Anti-WNK3 (aa 1-100) polyclonal antibody

Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl-];) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl-];,respectively. Cell shrinkage and a decrease in [C1]; increase the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl-]; activate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl-];. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC. is not sensitive to [Cl-]; but can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl-]; but is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK's carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC.

Background: The renal distal tubule fine-tunes renal epithelial calcium transport. Dietary intake of salt and fluid varies day-to-day and the kidney adapts accordingly to maintain homeostasis. The alternations in salt and fluid balance affect calcium and magnesium transport in the distal tubule, but the mechanisms are not fully understood. Methods: Sprague-Dawley rats were grouped into high-salt, low-salt and dehydration treatment. Daily intake, water consumption and urine output were recorded. At the end of the experiment, blood and urine samples were collected for hormonal and biochemical tests. Genetic analysis, immunoblotting and immunofluorescence studies were then performed to assess the alterations of calcium and magnesium transport-related molecules. Results: High-salt treatment increased urinary sodium, calcium and magnesium excretion. Low-salt treatment and dehydration were associated with decreased urinary excretion of all electrolytes. High-salt treatment was associated with increased intact parathyroid hormone levels. A significant increase in gene expression of TRPV5, TRPV6, calbindin-D28k and TRPM6 was found during high-salt treatment, while low salt and dehydration diminished expression. These findings were confirmed with immunofluorescence studies. High-salt and low-salt intake or dehydration did not cause any significant changes in WNK1, WNK3 and WNK4. Conclusions: Alternations in salt and water intake affect renal calcium and magnesium handling. High-salt intake increases the distal delivery of the divalent cations which up-regulates distal tubule calcium and magnesium transport molecules, while the opposite effects are associated with low-salt intake or dehydration. Copyright (C) 2013 S. Karger AG, Basel