Despite the tremendous success of solid organ transplantation in past 20 years, long-term kidney outcomes have been limited by chronic calcineurin inhibitor (CNI)-mediated fibrosis. CNIs including cyclosporine A (CsA) and tacrolimus (TAC) are the backbone of anti-rejection therapy in transplantation. However, they represent a major public health concern since CNI-induced kidney fibrosis is an important cause of renal failure after solid organ transplantation. Increasing evidence suggests that Nox2 is at the crossroads of CNI-induced renal hypoxia and fibrosis. Nox2 is the classical phagocytic isoform of the NADPH oxidase enzyme responsible for "oxidative burst", the mechanism that eliminates internalized pathogens. We hypothesize that independent from its role in phagocytes, Nox2 is a key regulator of CNI-induced kidney hypoxia and fibrosis. In support of this hypothesis, observations from our laboratory demonstrate that (a) Nox2 is inducible in renal tubular epithelial cells during CsA-induced epithelial-to-mesenchymal transition (EMT) (b) tubulointerstitial Nox2 is upregulated in kidneys undergoing CsA-induced fibrosis (c) inhibition of Nox activity with apocynin and diphenyleneiodonium (DPI) is associated with prevention of CsA-induced fibrosis and hypoxia measured by blood oxygen-level-dependent MRI (BOLD MRI) in rats. Based on these observations, and the availability of Nox2 knockout mice to rigorously test our hypotheses, we propose the following studies.
In Specific Aim 1, we will characterize the molecular mechanisms that regulate Nox2 activity during CNI-induced EMT and matrix remodeling in renal tubular epithelial cells. Next, we will define the specific role of Nox2 in CNI-induced renal fibrosis. We will perform these studies using wild type, Nox2, TGFb1 and Smad2/3-null mice and tubular cells.
In Specific Aim 2, we will characterize the effects of chronic CsA therapy on medullary and cortical perfusion and oxygenation using noninvasive MR-imaging with BOLD and contrast-enhanced perfusion studies in mice. We will confirm these studies with molecular markers of tissue hypoxia including pimonidazole and HIF-1a. Last, we will determine if the absence of Nox2 improves CsA-induced hypoperfusion and hypoxia in mice.
In Specific Aim 3, we will confirm the validity of the above molecular and small animal investigations in a pilot clinical study of 20 nonkidney organ transplant recipients. The study is designed to determine whether intrarenal Nox2 is associated with chronic CNI nephrotoxicity. The secondary objective is to determine whether intrarenal oxygenation measured by BOLD MRI can predict disease progression. In summary, we outline research studies that rigorously assess the role of Nox2 in CNI-induced renal fibrosis. In addition, will propose contemporary and complementary clinical and translational strategies to examine the molecular mechanisms that regulate Nox2 activity during CNI-mediated fibrogenesis. If successful, the results of our studies will provide a significant step forward in the design of new diagnostic, monitoring and treatment options aimed to offset the deleterious effects of immunosuppressant therapy and improve long-term kidney outcomes.
Chronic kidney failure from calcineurin inhibitors is an important public health concern in organ transplant recipients. We propose in vitro, animal and human studies that examine the specific role of Nox2 as a mediator of kidney fibrosis. We also propose noninvasive imaging technologies to diagnose and monitor renal scarring from these drugs. If successful these studies could lead to the development of treatment targets and diagnostic/monitoring methods for the prevention of kidney fibrosis in organ transplant recipients.
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