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Abstract In diabetic nephropathy (DN), glomerular endothelial cells (GECs) and podocytes undergo pathological alterations, which are influenced by metabolic changes characteristic of diabetes, including hyperglycaemia (HG) and elevated methylglyoxal (MGO) levels. However, it remains insufficiently understood what effects these metabolic factors have on GEC and podocytes and to what extent the interactions between the two cell types can modulate these effects. To address these questions, we established a co-culture system in which GECs and podocytes were grown together in close proximity, and assessed transcriptional changes in each cell type after exposure to HG and MGO. We found that HG and MGO had distinct effects on gene expression and that the effect of each treatment was markedly different between GECs and podocytes. HG treatment led to upregulation of “immediate early response” genes, particularly those of the EGR family, as well as genes involved in inflammatory responses (in GECs) or DNA replication/cell cycle (in podocytes). Interestingly, both HG and MGO led to downregulation of genes related to extracellular matrix organisation in podocytes. Crucially, the transcriptional responses of GECs and podocytes were dependent on their interaction with each other, as many of the prominently regulated genes in co-culture of the two cell types were not significantly changed when monocultures of the cells were exposed to the same stimuli. Finally, the changes in the expression of selected genes were validated in BTBR ob/ob mice, an established model of DN. This work highlights the molecular alterations in GECs and podocytes in response to the key diabetic metabolic triggers HG and MGO, as well as the central role of GEC-podocyte crosstalk in governing these responses.

Abstract Background Hypoxia in ischemic disease impairs Ca2+ homeostasis and may promote angiogenesis. The therapeutic efficacy of mesenchymal stromal cells (MSCs) in peripheral arterial occlusive disease is well established, yet its influence on cellular Ca2+ homeostasis remains to be elucidated. We addressed the influence of ATP-binding cassette subfamily B member 5 positive mesenchymal stromal cells (ABCB5+ MSCs) on Ca2+ homeostasis in hypoxic human umbilical vein endothelial cells (HUVECs) in vitro and in vivo. Methods Hypoxia was induced in HUVECs by Cobalt (II) chloride (CoCl2) or Deferoxamine (DFO). Dynamic changes in the cytosolic- and endoplasmic reticulum (ER) Ca2+ and changes in reactive oxygen species were assessed by appropriate fluorescence-based sensors. Metabolic activity, cell migration, and tube formation were assessed by standard assays. Acute-on-chronic ischemia in Apolipoprotein E knock-out (ApoE−/−) mice was performed by double ligation of the right femoral artery (DFLA). ABCB5+ MSC cells were injected into the ischemic limb. Functional recovery after DFLA and histology of gastrocnemius and aorta were assessed. Results Hypoxia-induced impairment of cytosolic and ER Ca2+ were restored by ABCB5+ MSCs or their conditioned medium. Similar was found for changes in intracellular ROS production, metabolic activity, migratory ability and tube formation. The restoration was paralleled by an increased expression of the Ca2+ transporter Sarco-/endoplasmic reticulum ATPase 2a (SERCA2a) and the phosphorylation of Phospholamban (PLN). In acute-on-chronic ischemia, ABCB5+ MSCs treated mice showed a higher microvascular density, increased SERCA2a expression and PLN phosphorylation relative to untreated controls. Conclusions ABCB5+ MSCs therapy can restore cellular Ca2+ homeostasis, which may beneficially affect the angiogenic function of endothelial cells under hypoxia in vitro and in vivo.

