The phagocytosis dysfunction in lupus nephritis is related to monocyte/macrophage CPT1a.

Macrophages must remove apoptotic cells to shield tissues from the deleterious components of dying cells. The development of chronic inflammation and autoimmune symptoms in systemic lupus is influenced by a deficiency in phagocytosis of apoptotic cells but the underlying mechanism is still unknown. Modifications in monocyte/macrophage phenotype brought on by an increase in their inflammatory phenotype would cause them to decrease the expression of CPT1a, which would reduce their ability to phagocytose, aggravating kidney damage in lupus nephritis. We aim to demonstrate that the deficiency of CPT1A in the immunological system determines lupus. For this purpose, we will monitor CPT1a expression in blood monocytes and phagocytosis and CPT1a expression of macrophages isolated from kidneys and the inflammatory state in kidneys in two experimental models of lupus nephritis such as lupus induced pristane model and in the OVA-IC in vivo model. Additionally, we will test if reestablishing CPT1a expression in tissue macrophages restores the lost phagocytic function. We evidenced that blood monocytes and macrophages isolated from kidneys in the two in vivo models have a reduced expression of CPT1a and a reduced phagocytosis. Phagocytosis could be restored only if macrophage administration leads to an increase in CPT1a expression in kidney macrophages. A new cell therapy to reduce kidney nephritis in lupus could be developed based on these results.

Development of Cell Therapies for Renal Disease and Regenerative Medicine.

The incidence of renal disease is gradually increasing worldwide, and this condition has become a major public health problem because it is a trigger for many other chronic diseases. Cell therapies using multipotent mesenchymal stromal cells, hematopoietic stem cells, macrophages, and other cell types have been used to induce regeneration and provide a cure for acute and chronic kidney disease in experimental models. This review describes the advances in cell therapy protocols applied to acute and chronic kidney injuries and the attempts to apply these treatments in a clinical setting.

NGAL release from peripheral blood mononuclear cells protects against acute kidney injury and prevents AKI induced fibrosis.

We propose the use of a peripheral blood mononuclear cell therapy based on cell NGAL release to be used in the clinical setting for acute kidney injury (AKI) and the derived fibrosis. First, we designed a procedure whereby PBMC overexpress NGAL and anti-inflammatory agents when subjected to repetitive anoxia/reoxygenation (PBMC (A/R)). Using an in vivo AKI model, we observed that PBMC(A/R) reduces BUN and creatinine levels in blood and inflammation, enhances anti-inflammation, induces proliferation of tubular epithelial cells and reduces AKI-induced fibrosis. Flow cytometry analysis evidenced that monocytes are the only cells accumulated in the injured kidney and phenotype analysis of freshly isolated kidney macrophages, revealed that the healing phenotype is maintained the time needed for recovery. NGAL release from PBMC(A/R) determines the beneficial effect of the therapy since administration of a NGAL antibody previous to the therapy or injection of PBMC(A/R) obtained from NGAL KO animals abolished the beneficial effects. CD11b-NGAL positive cells were enhanced in tissue after PBMC (A/R) therapy and were produced by the injected monocytes. In an in vitro model with tubular epithelial cells (NRK52e) we proved that NGAL release by PBMC(A/R) induced epithelial proliferation and activation of PI3K/Akt pathway.

Mitochondrial Transplantation Enhances Phagocytic Function and Decreases Lipid Accumulation in Foam Cell Macrophages.

Macrophages have mechanisms for eliminating cholesterol from cells. If excess cholesterol is not eliminated from the macrophages, then transformation into a foam cell may occur. Foam cells are a hallmark of the atherosclerotic lesions that contribute to the development and rupture of atherosclerotic plaques. Several in vitro and in vivo studies have shown changes in the macrophage phenotype and improved phagocytosis after the acquisition of functional mitochondria. However, the effect of mitochondrial transplantation on promoting phagocytosis and phenotypic changes in lipid-loaded macrophages leading to foam cells has not been studied. We aimed to prove that the transplantation of healthy mitochondria to highly cholesterol-loaded macrophages induces macrophage phagocytosis and reduces the macrophage shift towards foam cells. For this purpose, using a murine macrophage cell line, RAW264.7, we determined if mitochondria transplantation to 7-ketocholesterol (7-KC)-loaded macrophages reduced lipid accumulation and modified their phagocytic function. We evidenced that mitochondrial transplantation to 7-KC-loaded macrophages reestablished phagocytosis and reduced lipid content. In addition, CPT1a expression and anti-inflammatory cytokines were restored after mitochondrial transplantation. We have developed a potential therapeutic approach to restore foam cell functionality.

