[Long-Term Survival by Low-Dose Imatinib after Recurrence of GIST].

A man in his 70s was observed to have GIST recurrence 19 months after surgery. Chemotherapy was initiated with imatinib 400 mg/day orally. The dose was eventually reduced to 100 mg/day to avoid side effects. Tumor reduction was confirmed 3 months after treatment initiation. Currently, 84 months after the onset of GIST, the patient survives with continuous intake of the same dose of oral imatinib. We were able to observe long-term survival in a patient with recurrent GIST after the administration of a small dose of imatinib.

Structural Development of Salicylanilide-Based SPAK Inhibitors as Candidate Antihypertensive Agents.

Hypertension is an important target for drug discovery. We have focused on the with-no-lysine kinase (WNK)-oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related proline-alanine-rich protein kinase (SPAK)-NaCl cotransporter (NCC) signal cascade as a potential target, and we previously developed a screening system for inhibitors of WNK-OSR1/SPAK-NCC signaling. Herein we used this system to examine the structure-activity relationship (SAR) of salicylanilide derivatives as SPAK kinase inhibitors. Structural design and development based on our previous hit compound, aryloxybenzanilide derivative 2, and the veterinary anthelmintic closantel (3) led to the discovery of compound 10 a as a potent SPAK inhibitor with reduced toxicity. Compound 10 a decreased the phosphorylation level of NCC in mouse kidney in vivo, and appears to be a promising lead compound for a new class of antihypertensive drugs.

Structural development of N-(4-phenoxyphenyl)benzamide derivatives as novel SPAK inhibitors blocking WNK kinase signaling.

We report here structural development of N-(4-phenoxyphenyl)benzamide derivatives as novel SPAK (STE20/SPS1-related proline/alanine-rich kinase) inhibitors. Abnormal activation of the signal cascade of with-no-lysine kinase (WNK) with OSR1 (oxidative stress-responsive kinase 1)/SPAK and NCC (NaCl cotransporter) results in characteristic salt-sensitive hypertension, and therefore inhibitors of the WNK-OSR1/SPAK-NCC cascade are candidates for antihypertensive drugs. Based on the structure of lead compound 2, we examined the SAR of N-(4-phenoxyphenyl)benzamide derivatives, and developed compound 20l as a potent SPAK inhibitor. Compounds 20l is a promising candidate for a new class of antihypertensive drugs.

Sodium-calcium exchanger 1 is the key molecule for urinary potassium excretion against acute hyperkalemia.

The sodium (Na+)-chloride cotransporter (NCC) expressed in the distal convoluted tubule (DCT) is a key molecule regulating urinary Na+ and potassium (K+) excretion. We previously reported that high-K+ load rapidly dephosphorylated NCC and promoted urinary K+ excretion in mouse kidneys. This effect was inhibited by calcineurin (CaN) and calmodulin inhibitors. However, the detailed mechanism through which high-K+ signal results in CaN activation remains unknown. We used Flp-In NCC HEK293 cells and mice to evaluate NCC phosphorylation. We analyzed intracellular Ca2+ concentration ([Ca2+]in) using live cell Ca2+ imaging in HEK293 cells. We confirmed that high-K+-induced NCC dephosphorylation was not observed without CaN using Flp-In NCC HEK29 cells. Extracellular Ca2+ reduction with a Ca2+ chelator inhibited high-K+-induced increase in [Ca2+]in and NCC dephosphorylation. We focused on Na+/Ca2+ exchanger (NCX) 1, a bidirectional regulator of cytosolic Ca2+ expressed in DCT. We identified that NCX1 suppression with a specific inhibitor (SEA0400) or siRNA knockdown inhibited K+-induced increase in [Ca2+]in and NCC dephosphorylation. In a mouse study, SEA0400 treatment inhibited K+-induced NCC dephosphorylation. SEA0400 reduced urinary K+ excretion and induced hyperkalemia. Here, we identified NCX1 as a key molecule in urinary K+ excretion promoted by CaN activation and NCC dephosphorylation in response to K+ load.

Renal TNFα activates the WNK phosphorylation cascade and contributes to salt-sensitive hypertension in chronic kidney disease.

