Macrophage migration inhibitory factor promotes renal injury induced by ischemic reperfusion.

Macrophage migration inhibitory factor (MIF) is pleiotropic cytokine that has multiple effects in many inflammatory and immune diseases. This study reveals a potential role of MIF in acute kidney injury (AKI) in patients and in kidney ischemic reperfusion injury (IRI) mouse model in MIF wild-type (WT) and MIF knockout (KO) mice. Clinically, plasma and urinary MIF levels were largely elevated at the onset of AKI, declined to normal levels when AKI was resolved and correlated tightly with serum creatinine independent of disease causes. Experimentally, MIF levels in plasma and urine were rapidly elevated after IRI-AKI and associated with the elevation of serum creatinine and the severity of tubular necrosis, which were suppressed in MIF KO mice. It was possible that MIF may mediate AKI via CD74/TLR4-NF-κB signalling as mice lacking MIF were protected from AKI by largely suppressing CD74/TLR-4-NF-κB associated renal inflammation, including the expression of MCP-1, TNF-α, IL-1ß, IL-6, iNOS, CXCL15(IL-8 in human) and infiltration of macrophages, neutrophil, and T cells. In conclusion, our study suggests that MIF may be pathogenic in AKI and levels of plasma and urinary MIF may correlate with the progression and regression of AKI.

Advanced glycation end products activate Smad signaling via TGF-beta-dependent and independent mechanisms: implications for diabetic renal and vascular disease.

While it is thought that advanced glycation end products (AGEs) act by stimulating transforming growth factor (TGF)-beta to mediate diabetic injury, we report that AGEs can activate TGF-beta signaling, Smads, and mediate diabetic scarring directly and independently of TGF-beta. AGEs activate Smad2/3 in renal and vascular cells at 5 min, peaking over 15-30 min before TGF-beta synthesis at 24 h and occurs in TGF-beta receptor I and II mutant cells. This is mediated by RAGE and ERK/p38 mitogen-activated protein kinases (MAPKs). In addition, AGEs also activate Smads at 24 h via the classic TGF-beta-dependent pathway. A substantial inhibition of AGE-induced Smad activation and collagen synthesis by ERK/p38 MAPK inhibitors, but not by TGF-beta blockade, suggests that the MAPK-Smad signaling crosstalk pathway is a key mechanism in diabetic scarring. Prevention of AGE-induced Smad activation and collagen synthesis by overexpression of Smad7 indicates that Smad signaling may play a critical role in diabetic complications. This is further supported by the findings that activation of Smad2/3 in human diabetic nephropathy and vasculopathy is associated with local deposition of AGEs and up-regulation of RAGE. Thus, AGEs act by activating Smad signaling to mediate diabetic complications via both TGF-beta-dependent and -independent pathways, shedding new light on the pathogenesis of diabetic organ injury.

Randomized controlled trials on the therapeutic effects of adult progenitor cells for myocardial infarction: meta-analysis.

OBJECTIVE: We performed a systematic meta-analysis to assess the therapeutic effects of progenitor cell therapy after myocardial infarction (MI). RESEARCH DESIGN/METHODS: Randomized controlled trials of progenitor cell therapy for MI were extracted from MEDLINE. We performed a prospective comparison of progenitor cell therapy versus placebo after acute or chronic MI, with changes in left ventricular ejection fraction (LVEF) as the primary endpoint. We conducted random-effects meta-analyses to pool these outcomes across the studies. RESULTS: A total of 980 patients from 18 studies were analysed. Seventeen trials used bone marrow-derived cells (BMCs). Overall, BMCs significantly increased LVEF, left ventricular end systolic volume and left ventricular end diastolic volume within six months of treatment, and the effect was sustained one year later. Following BMC transplantation regional myocardial anatomy displayed statistically and clinically significant improvements compared with controls, albeit without functional changes. Similar results were observed in the subgroup of patients with impaired LVEF at the baseline. The subgroup analysis suggested a benefit of BMCs on LVEF in acute but not chronic MI. LVEF enhancement seemed to correlate positively with dose and inversely with the storage duration of the BMCs. CONCLUSIONS: BMC transplantation for MI was able to deliver benefits over regular therapy even at an 18-month follow-up, particularly when used to treat acute MI. CD34(+) cell therapy holds promise for MI treatment in the future.

Susceptibility to primary sclerosing cholangitis is associated with polymorphisms of intercellular adhesion molecule-1.

