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.

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.

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.

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.

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.

[Utility of routine gastric biopsies and staining with methylene blue in the diagnosis of intestinal metaplasia in patients over 40 years]. / Utilidad de las biopsias gástricas rutinarias y de la tinción con azul de metileno en el diagnóstico de la metaplasia intestinal en mayores de 40 años.

OBJECTIVE: To evaluate the diagnostic utility of routine gastric biopsies taken at random versus targeted biopsies with methylene blue staining for the diagnosis of intestinal metaplasia. MATERIAL AND METHODS: We performed a validation study in 376 patients. We performed 2 antral biopsies, 2 gastric body biopsies, 1 biopsy for urease test and additional biopsies if demanded. One hundred and one patients underwent 2 biopsies after methylene blue staining. A total of 1,486 biopsies were analyzed. Frequencies of histological and endoscopic diagnosis and the validation of endoscopic diagnosis of intestinal metaplasia with and without staining against histology, which followed updated Sydney and Vienna criteria, were determined RESULTS: Metaplasia was found in 37.23% ofcases and in 6.38% was incomplete in antrum and body, moderate or severe. The endoscopic diagnosis had: sensitivity 46%, specificity 91%, positive predictive value (PPV) 75% and negative predictive value (NPV) 74%. The low sensitivity indicates the need for gastric biopsies. Staining had: sensitivity 95%, specificity 67%, PPV 84% and NPV 89%, with significant difference for staining (P < 0.05). The typical endoscopic appearance of intestinal metaplasia always coincided with staining and histology. Dysplasia was found in 0.53% ofcases, gastric cancer in 1.8% and Helicobacter pylori in 56%. CONCLUSION: Directed biopsy staining is superior to those taken at random for diagnosing metaplasia. Metaplasia was not found when endoscopy was normal and typical endoscopic appearance of metaplasia agreed with staining and histology.

Utilidad de las biopsias gástricas rutinarias y de la tinción con azul de metileno en el diagnóstico de la metaplasia intestinal en mayores de 40 años / [Utility of routine gastric biopsies and staining with methylene blue in the diagnosis of intestinal metaplasia in patients over 40 years].

OBJECTIVE: To evaluate the diagnostic utility of routine gastric biopsies taken at random versus targeted biopsies with methylene blue staining for the diagnosis of intestinal metaplasia. MATERIAL AND METHODS: We performed a validation study in 376 patients. We performed 2 antral biopsies, 2 gastric body biopsies, 1 biopsy for urease test and additional biopsies if demanded. One hundred and one patients underwent 2 biopsies after methylene blue staining. A total of 1,486 biopsies were analyzed. Frequencies of histological and endoscopic diagnosis and the validation of endoscopic diagnosis of intestinal metaplasia with and without staining against histology, which followed updated Sydney and Vienna criteria, were determined RESULTS: Metaplasia was found in 37.23

ofcases and in 6.38

was incomplete in antrum and body, moderate or severe. The endoscopic diagnosis had: sensitivity 46

. The low sensitivity indicates the need for gastric biopsies. Staining had: sensitivity 95

, with significant difference for staining (P < 0.05). The typical endoscopic appearance of intestinal metaplasia always coincided with staining and histology. Dysplasia was found in 0.53

ofcases, gastric cancer in 1.8

and Helicobacter pylori in 56

. CONCLUSION: Directed biopsy staining is superior to those taken at random for diagnosing metaplasia. Metaplasia was not found when endoscopy was normal and typical endoscopic appearance of metaplasia agreed with staining and histology.

Utilidad de las biopsias gástricas rutinarias y de la tinción con azul de metileno en el diagnóstico de la metaplasia intestinal en mayores de 40 años. / [Utility of routine gastric biopsies and staining with methylene blue in the diagnosis of intestinal metaplasia in patients over 40 years].

OBJECTIVE: To evaluate the diagnostic utility of routine gastric biopsies taken at random versus targeted biopsies with methylene blue staining for the diagnosis of intestinal metaplasia. MATERIAL AND METHODS: We performed a validation study in 376 patients. We performed 2 antral biopsies, 2 gastric body biopsies, 1 biopsy for urease test and additional biopsies if demanded. One hundred and one patients underwent 2 biopsies after methylene blue staining. A total of 1,486 biopsies were analyzed. Frequencies of histological and endoscopic diagnosis and the validation of endoscopic diagnosis of intestinal metaplasia with and without staining against histology, which followed updated Sydney and Vienna criteria, were determined RESULTS: Metaplasia was found in 37.23

ofcases and in 6.38

was incomplete in antrum and body, moderate or severe. The endoscopic diagnosis had: sensitivity 46