The inappropriate over-activation of the with-no-lysine kinase (WNK)-STE20/SPS1-related proline/alanine-rich kinase (SPAK)-sodium chloride cotransporter (NCC) phosphorylation cascade increases sodium reabsorption in distal kidney nephrons, resulting in salt-sensitive hypertension. Although chronic kidney disease (CKD) is a common cause of salt-sensitive hypertension, the involvement of the WNK phosphorylation cascade is unknown. Moreover, the effect of immune systems on WNK kinases has not been investigated despite the fact that immune systems are important for salt sensitivity. Here we demonstrate that the protein abundance of WNK1, but not of WNK4, was increased at the distal convoluted tubules in the aristolochic acid nephropathy mouse model of CKD. Accordingly, the phosphorylation of both SPAK and NCC was also increased. Moreover, a high-salt diet did not adequately suppress activation of the WNK1-SPAK-NCC phosphorylation cascade in this model, leading to salt-sensitive hypertension. WNK1 also was increased in adenine nephropathy, but not in subtotal nephrectomy, models of CKD. By comparing the transcripts of these three models focusing on immune systems, we hypothesized that tumor necrosis factor (TNF)-α regulates WNK1 protein expression. In fact, TNF-α increased WNK1 protein expression in cultured renal tubular cells by reducing the transcription and protein levels of NEDD4-2 E3-ligase, which degrades WNK1 protein. Furthermore, the TNF-α inhibitor etanercept reversed the reduction of NEDD4-2 expression and upregulation of the WNK1-SPAK-NCC phosphorylation cascade in distal convoluted tubules in vivo in the aristolochic acid nephropathy model. Thus, salt-sensitive hypertension is induced in CKD via activation of the renal WNK1- SPAK-NCC phosphorylation cascade by TNF-α, reflecting a link with the immune system.

CRISPR-Cas9/phosphoproteomics identifies multiple noncanonical targets of myosin light chain kinase.

Prior studies have implicated myosin light chain kinase (MLCK) in the regulation of aquaporin-2 (AQP2) in the renal collecting duct. To discover signaling targets of MLCK, we used CRISPR-Cas9 to delete the MLCK gene (Mylk) to obtain MLCK-null mpkCCD cells and carried out comprehensive phosphoproteomics using stable isotope labeling with amino acids in cell culture for quantification. Immunocytochemistry and electron microscopy demonstrated a defect in the processing of AQP2-containing early endosomes to late endosomes. The phosphoproteomics experiments revealed that, of the 1,743 phosphopeptides quantified over multiple replicates, 107 were changed in abundance by MLCK deletion (29 decreased and 78 increased). One of the decreased phosphopeptides corresponded to the canonical target site in myosin regulatory light chain. Network analysis indicated that targeted phosphoproteins clustered into distinct structural/functional groups: actomyosin, signaling, nuclear envelope, gene transcription, mRNA processing, energy metabolism, intermediate filaments, adherens junctions, and tight junctions. There was significant overlap between the derived MLCK signaling network and a previously determined PKA signaling network. The presence of multiple proteins in the actomyosin category prompted experiments showing that MLCK deletion inhibits the normal effect of vasopressin to depolymerize F-actin, providing a potential explanation for the AQP2 trafficking defect. Changes in phosphorylation of multiple proteins in the nuclear envelope prompted measurement of nuclear size, showing a significant increase in average nuclear volume. We conclude that MLCK is part of a multicomponent signaling pathway in both the cytoplasm and nucleus that includes much more than simple regulation of conventional nonmuscle myosins through myosin regulatory light chain phosphorylation.

Nationwide in-hospital mortality following major fractures among hemodialysis patients and the general population: An observational cohort study.

