Role of Protein Disulfide Isomerase in Redox Homeostasis and Regulation of Renal Angiotensin II at1 Receptor Function and Blood Pressure in Rats



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Kidney angiotensin II AT 1 receptors (AT1R) plays pivotal role in the regulation of blood pressure. Protein Disulfide Isomerase (PDI) via oxidative mechanism in vasculature causes hypertension. However, the role and mechanism of PDI, in the kidney, in regulating oxidative stress and blood pressure is not known. To examine this, adult male Sprague-Dawley rats were administered with PDI inhibitor bacitracin for 2-weeks. Thereafter, blood pressure and renal AT1R function in response to AT1R blocker candesartan in anesthetized rats were measured. Second set of rats, treated as above, were used for biochemical studies in the urine, plasma and kidney samples from these rats. Renal cells in cultures also were used to examine the mechanistic aspects.
Systolic and diastolic blood pressures were increased in bacitracin-treated compared to vehicle-treated rats. Candesartan response on diuresis and natriuresis were increased in vehicle-treated rats, which further increased in bacitracin-treated rats suggesting exaggerated AT1R function in these rats. These were associated with higher renin activities in the plasma and renal tissues in bacitracin-treated rats. Furthermore, urinary 8-isoprostane and kidney injury molecule-1 levels were higher whereas urinary antioxidant capacity and nitrate/nitrite levels were lower in bacitracin-treated rats, suggesting a higher systemic oxidative stress levels in these rats. Also, renal protein carbonyls and nitrotyrosine levels were higher in bacitracin-treated rats, suggesting oxidative stress burden in the kidneys of bacitracin-treated rats. Moreover, PDI activity decreased while its protein levels increased in renal tissues of bacitracin-treated rats. Nuclear levels of Nrf2, a transcription factor that regulates antioxidant enzymes genes, were decreased in bacitracin-treated rats. This was associated with lower antioxidant enzymes activities, superoxide dismutase (SOD) and glutathione S-transferase (GST), in these rats. Further, tissue levels of Keap1, an Nrf2 suppressor, and tyrosine 216 phosphorylated GSK3β protein (GSK3β-pY216), that exports Nrf2 from the nuclei to cytosol, were increased in bacitracin-treated rats. Further, DCFH (hydrogen peroxide probe) fluorescence levels were not changed in bacitracin-treated compared to control-cells, but increased with SOD-mimetic tempol, suggesting increased levels of superoxide in these cells. Nuclear Nrf2 levels were decreased in bacitracin-treated compared to control-cells, which was associated with reduced GST activity in these cells. These effects were attenuated with tempol. PDI protein levels were reduced in bacitracin-treated cells, which increased with tempol to the levels of control cells. In addition, membranous AT1 receptor proteins abundance was increased in bacitracin-treated cells, which reduced with tempol. Further, the levels of Nrf2 were higher in the Keap1 immunoprecipitants from bacitracin alone treated cells and bacitracin + tempol treated cells. Furthermore, the levels of thiols (-SH) in the Keap1 immunoprecipitants were higher in bacitracin treated cells, suggesting increased Keap1-SH binds to Nrf2 and reduced Nrf2 nuclear translocation. Taken together, these studies suggest that renal PDI, as an antioxidant, by regulating Keap1/Nrf2 pathway, regulates redox balance, renal AT1 receptor function and blood pressure. PDI may provide an alternative and a promising future therapeutic target to combat hypertension and associated cardiovascular diseases.



Protein Disulfide Isomerase, AT1 receptors, Blood pressure, NRF2


Portions of this document appear in: Wang, Xitao, and Mohammad Asghar. "Protein disulfide isomerase regulates renal AT1 receptor function and blood pressure in rats." American Journal of Physiology-Renal Physiology 313, no. 2 (2017): F461-F466.