Therapeutic miR-21 Silencing Ameliorates Diabetic Kidney Disease in Mice.

Diabetic nephropathy is the main cause of end-stage renal disease. MicroRNAs are powerful regulators of the genome, and global expression profiling revealed miR-21 to be among the most highly regulated microRNAs in kidneys of mice with diabetic nephropathy. In kidney biopsies of diabetic patients, miR-21 correlated with tubulointerstitial injury. In situ PCR analysis showed a specific enrichment of miR-21 in glomerular cells. We identified cell division cycle 25a (Cdc25a) and cyclin-dependent kinase 6 (Cdk6) as novel miR-21 targets in mesangial cells. miR-21-mediated repression of Cdc25a and Cdk6 resulted in impaired cell cycle progression and subsequent mesangial cell hypertrophy. miR-21 increased podocyte motility by regulating phosphatase and tensin homolog (Pten). miR-21 antagonism in vitro and in vivo in streptozotocin-induced diabetic mice decreased mesangial expansion, interstitial fibrosis, macrophage infiltration, podocyte loss, albuminuria, and fibrotic- and inflammatory gene expression. In conclusion, miR-21 antagonism rescued various functional and structural parameters in mice with diabetic nephropathy and, thus, might be a viable option in the treatment of patients with diabetic kidney disease.

Tubular Epithelial NF-κB Activity Regulates Ischemic AKI.

NF-κB is a key regulator of innate and adaptive immunity and is implicated in the pathogenesis of AKI. The cell type-specific functions of NF-κB in the kidney are unknown; however, the pathway serves distinct functions in immune and tissue parenchymal cells. We analyzed tubular epithelial-specific NF-κB signaling in a mouse model of ischemia-reperfusion injury (IRI)-induced AKI. NF-κB reporter activity and nuclear localization of phosphorylated NF-κB subunit p65 analyses in mice revealed that IRI induced widespread NF-κB activation in renal tubular epithelia and in interstitial cells that peaked 2-3 days after injury. To genetically antagonize tubular epithelial NF-κB activity, we generated mice expressing the human NF-κB super-repressor IκBαΔN in renal proximal, distal, and collecting duct epithelial cells. Compared with control mice, these mice exhibited improved renal function, reduced tubular apoptosis, and attenuated neutrophil and macrophage infiltration after IRI-induced AKI. Furthermore, tubular NF-κB-dependent gene expression profiles revealed temporally distinct functional gene clusters for apoptosis, chemotaxis, and morphogenesis. Primary proximal tubular cells isolated from IκBαΔN-expressing mice and exposed to hypoxia-mimetic agent cobalt chloride exhibited less apoptosis and expressed lower levels of chemokines than cells from control mice did. Our results indicate that postischemic NF-κB activation in renal tubular epithelia aggravates tubular injury and exacerbates a maladaptive inflammatory response.

The viral dsRNA analogue poly (I:C) induces necrotizing enterocolitis in neonatal mice.

BACKGROUND: Necrotizing enterocolitis (NEC) is a life-threatening gastrointestinal disease in premature infants with high mortality and morbidity with uncertain pathogenesis. Recent research focused on the role of intraluminal bacteria and lipopolysaccharide (LPS). However, an additional role of viral agents in the pathogenesis of NEC has recently been postulated. We assessed the role of polyinosinic:polycytidylic acid (pIC) mimicking viral dsRNA in contributing to the development of NEC in neonatal mice. METHODS: Four-d-old C57BL/6J pups were stressed by asphyxia and hypothermia twice daily. Animals were either fed by formula only (FO), formula containing LPS or pIC. After 72 h, mice were euthanized, intestines harvested, and the severity of NEC was assessed. RESULTS: Breastfed mice showed no evidence of NEC. Very mild NEC-like lesions were observed in mice fed by FO. Supplementation of LPS or pIC to the formula led to increased intestinal tissue damage and inflammation compared with FO in a similar manner. CONCLUSION: Our study demonstrates the ability of viral factors to induce NEC in neonatal mice even in the absence of LPS. Furthermore, we present a new mouse model of pIC-induced NEC which may be used to obtain further mechanistic insights in the pathogenesis of this disease.

