with actin from cell lysates of rat kidney papilla. In addition, surface plasmon resonance analyses showed that the C-terminal fragment of AQP2 strongly bound to actin and the K(D) value was 3.18x10(-8)M. In this experiment we have elucidated the direct binding of channel protein AQP2 to cytoskeletal protein actin, providing a novel mechanism for trafficking of not only AQP2 but also recycling channel proteins.

Aquaporin-2 (AQP2) is one of the water-channel proteins expressed in principal cells of kidney collecting ducts, where it is stored in the intracellular compartment. Previous studies have demonstrated that AQP2 vesicles constitute a distinct intracellular compartment partially overlapping with early endosomes. In this report, we performed in vitro experiments using the renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, stably expressing AQP2 (MDCK-hAQP2). In nonpolarized cells, AQP2 vesicles were scattered in the cytoplasm and did not colocalize with Golgi 58K or TGN38. Small portions of AQP2 vesicles were positive for the lysosome marker cathepsin D. An early endosome antigen (EEA1) localized around AQP2 vesicles in close proximity, suggesting involvement of the endosomal system in the trafficking of AQP2. AQP2 vesicles are distinct from other recycling molecules, such as glucose transporter 4 (GLUT4) and endocytosed transferrin. In polarized MDCK-hAQP2 cells, AQP2 vesicles were localized in the subapical recycling compartment and distinct from the Golgi apparatus, trans-Golgi network, lysosome, and early endosome in the nonstimulated state. When the cells were treated with forskolin, translocation of AQP2 to the apical membrane was observed. Washout of forskolin induced retrieval of AQP2 into the cytoplasm, and AQP2 was transiently colocalized with EEA1-positive endosomes. Then, AQP2 moved from EEA1-positive endosomes to the subapical AQP2-storage compartment, which is sensitive to wortmannin and LY294002. These results suggest that AQP2 resides in a recycling compartment at the apical side in polarized MDCK-hAQP2 cells, and its retrieval uses the apical endosomal system and the phosphatidylinositol 3-kinase-dependent pathway.

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.

Mutations in the WNK4 gene cause pseudohypoaldosteronism type II (PHAII), an autosomal-dominant disorder of hyperkalemia and hypertension. The target molecules of this putative kinase and the molecular mechanisms by which the mutations cause the phenotypes are currently unknown. Although recent reports found that expression of WNK4 in Xenopus oocytes causes inhibition of the thiazide-sensitive NaCl cotransporter and the renal K channel ROMK, there may be additional targets of WNK4. For example, an increase in paracellular chloride permeability has been postulated to be a mediator of PHAII pathogenesis, a possibility supported by the localization of WNK4 at tight junctions in vivo . To determine the validity of this hypothesis, we measured transepithelial Na and Cl permeability in Madin-Darby canine kidney II cells stably expressing wild-type or a pathogenic mutant of WNK4. We found that transepithelial paracellular Cl permeability was increased in cells expressing a disease-causing mutant WNK4 (D564A) but that Na permeability was decreased slightly. Furthermore, WNK4 bound and phosphorylated claudins 1-4, major tight-junction membrane proteins known to be involved in the regulation of paracellular ion permeability. The increases in phosphorylation of claudins were greater in cells expressing the mutant WNK4 than in cells expressing wild-type protein. These results clearly indicate that the pathogenic WNK4 mutant possesses a gain-of-function activity and that the claudins may be important molecular targets of WNK4 kinase. The increased paracellular “chloride shunt” caused by the mutant WNK4 could be the pathogenic mechanism of PHAII.

Volume-sensitive outwardly rectifying (VSOR) Cl- channels are activated during osmotic swelling and involved in the subsequent volume regulation in most animal cells. To test the hypothesis that the ClC-3 protein is the molecular entity corresponding to the VSOR Cl- channel in cardiomyocytes, the properties of VSOR Cl- currents in single ventricular myocytes isolated from ClC-3-deficient (Clcn3(-/-)) mice were compared with those of the same currents in ClC-3-expressing wild-type (Clcn3(+/+)) and heterozygous (Clcn3(+/-)) mice. Basal whole-cell currents recorded under isotonic conditions in ClC-3-deficient and -expressing cells were indistinguishable. The biophysical and pharmacological properties of whole-cell VSOR Cl- currents in ClC-3-deficient cells were identical in ClC-3-expressing cells. The VSOR Cl- current density, which is an indicator of the plasmalemmal expression of functional channels, was essentially the same in cells isolated from these 3 types of mice and C57BL/6 mice. Activation of protein kinase C (PKC) by a phorbol ester was found to upregulate VSOR Cl- currents in ClC-3-deficient and -expressing cardiomyocytes. This effect is opposite to the reported downregulatory effect of PKC activators on ClC-3-associated Cl- currents. We thus conclude that functional expression of VSOR Cl- channels in the plasma membrane of mouse cardiomyocytes is independent of the molecular expression of ClC-3.

