Human Alpha-1 Antitrypsin Attenuates ENaC and MARCKS and Lowers Blood Pressure in Hypertensive Diabetic db/db Mice

Abstract

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.

Extracellular Vesicles: Investigating the Pathophysiology of Diabetes-Associated Hypertension and Diabetic Nephropathy

“…Several epithelial transport mechanisms have been studied in the context of the induction and maintenance of salt-sensitive hypertension. Multiple studies have shown db/db mice develop hypertension upon salt-loading [ 41 , 42 , 43 , 44 ]. A study by Scindia et al showed that metformin mitigated high blood pressure in hypertensive diabetic db/db mice in a mechanism involving the attenuation of renal ENaC [ 43 ].…”

“…A study by Scindia et al showed that metformin mitigated high blood pressure in hypertensive diabetic db/db mice in a mechanism involving the attenuation of renal ENaC [ 43 ]. A study by Lugo et al showed that alpha-1 antitrypsin (AAT) treatment by intraperitoneal injection normalized blood pressure in salt-loaded hypertensive diabetic db/db mice in a mechanism involving the attenuation of renal ENaC and MARCKS protein expression [ 42 ].…”

“…A study by Musante et al investigated the alterations in protease profiles in uEVs in diabetic nephropathy patients [ 56 ]. Lugo et al showed that multiple hexosylceramides and other classes of lipids are increased in uEVs released from hypertensive diabetic db/db mice compared to control animals [ 42 ]. A study by Scindia et al showed that uEVs from diabetic db/db mice are enriched in the cysteine protease cathepsin B, and the amount is attenuated after treating the mice with metformin compared to the vehicle [ 43 ].…”

“…Db/db mice have become an appreciated model to investigate the mechanisms associated with the development of hypertension in the diabetic kidney. These mice develop profound hypertension after salt loading and have elevated ENaC protein expression and proteolysis [ 42 , 43 ]. Also, this model can be useful in investigating the crosstalk between multiple organ systems, including the cardiovascular and renal systems.…”

“…Multiple studies have shown EV marker proteins are differentially enriched in uEVs isolated from urine samples during the inactive and active cycles of adult mice. A study by Lugo et al showed differences in the amount of annexin A2, GAPDH, TSG101, and caveolin-1 proteins, as well as lipids including specific hexosylceramides, monoacylglycerols, phosphatidylcholines, lysophosphatidylethanolamine, phosphatidylethanolamine, and phosphatidylglycerol enriched in uEVs from male and female diabetic db/db mice treated with human AAT (hAAT) or the vehicle [ 42 ]. A study by Lopez et al showed differences in the amounts of syntein and flotillin proteins, as well as lipids including specific phosphatidylethanolamines, triacylglycerols, and phosphatidylcholines enriched in uEVs from aged male mice with spontaneous hypertension [ 15 ].…”