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Background: Sepsis is a life-threatening condition often associated with a high incidence of multiple organs injury. Several papers suggested the immune response by itself, with the production of humoral inflammatory mediators, is crucial in determining organ injury. However, little is known of how sepsis directly induces organ injury at the cellular levels. To assess this point, we set up an in vitro study to investigate the response of renal tubular cells (RTCs), monocytes (U937) and hepatocytes (HepG2) after 24 h-incubation with septic patients' plasma., Methods: We enrolled 26 septic patients ("test" group). We evaluated cell viability, apoptosis and necrosis by flow cytometer. Caspase-3,-8,-9 and cytochrome-c concentrations have been analyzed using the Human enzyme-linked immunosorbent assay kit., Results: We found that a decrease of cell viability in all cell lines tested was associated to the increase of apoptosis in RTCs and U937 (p < 0.0001) and increase of necrosis in HepG2 (p < 0.5). The increase of apoptosis in RTCs and U937 cells was confirmed by higher levels of caspase-3 (p < 0.0001). We showed that apoptosis in both RTCs and U937 was triggered by the activation of the intrinsic pathway, as caspase-9 and cytochrome-c levels significantly increased (p < 0.0001), while caspase-8 did not change. This assumption was strengthened by the significant correlation of caspase-9 with both cytochrome-c (r = 0.73 for RTCs and r = 0.69 for U937) and caspase-3 (r = 0.69 for RTCs and r = 0.63 for U937)., Conclusion: Humoral mediators in septic patients' plasma induce apoptosis. This fact suggests that apoptosis inhibitors should be investigated as future strategy to reduce sepsis-induced organ damages., (© 2018 S. Karger AG, Basel.)

Background: The renal assist device (RAD) is a blood purification system containing viable renal tubular epithelial cells (TECs) that has been proposed for the treatment of acute kidney injury (AKI) and multiple organ failure. Perfluorocarbons (PFCs) are oxygen carriers used for organ preservation in transplantation. The aim of this study was to investigate the effect of PFCs on hypoxia- and sepsis-induced TEC injury and on renal CD133+ progenitor differentiation in a microenvironment similar to the RAD., Methods: TECs were seeded in a polysulphone hollow fibre under hypoxia or cultured with plasma from 10 patients with sepsis-associated AKI in the presence or absence of PFCs and were tested for cytotoxicity (XTT assay), apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling assay, caspases, enzyme-linked immunosorbent assay, Fas/Fas Ligand pathway activation), mitochondrial activity, cell polarity [transepithelial electrical resistance (TEER)] and adenosine triphosphate production. The effect of PFCs on proliferation and differentiation of human CD133+ progenitors was also studied., Results: In the presence of PFCs, TECs seeded into the polysulphone hollow fibre showed increased viability and expression of insulin-like growth factor 1, hepatocyte growth factor and macrophage-stimulating protein. Plasma from septic patients induced TEC apoptosis, disruption of oxidative metabolism, alteration of cell polarity and albumin uptake, down-regulation of the tight junction protein ZO-1 and the endocytic receptor megalin on the TEC surface. These detrimental effects were significantly reduced by PFCs. Moreover, PFCs induced CD133+ renal progenitor cell proliferation and differentiation towards an epithelial/tubular-like phenotype., Conclusions: PFCs improved the viability and metabolic function of TECs seeded within a polysulphone hollow fibre and subjected to plasma from septic AKI patients. Additionally, PFCs promoted differentiation towards a tubular/epithelial phenotype of CD133+ renal progenitor cells.