Circulating ADAM17 Level Reflects Disease Activity in Proteinase-3 ANCA-Associated Vasculitis.

ANCA-associated vasculitides are characterized by inflammatory destruction of small vessels accompanied by enhanced cleavage of membrane-bound proteins. One of the main proteases responsible for ectodomain shedding is disintegrin and metalloproteinase domain-containing protein 17 (ADAM17). Given its potential role in aggravating vascular dysfunction, we examined the role of ADAM17 in active proteinase-3 (PR3)-positive ANCA-associated vasculitis (AAV). ADAM17 concentration was significantly increased in plasma samples from patients with active PR3-AAV compared with samples from patients in remission or from other controls with renal nonvascular diseases. Comparably, plasma levels of the ADAM17 substrate syndecan-1 were significantly enhanced in active AAV. We also observed that plasma-derived ADAM17 retained its specific proteolytic activity and was partly located on extracellular microparticles. Transcript levels of ADAM17 were increased in blood samples of patients with active AAV, but those of ADAM10 or tissue inhibitor of metalloproteinases 3, which inhibits ADAMs, were not. We also performed a microRNA (miR) screen and identified miR-634 as significantly upregulated in blood samples from patients with active AAV. In vitro, miR-634 mimics induced a proinflammatory phenotype in monocyte-derived macrophages, with enhanced expression and release of ADAM17 and IL-6. These data suggest that ADAM17 has a prominent role in AAV and might account for the vascular complications associated with this disease.

Cytochrome P450 subfamily 2J polypeptide 2 expression and circulating epoxyeicosatrienoic metabolites in preeclampsia.

BACKGROUND: Preeclampsia is a multisystem disorder of pregnancy, originating in the placenta. Cytochrome P450 (CYP)-dependent eicosanoids regulate vascular function, inflammation, and angiogenesis, which are mechanistically important in preeclampsia. METHODS AND RESULTS: We performed microarray screening of placenta and decidua (maternal placenta) from 25 preeclamptic women and 23 control subjects. The CYP subfamily 2J polypeptide 2 (CYP2J2) was upregulated in preeclamptic placenta and decidua. Reverse-transcription polymerase chain reaction confirmed the upregulation, and immunohistochemistry localized CYP2J2 in trophoblastic villi and deciduas at 12 weeks and term. The CYP2J2 metabolites, 5,6-epoxyeicosatrienoic acid (EET), 14,15-EET, and the corresponding dihydroxyeicosatrienoic acids, were elevated in preeclamptic women compared with controls in the latter two thirds of pregnancy and after delivery. Stimulating a trophoblast-derived cell line with the preeclampsia-associated cytokine tumor necrosis factor-α enhanced CYP2J2 gene and protein expression. In 2 independent rat models of preeclampsia, reduced uterine-perfusion rat and the transgenic angiotensin II rat, we observed elevated EET, dihydroxyeicosatrienoic acid, and preeclamptic features that were ameliorated by the CYP epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MsPPOH). Uterine arterial rings of these rats also dilated in response to MsPPOH. Furthermore, 5,6-EET could be metabolized to a thromboxane analog. In a bioassay, 5,6-EET increased the beating rate of neonatal cardiomyocytes. Blocking thromboxane synthesis reversed that finding and also normalized large-conductance calcium-activated potassium channel activity. CONCLUSIONS: Our data implicate CYP2J2 in the pathogenesis of preeclampsia and as a potential candidate for the disturbed uteroplacental remodeling, leading to hypertension and endothelial dysfunction.

The direct renin inhibitor aliskiren localizes and persists in rat kidneys.

