Intracellular Chloride and Scaffold Protein Mo25 Cooperatively Regulate Transepithelial Ion Transport through WNK Signaling in the Malpighian Tubule

Abstract

With No Lysine kinase (WNK) signaling regulates mammalian renal epithelial ion transport to maintain electrolyte and BP homeostasis. Our previous studies showed a conserved role for WNK in the regulation of transepithelial ion transport in the Malpighian tubule. Using assays and transgenic lines, we examined two potential WNK regulators, chloride ion and the scaffold protein mouse protein 25 (Mo25), in the stimulation of transepithelial ion flux., autophosphorylation of purified WNK decreased as chloride concentration increased. In conditions in which tubule intracellular chloride concentration decreased from 30 to 15 mM as measured using a transgenic sensor, WNK activity acutely increased. WNK activity in tubules also increased or decreased when bath potassium concentration decreased or increased, respectively. However, a mutation that reduces chloride sensitivity of WNK failed to alter transepithelial ion transport in 30 mM chloride. We, therefore, examined a role for Mo25. In kinase assays, Mo25 enhanced the activity of the WNK downstream kinase Fray, the fly homolog of mammalian Ste20-related proline/alanine-rich kinase (SPAK), and oxidative stress-responsive 1 protein (OSR1). Knockdown of in the Malpighian tubule decreased transepithelial ion flux under stimulated but not basal conditions. Finally, whereas overexpression of wild-type , with or without, did not affect transepithelial ion transport, overexpressed with chloride-insensitive increased ion flux. Cooperative interactions between chloride and Mo25 regulate WNK signaling in a transporting renal epithelium.

The chloride anion as a signalling effector

“…However, other authors (Miyazaki et al ., ; Hiraoka et al ., ; Ohsawa et al ., ) found a reduction of extracellular Cl − ‐induced p38 and JNK activation. Something similar occurs with WNK Cl − sensors, which are negatively modulated by increased [Cl − ] (Sun et al ., ). However, SGK1 showed a response to Cl − similar to that seen for IL‐1β, in which the activity increased with [Cl − ]; in both cases in the presence of nigericin and tributyltin (Clauzure et al ., ; Zhang et al ., ).…”

Three-dimensional architecture of pericardial nephrocytes in Drosophila melanogaster revealed by FIB/SEM tomography

“…Pterygote insects, including the fruit fly (Drosophila melanogaster), are the exception in that they do not possess filtration epithelia; instead, they produce urine via epithelial transport of the modulating tubules that are called Malpighian tubules ( Fig. 1e) (Kolosov et al 2018;Sun et al 2018).…”

Kidney Is Essential for Blood Pressure Modulation by Dietary Potassium

“…Piala et al reported that chloride binds and regulates WNK activity [88], supporting the hypothesis that chloride and WNKs serve as the secondary messengers to regulate apical membrane NaCl transport. At a higher intracellular concentration, chloride binds to the WNK catalytic domain and prevents kinase autophosphorylation and activation [88][89][90][91][92]. The intracellular chloride concentration ([Cl − ] i ) is dependent on the rate of chloride entry into and exit out of cells.…”