Background: Cardiorenal syndrome type 1 (CRS type 1) is characterized by a rapid worsening of cardiac function leading to acute kidney injury (AKI). Its pathophysiology is complex and not completely understood. In this study, we examined the role of apoptosis and the caspase pathways involved., Material and Methods: We enrolled 40 acute heart failure (AHF) patients, 11 of whom developed AKI characterizing CRS type 1. We exposed the human cell line U937 to plasma from the CRS type 1 and AHF groups and then we evaluated apoptotic activity by annexin-V evaluation, determination of caspase-3, -8 and -9 levels, and BAX, BAD, and FAS gene expression., Results: We observed significant upregulation of apoptosis in monocytes exposed to CRS type 1 plasma compared to AHF, with increased levels of caspase-3 (p < 0.01), caspase-9 (p < 0.01), and caspase-8 (p < 0.03) showing activation of both intrinsic and extrinsic pathways. Furthermore, monocytes exposed to CRS type 1 plasma had increased gene expression of BAX and BAD (intrinsic pathways) (p = 0.010 for both). Furthermore, strong significant correlations between the caspase-9 levels and BAD and BAX gene expression were observed (Spearman ρ = - 0.76, p = 0.011, and ρ = - 0.72, p = 0.011)., Conclusion: CRS type 1 induces dual apoptotic pathway activation in monocytes; the two pathways converged on caspase-3. Many factors may induce activation of both intrinsic and extrinsic apoptotic pathways in CRS type 1 patients, such as upregulation of proinflammatory cytokines and hypoxia/ischemia. Further investigations are necessary to corroborate the present findings, and to better understand the pathophysiological mechanism and consequent therapeutic and prognostic implications for CRS type 1., (© 2018 S. Karger AG, Basel.)

Cardiomyocyte apoptosis plays a pivotal role in the pathogenesis of heart failure. It may be induced by different stimuli, and it seems to be perpetuated by oxidative stress and inflammation. In this scenario, heart failure may trigger various cell-mediated and humoral pathways affecting distant organs, such as kidneys, contributing to higher therapeutic costs, morbidity and mortality. The term Cardiorenal Syndromes describes this condition and represents an important model for exploring the pathophysiology of cardiac and renal dysfunction. In this review, we have analyzed the mechanisms of organ interaction and the role of apoptosis in heart-kidney crosstalk, in particular its role in the pathogenesis of the different types of Cardiorenal Syndromes.

Erythrocytes (RBCs) have a highly specialized and organized membrane structure and undergo programmed cell death, known as eryptosis. Our preliminary data show a significant increase in the eryptosis during peritoneal dialysis (PD)-associated peritonitis. The objectives of the present study were assessment of the incrementation of eryptosis in PD patients with peritonitis, evaluation of the relationship between systemic eryptosis in peritonitis and specific peritonitis biomarkers in PD effluent (PDE), and confirmation of the induction of eryptosis by peritonitis in a vitro setting. We enrolled 22 PD patients with peritonitis and 17 healthy subjects (control group, CTR). For the in vivo study, eryptosis was measured in freshly isolated RBCs. For the in vitro study, healthy RBCs were exposed to the plasma of 22 PD patients with peritonitis and the plasma of the CTR group for 2, 4, and 24 h. Eryptosis was evaluated by flow cytometric analyses in vivo and in vitro. PDE samples were collected for biomarkers analysis.The percentage of eryptotic RBCs was significantly higher in PD patients with peritonitis than in CTR (PD patients with peritonitis: 7.7; IQR 4.3–14.2, versus CTR: 0.8; IQR 0.7–1.3; p < 0.001). We confirmed these in vivo results by in vitro experiments: healthy RBCs incubated with plasma from PD patients with peritonitis demonstrated a significant increase in eryptosis compared to healthy RBCs exposed to plasma from the control group at all times. Furthermore, significant positive correlations were observed between eryptosis level and all analyzed peritoneal biomarkers of peritonitis. We investigated a potential connection between systemic eryptosis and peritoneal biomarkers of peritonitis. Up-regulation of inflammatory markers could explain the increased rate of systemic eryptosis during PD-related peritonitis. [ABSTRACT FROM AUTHOR]

