Early modulation of inflammation by mesenchymal stem cell after acute kidney injury

Therapy with stem cells has showed to be promising for acute kidney injury (AKI), although how it works is still controversial. Modulation of the inflammatory response is one possible mechanism. Most of published data relies on early time and whether the protection is still maintained after that is not known. Here, we analyzed whether immune modulation continues after 24 h of reperfusion. MSC were obtained from male Wistar rats. After 3-5 passages, cells were screened for CD73, CD90, CD44, CD45, CD29 and CD 31. In addition, MSC were submitted to differentiation in adipocyte and in osteocyte. AKI was induced by bilaterally clamping of renal pedicles for 60 min. Six hours after injury, MSC (2 x 105 cells) were administered intravenously. MSC-treated animals presented the lowest serum creatinine compared to non-treated animals (24 h: 1.3 +/- 0.21 vs. 3.23 +/- 0.89 mg/dl, p<0.05). The improvement in renal function was followed by a lower expression of IL-1b, IL-6 and TNF-alpha and higher expression of IL-4 and IL-10. However, 48 h after reperfusion, this cytokine profile has changed. The decrease in Th1 cytokines was less evident and IL-6 was markedly up regulated. PCNA analysis showed that regeneration occurs faster in kidney tissues of MSC-treated animals than in controls at 24 h. And also ratio of Bcl-2/Bad was higher at treated animals after 24 and 48 h. Our data demonstrated that the immunomodulatory effects of MSC occur at very early time point, changing the inflammation profile toward a Th2 profile. (C) 2009 Elsevier B.V. All rights reserved.

A role for regulatory T cells in renal acute kidney injury

Ischemia reperfusion injury (IRI) is a potential contributor for the development of chronic allograft nephropathy. T cells are important mediators of injury, even in the absence of alloantigens. We performed a depletion of TCD4(+)CTLA4(+)Foxp3(+) cells with anti-CD25(PC61), a treatment with anti-GITR (DTA-1) and rat-IgG, followed by 45 min of ischemia and 24/72 h of reperfusion, and then analyzed blood urea, kidney histopathology and gene expression in kidneys by QReal Time PCR. After 24 h of reperfusion, depletion of TCD4(+)CTLA4(+)Foxp3(+) cells reached 30.3%(spleen) and 67.8%(lymph nodes). 72 h after reperfusion depletion reached 43.1%(spleen) and 90.22%(lymph nodes) and depleted animals presented with significantly poorer renal function, while DTA-1 (anti-GITR)-treated ones showed a significant protection, all compared to serum urea from control group (IgG: 150.10 +/- 50.04; PC61: 187.23 +/- 31.38; DTA-1: 64.53 +/- 25.65, mg/dL, p<0.05). These data were corroborated by histopathology. We observed an increase of HO-1 expression in animals treated with DTA-1 at 72 h of reperfusion with significant differences. Thus, our results suggest that PC61 (anti-CD25) mAb treatment is deleterious, while DTA-1 (anti-GITR) mAb treatment presents a protective role in the renal IRI, indicating that some regulatory populations of T cells might have a role in IRI. (C) 2009 Elsevier B.V. All rights reserved.

INSULIN REGULATES CYTOKINES AND INTERCELLULAR ADHESION MOLECULE-1 GENE EXPRESSION THROUGH NUCLEAR FACTOR-kappa B ACTIVATION IN LPS-INDUCED ACUTE LUNG INJURY IN RATS

Diabetic patients have increased susceptibility to infection, which may be related to impaired inflammatory response observed in experimental models of diabetes, and restored by insulin treatment. The goal of this study was to investigate whether insulin regulates transcription of cytokines and intercellular adhesion molecule 1 (ICAM-1) via nuclear factor-kappa B (NF-kappa B) signaling pathway in Escherichia coli LIPS-induced lung inflammation. Diabetic male Wistar rats (alloxan, 42 mg/kg, iv., 10 days) and controls were instilled intratracheally with saline containing LPS (750 mu g/0.4 mL) or saline only. Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU, s.c.) 2 h before LIPS. Analyses performed 6 h after LPS included: (a) lung and mesenteric lymph node IL-1 beta, TNF-alpha, IL-10, and ICAM-1 messenger RNA (mRNA) were quantified by real-time reverse transcriptase-polymerase chain reaction; (b) number of neutrophils in the bronchoalveolar lavage (BAL) fluid, and concentrations of IL-1 beta, TNF-alpha, and IL-10 in the BAL were determined by the enzyme-linked immunosorbent assay; and (c) activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were quantified by Western blot analysis. Relative to controls, diabetic rats exhibited a reduction in lung and mesenteric lymph node IL-1 beta (40%), TNF-alpha (similar to 30%), and IL-10 (similar to 40%) mRNA levels and reduced concentrations of IL-1 beta (52%), TNF-alpha (62%), IL-10 (43%), and neutrophil counts (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were almost suppressed in diabetic rats. Treatment of diabetic rats with insulin completely restored mRNA and protein levels of these cytokines and potentiated lung ICAM-1 mRNA levels (30%) and number of neutrophils (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were partially restored by insulin treatment. In conclusion, data presented suggest that insulin regulates transcription of proinflammatory (IL-1 beta, TNF-alpha) and anti-inflammatory (IL-10) cytokines, and expression of ICAM-1 via the NF-kappa B signaling pathway.

