Role of Nox2 in CNI-induced Renal Fibrosis



Status:Active, not recruiting
Conditions:Renal Impairment / Chronic Kidney Disease
Therapuetic Areas:Nephrology / Urology
Healthy:No
Age Range:18 - 70
Updated:1/5/2019
Start Date:November 2012
End Date:August 2019

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The Role of Nox2 in CNI-Induced Renal Fibrosis

Calcineurin Inhibitors (CNI) are drugs used to suppress the immune system when a person has a
solid organ transplant. Although these drugs keep the transplanted organ from being rejected
they are toxic to kidneys, or nephrotoxic. CNIs cause damage, called fibrosis, to kidneys.

Fibrosis is a type of scarring that occurs in kidney tissue. Fibrosis can eventually lead to
kidney failure. One of the pathways that cause fibrosis is a chronic lack of oxygen to the
kidney tissue called "hypoxia". There is a protein called Nox2 that may be involved in how
this hypoxia happens in the kidney. The Department of Medicine-Nephrology at the University
of Wisconsin is conducting a research study to see how much of the Nox2 protein is present in
kidneys that may have fibrosis caused by CNIs and whether a certain type of Magnetic
Resonance Imaging (MRI) can be used to tell in advance if the disease caused by CNIs is
getting worse. Study hypothesis: MRI, a non-invasive technique, can be used to determine
whether CNI induced kidney disease is getting worse. Additionally, the study aims to
determine the role of Nox2 in CNI nephrotoxicity.

Calcineurin Inhibitors (CNIs) and Renal Fibrosis. CNIs including CsA and tacrolimus are the
cornerstone of maintenance immunosuppression in solid organ transplantation. These drugs are
so effective that newer generation CNIs such as Voclosporin (Isotechnika Pharma, Inc,
Edmonton, Alberta) are in development. They play their immunosuppressive role by inhibiting
the activity of calcineurin, a serine phosphatase that normally dephosphorylates nuclear
factor of activated T cells (NFAT). Dephosphorylated NFAT translocates in the nucleus and
induces the transcription of interleukin-2, an important cytokine for the activation and
proliferation of T-lymphocytes.

Despite their beneficial actions in transplantation and many autoimmune disorders, the
clinical use of CNIs is limited by their chronic nephrotoxicity. This represents a
significant public health problem since the 10-year incidence of chronic CNI nephrotoxicity
is 100% in renal transplant recipients. Similarly, chronic CNI nephrotoxicity is the dominant
causative factor for kidney failure in nonkidney organ transplant recipients. In this group,
the 5-year risk of ESRD ranges from 7 to 21% and is associated with a 4-time greater risk of
death. The pathological features of chronic CNI nephrotoxicity include progressive and
irreversible interstitial fibrosis, tubular atrophy and arteriolar hyaline changes. De novo
or progressive arteriolar hyaline thickening (AH) is the most pathognomonic lesion of chronic
CNI nephrotoxicity. This lesion consists of vacuolization of endothelial and smooth muscle
cells and focal or circular lumpy protein deposits in the arteriolar wall, which usually
replaces necrotic smooth muscle cells and eventually narrow the vascular lumen. This
arteriolopathy is important for the development of fibrosis. Typically, fibrosis follows a
"striped pattern" from the medulla to the cortex and is associated with vacuolization of the
cytoplasm in tubular epithelial cells.

Although tacrolimus is superior to CsA in improving graft survival and preventing acute
rejection in kidney transplantation, the individual nephrotoxicity profiles of these drugs
are similar. At cellular and molecular levels, CNIs result in matrix accumulation and EMT via
TGF-beta1 dependent and independent pathways. EMT is an important profibrotic process and a
surrogate marker of native and transplant kidney fibrosis. TGF-beta1 is the primary cytokine
that initiates and maintains EMT by activating signaling pathways and transcriptional
regulators such as Smad 2/3 molecules. During EMT, tubular epithelial cells are transformed
into myofibroblasts through a stepwise process involving loss of cell-cell adhesion molecules
(e.g. E-cadherin) and de novo expression of mesenchymal markers (e.g. alpha-SMA). These
events are followed by tubular basement membrane disruption, cell migration and fibroblast
invasion in the interstitium with production of profibrotic molecules including collagen and
fibronectin. A better understanding of the molecular mechanisms that regulate CNIinduced EMT
and fibrosis will pave the way for the development of antifibrotic strategies.

Objective evaluation of renal fibrosis in vivo is a key step in this direction. This was
recently achieved by quantitative assessment of renal allograft fibrosis using computerized
digital analyses of Sirius Red. These studies showed that fibrosis was a prognostic indicator
of long-term kidney allograft function and loss. However, the role of this technology in
predicting outcomes in patients with chronic CNI toxicity is unknown.