Background: Cell-free plasma DNA (cfDNA) is circulating extracellular DNA arising from cell death mechanisms (apoptosis, necrosis, etc.). It is commonly existent in healthy individuals, but its ranks increase in diverse clinical circumstances, such as kidney disease, sepsis, myocardial infarction, trauma and cancer. In patients with advanced chronic kidney disease, cfDNA is connected to inflammation, and it has been associated with higher mortality. Caspase-3 plays a dominant role in apoptosis, a mechanism of programmed cell death involved in the pathogenesis and progression of chronic kidney disease (CKD). The aim of this pilot study was the evaluation of cfDNA levels and caspase-3 concentrations in patients with chronic kidney disease, in order to investigate the potential role of these molecules, deriving from inflammatory and apoptotic mechanisms, in the progression of renal damage. Methods: We compared cfDNA and caspase-3 levels in 25 CKD patients and in 10 healthy subjects, evaluating their levels based on CKD stage. We also explored correlations between cfDNA and caspase-3 levels in CKD patients and between cfDNA and caspase-3 levels and serum creatinine and urea in this population. Results: We observed that cfDNA and caspase-3 levels were higher in patients with CKD compared to healthy subjects, in particular in patients with advanced renal disease (CKD stage 5). A positive correlation between cfDNA and caspase-3 levels and between cfDNA and caspase-3 and creatinine and urea were also noticed. Conclusions: Patients with chronic kidney disease show higher levels of cfDNA and caspase-3 levels compared to the control group. Based on these preliminary results, we speculated that the worsening of renal damage and the increase in uremic toxin concentration could be associated with higher levels of cfDNA and caspase-3 levels, thus reflecting the potential role of inflammation and apoptosis in the progression of CKD. Future studies should focus on the validation of these promising preliminary results. [ABSTRACT FROM AUTHOR]

Background: Peritonitis and exit site infections are the main complications of patients treated with peritoneal dialysis (PD). Erythrocytes (red blood cells—RBCs) are very sensitive cells, and they are characterized by eryptosis (programmed cell death). The purpose of this research was to assess eryptosis in PD patients with PD-related peritonitis and its connection to inflammatory markers in vivo and in vitro. Material and Methods: In this study, we included 65 PD patients: 34 PD patients without systemic inflammation nor PD-related peritonitis in the previous 3 months, and 31 PD patients with an acute episode of PD-related peritonitis. We measured C-reactive protein (CRP) and cytokine (IL-1β, IL-6, and IL-18) levels as systemic inflammatory markers. Eryptosis was evaluated by flow cytometric analyses in freshly isolated RBCs. The induction of eryptosis due to in vitro exposure to IL-1β, IL-6, and IL-18 was verified. Results: Eryptosis was significantly higher in PD patients with peritonitis (9.6%; IQR 4.2–16.7), compared to the those in the other group (2.7%; IQR 1.6–3.9) (p < 0.0001). Significant positive correlations were noticed between eryptosis and CRP, IL-1β, and IL-6. RBCs, incubated with greater concentrations of all cytokines in vitro, resulted in significantly higher occurrences of eryptosis in comparison with those incubated with lower concentration and with untreated cell (p < 0.05), and for those with extensive exposure (p < 0.05). Conclusion: In conclusion, we investigated a potential relationship between systemic eryptosis and the in vivo and in vitro inflammatory damage of the peritoneal membrane during peritonitis. Thus, the presented results revealed that upregulated inflammatory markers and immune system dysregulation could be the cause of high levels of systemic eryptosis during PD-related peritonitis. [ABSTRACT FROM AUTHOR]