The aims of this study were to 1) determine whether renal localization of aliskiren and its antihypertensive and renoprotective effects persist after administration of the drug is stopped and 2) define the renal localization of aliskiren by light microscopy autoradiography. Hypertensive double transgenic rats (dTGR) overexpressing genes for human renin and angiotensinogen were treated with aliskiren (3 mg·kg(-1)·day(-1) sc; osmotic minipumps) or enalapril (18 mg/l in drinking water). After a 2-wk treatment, dTGR were assigned to either continued treatment with aliskiren ("continued"), or to cessation of their respective treatment ("stopped") for a 3-wk washout. One week of treatment with aliskiren and enalapril reduced blood pressure and albuminuria vs. baseline. After cessation of either treatment, blood pressure had returned to pretreatment levels and albuminuria remained relatively low for 1 wk, but rose thereafter similarly in both groups. In contrast, renal mRNA for transforming growth factor-ß and renal collagen IV was reduced by aliskiren (continued and stopped groups), but not after cessation of enalapril. Similar patterns were found for collagen IV protein expression. Even 3 wk after stopping aliskiren treatment, renal levels of the drug exceeded its IC50, whereas enalaprilat was not detected. To localize aliskiren accumulation, Wistar rats were treated with [(3)H]-aliskiren for 7 days. Autoradiography demonstrated specific labeling in glomeruli, arterioles, and afferent arterioles as well as in the distal nephron. Labeling could still be observed even after 7 days' washout. These results suggest that the renophilic properties of aliskiren are different from enalapril and could have contributed to the renoprotective mechanism of this renin inhibitor.

NKG2D stimulated T-cell autoreactivity in giant cell arteritis and polymyalgia rheumatica.

OBJECTIVE: To investigate functional expression of NKG2D on CD4 and CD8 T-cells in patients with giant cell arteritis (GCA) and polymyalgia rheumatica (PMR). METHODS: Peripheral blood was drawn from patients with GCA (n=16), PMR (n=78) and healthy controls (HC, n=64). Tissue samples were obtained from GCA patients and controls. Proliferation and cytokine production assays were performed using CFSE and intracellular IFN-γ or TNF-α staining, respectively, and flow cytometry analysis. Immunofluorescence and immunohistology were applied to analyse the presence of NKG2D-expressing T-cells and NKG2D-ligands in temporal arteries, respectively. mRNA levels of NKG2D-ligands were determined by RT-PCR. RESULTS: In both GCA and PMR patients, NKG2D was preferentially expressed on senescent CD4CD28(-) and CD8CD28(-), as well as on CD8CD28 T-cells. Frequencies of senescent T-cells were increased in GCA and PMR patients compared to HC. In GCA tissue samples, infiltrating T-cells were predominately CD28(-). NKG2D expressing T-cells concentrated around the vasa vasorum of the adventitia. Antigenic stimulation induced rapid up-regulation of NKG2D on CD4CD28(-) and CD4CD28 T-cells, whereas TNF-α and interleukin-15 enhanced NKG2D expression on senescent CD4 and CD8 T-cells only. NKG2D cross-linkage augmented anti-CD3 triggered proliferation, IFN-γ and TNF-α production of CD8 T-cells. In CD4CD28(-) T-cells, NKG2D ligation resulted in increased IFN-γ production only. NKG2D ligands were expressed in temporal arteries from GCA patients, particularly in the adventitial and medial layers of affected vessels. CONCLUSIONS: NKG2D is functionally expressed on CD4CD28(-) and CD8 T-cells in GCA and PMR. NKG2D-ligands are present in temporal arteries and may co-stimulate NKG2D expressing T-cells.

Effects of chronic SDMA infusion on glomerular filtration rate, blood pressure, myocardial function and renal histology in C57BL6/J mice.

BACKGROUND: Symmetrical dimethylarginine (SDMA), the structural isomer of the nitric oxide synthase inhibitor asymmetrical dimethylarginine, has long been regarded as an inert substance. Recent epidemiological and preclinical data suggest that it might be involved in the pathophysiology of renal and cardiovascular diseases. Therefore, we aimed to investigate the effect of chronic SDMA infusion on renal and cardiac function in mice. METHODS: Eight-week-old male C57Bl/6 mice received vehicle-controlled infusion of SDMA (250 µmol/kg/days) for 28 days using osmotic minipumps (n = 24/group). The following parameters were monitored: glomerular filtration rate (GFR; fluoresceinyl thiocarbamoyl-inulin excretion kinetic), cardiac function (echocardiography) and blood pressure (tail cuff). Blood samples for SDMA determination were obtained at baseline, 2 and 4 weeks. Mice were euthanized at 4 weeks to obtain tissue for renal histology. RESULTS: Chronic SDMA infusion led to a significant increase of SDMA levels from 0.26 ± 0.10 to 3.49 ± 1.66 µmol/L (P < 0.001) at 4 weeks. Despite this SDMA increase, the GFR did not change (1224 ± 351 versus 1017 ± 345 mL/min/g body weight, n.s.) at 4 weeks, when compared with baseline. We did not find any histological changes, particularly no effect on fibrosis or endothelias nitric oxide synthase expression. There was neither an effect of SDMA on systolic blood pressure (106 ± 12 versus 111 ± 18 mmHg, n.s.) nor on ejection fraction (54.2 ± 1.7 versus 58.4 ± 1.9%, n.s.). CONCLUSIONS: Based on our experiments, it seems unlikely that chronically elevated SDMA alone has an effect on renal and cardiac function in otherwise healthy mice. Future studies have to clarify the potential pathophysiological role of SDMA in cardiovascular disease.