BACKGROUND/AIMS: Intercellular adhesion molecule-1 (ICAM-1, CD54) gene polymorphisms have been implicated in the susceptibility to a range of inflammatory diseases, including inflammatory bowel disease (IBD). Primary sclerosing cholangitis (PSC) is an immune-mediated chronic cholestatic liver disease associated with IBD. ICAM-1 is expressed on proliferating bile ducts and interlobular bile ducts in late stage PSC and serum levels of soluble intercellular adhesion molecules are increased in PSC. The aim of this study was to analyse ICAM-1 gene polymorphisms in PSC patients. METHODS: In this study, 104 patients with PSC and 213 healthy controls were recruited from Oxfordshire Caucasians. PCR with sequence-specific primers (PCR-SSP) was used to detect both ICAM-1 biallelic polymorphisms G241R and K469E. The results were controlled for the HLA haplotypes associated with PSC. RESULTS: The E/E frequency of K469E in PSC was 12% (12/104), significantly lower than that in controls (24%, 51/213;P = 0.009; Pc = 0.02; OR, 0.41). The occurrence of the haplotype G241-E469/G241-E469 in PSC was 4% (4/104), significantly lower than the control group (13%, 28/213; P = 0.01; Pc = 0.04; OR, 0.26). There was no difference between PSC and control groups in the frequencies of the genotype R241G or in allele frequencies of K469E. CONCLUSIONS: The E469E homozygote status for ICAM-1 is associated with protection against PSC.

Role of TGF-beta signaling in extracellular matrix production under high glucose conditions.

BACKGROUND: Hyperglycemia has been shown to play an important role in diabetic renal and vascular complications. Some studies show that high glucose may mediate diabetic complications by stimulating extracellular matrix (ECM) production. We hypothesize that this may be mediated by activating transforming growth factor-beta (TGF-beta)/Smads signaling. METHODS: Renal and vascular cells were cultured under high glucose conditions in the presence or absence of a neutralizing TGF-beta antibody and examined for activation of Smad signaling and collagen production. The regulating role of Smad signaling in high glucose-induced collagen synthesis was determined by inducing overexpression of the inhibitory Smad7 in a stable Smad7-expressing tubular cell line. RESULTS: Activation of Smad signaling, as evidenced by Smad2 and Smad3 nuclear translocation and phosphorylation, was found in renal and vascular cells at 24 hours after high glucose stimulation (up to 55% increased). This was associated with de novo synthesis of collagen I at day 3 by all cell types. High glucose-induced activation of Smad signaling and collagen synthesis were TGF-beta-dependent since these were associated with a significant increase in TGF-beta production at 24 hours (P < 0.01) and were blocked by a neutralizing TGF-beta antibody. Importantly, overexpression of Smad7 resulted in marked inhibition of high glucose-induced Smad2 and Smad3 activation and type I collagen synthesis, suggesting that Smad signaling is a key pathway in high glucose-mediated renal and vascular scarring. CONCLUSION: High glucose acts by activating the TGF-beta dependent Smad signaling pathway to stimulate collagen synthesis by renal and vascular cells. Smad signaling plays a critical role in regulating high-glucose-mediated diabetic renal and vascular complications.

Smad7 inhibits fibrotic effect of TGF-Beta on renal tubular epithelial cells by blocking Smad2 activation.

It has been shown that transforming growth factor-beta (TGF-beta) is a potent mediator in renal fibrosis and that Smad proteins are critical intracellular mediators in TGF-beta signaling. It is here reported that TGF-beta mediates renal fibrogenesis in tubular epithelial cells (TEC) in association with the activation of Smad2 and that overexpression of Smad7 blocks this fibrotic process. Using a normal rat kidney tubular epithelial cell line (NRK52E), it was determined that TGF-beta1 induces Smad2 phosphorylation and nuclear localization in both a dose- and time-dependent manner. The activation of Smad2 was evident at 5 min (20%), peaked at 15 to 30 min (85%), and declined to baseline levels by 2 h (5 to 10%). This was associated with de novo expression of collagens I, III, and IV and the transformation of TEC into a "myofibroblast" phenotype with de novo expression of alpha-smooth muscle actin (alpha-SMA) and with the loss of E-cadherin (>50%). To investigate a negative regulatory role of Smad7 in renal fibrosis, the Smad 7 gene was stably transfected and its expression was tightly controlled by doxycycline into NRK52E cells. Overexpression of Smad7 induced by doxycycline results in marked inhibition of TGF-beta-induced Smad2 activation (90% downward arrow) with the prevention of collagen synthesis and myofibroblast transformation. Thus, Smad2 activation occurs in the fibrogenic response of TEC to TGF-beta, and this process is blocked by overexpression of Smad7. This indicates that Smad signaling is a key pathway of TGF-beta-mediated renal fibrosis and suggests that treatments targeting the inactivation of Smad2 by overexpression of Smad7 may provide a new therapeutic strategy for renal fibrosis.

