Immunolocalization of the AT-1R Ang II Receptor in Human Kidney Cancer

Figures

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  Figure 1. Expression of AT-1R mRNA in the kidney tissue. (a) The total RNA was extracted from 42 tumoral kidney tissues and 40 normal kidney tissues, and real-time qPCR analyses targeting the expression of AT-1R were performed (statistical analysis by Mann–Whitney U test: p < 0.001). (b) Tumoral kidney tissues were classified as G1 (n = 6), G2 (n = 9), G3 (n = 13), and G4 (n = 14). Gene expressions were normalized by individual β-actin. Values refer to the mean data ± SD (statistical analysis by ANOVA: p < 0.001). Different letters indicate significantly different values under the post hoc Bonferroni test.

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  Figure 2. Expression of the AT1-R protein in the kidney tissue. (a) SDS analysis, followed by immunoblotting, performed on matched pairs of normal (N1, N2, N3, and N4) and cancerous (C1 (G1), C2 (G2), C3 (G3), and C4 (G4)) kidney samples. In this experiment, the same protein concentration (100 µg) was loaded on the gel. A band of molecular mass of approximately 41 kDa (arrow) was detected. For each sample lane, the densitometric quantification of the AT-1R band refers to the mean data from n = 3 different blots. Mean values have been normalized concerning the corresponding β-actin band. (b) Densitometric quantification of the AT-1R band obtained by immunoblotting analysis performed on 40 normal (N) and 42 tumoral kidney tissues classified as G1 (n = 6), G2 (n = 9), G3 (n = 13), and G4 (14). Values refer to the mean data ± SD from different blots and have been normalized concerning the corresponding β-actin band. Molecular weight markers are shown on the left (statistical analysis: p < 0.001 by one-way ANOVA). Values with shared letters are not significantly different according to Bonferroni–Dunn post hoc tests.

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  Figure 3. The expression of AT-1R in normal kidney and kidney cancer tissues with immunostaining of paraffin-embedded human kidney. (a) AT-1R expression in the kidney at low magnification (400×). AT-1R is expressed in the proximal tubule cells, collecting ducts, and vascular structures. (b) AT-1R is expressed in epithelial cells (600×). (Right) Binary images obtained from (a,b) after thresholding showed the regions stained by the antibody.

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  Figure 4. The expression of AT-1R in normal and cancerous kidney tissues. (a) The expression of AT-1R in renal cancer G1 (200×). (b) Lesser expression of AT-1R in G4 renal cancer both in epithelioid and spindle cells (upper and left portion of the picture (200×)). (Right) Binary images obtained from a and b after thresholding, which show the regions stained by the antibody. (c) Distribution of AT-1R-positive cells in the kidney tumors of differing nuclear gradings. Values refer to the mean data ± SD (statistical analysis: p < 0.001 by one-way ANOVA). Values with shared letters are not significantly different according to Bonferroni–Dunn post hoc tests. (d) Distribution of AT-1R expression values (expressed as the sum of gray levels) into the two groups of patients’ lymph node metastasis statuses. Horizontal long bars represent the mean values, while shorter bars represent standard deviations (Kruskal–Wallis test: p < 0.006).

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  Figure 5. Effects of increasing doses of Ang II upon the proliferation of human cultured normal and cancerous kidney cells and the effects of losartan or CGP4221A. Normal and cancerous kidney cells were stimulated with increasing concentrations of Ang II (0, 0.1, 1, 10, and 100 nM) for 24 h in serum-deprived medium, or the cells were pretreated with 1 µM losartan or 1 mM CGP4221A for 45 min. Proliferation was analyzed by a spectrophotometric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol-2H-tetrazolium bromide (MTT) assay. The data are means ± S.D. of five different experiments (n = 5). Values with shared letters are not significantly different according to Bonferroni–Dunn post hoc tests.

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  Figure 6. Effects of Ang II on ERK1/2 phosphorylation in human cultured normal and cancerous kidney cells. Normal (up) and cancerous (down) cells were incubated (a) without or with 0.1, 10, and 100 nM Ang II for the indicated times or (b) with 100 nM Ang II for the indicated time. Lysates were analyzed by western blotting with anti-total-ERK1/2 or with anti-phosphorylated-ERK1/2. (c,d) Densitometric analysis of phosphorylated-ERK1/2 normalized to unphosphorylated ERK1/2. The data are means ± S.D. of five different experiments (n = 5). Values with shared letters are not significantly different according to Bonferroni–Dunn post hoc tests.