Abnormal proliferation of cyst-lining epithelium and increased intra-cystic fluid secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) are thought to contribute to cyst growth in autosomal dominant polycystic kidney disease (ADPKD). Histone deacetylase 6 (HDAC6) expression and activity are increased in certain cancers, and neurodegenerative diseases, and in Pkd1-mutant renal epithelial cells. Inhibition of HDAC6 activity with specific inhibitors slows cancer growth. Here we studied the effect of tubacin, a specific HDAC6 inhibitor, on cyst growth in polycystic kidney disease. Treatment with tubacin prevented cyst formation in MDCK cells, an in vitro model of cystogenesis. Cyclic AMP stimulates cell proliferation and activates intra-cystic CFTR-mediated chloride secretion in ADPKD. Treatment with tubacin down-regulated cyclic AMP levels, inhibited cell proliferation, and inhibited cyclic AMP-activated CFTR chloride currents in MDCK cells. We also found that tubacin reduced cyst growth by inhibiting proliferation of cyst-lining epithelial cells, down-regulated cyclic AMP levels, and improved renal function in a Pkd1-conditional mouse model of ADPKD. Thus, HDAC6 could play a role in cyst formation and could serve as a potential therapeutic target in ADPKD.

cell line represents absorptive polarized intestinal epithelial cells that express multiple forms of Na ϩ /H ϩ exchanger (NHE) in their plasma membranes. Caco-2 cells express the major apical NHE isoform NHE3, but low NHE3 expression together with inefficient transfection often hamper intended studies. In this study, we examined whether SK-CO15 cells could be used to study NHE3 regulation. SK-CO15 cells grown on Transwell inserts developed polarized epithelial cells with microvilli. The transfection efficiency of SK-CO15 cells was markedly higher compared with Caco-2 cells, an advantage in gene transfer and knockout. SK-CO15 cells expressed NHE1, NHE2, and NHE3. NHE3 expression was significantly greater in these cells than Caco-2, and NHE3 comprised more than half of total NHE activity. Apical expression of NHE3 in SK-CO15 cells was confirmed by confocal immunofluorescence and surface biotinylation. NHE regulatory factors NHERF1 and NHERF2, which are important for regulation of NHE3 activity, were expressed in these cells. Stimulatory response of NHE3 in SK-CO15 cells was assessed by dexamethasone and lysophosphatidic acid (LPA). Treatment with dexamethasone for 24 -48 h increased NHE3 expression and activity. Similarly to Caco-2 cells, SK-CO15 cells lacked the expression of the LPA receptor LPA 5, but exogenous expression of LPA5 resulted in acute stimulation of NHE3. Forskolin acutely inhibited NHE3 activity in SK-CO15 cells, further attesting the validity of these cells. We conclude that SK-CO15 cells with the amenity for transfection and high endogenous NHE3 expression are a new and better cell model for NHE3 regulatory investigation than widely used Caco-2 cells.intestine; epithelia POLARIZED INTESTINAL EPITHELIAL cells form an interface separating the internal from external environments and maintain homeostasis between intestinal lumen and the body interior. The plasma membranes of polarized epithelial cells are divided into apical and basolateral domains with asymmetric distribution of cytoplasmic organelles by vectorial sorting mechanisms (27). Na ϩ /H ϩ exchange is a major route of Na ϩ absorption in the small intestine and colon (9). Intestinal epithelial cells express multiple forms of Na ϩ /H ϩ exchangers (NHEs; Slc9a) among which the type 3, NHE3 (Slc9a3), is the primary brush-border NHE. The functions and mechanisms of NHE3 regulation by hormones and growth factors have been investigated since its cloning in the early 1990s using several cell model systems. Among these, PS120 Chinese hamster lung fibroblasts and AP1 Chinese hamster ovarian cells provide an ideal cell system for reductionist approach to characterize NHEs (26, 28). However, the nonepithelial origins of PS120 and AP1 cells often led to question the physiological validity of the findings. Originated from human intestines, T84 and HT29 human colon carcinoma epithelial cell lines are not suitable, as these cells represent secretory crypt epithelial cells (5, 6). The Caco-2 human adenocarcinoma cell line, on the other hand, express severa...

