A. Liantonio¹, G. Gramegna¹, G. M. Camerino¹, M. M. Dinardo¹, G. Procino²,D. R. Lasorsa²,L. Mastrofrancesco², A. Laghezza³, G. Fracchiolla³, F. Loiodice³, M. G. Perrone³, A. Lopedota³, S. Conte¹, R. Penza⁴, G. Valenti², M. Svelto², D. Conte Camerino^1
 
1Section of Pharmacology, Department of Pharmacobiology, Faculty of Pharmacy, University of Bari, Italy, ²Department of General and Environmental Physiology, University of Bari, Italy, ³Department of Medicinal Chemistry, Faculty of Pharmacy, University of Bari, Italy, ⁴Department of BioMedicine of Developing Age, University of Bari, Italy
 

The human kidney-specific chloride channels ClC-Ka (rodent ClC-K1) and ClC-Kb (rodent ClC-K2), and their auxiliary subunit barttin, are important determinants of renal function, participating to urine concentration (Krämer et al., 2008). Importantly, recent data point out to a possible role of CLC-K channels in blood-pressure regulation. Indeed, although with a large variations between the various ethnic populations, CLCNKA, CLCNKB andBSND are today proposed as candidate genes for hypertension (Lang et al., 2005; Sanada et al., 2011) and molecules capable of inhibiting CLC-K channels are proposed as potential drugs for the treatment of hypertension (Fong., 2004). We recently defined the key structural features responsible for blocking or activating effects of various ligands on heterologously expressed CLC-K and identified a series of benzofuran derivatives as new high affinity blockers for these chloride channels types (Liantonio et al., 2008). Here we tested the hypothesis that CLC-K channelscould represent new drug targets for inducing diuretic effects. To this purpose, we acutely administrated by gavage CLC-K blockers, the benzofuran derivatives MT-189 and RT-93 (10, 50, 100 mgKg^-1), to Wistar rats and by using a plethora of bioanalytical, biochemical and molecular biology assays determined a series of pharmacodynamic and pharmakinetic parameters. MT-189 and RT-93 dose-dependently increased urine volume without affecting electrolyte balance. Plasma drugs concentration values indicate a good bioavailability. Benzofuran derivatives treatment did not affect kidney CLC-K mRNA level as well as inner medulla osmolality while caused a significant vasopressin-independent down-regulation of AQP-2 at protein and transcriptional levels. The evaluation of furosemide effects allowed us also to reveal that ClC-K1 expression is early regulated on dependence of the salt load state. Our findings indicate that the polyuria observed in rats treated with benzofuran derivatives was water diuresis, indirectly supporting a cross-talk between chloride and water transport in nephron. The co-existence of CLC-K channels in the kidney and inner ear arise the question whether pharmacological block mediated by benzofuran derivatives impair auditory function. As preliminary investigation in this context, by performing in vitroexperiments aimed toinvestigate the drugs capability of crossing the blood-inner ear barrier, we also demonstrated that MT-189 and RT-93 represent substrates for P-glycoprotein (P-gp). Based on the fact that P-gp acts as an efflux pump in the inner ear, we concluded that P-gp could prevent ototoxicity. Furthermore, it could also be supposed that benzofuran derivatives using these transport proteins were efficiently secreted into the proximal tubule lumen, thus reaching the pharmacological target in part by this way, as CLC-K channels are also expressed into the luminal membrane of nephron. Accordingly, benzofuran derivatives were found in urine. In conclusion, the ability of these newly synthesized drugs to favor the excretion of an increased urine volume without causing an electrolyte imbalance indicates that CLC-K channels represent new drug targets with a high therapeutic potential. Inhibition of CLC-K could ensure a specific mechanism of action in hypertensive patients carrying CLC-K gene polymorphisms, thus opening the way to a personalized therapy in this field.

Krämer et al. (2008). Nat Clin Pract Nephrol. 4, 38-46.
Lang et al. (2005). Clin Exp Nephrol. 9, 91-99.
Sanada et al. (2011). Curr Hypertens Rep. 13, 55-66.
Fong (2004). EMBO Rep 5, 565-566.
Liantonio et al., (2008). Proc Natl Acad Sci U S A.105, 1369-1373.