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.

Cdc14 activation requires coordinated Cdk1-dependent phosphorylation of Net1 and PP2A-Cdc55 at anaphase onset.

Exit from mitosis and completion of cytokinesis require the inactivation of mitotic cyclin-dependent kinase (Cdk) activity. In budding yeast, Cdc14 phosphatase is a key mitotic regulator that is activated in anaphase to counteract Cdk activity. In metaphase, Cdc14 is kept inactive in the nucleolus, where it is sequestered by its inhibitor, Net1. At anaphase onset, downregulation of PP2ACdc55 phosphatase by separase and Zds1 protein promotes Net1 phosphorylation and, consequently, Cdc14 release from the nucleolus. The mechanism by which PP2ACdc55 activity is downregulated during anaphase remains to be elucidated. Here, we demonstrate that Cdc55 regulatory subunit is phosphorylated in anaphase in a Cdk1-Clb2-dependent manner. Interestingly, cdc55-ED phosphomimetic mutant inactivates PP2ACdc55 phosphatase activity towards Net1 and promotes Cdc14 activation. Separase and Zds1 facilitate Cdk-dependent Net1 phosphorylation and Cdc14 release from the nucleolus by modulating PP2ACdc55 activity via Cdc55 phosphorylation. In addition, human Cdk1-CyclinB1 phosphorylates human B55, indicating that the mechanism is conserved in higher eukaryotes.

SILAC-based phosphoproteomics reveals new PP2A-Cdc55-regulated processes in budding yeast.

Background: Protein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2ACdc55 phosphatase has been extensively related to cell cycle events in budding yeast; however, few PP2ACdc55 substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2ACdc55 phosphatase and new PP2A-related processes in mitotic arrested cells. Results: We identified 62 statistically significant PP2ACdc55 substrates involved mainly in actin-cytoskeleton organization. In addition, we validated new PP2ACdc55 substrates such as Slk19 and Lte1, involved in early and late anaphase pathways, and Zeo1, a component of the cell wall integrity pathway. Finally, we constructed docking models of Cdc55 and its substrate Mob1. We found that the predominant interface on Cdc55 is mediated by a protruding loop consisting of residues 84-90, thus highlighting the relevance of these aminoacids for substrate interaction. Conclusions: We used phosphoproteomics of Cdc55-deficient cells to uncover new PP2ACdc55 substrates and functions in mitosis. As expected, several hyperphosphorylated proteins corresponded to Cdk1-dependent substrates, although other kinases' consensus motifs were also enriched in our dataset, suggesting that PP2ACdc55 counteracts and regulates other kinases distinct from Cdk1. Indeed, Pkc1 emerged as a novel node of PP2ACdc55 regulation, highlighting a major role of PP2ACdc55 in actin cytoskeleton and cytokinesis, gene ontology terms significantly enriched in the PP2ACdc55-dependent phosphoproteome.

Mitotic Exit Function of Polo-like Kinase Cdc5 Is Dependent on Sequential Activation by Cdk1.

To complete mitosis, Saccharomyces cerevisiae needs to activate the mitotic phosphatase Cdc14. Two pathways contribute to Cdc14 regulation: FEAR (Cdc14 early anaphase release) and MEN (mitotic exit network). Cdc5 polo-like kinase was found to be an important mitotic exit component. However, its specific role in mitotic exit regulation and its involvement in Cdc14 release remain unclear. Here, we provide insight into the mechanism by which Cdc5 contributes to the timely release of Cdc14. Our genetic and biochemical data indicate that Cdc5 acts in parallel with MEN during anaphase. This MEN-independent Cdc5 function requires active separase and activation by Cdk1-dependent phosphorylation. Cdk1 first phosphorylates Cdc5 to activate it in early anaphase, and then, in late anaphase, further phosphorylation of Cdc5 by Cdk1 is needed to promote its MEN-related functions.