Adult-onset autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations in either the or gene, leading to malfunction of their gene products, polycystin 1 or 2. Histone deacetylase 6 (HDAC6) expression and activity are increased in mutant renal epithelial cells. Here we studied the effect of ACY-1215, a specific HDAC6 inhibitor, on cyst growth in ADPKD. Treatment with ACY-1215 slowed cyst growth in a mouse model of ADPKD that forms massive cysts within 3 wk after knockout of polycystin 1 function. It also prevented cyst formation in MDCK.2 cells, an in vitro model of cystogenesis, and in an ADPKD cell line derived from the proximal tubules from amouse (PN cells). In PN cells ACY-1215 also reduced the size of already established cysts. We found that ACY-1215 lowered cAMP levels and protein expression of adenylyl cyclase 6. Our results suggest that HDAC6 could potentially serve as a therapeutic target in ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) is associated with progressive enlargement of multiple renal cysts, often leading to renal failure that cannot be prevented by a current treatment. Two proteins encoded by two genes are associated with ADPKD: PC1 (), primarily a signaling molecule, and PC2 (), a Ca channel. Dysregulation of cAMP signaling is central to ADPKD, but the molecular mechanism is unresolved. Here, we studied the role of histone deacetylase 6 (HDAC6) in regulating cyst growth to test the possibility that inhibiting HDAC6 might help manage ADPKD. Chemical inhibition of HDAC6 reduced cyst growth in PC1-knock-out mice. In proximal tubule-derived, PC1-knock-out cells, adenylyl cyclase 6 and 3 (AC6 and -3) are both expressed. AC6 protein expression was higher in cells lacking PC1, compared with control cells containing PC1. Intracellular Ca was higher in PC1-knock-out cells than in control cells. HDAC inhibition caused a drop in intracellular Ca and increased ATP-simulated Ca release. HDAC6 inhibition reduced the release of Ca from the endoplasmic reticulum induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca-ATPase. HDAC6 inhibition and treatment of cells with the intracellular Ca chelator 1,2-bis(2-aminophenoxy)ethane-,,','-tetraacetic acid tetrakis(acetoxymethyl ester) reduced cAMP levels in PC1-knock-out cells. Finally, the calmodulin inhibitors W-7 and W-13 reduced cAMP levels, and W-7 reduced cyst growth, suggesting that AC3 is involved in cyst growth regulated by HDAC6. We conclude that HDAC6 inhibition reduces cell growth primarily by reducing intracellular cAMP and Ca levels. Our results provide potential therapeutic targets that may be useful as treatments for ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) is associated with progressive enlargement of cysts, leading to a decline in function and renal failure that cannot be prevented by current treatments. Mutations in and, encoding the polycystin 1 and 2 proteins, induce growth-related pathways, including heat shock proteins, as occurs in some cancers, raising the prospect that pharmacological interventions that target these pathways might alleviate or prevent ADPKD. Here, we demonstrate a role for VX-809, a corrector of cystic fibrosis transmembrane conductance regulator (CFTR), conventionally used to manage cystic fibrosis in reducing renal cyst growth. VX-809 reduced cyst growth in -knockout mice and in proximal, tubule-derived, cultured knockout cells. VX-809 reduced both basal and forskolin-activated cAMP levels and also decreased the expression of the adenylyl cyclase AC3 but not of AC6. VX-809 also decreased resting levels of intracellular Ca but did not affect ATP-stimulated Ca release. Notably, VX-809 dramatically decreased thapsigargin-induced release of Ca from the endoplasmic reticulum (ER). VX-809 also reduced the levels of heat shock proteins Hsp27, Hsp70, and Hsp90 in mice cystic kidneys, consistent with the restoration of cellular proteostasis. Moreover, VX-809 strongly decreased an ER stress marker, the GADD153 protein, and cell proliferation but had only a small effect on apoptosis. Given that administration of VX-809 is safe, this drug potentially offers a new way to treat patients with ADPKD.

The missing phenylalanine at position 508, located in nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane regulator (CFTR), is the most common cystic fibrosis mutation. Severe disease-causing mutations also occur in NBD2. To provide information on potential therapeutic strategies for mutations in NBD2, we used a combination of biochemical, cell biological and electrophysiological approaches and newly created cell lines to study two disease-causing NBD2 mutants, N1303K and S1235R. We observed that neither was sensitive to E64, a cysteine protease inhibitor. However, further investigation showed that when treated with a combination of correctors, C4 + C18, both mutants also responded to E64. Further exploration to assess aggresome throughput using the autophagy regulator LC3 as a marker showed that, in the absence of correctors, N1303K showed a stalled throughput of LC3-II to the aggresome. The throughput became active again after treatment with the corrector combination C4 + C18. Confocal microscopic studies showed that the N1303K and S1235R mutant proteins both co-localized with LC3, but this co-localization was abolished by the corrector combination and, to a lesser extent, by VX-809. Both the corrector combination and VX-809 increased the CFTR chloride channel function of both mutants. We conclude that correctors have a dual effect, particularly on N1303K: they improve trafficking and function at the plasma membrane and reduce the association with autophagosomes. After treatment with correctors persistent degradation by the autophagosome may limit restoration of function. Thus, mutations in NBD2 of CFTR, in contrast to ΔF508-CFTR, may require additional personalized strategies to rescue them.

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Edited by Phyllis I. Hanson Autosomal-dominant polycystic kidney disease (ADPKD) induces a secretory phenotype, resulting in multiple fluid-filled cysts. We have previously demonstrated that VX-809, a corrector of the cystic fibrosis transmembrane conductance regulator (CFTR), reduces cyst growth. Here, we show that in normal mice CFTR is located within the cells and also at the apical and basolateral membranes. However, in polycystic kidney disease (pkd1)-knockout mice, CFTR was located at the plasma membrane, consistent with its role in cAMP-dependent fluid secretion. In cystic mice, VX-809 treatment increased CFTR levels at the apical membrane and reduced its association with the endoplasmic reticulum. Surprisingly, VX-809 treatment significantly increased CFTR's co-localization with the basolateral membrane in cystic mice. Na ؉ /H ؉ exchanger 3 (NHE3) is present in pkd1-knockout and normal mice and in proximal tubule-derived, cultured pkd1-knockout cells. VX-809 increased the expression, activity, and apical plasma membrane localization of NHE3. Co-localization of epithelial sodium channel (ENaC) with the plasma membrane was reduced in cysts in pkd1-knockout mice, consistent with an inability of the cysts to absorb fluid. Interestingly, in the cystic mice, VX-809 treatment increased ENaC levels at the apical plasma membrane consistent with fluid absorption. Thus, VX-809 treatment of pkd1-null mouse kidneys significantly affected CFTR, NHE3, and ENaC, altering the cyst phenotype from one poised toward fluid secretion toward one more favorable for absorption. VX-809 also altered the location of CFTR but not of NHE3 or ENaC in normal mice. Given that VX-809 administration is safe, it may have potential utility for treating patients with ADPKD.