Infusion of Phagocytic Macrophages Overexpressing CPT1a Ameliorates Kidney Fibrosis in the UUO Model.

Phagocytosis is an inherent function of tissue macrophages for the removal of apoptotic cells and cellular debris during acute and chronic injury; however, the dynamics of this event during fibrosis development is unknown. We aim to prove that during the development of kidney fibrosis in the unilateral ureteral obstruction (UUO) model, there are some populations of macrophage with a reduced ability to phagocytose, and whether the infusion of a population of phagocytic macrophages could reduce fibrosis in the murine model UUO. For this purpose, we have identified the macrophage populations during the development of fibrosis and have characterized their phagocytic ability and their expression of CPT1a. Furthermore, we have evaluated the therapeutic effect of macrophages overexpressing CPT1a with high phagocytic skills. We evidenced that the macrophage population which exhibits high phagocytic ability (F4/80low-CD11b) in fibrotic animals decreases during the progression of fibrosis while the macrophage population with lower phagocytic ability (F4/80high-CD11b) in fibrotic conditions, conversely, increases and CPT1a macrophage cell therapy with a strengthening phagocytic ability is associated with a therapeutic effect on kidney fibrosis. We have developed a therapeutic approach to reduce fibrosis in the UUO model by enrichment of the kidney resident macrophage population with a higher proportion of exogenous phagocytic macrophages overexpressing CPT1a.

The influenza virus NS1A binding protein gene modulates macrophages response to cytokines and phagocytic potential in inflammation.

Macrophages show remarkable phenotypic plasticity in response to environmental signals. Although it is generally less considered, cytoskeletal changes in macrophages influence their phenotype, including phagocytosis and secretion of soluble cytokines. Influenza virus NS1A-binding protein (Ivns1abp) belongs to the Kelch family of proteins that play a central role in actin cytoskeleton dynamics by directly associating with F-actin and by protecting against actin derangement. Due to its role in cytoskeleton preservation, the Ivns1abp gene might be a critical regulator of the macrophage phenotype and function under inflammatory conditions. In this study, we determine that the modulation of the Ivns1abp gene in macrophages could modify resistance to macrophages against inflammation and maintain functional phagocytosis. Our results indicate that inflammatory insults inhibit the Ivns1abp gene, whereby phagocytosis is inhibited and the ability of macrophages to induce proliferation and repair of damaged cells is compromised. Furthermore, our results show that inflammatory insults alter the activity of the transcription factor c-myc, a factor which directly modulates the expression of the Ivns1abp gene. In conclusion, this study demonstrates a central role of lvns1abp in promoting and preserving a reparative macrophage phenotype and resistance to this inflammatory environment.

Macrophage Phenotype and Fibrosis in Diabetic Nephropathy.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease globally. The primary initiating mechanism in DN is hyperglycemia-induced vascular dysfunction, but its progression is due to different pathological mechanisms, including oxidative stress, inflammatory cells infiltration, inflammation and fibrosis. Macrophages (Mφ) accumulation in kidneys correlates strongly with serum creatinine, interstitial myofibroblast accumulation and interstitial fibrosis scores. However, whether or not Mφ polarization is involved in the progression of DN has not been adequately defined. The prevalence of the different phenotypes during the course of DN, the existence of hybrid phenotypes and the plasticity of these cells depending of the environment have led to inconclusive results. In the same sense the role of the different macrophage phenotype in fibrosis associated or not to DN warrants additional investigation into Mφ polarization and its role in fibrosis. Due to the association between fibrosis and the progressive decline of renal function in DN, and the role of the different phenotypes of Mφ in fibrosis, in this review we examine the role of macrophage phenotype control in DN and highlight the potential factors contributing to phenotype change and injury or repair in DN.

CPT1a gene expression reverses the inflammatory and anti-phagocytic effect of 7-ketocholesterol in RAW264.7 macrophages.