Background: Red blood cells (RBCs) undergo programmed cell death known as eryptosis. Triggers of eryptosis include increased cytosolic Ca(2+) concentration, oxidative stress, osmotic shock, energy depletion and several uremic toxins. Little is known about the pathogenesis of eryptosis in peritoneal dialysis (PD) patients; furthermore, its relevance in worsening clinical conditions in these patients is still not completely defined. Objectives: We investigated eryptosis levels in PD patients and its association with inflammatory and clinical parameters. Material and Methods: A total of 46 PD patients and 17 healthy subjects (CTR) were enrolled. All eryptosis measurements were made in freshly isolated RBCs using the flow cytometer. Results: Eryptosis was significantly higher in PD patients than that in CTR (p < 0.001). Eryptosis levels did not differ significantly between PD patients with and without diabetes, with and without hypertension, and with and without cardiovascular disease. Eryptosis showed no significant differences between patients treated with continuous ambulatory PD/automated PD, with Kt/Vurea value ≤1.7 and >1.7, with a negative or positive history of peritonitis. On the contrary, eryptosis showed significantly lower levels in PD patients with weekly creatinine clearance ≥45 L/week/1.73 m2 (2.8%, 1.7–4.9 vs. 5.6%, 5.0–13.5; p= 0.049). Eryptosis showed significantly lower levels in PD patients with residual diuresis (n = 23) than that in patients without (3.7%, 2.6–5.6 vs. 5%, 3.1–16; p = 0.03). In these 23 patients, significant negative correlations between percentage of eryptosis and residual glomerular filtration rate (rGFR; Spearman's rho = –0.51, p = 0.01) and diuresis volume (Spearman's rho = –0.43, p = 0.05) were found. Conclusions: The present study demonstrated higher eryptosis levels in PD patients compared to corresponding levels in CTR. Furthermore, important PD comorbidity and main PD parameters do not influence eryptosis. Importantly, our data have reported an increase in eryptosis levels with progressive residual diuresis and rGFR loss, probably due to decreased uremic toxins clearance. [ABSTRACT FROM AUTHOR]

Gram-negative sepsis is a major cause of morbidity and mortality in critical ill patients. Recent findings in molecular biology and in signaling pathways have enhanced our understanding of its pathogenesis and opened up opportunities of innovative therapeutic approaches. Endotoxin plays a pivotal role in the pathogenesis of multi-organ dysfunction in the setting of gram-negative sepsis. Indeed, heart and kidney impairments seem to be induced by the release of circulating pro-inflammatory and pro-apoptotic mediators triggered by endotoxin interaction with immune cells. These molecules are responsible for cellular apoptosis, autophagy, cell cycle arrest, and microRNAs activation. Therefore, the early identification of sepsis-associated acute kidney injury and heart dysfunction may improve the patient clinical outcome. In this report, we will consider the role of endotoxin in the pathogenesis of sepsis, its effects on both cardiac and renal functions, and the interactions between these 2 systems in the setting of cardiorenal syndromes (CRS), particularly in CRS type 5. Finally, we will discuss the possible role of extracorporeal therapies in reducing endotoxin levels. [ABSTRACT FROM AUTHOR]

Background/Aims: Cardiorenal Syndrome Type 5 (CRS Type 5) is characterized by concomitant cardiac and renal dysfunction in the setting of different systemic disorders, such as sepsis. In this study, we investigated the possible relationship between endotoxin levels, renal cell death and inflammation in septic patients with CRS Type 5. Methods: We enrolled 11 patients with CRS Type 5. CRS Type 5 was defined according to the current classification system. AKI was defined by Acute Kidney Injury Network (AKIN) criteria. Acute cardiac dysfunction was documented by echocardiography as acute left and/or right ventricular dysfunction leading to decreased ejection fraction. Endotoxin activity was measured by the Endotoxin Activity Assay (EAA). Plasma from CRS Type 5 patients was incubated with renal tubular cells (RTCs) and cell death levels were evaluated. Plasma cytokines levels were measured as well. Results: Accordingly to EAA levels, patients were divided into two groups: 45.4% of patients had low endotoxin activity level (negative EAA), while 54.5% of patients showed high endotoxin activity (positive EAA). RTCs incubated with plasma from EAA positive patients showed significantly higher apoptosis levels and higher caspase-3 activation compared to cells incubated with plasma from EAA negative patients, and a significant positive correlation was observed between EAA levels and RTC apoptosis levels. Furthermore, IL-6 and IFN-γ levels were significantly higher in CRS Type 5 patients with positive EAA. Conclusion: Our data suggest a possible relationship between endotoxin levels and renal cell death in septic patients with CRS Type 5. Furthermore, this study highlights the presence of renal apoptosis, the immune deregulation and the strong inflammation in CRS Type 5 patients, especially in those with high endotoxin activity. [ABSTRACT FROM AUTHOR]