Targeted positioning of water channel aquaporin-2 (AQP2) strictly regulates body water homeostasis. Trafficking of AQP2 to the apical membrane is critical for the reabsorption of water in renal collecting ducts. Besides the cAMP-mediated effect of vasopressin on AQP2 trafficking to the apical membrane, other signaling cascades also induce this sorting. Recently, AQP2-binding proteins that directly regulate this trafficking have been uncovered: SPA-1, a GTPase-activating protein (GAP) for Rap1, and cytoskeletal protein actin. This review summarizes recent advances related to the trafficking mechanism of AQP2 and its defect causing nephrogenic diabetes insipidus (NDI). Keywords:: aquaporin-2, PKA phosphorylation, missorting, Rho, cytoskeleton

The Wnt-beta-catenin pathway plays key roles in embryogenesis. Wnt-4 is known to be expressed in the mesonephric duct in embryonic development. It is tempting to speculate that the Wnt-4-beta-catenin pathway contributes to the recovery from acute renal failure (ARF). This study used an in vivo model of ARF rats to clarify the significance of the Wnt-4-beta-catenin pathway in ARF. ARF was induced by clamping the rat left renal artery for 1 h. At 3, 6, 12, 24, 48, and 72 h after reperfusion, whole kidney homogenate and total RNA were extracted for examination by Western blot analysis and real-time RT-PCR. Wnt-4 mRNA and protein expression were strongly increased at 3 to 12 h and 6 to 24 h after ischemia, respectively. In immunohistologic examination, Wnt-4 was expressed in the proximal tubules and co-expressed with aquaporin-1, GM130, and PCNA. Cyclin D1 and cyclin A were expressed at 24 to 48 h after reperfusion. In addition, the overexpression of Wnt-4 and beta-catenin promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 in LLC-PK1 cells. Taken together, these data suggest that the Wnt-4-beta-catenin pathway plays a key role in the cell cycle progression of renal tubules in ARF. The Wnt-4-beta-catenin pathway may regulate the transcription of cyclin D1 and control the regeneration of renal tubules in ARF.

Objective: To clarify whether combination treatment with desmopressin (DDAVP) and thiazide was clinically effective in a patient with congenital nephrogenic diabetes insipidus (CNDI), we evaluated the treatment in a 7-year-old boy with CNDI who had demonstrated a partial response to DDAVP. Method: Both volume of urine and the presence of nocturia were determined during treatment. Result: Neither the usual therapy of a low-salt diet and a thiazide nor intranasal therapy with a large dose of DDAVP was effective. However, combination treatment resulted in a decrease in urinary volume and the disappearance of nocturia. Conclusion: DDAVP coupled with thiazide may be useful for CNDI in patients who have shown a partial response to DDAVP.

We describe a case of a novel mutant vasopressin 2 receptor (V2R)-dependent nephrogenic diabetes insipidus (NDI) with bilateral non-obstructive hydronephrosis in a middle aged man. This could be distinguished from aquaporin 2 (AQP2)-dependent NDI by the response of factor VIII and von Willebrand factor (vWF) to 1-deamino-8-D-arginine vasopressin (DDAVP) administration. A 47-year-old man was admitted to hospital because of polyuria, which had been present from infancy and was suspected of causing non-obstructive hydronephrosis. His mother's father, the older brother of his mother and his second daughter also all had polyuria. Sodium concentration, osmolality and vasopressin in blood were high, while sodium concentration and osmolality in urine were low. There were no changes in urine osmolality, factor VIII and vWF in response to DDAVP infusion. Neither was heart rate, diastolic blood pressure nor facial flushing affected. These findings suggested this case was V2R-dependent NDI rather than AQP2-dependent NDI. Molecular genetic analysis demonstrated that the patient had a V2R missense mutation involving a substitution of cysteine for arginine at position 104 (R104C) located in the first extracellular loop of the V2R. It was also found that the patient's mother and his second daughter were heterozygous for this R104C mutation.

