Abstract Title

Prolonged High Glucose Treatment Increased Orai1 Protein Expression through Inhibition of Lysosomal Pathway in Human Mesangial Cells

Presenter Name

Sarika Chaudhari

Abstract

The Orai1-mediated store operated calcium entry (SOCE) is associated with many physiological and pathological processes in a variety of cells, including glomerular mesangial cells (MCs). We have previously demonstrated that prolonged treatment of MCs with high glucose (HG) significantly increased Orai1 expression at protein level, but not at mRNA level. These findings suggest that a post-transcriptional mechanism(s) contributes to the HG effect. The aim of the present study was to identify if proteosomal and/or lysosomal pathways were involved in the increased abundance of Orai1 protein in response to HG. Cultured human MCs were with and without treatment with 10 μM MG132 (an inhibitor of proteosomal pathway) or 10 mM ammonium chloride (an inhibitor of lysosomal pathway) in the presence of normal glucose (NG, 5.6 mM) or HG (25 mM) for 6 days. Western blots of the whole cell lysates were conducted to evaluate Orai1 expression level. Fura-2 fluorescence ratiometry was performed to study the intracellular calcium changes in the human MCs. We found that both MG132 and ammonium chloride increased abundance of Orai1 protein in MCs incubated with NG. The MG132 response was further increased by HG treatment. However, HG failed to cause additional increase in Orai1 protein expression in ammonium chloride-treated cells. Furthermore, fura-2 fluorescence ratiometry study showed that both MG132 and ammonium chloride increased the cyclopiazonic acid (25 μM)-stimulated SOCE. Simultaneous treatment with HG only enhanced the MG132 response, but not the ammonium chloride response. Taken together, our results indicate that HG increased expression of Orai1 protein by inhibiting its degradation through the lysosomal pathway.

Presentation Type

Poster

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Prolonged High Glucose Treatment Increased Orai1 Protein Expression through Inhibition of Lysosomal Pathway in Human Mesangial Cells

The Orai1-mediated store operated calcium entry (SOCE) is associated with many physiological and pathological processes in a variety of cells, including glomerular mesangial cells (MCs). We have previously demonstrated that prolonged treatment of MCs with high glucose (HG) significantly increased Orai1 expression at protein level, but not at mRNA level. These findings suggest that a post-transcriptional mechanism(s) contributes to the HG effect. The aim of the present study was to identify if proteosomal and/or lysosomal pathways were involved in the increased abundance of Orai1 protein in response to HG. Cultured human MCs were with and without treatment with 10 μM MG132 (an inhibitor of proteosomal pathway) or 10 mM ammonium chloride (an inhibitor of lysosomal pathway) in the presence of normal glucose (NG, 5.6 mM) or HG (25 mM) for 6 days. Western blots of the whole cell lysates were conducted to evaluate Orai1 expression level. Fura-2 fluorescence ratiometry was performed to study the intracellular calcium changes in the human MCs. We found that both MG132 and ammonium chloride increased abundance of Orai1 protein in MCs incubated with NG. The MG132 response was further increased by HG treatment. However, HG failed to cause additional increase in Orai1 protein expression in ammonium chloride-treated cells. Furthermore, fura-2 fluorescence ratiometry study showed that both MG132 and ammonium chloride increased the cyclopiazonic acid (25 μM)-stimulated SOCE. Simultaneous treatment with HG only enhanced the MG132 response, but not the ammonium chloride response. Taken together, our results indicate that HG increased expression of Orai1 protein by inhibiting its degradation through the lysosomal pathway.