T-type calcium channels and human mesangial cell proliferation
Aberrant proliferation of human mesangial cells (MC) is a critical step in the pathogenesis of mesangioproliferative renal diseases. The T-type calcium channel (T-CaCN) has been proposed to play an important role in the proliferation of a number of non-excitable cell types, but T-CaCN expression and functional significance in MC is not known. The aim of this thesis was to investigate the hypothesis that T-CaCN may play an important role in the proliferation of MC in primary culture. Expression of mRNA encoding the α1H isoform (Cav3.2 clone) (but not the α1G nor α1I T-CaCN isoforms) was demonstrated in human MC by RT-PCR. Expression of α1H T-CaCN protein was difficult to assess directly due to the lack of a highly sensitive and specific antibody, despite attempts at upregulation of the protein. Electrophysiological studies of MC demonstrated an inward calcium current in a proportion of cells with characteristics consistent with T-CaCN. Culture of MC with the T-CaCN antagonists mibefradil, Ni2+ and TTL-1177 resulted in a significant reduction in the growth velocity of MC. This effect was not seen upon incubation with verapamil, an L-type calcium channel antagonist. DNA synthesis in MC treated with each of the T-CaCN antagonists was significantly reduced by up to 50% as shown by BrdU incorporation. This anti-proliferative effect was not associated with direct drug-induced cytotoxicity or apoptosis. FACS analysis of MC incubated with T-CaCN antagonists illustrated an increased proportion of cells remaining in G1 and not progressing into S-phase. Treatment of cultured MC with TTL-1177 resulted in a significant reduction in the signalling protein p-ERK within 30 minutes, an effect not seen with verapamil, suggesting a possible mechanism needing further investigation. MC transfection with siRNA targeting the α1H isoform resulted in significant knockdown of T-CaCN α1H mRNA and a reduction in the growth velocity of cultured MC of approximately 50% compared to control siRNA transfection, with an associated 37% reduction in DNA synthesis. These results demonstrate evidence for an important role for T-type calcium channels in the proliferation of human mesangial cells, justifying further study in in greater detail. This could potentially lead to a novel therapy in the treatment of proliferative renal diseases.
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