Ligand-regulatable erythropoietin production by plasmid injection and in vivo electroporation. Background The development of an in vivo gene transfer approach to deliver physiologic levels of recombinant proteins to the systemic circulation would represent a significant advance in the treatment of protein deficiency disorders. However, the ability to regulate transgene expression is of paramount importance for safe and effective gene transfer therapy. Methods We developed two plasmids, one encoder of chimeric GeneSwitch protein, and the other an inducible transgene for human erythropoietin (Epo). The level of secretion of Epo into the serum was modulated by intraperitoneal administration of mifepristone (MFP). Rats were divided into four groups: one group administered Epo plasmid with MFP for 50 days, a second group administered Epo plasmid with MFP for 15 days and then again from day 30 to day 50, a third group administered Epo plasmid without MFP, and a fourth group administered control plasmid. A pair of electrodes was inserted into the muscle of the right thigh, 100 mg of each plasmid was injected, and in vivo electroporation (8 pulses at 100 V for 50 msec) was performed. Results The presence of vector-derived Epo mRNA was detected by RT-PCR only in the Epo plasmid and MFP(+) groups. The hematocrit levels increased continuously, from the pre-injection level of 41.2% to 55.0% on day 30 and 53.8% on day 50 in the Epo plasmid and MFP(+) groups. In the MFP re-challenged group, the hematocrit levels rose up to day 15, fell after 20 to 30 days, and then rose again after MFP re-administration. The serum Epo levels increased only in the Epo plasmid and MFP(+) groups. There were no significant changes in hematocrit levels and Epo levels in the Epo plasmid and MFP(-) group. Conclusion Epo gene transfer with the GeneSwitch system by in vivo electroporation is a useful procedure for efficient drug-regulated delivery of Epo.

Smad7 mediates transforming growth factor-β–induced apoptosis in mesangial cells. Background In addition to inhibiting cell growth, transforming growth factor-β (TGF-β) has recently been reported to induce apoptosis in various cell lines. Smad proteins are the downstream effectors of TGF-β signaling. Among them, Smad7 exerts negative feedback control over the action of TGF-β. However, we do not know how the Smad proteins contribute to TGF-β–induced apoptosis in mesangial cells. To investigate the function of Smad proteins, we examined the effect of Smad overexpression using adenoviral vector in mesangial cells. Methods Primary cultured rat mesangial cells were transfected with Smad7-promoter-luciferase-plasmid by electroporation. Smad7 promoter activity was investigated by luciferase assay. The apoptotic phenomena elicited by TGF-β and Smad7 overexpression were investigated using adenoviral vector (AdCMV-Smad7). Apoptosis was detected by the cell death detection ELISA assay, CPP32/caspase-3 assay, and nucleosomal DNA laddering. Results TGF-β significantly increased the protein expression and the promoter activity of Smad7 in rat mesangial cells. Overexpression of Smad7 induced DNA fragmentation and significant increases in cell death ELISA and CPP32/caspase-3 assay. On the other hand, overexpression of Smad2 and Smad3 did not elicit any significant increases in CPP32/caspase-3 activity. Furthermore, the antisense oligonucleotide to Smad7 prevented the TGF-β–induced apoptosis. Overexpression of Smad7 did not affect nuclear factor-κB activity in mesangial cells. Conclusions These data indicate that TGF-β–induced apoptosis in mesangial cells is mediated through the activation of caspase-3 by Smad7, but not by Smad2 or Smad3. Our results provide new clarification on the function of Smad7 in TGF-β signaling in mesangial cells.

Recently, a new member of aquaporins was reported as AQP10 [Biochem. Biophys. Res. Commun. 287 (2001) 814], which is incompletely spliced to lose the sixth transmembrane domain and has poor water and no glycerol/urea permeabilities. Independently, we identified a similar clone in human. Our AQP10 consists of 301 amino acids with a highly conserved sixth transmembrane domain. AQP10 has higher identity with aquaglyceroporins (50% with AQP9, 48% with AQP3, 42% with AQP7) and lower identity with other aquaporins (32% with AQP1 and AQP8). AQP10 is expressed only in the small intestine with (approximately 2 kb). RNase protection assay revealed the absence of the unspliced form, supporting the authenticity of our clone. When expressed in Xenopus oocytes, AQP10 stimulated osmotic water permeability sixfold in a mercury-sensitive manner. Glycerol and urea uptakes were also stimulated, while adenine uptake was not. The genome structure of AQP10 is similar to those of other aquaglyceroporins (AQP3, AQP7, AQP9) with six exons. We conclude that AQP10 represents a new member of aquaglyceroporins functionally as well as structurally.