BACKGROUND: Macrophage are specialized cells that contributes to the removal of detrimental contents via phagocytosis. Lipid accumulation in macrophages, whether from phagocytosis of dying cells or from circulating oxidized low-density lipoproteins, alters macrophage biology and functionality. It is known that carnitine palmitoyl transferase 1-a (CPT1a) gene encodes an enzyme involved in fatty acid oxidation and, therefore, lipid content. However, the potential of CPT1a to activate macrophage phagocytic function have not been elucidated. METHODS: Using a murine macrophage cell line, RAW264.7, we determine if intracellular accumulation of 7-ketocholesterol (7-KC) modulates macrophage phagocytic function through CPT1a gene expression. In addition, the effects of CPT1a genetic modification on macrophage phenotype and phagocytosis has been studied. RESULTS: Our results revealed that CPT1a gene expression decreased by the accumulation of 7-KC at the higher dose of 7-KC. This was concomitant with an impair ability to phagocytize bioparticles and an inflammatory phenotype. GW3965 treatment, which have shown to facilitate the efflux of cholesterol, eliminated the intracellular lipid droplets of 7-KC-laden macrophages, increased the gene expression of CPT1a, diminished the gene expression of the inflammatory marker iNOS and restored macrophage phagocytosis. Furthermore, CPT1a Knockdown per se was detrimental for macrophage phagocytosis whereas transcriptional activation of CPT1a heightened the uptake of bioparticles. CONCLUSIONS: Altogether, our findings indicate that downregulation of CPT1a by lipid content modulates macrophage phagocytosis and inflammatory phenotype.

Urinary Neuropilin-1: A Predictive Biomarker for Renal Outcome in Lupus Nephritis.

At present, Lupus Nephritis (LN) is still awaiting a biomarker to better monitor disease activity, guide clinical treatment, and predict a patient's long-term outcome. In the last decade, novel biomarkers have been identified to monitor the disease, but none have been incorporated into clinical practice. The transmembrane receptor neuropilin-1 (NRP-1) is highly expressed by mesangial cells and its genetic deletion results in proteinuric disease and glomerulosclerosis. NRP-1 is increased in kidney biopsies of LN. In this work we were interested in determining whether urinary NRP-1 levels could be a biomarker of clinical response in LN. Our results show that patients with active LN have increased levels of urinary NRP-1. When patients were divided according to clinical response, responders displayed higher urinary and tissue NRP-1 levels at the time of renal biopsy. Areas under the receiver operating characteristic curve, comparing baseline creatinine, proteinuria, urinary NRP-1, and VEGFA protein levels, showed NRP-1 to be an independent predictor for clinical response. In addition, in vitro studies suggest that NRP-1could promote renal recovery through endothelial proliferation and migration, mesangial migration and local T cell cytotoxicity. Based on these results, NRP-1 may be used as an early prognostic biomarker in LN.

CPT1a downregulation protects against cholesterol-induced fibrosis in tubular epithelial cells by downregulating TGFß-1 and inflammasome.

BACKGROUND: Dyslipidemia causes renal damage; however, the detailed molecular mechanism has not been clarified. It is known that carnitine palmitoyl transferase 1-a (CPT1a) gene encodes an enzyme involved in fatty acid oxidation and, therefore, lipid content. In the present study, we investigated whether the accumulation of lipids induced by 7-ketocholesterol (7-KC) in tubular epithelial cells produce a fibrotic and inflammatory response through CPT1a. METHODS: Using an epithelial cell line, NRK-52E, we determine if intracellular accumulation of 7-KC modulates profibrotic and inflammatory events through CPT1a gene expression. In addition, the direct effects of CPT1a genetic modification has been studied. RESULTS: Our results revealed that high levels of 7-KC induce increased expression of CPT1a, TGF-ß1, α- SMA and NLRP3 that was correlated with lipid content. GW3965 treatment, which have shown to facilitate the efflux of cholesterol, eliminated the intracellular lipid droplets of 7-KC laden cells and decreased the expression of CPT1a, TGF-ß1, α- SMA and NLRP3. Furthermore, CPT1a Knockdown and C75 pre-treatment increased lipid content but decreased TGF-ß1, α- SMA and NLRP3. CONCLUSIONS: Our findings reveal that the profibrotic effect of 7-KC on renal epithelial cells are mediated by CPT1a overexpression, which acts on TGF-ß1, α-SMA and NLRP3. Thus, CPT1a downregulation protects against 7-KC-induced fibrosis in tubular epithelial cells by downregulating TGF-ß1, α- SMA and NLRP3.