BackgroundThe acute respiratory distress syndrome (ARDS) is characterized by pulmonary epithelial and endothelial barrier dysfunction and injury. In severe forms of ARDS, extracorporeal membrane oxygenation (ECMO) is often the last option for life support. Endothelial progenitor (EPC) and mesenchymal stem cells (MSC) can regenerate damaged endothelium and thereby improve pulmonary endothelial dysfunction. However, we still lack sufficient knowledge about how ECMO might affect EPC- and MSC-mediated regenerative pathways in ARDS. Therefore, we investigated if ECMO impacts EPC and MSC numbers in ARDS patients.MethodsPeripheral blood mononuclear cells from ARDS patients undergoing ECMO (n = 16) and without ECMO support (n = 12) and from healthy volunteers (n = 16) were isolated. The number and presence of circulating EPC and MSC was detected by flow cytometry. Serum concentrations of vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2) were determined.ResultsIn the ECMO group, MSC subpopulations were higher by 71% compared to the non-ECMO group. Numbers of circulating EPC were not significantly altered. During ECMO, VEGF and Ang2 serum levels remained unchanged compared to the non-ECMO group (p = 0.16), but Ang2 serum levels in non-survivors of ARDS were significantly increased by 100% (p = 0.02) compared to survivors.ConclusionsECMO support in ARDS is specifically associated with an increased number of circulating MSC, most likely due to enhanced mobilization, but not with a higher numbers of EPC or serum concentrations of VEGF and Ang2.

We have previously reported that the CNDP1 (CTG)5 allele affords protection against diabetic nephropathy (DN) in patients with Type 2 diabetes (T2DM) of Caucasian origin. Because the incidence of ESRD attributable to both Type 1 diabetes (T1DM) and T2DM is higher among South Asian than Caucasian people, the present study assessed relevant CNDP1 polymorphisms and their association with metabolic parameters in the Chinese Han population. To this end, the (CTG)n allele distribution along with 5 relevant SNPs in the CNDP1 gene, previously reported to be associated with DN in non (CTG)5 carriers of Afro-American ethnicity, were determined in 663 healthy individuals. The (CTG)6 homozygous genotype was the most prevalent (84.5%) genotype in the Chinese Han population. The (CTG)5 and (CTG)4 alleles were present in a small minority of individuals accounting for 15.2% and 0.3% of genotypes with at least one (CTG)5 or one (CTG)4 allele, respectively. Only 0.5% of individuals carried the homozygous (CTG)5 genotype and individuals carrying the homozygous (CTG)4 genotype were not found. The minor allele frequencies (MAFs) of the 5 SNP were 0.197 (C allele for rs4892247), 0.0855 (C allele for rs62099905), 0.085 (G allele for rs62099906), 0.066 (T allele for rs62099907), and 0.18 (A allele for rs72979715). All the SNPs except rs4892247 genotypes were in the Hardy-Weinberg equilibrium. Neither the (CTG)n polymorphism nor the latter three SNPs reached significance when compared with different metabolic parameters. In contrast, individuals with the TT genotype of rs62099905 presented lower fasting blood glucose but higher HbA1c levels. In conclusion, the rs62099905 in the CNDP1 gene is associated with serum glucose levels in the healthy Chinese Han population, while for the CNDP1 (CTG)n polymorphism, no association with serological parameters was found.

BackgroundCell-based therapies with bone marrow-derived progenitor cells (BMDPC) lead to an improved clinical outcome in animal sepsis models. In the present study we evaluated the ability of granulocyte macrophage-colony stimulating factor (GM-CSF) to mobilize BMDPC in a lipopolysaccharide (LPS)-induced sepsis model and thereby its potential as a novel treatment strategy.MethodsMale Wistar rats received LPS (25μg/kg/h for 4 days) intravenously and were subsequently treated with GM-CSF 12.5μg/kg (0h,24h,48h,72h). As control groups, rats were infused with sodium chloride or GM-CSF only. Clinical and laboratory parameters, proinflammatory plasma cytokines as well as BMDPC counts were analyzed. Cytokine release by isolated peripheral blood mononuclear cells from rat spleen upon incubation with LPS, GM-CSF and a combination of both were investigated in vitro.ResultsIn vivo, rats receiving both LPS and GM-CSF, showed a reduced weight loss and increased mobilization of BMDPC. At the same time, this regime resulted in an increased release of proinflammatory cytokines (IL-6, IL-8) and a significantly increased mortality. In vitro, the combination of LPS and GM-CSF showed a significantly increased IL-6 release upon incubation compared to incubation with LPS or GM-CSF alone.ConclusionsGM-CSF did not have a beneficial effect on the clinical course in our LPS-induced sepsis model. It synergistically promoted inflammation with LPS and probably thereby impaired survival.

