Volume 132, Issue 3, Pages 1104-1116 (March 2007) Octreotide Inhibits Hepatic Cystogenesis in a Rodent Model of Polycystic Liver Disease by Reducing Cholangiocyte Adenosine 3′,5′-Cyclic Monophosphate  Tatyana V. Masyuk, Anatoliy I. Masyuk, Vicente E. Torres, Peter C. Harris, Nicholas F. Larusso  Gastroenterology  Volume 132, Issue 3, Pages 1104-1116 (March 2007) DOI: 10.1053/j.gastro.2006.12.039 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Detection of SSTR2, SSTR3, and SSTR5 in normal and PCK rats by reverse-transcription polymerase chain reaction and confocal microscopy. (A) Reverse-transcription polymerase chain reaction analysis of SSTR2, SSTR3, and SSTR5 expression in freshly isolated bile ducts of normal and PCK rats. Brain messenger RNA was used as a positive control. Two bands of SSTR2 corresponding to 2 isoforms of receptor, SSTR2A and SSTR2B, were detected. (B) Representative liver sections of normal (original magnification 100×) and PCK (original magnification 63×) rats show positive staining for SSTR2, SSTR3, and SSTR5 in normal bile ducts (arrows) and in PCK cystic epithelia, as detected by immunofluorescent confocal microscopy. In the absence of respective primary antibodies, no staining was observed. Gastroenterology 2007 132, 1104-1116DOI: (10.1053/j.gastro.2006.12.039) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Effect of octreotide treatment on cAMP levels in bile ducts of normal and PCK rats. (A) In PCK rats, octreotide (OCT) reduces elevated cAMP concentration but does not affect its basal level in normal rats. (B) In both normal and PCK rats, pretreatment with forskolin results in substantial elevation of cAMP levels. In the presence of octreotide, forskolin-stimulated cAMP production was decreased. Note the difference in scales. *P < .05. Gastroenterology 2007 132, 1104-1116DOI: (10.1053/j.gastro.2006.12.039) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Bile ducts of normal and PCK rats grown in 3-D culture. (A) Representative images of cystic structures formed by bile ducts isolated from normal and PCK rats. Bars = 500 μm. (B) Quantitative assessment of circumferential areas of normal and PCK cystic structures over time. (C) Scanning electron microscopy shows that cysts derived from bile ducts of normal and PCK rats retain their in vivo morphology. In particular, cilia in the PCK cystic structures were ∼2 times shorter than in normal cystic structures. Bars = 100 μm (left panels), 10 μm (middle panels), and 1 μm (right panels). *P < .001 as compared with normal bile ducts. Gastroenterology 2007 132, 1104-1116DOI: (10.1053/j.gastro.2006.12.039) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Effect of octreotide treatment on cyst expansion and cAMP levels in the PCK rat in 3-D culture. (A) Representative images of cystic structures formed by PCK bile ducts in the absence or presence of octreotide. Bars = 500 μm. (B) In the presence of octreotide, cAMP levels were significantly lower. Note that cAMP levels in bile ducts growing in 3-D culture were higher than in freshly isolated bile ducts, reflecting the presence of forskolin in culture media. (C) Quantitative assessment of circumferential areas of cystic structures showed that octreotide treatment halted cyst expansion. *P < 05. Gastroenterology 2007 132, 1104-1116DOI: (10.1053/j.gastro.2006.12.039) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Role of cAMP in liver cyst progression. (A) Representative images of PCK bile ducts grown in 3-D culture. They were treated with octreotide for 3 days. Then octreotide was withdrawn from culture media and supplemented with forskolin for an additional 3 days. Bars = 500 μm. (B) Octreotide prevented progressive expansion of PCK cystic structures. Addition of forskolin significantly increased circumferential areas of bile ducts. (C) cAMP levels in PCK bile ducts grown in 3-D culture for 6 days in the presence of octreotide or forskolin. *P < .05. Gastroenterology 2007 132, 1104-1116DOI: (10.1053/j.gastro.2006.12.039) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Effect of octreotide treatment on cAMP levels, liver weight, hepatic cyst volume, and hepatic fibrosis in vivo in PCK rats. (A) Time-dependent and (B) dose-dependent effect of octreotide treatment. (C) Representative liver sections from PCK rats treated with saline or octreotide. Bars = 250 μm. *P < .05. Gastroenterology 2007 132, 1104-1116DOI: (10.1053/j.gastro.2006.12.039) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 Effect of octreotide treatment on cAMP levels, kidney weight, renal cyst volume, and renal fibrosis in vivo in PCK rats. (A) Time-dependent and (B) dose-dependent effect of octreotide treatment. (C) Representative kidney sections from PCK rats treated with saline or octreotide. Bars = 500 μm. *P < .05. Gastroenterology 2007 132, 1104-1116DOI: (10.1053/j.gastro.2006.12.039) Copyright © 2007 AGA Institute Terms and Conditions

Figure 8 Octreotide suppresses hepatic and renal cystic epithelial cell proliferation in time- and dose-dependent fashion in PCK rats in vivo. (A and D) Time-dependent and (B and E) dose-dependent effects of octreotide treatment on PCNA expression in liver and kidney. Representative (C) liver and (F) kidney sections of PCK rats treated with saline or octreotide for 12 weeks (original magnification 100×). Many PCNA-positive (bright red spots) nuclei were seen in saline-treated rats, while only few were detected after octreotide treatment. Gastroenterology 2007 132, 1104-1116DOI: (10.1053/j.gastro.2006.12.039) Copyright © 2007 AGA Institute Terms and Conditions