Uncontrolled hypertension can lead to dire outcomes, including chronic kidney disease and end stage renal disease. Lu et al. found that by knocking out A20 from mice, blood pressure was significantly elevated. Due to this, we predict that A20 is a critical player in regulating immune-mediated renal hypertension — and that upregulating A20 expression could protect against hypertension. We tested two known activators of A20, delta tocotrienol (DT) and gibberellic acid (GA3). We treated HK2 cells with both activators and found that DT was able to significantly induce A20 expression. However, the same effect was not seen in vivo after treatment of either activator. We intend to explore the use of other A20 activators, including adiponectin and using AAV9, to understand the capacity of A20 activation in limiting hypertension severity.
Although much research has been done to elucidate the immune system’s effect on hypertension and T-cell’s contribution towards hypertensive states, it is still unclear how we can go about manipulating the immune system to safely alleviate high blood pressure. Recently, A20 has become a protein of interest that can lessen inflammation and potentially reduce blood pressure via TNF inactivation. In this research proposal, we hope to identify an A20 activator in hope of resolving immune mediated hypertension. In vitro: HK2 cells were treated with 10 µg DT and 30 µg GA3 for 12 or 24 hours. Total RNA was isolated from the HK2 cells and reverse transcribed to cDNA. RT-PCR was run for GAPDH, A20, TNF, and HO-1. In vivo: 129/SvEv mice were treated with DT or GA3 subcutaneously for 0, 7 or 14 days. Mice were harvested on the 14th day of treatment, and body, kidney, and spleen weights were recorded. Immune cells were sorted from the spleen, including dendritic cells. RNA was isolated from these sorted immune cells and kidney tissue. RT-PCR was run for GAPDH, A20, and TNF.
In HK2 cells, DT was able to significantly induce A20 and HO-1 expression 24 hours after treatment. HO-1 is a stress response protein and protects against oxidative stress. The gene expression of TNFα, a proinflammatory cytokine, was not affected. This indicates a potential protective and promising effect from DT. GA3 did not have a significant effect on A20, HO-1, or TNFα expression in vitro. In wildtype 129/SvEv mice, neither DT nor GA3 were able to successfully induce A20 expression in the kidney, dendritic cells, nor other immune cells. This indicates that perhaps these compounds are not optimal for A20 induction, and that other compounds or methods of administration should be considered. Currently, we are working on two different ways to induce A20 expression. Hand et al. found that adiponectin can induce A20 expression in adipose tissue. We are currently treating C57BL/6J mice with adiponectin to see whether A20 expression is induced in kidney tissue. In addition, adeno-associated virus (AAV) vector is a novel gene therapy that could serve as a vehicle to deliver transgenes. We are hoping to use an AAV vector to deliver A20 in mice with hypertension to see if and to what extent blood pressure can be reduced.