'The ideal mesh?'.

Currently, more than 200 different textile constructions, so-called 'meshes', are available for use world-wide in the more than 20 million operations performed annually for the reinforcement of tissues. As any reintervention at the mesh-tissue compound is a surgical challenge, sometimes resulting in almost untreatable defects, huge efforts are being made to improve the biological and functional performance of the meshes. Based on numerous experimental and clinical studies in the past 20 years, our understanding of them has improved markedly. This includes the biomechanical aspects and the histopathological evaluation of the recipient tissue. Sufficiently large pores as well as structural stability in case of mechanical strain have been identified to be crucial to reduce excessive inflammation and fibrosis. Furthermore, large pores prevent bridging of the foreign body reaction through the pore and thereby help to reduce clinical adverse events as erosion, shrinkage or pain. However, with regard to the many different indications for meshes, there will never be one single ideal mesh for all purposes. To achieve an optimal performance, every construction should be designed according to the specific functional requirements, charging the surgeon to identify the best mesh for his purpose.

Quantifying the impact of gut microbiota on inflammation and hypertensive organ damage.

AIMS: Hypertension (HTN) can lead to heart and kidney damage. The gut microbiota has been linked to HTN, although it is difficult to estimate its significance due to the variety of other features known to influence HTN. In the present study, we used germ-free (GF) and colonized (COL) littermate mice to quantify the impact of microbial colonization on organ damage in HTN. METHODS AND RESULTS: 4-week-old male GF C57BL/6J littermates were randomized to remain GF or receive microbial colonization. HTN was induced by subcutaneous infusion with angiotensin (Ang) II (1.44 mg/kg/day) and 1% NaCl in the drinking water; sham-treated mice served as control. Renal damage was exacerbated in GF mice, whereas cardiac damage was more comparable between COL and GF, suggesting that the kidney is more sensitive to microbial influence. Multivariate analysis revealed a larger effect of HTN in GF mice. Serum metabolomics demonstrated that the colonization status influences circulating metabolites relevant to HTN. Importantly, GF mice were deficient in anti-inflammatory faecal short-chain fatty acids (SCFA). Flow cytometry showed that the microbiome has an impact on the induction of anti-hypertensive myeloid-derived suppressor cells and pro-inflammatory Th17 cells in HTN. In vitro inducibility of Th17 cells was significantly higher for cells isolated from GF than conventionally raised mice. CONCLUSION: The microbial colonization status of mice had potent effects on their phenotypic response to a hypertensive stimulus, and the kidney is a highly microbiota-susceptible target organ in HTN. The magnitude of the pathogenic response in GF mice underscores the role of the microbiome in mediating inflammation in HTN.

Reduction of activated macrophages after ischaemia-reperfusion injury diminishes oxidative stress and ameliorates renal damage.