, specificity 91

, positive predictive value (PPV) 75

and negative predictive value (NPV) 74

. The low sensitivity indicates the need for gastric biopsies. Staining had: sensitivity 95

, specificity 67

, PPV 84

and NPV 89

, with significant difference for staining (P < 0.05). The typical endoscopic appearance of intestinal metaplasia always coincided with staining and histology. Dysplasia was found in 0.53

ofcases, gastric cancer in 1.8

and Helicobacter pylori in 56

. CONCLUSION: Directed biopsy staining is superior to those taken at random for diagnosing metaplasia. Metaplasia was not found when endoscopy was normal and typical endoscopic appearance of metaplasia agreed with staining and histology.

[Score BISAP validation as a prognostic system in acute pancreatitis]. / Validación del score de BISAP como sistema pronóstico en pancreatitis aguda.

BACKGROUND: The BISAP score is a simple system, which englobes clinical features (laboratory and imagenology tests) allowing to predict the mortality in acute pancreatitis within the first 24 hours of hospitalization. OBJECTIVE: To determine the validity of the BISAP score in the prediction of prognosis and severity of acute pancreatitis. METHOD: In order to validate the BISAP score, a study was performed in 57 patients with a diagnosis of acute pancreatitis at the moment of admission. The reference test was the Atlanta criteria which confirmed severe clinical course beyond the fifth day, in many cases supported by CT. RESULTS: 71.9% were women. The mean age was 45.33 years. Biliary lithiasis was the most frequent cause (66.7%). According to the reference test 71.9% were mild and 28.1% severe. According to the BISAP score 77.2% mild and 22.8% severe. The sensitivity and specificity for the BISAP score was of 75% and 97.56% respectively. The predictive positive value was 92.31% and the predictive negative value was 90.91%. CONCLUSIONS: The BISAP score allows to predict the severity of the acute pancreatitis.

Validación del Score de BISAP como Sistema Pronóstico en Pancreatitis Aguda / Score Bisap validation as a prognostic system in acute pancreatitis

INTRODUCCIÓN: El score de BISAP es un sistema no complicado y factible de realizar en hospitales de referencia como el nuestro, engloba variables clínicas, de laboratorio y de imagen, permitiendo predecir la mortalidad por pancreatitis aguda, dentro de las 24 horas de inicio del cuadro clínico. OBJETIVO: Determinar la validez del score de BISAP en la gravedad y el pronóstico de la Pancreatitis Aguda. MÉTODO: Estudio de validación del score de BISAP en 57 pacientes consecutivos con diagnóstico de pancreatitis aguda al ingreso. Prueba de referencia: los criterios de Atlanta confirmados por evolución clínica grave más allá del quinto día, apoyados en muchos casos por TAC. RESULTADOS: El 71.9% fueron de sexo femenino. La media de edad fue de 45.33 años. La litiasis biliar fue la causa más frecuente (66.7%). Según la prueba de referencia, el 71.9% de casos fueron leves y el 28.1% graves. Según el score de BISAP el 77.2% leves y el 22.8% graves. La sensibilidad y especificidad para el score de BISAP fue del 75% y del 97.56% respectivamente. El valor predictivo positivo fue del 92.31% y el valor predictivo negativo, del 90.91%. CONCLUSIONES: El score de BISAP permite predecir la severidad en la pancreatitis aguda.

BACKGROUND: The BISAP score is a simple system, which englobes clinical features (laboratory and imagenology tests) allowing to predict the mortality in acute pancreatitis within the first 24 hours of hospitalization. OBJECTIVE: To determine the validity of the BISAP score in the prediction of prognosis and severity of acute pancreatitis. METHOD: In order to validate the BISAP score, a study was performed in 57 patients with a diagnosis of acute pancreatitis at the moment of admission. The reference test was the Atlanta criteria which confirmed severe clinical course beyond the fifth day, in many cases supported by CT. RESULTS: 71.9% were women. The mean age was 45.33 years. Biliary lithiasis was the most frequent cause (66.7%). According to the reference test 71.9% were mild and 28.1% severe. According to the BISAP score 77.2% mild and 22.8% severe. The sensitivity and specificity for the BISAP score was of 75% and 97.56% respectively. The predictive positive value was 92.31% and the predictive negative value was 90.91%. CONCLUSIONS: The BISAP score allows to predict the severity of the acute pancreatitis.