BACKGROUNDS: End-stage kidney disease (ESKD) is associated with increased risk of fracture and subsequent morbidity and mortality. However, fracture site-specific mortality in ESKD patients have yet to be elucidated in comparison with the general population. METHODS: In this population-based cohort derived from the Diagnosis Procedure Combination database of Japan from 2012 to 2014, we included 9320 ESKD patients undergoing hemodialysis and 547,726 patients without ESKD who were hospitalized for five major fractures, including hip (proximal femur), spine, forearm, upper arm, and leg (distal femur and proximal tibia). Overall and site-specific risks of in-hospital death were determined by logistic regression models. RESULTS: The age- and sex-adjusted mortality rates were 4.91% (95% confidence interval [CI], 4.46-5.37) and 1.02% (95% CI, 0.99-1.06) in the hemodialysis and general population groups, respectively. The multivariate odds ratio (OR) of death in hemodialysis patients versus the general population was 2.48 (95% CI, 2.25-2.74) for overall fractures, and was particularly high for a subgroup of upper arm fracture (OR 4.82, 95% CI, 3.19-7.28). The site-specific odds of death (95% CI) among hip, spine, forearm, upper arm, and leg (reference) fractures were 1.77 (0.98-3.18), 1.48 (0.79-2.75), 0.19 (0.04-0.86), and 2.01 (1.01-4.01) in hemodialysis patients, and 1.28 (1.13-1.45), 1.00 (0.88-1.14), 0.13 (0.10-0.17), and 0.83 (0.70-0.97) in the general population, respectively. CONCLUSION: Hemodialysis patients experienced a 4.8-fold higher mortality rate after fractures than the general population. Mortality after upper arm fracture was specifically high in patients on hemodialysis, likely due to the involvement of vascular access located on the fractured arm.

A case of tracheal obstruction caused by reflux and aspiration of semi-solid nutrients via the nasogastric tube.

INTRODUCTION: Semi-solid nutrients have several advantages, including reduced cases of diarrhea and aspiration pneumonia, and are usually administered via percutaneous endoscopic gastrostomy owing to its high viscosity. Administering semi-solid nutrients via a nasogastric tube was recently introduced in clinical practice; however, its safety has not been well confirmed. PRESENTATION OF CASE: An 82-year-old man with a right occipital hemorrhage and severe diarrhea consulted the nutritional support team. Administrations of semi-solid nutrients (HINE E-GEL®) via the nasogastric tube was initiated, which gradually alleviated his symptoms. Fourteen days after initiation, he suddenly had pulmonary failure owing to a tracheal obstruction caused by the reflux and aspiration of semi-solid nutrients. Intubation and subsequent reflex cough expectorated sputum with gel-form particles, which quickly stabilized his pulmonary condition. After this, his hospital course was stable, and he was referred to another hospital for further rehabilitation. DISCUSSION: Semi-solid nutrients administered via the nasogastric tube have different ingredients compared with those administered via percutaneous endoscopic gastrostomy. HINE E-GEL®, for example, contains pectin and calcium phosphate that changes from liquid to semi-solid inside the stomach via chemical reactions under acidic conditions. Data on the viscosity of HINE E-GEL® in vivo are insufficient. Uncertainty regarding the form and viscosity of HINE E-GEL® inside the stomach complicates clinical practice. CONCLUSIONS: Although semi-solid nutrients have several advantages, including reduced diarrhea and gastroesophageal reflux, evidence on semi-solid nutrients via the nasogastric tube is insufficient. It should be noted that semi-solid nutrient reflux can be more fatal than liquid nutrients.

Tacrolimus ameliorates the phenotypes of type 4 Bartter syndrome model mice through activation of sodium-potassium-2 chloride cotransporter and sodium-chloride cotransporter.

Type 4 Bartter syndrome (BS) is caused by genetic mutations in barttin, which is coded for by BSND. Barttin serves as the ß-subunit of the ClC-K chloride (Cl-) channel, which is widely expressed in distal nephrons. Type 4 BS is characterized by severely impaired reabsorption of salt, which may cause polyuria, hypokalemia, and metabolic alkalosis. Calcineurin inhibitors reportedly induce renal salt retention and hyperkalemia by enhancing the phosphorylation of the sodium (Na+)-potassium (K+)-2Cl- cotransporter (NKCC2) and Na+-Cl- cotransporter (NCC). In addition, we have previously reported that tacrolimus, a calcineurin inhibitor, increases the levels of phosphorylated NCC. In this study, we administered tacrolimus to barttin hypomorphic (Bsndneo/neo) mice, a murine model of type 4 BS that exhibits polyuria, hypokalemia, and metabolic alkalosis. Administration of tacrolimus increased the serum K+ level and suppressed urinary K+ excretion. Furthermore, after treatment with tacrolimus, Bsndneo/neo mice increased levels of phosphorylated NCC and NKCC2. We conclude that tacrolimus partially improves clinical phenotypes of Bsndneo/neo mice, and that calcineurin inhibitors might be effective for treating type 4 BS.