BACKGROUND: Macrophages are major effectors of the local inflammatory response syndrome (LIRS) and influence the extent of ischaemia/reperfusion injury, thereby impacting organ function. Several subgroups of macrophages exist, representing distinct modes of action. The specific role of the subset expressing Fc gamma receptor (FcγR) 1 in the activated state of macrophages is poorly defined. METHODS: We induced a LIRS via 30 min of ischaemia in uninephrectomized rats, transgenic for the human FcγR1. Six hours after reperfusion, the treatment group was injected with a recombinant immunotoxin (IT) H22(scFv)-ETA' targeted against human FcγR1, which induced apoptosis of target cells. The placebo group received normal saline (NS). Contralateral kidneys served as healthy controls (Ctr). After 24 h of reperfusion, the animals were analysed. RESULTS: Targeted treatment with IT resulted in preserved renal function [NS versus IT treatment and baseline (creatinine: 69.2 ± 2.6, 54.7 ± 3.4 and 27.3 ± 1.0 µmol/L; P < 0.001)]. The number of all infiltrating monocytes were significantly reduced (CD68-positive cells per view field: NS 3.8 ± 0.4, IT 2.5 ± 0.2 and Ctr 1.2 ± 0.4; P < 0.05), renal histology improved and there was a reduced expression of renal fibronectin (NS 4.0 ± 0.4, IT 2.3 ± 0.2 and Ctr 1.1 ± 0.1; P < 0.001). Following IT administration, we also observed less expression of renal monocyte chemoattractant protein-1-positive cells per view field (NS 19.0 ± 1, IT 10.1 ± 0.8 and Ctr 2.0 ± 0.3; P < 0.001) as well as reduced systemic and local oxidative stress [serum malondialdehyde (MDA): NS 340 ± 30, IT 224 ± 36 versus baseline 140 ± 5 nmol/mL; P < 0.01]; renal MDA arbitrary units of fluorescence intensity: NS 3.7 ± 0.2, IT 1.8 ± 0.3 and Ctr 0.4 ± 0.2; P < 0.001. CONCLUSIONS: Reduction of FcγR1-up-regulated monocytic cells leads to preserved renal function and morphology in a rat model of ischaemia-triggered LIRS. Our results show that targeting activated macrophages is a valuable approach for ameliorating ischaemia-induced tissue injury.

The TIM-1:TIM-4 pathway enhances renal ischemia-reperfusion injury.

CD4+ T cells contribute to the pathogenesis of ischemia-reperfusion injury, which is the primary cause of delayed graft failure after kidney transplantation. The TIM-1:TIM-4 pathway participates in the activation/differentiation of CD4+ T cells, suggesting that it may modulate ischemia-reperfusion injury. Here, we studied the role of TIM-1 in a murine uninephrectomized renal ischemia-reperfusion injury model. Blocking the TIM-1:TIM-4 pathway with an antagonistic monoclonal antibody protected renal function and diminished reperfusion injury resulting from 30 minutes of ischemia. Histologic examination showed significantly less evidence of renal damage as evidenced by diminished tubular necrosis, preservation of the brush border, fewer cast formations, and less tubular dilation. Blocking TIM-1 also reduced the number of apoptotic cells and diminished local inflammation within ischemic kidneys, the latter shown by decreased recruitment of macrophages, neutrophils, and CD4+ T cells and by reduced local production of proinflammatory cytokines. Furthermore, TIM-1 blockade significantly improved survival after ischemia-reperfusion injury. Taken together, these data suggest that the TIM-1:TIM-4 pathway enhances injury after renal ischemia-reperfusion injury and may be a therapeutic target.

Prorenin receptor is essential for podocyte autophagy and survival.

The prorenin receptor (PRR) is highly expressed in podocytes, but its role in the maintenance of podocyte function is unknown. Here we generated podocyte-specific PRR-knockout mice and found that these animals died between 2 to 3 wk after birth. Within 14 d, PRR-knockout mice developed nephrotic syndrome, albuminuria with podocyte foot-process fusion, and cytoskeletal changes. Podocyte-specific PRR deletion also led to disturbed processing of multivesicular bodies and enrichment of autophagosomal (LC3) and lysosomal (LAMP2) markers, indicating a functional block in autophagosome-lysosome fusion and an overload of the proteasomal protein-degradation machinery. In vitro, PRR knockdown and pharmacologic blockade of vacuolar H(+)-ATPases, which associate with the PRR, increased vesicular pH, led to accumulation of LC3-positive and LAMP2-positive vesicles and altered the cytoskeleton. Taken together, these results suggest that the PRR is essential for podocyte function and survival by maintaining autophagy and protein-turnover machinery. Furthermore, PRR contributes to the control of lysosomal pH, which is important for podocyte survival and cytoskeletal integrity.

Acute administration of recombinant Angiopoietin-1 ameliorates multiple-organ dysfunction syndrome and improves survival in murine sepsis.

