Short‐ and Long‐Term Furosemide Treatment Triggers WNK Body Formation in the Rat Distal Nephron

Abstract

The diuretic furosemide induces water and electrolyte excretion by inhibition of the type 2 Na+, K+, 2Cl−-cotransporter in the thick ascending limb (TAL) of the kidney. By virtue of its rapid onset of action, furosemide is paramount in the management of acute fluid and electrolyte disorders. It is not a preferred agent in conditions such as chronic hypertension, as an induction of salt reabsorption downstream of the TAL occurs in long-term treatment, causing furosemide resistance. This is partly mediated by a kinase cascade located in the distal convoluted tubule (DCT) involving with no lysine (WNK) kinases, which phosphorylate Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1). Phosphorylated SPAK/OSR1 activate their substrate, the Na+, Cl−-cotransporter (NCC), increasing salt reabsorption. We aimed to examine the effects of furosemide on the distribution pattern of WNKs and SPAK/OSR1 in the DCT. A particular property of WNK and SPAK/OSR1 kinases is that they assemble in discrete cytoplasmic domains (“WNK bodies”) in several other models of NCC activation, especially in hypokalemic settings; we postulated that this effect is inducible by furosemide. Wistar rats were treated with intraperitoneal furosemide for 2 h or daily for 7 d. Kidney sections were examined by a range of microscopic techniques including super-resolution fluorescence microscopy and electron microscopy. In immunogold stainings, SPAK/WNK kinases concentrated in spherical, electron-hypodense, non-membrane-bound structures after 2 h and 7 d treatments. To assess their distribution along the nephron, we performed immunofluorescent stainings of SPAK and marker proteins of the aldosterone-insensitive, early DCT (DCT1) or aldosterone-sensitive distal nephron (ASDN). WNK bodies were similarly enriched in both. In further immunostainings, we detected the active, phosphorylated isoforms of WNKs and SPAK/OSR1 within WNK bodies. In ultrastructural and STED evaluations, we could consistently show an adjacency of WNK bodies to the Golgi apparatus and microtubules (see images below). Our work demonstrates that WNK bodies can form within 2 hours in vivo and that WNK bodies arising as a consequence of furosemide treatment are present in the DCT1 and ASDN. Their consistent association with structures of cellular trafficking (Golgi and microtubules) is intriguing regarding several functional possibilities: the reported enrichment of proteasomes in WNK bodies suggests the centralization of kinase degradation, while the presence of active kinase isoforms in WNK bodies are consistent with the notion that WNK-SPAK signaling may occur in the WNK body environment. Our finding of phosphorylated WNKs and SPAK/OSR1 therein is at odds with parts of the published literature; this discrepancy highlights the need for conclusive studies regarding the phosphorylation status of WNK body components. Support or Funding Information Deutsche Forschungsgemeinschaft (MU 2924/2-1,2 und BA 700/22-1,2; INST 335/596-1 FUGG) WNK bodies in rat DCT after 7 days of furosemide treatment A Electron micrograph of a WNK body (asterisk) with Golgi dictyosomes (arrow) and microtubules (arrowheads) in the neighboring cytoplasm. B Immunofluorescent staining of Golgi protein Gm130 and WNK body component SPAK. SPAK has accumulated in the vicinity of Gm130-positive structures surrounding the nucleus of a DCT cell (dashed line = nuclear membrane). C Immunofluorescent staining of tubulin and WNK body component WNK4. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.