Exploring macrophage cell therapy on Diabetic Kidney Disease.

Alternatively activated macrophages (M2) have regenerative properties and shown promise as cell therapy in chronic kidney disease. However, M2 plasticity is one of the major hurdles to overcome. Our previous studies showed that genetically modified macrophages stabilized by neutrophil gelatinase-associated lipocalin (NGAL) were able to preserve their M2 phenotype. Nowadays, little is known about M2 macrophage effects in diabetic kidney disease (DKD). The aim of the study was to investigate the therapeutic effect of both bone marrow-derived M2 (BM-фM2) and ф-NGAL macrophages in the db/db mice. Seventeen-week-old mice with established DKD were divided into five treatment groups with their controls: D+BM-фM2; D+ф-BM; D+ф-NGAL; D+ф-RAW; D+SHAM and non-diabetic (ND) (db/- and C57bl/6J) animals. We infused 1 × 106 macrophages twice, at baseline and 2 weeks thereafter. BM-фM2 did not show any therapeutic effect whereas ф-NGAL significantly reduced albuminuria and renal fibrosis. The ф-NGAL therapy increased the anti-inflammatory IL-10 and reduced some pro-inflammatory cytokines, reduced the proportion of M1 glomerular macrophages and podocyte loss and was associated with a significant decrease of renal TGF-ß1. Overall, our study provides evidence that ф-NGAL macrophage cell therapy has a therapeutic effect on DKD probably by modulation of the renal inflammatory response caused by the diabetic milieu.

Lipocalin-2 abrogates epithelial cell cycle arrest by PPARγ inhibition.

Macrophage-epithelial cross-talk regulates cell cycle progression and represents an important factor in rescuing epithelial cells from cell cycle arrest in order to maintain a healthy epithelial phenotype. However, the underlying mechanisms are still not well defined. We provide evidence that macrophage-secreted lipocalin-2 (Lcn-2) plays a key role during this process. In a co-culture setup using cell cycle arrested NRK52e renal epithelial cells and primary bone marrow-derived macrophages, Lcn-2 restores proliferation through inhibition of peroxisome proliferator-activated receptor (PPAR)-γ. Lcn-2 overexpression in macrophages overcomes epithelial cell cycle arrest and enhances epithelial markers via megalin and the downstream activation of PI3K/Akt signalling pathway, whereas a knockdown of Lcn-2 in macrophages prevented this effect. Our results show that macrophage-secreting Lcn-2 is crucial in rescuing epithelial cells from cell cycle arrest and in promoting epithelial proliferation.

Cytoprotective Mechanisms in Fatty Liver Preservation against Cold Ischemia Injury: A Comparison between IGL-1 and HTK.

Institute Goeorges Lopez 1 (IGL-1) and Histidine-Tryptophan-Ketoglutarate (HTK) preservation solutions are regularly used in clinical for liver transplantation besides University of Wisconsin (UW) solution and Celsior. Several clinical trials and experimental works have been carried out comparing all the solutions, however the comparative IGL-1 and HTK appraisals are poor; especially when they deal with the underlying protection mechanisms of the fatty liver graft during cold storage. Fatty livers from male obese Zücker rats were conserved for 24 h at 4 °C in IGL-1 or HTK preservation solutions. After organ recovery and rinsing of fatty liver grafts with Ringer Lactate solution, we measured the changes in mechanistic target of rapamycin (mTOR) signaling activation, liver autophagy markers (Beclin-1, Beclin-2, LC3B and ATG7) and apoptotic markers (caspase 3, caspase 9 and TUNEL). These determinations were correlated with the prevention of liver injury (aspartate and alanine aminostransferase (AST/ALT), histology) and mitochondrial damage (glutamate dehydrogenase (GLDH) and confocal microscopy findings). Liver grafts preserved in IGL-1 solution showed a marked reduction on p-TOR/mTOR ratio when compared to HTK. This was concomitant with significant increased cyto-protective autophagy and prevention of liver apoptosis, including inflammatory cytokines such as HMGB1. Together, our results revealed that IGL-1 preservation solution better protected fatty liver grafts against cold ischemia damage than HTK solution. IGL-1 protection was associated with a reduced liver damage, higher induced autophagy and decreased apoptosis. All these effects would contribute to limit the subsequent extension of reperfusion injury after graft revascularization in liver transplantation procedures.

