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.

Background: We previously reported that survivin-2B, a splicing variant of survivin, was expressed in various types of tumors and that survivin-2B peptide might serve as a potent immunogenic cancer vaccine. The objective of this study was to examine the toxicity of and to clinically and immunologically evaluate survivin-2B peptide in a phase I clinical study for patients with advanced or recurrent breast cancer.

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.

In 2005, we initiated a clinical trial that examined the efficacy of the oncolytic virus HF10 to treat pancreatic cancer. Pancreatic cancer continues to have a high mortality rate, despite multimodal treatments for patients, and new therapeutic methods are greatly needed. The current mainstream methods for cancer treatment include biological therapeutics such as trastuzumab (Herceptin) for breast cancer or erlotinib (Tarceva) for non-small cell lung cancer. Oncolytic virus therapy is a new and promising treatment strategy for cancer. Oncolytic viruses are novel biological therapeutics for advanced cancer that appear to have a wide spectrum of anticancer activity with minimal human toxicity. To examine the efficacy of oncolytic virus therapy for pancreatic cancer, we initiated pilot studies by injecting six patients with non-resectable pancreatic cancer with three doses of HF10. All patients were monitored for 30 days for local and systemic adverse effects and were not administered any other therapeutics during this period. There were no adverse side-effects, and we observed some therapeutic potential based on tumor marker levels, survival, pathological findings and diagnostic radiography. The tumors were classified as stable disease in three patients, partial response in one patient and progressive disease in two patients.

The NaCl cotransporter (NCC) is essential for sodium reabsorption at the distal convoluted tubules (DCT), and its phosphorylation increases its transport activity and apical membrane localization. Although insulin has been reported to increase sodium reabsorption in the kidney, the linkage between insulin and NCC phosphorylation has not yet been investigated. This study examined whether insulin regulates NCC phosphorylation. In cultured mpkDCT cells, insulin increased phosphorylation of STE20/SPS1-related proline-alanine-rich kinase (SPAK) and NCC in a dose-dependent manner. This insulin-induced phosphorylation of NCC was suppressed in WNK4 and SPAK knockdown cells. In addition, Ly294002, a PI3K inhibitor, decreased the insulin effect on SPAK and NCC phosphorylation, indicating that insulin induces phosphorylation of SPAK and NCC through PI3K and WNK4 in mpkDCT cells. Moreover, acute insulin administration to mice increased phosphorylation of oxidative stress-responsive kinase-1 (OSR1), SPAK and NCC in the kidney. Time-course experiments in mpkDCT cells and mice suggested that SPAK is upstream of NCC in this insulin-induced NCC phosphorylation mechanism, which was confirmed by the lack of insulin-induced NCC phosphorylation in SPAK knockout mice. Moreover, insulin administration to WNK4 hypomorphic mice did not increase phosphorylation of OSR1, SPAK and NCC in the kidney, suggesting that WNK4 is also involved in the insulin-induced OSR1, SPAK and NCC phosphorylation mechanism in vivo. The present results demonstrated that insulin is a potent regulator of NCC phosphorylation in the kidney, and that WNK4 and SPAK are involved in this mechanism of NCC phosphorylation by insulin.