Grazia Maria Virzì

Introduction: The present study aimed to monitor peritoneal neutrophil gelatinase-associated lipocalin (pNGAL) during peritonitis episodes and to enhance its diagnostic value by evaluating pNGAL at scheduled times in parallel with white blood cell (WBC) count. In addition, we investigated possible correlations between pNGAL and the etiology of peritonitis, evaluating it as a possible marker of the clinical outcome. Methods: Twenty-two patients with peritoneal dialysis (PD)-related peritonitis were enrolled. Peritonitis was divided into Gram-positive, Gram-negative, polymicrobial, and sterile. WBC count and neutrophil gelatinase-associated lipocalin (NGAL) in PD effluent were measured at different times (days 0, 1, 5, 10, 15, and/or 20 and 10 days after antibiotic therapy discontinuation). NGAL was measured by standard quantitative laboratory-based immunoassay and by colorimetric NGAL dipstick (NGALds) (dipstick test). Results: We found strong correlations between peritoneal WBC, laboratory-based NGAL, and NGALds values, both overall and separated at each time point. On day 1, we observed no significant difference in WBC, both NGALds (p = 0.3, 0.9, and 0.2) between Gram-positive, Gram-negative, polymicrobial, and sterile peritonitis. No significant difference has been found between de novo versus relapsing peritonitis for all markers (p > 0.05). We observed a parallel decrease of WBC and both NGAL in patients with favorable outcomes. WBC count and both pNGAL resulted higher in patients with negative outcomes (defined as relapsing peritonitis, peritonitis-associated catheter removal, peritonitis-associated hemodialysis transfer, peritonitis-associated death) at day 10 (p = 0.04, p = 0.03, and p = 0.05, respectively) and day 15 (p = 0.01, p = 0.04, and tendency for p = 0.005). There was a tendency toward higher levels of WBC and NGAL in patients with a negative outcome at day 5. No significant difference in all parameters was proven at day 1 (p = 0.3, p = 0.9, p = 0.2) between groups. Conclusion: This study confirms pNGAL as a valid and reliable biomarker for the diagnosis of PD-peritonitis and its monitoring. Its trend is parallel to WBC count during peritonitis episodes, in particular, patients with unfavorable outcomes.

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.

INTRODUCTION AND AIMS: Bacterial peritonitis is a frequent complication during treatment of end-stage kidney disease by Peritoneal Dialysis (PD); severe or repeated episodes of peritonitis cause long-term complications that potentially lead to peritoneal membrane failure. Lipopolysaccharide (LPS) or endotoxin is an abundant glycolipid present on the outer membrane of Gram-negative bacteria and it may contribute to the progression of a local infection to sepsis. Previous studies demonstrated that LPS level is related to the degree of systemic inflammation and atherosclerosis in PD patients. The aim of this study was to evaluate LPS levels in PD patients, in particular, we investigated LPS levels in PD with Gram positive/ negative peritonitis.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disease with variable rate of progression. It is associated with inter- and intra-familial variability. Neutrophil gelatinase-associated lipocalin (NGAL) has been implicated in pathological conditions and it is proposed as a biomarker for CKD progression. Our aim was to evaluate whether NGAL could be a good marker for progression of ADPKD, as we hypothesized. ADPKD patients with confirmed mutations (PKD1 n=33; PKD2 n=17) were enrolled and followed in a prospective study. Creatinine (sCr) and NGAL values were measured at baseline and on follow-up. Plasma NGAL was measured by Triage point of care test. CKD progression was defined as 15% decrease in eGFR from baseline to follow-up. Patients were divided into 2 groups based on median baseline NGAL and compared by the Kaplan-Meier curve. We enrolled 50 ADPKD pts (60%M age 41 yrs); mean sCr 1.30.7 mg/dl and median eGFR was 62 mL/min/1.73 m2. NGAL values are inversely correlated with baseline eGFR (r=-0.64, p<0.001). There was weak correlation between baseline NGAL and subsequent change in eGFR (r=0.28, p=0.05). 9/50 of patients had NGAL below limits of detection (60 pg/ml); median NGAL level was 79 pg/ml. At follow-up, 12 patients (24%) had progression as defined. No statistically significant relationship between higher NGAL and ADPKD progression was observed. We did not observe a relationship between NGAL and CKD progression in ADPKD patients; however 18% of patients had undetectable plasma NGAL levels. This raises doubt about the utility of the current NGAL assay as a biomarker for CKD progression in this population.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of chronic kidney disease (CKD) in adults. It is associated with both inter- and intra- familial variability, which can be explained by genetic heterogeneity, modifier genes and a genetic background. NGAL, a 25-kDa protein released from tubular cells after harmful stimuli, is a new biomarker. Recent studies suggest a possible role for NGAL in CKD. The aim of this study was to investigate NGAL in the context of genotype-phenotype correlation in ADPKD. We measured plasma NGAL, creatinine (Cr), and urea in 18 ADPKD patients on dialysis (PKD1_TRS), 29 of their relatives with CKD stage II-III (PKD1_AMB), 33 wild-type relatives (PKD1_WT), 12 subjects in CKD stage II-III with confirmed mutation in PKD2 and 30 healthy controls (CTR). Cr, urea and CysC were measured according to standard methods. Plasma NGAL was measured using point-of-care test. Estimated glomerular filtration rate (eGFR) was calculated with a 4-variable standardized-MDRD formula. Plasma NGAL, CysC, Cr and urea levels in PKD1_TRS were significantly higher than CTR (all with p0.05). NGAL levels were not significantly different in ADPKD patients in CKD stage II-III with mutation in PKD1 or PKD2 (86 pg/mL, IQR 60-109 vs 82 pg/mL,IQR 63-169). NGAL correlated well (all with p<0.001) with CysC (r=0.95), Cr (r=0.89), urea (r=0.76) and inversely with eGFR (r=-0.81). A strong correlation between CysC and residual renal function was observed (CysC/eGFR: r=-0.86; CysC/Cr: r=0.7; CysC/Urea: r=0.79, all with p<0.001). This is the first study to evaluate plasma NGAL in the context of genotype-phenotype correlation. In ADPKD, NGAL levels were higher in patients already on Renal Replacement Therapy (RRT) compared to their affected relatives not on RRT. WT relatives had normal NGAL and CysC levels: these could be considered a better CTR group because they share the same genetic background with ADPKD subjects. No differences were found in NGAL levels between PKD1 and PKD2 patients in CKD stage II-III. Furthermore, the results indicate that NGAL correlates closely with renal function and CysC levels.