The aim of this multi-centre prospective study was to evaluate the efficacy, tolerability, and safety of the combination of sodium cromoglycate (SCG) and salbutamol (administered as a nebulized solution), compared to SCG alone and salbutamol alone, in the management of severe, intractable asthma in childhood. The study was an open, randomized, cross-over trial of 12 weeks' duration in children with moderate-to-severe intractable asthma. All treatments were administered twice daily by powered nebulizer. The primary outcome measure was the change in asthma severity, as measured by the mean asthma score during the last 2 weeks of a baseline period and the last 2 weeks of each treatment. Secondary outcome measure was the patient's opinion of the effectiveness of treatment. The change in asthma scores from baseline values were significantly greater with the combination treatment compared to each component administered separately. The mean difference in asthma score between the combination and salbutamol was: -7.5; 95% CI, -11.70 to -3.29 (p < 0.0001). The mean difference between the combination and SCG was: -8.53; 95% CI, -14.03 to -3.25 (p < 0.0001). Patients were also significantly in favor of combination treatment (p < 0.001 vs. salbutamol; p < 0.01 vs. SCG). Two patients reported adverse effects. We concluded that regular twice-daily inhalation of a combination of SCG and salbutamol gave better control of symptoms than previous treatments in patients with severe, intractable asthma. Few adverse effects with this therapy suggest that it is extremely useful, safe, and effective.

Mutations in the aquaporin-2 gene (AQP2), encoding the vasopressin-regulated water channel of the renal collecting duct, are responsible for the autosomal recessive or dominant forms of congenital nephrogenic diabetes insipidus. We describe two new families with normal hypotensive and coagulation responses following the administration of desamino-8-D-arginine AVP, a clinical suggestion of normal vasopressin-2 receptors. The patients were compound heterozygotes for point mutations at nucleotide position 170 (CAG to CCG; Q57P) and at position 299 (GGA to GTA; G100V) in exon 1 of the AQP2 gene. Expression of the G57P and G100V AQP2 proteins in Xenopus oocytes showed only 1.3-fold and 1.2-fold increase, respectively, in the water permeability in contrast to 8.0-fold increase in oocytes injected with wild-type cRNA. Immunoblots of oocyte lysate revealed the intensities of the 29-kDa bands were comparable among oocytes injected with wild-type and mutant cRNAs. Immunocytochemistry showed the plasma membrane was not stained in oocytes injected with cRNA of Q57P and of G100V. These results provide evidence that the Q57P and G100V mutations in congenital nephrogenic diabetes insipidus are attributable to the misrouting of AQP2.

Background: CLC-3 is a member of the CLC chloride channel family and is widely expressed in mammalian tissues. To determine the physiological role of CLC-3, we generated CLC-3-deficient mice (Clcn3–/–) by targeted gene disruption. Results: Together with developmental retardation and higher mortality, the Clcn3–/– mice showed neurological manifestations such as blindness, motor coordination deficit, and spontaneous hyperlocomotion. In histological analysis, the Clcn3–/– mice showed a pattern of progressive degeneration of the retina, hippocampus and ileal mucosa, which resembled the phenotype observed in cathepsin D knockout mice. The defect of cathepsin D results in a lysosomal accumulation of ceroid lipofuscin containing the mitochondrial F1F0 ATPase subunit c. In immunohistochemistry and Western blot analysis, we found that the subunit c was heavily accumulated in the lysosome of Clcn3–/– mice. Furthermore, we detected an elevation in the endosomal pH of the Clcn3–/– mice. Conclusions: These results indicated that the neurodegeneration observed in the Clcn3–/– mice was caused by an abnormality in the machinery which degrades the cellular protein and was associated with the phenotype of neuronal ceroid lipofuscinosis (NCL). The elevated endosomal pH could be an important factor in the pathogenesis of NCL.

