Tubular expression of heat-shock protein 27 inhibits fibrogenesis in obstructive nephropathy.

Morphological changes that occur during kidney injury involve actin skeleton remodeling. Here we tested whether heat-shock protein 27 (HSP27), a small stress response protein involved in cytoskeletal remodeling, protects the kidney from tubulointerstitial fibrosis in obstructive nephropathy. Tubular cell HSP27 immunostaining was significantly increased in human kidneys with ureteropelvic junction obstruction, supporting the clinical relevance of our studies. To develop an animal model for mechanistic studies, we generated transgenic mice that specifically overexpress human HSP27 in renal tubules, under the kidney androgen-regulated protein promoter, and determined the effects of HSP27 overexpression on epithelial-to-mesenchymal transition and tubulointerstitial fibrosis following unilateral ureteral obstruction. This was associated with decreased fibrogenesis as evidenced by significant declines in phosphorylated p38MAPK, collagen III, α-smooth muscle actin, 4-hydroxynonenal, and reduced trichrome staining following obstruction. Notably, E-cadherin and ß-catenin remained at the cell membrane of tubular cells in transgenic mice with an obstructed ureter. Monocyte/macrophage infiltration, however, was not significantly affected in these transgenic mice. Thus, tubular HSP27 inhibits fibrogenesis in obstructive nephropathy. Further studies are needed to determine pathways regulating the interactions between HSP27 and the E-cadherin-ß-catenin complex.

Heat shock protein 27 (HSP27): biomarker of disease and therapeutic target.

Heat shock protein 27 (HSP27) is a multidimensional protein which acts as a protein chaperone and an antioxidant and plays a role in the inhibition of apoptosis and actin cytoskeletal remodeling. In each of these capacities, HSP27 has been implicated in different disease states playing both protective and counter-protective roles. The current review presents HSP27 in multiple disease contexts: renal injury and fibrosis, cancer, neuro-degenerative and cardiovascular disease, highlighting its role as a potential biomarker and therapeutic target.

Serum HSP27 is associated with medullary perfusion in kidney allografts.

BACKGROUND: Heat shock protein 27 (HSP27) is a small HSP up-regulated in response to stress in the kidney. The relationship between HSP27 and intrarenal oxygenation in patients with native and transplant kidney disease is unknown. METHODS: We compared HSP27 levels, intrarenal oxygenation measured by blood oxygen-level dependent (BOLD) imaging using R(2)* values, and perfusion determined by arterial spin labeling (ASL) magnetic resonance imaging (MRI), between patients with native and transplant kidney disease (n=28). RESULTS: There were no statistical differences in mean age (53.9 vs. 47.1 years), kidney function (63.6 vs. 50.7 ml/min per 1.73 m(2)), mean arterial blood pressure (91.6 vs. 91.1 mm Hg), hematocrit (40.6% vs. 39.3%), diuretic or angiotensin-converting enzyme inhibitor use, serum or urine levels of hydrogen peroxide, nitric oxide, F(2) isoprostanes and HSP27 between native and transplant kidneys. BOLD-MRI studies demonstrated comparable patterns in intrarenal oxygen bioavailability (medullary R(2)* 18.1 vs. 18.3/s and cortical R(2)* 12 vs. 11.7/s, respectively). However, medullary perfusion was significantly lower in transplant kidneys (36.4 vs. 78.7 ml/100 g per minute, p=0.0002). There was a linear relationship between serum HSP27 concentrations and medullary perfusion in kidney allografts (HSP27 concentration [ng/mL] = 0.78 + 0.09 medullary perfusion, R(2)=0.43, p=0.01). CONCLUSIONS: Our study demonstrates that medullary perfusion is significantly lower in kidney allografts compared with native kidneys with comparable renal function. We further noted a direct association between serum HSP27 levels and medullary perfusion after transplantation. Additional studies are needed to examine the role of HSP27 as a biomarker of kidney disease progression.

Mycophenolic acid may delay allograft fibrosis by inhibiting transforming growth factor-beta1-induced activation of Nox-2 through the nuclear factor-kappaB pathway.