Microencapsulated macrophages releases conditioned medium able to prevent epithelial to mesenchymal transition.

Epithelial to mesenchymal transition (EMT) has emerged as a key process in the development of renal fibrosis. In fact, EMT-derived fibroblasts contribute to the progression of chronic renal disease. In addition, anti-inflammatory M2 macrophages have exhibited a great influence on renal fibrosis. However, because of the high impact that the inputs of different environmental cytokines have on their phenotype, macrophages can easily lose this property. We aim to known if microencapsulated macrophages on M2-inducing alginate matrices could preserve macrophage phenotype and thus release factors able to act on epithelial cells to prevent the epithelial differentiation towards mesenchymal cells. We reproduced an in vitro model of EMT by treating adipose-derived stem cells with all-trans retinoic acid (ATRA) and induced their transformation toward epithelia. Dedifferentiation of epithelial cells into a mesenchymal phenotype occurred when ATRA was retired, thus simulating EMT. Results indicate that induction of M2 phenotype by IL-10 addition in the alginate matrix produces anti-inflammatory cytokines and increases the metabolic activity and the viability of the encapsulated macrophages. The released conditioned medium modulates EMT and maintains healthy epithelial phenotype. This could be used for in vivo cell transplantation, or alternatively as an external releaser able to prevent epithelial to mesenchymal transformation for future anti-fibrotic therapies.

The Relevance of the UPS in Fatty Liver Graft Preservation: A New Approach for IGL-1 and HTK Solutions.

The 26S proteasome is the central proteolytic machinery of the ubiquitin proteasome system (UPS), which is involved in the degradation of ubiquitinated protein substrates. Recently, UPS inhibition has been shown to be a key factor in fatty liver graft preservation during organ cold storage using University of Wisconsin solution (UW) and Institute Georges Lopez (IGL-1) solutions. However, the merits of IGL-1 and histidine-tryptophan-ketoglutarate (HTK) solutions for fatty liver preservation have not been compared. Fatty liver grafts from obese Zücker rats were preserved for 24 h at 4 °C. Aspartate aminotransferase and alanine aminotransferase (AST/ALT), glutamate dehydrogenase (GLDH), ATP, adenosine monophosphate protein kinase (AMPK), e-NOS, proteasome activity and liver polyubiquitinated proteins were determined. IGL-1 solution prevented ATP breakdown during cold-storage preservation of steatotic livers to a greater extent than HTK solution. There were concomitant increases in AMPK activation, e-NOS (endothelial NOS (NO synthase)) expression and UPS inhibition. UPS activity is closely related to the composition of the solution used to preserve the organ. IGL-1 solution provided significantly better protection against ischemia-reperfusion for cold-stored fatty liver grafts than HTK solution. The effect is exerted through the activation of the protective AMPK signaling pathway, an increase in e-NOS expression and a dysregulation of the UPS.

Macrophage Overexpressing NGAL Ameliorated Kidney Fibrosis in the UUO Mice Model.

BACKGROUND/AIMS: Alternatively activated macrophages (AAM) have regenerative and anti-inflammatory characteristics. Here, we sought to evaluate whether AAM cell therapy reduces renal inflammation and fibrosis in the unilateral ureteral obstruction (UUO) mice model. METHODS: We stabilized macrophages by adenoviral vector NGAL (Neutrophil gelatinase-associated lipocalin-2) and infused them into UUO mice. To ascertain whether macrophages were capable of reaching the obstructed kidney, macrophages were stained and detected by in vivo cell tracking. RESULTS: We demonstrated that some infused macrophages reached the obstructed kidney and that infusion of macrophages overexpressing NGAL was associated with reduced kidney interstitial fibrosis and inflammation. This therapeutic effect was mainly associated with the phenotype and function preservation of the transferred macrophages isolated from the obstructed kidney Conclusions: Macrophage plasticity is a major hurdle for achieving macrophage therapy success in chronic nephropathies and could be overcome by transferring lipocalin-2.