Tolvaptan activates the Nrf2/HO-1 antioxidant pathway through PERK phosphorylation.

Tolvaptan, a vasopressin type 2 receptor antagonist initially developed to increase free-water diuresis, has been approved for the treatment of autosomal dominant polycystic kidney disease in multiple countries. Furthermore, tolvaptan has been shown to improve the renal functions in rodent models of chronic kidney disease (CKD); however, the underlying molecular mechanisms remain unknown. CKD is characterized by increased levels of oxidative stress, and an antioxidant transcription factor-nuclear factor erythroid 2-related factor 2 (Nrf2)-has been gaining attention as a therapeutic target. Therefore, we investigated the effects of tolvaptan and a well-known Nrf2 activator, bardoxolone methyl (BARD) on Nrf2. To determine the role of tolvaptan, we used a renal cortical collecting duct (mpkCCD) cell line and mouse kidneys. Tolvaptan activated Nrf2 and increased mRNA and protein expression of antioxidant enzyme heme oxygenase-1 (HO-1) in mpkCCD cells and the outer medulla of mouse kidneys. In contrast to BARD, tolvaptan regulated the antioxidant systems via a unique mechanism. Tolvaptan activated the Nrf2/HO-1 antioxidant pathway through phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK). As a result, tolvaptan and BARD could successfully generate synergistic activating effects on Nrf2/HO-1 antioxidant pathway, suggesting that this combination therapy can contribute to the treatment of CKD.

Failure to sense energy depletion may be a novel therapeutic target in chronic kidney disease.

The kidneys consume a large amount of energy to regulate volume status and blood pressure and to excrete uremic toxins. The identification of factors that cause energy mismatch in the setting of chronic kidney disease (CKD) and the development of interventions aimed at improving this mismatch are key research imperatives. Although the critical cellular energy sensor 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is known to be inactivated in CKD, the mechanism of AMPK dysregulation is unknown. In a mouse model of CKD, metabolome analysis confirmed a decrease in AMPK activation in the kidneys despite a high AMP: ATP ratio, suggesting that AMPK did not sense energy depletion. Similar AMPK inactivation was found in heart and skeletal muscle in CKD mice. Several uremic factors were shown to inactivate AMPK in vitro and in ex vivo preparations of kidney tissue. The specific AMPK activator A-769662, which bypasses the AMP sensing mechanism, ameliorated fibrosis and improved energy status in the kidneys of CKD mice, whereas an AMP analog did not. We further demonstrated that a low-protein diet activated AMPK independent of the AMP sensing mechanism, leading to improvement in energy metabolism and kidney fibrosis. These results suggest that a failure to sense AMP is the key mechanism underlying the vicious cycle of energy depletion and CKD progression and direct AMPK activation may be a novel therapeutic approach in CKD.

Decreased KLHL3 expression is involved in the pathogenesis of pseudohypoaldosteronism type II caused by cullin 3 mutation in vivo.