INTRODUCTION: Endothelial activation leading to vascular barrier breakdown plays an essential role in the pathophysiology of multiple-organ dysfunction syndrome (MODS) in sepsis. Increasing evidence suggests that the function of the vessel-protective factor Angiopoietin-1 (Ang-1), a ligand of the endothelial-specific Tie2 receptor, is inhibited by its antagonist Angiopoietin-2 (Ang-2) during sepsis. In order to reverse the effects of the sepsis-induced suppression of Ang-1 and elevation of Ang-2 we aimed to investigate whether an intravenous injection of recombinant human (rh) Ang-1 protects against MODS in murine sepsis. METHODS: Polymicrobiological abdominal sepsis was induced by cecal ligation and puncture (CLP). Mice were treated with either 1 µg of intravenous rhAng-1 or control buffer immediately after CLP induction and every 8h thereafter. Sham-operated animals served as time-matched controls. RESULTS: Compared to buffer-treated controls, rhAng-1 treated septic mice showed significant improvements in several hematologic and biochemical indicators of MODS. Moreover, rhAng-1 stabilized endothelial barrier function, as evidenced by inhibition of protein leakage from lung capillaries into the alveolar compartment. Histological analysis revealed that rhAng-1 treatment attenuated leukocyte infiltration in lungs and kidneys of septic mice, probably due to reduced endothelial adhesion molecule expression in rhAng-1 treated mice. Finally, the protective effects of rhAng-1 treatment were reflected by an improved survival time in a lethal CLP model. CONCLUSIONS: In a clinically relevant murine sepsis model, intravenous rhAng-1 treatment alone is sufficient to significantly improve a variety of sepsis-associated organ dysfunctions and survival time, most likely by preserving endothelial barrier function. Further studies are needed to pave the road for clinical application of this therapy concept.

The synthetic tie2 agonist peptide vasculotide protects against vascular leakage and reduces mortality in murine abdominal sepsis.

INTRODUCTION: Angiopoietin-1 (Angpt1), the natural agonist ligand for the endothelial Tie2 receptor, is a non-redundant endothelial survival and vascular stabilization factor that reduces endothelial permeability and inhibits leukocyte-endothelium interactions. Here we evaluate the efficacy of a novel polyethylene glycol (PEG)-clustered Tie2 agonist peptide, Vasculotide (VT), to protect against vascular leakage and mortality in a murine model of polymicrobial abdominal sepsis. METHODS: Polymicrobial abdominal sepsis in C57BL6 mice was induced by cecal-ligation-and-puncture (CLP). Mice were treated with different dosages of VT or equal volume of phosphate-buffered saline (PBS). Sham-operated animals served as time-matched controls. RESULTS: Systemic administration of VT induced long-lasting Tie2 activation in vivo. VT protected against sepsis-induced endothelial barrier dysfunction, as evidenced by attenuation of vascular leakage and leukocyte transmigration into the peritoneal cavity. Histological analysis revealed that VT treatment ameliorated leukocyte infiltration in kidneys of septic mice, probably due to reduced endothelial adhesion molecule expression. VT-driven effects were associated with significantly improved organ function and reduced circulating cytokine levels. The endothelial-specific action of VT was supported by additional in vitro studies showing no effect of VT on either cytokine release from isolated peritoneal macrophages, or migratory capacity of isolated neutrophils. Finally, administration of VT pre-CLP (Hazard Ratio 0.39 [95% Confidence interval 0.19-0.81] P < 0.001) and post-CLP reduced mortality in septic mice (HR 0.22 [95% CI 0.06-0.83] P < 0.05). CONCLUSIONS: We provide proof of principle in support of the efficacious use of PEGylated VT, a drug-like Tie2 receptor agonist, to counteract microvascular endothelial barrier dysfunction and reduce mortality in a clinically relevant murine sepsis model. Further studies are needed to pave the road for clinical application of this therapeutic concept.

Phenotypic Switch of Human Peritoneal Macrophages during Childhood.

