Mechanisms of high glucose-induced altered endothelin-1 signalling in glomerular mesangial cells

Hyperglycaemia is a key factor in the development of diabetic microvascular complications, including diabetic nephropathy. High glucose (HG) alters signalling of autocrine factors, including endothelin-1 (ET-1), to cause aberrant synthesis of extracellular matrix by glomerular mesangial cells and renders them unresponsive to vasoconstrictor actions. We hypothesized that protein kinase C (PKC) activation plays a pivotal role in HG-enhanced alpha1 (IV) collagen synthesis and reduced Ca2+ signalling in response to ET-1. Through expression of dominant-negative (DN) constructs, we showed that PKC-delta, -epsilon, and -zeta are required for HG-enhanced ET-1 activation of ERK1/2 leading to alpha1 (IV) collagen expression whereas PKC-alpha and -beta regulate alpha1 (IV) collagen synthesis independently of the ERK1/2 pathway. In normal glucose, ET-1-induced ERK1/2 phosphorylation involved a PKC-dependent EGF-R transactivation and caveolae signalling, ascertained by the use of a specific EGF-R inhibitor, caveolae-disrupting reagents, and by an inhibitory peptide that binds to the scaffolding domain of caveolin-1. Antisense oligodeoxynucleotide to a component of NADPH oxidase, p47phox, prevented the increased generation of ROS in HG and reversed the effect of HG-induced depressed Ca2+ signalling in response to ET-1. Inhibition of DAG-sensitive PKC likewise attenuated mesangial cell Ca2+ signalling. Therefore, HG causes NADPH oxidase generation of ROS, and consequent activation of DAG-sensitive PKC isozymes to reduce mesangial cell Ca2+ signalling. Overall, we found that during ET-1 signal transduction PKC participates in acute stimulation of ER1/2 through a mechanism involving EGF-R transactivation and caveolin-1 interaction and that sustained PKC activity in HG is responsible for alpha1 (IV) collagen expression and reduced Ca2+ signalling.