Advanced glycation end products induce tubular epithelial-myofibroblast transition through the RAGE-ERK1/2 MAP kinase signaling pathway.

Advanced glycation end products (AGEs) have been shown to play a role in tubular epithelial-myofibroblast transdifferentiation (TEMT) in diabetic nephropathy, but the intracellular signaling pathway remains unknown. We report here that AGEs signal through the receptor for AGEs (RAGE) to induce TEMT, as determined by de novo expression of a mesenchymal marker (alpha-smooth muscle actin, alpha-SMA) and loss of epithelial marker (E-cadherin), directly through the MEK1-ERK1/2 MAP kinase pathway, which is TGF-beta independent. This is supported by the following findings: AGEs induced de novo alpha-SMA mRNA expression as early as 2 hours followed by a loss of E-cadherin before TGF-beta mRNA expression at 24 hours and occurred in the absence of TGF-beta and AGE-induced activation of ERK1/2 MAP kinase at 15 minutes and TEMT at 24 hours were completely blocked by a neutralizing RAGE antibody, a soluble RAGE receptor, an ERK1/2 MAP kinase inhibitor (PD98059), and DN-MEK1, but not by a neutralizing TGF-beta antibody. Thus, this study demonstrates that AGEs activate the RAGE-ERK1/2 MAP kinase pathway to mediate the early TEMT process. The findings from this study suggest that targeting the RAGE or the ERK MAP kinase pathway may provide new therapeutic strategies for diabetic nephropathy and shed new light on the pathogenesis of diabetic nephropathy.

Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model.

TGF-beta is a key mediator in renal fibrosis. Kidney-targeted gene therapy with anti-TGF-beta strategies is expected to have therapeutic potential, but this has been hampered by concerns over the safety and practicability of viral vectors and the inefficiency of nonviral transfection techniques. The present study explored the potential role of TGF-beta/Smad signaling in renal fibrosis in vivo and developed a safe and effective gene therapy to specifically block TGF-beta signaling and renal fibrosis in a rat unilateral ureteral obstruction (UUO) model by transferring a doxycycline-regulated Smad7 gene or control empty vectors using an ultrasound-microbubble (Optison)-mediated system. The Smad7 transgene expression was tightly controlled by addition of doxycycline in the daily drinking water. Groups of six rats were sacrificed at day 7, and the transfection rate, Smad7 transgene expression, and tubulointerstitial fibrosis including alpha-smooth muscle actin and collagen matrix mRNA and protein expression were determined. Compared with the non-ultrasound treatment, the combination of ultrasound with Optison largely increased the transfection rate of FITC-ODN and Smad7 transgene expression up to a 1000-fold, and this was found in all kidney tissues. Compared with normal rats, Smad7 expression within the UUO kidney was significantly reduced, and this was associated with up to a sixfold increase in Smad2 and Smad3 activation and severe tubulointerstitial fibrosis. In contrast, treatment with inducible Smad7 resulted in a fivefold increase in Smad7 expression with complete inhibition of Smad2 and Smad3 activation and tubulointerstitial fibrosis in terms of tubulointerstitial myofibroblast accumulation (85% downward arrow ) and collagen I and III mRNA and protein expression (60 to 70% downward arrow ). In conclusion, the ultrasound-mediated inducible Smad7 gene transfer is a safe, effective, and controllable gene therapy. TGF-beta-mediated renal fibrosis is regulated positively by Smad2/3, but negatively by Smad7. Target blockade of TGF-beta/Smad signaling by expression of Smad7 may provide a new therapeutic potential for renal fibrosis.

Role of JAK/STAT pathway in IL-6-induced activation of vascular smooth muscle cells.