BACKGROUND: Pseudohypoaldosteronism type II (PHAII) is a hereditary hypertensive disease caused by mutations in four genes: WNK1, WNK4, Kelch-like3 (KLHL3), and cullin3 (CUL3). Recently, it was revealed that CUL3-KLHL3 E3 ligase complex ubiquitinates WNK1 and WNK4, leading to their degradation, and that a common pathogenesis of PHAII is defective WNK degradation due to CUL3-KLHL3 E3 ligase complex impairment. PHAII-causing CUL3 mutations mediate exon9 skipping, producing a CUL3 protein with a 57-amino acid deletion (Δ403-459). However, the pathogenic effects of KLHL3, an adaptor protein that links WNKs with CUL3, in PHAII caused by CUL3 mutation remain unclear. METHODS: To clarify detailed pathophysiological mechanisms underlying PHAII caused by CUL3 mutation in vivo, we generated and analyzed knock-in mice carrying the same CUL3 exon9 deletion (CUL3WT/Δex9) as that reported in PHAII patients. RESULTS: CUL3WT/Δex9 mice exhibited a PHAII-like phenotype. Interestingly, we confirmed markedly decreased KLHL3 expression in CUL3WT/Δex9 mice by confirming the true KLHL3 band in vivo. However, the expression of other KLHL family proteins, such as KLHL2, was comparable between WT and mutant mice. CONCLUSION: KLHL3 expression was decreased in CUL3WT/Δex9 mice. However, expression levels of other KLHL family proteins were comparable between the wild-type and mutant mice. These findings indicate that the decreased abundance of KLHL3 is a specific phenomenon caused by mutant CUL3 (Δexon9). Our findings would improve our understanding of the pathogenesis of PHAII caused by CUL3 mutation in vivo.

Metformin increases urinary sodium excretion by reducing phosphorylation of the sodium-chloride cotransporter.

OBJECTIVE: Metformin is an antidiabetic drug that is widely used to treat patients with diabetes mellitus. Recent studies have reported that treatment with metformin not only improved blood glucose levels but also reduced blood pressure. However, it remains unclear how metformin reduces blood pressure. We hypothesized that metformin affects sodium reabsorption in the kidneys. METHODS: Urinary sodium excretion and expression of renal sodium transporters were examined in 8-week-old male C57BL/6 mice with acute and chronic treatment of metformin. In addition, we examined metformin effects using ex vivo preparations of mice kidney slices. RESULTS: In this study, we demonstrated that metformin increased urinary sodium excretion by reducing phosphorylation of the thiazide-sensitive Na-Cl cotransporter (NCC) in acute and chronic metformin administration. We also confirmed reduction of phosphorylated NCC in an ex vivo study. The activity of other renal sodium transporters, such as NKCC2, ENaC, and NHE3 did not show significant changes. WNK-OSR1/SPAK kinase signals were not involved in this inactivation effect of metformin on NCC. CONCLUSION: Metformin increased urinary sodium excretion by reducing phosphorylation of NCC, suggesting its role in improving hypertension.

The proteasome inhibitor bortezomib attenuates renal fibrosis in mice via the suppression of TGF-ß1.

Kidney fibrosis and fibrogenesis significantly exacerbate chronic kidney disease (CKD) progression and are essential therapeutic targets. Bortezomib (BZM) is a proteasome inhibitor used for the treatment of multiple myeloma (MM). Several studies have demonstrated that BZM attenuates renal impairment in patients with MM, although this effect is generally considered to be the result of MM remission. Recently, several studies on BZM reported anti-fibrotic effects on liver and skin in experimental animal models. However, its effect on renal fibrosis has yet to be examined. Here, we investigated the anti-fibrotic effects of BZM in an experimental mouse model of fibrosis that uses aristolochic acid I (AA). Ten weeks of AA administration with BZM treatment twice a week significantly attenuated AA-induced renal dysfunction and albuminuria, reduced the expression of renal fibrosis-related proteins and kidney injury markers, such as αSMA, Kim1, and Ngal, and prevented renal fibrosis at the level of histopathology. Furthermore, pathological activation of TGFß1-Smad3 signaling and apoptosis, essential pathophysiological causes of AA-induced nephropathy (AAN), were ameliorated by BZM, suggesting this mechanism may be involved in improving fibrosis in AAN. In conclusion, BZM directly inhibits renal fibrosis in CKD via suppression of TGFß1-Smad3 signaling and is promising in terms of drug repositioning.

Systems-level identification of PKA-dependent signaling in epithelial cells.

