Hypertension may develop before or after the onset of diabetes and it is known to increase the risk of developing diabetic nephropathy. Alpha-1 antitrypsin (AAT) is a multi-functional protein with beneficial effects in various diseases but its role in reducing blood pressure in the diabetic kidney has not been thoroughly studied. Like blood pressure, epithelial sodium channels (ENaC) and its adaptor protein myristoylated alanine-rich C-kinase substrate (MARCKS) are regulated by circadian rhythms. Our hypothesis is that administration of human AAT (hAAT) reduces blood pressure in hypertensive diabetic mice by attenuating membrane expression of ENaC and its association with the actin cytoskeleton. First, we show hAAT administration results in reduced blood pressure in diabetic db/db mice compared to vehicle treatment in both the inactive and active cycles. Western blotting and immunohistochemistry analyses showed a reduction of ENaC and the actin cytoskeleton protein, MARCKS in the kidneys of diabetic db/db mice treated with hAAT compared to vehicle. hAAT treatment resulted in elevated amounts of extracellular vesicles present in the urine of diabetic db/db mice compared to vehicle treatment both in the inactive and active cycles. Multiple hexosylceramides, among other lipid classes increased in urinary EVs released from hAAT treated hypertensive diabetic mice compared to vehicle treated mice. Taken together, these data suggest hAAT treatment could normalize blood pressure in the diabetic kidney in a mechanism involving attenuation of renal ENaC and MARCKS protein expression and possibly ceramide metabolism to hexosylceramide in kidney cells.
The C-type natriuretic peptide receptor (NPRC) is expressed in many cell types and binds all natriuretic peptides with high affinity. Ligand binding results in the activation or inhibition of various intracellular signaling pathways. Although NPRC ligand binding has been shown to regulate various ion channels, the regulation of endothelial sodium channel (EnNaC) activity by NPRC activation has not been studied. The objective of this study was to investigate mechanisms of EnNaC regulation associated with NPRC activation in human aortic endothelial cells (hAoEC). EnNaC protein expression and activity was attenuated after treating hAoEC with the NPRC agonist cANF compared to vehicle, as demonstrated by Western blotting and patch clamping studies, respectively. NPRC knockdown studies using siRNA’s corroborated the specificity of EnNaC regulation by NPRC activation mediated by ligand binding. The concentration of multiple diacylglycerols (DAG) and the activity of protein kinase C (PKC) was augmented after treating hAoEC with cANF compared to vehicle, suggesting EnNaC activity is down-regulated upon NPRC ligand binding in a DAG-PKC dependent manner. The reciprocal cross-talk between NPRC activation and EnNaC inhibition represents a feedback mechanism that presumably is involved in the regulation of endothelial function and aortic stiffness.
The expression of the myristoylated alanine-rich C-kinase substrate (MARCKS) family of proteins in the kidneys plays an important role in the regulation of the renal epithelial sodium channel (ENaC) and hence overall blood pressure regulation. The function of MARCKS is regulated by post-translational modifications including myristoylation, phosphorylation, and proteolysis. Proteases known to cleave both ENaC and MARCKS have been shown to contribute to the development of high blood pressure, or hypertension. Here, we investigated protein expression and proteolysis of MARCKS, protein expression of multiple protein kinase C (PKC) isoforms, and protein expression and activity of several different proteases in the kidneys of diabetic db/db mice compared to wild-type littermate mice. In addition, MARCKS protein expression was assessed in cultured mouse cortical collecting duct (mpkCCD) cells treated with normal glucose and high glucose concentrations. Western blot and densitometric analysis showed less abundance of the unprocessed form of MARCKS and increased expression of a proteolytically cleaved form of MARCKS in the kidneys of diabetic db/db mice compared to wild-type mice. The protein expression levels of PKC delta and PKC epsilon were increased, while cathepsin B, cathepsin S, and cathepsin D were augmented in diabetic db/db kidneys compared to those of wild-type mice. An increase in the cleaved form of MARCKS was observed in mpkCCD cells cultured in high glucose compared to normal glucose concentrations. Taken together, these results suggest that high glucose may contribute to an increase in the proteolysis of renal MARCKS, while the upregulation of the cathepsin proteolytic pathway positively correlates with increased proteolysis of MARCKS in diabetic kidneys, where PKC expression is augmented.
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