Endogenous Hepatocyte Growth Factor Attenuates Inflammatory Response in Glycerol-Induced Acute Kidney Injury

Background: Hepatocyte growth factor (HGF) is overexpressed after acute kidney injury (AKI). The aim of this study was to evaluate the role of endogenous HGF in the progression of the inflammatory response in glycerol-induced AKI (Gly-AKI) in rats. Methods: Renal and systemic HGF expressions were evaluated during the development of Gly-AKI. Subsequently, the blockade of endogenous HGF was analyzed in rats treated with anti-HGF antibody concomitant to glycerol injection. Apoptosis, cell infiltration and chemokine and cytokine profiles were investigated. Results: We detected an early peak of renal and plasma HGF protein expressions 3 h after glycerol injection. The pharmacological blockade of the endogenous HGF exacerbated the renal impairment, the tubular apoptosis, the renal expression of monocyte chemoattractant protein-1 and the macrophage, CD43+, CD4+ and CD8+ T lymphocytes renal infiltration. The analysis of mRNA expressions of Th1 (t-bet, TNF-alpha, IL-1 beta) and Th2 (gata-3, IL-4, IL-10) cytokines showed a Th1-polarized response in Gly-AKI rats that was aggravated with the anti-HGF treatment. Conclusion: Endogenous HGF attenuates the renal inflammatory response, leukocyte infiltration and Th1 polarization after glycerol injection. The control of cellular immune response may partly explain the protective effect of endogenous HGF in the development of Gly-AKI. Copyright (C) 2008 S. Karger AG, Basel

Administration of Neural Precursor Cells Ameliorates Renal Ischemia-Reperfusion Injury

In this study we evaluated whether administration of stem cells of neural origin (neural precursor cells, NPCs) could be protective against renal ischemia-reperfusion injury (IRI). We hypothesized that stem cell outcomes are not tissue-specific and that NPCs can improve tissue damage through paracrine mechanisms, especially due to immunomodulation. To this end, Wistar rats (200-250 g) were submitted to 1-hour ischemia and treated with NPCs (4 x 10(6) cells/animal) at 4 h of reperfusion. To serve as controls, ischemic animals were treated with cerebellum homogenate harvested from adult rat brain. All groups were sacrificed at 24 h of reperfusion. NPCs were isolated from rat fetus telencephalon and cultured until neurosphere formation (7 days). Before administration, NPCs were labeled with carboxyfluorescein diacetate succinimydylester (CFSE). Kidneys were harvested for analysis of cytokine profile and macrophage infiltration. At 24 h, NPC treatment resulted in a significant reduction in serum creatinine (IRI + NPC 1.21 + 0.18 vs. IRI 3.33 + 0.14 and IRI + cerebellum 2.95 + 0.78mg/dl, p < 0.05) and acute tubular necrosis (IRI + NPC 46.0 + 2.4% vs. IRI 79.7 + 14.2%, p < 0.05). NPC-CFSE and glial fibrillary acidic protein (GFAP)-positive cells (astrocyte marker) were found exclusively in renal parenchyma, which also presented GFAP and SOX-2 (an embryonic neural stem cell marker) mRNA expression. NPC treatment resulted in lower renal proinflammatory IL1-beta and TNF-alpha expression and higher anti-inflammatory IL-4 and IL-10 transcription. NPC-treated animals also had less macrophage infiltration and decreased serum proinflammatory cytokines (IL-1 beta, TNF-alpha and INF-gamma). Our data suggested that NPC therapy improved renal function by influencing immunological responses. Copyright (C) 2009 S. Karger AG, Basel

A Role for galectin-3 in renal tissue damage triggered by ischemia and reperfusion injury