CNIs and Renal Hypoxia. Accumulating evidence suggests that chronic tubulointerstitial
hypoxia is a final common pathway to end-stage renal disease. CNIs result in decreased nitric
oxide (NO) production and bioavailability, thereby leading to decreased vasodilation and
unopposed vasoconstriction and hypoxia. In the chronic setting, CNI-mediated arteriolopathy
and narrowing of the arteriolar lumen contributes to the development of striped interstitial
fibrosis, loss of peritubular capillaries, tubular atrophy and glomerular sclerosis. In turn,
hypoxia of the tubulointerstitial compartment may lead to the formation of free radicals or
reactive oxygen species (ROS) causing cellular injury and death, promoting EMT and renal
fibrosis, setting in train a vicious cycle that will end in end-stage renal disease.

A better understanding of the cellular and molecular mechanisms that result in CNI-induced
renal hypoxia would lead to the development of diagnostic and treatment strategies that delay
or prevent CNI-induced renal fibrosis. A major limitation of studies addressing intrarenal
oxygenation is the lack of noninvasive technologies to quantitate and monitor the
bioavailability of oxygen in the kidney. Blood oxygen level-dependent MRI or BOLD MRI is an
innovative imaging method that uses deoxyhemoglobin as an endogenous contrast agent to
determine tissue oxygen bioavailability. The relationship between paramagnetic
deoxyhemoglobin and T2 relaxation was first demonstrated in 1982. This technique was recently
used, including by our group, to assess intra-renal oxygen bioavailability in patients with
native and transplant kidney disease.

We now propose to use this technology for the monitoring of intrarenal oxygenation in
patients with chronic CNI toxicity. We further suggest combining BOLD MRI with molecular
assessment of renal hypoxia. Hypoxia Inducible Factor one alpha is an oxygen response system
that has recently been used as a molecular marker of renal hypoxia. HIF molecules are
regulated at the protein level by oxygen-dependent enzymes and therefore allow for tissue
hypoxia detection. In the kidney allograft for example, the strong correlation between
HIF-1alpha staining and clinical/subclinical rejection suggests that hypoxia is involved in
the pathogenesis of the immune-mediated renal injury and that HIF-1 alpha
immunohistochemistry could enhance the specific diagnosis of acute rejection.

Pimonidazole is also a molecular biomarker of hypoxia that once delivered in vivo, binds to
thiol groups at oxygen tensions below 10 mmHg and is visualized with commercially available
antipimonidazole antibodies. It is a small, effective and nontoxic radiosensitizer used as a
hypoxia marker for human squamous cell carcinomas of the cervix, head, and neck. In the
kidney, pimonidazole staining has been used to examine tissue hypoxia in experimental models
of chronic renal disease and acute CNI nephrotoxicity. The role of BOLD MRI, HIF-1 alpha and
pimonidazole in evaluating the mechanisms of chronic CNI-induced hypoxia has not been
evaluated.

CNIs and Nox2. CsA is a pro-oxidant molecule as supported by experimental studies suggesting
that vitamin E inhibits CsA-induced lipid peroxidation and renal damage. Similarly, catalase,
an enzyme that specifically breaks down the reactive hydrogen peroxide (H2O2) into H2O and O2
and acts as a scavenger of ROS, reduced CsA-associated renal tubular epithelial cell
senescence. In addition, rat proximal tubular epithelial cells exposed to CsA accumulate
intracellular ROS and lipid peroxidation products, along with an altered glutathione redox
state. However, the molecular mechanisms that regulate CNI-induced generation of ROS remain
unclear.

Originally named gp91phox, Nox2 is the classical phagocytic NADPH oxidase, an enzyme that is
naturally involved in the immune response including the "oxidative burst". Nox2 is also the
original identified example of a system that generates ROS as the primary function of the
enzyme and not as a byproduct like the mitochondrion or peroxisomes. It is one of the seven
currently known Nox isoforms, is constitutively associated with the transmembrane stabilizing
protein p22phox and requires the recruitment of cytosolic components p47phox, p67phox, and
p40phox for function.

In summary, CNIs, the backbone of anti-rejection therapies are also profibrotic molecules,
making it difficult to effectively manage immunosuppression and long-term outcomes. We
outline research studies that rigorously assess the role of Nox2 in CNI-induced renal
fibrosis. In addition, we 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 in organ transplantation.

Inclusion Criteria:

- liver, heart, lung or pancreas transplant recipients

- suspected CNI induced nephrotoxicity

Exclusion Criteria:

- minors

- pregnant women

- prisoners

- institutionalized individuals or other vulnerable groups

- history of allergic reactions or adverse reactions to Pimonidazole

- Claustrophobia

- hazardous metallic implants

- cardiac pacemakers
We found this trial at
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sites
600 Highland Ave
Madison, Wisconsin 53792
(608) 263-6400
Principal Investigator: Arjang Djamali, MD,MS,FASN
Phone: 608-265-5489
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Madison, Wisconsin 53705
Principal Investigator: Arjang Djamali, MD,MS,FASN
Phone: 608-265-5489
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