Gene transfer of Smad7 using electroporation of adenovirus prevents renal fibrosis in post-obstructed kidney. Unilateral ureteral obstruction (UUO) leads to interstitial fibrosis of the obstructed kidney, and TGF-β is considered to play an important role in this fibrotic process. Smad7 has been recently identified as an antagonist of TGF-β signaling. To investigate whether this novel molecule can be exploited for therapy of renal fibrosis, we determined the effect of exogenous Smad7, introduced by a recombinant adenovirus vector combined with in vivo electroporation (EP), on UUO-induced renal fibrosis in rats. Methods A model of UUO was made in SD rats by ligating their left ureters. The next day, the rats were divided into four groups and adenovirus was injected into the extended pelvic space (two groups received AdCMV-LacZ and two groups received AdCMV-Smad7). Then, EP was performed in one group of AdCMV-LacZ-injected rats and one group of AdCMV-Smad7-injected rats. The renal tissues were obtained 3, 5, 10, and 14 days after the UUO operation. We detected the efficiency of transgene by immunoblots of renal cortical and medullary tissues and immunohistochemical studies for Smad7 and FLAG (the FLAG gene was introduced in the AdCMV-Smad7 as a marker). The renal fibrosis was monitored by histological scoring of Masson stainings. Results In immunoblotting, both Smad7 and FLAG were clearly detected in the renal medullary tissue of the rats given AdCMV-Smad7 with EP. In contrast, immunoblots of renal cortical tissue did not demonstrate positive bands. In immunohistological study, Smad7 was stained in the renal medulla in the rats given AdCMV-Smad7 with EP. In the rats given AdCMV-Smad7 without EP, only a weak signal was detected in renal medullary tissue. The rats given AdCMV-Smad7 with EP demonstrated significantly more suppression of renal fibrosis than rats treated with AdCMV-LacZ. The rats treated with AdCMV-Smad7 without EP did not demonstrate significant suppression of renal fibrosis. Conclusion These data indicate that gene transfer of Smad7 prevents UUO-induced renal fibrosis, suggesting that Smad7 may be applicable for the treatment of renal fibrosis. In vivo electroporation of adenovirus may be a powerful tool for gene delivery in renal tissue.

Estimating fluid balance in haemodialysis patients is essential when determining dry weight, but limited methods are currently available. B-type natriuretic peptide (BNP) is a useful surrogate marker in patients with congestive heart failure (CHF), but whether its validity could be generalized to haemodialysis patients has not been studied well.A total of 457 haemodialysis patients at a dialysis centre were analyzed. Determinants of BNP were assessed in connection with ultrasound cardiography (UCG) records, Kt/V, ultrafiltration rate (UFR), and demographic factors. All-cause death and cardiovascular (CV) events were recorded as the main outcome.Among the UCG records, left atrial diameter (LAD), left ventricular ejection fraction (LVEF), were determinants of log-transformed (ln) BNP; UFR, age and sex were also significant. There was a positive correlation between BNP and LAD (r = 0.285, P 0.001). Receiver operating characteristic (ROC) analysis revealed that BNP had 90% and 80% sensitivity to predict the presence of LA enlargement of 77.9 pg/mL and 133.2 pg/mL, respectively. Higher BNP and lower LVEF were associated with higher risk for developing all-cause death and CVD. In the adjusted model, patients with BNP higher than 471 pg/mL had hazard ratio of 2.18 (95% confidence interval (CI) 1.20-3.96, P = 0.01), compared to those with BNP109 pg/mL.B-type natriuretic peptide was determined by LAD, LVEF, UFR, age and sex. BNP and LAD had positive correlation and BNP could become a useful tool for estimating the presence of LA enlargement. BNP and LVEF was a strong risk factor for predicting all-cause death and CV events among patients undergoing haemodialysis.

A 64-year-old Japanese man with renal dysfunction caused by autosomal dominant polycystic kidney disease (ADPKD) was admitted to our hospital for evaluation of right back pain, fever, inflammation, and pleural effusion. Diagnostic investigations for tuberculous pleuritis were all negative. Although no radiographic abnormality suggesting hepatic cyst infection was detected by computed tomography, hepatic cyst drainage demonstrated purulent contents indicative of cyst infection. Conglutination of the cyst by minocycline 100 mg was performed five times in addition to drainage. After drainage, the symptoms of inflammation, right back pain and right pleural effusion subsided. Renal function and anemia, which had been resistant to darbepoetin treatment, also improved after the procedure. These results suggested that the infected hepatic cyst was associated with the patient's symptoms, exacerbation of renal dysfunction and anemia. The pleural effusion was due to the propagation of inflammation from the cyst infection. This is the first report of an infected hepatic cyst in an ADPKD patient presenting with and diagnosed by right pleural effusion.

