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.

Involvement of selective autophagy mediated by p62/SQSTM1 in KLHL3-dependent WNK4 degradation.

We reported that kelch-like protein 3 (KLHL3)-Cullin3 E3 ligase ubiquitinates with-no-lysine kinase 4 (WNK4) and that impaired WNK4 ubiquitination causes pseudohypoaldosteronism type II, a hereditary hypertensive disease. However, we also found that KLHL3-induced WNK4 degradation could not be inhibited completely by a proteasome inhibitor. Rather, on exposure, for 24 h, of HEK293T cells expressing WNK4 and KLHL3 to a proteasome inhibitor, epoxomicin, the WNK4 protein level was further decreased. As proteasome inhibition is known to activate p62-mediated selective autophagy, we investigated whether WNK4 degradation induced by KLHL3 is also mediated by such an autophagic mechanism. 3-Methyladenine, an autophagy inhibitor, blocked the epoxomicin-induced decrease in WNK4. Co-immunoprecipitation assays revealed that KLHL3 formed a complex not only with WNK4 but also with p62 via its kelch repeat domain. Under proteasome inhibition, p62 overexpression decreased KLHL3 and WNK4 protein levels, and p62 knockdown dramatically increased KLHL3 and WNK4 protein levels. Based on immunofluorescent staining, transiently overexpressed WNK4 showed punctate localization in the cytoplasm where it co-localized with KLHL3, p62 and light chain 3, a marker of autophagosomes. Thus, WNK4 was degraded not only by proteasomes but also by p62-KLHL3-mediated selective autophagy, which may be involved in WNK regulation under certain pathophysiological conditions.

Unusual bleeding from hepaticojejunostomy controlled by adult variable stiffness colonoscopy: report of a case and literature review.

We herein present a case of a 59-year-old man who had undergone pylorus preserving pancreaticoduodenectomy with regional lymph node dissection prior to episodes of melena. Series of conventional endoscopic investigations failed to identify the bleeding source. Enhanced computed tomography scan revealed complete obstruction of the main portal vein with numerous collateral veins running towards the hepatic hilus. Comprehensively, hemorrhage from the jejunal varices caused by postoperative portal hypertension was highly suspected. As the jejunal loop was out of reach, adult variable-stiffness colonoscope (AVSC) was utilized to solve the Roux-en-Y anatomy. Numerous telangiectasis and small varices at hepaticojejunostomy were observed and in the mean time, bleeding was noticed and endoclips were placed without any delay. Ectopic variceal bleeding in jejunal loop after pancreaticoduodenectomy is difficult to manage. We believe that AVSC is an alternative device when specialized jejunal endoscopy is not available.

Primary papillary hyperplasia of the gallbladder mimicking gallbladder cancer.

Primary papillary hyperplasia of the gallbladder (PPHG) is a rare entity. PPHG is a benign diffuse mucosal projection without any background chronic inflammation-related disease of the gallbladder or bile ducts. Reported cases of PPHG are limited in that its characteristics are not well defined. We herein report a case of PPHG mimicking gallbladder cancer in radiologic investigations and present a review of the literature. Also coincident erythroderma is discussed.

Copper-deficiency anemia after esophagectomy: A pitfall of postoperative enteral nutrition through jejunostomy.

INTRODUCTION: Copper deficiency leads to functional disorders of hematopoiesis and neurological system. There have been some reports of copper deficiency occurring to the patients on enteral nutrition through a jejunostomy in long-term-care hospitals. However, it is extremely rare to find patients with copper deficiency several months after esophagectomy, regardless of enteral nutrition through the jejunostomy. To the best of our knowledge, this is the first case report of a patient who experienced copper-deficiency anemia after esophagectomy and subsequent enteral nutrition through the jejunostomy. PRESENTATION OF CASE: A 73-year-old man presented with pulmonary failure after esophagectomy for esophageal cancer with video-assisted thoracoscopic surgery, and needed long-term artificial ventilator support. Nutritional management included enteral nutrition through a jejunostomy from the early postoperative period. Copper-deficiency anemia was detected 3 months postoperatively; therefore, copper supplementation with cocoa powder was performed, and both serum copper and hemoglobin levels subsequently recovered. DISCUSSION: Copper-deficiency anemia has already been reported to occur in patients receiving enteral nutrition in long-term care hospitals. However, this is the first case report of copper deficiency after esophagectomy despite administration of standard enteral nutrition through the jejunostomy for several months. CONCLUSION: It is extremely rare to find copper-deficiency anemia several months after esophagectomy followed by enteral nutrition through the jejunostomy. However, if anemia of unknown origin occurs in such patients, copper-deficiency anemia must be considered among the differential diagnoses.

