Shannon Reese

Epithelial-to-mesenchymal transition and oxidative stress in chronic allograft nephropathy.

Epithelial‐to‐mesenchymal transition (EMT) and oxidative stress contribute to kidney tissue fibrosis in various forms of native kidney disease. However, their role in chronic allograft nephropathy (CAN) remains somewhat uncertain. To address this question, kidney transplants were performed in 3‐month‐old rats, using the Fisher 344 → Lewis model of CAN. Six‐month posttransplant, kidney allografts displayed significant tubular atrophy, interstitial fibrosis and vascular wall thickening. Allograft recipients had significantly higher levels of serum creatinine (4.7 ± 1.3 versus 0.59 ± 0.08 mg/dL, p = 0.03) and proteinuria (380 ± 102 versus 30.2 ± 8 mg/dL, p = 0.04) compared to syngeneic grafts. Semiquantitative PCR, immunoblot and immunohistochemical analyses demonstrated increased α‐smooth muscle actin (α‐SMA) mRNA and protein levels coupled with reduced E‐cadherin mRNA and protein immunoreactivity, confirming the presence of CAN‐associated EMT. Allograft α‐SMA levels were increased as early as 1–2 weeks posttransplant. Immunohistochemical studies for collagen type I and III, superoxide anion (O2−), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) confirmed that tubular O2−, eNOS and iNOS, and interstitial collagen I, III and O2− levels were significantly increased in CAN‐associated EMT. In conclusion, these observations suggest that CAN‐associated EMT may be a link between oxidative stress and allograft fibrosis.

Nox-2 Is a Modulator of Fibrogenesis in Kidney Allografts

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.

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.

Heat shock protein 27 in chronic allograft nephropathy: a local stress response.

Background. Heat shock protein (HSP) 27 plays a cytoprotective role through its antioxidant, antiapoptotic, and actin-stabilizing properties during cell stress. The authors hypothesized that HSP27 is involved in chronic allograft nephropathy (CAN), a chronic state of inflammation and stress. Methods. The authors used the Fisher 344-to-Lewis model of CAN. Transplants were performed in 3-month-old recipient rats. HSP27 mRNA and protein levels were determined using semiquantitative polymerase chain reaction, microarray (stress-toxicity, GEArray) analyses, gene sequencing, immunoblotting, and immunohistochemical analyses at 10 days and 6 months posttransplant. P38 mitogen-activated protein kinase (MAPK), manganese (Mn) superoxide dismutase (SOD), copper-zinc (CuZn) SOD, FasL, Bax, hypoxia-inducible factor (HIF)-1&agr;, and CD3+ lymphocytes were studied in parallel as selective biomarkers of oxidative stress (OS), apoptosis, hypoxia, and graft-infiltrating immune cells. Results. Six months after transplantation, kidney allografts displayed histologic and functional features of CAN, including tubular atrophy, interstitial fibrosis, glomerulosclerosis, and increased proteinuria and serum creatinine levels. HSP27 mRNA and protein levels in CAN were reduced by 50% and 85%, respectively (P=0.04). Immunohistochemical analyses revealed a “shift” in HSP27 from the medulla to the cortex in allografts with CAN. Bax, phosphorylated p38-MAPK, HIF-1&agr;, and MnSOD followed a parallel relocation pattern. CD3+ lymphocyte density and tubular FasL expression were also greater in the cortex of allografts with CAN. Time-course analyses revealed that most of these changes were present as early as 10 days posttransplant. Conclusions. The shift of HSP27 from the medulla to the cortex, combined with greater CD3+, p38-MAPK, Bax, FasL, HIF-1&agr;, and MnSOD immunoreactivity in this area of the kidney, likely represents an allograft-level response to CAN-related OS-hypoxia.

Enteral Feeding Preserves Mucosal Immunity Despite In Vivo MAdCAM-1 Blockade of Lymphocyte Homing

ObjectiveTo determine the influence of route of nutrition on gut mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) expression and the effect of MAdCAM-1 blockade on gut-associated lymphoid tissue (GALT) lymphocyte populations and established respiratory antibacterial immunity. Summary Background DataLymphocytes, sensitized to antigens in Peyer’s patches, migrate via mesenteric lymph nodes and home to intestinal lamina propria. MAdCAM-1 located on endothelial cells regulates this trafficking. Experimentally, parenteral nutrition (PN) decreases GALT cell mass and mucosal immunity when compared with enteral feeding. MethodsIn experiment 1, MAdCAM-1 expression was quantified in 32 mice after 4 days of feeding chow, a complex diet, intragastric (IG)-PN, or PN. In experiment 2, MAdCAM-1 was measured in 102 mice 0, 4, 8, 12, 24, 48, or 72 hours after starting PN and at 0, 4, 8, 12, 24, or 48 hours after reinstituting chow following 5 days of PN. In experiment 3, 56 mice received chow, PN, chow + MECA-367 (anti-MAdCAM-1 mAb), or chow + Isotype control Ab (IsoAb) for 5 days, followed by Peyer’s patches, lamina propria, and intraepithelial lymphocyte yield with respiratory and intestinal IgA levels. In experiment 4, 10 days after Pseudomonas immunization, mice received chow + MECA-367 or chow + IsoAb for 4 days followed by 1.2 × 108Pseudomonas intratracheally. ResultsDiet and route affect MAdCAM-1 expression (chow > complex diet > IG-PN > PN). Decreased MAdCAM-1 expression occurred within hours of starting PN in Peyer’s patches, but not mesenteric lymph nodes or the intestine, and recovered quickly with enteral refeeding. MAdCAM-1 blockade reduced all GALT populations. Blockade had little effect on IgA levels and partially impaired the late response of established respiratory immunity. ConclusionsEnteral feeding affects MAdCAM-1 expression. Complete MAdCAM-1 blockade reduces GALT lymphocytes to PN levels, but the chow feeding stimulus preserves IgA and early antibacterial resistance, implying the existence of non-MAdCAM-1 mechanisms to preserve mucosal immunity.