By analysing the pathogenesis of a hereditary hypertensive disease, PHAII (pseudohypoaldosteronism type II), we previously discovered that WNK (with-no-lysine kinase)–OSR1/SPAK (oxidative stress-responsive 1/Ste20-like proline/alanine-rich kinase) cascade regulates NCC (Na–Cl co-transporter) in the DCT (distal convoluted tubules) of the kidney. However, the role of WNK4 in the regulation of NCC remains controversial. To address this, we generated and analysed WNK4−/− mice. Although a moderate decrease in SPAK phosphorylation and a marked increase in WNK1 expression were evident in the kidneys of WNK4−/− mice, the amount of phosphorylated and total NCC decreased to almost undetectable levels, indicating that WNK4 is the major WNK positively regulating NCC, and that WNK1 cannot compensate for WNK4 deficiency in the DCT. Insulin- and low-potassium diet-induced NCC phosphorylation were abolished in WNK4−/− mice, establishing that both signals to NCC were mediated by WNK4. As shown previously, a high-salt diet decreases phosphorylated and total NCC in WNK4+/+ mice via AngII (angiotensin II) and aldosterone suppression. This was not ameliorated by WNK4 knock out, excluding the negative regulation of WNK4 on NCC postulated to be active in the absence of AngII stimulation. Thus, WNK4 is the major positive regulator of NCC in the kidneys., The analyses of WNK4 (with-no-lysine kinase 4) knockout mice help to end a long-standing controversy about the role of WNK4 on NCC (Na–Cl co-transporter) regulations in the kidney. WNK4 is a strong positive regulator of NCC.

Chronic kidney disease is a risk factor for cardiovascular mortality and morbidity of cardiovascular events (CVEs). We obtained baseline data regarding blood biochemistry, ankle-brachial index (ABI), brachial-ankle pulse wave velocity (baPWV) and echocardiographic parameters from 300 patients on hemodialysis in 2005. We also measured ABI and baPWV annually from June 2005 until June 2012 and calculated rates of changes in ABI and baPWV to identify factors associated with CVEs. Seventy-three patients died of cardiovascular disease and 199 CVEs occurred in 164 patients during the study period. Cardiac, cerebrovascular and peripheral artery disease (PAD) events occurred in 124, 43 and 32 patients, respectively, and 30 patients had more than two types of CVEs. Analysis using the Cox proportional hazards model showed that a higher rate of decline in ABI (hazard ratio [HR], 4.034; P0.001) was the most significant risk factor for decreased patient survival. Multivariate Cox analysis revealed that a higher rate of ABI decline (HR, 2.342; P0.001) was a significant risk factor for cardiac events, and that a lower baseline ABI was a risk factor for cerebrovascular (HR, 0.793; P = 0.03) and PAD (HR, 0.595; P0.0001) events. Our findings suggested that the rate of a decline in ABI and the baseline ABI value are potent correlation factors for survival and CVE morbidity among patients on hemodialysis in Japan.

The development of an in vivo gene transfer approach to deliver physiologic levels of recombinant proteins to the systemic circulation would represent a significant advance in the treatment of protein deficiencies-disorders. However, the ability to regulate transgene expression will become paramount for safety and efficacy in gene transfer therapy. We have described the construction of an efficient and ligand-dependently regulated erythropoietin (EPO) production system using naked plasmid and in vivo electroporation. Two plasmids, one encoding the chimeric GeneSwitch protein and the other encoding an inducible transgene for human EPO, were developed. Modulation of the level of secretion of EPO into the serum was achieved by intraperitoneal administration of mifepristone (MFP). Rats were divided into 4 groups: one group received EPO plasmid with MFP for 30 days, a second group received with EPO plasmid MFP for 9 days, a third group received EPO plasmid without MFP, and a fourth group received control plasmid. A pair of electrodes was inserted into the muscle of the right thigh and 100 μg of each plasmid was injected. In vivo electrporation (8 times at 100 V for 50 milliseconds) was performed. The presence of vector-derived EPO mRNA was detected by reverse transcriptase-polymerase chain reaction only in the EPO and MFP(+) group. The hematocrit levels increased continuously from the preinjection level of 42.7% to 53.8% on day 28 in the EPO and MFP(+) group. The serum EPO levels increased only in the EPO and MFP(+) group. There was no significant change in hematocrit levels and EPO levels in the EPO and MFP(−) group. These results demonstrate that EPO gene transfer with the GeneSwitch system by in vivo electroporation is a useful procedure for efficient and drug-dependent regulated delivery of EPO. © 2001 by the National Kidney Foundation, Inc.

