Abstract
The physiological role of the renal ClC-Ka/ClC-K1 channels is to confer a high Cl- permeability to the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla that drives water reabsorption from collecting ducts. Here, we investigated by whole-cell patch-clamp measurements on HEK293 cells co-expressing ClC-Ka (tagged with GFP) and the accessory subunit barttin (tagged with m-Cherry) the effect of a natural diuretic extract from roots of Dandelion (DRE), and other compounds activating PKC, such as ATP, on ClC-Ka activity and its membrane localization. Treatment with 400 µg/ml DRE significantly inhibited Cl- currents time-dependently within several minutes. Of note, the same effect on Cl- currents was obtained upon treatment with 100 µM ATP. Pretreatment of cells with either the intracellular Ca2+ chelator BAPTA-AM (30 μM) or the PKC inhibitor Calphostin C (100 nM) reduced the inhibitory effect of DRE. Conversely, 1 µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of DRE on ClC-Ka. Finally, we found that pretreatment with 30 µM Heclin, an E3 ubiquitin ligase inhibitor, did not revert DRE-induced Cl- current inhibition. In agreement with this, live-cell confocal analysis showed that DRE treatment did not induce ClC-Ka internalization. In conclusion, we demonstrate for the first time that the activity of ClC-Ka in renal cells could be significantly inhibited by the activation of PKC elicited by classical maneuvers, such as activation of purinergic receptors, or by exposure to herbal extracts that activates a PKC-dependent pathway. Overall, we provide both new information regarding the regulation of ClC-Ka and a proof-of-concept study for the use of DRE as new diuretic.
Highlights
In the mammalian kidney, the loop of Henle plays a crucial role in the urine concentrating mechanism and in blood pressure regulation
ClC-K1 KO mice failed to concentrate urine after 24-h water deprivation or intraperitoneal injection of dDAVP11. These findings suggested that ClC-Ka and ClC-Kb have different physiological functions in the kidney, with ClC-Ka contributing mostly to the high osmolarity of the medulla and ClC-Kb retrieving most of the chloride that is left in the urine, making ClC-Ka, rather than ClC-Kb, a very interesting target for diuretics
Dandelion Root Extract (DRE) time-dependently inhibits the Cl− current in HEK293 cells co-expressing ClC-Ka and barttin
Summary
The loop of Henle plays a crucial role in the urine concentrating mechanism and in blood pressure regulation. In the thick ascending limb (TAL), the functional cooperation between the apical Na+/2Cl−/K+ cotransporter (NKCC2) and the inwardly rectifying potassium channel (ROMK1) with the basolateral complex of ClC-Kb/barttin results in a transcellular and electrogenic NaCl reabsorption. This salt movement toward the interstitium generates the cortico-medullary osmotic gradient needed for water reabsorption in the collecting ducts, where the vasopressin-dependent AQP2 translocation on the luminal membrane of collecting duct cells renders collecting ducts permeable to water. At the best of our knowledge here we reported the first evidence regarding the regulation of ClC-Ka by a PKC-associated signaling pathway with several physiological and translational implications
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