Angela J. Stroope

MicroRNA15a modulates expression of the cell-cycle regulator Cdc25A and affects hepatic cystogenesis in a rat model of polycystic kidney disease

Hyperproliferation of bile duct epithelial cells due to cell-cycle dysregulation is a key feature of cystogenesis in polycystic liver diseases (PCLDs). Recent evidence suggests a regulatory role for microRNAs (miRNAs) in a variety of biological processes, including cell proliferation. We therefore hypothesized that miRNAs may be involved in the regulation of selected components of the cell cycle and might contribute to hepatic cystogenesis. We found that the cholangiocyte cell line PCK-CCL, which is derived from the PCK rat, a model of autosomal recessive polycystic kidney disease (ARPKD), displayed global changes in miRNA expression compared with normal rat cholangiocytes (NRCs). More specific analysis revealed decreased levels of 1 miRNA, miR15a, both in PCK-CCL cells and in liver tissue from PCK rats and patients with a PCLD. The decrease in miR15a expression was associated with upregulation of its target, the cell-cycle regulator cell division cycle 25A (Cdc25A). Overexpression of miR15a in PCK-CCL cells decreased Cdc25A levels, inhibited cell proliferation, and reduced cyst growth. In contrast, suppression of miR15a in NRCs accelerated cell proliferation, increased Cdc25A expression, and promoted cyst growth. Taken together, these results suggest that suppression of miR15a contributes to hepatic cystogenesis through dysregulation of Cdc25A.

Cholangiocyte primary cilia are chemosensory organelles that detect biliary nucleotides via P2Y12 purinergic receptors

Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, contain primary cilia, which are mechano- and osmosensory organelles detecting changes in bile flow and osmolality and transducing them into intracellular signals. Here, we asked whether cholangiocyte cilia are chemosensory organelles by testing the expression of P2Y purinergic receptors and components of the cAMP signaling cascade in cilia and their involvement in nucleotide-induced cAMP signaling in the cells. We found that P2Y(12) purinergic receptor, adenylyl cyclases (i.e., AC4, AC6, and AC8), and protein kinase A (i.e., PKA RI-beta and PKA RII-alpha regulatory subunits), exchange protein directly activated by cAMP (EPAC) isoform 2, and A-kinase anchoring proteins (i.e., AKAP150) are expressed in cholangiocyte cilia. ADP, an endogenous agonist of P2Y(12) receptors, perfused through the lumen of isolated rat intrahepatic bile ducts or applied to the ciliated apical surface of normal rat cholangiocytes (NRCs) in culture induced a 1.9- and 1.5-fold decrease of forskolin-induced cAMP levels, respectively. In NRCs, the forskolin-induced cAMP increase was also lowered by 1.3-fold in response to ATP-gammaS, a nonhydrolyzed analog of ATP but was not affected by UTP. The ADP-induced changes in cAMP levels in cholangiocytes were abolished by chloral hydrate (a reagent that removes cilia) and by P2Y(12) siRNAs, suggesting that cilia and ciliary P2Y(12) are involved in nucleotide-induced cAMP signaling. In conclusion, cholangiocyte cilia are chemosensory organelles that detect biliary nucleotides through ciliary P2Y(12) receptors and transduce corresponding signals into a cAMP response.

Pasireotide is more effective than octreotide in reducing hepatorenal cystogenesis in rodents with polycystic kidney and liver diseases.

In polycystic liver (PLD) and kidney (PKD) diseases, increased cyclic adenosine monophosphate (cAMP) levels trigger hepatorenal cystogenesis. A reduction of the elevated cAMP by targeting somatostatin receptors (SSTRs) with octreotide (OCT; a somatostatin analog that preferentially binds to SSTR2) inhibits cyst growth. Here we compare the effects of OCT to pasireotide (PAS; a more potent somatostatin analog with broader receptor specificity) on: (1) cAMP levels, cell cycle, proliferation, and cyst expansion in vitro using cholangiocytes derived from control and PCK rats (a model of autosomal recessive PKD [ARPKD]), healthy human beings, and patients with autosomal dominant PKD (ADPKD); and (2) hepatorenal cystogenesis in vivo in PCK rats and Pkd2WS25/‐ mice (a model of ADPKD). Expression of SSTRs was assessed in control and cystic cholangiocytes of rodents and human beings. Concentrations of insulin‐like growth factor 1 (IGF1) and vascular endothelial growth factor (VEGF) (both involved in indirect action of somatostatin analogs), and expression and localization of SSTRs after treatment were evaluated. We found that PAS was more potent (by 30%‐45%) than OCT in reducing cAMP and cell proliferation, affecting cell cycle distribution, decreasing growth of cultured cysts in vitro, and inhibiting hepatorenal cystogenesis in vivo in PCK rats and Pkd2WS25/‐ mice. The levels of IGF1 (but not VEGF) were reduced only in response to PAS. Expression of SSTR1 and SSTR2 (but not SSTR3 and SSTR5) was decreased in cystic cholangiocytes compared to control. Although both OCT and PAS increased the immunoreactivity of SSTR2, only PAS up‐regulated SSTR1; neither drug affected cellular localization of SSTRs. Conclusion: PAS is more effective than OCT in reducing hepatorenal cystogenesis in rodent models; therefore, it might be more beneficial for the treatment of PKD and PLD. (HEPATOLOGY 2013)

Activation of Trpv4 reduces the hyperproliferative phenotype of cystic cholangiocytes from an animal model of ARPKD

