Human renal adipose tissue from normal and tumor kidney: its influence on renal cell carcinoma

Abstract

Tumor cells can interact with neighboring adipose tissue. We evaluated components present in human adipose explants from normal (hRAN) and kidney cancer (hRAT) tissue, and we evaluated the effects of conditioned media (CMs) from hRAN and hRAT on proliferation, adhesion and migration of tumor and non-tumor human renal epithelial cell lines. In addition, we evaluated the expression of AdipoR1, ObR, CD44, vimentin, pERK and pPI3K on cell lines incubated with CMs. hRAN were obtained from healthy operated donors, and hRAT from patients who underwent a nephrectomy. hRAT showed increased levels of versican, leptin and ObR; and decreased levels of perilipin, adiponectin and AdipoR1, compared to hRAN. Cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRAT-CMs vs. hRAN- or control-CMs. Surprisingly, HK-2, 786-O and ACHN cells showed a significant decrease in cell migration after incubation with hRAN-CMs vs. control-CMs. No difference in proliferation of cell lines was found after 24 or 48 h of treatment with CMs. AdipoR1 in ACHN and Caki-1 cells decreased significantly after incubation with hRAT-CMs vs. hRAN-CMs and control-CMs. ObR and CD44 increased in tumor line cells, and vimentin increased in non-tumor cells, after incubation with hRAT-CMs vs. hRAN-CMs and control-CMs. We observed an increase in the expression of pERK and pPI3K in HK-2, 786-O and ACHN, incubated with hRAT-CMs. In conclusion, results showed that adipose microenvironment can regulate the behavior of tumor and non tumor human renal epithelial cells.

Keywords: cancer; epithelial-stromal interactions; human renal adipose tissue; migration; renal epithelial cells.

Figure 1. Relative fold expression of versican, adiponectin and leptin gene expression from hRAN and hRAT.

The mRNA profiles of versican, adiponectin and leptin from different adipose tissue were analyzed by qRT-PCR and normalized by their relative ratio to GAPDH. Data are mean ± SEM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ^* p < 0.05.

Figure 2. Adiponectin, ADAMTS1 and perilipin 1 in hRAN- and hRAT-CMs.

Adiponectin, ADAMTS1 and perilipin 1 expression was evaluated by Western blot. Images were analyzed by densitometry. Horizontal bars represent the geometric mean of each data set. Vertical bars indicate SEM. ^* p < 0.05 hRAN-CMs vs. hRAT-CMs.

Figure 3. ObR, AdipoR1, adiponectin and perilipin 1 expression in the different adipose tissues.

ObR, AdipoR1, adiponectin and perilipin 1 expression was evaluated by immunohistochemistry in serial cuts of hRAN and hRAT. DAB staining quantification in the three tissue types was performed with Image J software (NIH). Histograms show mean ± SEM of six independent experiments. (a. u.: arbitrary units). ^* p < 0.01 hRAN vs. hRAT. Representative photographs of hRAN- and hRAT-staining. Magnification: ×100.

Figure 4. Figure 4: Effect of CMs from hRAN and hRAT on proliferation of HK-2, 786-O, ACHN and Caki-1 cell lines.

HK-2, 786-O, ACHN and Caki-1 cell lines were incubated with hRAN- (n = 13), hRAT- (n = 14) or control-CMs for 24 (A) or 48 h (B). Proliferation was measured by MTT assays. Data are shown as the mean ± SEM (n = 4–5 experiments by triplicate).

Figure 5. Effect of CMs from hRAN and hRAT on HK-2, 786-O, ACHN and Caki-1 cell lines attachment.

HK-2, 786-O, ACHN and Caki-1 cell lines were plated at a density of 5 × 10⁴ cells/well in wells preincubated ON with hRAN- (n = 10–13), hRAT- (n = 10–13) or control-CMs and adherent cells were quantified by MTT. Data are shown as the mean ± SEM (n = 3 experiments by triplicate). ^* p < 0.05 hRAT-CMs vs. hRAN-CMs and control-CMs; ^** p < 0.05 hRAN-CMs vs. control-CMs.

Figure 6. Effect of CMs from hRAN and hRAT on migration of HK-2, 786-O, ACHN and Caki-1 cell lines.

Wound healing assay (A) HK-2, 786-O, ACHN and Caki-1 cell lines were grown with hRAN- (n = 10–13), hRAT- (n = 10–14) or control-CMs by additional 24 hs. After that, cells were wounded, washed twice with PBS and hRAN- (n = 13), hRAT- (n = 14) or control-CMs were added. Images were captured at the wound instant (0 h), after 6 and 12 hs. Representative light microscopic images (4×) are shown. HK-2, 786-O, ACHN and Caki-1 cell lines were incubated with hRAN-, hRAT- or control-CMs. The histogram shows the ratio of 12 hs/0 hs (HK-2) or 6 hs/0 hs (786-O, ACHN and Caki-1) cutting area and is plotted as mean ± SEM (n = 4 experiments by duplicate). ^* p < 0.001 hRAT-CMs vs. hRAN-CMs and control-CMs; ^** p < 0.05 hRAN-CMs vs. control-CMs. Transmigration assay (B): HK-2, 786-O, ACHN and Caki-1 cells were incubated with hRAN- (n = 10), hRAT- (n = 10) or control-CMs allowed to migrate across the porous membrane for 24 hs. The membranes were viewed under 20× magnification and migrated cells were counted in 5 randomly chosen fields per membrane. Data are shown as the mean ± SEM (n = 3 experiments by duplicate). ^* p < 0.001 hRAT-CMs vs. hRAN-CMs and control-CMs; ^** p < 0.05 hRAN-CMs vs. control-CMs.

Figure 7

Effect of CMs from hRAN and hRAT on: AdipoR1 (A); ObR (B), CD44 (C) and vimentin (D) expression was evaluated in HK-2, 786-O, ACHN and Caki-1 cell lines. HK-2, 786-O, ACHN and Caki-1 cells were grown on 6 well plates, incubated for 24 hs with the different CMs and then lysed. Expression of the different proteins was measured by Western blot. β-actin was used as internal control. Images were analyzed by densitometry Horizontal bars represent the geometric mean of each data set. Vertical bars indicate SEM. ^* p < 0.001 cells incubated with hRAT-CMs vs. hRAN-CMs and control-CMs.

Figure 8

Effect of CMs from hRAN and hRAT on: pERK (A) and pPI3K (B) expression was evaluated in HK-2, 786-O, ACHN and Caki-1 cell lines. HK-2, 786-O, ACHN and Caki-1 cells were grown on 6 well plates, incubated for 24 hs with the different CMs and then lysed. Expression of the different proteins was measured by Western blot. ERK (A) and PI3K (B) were used as internal controls. Images were analyzed by densitometry Horizontal bars represent the geometric mean of each data set. Vertical bars indicate SEM. ^* p < 0.001 cells incubated with hRAT-CMs vs. hRAN-CMs and control-CMs.