Self-Aggregation, Enhanced Guest-Binding Behavior, and Anion-Induced Agglutination of Cyclophane Dimers Linked with Florescent Perylene Diimide.

Cationic, anionic, and nonionic cyclophane dimers, 1a, 1b, and 1c, covalently linked with fluorescent perylene diimide were synthesized, respectively, as a host for aggregation-induced multivalent effects on guest-binding. These PDI-linked cyclophane dimers 1a and 1b, especially 1c, formed self-aggregates in H2O-rich solvents through π-stacking interactions. Critical aggregation concentrations of 1a, 1b, and 1c in H2O were 4.0 × 10-6, 4.0 × 10-6, and less than 0.1 × 10-6 M, respectively. The self-aggregates of the PDI-linked cyclophane dimers in H2O were several hundred nanometers in size by DLS and TEM measurements. In an aggregated state, 1a exhibited guest-binding abilities toward hydrophobic guests, such as 6-p-toluidinonaphthalene-2-sulfonate and 6-anilino-naphthalene-2-sulfonate. Moreover, anion-induced agglutination of 1a was observed upon the addition of phosphate ions, and its effectiveness was as follows: ATP > ADP > H2P2O72- > AMP ≈ PO43- > HPO42- > H2PO4-. The contribution of the adenine base of the nucleotides as a guest moiety used for effective agglutination was confirmed by 1H NMR spectroscopy.

A Significant Increase in the Serum Carbohydrate Antigen 19-9 Level Accompanied by Acute Cholecystitis and Choledocholithiasis: A Case Report and Review of the Literature.

An 80-year-old man with jaundice and fatigue was referred to our hospital. A laboratory examination revealed increased levels of hepatobiliary enzymes, and CA19-9 levels increased to 29,512 U/mL. Based on the findings of imaging examination and laboratory data, the patient was diagnosed with acute cholecystitis and choledocholithiasis. The possibility of malignancy could not be ruled out because of the high levels of CA19-9. Antibiotic administration was commenced, and the common bile duct stone was endoscopically removed. One month after treatment, the CA19-9 level decreased to within the normal range. One year after treatment, imaging examinations did not reveal any malignancy.

Formal Total Synthesis of Manzacidin B via Sequential Diastereodivergent Henry Reaction.

A formal total synthesis of manzacidin B is described. ß,ß-Disubstituted γ-hydroxy-ß-aminoalcohol, the key structure of manzacidin B, is stereoselectively constructed via sequential Henry reactions. By taking advantage of noncovalent interactions, such as intramolecular hydrogen bonding and chelation, we could diastereodivergently control the stereoselectivity of the Henry reaction.

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.

Drug-Repositioning Screening for Keap1-Nrf2 Binding Inhibitors using Fluorescence Correlation Spectroscopy.

The Kelch-like ECH-associating protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. The Cul3/Keap1 E3 ubiquitin ligase complex interacts with Nrf2, leading to Nrf2 ubiquitination and degradation. In this study, we focused on the disruption of the Keap1-Nrf2 interaction to upregulate Nrf2 expression and the transcription of ARE-controlled cytoprotective oxidative stress response enzymes, such as HO-1. We completed a drug-repositioning screening for inhibitors of Keap1-Nrf2 protein-protein interactions using a newly established fluorescence correlation spectroscopy (FCS) screening system. The binding reaction between Nrf2 and Keap1 was successfully detected with a KD of 2.6 µM using our FCS system. The initial screening of 1,633 drugs resulted in 12 candidate drugs. Among them, 2 drugs significantly increased Nrf2 protein levels in HepG2 cells. These two promising drugs also upregulated ARE gene promoter activity and increased HO-1 mRNA expression, which confirms their ability to dissociate Nrf2 and Keap1. Thus, drug-repositioning screening for Keap1-Nrf2 binding inhibitors using FCS enabled us to find two promising known drugs that can induce the activation of the Nrf2-ARE pathway.

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.

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.

Kelch-Like Protein 2 Mediates Angiotensin II-With No Lysine 3 Signaling in the Regulation of Vascular Tonus.

