The role of oxidant stress in the pathophysiology of cardiovascular disease has long been a subject of considerable interest. Although end stage renal disease is relatively uncommon, milder degrees of chronic kidney disease [CKD] such as CKD 3 are quite common and have been implicated as important determinant in cardiovascular morbidity and mortality. Oxidant stress plays a key role in the development of renal-failure- associated cardiomyopathy (also known as uremic cardiomyopathy), both experimentally and clinically. The ?Na/K-ATPase,? a sodium-potassium pump, has been shown by our group to affect cellular signaling via the ?Na/K-ATPase signaling,? which amplifies oxidative stress. We have specifically shown that this pathway is critical to the pathophysiology of several experimental models of disease including obesity/metabolic syndrome and experimental uremic cardiomyopathy. We and others have also observed that the adipocyte itself is an important source of oxidant stress in models with obesity/metabolic syndrome and that mediators directly tied to the cellular phenotype of these adipocytes play a causal role in the cardiovascular conditions associated with obesity/metabolic syndrome. This led us to believe that adipocytes could play a central role in uremic cardiomyopathy. Therefore, we hypothesize that adipocytes create systemic oxidant stress through the Na/K- ATPase feed-forward oxidant amplification loop in uremic cardiomyopathy and serve as a therapeutic target for this condition. Our group has developed a cell permeant peptide, NaKtide, from the N domain of the ?1 subunit of the Na/K-ATPase, which inhibits Na/K-ATPase-ROS amplification. Our preliminary results show that the NaKtide, targeted specifically to adipocyte, attenuates oxidative stress and inflammatory cytokines, in addition to improving metabolic parameters. Our experimental approach includes studies to determine the role of adipocyte Na/K-ATPase signaling in the development of experimental uremic cardiomyopathy using a partial nephrectomy (PNx) mouse model with and without dietary manipulations (Aim 1). We will also target the NaKtide to the adipocyte using ?lentiviral gene transfer? strategy (Aim 1) to determine the role of adipocyte-derived Na/K- ATPase/Src signaling in the progression of uremic cardiomyopathy. To test the off-target effects of NaKtide, we will also use lentiviral vectors with tissue specific promoters, including heart and kidney to targeted NaKtide specially in these tissues. Further, we will target c-Src-shRNA to adipocytes using lentivirus vector, as an alternate strategy of inhibiting Na/K-ATPase signaling, to demonstrate the role of c-Src as a downstream mediator of Na/K-ATPase in exacerbating oxidative stress and eventually uremic cardiomyopathy.
In Aim 2, we will employ in vitro protocols for primary adipocytes, isolated from Sham or PNx operated C57BL6 mice and perform RNASeq analysis to study the adipocyte phenotypic alterations and pathways associated with uremic cardiomyopathy phenotype. We will also use primary adipocytes isolated from C57BL6 and Na/K-ATPase ?1+/- mice which will be exposed to uremic toxins, subsequently activating Na/K-ATPase signaling, to determine the activation of specific downstream molecular pathways in vitro that mimic in vivo outcomes. These experiments will allow us to determine if the Na/K-ATPase signaling and/or adipocytes are potential targets for disease intervention. These studies if proven may provide a basis for the future studies to ameliorate uremic cardiomyopathy phenotype.
The ?Na/K-ATPase,? a sodium-potassium pump, has been shown by our group to affect cellular signaling via the ?Na/K-ATPase/Src/ROS amplification loop,? which amplifies oxidative stress. This proposal will determine, for the first time, that adipocytes creates systemic oxidant stress through the Na/K-ATPase feed-forward oxidant amplification loop in uremic cardiomyopathy and serve as a therapeutic target for this condition. In this proposal, we will also determine whether the Na/K-ATPase signaling antagonist, NaKtide peptide, targeted specifically to adipocytes is beneficial in the prevention and/or reversal of uremic cardiomyopathy. This proposal will allow us to determine if the Na/K-ATPase- Src feed-forward oxidant-amplification loop and/or adipocytes are potential targets for disease intervention.