Ischemic-reperfusion injury (IRI) triggers an inflammatory response involving neutrophils/macrophages, lymphocytes and endothelial cells. Galectin-3 is a multi-functional lectin with a broad range of action such as promotion of neutrophil adhesion, induction of oxidative stress, mastocyte migration and degranulation, and production of pro-inflammatory cytokines. The aim of this study was evaluate the role of galectin-3 in the inflammation triggered by IRI. Galectin-3 knockout (KO) and wild type (wt) mice were subjected to 45 min of renal pedicle occlusion. Blood and kidney samples were collected at 6, 24, 48 and 120 h. Blood urea was analyzed enzymatically, while MCP-1, IL-6 and IL-1 beta were studied by real-time PCR. Reactive oxygen species (ROS) was investigated by flow cytometry. Morphometric analyses were performed at 6, 24, 48 and 120 h after reperfusion. Urea peaked at 24 h, being significantly lower in knockout animals (wt = 264.4 +/- 85.21 mg/dl vs. gal-3 KO = 123.74 +/- 29.64 mg/dl, P = 0.001). Galectin-3 knockout animals presented less acute tubular necrosis and a more prominent tubular regeneration when compared with controls concurrently with lower expression of MCP-1, IL-6, IL-1 beta, less macrophage infiltration and lower ROS production at early time points. Galectin-3 seems to play a role in renal IRI involving the secretion of macrophage-related chemokine, pro-inflammatory cytokines and ROS production.

TLR2 and TLR4 expression after kidney ischemia and reperfusion injury in mice treated with FTY720

Ischemia and reperfusion injury (IR) is an antigen independent inflammatory process that causes tissue damage. After IR, kidneys up-regulate leukocyte adhesion molecules and toll-like receptors (TLRs). Moreover, injured kidneys can also secrete factors (i.e. heat shock protein) which bind to TLRs and trigger intracellular events culminating with the increase in the gene expression of inflammatory cytokines. FTY720 is an immunomodulatory compound and protects at least in part kidneys submitted to IR. The mechanisms associated with FTY720`s beneficial effects on kidneys after IR remain elusive. We investigated whether FTY720 administration in mice submitted to kidney IR is associated with modulation of TLR2 and TLR4 expression. C57BL/6 mice submitted to 30 min of renal pedicles clamp were evaluated for serum parameters (creatinine, urea and nitric oxide), kidney histology, spleen and kidney infiltrating cells expression of TLR2 and TLR4, resident kidney cells expression of TLR2 and TLR4 and IL-6 protein expression in kidney. FTY720-treated mice presented decrease in serum creatinine, urea and nitric oxide, diminished expression of TLR2 and TLR4 both in spleen and kidney infiltrating cells, and reduced kidney IL-6 protein expression in comparison with IR non-treated mice. However, acute tubular necrosis was present both in IR non-treated and IR + FTY720-treated groups. Also, FTY720 did not prevent TLR2 and TLR4 expression in kidney resident cells. In conclusion, FTY720 can promote kidney function recovery after IR by reducing the inflammatory process. Further studies are needed in order to establish whether TLR2 and TLR4 down regulation should be therapeutically addressed as protective targets of renal function and structure after IR. (C) 2011 Elsevier B.V. All rights reserved.

Bradykinin inducible receptor is essential to lipopolysaccharide-induced acute lung injury in mice

Lipopolysaccharides from gram-negative bacteria are amongst the most common causative agents of acute lung injury, which is characterized by an inflammatory response, with cellular infiltration and the release of mediators/cytokines. There is evidence that bradykinin plays a role in lung inflammation in asthma but in other types of lung inflammation its role is less clear. In the present study we evaluated the role of the bradykinin B(1) receptor in acute lung injury caused by lipopolysaccharide inhalation and the mechanisms behind bradykinin actions participating in the inflammatory response. We found that in C57BI/6 mice, the bradykinin B(1) receptor expression was up-regulated 24 h after lipopolysaccharide inhalation. At this time, the number of cells and protein concentration were significantly increased in the bronchoalveolar lavage fluid and the mice developed airway hyperreactivity to methacholine. In addition, there was an increased expression of tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma and chemokines (monocytes chemotactic protein-1 and KC) in the bronchoalveolar lavage fluid and in the lung tissue. We then treated the mice with a bradykinin B, receptor antagonist, R-954 (Ac-Orn-[Oic(2), alpha-MePhe(5), D-beta Nal(7), Ile(8)]desArg(9)-bradykinin), 30 min after lipopolysaccharide administration. We observed that this treatment prevented the airway hyperreactivity as well as the increased cellular infiltration and protein content in the bronchoalveolar lavage fluid. Moreover, R-954 inhibited the expression of cytokines/chemokines. These results implicate bradykinin, acting through B(1) receptor, in the development of acute lung injury caused by lipopolysaccharide inhalation. (C) 2010 Elsevier B.V. All rights reserved.