Background: Autosomal dominant polycystic kidney disease (ADPKD) is an inherited condition caused by PKD1 and PKD2 mutations. Complete analysis of both genes is typically required in each patient. In this study, we explored the utility of High‐Resolution Melt (HRM) as a tool for mutation analysis of the PKD2 gene in ADPKD families.Methods: HRM is a mismatch‐detection method based on the difference of fluorescence absorbance behavior during the melting of the DNA double strand to denatured single strands in a mutant sample as compared to a reference control. Our families were previously screened by linkage analysis. Subsequently, HRM was used to characterize PKD2‐linked families. Amplicons that produced an overlapping profile sample versus wild‐type control were not further evaluated, while those amplicons with profile deviated from the control were consequently sequenced.Results: We analyzed 16 PKD2‐linked families by HRM analysis. We observed ten different variations: six single‐nucleotide polymorphisms and four mutations. The mutations detected by HRM and confirmed by sequencing were as follows: 1158T>A, 2159delA, 2224C>T, and 2533C>T. In particular, the same haplotype block and nonsense mutation 2533C>T was found in 8 of 16 families, so we suggested the presence of a founder effect in our province.Conclusions: We have developed a strategy for rapid mutation analysis of the PKD2 gene in ADPKD families, which utilizes an HRM‐based prescreening followed by direct sequencing of amplicons with abnormal profiles. This is a simple and good technique for PKD2 genotyping and may significantly reduce the time and cost for diagnosis in ADPKD.

Organ failure in the heart or kidney can initiate various complex metabolic, cell-mediated and humoral pathways affecting distant organs, contributing to the high therapeutic costs and significantly higher morbidity and mortality. The universal outreach of cells in an injured state has myriad consequences to distant organ cells and their milieu. Heart performance and kidney function are closely interconnected and communication between these organs occurs through a variety of bidirectional pathways. The term cardiorenal syndrome (CRS) is often used to describe this condition and represents an important model for exploring the pathophysiology of cardiac and renal dysfunction. Clinical evidence suggests that tissue injury in both acute kidney injury and heart failure has immune-mediated inflammatory consequences that can initiate remote organ dysfunction. Acute cardiorenal syndrome (CRS type 1) and acute renocardiac syndrome (CRS type 3) are particularly relevant in high-acuity medical units. This review briefly summarizes relevant research and focuses on the role of signaling in heart–kidney crosstalk in the critical care setting.

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.

Cardiorenal syndrome (CRS) type 4, or chronic renocardiac syndrome, has been defined as ‘chronic abnormalities in renal function leading to cardiac disease' and recognizes the extreme burden of cardiovascular (CV) disease (CVD) risk in patients with chronic kidney disease (CKD). CKD is common and increasingly recognized as a risk factor for CVD. Although during the past 10 years CV morbidity and mortality have decreased markedly in the general population, their rates still remain high among CKD patients. For this reason, CKD patients should be evaluated thoroughly for CV risk factors which require an aggressive management, given the significant implications of CRS type 4 at both individual and societal level. We will review the management of the most important conventional and nonconventional CVD risk factors related to CKD.

There is a bidirectional and complex relationship between the heart and kidneys. This interaction is physical, chemical as well as biological and is also reflected in a strong connection between renal and cardiovascular diseases. Cardiorenal syndrome type 4 (CRS type 4) is characterized by primary chronic kidney disease (CKD) leading to an impairment of cardiac function, with ventricular hypertrophy, diastolic dysfunction, and/or increased risk of adverse cardiovascular events. The incidence of CKD is increasing, and CRS type 4 is becoming a major public health problem associated with a high morbidity and mortality. In this study, we briefly review the epidemiology and pathophysiology of CRS type 4, the role of biomarkers in its early identification, and its management.