INTRODUCTION: Human peritoneal macrophages are resident in the abdominal cavity where they support the specific microenvironmental regulation. We have previously observed a phenotypic switch of murine macrophages during infancy that was associated with a functional development. To investigate the age related changes in human peritoneal macrophages, we analyzed peritoneal macrophages of children undergoing laparoscopic procedures. MATERIALS AND METHODS: Immunologically healthy children who received minimally invasive surgery in our department were included in this study. In all cases, the written consent was obtained. At the beginning of laparoscopy, physiologic NaCl-solution was instilled and manually removed through the umbilical trocar to gain macrophages. Lavage cells were processed for flow cytometry analysis. CD14+ myeloid cells were monitored for specific lineage marker expression. RESULTS: A total of 21 donors (age: 7 days-18 years) were included and divided into three groups. In all age groups, 97% of myeloid cells expressed CD11b. 70% of these expressed CD14. Three subsets of CD14 cells were detected on the basis of CD14/CD16 expression (CD14 + CD16dim, CD14 + CD16inter, and CD14 + CD16high). In neonates, >80% belonged to the CD14 + CD16high subset, reducing to 30% in adolescents. In none of the cases, the M2 markers CD23 and CD25 were expressed. CONCLUSION: This is the first study showing that lineage marker expression of peritoneal macrophages in neonates differs from that in adults. The knowledge about neonatal tissue resident macrophages might help to understand their complex interaction and to use specific macrophage properties for therapeutic approaches.

Regulatory T cells ameliorate angiotensin II-induced cardiac damage.

BACKGROUND: Hypertensive target organ damage, especially cardiac hypertrophy with heart failure and arrhythmia, is a major source of morbidity and mortality. Angiotensin II, a major mediator of hypertension and cardiac damage, has proinflammatory properties. Inflammation and activation of the immune system play a pivotal role in pathogenesis of hypertensive target organ damage. However, the role of immunosuppressive CD4+CD25+ regulatory T (Treg) cells in the pathogenesis of hypertensive target organ damage is unexplored. METHODS AND RESULTS: We conducted adoptive transfer of Treg cells into angiotensin II-infused hypertensive mice. Treg cell recipients exhibited improved cardiac hypertrophy and less cardiac fibrosis despite sustained hypertension. Amelioration of cardiac morphology was accompanied by an improvement in arrhythmogenic electric remodeling, indicating the functional significance of the enhanced cardiac morphology. Delocalization of the connexin 43 gap junction protein is one of the major pathomechanisms in electric remodeling. Pronounced connexin 43 immunoreactivity was found at the lateral borders of cardiomyocytes in angiotensin II-treated mice. In contrast, connexin 43 was restricted to the intercalated disk regions in sham controls. Surprisingly, angiotensin II+Treg-treated mice showed normal connexin 43 gap junction protein localization. Adoptive Treg cell transfer resulted in a marked reduction in cardiac CD4+, CD8+, and CD69+ cell and macrophage infiltration. CONCLUSIONS: Immunosuppressive effects of transferred Treg cells ameliorated cardiac damage and accounted for the improved electric remodeling independently of blood pressure-lowering effects. Our results provide new insights into the pathogenesis of hypertensive cardiac damage and could therefore lead to new therapeutic approaches that involve manipulation of the immune system.

Renal urokinase-type plasminogen activator (uPA) receptor but not uPA deficiency strongly attenuates ischemia reperfusion injury and acute kidney allograft rejection.

Central mechanisms leading to ischemia induced allograft rejection are apoptosis and inflammation, processes highly regulated by the urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR). Recently, up-regulation of uPA and uPAR has been shown to correlate with allograft rejection in human biopsies. However, the causal connection of uPA/uPAR in mediating transplant rejection and underlying molecular mechanisms remain poorly understood. In this study, we evaluated the role of uPA/uPAR in a mice model for kidney ischemia reperfusion (IR) injury and for acute kidney allograft rejection. uPAR but not uPA deficiency protected from IR injury. In the allogenic kidney transplant model, uPAR but not uPA deficiency of the allograft caused superior recipient survival and strongly attenuated loss of renal function. uPAR-deficient allografts showed reduced generation of reactive oxygen species and apoptosis. Moreover, neutrophil and monocyte/macrophage infiltration was strongly attenuated and up-regulation of the adhesion molecule ICAM-1 was completely abrogated in uPAR-deficient allografts. Inadequate ICAM-1 up-regulation in uPAR(-/-) primary aortic endothelial cells after C5a and TNF-alpha stimulation was confirmed by in vitro experiments. Our results demonstrate that the local renal uPAR plays an important role in the apoptotic and inflammatory responses mediating IR-injury and transplant rejection.