The vasopressin-regulated water channel aquaporin-2 (AQP2) is known to tetramerize in the apical membrane of the renal tubular cells and contributes to urine concentration. We identified three novel mutations, each in a single allele of exon 4 of the AQP2 gene, in three families showing autosomal dominant nephrogenic diabetes insipidus (NDI). These mutations were found in the C-terminus of AQP2: a deletion of G at nucleotide 721 (721 delG), a deletion of 10 nucleotides starting at nucleotide 763 (763–772del), and a deletion of 7 nucleotides starting at nucleotide 812 (812–818del). The wild-type AQP2 is predicted to be a 271–amino acid protein, whereas these mutant genes are predicted to encode proteins that are 330–333 amino acids in length, because of the frameshift mutations. Interestingly, these three mutant AQP2s shared the same C-terminal tail of 61 amino acids. In Xenopus oocytes injected with mutant AQP2 cRNAs, the osmotic water permeability (Pf) was much smaller than that of oocytes with the AQP2 wild-type (14%–17%). Immunoblot analysis of the lysates of the oocytes expressing the mutant AQP2s detected a band at 34 kD, whereas the immunoblot of the plasma-membrane fractions of the oocytes and immunocytochemistry failed to show a significant surface expression, suggesting a defect in trafficking of these mutant proteins. Furthermore, coinjection of wild-type cRNAs with mutant cRNAs markedly decreased the oocyte Pf in parallel with the surface expression of the wild-type AQP2. Immunoprecipitation with antibodies against wild-type and mutant AQP2 indicated the formation of mixed oligomers composed of wild-type and mutant AQP2 monomers. Our results suggest that the trafficking of mutant AQP2 is impaired because of elongation of the C-terminal tail, and the dominant-negative effect is attributed to oligomerization of the wild-type and mutant AQP2s. Segregation of the mutations in the C-terminus of AQP2 with dominant-type NDI underlies the importance of this domain in the intracellular trafficking of AQP2.

Hyperosmolality activates Akt and regulates apoptosis in renal tubular cells . Background The novel serine-threonine kinase Akt is a critical enzyme in cell survival. We investigated the roles of the Akt pathway and apoptotic signals in ( 1 ) Madin-Darby canine kidney (MDCK) cells in a hyperosmotic condition in vitro and ( 2 ) in the inner medulla of dehydrated rat in vivo. Methods The in vivo experiments were performed in 24- and 48-hour water-restricted rats. Hyperosmolality-stimulated Akt phosphorylation was examined in MDCK cells. Phosphatidylinositol 3-kinase (PI3-K) inhibitors, the dominant-negative mutant of PI3-K, the dominant-negative mutant of Akt, and the dominant-active form of Akt were used to examine the roles of the PI3-K/Akt pathways in renal tubular cell apoptosis. Results The amount of phosphorylated Akt protein was increased in the inner medulla of dehydrated rats. Hyperosmolality induced by the addition of NaCl, urea, and raffinose phosphorylated Akt in MDCK cells in an osmolality-dependent manner. PI3-K inhibitors and the dominant-negative mutant of PI3-K inhibited the hyperosmolality-induced phosphorylation of Akt. Raising the media osmolality from a normal level to 500 or 600 mOsm/kg H 2 O final osmolality elicited apoptotic changes such as nucleosomal laddering of DNA and an increment of caspase-3 activity and increased activity in the cell death enzyme-linked immunosorbent assay. Dominant-active Akt prevented the mild hyperosmolality-induced apoptosis, while inhibition of the PI3-K/Akt pathways promoted apoptosis. Conclusion The Akt pathway is activated by hyperosmolality in vitro and in vivo, and activation of Akt prevents the mild hyperosmolality-induced apoptotic changes in MDCK cells. PI3-K/Akt pathways are involved in a hypertonic condition that confers the balance between cell survival and apoptosis.