Macrophage-derived Lipocalin-2 contributes to ischemic resistance mechanisms by protecting from renal injury.

Renal ischemia-reperfusion injury triggers an inflammatory response associated to infiltrating macrophages which determines the further outcome of disease. Brown Norway rats are known to show endogenous resistance to ischemia-induced renal damage. By contrast, Sprague Dawley rats exhibit a higher susceptibility to ischemic injury. In order to ascertain cytoprotective mechanisms, we focused on the implication of lipocalin-2 protein in main resistance mechanisms in renal ischemia/reperfusion injury by using adoptive macrophage administration, genetically modified ex vivo either to overexpress or to knockdown lipocalin-2. In vitro experiments with bone marrow-derived macrophages both from Brown Norway rats and from Sprague Dawley rats under hypoxic conditions showed endogenous differences regarding cytokine and lipocalin-2 expression profile in the two strains. Most interestingly, we observed that macrophages of the resistant strain express significantly more lipocalin-2. In vivo studies showed that tubular epithelial cell apoptosis and renal injury significantly increased and reparative markers decreased in Brown Norway rats after injection of lipocalin-2-knockdown macrophages, while the administration of lipocalin-2-overexpressing cells significantly decreased Sprague Dawley susceptibility. These data point to a crucial role of macrophage-derived lipocalin-2 in endogenous cytoprotective mechanisms. We conclude that expression of lipocalin-2 in tissue-infiltrating macrophages is pivotal for kidney-intrinsic cytoprotective pathways during ischemia reperfusion injury.

Carbonic Anhydrase Protects Fatty Liver Grafts against Ischemic Reperfusion Damage.

Carbonic anhydrases (CAs) are ubiquitous metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton. CAs are involved in numerous physiological and pathological processes, including acid-base homeostasis, electrolyte balance, oxygen delivery to tissues and nitric oxide generation. Given that these processes are found to be dysregulated during ischemia reperfusion injury (IRI), and taking into account the high vulnerability of steatotic livers to preservation injury, we hypothesized a new role for CA as a pharmacological agent able to protect against ischemic damage. Two different aspects of the role of CA II in fatty liver grafts preservation were evaluated: 1) the effect of its addition to Institut Georges Lopez (IGL-1) storage solution after cold ischemia; 2) and after 24h of cold storage followed by two hours of normothermic ex-vivo perfusion. In all cases, liver injury, CA II protein concentration, CA II mRNA levels and CA II activity were determined. In case of the ex-vivo perfusion, we further assessed liver function (bile production, bromosulfophthalein clearance) and Western blot analysis of phosphorylated adenosine monophosphate activated protein kinase (AMPK), mitogen activated protein kinases family (MAPKs) and endoplasmic reticulum stress (ERS) parameters (GRP78, PERK, IRE, eIF2α and ATF6). We found that CA II was downregulated after cold ischemia. The addition of bovine CA II to IGL-1 preservation solution efficiently protected steatotic liver against cold IRI. In the case of reperfusion, CA II protection was associated with better function, AMPK activation and the prevention of ERS and MAPKs activation. Interestingly, CA II supplementation was not associated with enhanced CO2 hydration. The results suggest that CA II modulation may be a promising target for fatty liver graft preservation.

miRNA let-7e modulates the Wnt pathway and early nephrogenic markers in mouse embryonic stem cell differentiation.

This study indicates that embryonic stem cells [ESCs] cultured with retinoic acid and activin A significantly upregulate the miRNA let-7e. This specific miRNA modulates the Wnt pathway and the expression of early nephrogenic markers under these differentiation conditions. The differentiation markers WT1, Pax2 and Wnt4 were downregulated when miRNA let-7e was silenced, thus indicating the role of miRNA let-7e in the differentiation process. PKCß, GSK3ß phosphorylation (GSK3ß(P)) and ß-catenin expression was reduced in differentiated cells and reversed by miRNA let-7e silencing. Addition of a PKCß inhibitor to the miRNA let-7e silenced cells abolished let-7e-derived effects in differentiation markers, and reversed the increase in GSK3ß(P) and ß-catenin, thus indicating that miRNA let-7e is involved in differentiation via the modulation of GSK3ß phosphorylation and ß-catenin production.