G protein stimulatory α-subunit (Gαs)-coupled heptahelical receptors regulate cell processes largely through activation of protein kinase A (PKA). To identify signaling processes downstream of PKA, we deleted both PKA catalytic subunits using CRISPR-Cas9, followed by a "multiomic" analysis in mouse kidney epithelial cells expressing the Gαs-coupled V2 vasopressin receptor. RNA-seq (sequencing)-based transcriptomics and SILAC (stable isotope labeling of amino acids in cell culture)-based quantitative proteomics revealed a complete loss of expression of the water-channel gene Aqp2 in PKA knockout cells. SILAC-based quantitative phosphoproteomics identified 229 PKA phosphorylation sites. Most of these PKA targets are thus far unannotated in public databases. Surprisingly, 1,915 phosphorylation sites with the motif x-(S/T)-P showed increased phosphooccupancy, pointing to increased activity of one or more MAP kinases in PKA knockout cells. Indeed, phosphorylation changes associated with activation of ERK2 were seen in PKA knockout cells. The ERK2 site is downstream of a direct PKA site in the Rap1GAP, Sipa1l1, that indirectly inhibits Raf1. In addition, a direct PKA site that inhibits the MAP kinase kinase kinase Map3k5 (ASK1) is upstream of JNK1 activation. The datasets were integrated to identify a causal network describing PKA signaling that explains vasopressin-mediated regulation of membrane trafficking and gene transcription. The model predicts that, through PKA activation, vasopressin stimulates AQP2 exocytosis by inhibiting MAP kinase signaling. The model also predicts that, through PKA activation, vasopressin stimulates Aqp2 transcription through induction of nuclear translocation of the acetyltransferase EP300, which increases histone H3K27 acetylation of vasopressin-responsive genes (confirmed by ChIP-seq).

KLHL3 Knockout Mice Reveal the Physiological Role of KLHL3 and the Pathophysiology of Pseudohypoaldosteronism Type II Caused by Mutant KLHL3.

Mutations in the with-no-lysine kinase 1 (WNK1), WNK4, kelch-like 3 (KLHL3), and cullin3 (CUL3) genes are known to cause the hereditary disease pseudohypoaldosteronism type II (PHAII). It was recently demonstrated that this results from the defective degradation of WNK1 and WNK4 by the KLHL3/CUL3 ubiquitin ligase complex. However, the other physiological in vivo roles of KLHL3 remain unclear. Therefore, here we generated KLHL3-/- mice that expressed ß-galactosidase (ß-Gal) under the control of the endogenous KLHL3 promoter. Immunoblots of ß-Gal and LacZ staining revealed that KLHL3 was expressed in some organs, such as brain. However, the expression levels of WNK kinases were not increased in any of these organs other than the kidney, where WNK1 and WNK4 increased in KLHL3-/- mice but not in KLHL3+/- mice. KLHL3-/- mice also showed PHAII-like phenotypes, whereas KLHL3+/- mice did not. This clearly demonstrates that the heterozygous deletion of KLHL3 was not sufficient to cause PHAII, indicating that autosomal dominant type PHAII is caused by the dominant negative effect of mutant KLHL3. We further demonstrated that the dimerization of KLHL3 can explain this dominant negative effect. These findings could help us to further understand the physiological roles of KLHL3 and the pathophysiology of PHAII caused by mutant KLHL3.

Photoinduced bending of rod-like millimetre-size crystals of a rhodium dithionite complex with n-pentyl moieties.

Rod-like millimetre-size crystals of a newly prepared rhodium dithionite complex with n-pentyl moieties bend upon photoirradiation and return to the initial shape upon heating; the roles of the flexible n-pentyl moieties as well as the photoreactive dithionite unit (µ-O2SSO2) are disclosed by single crystal X-ray diffraction.

Generation and analysis of knock-in mice carrying pseudohypoaldosteronism type II-causing mutations in the cullin 3 gene.