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.

KLHL2 interacts with and ubiquitinates WNK kinases.

Mutations in the WNK1 and WNK4 genes result in an inherited hypertensive disease, pseudohypoaldosteronism type II (PHAII). Recently, the KLHL3 and Cullin3 genes were also identified as responsible genes for PHAII. Although we have reported that WNK4 is a substrate for the KLHL3-Cullin3 E3 ligase complex, it is not clear whether all of the WNK isoforms are regulated only by KLHL3. To explore the interaction of WNKs and other Kelch-like proteins, we focused on KLHL2 (Mayven), a human homolog of Drosophila Kelch that shares the highest similarity with KLHL3. We found that KLHL2, as well as KLHL3, was co-immunoprecipitated with all four WNK isoforms. The direct interaction of KLHL2 with WNKs was confirmed on fluorescence correlation spectroscopy. Co-expression of KLHL2 and Cullin3 decreased the abundance of WNK1, WNK3 and WNK4 within HEK293T cells, and a significant increase of WNK4 ubiquitination by KLHL2 and Cullin3 was observed both in HEK293T cells and in an in vitro ubiquitination assay. These results suggest that KLHL2-Cullin3 also functions as an E3-ligase for WNK isoforms within the body.

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 case of vaginal metastasis of rectal cancer post -operation].

Vaginal metastasis is extremely rare, as is metastatic colorectal cancer. A 78-year-old woman was diagnosed with rectal cancer and uterine fibroid. Low anterior resection and simple hysterectomy was performed and the final diagnosis was fStage IIIa. Adjuvant chemotherapy was not performed. One year after the surgery, she was referred to our hospital with the chief complaint of hematuria. A tumor was observed in the posterior wall of the vagina. We performed vaginal mucosal resection and the pathological diagnosis was metastasis of rectal cancer. One year and 5 months after the surgery, we performed left inguinal node dissection and the pathological diagnosis was metastasis of rectal cancer. The patient has remained disease-free for 3 years and 5 months without adjuvant chemotherapy after resection of the vaginal metastasis.

Phosphorylation of Na-Cl cotransporter by OSR1 and SPAK kinases regulates its ubiquitination.

Na-Cl cotransporter (NCC) is phosphorylated in its amino terminus based on salt intake under the regulation of the WNK-OSR1/SPAK kinase cascade. We have observed that total protein abundance of NCC and its apical membrane expression varies in the kidney based on the phosphorylation status. To clarify the mechanism, we examined NCC ubiquitination status in mice fed low, normal and high salt diets, as well as in a model mouse of pseudohypoaldosteronism type II (PHAII) where NCC phosphorylation is constitutively elevated. Low-salt diet decreased NCC ubiquitination, while high-salt diet increased NCC ubiquitination in the kidney, and this was inversely correlated with total and phosphorylated NCC abundance. In the PHAII model, the ubiquitination of NCC in kidney was also lower when compared to that in wild-type littermates. To evaluate the relationship between phosphorylation and ubiquitination of NCC, we expressed wild-type, phospho-deficient and -mimicking NCC in COS7 cells, and the ubiquitination of immunoprecipitated total and biotinylated surface NCC was evaluated. NCC ubiquitination was increased in the phospho-deficient NCC and decreased in phospho-mimicking NCC in both total and surface NCC. Thus, we demonstrated that NCC phosphorylation decreased NCC ubiquitination, which may contribute to the increase of NCC abundance mostly on plasma membranes.

[mFOLFOX6 and FOLFIRI/bevacizumab treatment in a patient on hemodialysis with metastatic colon cancer].

A 58-year-old woman, who was undergoing peritoneal dialysis( PD) for chronic kidney disease (CKD) and had been operated by sigmoidectomy for early colonic cancer, was diagnosed as peritoneal recurrence of the colonic cancer. Her treatment for CKD was switched from PD to hemodialysis. She was administered mFOLFOX6 therapy(reducing the dose to 70%). Hemodialysis was performed 1 hour after administration of oxaliplatin on day 1 and repeated two days later after the completion of drug administration. No serious adverse events were observed. After 10 courses of mFOLFOX6, an ovarian metastasis was appeared. We then changed the regimen to FOLFIRI (70% dose)/bevacizumab (BV). Neutropenia (grade 4) was observed after the second treatment. After some rest, 21 courses of FOLFIRI/BV therapy were performed safely by reducing the dose to 60%. We thought that a reduced dose of FOLFIRI/BV therapy appeared to be safe for a patient with chronic kidney disease who is on hemodialysis.