Within the last years, the use of stem cells (embryonic, induced pluripotent stem cells, or hematopoietic stem cells), Progenitor cells (e.g., endothelial progenitor cells), and most intensely mesenchymal stromal cells (MSC) has emerged as a promising cell-based therapy for several diseases including nephropathy. For patients with end-stage renal disease (ESRD), dialysis or finally organ transplantation are the only therapeutic modalities available. Since ESRD is associated with a high healthcare expenditure, MSC therapy represents an innovative approach. In a variety of preclinical and clinical studies, MSC have shown to exert renoprotective properties, mediated mainly by paracrine effects, immunomodulation, regulation of inflammation, secretion of several trophic factors, and possibly differentiation to renal precursors. However, studies are highly diverse; thus, knowledge is still limited regarding the exact mode of action, source of MSC in comparison to other stem cell types, administration route and dose, tracking of cells and documentation of therapeutic efficacy by new imaging techniques and tissue visualization. The aim of this review is to provide a summary of published studies of stem cell therapy in acute and chronic kidney injury, diabetic nephropathy, polycystic kidney disease, and kidney transplantation. Preclinical studies with allogeneic or xenogeneic cell therapy were first addressed, followed by a summary of clinical trials carried out with autologous or allogeneic hMSC. Studies were analyzed with respect to source of cell type, mechanism of action etc.

[This corrects the article DOI: 10.1371/journal.pone.0146831.].

BACKGROUND AND AIMS:The proportion of serum carnosinase (CN-1) recognized by RYSK173 monoclonal antibody negatively correlates with CN-1 activity. We thus hypothesized that the epitope recognized by RYSK173 is accessible only in a catalytically incompetent conformation of the zinc dependent enzyme and we mapped its position in the CN-1 structure. Since patients with kidney failure are often deficient in zinc and other trace elements we also assessed the RYSK173 CN-1 proportion in serum of these patients and studied the influence of hemodialysis hereon in relation to Zn2+ and Cu2+ concentration during hemodialysis. METHODS AND RESULTS:Epitope mapping using myc-tagged CN-1 fragments and overlapping peptides revealed that the RYSK173 epitope directly contributes to the formation of the dinuclear Zn center in the catalytic domain of homodimeric CN-1. Binding of RYSK173 to CN-1 was however not influenced by addition of Zn2+ or Cu2+ to serum. In serum of healthy controls the proportion of CN-1 recognized by RYSK173 was significantly lower compared to end-stage renal disease (ESRD) patients (1.12 ± 0.17 vs. 1.56 ± 0.40% of total CN-1; p

Background and purposeCatechol containing compounds have anti-inflammatory properties, yet for catecholamines these properties are modest. Since we have previously demonstrated that the synthetic dopamine derivative N-octanoyl dopamine (NOD) has superior anti-inflammatory properties compared to dopamine, we tested NOD in more detail and sought to elucidate the molecular entities and underlying mechanism by which NOD down-regulates inflammation.Experimental approachGenome wide gene expression profiling of human umbilical vein endothelial cells (HUVECs) was performed after stimulation with TNF-α or in the combination with NOD. Confirmation of these differences, NFκB activation and the molecular entities that were required for the anti-inflammatory properties were assessed in subsequent experiments.Key resultsDown regulation of inflammatory genes by NOD occurred predominantly for κB regulated genes, however not all κB regulated genes were affected. These findings were explained by inhibition of RelA phosphorylation at Ser276. Leukocyte adherence to TNF-α stimulated HUVECs was inhibited by NOD and was reflected by a diminished expression of adhesion molecules on HUVECs. NOD induced HO-1 expression, but this was not required for inhibition of NFκB. The anti-inflammatory effect of NOD seems to involve the redox active catechol structure, although the redox active para-dihydroxy benzene containing compounds also displayed anti-inflammatory effects, provided that they were sufficiently hydrophobic.Conclusions and implicationsThe present study highlighted important mechanisms and molecular entities by which dihydroxy benzene compounds exert their potential anti-inflammatory action. Since NOD does not have hemodynamic properties, NOD seems to be a promising candidate drug for the treatment of inflammatory diseases.

