Ginkgolide B inhibits renal cyst development in in vitro and in vivo cyst models

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disease characterized by massive enlargement of fluid-filled cysts in the kidney. However, there is no effective therapy yet for this disease. To examine whether ginkgolide B, a natural compound, inhibits cyst development, a Madin-Darby canine kidney (MDCK) cyst model, an embryonic kidney cyst model, and a PKD mouse model were used. Interestingly, ginkgolide B significantly inhibited MDCK cyst formation dose dependently, with up to 69% reduction by 2 μM ginkgolide B. Ginkgolide B also significantly inhibited cyst enlargement in the MDCK cyst model, embryonic kidney cyst model, and PKD mouse model. To determine the underlying mechanisms, the effect of ginkgolide B on MDCK cell viability, proliferation, apoptosis, chloride transporter CFTR activity, and intracellular signaling pathways were also studied. Ginkgolide B did not affect cell viability, proliferation, and expression and activity of the chloride transporter CFTR that mediates cyst fluid secretion. Ginkgolide B induced cyst cell differentiation and altered the Ras/MAPK signaling pathway. Taken together, our results demonstrate that ginkgolide B inhibits renal cyst formation and enlargement, suggesting that ginkgolide B might be developed into a novel candidate drug for ADPKD.

Fig. 1.

Ginkgolide B (GB) inhibits Madin-Darby canine kidney (MDCK) cell cyst formation. A: representative light micrographs of MDCK cells cultured in collagen gels. Light micrographs were taken on day 6 after cell seeding. MDCK cells were cultured without forskolin (FSK; top) or with 2 μM GB and without FSK (the second from top) or with 10 μM FSK (third from top) or 10 μM forskolin plus 2 μM GB (bottom). Scale bar = 50 μm. B: MDCK cyst formation rate. Open bars show the total numbers of colonies (cysts colonies plus noncyst colonies) per well on day 6 after MDCK cells were incubated without (control) or with GB at indicated concentrations in the presence of 10 μM FSK. Filled bars show the numbers of cysts with a diameter >50 μm (means ± SD; n = 3). *P < 0.05 vs. control.

Fig. 2.

GB reversibly inhibits MDCK cyst growth. A: representative light micrographs of MDCK cyst growth in collagen gels. Light micrographs were taken at indicated days after cell seeding. MDCK cells were exposed continuously to 10 μM FSK (top). In some experiments, MDCK cysts (established on day 4 with FSK stimulation) were treated with GB from day 4 to day 12 (middle) or from day 4 to day 8 (bottom) in the presence of FSK. Scale bar = 200 μm. Thick lines indicate the culture time with GB. B: MDCK cell cyst enlargement shown as cyst diameters for GB at indicated concentration (means ± SD; >30 cysts analyzed/time point). *P < 0.05 vs. control. C: inhibition was reversible as shown by exposure to GB from day 4 to day 8 followed by washout (means ± SD, >30 cysts analyzed/time point). White bar and ○ represent GB group treated from day 4 to day 8; black bar and ● represent GB group treated from day 4 to day 12. *P < 0.05 vs. GB group treated from day 4 to day 12.

Fig. 3.

GB retards cyst development in embryonic kidney cyst model. A: representative light micrographs of embryonic kidneys cultured in Transwells and maintained from day 0 to day 4. Embryonic day 13.5 (E13.5) kidneys were exposed to 100 μM 8-bromo (Br)-cAMP as a positive control (top) or were treated with 2 μM GB from day 0 to day 4 (middle) or from day 0 to day 2 (bottom) in the presence of 100 μM 8-Br-cAMP. Each series of photographs shows the same kidney on successive days in culture. Scale bar = 1 mm. Thick solid lines indicate the culture time with GB. B: inhibition of 8-Br-cAMP-induced cyst growth by GB at indicated concentrations. Images show embryonic kidneys before (day 0) and after (day 4) continuous GB treatment. Scale bar = 1 mm. C: fractional cyst area (%) in positive control and GB-treated group (means ± SD; n = 6–10). *P < 0.05 vs. positive control.