[A case report of chemoradiotherapy combined with S-1 responding to squamous cell carcinoma of the anal canal].

A 79-year-old woman was reffered to our hospital with the chief complaint of hematochezia. Type-2 tumor was found on anal canal by colonoscopy, and pathologic examination revealed a poorly differentiated squamous cell carcinoma. Computed tomography of the abdomen demonstrated obturatory node metastasis. The patient was diagnosed as having squamous cell carcinoma of the anal cana (l cStage III). After four months from chemoradiation (66 Gy/33 Fr plus S-1), the ulcer side was improved completely to epithelization, and abdominal CT scan showed a remarkable reduction of obturatory node metastasis. She was obtained a complete response. Now the patient is disease-free for ten months after chemoradiation.

[Curative resection of gallbladder cancer with simultaneous liver metastasis].

A 67-year-old man visited our hospital for further check-up of biliary tract disease since his two brothers suffered from biliary tract cancer. Abdominal CT scan revealed a wall thickning at the fundus of gallbladder and its vascularity was rich. Chronic cholecystitis was diagnosed, however, cancer was highly suspected. Cholecystectomy was performed and the frozen section of the gallbladder was compatible for cancer. Therefore, segment-4a and -5 liver resections with regeonal lymph node dissection were added. Although preoperative radiological findings were free of liver metastasis, the resected liver specimen included a nodule of 1 cm in segment-5. Extrahepatic bile duct was not resected because the stump of the cystic duct was free from cancer. The final pathological diagnosis according to the TNM classification was pT3N1M1, Stage IV. We considered the patient to be in the high-risk group of recurrence, adjuvant chemotherapy using both gemcitabine and S-1 was performed. S-1 (80 mg/body/day) was scheduled on day 1-14, and gemcitabine (1,000 mg/body) was scheduled on day 8, day 15. The treatment was continued for two years (a total of 28 courses) without experiencing advese events. The patient is cancer free by means of radiological and hematological studies. Gallbladder cancer with liver metastasis in segment-4a and/or -5 can be considered as "local" metastasis, which a liver resection and adjuvant therapy may lead to a good prognosis.

Calcium-alkali syndrome-like symptoms manifested by daily alphacalcidol and thiazide.

A 73-year-old man was admitted with complaints of a 2-month history of generalized weakness and numbness. Laboratory examination revealed hypercalcemia, metabolic alkalosis, and kidney injury, similar to the traditional milk-alkali syndrome. The clinical history and the response to therapy indicated that alphacalcidol and thiazide taken daily were the cause. Recently, it has been recommended the term "milk-alkali syndrome" be replaced by "calcium-alkali syndrome", which broadens the definition of the condition. This case suggests that the calcium-alkali syndrome can occur without calcium and alkali, but rather with alphacalcidol and a thiazide diuretic.

Assessment and significance of abdominal aortic calcification in chronic kidney disease.

BACKGROUND: Abdominal aortic calcification is a common complication and a predictor of cardiovascular mortality in dialysis patients. However, abdominal aortic calcification in pre-dialysis chronic kidney disease (CKD) is poorly understood. METHODS: A cohort study of 101 adult Japanese patients (mean age 66.6 +/- 11.3 years old) with pre-dialysis CKD (18, 29 and 54 in stages 3, 4 and 5, respectively) was performed. At entry, a non-contrast computed tomography scan was used to determine the abdominal aortic calcification index (ACI). Clinical characteristics and laboratory variables were also assessed. The patients were followed for a mean period of 48 +/- 12 months. RESULTS: Among the subjects, 82% had abdominal aortic calcification (50, 83 and 91% for CKD stages 3, 4 and 5, respectively), and the median ACI was 16.7% (8.5, 20.0 and 21.4%, respectively). Multivariate logistic regression analyses identified older age, presence of diabetes and decreased estimated glomerular filtration rate (e-GFR) as independent predictors of the presence (ACI > 0%) and extent (ACI >or= 20%) of aortic calcification. Multivariate Cox proportional hazards analysis identified ACI >or= 20% and diabetes as independent predictors for de novo cardiovascular events in CKD stages 4 and 5. CONCLUSION: Decreased GFR may be associated with the presence and extent of abdominal aortic calcification, and a high level of calcification may be associated with de novo cardiovascular events in pre-dialysis CKD, suggesting that elucidation of the mechanism through which CKD contributes to vascular calcification may lead to an improved prognosis in patients with pre-dialysis CKD.

[Two cases of pancreatic cancer with fairly good prognosis].