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

BackgroundPin 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).ResultsAfter 2 weeks of UUO, immunoblot analyses demonstrated that juglone (0.25 and 1 mg/kg/24 h) inhibited the deposition of matrix (α-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 (α-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 μM) significantly decreased α-SMA and p-smad levels compared to vehicle.ConclusionsJuglone 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.

Nox2 is a Mediator of Chronic CsA Nephrotoxicity

We hypothesized that Nox2, the classical phagocytic NADPH oxidase, plays an important role in calcineurin inhibitor (CNI)‐induced renal fibrosis. We tested this hypothesis in vitro, in animal and in human studies. Cyclosporine A (CsA) and tacrolimus (TAC) were associated with greater levels of Nox2 mRNA and epithelial to mesenchymal transition (EMT) in NRK52E cells. CsA increased Nox2, α‐SMA and phosphorylated‐p38MAPK, Smad3 and NFκB proteins. Nox2 upregulation and EMT were inhibited in TGF‐β1 knockout cells suggesting that TGF‐β1 is required for Nox2 activation. Fisher344 rats treated with high dose CsA showed increased Nox2 in the tubulointerstitium and greater Nox2, α‐SMA, phosphorylated Smad3 and nitrotyrosine by immunoblot analyses. Inhibition of Nox2 by coadministration of apocynin or diphenyleneiodonium was associated with reduced fibrogenesis. We validated these findings by treating wild type and Nox2 null (B6.129S‐CybbTm1Din/J) mice with high dose CsA. Western blot analyses confirmed the absence of Nox2 and significantly lower levels of α‐SMA and 4‐hydroxynonenal (HNE) in CsA‐treated knockout mice. These findings were clinically relevant since Nox2 and α‐SMA were increased in the tubulointerstitium of kidneys from 15 liver transplant recipients with biopsy‐confirmed chronic CsA or TAC nephrotoxicity. In conclusion, specific Nox2 inhibition strategies may improve chronic CNI nephrotoxicity in solid organ transplantation.

Mycophenolic Acid May Delay Allograft Fibrosis by Inhibiting Transforming Growth Factor-β1-induced Activation of Nox-2 Through the Nuclear Factor-κb 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-&bgr;1-induced EMT in normal rat kidney epithelial cells (NRK52E). Results. We first demonstrated that Nox-2 and &agr;-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, &agr;-SMA, and E-cadherin showed that similar to patients with IFTANOS, rat allografts had greater tubulointerstitial staining for Nox-2 and &agr;-SMA. MPA therapy prevented these changes. Immunoblot analyses examining Nox-2 signaling (phospho-nuclear factor [NF]-&kgr;B), redox signaling (phospho-smad2), and fibrosis (&agr;-SMA and fibronectin) demonstrated that MPA treatment prevented the up-regulation of Nox-2, inhibited p-NF-&kgr;B and p-smad2, and down-regulated &agr;-SMA and fibronectin levels. Finally, we examined Nox-2 signaling in vitro and confirmed that MPA inhibited phospho-NF-&kgr;B, Nox-2, phospho-smad2, and &agr;-SMA during transforming growth factor-&bgr;1-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-&kgr;B pathway in the tubular epithelial cells, suggesting a novel role for this drug independent of its immunosuppressive properties.

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 β-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-β-catenin complex.

Characterization of Transfusion‐Elicited Acute Antibody‐Mediated Rejection in a Rat Model of Kidney Transplantation

Animal models of antibody‐mediated rejection (ABMR) may provide important evidence supporting proof of concept. We elicited donor‐specific antibodies (DSA) by transfusion of donor blood (Brown Norway RT1n) into a complete mismatch recipient (Lewis RT1l) 3 weeks prior to kidney transplantation. Sensitized recipients had increased anti‐donor splenocyte IgG1, IgG2b and IgG2c DSA 1 week after transplantation. Histopathology was consistent with ABMR characterized by diffuse peritubular capillary C4d and moderate microvascular inflammation with peritubular capillaritis + glomerulitis > 2. Immunofluorescence studies of kidney allograft tissue demonstrated a greater CD68/CD3 ratio in sensitized animals, primarily of the M1 (pro‐inflammatory) phenotype, consistent with cytokine gene analyses that demonstrated a predominant T helper (TH)1 (interferon‐γ, IL‐2) profile. Immunoblot analyses confirmed the activation of the M1 macrophage phenotype as interferon regulatory factor 5, inducible nitric oxide synthase and phagocytic NADPH oxidase 2 were significantly up‐regulated. Clinical biopsy samples in sensitized patients with acute ABMR confirmed the dominance of M1 macrophage phenotype in humans. Despite the absence of tubulitis, we were unable to exclude the effects of T cell–mediated rejection. These studies suggest that M1 macrophages and TH1 cytokines play an important role in the pathogenesis of acute mixed rejection in sensitized allograft recipients.