The Role of Nox2 in CNI-Induced Renal Fibrosis

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.