With an eye to academia or vocation? A draft to a research plan

There is a profound need to increase the quality and status of vocational education and train-ing (VET) and vocational education and training education – Teacher training (VTE-TT). As a consequence of several reforms the requirements of academic achievements in these different education systems are distinctly higher than before. These high ambitions cause problems and the actors in school and teacher education has got new challenges. Furthermore, vocational education teachers are supposed to prepare their pupils both to a pro-fessional career and to future academic studies. Accordingly, Vocational Education Teacher Trainers are supposed to prepare their students both to a career as vocational teachers and for further academic studies. This makes imprints in the field of VET and VTE-TT. The aim of this paper is to present a coming study about how actors in VET and VTE-TT think and act in relation to their changed assignment.

'Ultrasound is an invaluable third eye, but it can't see everything' : a qualitative study with obstetricians in Australia

Background: Obstetric ultrasound has come to play a significant role in obstetrics since its introduction in clinical care. Today, most pregnant women in the developed world are exposed to obstetric ultrasound examinations, and there is no doubt that the advantages of obstetric ultrasound technique have led to improvements in pregnancy outcomes. However, at the same time, the increasing use has also raised many ethical challenges. This study aimed to explore obstetricians' experiences of the significance of obstetric ultrasound for clinical management of complicated pregnancy and their perceptions of expectant parents' experiences. Methods: A qualitative study was undertaken in November 2012 as part of the CROss-Country Ultrasound Study (CROCUS). Semi-structured individual interviews were held with 14 obstetricians working at two large hospitals in Victoria, Australia. Transcribed data underwent qualitative content analysis. Results: An overall theme emerged during the analyses, 'Obstetric ultrasound - a third eye', reflecting the significance and meaning of ultrasound in pregnancy, and the importance of the additional information that ultrasound offers clinicians managing the surveillance of a pregnant woman and her fetus. This theme was built on four categories: I:'Everyday-tool' for pregnancy surveillance, II: Significance for managing complicated pregnancy, III: Differing perspectives on obstetric ultrasound, and IV: Counselling as a balancing act. In summary, the obstetricians viewed obstetric ultrasound as an invaluable tool in their everyday practice. More importantly however, the findings emphasise some of the clinical dilemmas that occur due to its use: the obstetricians' and expectant parents' differing perspectives and expectations of obstetric ultrasound examinations, the challenges of uncertain ultrasound findings, and how this information was conveyed and balanced by obstetricians in counselling expectant parents. Conclusions: This study highlights a range of previously rarely acknowledged clinical dilemmas that obstetricians face in relation to the use of obstetric ultrasound. Despite being a tool of considerable significance in the surveillance of pregnancy, there are limitations and uncertainties that arise with its use that make counselling expectant parents challenging. Research is needed which further investigates the effects and experiences of the continuing worldwide rapid technical advances in surveillance of pregnancies.

The differential roles of D-Pax2 variants in regulating Drosophila eye and bristle development

The ability to appropriately interact with the environment is crucial to an organism’s survival. The establishment of functional sensory systems, such as the bristles and eyes in Drosophila, is a critical event during the development of the organism. The transcription factor D Pax2 is involved in the differentiation of the shaft and glial cells in the developing bristle (Kavaler et al., Dev, 126:2261-2272, 1999) and of the cone and primary pigment cells in the developing eye (Fu and Noll, Genes Dev, 11:389-405, 1997). How D-Pax2 contributes to distinct differentiative pathways in different cell types is not known. Recent work by Anna Czechowski and Katherine Harmon (personal communication) identified a mutation in the D-Pax2 gene that introduced a stop codon at the end of exon 9, effectively truncating the protein. This mutation affects bristle, but not eye, development. We thus suspected regions after exon 9 are required for D-Pax2 function only in the bristles and may also be associated with alternative splicing of the D Pax2 transcript. We plan to assess the role of the carboxy terminal region of the protein by establishing transgenic lines bearing rescue constructs of D-Pax2 with either the complete coding sequence or with deletions of specific exons. To date, we have generated the first rescue construct bearing the complete coding region of the gene driven by a 3 KB upstream regulatory region of D-Pax2 and are currently generating transgenic fly lines with this construct.