PMC

(A) Compared to the release of IL-8 from Mz-ChA1 cells under basal conditions, cells exposed to repetitive stretch cycles for 25 hrs (Stretch) had significantly elevated levels of IL-8. *p <0.05 basal vs stretch (B) Compared to the release of IL-8 from Mz-ChA1 cells under basal conditions, cells cultured under hypoxic conditions for 24 hrs (1% O₂; Hypoxia) had a significantly greater release of IL-8. *p<0.05 basal vs hypoxia

ELISA analyses found concentrations of CXCR2 chemokines were significantly higher in human liver cyst fluids (hLCF) than human renal cyst fluids (hRCF). The analysis specifically included IL-8 (left panel), ENA78 (middle panel) and GROα (right panel). Individual values are represented with diamonds and the means are represented with the bar. (*p<0.05 in liver versus kidney samples).

Cell stretching induced a significant increase in the IL-8 promoter activity in Mz-ChA1 cells. When the –251 residue was mutated from A to T, the promoter activity was not different under basal conditions but had a reduced response to cell stretching. From an analysis of variance, bars without a shared letter (e.g. A, B, C) were significantly different (p<0.05).

Primary cultures of isolated liver cyst epithelial cells from pkd2(WS25/−) mice grown on semi-permeable supports form high resistance monolayers (R_t>1,000 Ω·cm²). Cytokine array analysis of media collected after 72 hours of incubation showed a parallel pattern of cytokine and growth factor release into both the apical and basal media. Boxes show internal array controls.

(A) AP-1, c/EBP and NF-κB binding sites were mutated individually, in tandem or in triplicate in the −1,481/+44 promoter. Mutated sites were denoted by X’s in the specific sites. (B) Stretching of Mz-ChA1 cells (n=4) roughly doubled the IL-8 promoter activity. Mutation of the AP-1 or NF-κB sites, individually or in tandem, resulted in a profound loss of promoter activity under both basal and stretched conditions. Mutation of the c/EBP site resulted in a modest decrease in the basal activity and a complete loss of the stretch-induced increase. (C) Subjecting Mz-ChA1 cells (n=4) to hypoxia also induced a doubling in the IL-8 promoter activity. This response to hypoxia was largely lost when the binding sites for AP-1, NF-κB were mutated.

(A) A full length IL-8 promoter (−1,481/+44 base pairs) was truncated to generate −272/+44, −98/+44 and −50/+44 IL-8 promoter constructs. (B) Stretching of Mz-ChA1 cells (n=8) for 25 hours doubled the IL-8 promoter activity when compared to control cells. The −272/+44 truncated promoter showed a modest decrease in the basal promoter activity and a diminished stretch response. The basal and stretch-induced promoter activity in the −98/+44 construct and −50/+44 construct were essentially abolished. (C) Subjecting Mz-ChA1 cells (n=6) to hypoxia (1% O₂ for 8 hrs) doubled the IL-8 promoter activity. Under basal conditions, the promoter activity of the −272/+44 construct was unchanged but there was a loss of the hypoxia response. The basal and hypoxia-induced promoter activity in the −98/+44 construct and −50/+44 construct were essentially abolished.

(A) Cytokine arrays established the profile of cytokines and growth factors present in human ADPKD liver (left panel) and kidney (right panel) cyst fluids. Some factors (e.g. angiogenin) were detected in both liver and kidney cyst fluids. Some factors were specifically elevated in either kidney cyst fluids (e.g. leptin, MIP-1δ) or liver cyst fluids (e.g. IL-8). Extended exposures also revealed the presence of ENA78 and GRO in liver cyst fluids. Internal positive controls are shown enclosed in a rectangle. provides a relative densitometric analysis of liver (n=5) versus kidney (n=6) cyst fluid arrays.

(A) Treatment of HMEC-1 cells with increasing doses of IL-8 for 24 hrs resulted in a significant increase in cell proliferation at 0.1 ng/ml and a maximal response at 100 ng/ml IL-8 when compared to unsupplemented control (Con) cells. From an analysis of variance, bars without a shared letter (e.g. A, B, C, D) are significantly different from each other. (B) Compared to unsupplemented HMEC-1 cells, cells treated with 10% human liver cyst fluid markedly induced cell proliferation. Pretreatment with 50 nM SB225002 inhibited the proliferative response in paired assays (*p<0.05 vehicle vs SB225002). Treatment with 10% human renal cyst fluid also induced HMEC-1 cell proliferation. From four studies, pretreatment with 50 nM SB225002 did not significantly block the proliferative effect of 10% human renal cyst fluid.

Treatment of Mz-ChA1 cells with IL-8 for 24 hrs induced cell proliferation. (A) Western blot analysis of PCNA showed Mz-ChA1 cells had modest responses with 0.01 to 1.0 ng/ml IL-8 and marked proliferative responses when treated with 10 to 100 ng/ml IL-8. Actin served as a loading control. Blot is representative of three separate experiments. (B) Alamar Blue reduction assays showed a modest proliferative response from 1 ng/ml IL-8 and an apparent maximal response at 100 ng/ml. From an analysis of variance, bars without a shared letter (e.g. A, B, C, D) are significantly different from each other. (C) Stimulation with 10% human liver cyst fluid markedly induced proliferation of unsupplemented Mz-ChA1 cells. This response was significantly inhibited by pretreatment with 50 nM SB225002 (*p <0.05 vehicle vs SB225002). Addition of 10% renal cyst fluid also induced the proliferation of Mz-ChA1 cells. This response, however, was not inhibited by pretreatment with SB225002.