Blood oxygen level-dependent (BOLD) magnetic resonance imaging (MRI) uses deoxyhemoglobin as an endogenous contrast agent for the noninvasive assessment of tissue oxygen bioavailability. We hypothesized that intrarenal oxygenation was impaired in patients with chronic allograft nephropathy (CAN). Ten kidney-transplant recipients with CAN and nine healthy volunteers underwent BOLD-MRI. Medullary R2* (MR2*) and cortical R2* (CR2*) levels (measures directly proportional to tissue deoxyhemoglobin levels) were determined alongside urine and serum markers of oxidative stress (OS): hydrogen peroxide (H(2)O(2)), F(2)-isoprostanes, total nitric oxide (NO), heat shock protein 27 (HSP27), and total antioxidant property (TAOP). Mean MR2* and CR2* levels were significantly decreased in CAN (increased local oxyhemoglobin concentration) compared with healthy volunteers (20.7 +/- 1.6 vs. 23.1 +/- 1.8/s, P = 0.03 and 15.9 +/- 1.9 vs. 13.6 +/- 2.3/s, P = 0.05, respectively). There was a significant increase in serum and urine levels of H(2)O(2) and serum HSP27 levels in patients with CAN. Conversely, urine NO levels and TAOP were significantly increased in healthy volunteers. Multiple linear regression analyses showed a significant association between MR2* and CR2* levels and serum/urine biomarkers of OS. BOLD-MRI demonstrated significant changes in medullary and cortical oxygen bioavailability in allografts with CAN. These correlated with serum/urine biomarkers of OS, suggesting an association between intrarenal oxygenation and OS.

Epithelial-to-mesenchymal transition (EMT) and ox-− levels were significantly increased in CAN-associated EMT. In conclusion, these observations suggest that CANassociated EMT may be a link between oxidative stress and allograft fibrosis.

We studied the role of classical phagocytic NADPH oxidase (Nox) in the pathogenesis of kidney allograft tubulointerstitial fibrosis. Immunofluorescence studies showed that Nox-2 and p22phox (electron transfer subunits of Nox) colocalized in the tubulointerstitium of human kidney allografts. Tubular Nox-2 also colocalized with α -SMA in areas of injury, suggestive of epithelial-to-mesenchymal transition (EMT). Interstitial macrophages (CD68+) and myofibroblasts (α -SMA+) expressed Nox-2 while graft infiltrating T cells (CD3+) and mature fibroblasts (S100A4+) were Nox-2−. These results were confirmed in the Fisher-to-Lewis rat kidney transplant model. Areas of tubulitis were associated with Nox-2 and α -SMA, suggestive of EMT. Immunoblot analyses showed that Nox-2 upregulation was associated with oxidative stress (nitrotyrosine) and fibrogenesis (α -SMA and phospho-Smad2) at 3 weeks and 6 months. Allografts treated with Nox inhibitors (DPI or apocynin) for 1 week showed reduced fibronectin and phospho-Smad2 and increased E-cadherin levels. Cyclosporine A, TGF-β1 and angiotensin II increased Nox-2 mRNA levels 2- to 7-fold in vitro (NRK52E cells). Treatment with specific Nox inhibitors (DPI or apocynin) prevented the downregulation of E-cadherin and upregulation of fibronectin transcripts. In aggregate, these studies suggest that Nox-2 is involved in the pathogenesis of allograft tubulointerstitial fibrosis via activation transcription factor Smad2, EMT and myofibroblasts.

We hypothesized that heat shock protein 27 (HSP27), a small heat shock protein with actin-remodeling properties, is involved in the pathogenesis of kidney tubulointerstitial fibrosis. We first examined its expression in the rat unilateral ureteral obstruction (UUO) model of kidney fibrosis and epithelial-to-mesenchymal transition (EMT). Immunoblot analyses showed that UUO resulted in significant upregulation of TGF-β1, α-smooth muscle actin (α-SMA), total and phosphorylated HSP27, and phosphorylated p38MAPK. Immunofluorescence studies showed that HSP27 costained with TGF-β1, α-SMA, and E-cadherin in areas of tubulointerstitial injury. We next attempted to translate these studies in an in vitro model of EMT using rat proximal tubular epithelial cells (NRK52E). TGF-β1 (20 ng/ml) treatment resulted in EMT (upregulation of α-SMA and downregulation of E-cadherin) and significant upregulation of total and phosphorylated HSP27 and p38MAPK after 3 days. Real-time PCR analyses showed that HSP27, vimentin, and fibronectin increased whereas E-cadherin mRNA levels decreased. Double-staining immunofluorescence studies showed intracytoplasmic colocalization of HSP27 with both F-actin and E-cadherin in cells undergoing EMT. HSP27 overexpression by transient transfection significantly increased E-cadherin while decreasing E-cadherin repressor Snail levels. In aggregate, these studies show that HSP27 is involved in the pathogenesis of TGF-β1-induced EMT and chronic tubulointerstitial fibrosis. HSP27 overexpression may delay injury by upregulating E-cadherin through downregulation of Snail.

The shift of HSP27 from the medulla to the cortex, combined with greater CD3, p38-MAPK, Bax, FasL, HIF-1alpha, and MnSOD immunoreactivity in this area of the kidney, likely represents an allograft-level response to CAN-related OS-hypoxia.