BACKGROUND: We evaluated the role of renal tubular Nox-2 in the pathogenesis of epithelial-to-mesenchymal transition (EMT) in kidney allografts. METHODS: We examined this question in the human kidney allografts with interstitial fibrosis and tubular atrophy not otherwise specified (IFTANOS), in the Fisher to Lewis rat transplant model, and in the in vitro model of transforming growth factor-beta1-induced EMT in normal rat kidney epithelial cells (NRK52E). RESULTS: We first demonstrated that Nox-2 and alpha-smooth muscle actin (SMA) were increased in renal tubules from kidney transplant recipients on calcineurin inhibitors, mycophenolic acid (MPA), and prednisone with IFTANOS, suggestive of EMT (n=6). Next, we examined Nox-2 expression and fibrogenesis in syngeneic transplants, allogeneic transplants treated with MPA 40 mg/kg per 24 hr, and untreated allogeneic transplants for 6 months (n=14 in each group). Immunofluorescent and immunohistochemical studies for Nox-2, alpha-SMA, and E-cadherin showed that similar to patients with IFTANOS, rat allografts had greater tubulointerstitial staining for Nox-2 and alpha-SMA. MPA therapy prevented these changes. Immunoblot analyses examining Nox-2 signaling (phospho-nuclear factor [NF]-kappaB), redox signaling (phospho-smad2), and fibrosis (alpha-SMA and fibronectin) demonstrated that MPA treatment prevented the up-regulation of Nox-2, inhibited p-NF-kappaB and p-smad2, and down-regulated alpha-SMA and fibronectin levels. Finally, we examined Nox-2 signaling in vitro and confirmed that MPA inhibited phospho-NF-kappaB, Nox-2, phospho-smad2, and alpha-SMA during transforming growth factor-beta1-induced EMT of NRK52E cells while reducing Nox-2, vimentin, and fibronectin mRNA levels. CONCLUSIONS: MPA may down-regulate Nox-2 activation and EMT through the NF-kappaB pathway in the tubular epithelial cells, suggesting a novel role for this drug independent of its immunosuppressive properties.

The Pin 1 inhibitor juglone attenuates kidney fibrogenesis via Pin 1-independent mechanisms in the unilateral ureteral occlusion model.

BACKGROUND: Pin 1 is a peptidyl-prolyl isomerase inhibitor related to cyclophilin A and FK506 binding protein (FKBP). Juglone (5-hydroxy-1,4-naphthoquinone) is a natural inhibitor of Pin 1 with anti-inflammatory and antifibrotic properties. We evaluated the role of Pin 1 in renal fibrogenesis by evaluating the effects of juglone on epithelial to mesenchymal transition (EMT) and fibrogenesis in the rat unilateral ureteral obstruction (UUO) model and normal rat tubular epithelial cells (NRK52E). RESULTS: After 2 weeks of UUO, immunoblot analyses demonstrated that juglone (0.25 and 1 mg/kg/24 h) inhibited the deposition of matrix (alpha-smooth muscle actin (SMA), collagen type III and vimentin) and the activation of signaling pathways involved in fibrogenesis (phospho-smad2) and stress response (phospho-heat shock protein (HSP)27). Juglone also reduced EMT (alpha-SMA and E-cadherin dual staining) and oxidative stress (Mn superoxide dismutase (SOD) and NAPDH oxidase 2 (Nox-2) dual staining) in the obstructed kidney. There was no difference in Pin 1 levels between treatment and control groups. Pin 1 activity was significantly decreased in obstructed kidneys regardless of treatment status. In vitro, juglone (1 muM) significantly decreased alpha-SMA and p-smad levels compared to vehicle. CONCLUSIONS: Juglone attenuates fibrogenesis via Pin 1-independent mechanisms in the UUO model. The antifibrotic effects of juglone may result from the inhibition of smad2 and oxidative stress.

Epithelial-to-mesenchymal transition and chronic allograft tubulointerstitial fibrosis.

Chronic allograft tubular atrophy/interstitial fibrosis (TA/IF) is a major cause of late allograft loss. A major challenge to the future of kidney transplantation is to dissect the identifiable causes of chronic allograft TA/IF and to develop cause-specific treatment strategies. Emerging evidence suggests that epithelial-to-mesenchymal transition (EMT) is an important event in native and transplant kidney injury, including chronic allograft TA/IF. During EMT, tubular epithelial cells are transformed into myofibroblasts through a stepwise process including loss of cell-cell adhesion and E-cadherin expression, de novo alpha-smooth muscle actin expression, actin reorganization, tubular basement membrane disruption, cell migration, and fibroblast invasion with production of profibrotic molecules such as collagen types I and III and fibronectin. We examined in this review the molecular and cellular pathways of EMT and their involvement in chronic allograft tubulointerstitial fibrosis. We examined the role of alloimmune T cells and oxidative stress in this context and evaluated EMT as a marker of disease progression. Potential therapeutic options are discussed. In conclusion, there is enough evidence demonstrating that EMT is involved in the pathogenesis of chronic allograft tubulointerstitial fibrosis. However, the extent of its contribution to allograft fibrogenesis remains unknown, and only interventional trials will enable us to clarify this question. Furthermore, additional data are required to determine whether EMT may be used as a surrogate marker of disease progression in kidney transplant recipients.

HSP27 is involved in the pathogenesis of kidney tubulointerstitial fibrosis.

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-beta1, alpha-smooth muscle actin (alpha-SMA), total and phosphorylated HSP27, and phosphorylated p38MAPK. Immunofluorescence studies showed that HSP27 costained with TGF-beta1, alpha-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-beta1 (20 ng/ml) treatment resulted in EMT (upregulation of alpha-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-beta1-induced EMT and chronic tubulointerstitial fibrosis. HSP27 overexpression may delay injury by upregulating E-cadherin through downregulation of Snail.

BOLD-MRI assessment of intrarenal oxygenation and oxidative stress in patients with chronic kidney allograft dysfunction.

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.