Recently, the kelch-like protein 3 (KLHL3)-Cullin3 complex was identified as an E3 ubiquitin ligase for with no lysine (WNK) kinases, and the impaired ubiquitination of WNK4 causes pseudohypoaldosteronism type II (PHAII), a hereditary hypertensive disease. However, the involvement of WNK kinase regulation by ubiquitination in situations other than PHAII has not been identified. Previously, we identified the WNK3-STE20/SPS1-related proline/alanine-rich kinase-Na/K/Cl cotransporter isoform 1 phosphorylation cascade in vascular smooth muscle cells and found that it constitutes an important mechanism of vascular constriction by angiotensin II (AngII). In this study, we investigated the involvement of KLHL proteins in AngII-induced WNK3 activation of vascular smooth muscle cells. In the mouse aorta and mouse vascular smooth muscle (MOVAS) cells, KLHL3 was not expressed, but KLHL2, the closest homolog of KLHL3, was expressed. Salt depletion and acute infusion of AngII decreased KLHL2 and increased WNK3 levels in the mouse aorta. Notably, the AngII-induced changes in KLHL2 and WNK3 expression occurred within minutes in MOVAS cells. Results of KLHL2 overexpression and knockdown experiments in MOVAS cells confirmed that KLHL2 is the major regulator of WNK3 protein abundance. The AngII-induced decrease in KLHL2 was not caused by decreased transcription but increased autophagy-mediated degradation. Furthermore, knockdown of sequestosome 1/p62 prevented the decrease in KLHL2, suggesting that the mechanism of KLHL2 autophagy could be selective autophagy mediated by sequestosome 1/p62. Thus, we identified a novel component of signal transduction in AngII-induced vascular contraction that could be a promising drug target.

WNK4 is the major WNK positively regulating NCC in the mouse kidney.

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.

Decrease of WNK4 ubiquitination by disease-causing mutations of KLHL3 through different molecular mechanisms.

Recently, we demonstrated that WNK4 is a substrate for KLHL3-Cullin3 (CUL3) E3 ubiquitin ligase complexes and that impaired WNK4 ubiquitination is a common mechanism for pseudohypoaldosteronism type II (PHAII) caused by WNK4, KLHL3, and CUL3 mutations. Among the various KLHL3 mutations that cause PHAII, we demonstrated that the R528H mutation in the Kelch domain decreased the binding to WNK4, thereby causing less ubiquitination and increased intracellular levels of WNK4. However, the pathogenic mechanisms of PHAII caused by other KLHL3 mutants remain to be determined. In this study, we examined the pathogenic effects of three PHAII-causing mutations in different KLHL3 domains; the protein levels of these mutants significantly differed when they were transiently expressed in HEK293T cells. In particular, S410L expression was low even with increased plasmid expression. The cycloheximide chase assay revealed that an S410L mutation in the Kelch domain significantly decreased the intracellular stability. Mutations in E85A in the BTB domain and C164F in the BACK domain decreased the binding to CUL3, and S410L as well as R528H demonstrated less binding to WNK4. In vitro and in vivo assays revealed that these mutants decreased the ubiquitination and increased the intracellular levels of WNK4 compared with wild-type KLHL3. Therefore, the KLHL3 mutants causing PHAII investigated in this study exhibited less ability to ubiquitinate WNK4 because of KLHL3's low stability and/or decreased binding to CUL3 or WNK4.

Impaired KLHL3-mediated ubiquitination of WNK4 causes human hypertension.

Mutations in WNK kinases cause the human hypertensive disease pseudohypoaldosteronism type II (PHAII), but the regulatory mechanisms of the WNK kinases are not well understood. Mutations in kelch-like 3 (KLHL3) and Cullin3 were also recently identified as causing PHAII. Therefore, new insights into the mechanisms of human hypertension can be gained by determining how these components interact and how they are involved in the pathogenesis of PHAII. Here, we found that KLHL3 interacted with Cullin3 and WNK4, induced WNK4 ubiquitination, and reduced the WNK4 protein level. The reduced interaction of KLHL3 and WNK4 by PHAII-causing mutations in either protein reduced the ubiquitination of WNK4, resulting in an increased level of WNK4 protein. Transgenic mice overexpressing WNK4 showed PHAII phenotypes, and WNK4 protein was indeed increased in Wnk4(D561A/+) PHAII model mice. Thus, WNK4 is a target for KLHL3-mediated ubiquitination, and the impaired ubiquitination of WNK4 is a common mechanism of human hereditary hypertension.

A loss-of-function mutation in the PAS kinase Rim15p is related to defective quiescence entry and high fermentation rates of Saccharomyces cerevisiae sake yeast strains.

Sake yeast cells have defective entry into the quiescent state, allowing them to sustain high fermentation rates. To reveal the underlying mechanism, we investigated the PAS kinase Rim15p, which orchestrates initiation of the quiescence program in Saccharomyces cerevisiae. We found that Rim15p is truncated at the carboxyl terminus in modern sake yeast strains as a result of a frameshift mutation. Introduction of this mutation or deletion of the full-length RIM15 gene in a laboratory strain led to a defective stress response, decreased synthesis of the storage carbohydrates trehalose and glycogen, and impaired G(1) arrest, which together closely resemble the characteristic phenotypes of sake yeast. Notably, expression of a functional RIM15 gene in a modern sake strain suppressed all of these phenotypes, demonstrating that dysfunction of Rim15p prevents sake yeast cells from entering quiescence. Moreover, loss of Rim15p or its downstream targets Igo1p and Igo2p remarkably improved the fermentation rate in a laboratory strain. This finding verified that Rim15p-mediated entry into quiescence plays pivotal roles in the inhibition of ethanol fermentation. Taken together, our results suggest that the loss-of-function mutation in the RIM15 gene may be the key genetic determinant of the increased ethanol production rates in modern sake yeast strains.

