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Deprecated: Implicit conversion from float 233.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534 Sci+Rep 2020 ; 10 (1): 10268 Nephropedia Template TP
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Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka #MMPMID32581267
Gerbino A; De Zio R; Russo D; Milella L; Milano S; Procino G; Pusch M; Svelto M; Carmosino M
Sci Rep 2020[Jun]; 10 (1): 10268 PMID32581267show ga
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 microg/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 microM ATP. Pretreatment of cells with either the intracellular Ca(2+) chelator BAPTA-AM (30 muM) or the PKC inhibitor Calphostin C (100 nM) reduced the inhibitory effect of DRE. Conversely, 1 microM 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 microM 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.
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Sci+Rep 2020 ; 10 (1): 10268