Fig. 4.

GB slows cyst growth in a Pkd1^flox/−;Ksp-Cre mouse model of PKD. A: kidney weight indexes (age 4 days) of non-PKD mice (denoted wild-type) and Pkd1^flox/−;Ksp-Cre mice treated for 3 days with vehicle (control) or GB (means ± SD; 5 mice per group). *P < 0.01. B: liver weight indexes of the same mice as in A. C: representative central coronal kidney sections from Pkd1^flox/−;Ksp-Cre mice treated for 3 days with vehicle (left) or GB (16 mg·kg⁻¹·day⁻¹; right). Scale bar = 1 mm. D: fractional cyst area (%) in vehicle and GB-treated Pkd1^flox/−;Ksp-Cre mice (means ± SD; n = 5). **P < 0.01 vs. control.

Fig. 5.

GB promotes the tubulogenesis in MDCK cells and MDCK cysts. A: representative light micrographs of tubule-like structures induced from the MDCK cysts in collagen gels. Light micrographs were taken on day 12 after MDCK cysts (established on day 4 with FSK stimulation) cultured without 3T3 conditioned media (CM; left) or with GB and without 3T3 CM (second from left) or with 3T3 CM (third from left), or with 3T3 CM and GB (right). Scale bar = 100 μm. B: numbers of MDCK cells forming tubule-like structures without or with GB treatment (means ± SD; n = 3). *P < 0.05 vs. control. C: average values of the longest length of tubule-like structures on each 3T3 CM-treated MDCK cyst without or with GB treatment (means ± SD; n = >30). *P < 0.05 vs. control.

Fig. 6.

GB does not induce cytotoxicity and apoptosis in MDCK cells. A: cytotoxicity assayed by MTT (means ± SD; n = 3). MDCK cells were treated without or with GB at indicated concentrations for 24 h. Cell viability is showed as OD₄₉₀. B: apoptosis index of MDCK cells cultured with DMSO, gentamycin or GB (means ± SD; n = 3). *P < 0.05 vs. control. C: effect of GB on MDCK cell proliferation measured by CCK-8 kit and showed as OD₄₅₀. MDCK cells were treated without or with GB at indicated concentrations. D: effect of GB on FSK-stimulated MDCK cell proliferation. MDCK cells were treated without or with GB at indicated concentrations in the presence of 10 μM FSK (means ± SD; n = 3). *P < 0.05 vs. GB-treated groups.

Fig. 7.

Effect of GB on the function of CFTR. A: representative original fluorescence data from individual wells showing CFTR inhibitor control and GB-treated group. After addition of CFTR inhibitor or GB, I⁻ influx was induced by adding an I⁻-containing solution. B: effect of GB on short-circuit current in MDCK cell monolayer stimulated by 20 μM FSK. C: short-circuit current in MDCK cell monolayer treated without or with 10 μM GB for 1 or 48 h. GB was washed out for 1 h before measurements. CFTR chloride current was stimulated by 20 μM FSK.

Fig. 8.

Effect of GB on intracellular cAMP in MDCK cells. Intracellular cAMP concentration was measured by cAMP RIA kit (means ± SD; n = 6). MDCK cells were treated without or with GB in the presence of 10 μM FSK for 30 min.

Fig. 9.

GB regulates intracellular pathways in MDCK cells. A: representative Western blots of signaling proteins in MDCK cells treated with GB at indicated concentrations in the presence of 10 μM FSK for 60 min. Control refers to MDCK cells without GB and FSK treatment. Each lane was loaded with 20 μg protein. B: quantitative analysis of signaling protein expression in MDCK cells as described above. Relative level means the ratio of Western blotting band density in testing groups (with FSK or FSK plus GB) to that in control group (means ± SD; n = 6). #P < 0.05, ##P < 0.01 vs. control group. *P < 0.05, **P < 0.01 vs. FSK-treated group.