Angiotensin III increases monocyte chemoattractant protein-1 expression in cultured human proximal tubular epithelial cells

Abstract

Background/aims: We investigated whether angiotensin III (Ang III) is involved in monocyte recruitment through regulation of the chemokine monocyte chemoattractant protein-1 (MCP-1) in cultured human proximal tubular epithelial cells (HK-2 cells).

Methods: We measured MCP-1 levels in HK-2 cells that had been treated with various concentrations of Ang III and Ang II type-1 (AT1) receptor antagonists at various time points. The phosphorylation states of p38, c-Jun N-terminal kinases (JNK), and extracellular-signal-regulated kinases were measured in Ang III-treated cells to explore the mitogen-activated protein kinase (MAPK) pathway. MCP-1 levels in HK-2 cell-conditioned media were measured after pre-treatment with the transcription factor inhibitors curcumin or pyrrolidine dithiocarbamate.

Results: Ang III increased MCP-1 protein production in dose- and time-dependent manners in HK-2 cells, which was inhibited by the AT1 receptor blocker losartan. p38 MAPK activity increased significantly in HK-2 cells exposed to Ang III for 30 minutes, and was sustained at higher levels after 60 minutes (p < 0.05). Total phosphorylated JNK protein levels tended to increase 20 minutes after stimulation with Ang III. Pre-treatment with a p38 inhibitor, a JNK inhibitor, or curcumin significantly inhibited Ang III-induced MCP-1 production.

Conclusions: Ang III increases MCP-1 synthesis via stimulation of intracellular p38 and JNK MAPK signaling activity and subsequent activated protein-1 transcriptional activity in HK-2 cells.

Keywords: Angiotensin III; Chemokine CCL2; Kidney tubules; Mitogen-activated protein kinases; Transcription factors.

Figure 1.

Angiotensin III (Ang III)-induced monocyte chemoattractant protein-1 (MCP-1) production in HK-2 cells via the Ang II type-1 (AT1) receptor. HK-2 cells were treated with Ang II (10⁻⁶ M) and Ang III (10⁻⁹ to 10⁻⁶ M) for 48 hours in the presence or absence of the AT1 receptor antagonist losartan (10⁻⁷ M). MCP-1 protein in conditioned medium was quantified by enzyme-linked immunosorbent assay. Results are expressed as the percentage increase over untreated cells. Results are shown as mean ± standard error of mean from six independent experiments. ^ap < 0.05 vs. untreated cells, ^bp < 0.05 vs. Ang III (10⁻⁷ M)-treated cells.

Figure 2.

Angiotensin III (Ang III)-stimulated monocyte chemoattractant protein-1 (MCP-1) production in HK-2 cells. Cells were incubated for the indicated times in the presence or absence of Ang III. MCP-1 protein in culture medium was quantified by enzyme-linked immunosorbent assay. Results are shown as mean ± SEM from six independent experiments. Lactate dehydrogenase (LDH) release from Ang II-, Ang III-, or losartan-treated cells. LDH release is expressed as percentage of maximal LDH release induced by 1% Triton X-100 for 48 hours. ^ap < 0.05 vs. 8 hours MCP-1 level, ^bp < 0.05 vs, control cells.

Figure 3.

Lactate dehydrogenase (LDH) release from angiotensin (Ang) II-, Ang III-, or losartan-treated cells. LDH release is expressed as percentage of maximal LDH release induced by 1% Triton X-100 for 48 hours.

Figure 4.

Angiotensin III (Ang III, 10⁻⁷M) significantly stimulates p38 phosphorylation. Cells were incubated with Ang III (10⁻⁷ M) for various times, and (A) phosphorylated p38, (B) c-Jun N-terminal kinases (JNK), and (C) extracellular signal-regulated kinases (ERK) were detected by Western blot. Results are representative of three independent experiments with similar results. Con, control. ^ap < 0.05 vs. untreated cells.

Figure 5.

The effect of mitogen-activated protein kinase (MAPK) inhibitors on angiotensin III (Ang III)-induced monocyte chemoattractant protein-1 (MCP-1) production. HK-2 cells were pre-incubated with MAPK inhibitors for 30 minutes and then incubated with Ang III (10⁻⁷ M) for 48 hours. MCP-1 protein levels were measured by enzyme-linked immunosorbent assay. Results are expressed as percent increase compared to untreated cells. Results are shown as mean ± SEM from six independent experiments. ^ap < 0.05 vs. untreated cells, ^bp < 0.05 vs. Ang III-treated cells.

Figure 6.

Effects of the transcription factor inhibitors curcumin and pyrrolidine dithiocarbamate (PDTC) on angiotensin III (Ang III)-induced monocyte chemoattractant protein-1 (MCP-1) production. HK-2 cells were pre-incubated with curcumin or PDTC for 30 minutes and then incubated with Ang III (10⁻⁷ M) for 48 hours. MCP-1 protein levels were measured by enzyme-linked immunosorbent assay. Results are expressed as percentage increase over untreated cells. Results are shown as mean ± SEM from six independent experiments. ^ap < 0.05 vs. untreated cells, ^bp < 0.05 vs. Ang III-treated cells.