Does a proton pump inhibitor cause hypokalemia?

Proton pump inhibitors (PPIs) act only in the stomach, although the proton pump, H(+),K(+)-ATPase exists and contributes to H(+) and K(+) homeostasis in the kidney. We encountered two hypokalemic cases receiving omeprazole. These cases were women ages 69 and 80 years old. Their serum potassium levels decreased with accelerated urinary potassium excretion with the use of omeprazole, and recovered by potassium-supplement and the discontinuation of omeprazole. Because inhibitory effects of PPIs on H(+),K(+)-ATPase are exerted only in acidic condition, hypokalemia is not generally introduced by PPIs alone. However, in extreme alkalosis or impaired K(+)-recycling system, PPIs may cause hypokalemia unrelated to hypomagnesemia.

Successful treatment of hypertension in anuric hemodialysis patients with a direct Renin inhibitor, aliskiren.

OBJECTIVE: A direct renin-inhibitor (DRI), aliskiren, was administered to anuric patients to investigate whether it can be a new optional therapy against hypertension in hemodialysis (HD) patients. PATIENTS: The patients that received aliskiren comprised 8 males and 2 females with a mean ± SD age of 63 ± 8 years (43-72 years). They were exposed to dialysis therapy for 118 ± 73 months (8-251 months), with diabetes mellitus in 4 cases, chronic glomerulonephritis in 4 cases, and other diagnoses in 2 cases. METHODS: After the plasma renin activity (PRA) and plasma aldosterone concentration (PAC) were measured before an HD session, aliskiren, 150 mg as an initial dose, was administered to the patients. PRA and PAC were also examined a week after initiating aliskiren. The blood pressure (BP) levels at the start of each HD session for a period of 2 weeks (6 HD sessions) were compared between before and after administration of aliskiren. The change of doses in other antihypertensive agents was also counted. RESULTS: The averaged reduction of mean blood pressure was 4 ± 5 mmHg, and doses of antihypertensives other than aliskiren were reduced in 4 patients. Of the examined parameters, only the reduction rate of PRA x PAC seemed correlated with the BP lowering effect of aliskiren, which was calculated as the sum of the mean BP reduction in mmHg and drug reduction with 1 tablet (capsule)/day considered to be 10 mmHg. CONCLUSION: A DRI, aliskiren, was effective even in anuric dialysis patients, and monitoring of PRA and PAC was valuable for selecting cases responsive to aliskiren.

Measurement of glomerular filtration rate by rapid intravenous injection of a newly developed inulin fraction.

OBJECTIVE: Since the conventional drip-infusion method for measuring inulin clearance (Cin) has problems related to its accuracy and performance, we explored a more accurate and concise method by rapid intravenous injection of a newly developed inulin fraction (Inulead(®)), in which spot urine sampling was omitted and the administration period of inulin was shortened from 120 to 5 minutes. PATIENTS AND METHODS: Twenty seven patients (M/F: 15/12, 67.8 ± 12.9 years old) admitted to the Nephrology ward were enrolled in this study. Inulead(®), 1500 mg dissolved in 150 mL of saline, was intravenously administered in 5 minutes. Then, sequential blood samplings and urine collection were performed for 24 hours. Cins were calculated by the following three formulae: (1) a pharmacokinetic analysis using a two compartments model based on the plasma inulin concentration to determine Cin, which was the administered dose divided by the area under the curve (AUC) from 0 to ∞, (2) urinary inulin excretion divided by the AUC for 24 hours and (3) the Bayesian method using a three-point set of plasma inulin concentrations to predict the change of inulin concentration to determine Cin as in 1. These Cins were compared with levels of estimated GFR (eGFR), creatinine clearance (Ccr), serum ß2 microglobulin (ß2MG) and serum cystatin C (Cys C). RESULTS: Cins obtained by the above three methods were well correlated with each other (r. = 0.9088 (- )0.9998) and with eGFR (r. = 0.8286 - 0.8650), Ccr (r. = 0.821 - 0.864), 1/ß2MG (r. = 0.631 - 0.752) and 1/CysC (r. = 0.830 - 0.857). The averaged differences of each Cin from eGFR were distributed between - 4.4 and - 4.5 mL/min. CONCLUSION: Since the Cins by rapid inulin injection showed satisfactory correlation and differences with other GFR parameters, this method will be a good alternative to the drip infusion method, and may reduce the burden of patients and medical staff.