• Endothelin Receptor A Function in Progressive Kidney Disease

      Heimlich, Jonathan B.; Department of Physiology (2014-05)
      Endothelin-1 (ET-1) is a vasoactive peptide that regulates electrolyte and arterial blood pressure homeostasis. For this reason, ET-1 is increased under conditions of high dietary salt intake but also contributes to the progression of a variety of forms of kidney disease associated with hypoxia including sickle cell nephropathy. We previously found that ET-1 is an integral factor in glomerular injury seen during diabetic nephropathy through the activation of the ETA receptor. Recent studies also suggest ET-1 via the ETA receptor mediate the upregulation of reactive oxygen species (ROS) contributing to tissue damage. The overall aim of this work is to understand the role of ETA receptor activation in the development of sickle cell nephropathy and to determine the therapeutic potential of an ETA receptor antagonist in the treatment of renal manifestations associated with sickle cell disease (SCD). Since ET-1 is elevated and hypoxia is a prevalent occurrence in SCD, we utilized several strategies to elucidate the actions of ET-1 via the ETA receptor including ET-1 induction with a high salt diet and a hypoxic challenge to re-create an environment similar to SCD. The first aim was designed to determine whether ET-1 derived from endothelial cells contributes to oxidative stress in the glomerulus of mice subjected to a high salt diet and/or hypoxia. Hypoxia increased glomerular ET-1 mRNA expression in control, but not in vascular endothelial cell ET-1 knockout (VEET KO) mice. Increased superoxide formation was detected in the cortices of mice exposed to acute hypoxia. Under normoxic conditions, mice on a high salt diet had approximately 150% higher glomerular ET-1 mRNA expression compared to a normal salt diet. High salt diet administration also significantly increased glomerular ROS production in flox control, but not in glomeruli isolated from VEET KO mice. In C57BL6/J mice, the ETA receptor selective antagonist, ABT-627, significantly attenuated the increase in glomerular ROS production produced by high salt diet. In addition, chronic infusion of a sub-pressor dose of ET-1 via miniosmotic pumps in C57BL6/J mice significantly increased levels of glomerular ROS that were prevented by ETA antagonist treatment. Finally, three-hour exposure to hypoxia (8% O2) in control C57BL6/J mice significantly increased urinary protein excretion during the 24 hrs following hypoxia, but only in animals on a high salt diet. In conclusion, these data suggest that both hypoxia and a high salt diet increases glomerular ROS production via endothelial derived ET-1-ETA receptor activation and provide a potential mechanism for ET-1 induced nephropathy. The second aim was to test whether ET-1 acting via the ETA receptor contributes to renal injury in a mouse model of SCD. Humanized knockout, knockin sickle mice were used to describe the endothelin phenotype in the kidney. Sickle mice have increased ET-1 mRNA expression in both the cortex and glomeruli compared to heterozygous controls. Sickle animals have increased renal cortex ETA receptor mRNA expression but similar levels of ETB receptor mRNA expression. Radiolabeled ligand binding assays revealed sickle mice had increased ET-1 binding and subsequently increased ETA receptor binding in the renal vessels when compared to control mice. In response to PMA stimulation, sickle mice had increased glomerular ROS compared to controls, which could be prevented by treatment with ABT-627. Protein and nephrin excretion are two urinary markers of renal injury and were both found to be elevated in our mouse model of SCD. Treatment with ABT-627 resulted in significant decreases in both nephrin and protein excretion in SCD mice. Finally, 1 week of ETA antagonism also caused a significant decrease in mRNA expression of NADPH oxidase subunits as well as VEGF mRNA expression, both of which are thought to contribute to pathologic processes in chronic kidney disease. These data indicate a novel role for ET-1 in the progression of SCN, specifically via the ETA receptor in the glomerulus and suggest ETA antagonism to be a viable treatment strategy for SCN. Taken together, these studies reveal a prominent role for the pathologic activation of the ETA receptor in the progression of kidney diseases where ET-1 is upregulated and hypoxia is prevalent. ET-1 is known to contribute to other chronic kidney diseases such as diabetic nephropathy and significant benefit is found through ETA antagonism. These data offer a rationale for a novel treatment modality in renal disease associated with SCD.