BACKGROUND & AIMS In polycystic liver diseases, cyst formation involves cholangiocyte hyperproliferation. In polycystic kidney (PCK) rats, an animal model of autosomal-recessive polycystic kidney disease (ARPKD), decreased intracellular calcium [Ca(2+)](i) in cholangiocytes is associated with hyperproliferation. We recently showed transient receptor potential vanilloid 4 (Trpv4), a calcium-entry channel, is expressed in normal cholangiocytes and its activation leads to [Ca(2+)](i) increase. Thus, we hypothesized that pharmacologic activation of Trpv4 might reverse the hyperproliferative phenotype of PCK cholangiocytes. METHODS Trpv4 expression was examined in liver of normal and PCK rats, normal human beings, and patients with autosomal-dominant polycystic kidney disease or ARPKD. Trpv4 activation effect on cell proliferation and cyst formation was assessed in cholangiocytes derived from normal and PCK rats. The in vivo effects of Trpv4 activation on kidney and liver cysts was analyzed in PCK rats. RESULTS Trpv4 was overexpressed both at messenger RNA (8-fold) and protein (3-fold) levels in PCK cholangiocytes. Confocal and immunogold electron microscopy supported Trpv4 overexpression in the livers of PCK rats and ARPKD or autosomal-dominant polycystic kidney disease patients. Trpv4 activation in PCK cholangiocytes increased [Ca(2+)](i) by 30%, inhibiting cell proliferation by approximately 25%-50% and cyst growth in 3-dimensional culture (3-fold). Trpv4-small interfering RNA silencing blocked effects of Trpv4 activators by 70%. Trpv4 activation was associated with Akt phosphorylation and beta-Raf and Erk1/2 inhibition. In vivo, Trpv4 activation induced a significant decrease in renal cystic area and a nonsignificant decrease in liver cysts. CONCLUSIONS Taken together, our in vitro and in vivo data suggest that increasing intracellular calcium by Trpv4 activation may represent a potential therapeutic approach in PKD.

Inhibition of Cdc25A suppresses hepato-renal cystogenesis in rodent models of polycystic kidney and liver disease

BACKGROUND & AIMS In polycystic kidney disease and polycystic liver disease (PLD), the normally nonproliferative hepato-renal epithelia acquire a proliferative, cystic phenotype that is linked to overexpression of cell division cycle 25 (Cdc25)A phosphatase and cell-cycle deregulation. We investigated the effects of Cdc25A inhibition in mice and rats via genetic and pharmacologic approaches. METHODS Cdc25A(+/-) mice (which have reduced levels of Cdc25A) were cross-bred with polycystic kidney and hepatic disease 1 (Pkhd1(del2/del2)) mice (which have increased levels of Cdc25A and develop hepatic cysts). Cdc25A expression was analyzed in livers of control and polycystic kidney (PCK) rats, control and polycystic kidney 2 (Pkd2(ws25/-)) mice, healthy individuals, and patients with PLD. We examined effects of pharmacologic inhibition of Cdc25A with vitamin K3 (VK3) on the cell cycle, proliferation, and cyst expansion in vitro; hepato-renal cystogenesis in PCK rats and Pkd2(ws25/-)mice; and expression of Cdc25A and the cell-cycle proteins regulated by Cdc25A. We also examined the effects of the Cdc25A inhibitor PM-20 on hepato-renal cystogenesis in Pkd2(ws25/-) mice. RESULTS Liver weights and hepatic and fibrotic areas were decreased by 32%-52% in Cdc25A(+/-):Pkhd1(del2/del2) mice, compared with Pkhd1(del2/del2) mice. VK3 altered the cell cycle and reduced proliferation of cultured cholangiocytes by 32%-83% and decreased growth of cultured cysts by 23%-67%. In PCK rats and Pkd2(ws25/-) mice, VK3 reduced liver and kidney weights and hepato-renal cystic and fibrotic areas by 18%-34%. PM-20 decreased hepato-renal cystogenesis in Pkd2(ws25/-) mice by 15%. CONCLUSIONS Cdc25A inhibitors block cell-cycle progression and proliferation, reduce liver and kidney weights and cyst growth in animal models of polycystic kidney disease and PLD, and might be developed as therapeutics for these diseases.

Hepato-Renal Pathology in Pkd2ws25/− Mice, an Animal Model of Autosomal Dominant Polycystic Kidney Disease

Polycystic liver diseases, the most important of which are autosomal dominant and autosomal recessive polycystic kidney diseases, are incurable pathological conditions. Animal models that resemble human pathology in these diseases provide an opportunity to study the mechanisms of cystogenesis and to test potential treatments. Here we demonstrate that Pkd2ws25/- mice, an animal model of autosomal dominant polycystic kidney disease, developed hepatic cysts. As assessed by micro-computed tomography scanning of intact livers and by light microscopy of hepatic tissue, hepatic cystic volumes increased from 12.82+/-3.16% (5- to 8-month-old mice) to 21.58+/-4.81% (9- to 12-month-old mice). Renal cystogenesis was more severe at early stages of disease: in 5- to 7-month-old mice, cystic volumes represented 40.67+/-5.48% of kidney parenchyma, whereas in older mice cysts occupied 31.04+/-1.88% of kidney parenchyma. Mild fibrosis occurred only in liver, and its degree was unchanged with age. Hepatic cysts were lined by single or multiple layers of squamous cholangiocytes. Cystic cholangiocyte cilia were short and malformed, whereas in renal cysts they appeared normal. In Pkd2ws25/- mice, mitotic and apoptotic indices in both kidney and liver were increased compared with wild-type mice. In conclusion, Pkd2ws25/- mice exhibit hepatorenal pathology resembling human autosomal dominant polycystic kidney disease and represent a useful model to study mechanisms of cystogenesis and to evaluate treatment options.