BACKGROUND/AIMS: IL-6, an inducer of the acute-phase response, is linked with the development of vascular disease and atherosclerosis. One mechanism likely involves direct effects of IL-6 on vascular smooth muscle cells (VSMC), for IL-6 can induce VSMC proliferation and the release of monocyte chemoattractant protein-1 (MCP-1). We hypothesized that this stimulation occurs via the JAK (janus-activated kinase)/STAT (signal and transducers and activators of transcription) signaling pathway. METHODS: Rat VSMC were stimulated with IL-6 in the presence or absence of a JAK 2 inhibitor, and the activation of STAT 3 (by Western), MCP-1 (by ELISA) and DNA synthesis (by (3)H-thymidine incorporation) was determined. RESULTS: IL-6 rapidly induced phosphorylation of STAT 3 in a dose- and time-dependent manner with a peak expression at 30 min. IL-6 also stimulated MCP-1 protein production and DNA synthesis dose dependently. 50 microM of AG490, a specific JAK 2 inhibitor, partially inhibited STAT 3 activation and MCP-1 production, with near complete inhibition of DNA synthesis. CONCLUSION: The JAK/STAT pathway partially mediates IL-6-induced MCP-1 production and DNA synthesis in rat VSMC. These studies implicate a role of the JAK/STAT pathway in the development of vascular disease and atherosclerosis.

Uric acid stimulates monocyte chemoattractant protein-1 production in vascular smooth muscle cells via mitogen-activated protein kinase and cyclooxygenase-2.

Previous studies have reported that uric acid stimulates vascular smooth muscle cell (VSMC) proliferation in vitro. We hypothesized that uric acid may also have direct proinflammatory effects on VSMCs. Crystal- and endotoxin-free uric acid was found to increase VSMC monocyte chemoattractant protein-1 (MCP-1) expression in a time- and dose-dependent manner, peaking at 24 hours. Increased mRNA and protein expression occurred as early as 3 hours after uric acid incubation and was partially dependent on posttranscriptional modification of MCP-1 mRNA. In addition, uric acid activated the transcription factors nuclear factor-kappaB and activator protein-1, as well as the MAPK signaling molecules ERK p44/42 and p38, and increased cyclooxygenase-2 (COX-2) mRNA expression. Inhibition of p38 (with SB 203580), ERK 44/42 (with UO126 or PD 98059), or COX-2 (with NS398) each significantly suppressed uric acid-induced MCP-1 expression at 24 hours, implicating these pathways in the response to uric acid. The ability of both n-acetyl-cysteine and diphenyleneionium (antioxidants) to inhibit uric acid-induced MCP-1 production suggested involvement of intracellular redox pathways. Uric acid regulates critical proinflammatory pathways in VSMCs, suggesting it may have a role in the vascular changes associated with hypertension and vascular disease.

TGF-beta induces proangiogenic and antiangiogenic factors via parallel but distinct Smad pathways.

BACKGROUND: Angiogenesis has a key role in numerous disease processes. One of the most important angiogenic factors is vascular endothelial growth factor (VEGF-A), whereas thrombospondin-1 (TSP-1) is a major antiangiogenic factor. Recent studies have shown that VEGF-A as well as TSP-1 is regulated by transforming growth factor-beta1 (TGF-beta1), but the mechanism remains unclear. METHODS: We examined the role of TGF-beta1 and its signaling pathways in mediating expression of these two molecules. Rat proximal tubular cells (NRK52E) were stimulated with TGF-beta1 to induce VEGF-A and TSP-1 synthesis. To clarify roles of receptor-activated Smads (R-Smads), we blocked Smad signaling using overexpression of the inhibitory Smad, Smad7, and by using fibroblasts from wild-type or knockout mice. To confirm the antiantigenic role of Smads, soluble Flt-1 regulation in response to TGF-beta1 was also examined. In addition, the effect of conditioned media from NRK52E and Smad knockout cells was examined on endothelial cell proliferation. RESULTS: Induction of VEGF-A and TSP-1 by TGF-beta1 in NRK52E cells was associated with activation of pathway-restricted R-Smads (Smad2 and 3) and blocking these Smads by overexpression of Smad7 blocked their induction. By using of Smad knockout cells, Smad3 was shown to have a key role in the stimulation of VEGF-A expression whereas Smad2 was critical for TSP-1 expression. Consistent with the hypothesis that Smad2 has an antiangiogenic function, we also demonstrated that Smad2, but not Smad3, mediated the expression of VEGF-A antagonist, soluble VEGF-A receptor sFlt-1, in response to TGF-beta1. Conditioned media from NRK52E, which was stimulated by TGF-beta1 for 24 hours, did not induce endothelial cell proliferation. However, conditioned media from Smad2 knockout induced endothelial cell proliferation, whereas endothelial cell proliferation was inhibited by Smad3 knockout-derived conditioned media. CONCLUSION: R-Smads have distinct roles in mediating the expression of pro- and antiangiogenic growth factors in response to TGF-beta1.