Background: We tested the effect of uremia on red blood cell (RBC) eryptosis, CD14++/CD16 + monocytes and erythrophagocytosis. Design: RBC and monocytes from chronic kidney disease (CKD) stages 3/4 (P-CKD3/4) or hemodialysis (HD) patients and healthy controls (HCs) cells incubated with sera pools from patients with CKD stages 2/3 (S-CKD2/3) or 4/5 (S-CKD4/5) were evaluated to assess eryptosis, monocyte phenotypes and reactive oxygen species (ROS) by cytometer. Erythrophagocytosis was evaluated by subsequent co-incubation of preincubated HC-monocytes and autologous-RBC. Results: HC-eryptosis (1.3 ± 0.9%) was lower than in HD (4.3 ± 0.5%) and HC-RBC incubated with S-CKD4/5 (5.6 ± 1%). CD14++/CD16 + were augmented in P-CKD3/4 (34.6 ± 8%) and HC-monocytes incubated with S-CKD4/5 (26.4 ± 7%) than in HC (5.4 ± 1%). In these cells, ROS was increased (44.5 ± 9%; control 9.6 ± 2%) and inhibited by N-acetylcysteine (25 ± 13%). Erythrophagocytosis was increased in CD14++/CD16 + (60.8 ± 10%) than in CD14++/CD16 - (15.5 ± 2%). Conclusions: Sera pools from CKD patients increase eryptosis and promote a proinflammatory monocyte phenotype. Both processes increased erythrophagocytosis, thereby suggesting a novel pathway for renal anemia. [ABSTRACT FROM AUTHOR]

Background: The identification of highly reliable outcome predictors in severe sepsis is important to define disease severity, predict bedside prognosis and monitor response to treatment. Cell-free plasma DNA (cfDNA) has been recently proposed as a possible prognostic marker of clinical outcome in septic patients. In this study, we investigated the prognostic value of cfDNA in patients with sepsis and its possible correlation with caspase-3, IL-6 and IL-18 levels. Methods: We enrolled 34 patients admitted to the intensive care unit (ICU). Out of these 34, 27 patients were septic and 7 were non-septic. cfDNA was extracted from plasma and quantified by real time PCR. Plasma levels of caspase-3, IL-6 and IL- 18 were measured by ELISA. Results: We observed significantly higher levels of cfDNA in septic patients. No significant differences were found between cfDNA levels in patients with Gram+, Gram- and fungal infections. Out of the 27 septic patients, 12 developed acute kidney injury (AKI) requiring continuous renal replacement therapy (CRRT), and cfDNA levels resulted to be higher in this group. Out of the 27 septic patients, 11 had a negative outcome during the ICU stay. The cfDNA concentrations at admission were higher in non-survivors than in survivors. Caspase-3, IL-6 and IL-18 levels were significantly higher in septic patients when compared to these levels in non-septic patients and correlated with cfDNA levels. Conclusion: cfDNA can be considered a good prognostic marker of clinical outcome in septic patients. Its levels increase in case of AKI complicating sepsis, in particular if CRRT is needed, and are associated with poor outcome. Caspase-3, IL-6 and IL-18 levels are higher in septic patients and correlate to cfDNA concentrations. [ABSTRACT FROM AUTHOR]