We report two cases of advanced pancreatic cancer whose prognoses are fairly good with surgery and chemotherapy. Case 1: A 71-year-old male patient was diagnosed as pancreatic head cancer by abdominal ultrasound. The tumor size was about 2 cm in diameter. Whipple's procedure and regional lymphadenectomy were conducted. Pathological diagnosis was pT3N2 with s0 and rp1. Gemcitabine (GEM) was administered in a routine fashion at out-patient clinic. He is free of disease after three years and eight months. Case 2: A 63-year-old male patient was diagnosed as pancreatic head cancer although the mass was not so clearly visible by CT. Pylorus preserving pancreaticoduodenectomy was performed with D2 lymph node dissection. Pathological report was pT3N1 with s0 and rp0. GEM was started six months after the operation but continued for only six months. After the non-treatment interval of six months, GEM was restarted due to the sudden elevation of CA19-9. Soon the number dropped but instead of reaching normal range, it began to increase again. S-1 was added to the regimen which gave a great response. He is well after three and a half years. CA19-9 was almost being normal. Both patients had cancer within the pancreas without an invasion to the surrounding tissue. GEM is a standard regimen for adjuvant chemotherapy. However, S-1 may contribute to the outcome when GEM becomes powerless.

[A case of ruptured hepatocellular carcinoma originated from nonalcoholic steatohepatitis (NASH)].

A 69-year-old woman visited our emergency room because of sudden right lower quadrant abdominal pain. Abdominal CT revealed a ruptured tumor in the anterior segment of the liver. Emergency laparotomy was selected due to interventional radiology was not available. As the patient was in shock status with massive intraabdominal hemorrhage, ligation of the right hepatic artery and suturing of the tumor bleeding point was carried out. Given the patient's history and liver biopsy report, we concluded as hepatocellular carcinoma derived form NASH. Anterior resection of the liver was performed after the patient became stable. About one year later, tumor was detected in the lateral segment and was removed. The patient is free of disease for two and a half years after the initial operation. Carcinogenesis of the hepatocellular carcinoma within NASH is not well described. We herein report a case of ruptured hepatocellular carcinoma originated from NASH.

[Gallbladder carcinoma, progressed along cholecystoduodenal fistula--a case report].

A 75-year-old woman had an operation for gallstone ileus without cholecystectomy in other hospital and she was admitted to our hospital because of duodenal adenoma with severe atypia and small carcinoid in proximal duodenal wall. Distal gastrectomy and cholecystectomy were performed. Histological studies revealed the existence of cholecystoduodenal fistula and suggested the existence of gallbladder carcinoma progressed to the duodenal wall through the fistula. Cystic duct dissection and lymph nodes dissection were performed. It has been theorized that a cholecystoduodenal fistula may represent a significant risk factor in the development of gallbladder carcinoma because of the chronic reflux of duodenal contents which includes pancreatic juice. Our case may support this theory. In this case, we thought that the formation of gallbladder cancer could have been avoided if the cholecystectomy was performed in the first operation for gallstone ileus. It is very important that cholecystectomy should be performed when an existence of cholecystoduodenal fistula is highly suspected.

[Case of Henoch-Schönlein purpura nephritis successfully treated with tonsillectomy and steroid pulse therapy].

A 30-year-old Japanese man was admitted to our hospital because of fever, sore throat, abdominal pain, purpura skin lesion of the lower legs, and macrohematuria. On admission, his urine was positive (++) for protein; the sediment contained 100 red blood cells per high-power field, and the daily proteinuria level was 1.7 g. Renal biopsy was performed, and we diagnosed Henoch-Schönlein purpura nephritis (HSPN). Six months after the renal biopsy, the patient underwent a tonsillectomy. The pathological diagnosis of the resected tonsils was chronic tonsillitis. After tonsillectomy, the daily proteinuria had decreased to 0.1 g and the sediment contained only 10-19 red blood cells per high-power field. High-dose methylprednisolone therapy (500 mg/day for 3 days for three courses) was started two weeks after the tonsillectomy, followed by oral prednisolone at the initial dose of 30 mg on alternate days. The oral prednisolone was tapered gradually over 1 year. Antiplatelet drug (dipyridamole, 300 mg/day) and angiotensin II receptor antagonist (olmesartan, 10 mg/day) were also administered. This combination therapy resulted in a significant decrease in proteinuria and disappearance of microhematuria. The patient finally achieved clinical remission. Recent reports have shown that in patients with IgA nephropathy, combined tonsillectomy and methylprednisolone pulse therapy have an effect on clinical remission. In addition, it has been suggested that HSPN and IgA nephropathy represent a spectrum of clinical presentations of similar disorders. The result of this case indicated that this combination therapy had a favorable effect on clinical remission in adult patients with HSPN.