Abstract
Cyst enlargement in polycystic kidney disease (PKD) involves cAMP-activated proliferation of cyst-lining epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. This study aimed to investigate an inhibitory effect and detailed mechanisms of steviol and its derivatives on cyst growth using a cyst model in Madin-Darby canine kidney (MDCK) cells. Among 4 steviol-related compounds tested, steviol was found to be the most potent at inhibiting MDCK cyst growth. Steviol inhibition of cyst growth was dose-dependent; steviol (100 microM) reversibly inhibited cyst formation and cyst growth by 72.53.6% and 38.2±8.5%, respectively. Steviol at doses up to 200 microM had no effect on MDCK cell viability, proliferation and apoptosis. However, steviol acutely inhibited forskolin-stimulated apical chloride current in MDCK epithelia, measured with the Ussing chamber technique, in a dose-dependent manner. Prolonged treatment (24 h) with steviol (100 microM) also strongly inhibited forskolin-stimulated apical chloride current, in part by reducing CFTR protein expression in MDCK cells. Interestingly, proteasome inhibitor, MG-132, abolished the effect of steviol on CFTR protein expression. Immunofluorescence studies demonstrated that prolonged treatment (24 h) with steviol (100 microM) markedly reduced CFTR expression at the plasma membrane. Taken together, the data suggest that steviol retards MDCK cyst progression in two ways: first by directly inhibiting CFTR chloride channel activity and second by reducing CFTR expression, in part, by promoting proteasomal degradation of CFTR. Steviol and related compounds therefore represent drug candidates for treatment of polycystic kidney disease.
Highlights
Polycystic kidney disease (PKD) is an inherited disorder characterized by the presence of enlarging fluid-filled cysts, which disrupt the normal renal parenchyma and eventually leads to endstage renal failure [1,2]
Inhibitory effect of steviol and its derivatives on Madin-Darby canine kidney (MDCK) cyst formation and growth Before proceeding with the assay of cyst formation and growth, MDCK cell viability was evaluated in the presence of 50, 100, and 200 mM steviol and its derivatives, isosteviol, dihydroisosteviol, 16oxime isosteviol (Fig. 1A) using MTT assay
To determine the effects of steviol and derivatives on cyst formation in an in vitro model of PKD, MDCK cells seeded in collagen gel were exposed to 100 mM steviol, its 3 derivatives and 10 mM CFTRinh172 [28] in the presence of 10 mM forskolincontaining media
Summary
Polycystic kidney disease (PKD) is an inherited disorder characterized by the presence of enlarging fluid-filled cysts, which disrupt the normal renal parenchyma and eventually leads to endstage renal failure [1,2]. The exact mechanism of ADPKD pathogenesis is not known, studies have shown that an increase in cAMP level within the renal epithelial cells lining the cyst plays a central role in PKD cystogenesis. The increase in intracellular cAMP level stimulates renal epithelial cell proliferation and raises transepithelial fluid secretion into the cyst lumen [2,5,6]. This fluid secretion is driven by cAMP-activated transepithelial chloride transport via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel located at apical membrane (facing the lumen) of the cells lining the cyst [7]. CFTR chloride channel has been proposed as a potential target for PKD intervention
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