Diabetic nephropathy (DN) is a common microvascular complication of diabetes and the main cause of end-stage nephropathy (ESRD). Inflammation and fibrosis play key roles in the development and progression of diabetic nephropathy. By using in vivo and in vitro DN models, our laboratory has identified the protective role of carnosine (CAR) on renal tubules. Our results showed that carnosine restored the onset and clinical symptoms as well as renal tubular injury in DN. Furthermore, carnosine decreased kidney inflammation and fibrosis in DN mice. These results were consistent with high glucose (HG)-treated mice tubular epithelial cells (MTECs). Using web-prediction algorithms, cellular thermal shift assay (CETSA) and molecular docking, we identified glycine N-methyltransferase (GNMT) as a carnosine target. Importantly, we found that GNMT, a multiple functional protein that regulates the cellular pool of methyl groups by controlling the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH), was down-regulated significantly in the serum of Type 1 DM patients and renal tissues of DN mice. Moreover, using cultured TECs, we confirmed that the increased GNMT expression by transient transfection mimicked the protective role of carnosine in reducing inflammation and fibrosis. Conversely, the inhibition of GNMT expression abolished the protective effects of carnosine. In conclusion, carnosine might serve as a promising therapeutic agent for DN and GNMT might be a potential therapeutic target for DN. [ABSTRACT FROM AUTHOR]

Background. Here we compare the efficacy of cyclophosphamide (CYC) for treatment of crescentic nephritis (CGN) with the newer immunosuppressants 15-deoxyspergualin (DSG) and mycophenolate mofetil (MMF) in SCG/Kj mice, an inbred mouse strain that spontaneously develops CGN, systemic necrotizing vasculitis and antineutrophil cytoplasmic antibodies (ANCAs).Methods. Mice were randomly assigned to intraperitoneal treatment with either DSG (2?mg/kg/day), CYC (50?mg/kg/week), MMF (60 or 100?mg/kg/day) or vehicle (VEH, dextrose 5% 0.3?ml/day) beginning at the 10th week of life. ANCA, blood urea nitrogen (BUN) and proteinuria were determined in all animals regularly, and survival was calculated. Renal histology was obtained in the 18th week of life in the MMF- or VEH-treated groups and in the 24th week in DSG- or CYC-treated animals.Results. Mean survival in VEH-treated animals was 123 days. At that point, survival was 100% in the CYC- or DSG-treated animals (P<0.001). Survival in the MMF group (pooled data) was not significantly different from the VEH-treated animals [MMF, 117 days (95% CI 108127)]. BUN (18th week, CYC 439?mg/dl and DSG 366?mg/dl vs VEH 7328?mg/dl, P<0.001, MMF 6626?mg/dl), 24?h proteinuria (18th week, CYC 0.40.2?mg and DSG 0.70.6?mg vs VEH 2.73?mg, P<0.001, MMF 2.23?mg) crescent formation (18th week, VEH 429%, MMF 3911%; CYC 52% and DSG 227% vs VEH, P<0.05), glomerular immune complex deposition, and ANCA formation were significantly improved in CYC- and DSG- but not in MMF-treated animals when compared with controls.Conclusion. DSG and CYC, but not MMF, prolong life, limit renal damage and prevent autoantibody formation in SCG/Kj mice. [ABSTRACT FROM AUTHOR]