Pseudohypoaldosteronism type II (PHAII) is a hereditary hypertensive disease caused by mutations in four different genes: with-no-lysine kinases (WNK) 1 and 4, Kelch-like family member 3 (KLHL3), and cullin 3 (Cul3). Cul3 and KLHL3 form an E3 ligase complex that ubiquitinates and reduces the expression level of WNK4. PHAII-causing mutations in WNK4 and KLHL3 impair WNK4 ubiquitination. However, the molecular pathogenesis of PHAII caused by Cul3 mutations is unclear. In cultured cells and human leukocytes, PHAII-causing Cul3 mutations result in the skipping of exon 9, producing mutant Cul3 protein lacking 57 amino acids. However, whether this phenomenon occurs in the kidneys and is responsible for the pathogenesis of PHAII in vivo is unknown. We generated knock-in mice carrying a mutation in the C-terminus of intron 8 of Cul3, c.1207-1G>A, which corresponds to a PHAII-causing mutation in the human Cul3 gene. Heterozygous Cul3(G(-1)A/+) knock-in mice did not exhibit PHAII phenotypes, and the skipping of exon 9 was not evident in their kidneys. However, the level of Cul3 mRNA expression in the kidneys of heterozygous knock-in mice was approximately half that of wild-type mice. Furthermore, homozygous knock-in mice were nonviable. It suggested that the mutant allele behaved like a knockout allele and did not produce Cul3 mRNA lacking exon 9. A reduction in Cul3 expression alone was not sufficient to develop PHAII in the knock-in mice. Our findings highlighted the pathogenic role of mutant Cul3 protein and provided insight to explain why PHAII-causing mutations in Cul3 cause kidney-predominant PHAII phenotypes.

Impaired degradation of WNK by Akt and PKA phosphorylation of KLHL3.

Mutations in with-no-lysine kinase (WNK) 1, WNK4, Kelch-like 3 (KLHL3), and Cullin3 result in an inherited hypertensive disease, pseudohypoaldosteronism type II. WNK activates the Na-Cl cotransporter (NCC), increasing sodium reabsorption in the kidney. Further, KLHL3, an adapter protein of Cullin3-based E3 ubiquitin ligase, has been recently found to bind to WNK, thereby degrading them. Insulin and vasopressin have been identified as powerful activators of WNK signaling. In this study, we investigated effects of Akt and PKA, key downstream substrates of insulin and vasopressin signaling, respectively, on KLHL3. Mass spectrometry analysis revealed that KLHL3 phosphorylation at S433. Phospho-specific antibody demonstrated defective binding between phosphorylated KLHL3 and WNK4. Consistent with the fact that S433 is a component of Akt and PKA phosphorylation motifs, in vitro kinase assay demonstrated that Akt and PKA can phosphorylate KLHL3 at S433, that was previously reported to be phosphorylated by PKC. Further, forskolin, a representative PKA stimulator, increased phosphorylation of KLHL3 at S433 and WNK4 protein expression in HEK293 cells by inhibiting the KLHL3 effect that leads to WNK4 degradation. Insulin also increased phosphorylation of KLHL3 at S433 in cultured cells. In conclusion, we found that Akt and PKA phosphorylated KLHL3 at S433, and phosphorylation of KLHL3 by PKA inhibited WNK4 degradation. This could be a novel mechanism on how insulin and vasopressin physiologically activate the WNK signal.

PiCO2 monitoring of transferred jejunum perfusion using an air tonometry technique after hypopharyngeal cancer surgery.

This study aimed to investigate the usefulness of intraluminal PCO2 (PiCO2) monitoring by air tonometry for the assessment of the vascular condition of the transferred jejunum after surgery for hypopharyngeal cancer.PiCO2 in the transplanted jejunum of 24 patients was monitored using air tonometry after radical surgery for hypopharyngeal cancer from 2003 to 2010.All but 1 patient, who removed the catheter before monitoring began, were monitored safely. PiCO2 in the transferred jejunum correlated with arterial PCO2 (PaCO2) that was measured concurrently, and dissociation of PiCO2 from PaCO2 was observed in cases with vascular complication. In those cases without postoperative vascular complication, the PiCO2 value gradually increased for 3 hours but then decreased by 12 hours after surgery. Three patients experienced major vascular complication. All 3 patients had continuous elevation of PiCO2 >100 mm Hg, although vascular flow in 1 patient recovered by removal of a venous thrombosis and reanastomosis of the vein 7.5 hours after surgery. Four other patients who experienced elevation of PiCO2 had their skin suture released for decompression of their neck wound, resulting in a decrease in PiCO2 after treatment.The current results demonstrated that continuous monitoring of PiCO2 by air tonometry accurately reflects the vascular condition of the transferred jejunum, and this method is one of the best options for postoperative monitoring of jejunum blood perfusion.