Background/Aim: Bisphenol A (BPA) is used in the production of many plastics, which are used to build biomaterials that sometimes are in direct contact with blood. It is believed that the release of BPA into bloodstream may give rise to cytotoxic events for blood components. The aim of the present study was to perform an in vitro investigation of the observable cytotoxic effect of BPA, at increasing concentrations, on the monocyte cell line. Methods: We incubated in vitro monocyte cells (U937) for 24 h in cell line medium samples (RPMI 1640) at different concentrations of BPA. We then generated curves to evaluate viability, necrosis and apoptosis of monocytes against increasing concentrations of BPA. Results: The percentage values of concentrations of BPA corresponding to 50% of the viability and necrosis of the monocytes were 1.39 and 1.48 ng/ml, respectively. Based on our observations, we reported an increasing cytotoxic effect for higher concentrations. The apoptotic effect reached the maximum value at BPA concentration of 1.5 ng/ml; at still higher concentrations, we observed a predominantly necrotic cell death. Conclusion: Viability, necrosis and apoptosis of monocytes are strongly and positively correlated with BPA concentration. A direct contact of such compound with biological components of blood may lead to high levels of cytotoxicity, and require us to evaluate additional factors while judging the bio-incompatibility of BPA. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: The pathophysiology of Cardiorenal Syndrome Type 1 (CRS1) is widely studied, although the mechanisms by which renal tubular epithelial cells (TECs) cease to proliferate and embark upon terminal differentiation, following the initial insult of heart failure (HF), remain a key target. This study seeks to provide insight into the pathophysiological pathways in CRS1; we evaluated in vitro the effects of CRS1 plasma on TECs. Methods: We enrolled 40 acute HF patients and 15 controls (CTR) without HF or acute kidney injury (AKI). Ten out of 40 HF patients exhibited AKI at the time of admission for HF or developed AKI during hospitalization and were classified as CRS1. In vitro, cell viability, DNA fragmentation and caspase-3 levels were investigated in TECs incubated with HF, CRS1, and CTR plasma. We assessed inflammatory cytokines and NGAL expression at the gene and protein levels. Results: We observed a marked pro-apoptotic activity and a significantly increased in vitro level of apoptosis in TECs incubated with plasma from CRS1 patients compared to HF and CTR (p < 0.01). In the CRS1 group, the mRNA expression of IL-6, IL-18 and NGAL resulted significantly higher in TECs incubated with CRS1 plasma compared with those incubated with plasma from HF and CTR (p < 0.01). IL-6, IL-18, NGAL, and RANTES levels were significantly higher in TECs supernatant incubated with CRS1 plasma compared with HF patients and CTR plasma (p < 0.01). Conclusion: In vitro exposure to plasma from CRS1 patients altered the expression profile of TECs characterized by increases in proinflammatory mediators, release of tubular damage markers, and apoptosis. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: The uremic syndrome is characterized by a deterioration of kidney function due to the accumulation of uremic toxins. Currently, 100 different uremic toxins have been identified. Uremic toxins are particularly difficult to remove by conventional dialysis treatments and are the major causes of mortality in patients with chronic kidney disease (CKD). p-Cresol is a well-known uremic toxin which accumulates in uremic serum. Our aim was to evaluate the in vitro effect of p-cresol on apoptosis and necrosis in renal tubular cells (RTCs) to better understand the pathophysiological effect of this toxin on the kidney. Methods: We studied apoptosis and necrosis in RTCs, which were incubated for 24 h with increasing concentrations of p-cresol. A DNA ladder was noted in treated cells as a qualitative marker of the apoptotic process. Furthermore, we performed quantitative analysis of cell viability using a flow cytometer and assessed caspase-3 activity. Results: Incubation with p-cresol for 24 h resulted in a significant reduction in RTC viability. DNA isolated from RTCs incubated with increasing p-cresol concentrations for 24 h showed a 'ladder' pattern of apoptosis at p-cresol concentrations of 10, 5 and 2.5 mg/l. However, we did not observe any significant changes in apoptosis levels detected by annexin V and caspase-3 compared with untreated cells. Cytofluorimetric analysis of necrosis highlighted significantly higher cell death rates in RTCs incubated with the higher p-cresol concentrations (range 40-10 mg/l) compared with other concentrations (5-2.5 mg/l) and untreated cells (p < 0.05). Necrosis induction was stronger at higher p-cresol concentrations. Conclusion: It is necessary to develop new therapeutic and dialytic strategies to manage p-cresol concentrations in CKD. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]