B-cell lymphoma/leukaemia 10 and angiotensin II-induced kidney injury.

AIMS: B-cell lymphoma/leukaemia 10 (Bcl10) is a member of the CARMA-Bcl10-MALT1 signalosome, linking angiotensin (Ang) II, and antigen-dependent immune-cell activation to nuclear factor kappa-B signalling. We showed earlier that Bcl10 plays a role in Ang II-induced cardiac fibrosis and remodelling, independent of blood pressure. We now investigated the role of Bcl10 in Ang II-induced renal damage. METHODS AND RESULTS: Bcl10 knockout mice (Bcl10 KO) and wild-type (WT) controls were given 1% NaCl in the drinking water and Ang II (1.44 mg/kg/day) for 14 days. Additionally, Bcl10 KO or WT kidneys were transplanted onto WT mice that were challenged by the same protocol for 7 days. Kidneys of Ang II-treated Bcl10 KO mice developed less fibrosis and showed fewer infiltrating cells. Nevertheless, neutrophil gelatinase-associated lipocalin (Ngal) and kidney injury molecule (Kim)1 expression was higher in the kidneys of Ang II-treated Bcl10 KO mice, indicating exacerbated tubular damage. Furthermore, albuminuria was significantly higher in Ang II-treated Bcl10 KO mice accompanied by reduced glomerular nephrin expression and podocyte number. Ang II-treated WT mice transplanted with Bcl10 KO kidney showed more albuminuria and renal Ngal, compared to WT- > WT kidney-transplanted mice, as well as lower podocyte number but similar fibrosis and cell infiltration. Interestingly, mice lacking Bcl10 in the kidney exhibited less Ang II-induced cardiac hypertrophy than controls. CONCLUSION: Bcl10 has multi-faceted actions in Ang II-induced renal damage. On the one hand, global Bcl10 deficiency ameliorates renal fibrosis and cell infiltration; on the other hand, lack of renal Bcl10 aggravates albuminuria and podocyte damage. These data suggest that Bcl10 maintains podocyte integrity and renal function.

Novel role for inhibitor of differentiation 2 in the genesis of angiotensin II-induced hypertension.

BACKGROUND: Angiotensin (Ang) II-induced target-organ damage involves innate and acquired immunity. Mice deficient for the helix-loop-helix transcription factor inhibitor of differentiation (Id2(-/-)) lack Langerhans and splenic CD8a+ dendritic cells, have reduced natural killer cells, and have altered CD8 T-cell memory. We tested the hypothesis that an alteration in the number and quality of circulating blood cells caused by Id2 deletion would ameliorate Ang II-induced target-organ damage. METHODS AND RESULTS: We used gene-deleted and transgenic mice. We conducted kidney and bone marrow transplants. In contrast to Ang II-infused Id2(+/-), Id2(-/-) mice infused with Ang II remained normotensive and failed to develop albuminuria or renal damage. Bone marrow transplant of Id2(+/-) bone marrow to Id2(-/-) mice did not restore the blunted blood pressure response to Ang II. Transplantation of Id2(-/-) kidneys to Id2(+/-) mice also could not prevent Ang II-induced hypertension and renal damage. We verified the Ang II resistance in Id2(-/-) mice in a model of local tissue Ang II production by crossing hypertensive mice transgenic for rat angiotensinogen with Id2(-/-) or Id2(+/-) mice. Angiotensinogen-transgenic Id2(+/-) mice developed hypertension, albuminuria, and renal injury, whereas angiotensinogen-transgenic Id2(-/-) mice did not. We also found that vascular smooth muscle cells from Id2(-/-) mice showed an antisenescence phenotype. CONCLUSIONS: Our bone marrow and kidney transplant experiments suggest that alterations in circulating immune cells or Id2 in the kidney are not responsible for Ang II resistance. The present studies identify a previously undefined role for Id2 in the pathogenesis of Ang II-induced hypertension.