Role of Nox2 in CNI-induced Renal Fibrosis
Condition: Kidney Disease
MeSH terms: Kidney Diseases; Fibrosis
Study type: Observational

Summary: 
      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.
    

Detailed description: 
      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.
    

Intervention name(s): Hypoxyprobe-1
Intervention type(s): Biological
Intervention description(s): Hypoxyprobe-1 is a biological marker used to detect oxygen levels in tissue;subjects will receive and intravenous solution for 20 minutes containing 500mg/m^2 two-three hours prior to their standard of care biopsy
Other intervention name(s): Pimonidazole
Observational model: Other
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Study population will be chosen from non-kidney transplant patients scheduled for a
        standard of care kidney biopsy.
      
Keywords: Kidney fibrosis; Kidney hypoxia; Calcineurin inhibitors