The effects of maternal cigarette smoking on cadmium and lead levels, miRNA expression and biochemical parameters across the feto-placental unit.

Several miRNAs have been previously identified to be associated with cigarette smoke and/or the toxic metals cadmium (Cd) and lead (Pb). The aim of this study was to investigate the associations of maternal cigarette smoking with cadmium (Cd) and lead (Pb) levels, candidate miRNA expression and biochemical parameters across the feto-placental unit. miRNAs were isolated according to protocols provided by manufacturer from 72 healthy postpartum women using Qiagens' kits based on phenol/guanidine samples lysis and silica-membrane purification of total RNA. Candidate miRNAs (miR-1537, miR-190b, miR-16, miR-21, and miR-146a) were quantified by real-time PCR. Biochemical parameters were analyzed in plasma samples by standardized and harmonized enzymatic methods using appropriate calibrators, while CRP was determined by immunoturbidimetric method. Concentration of Cd and Pb in whole blood and placenta samples were measured by inductively coupled plasma mass spectroscopy. Cd levels in smokers were higher in all of the analyzed compartments of the feto-placental unit, Pb in maternal blood and placenta than non-smokers. Smokers also had a higher expression of miR-16 in maternal and miR-146a in cord plasma, and lower expression of miR-21 in the placenta in comparison to non-smokers. Urate concentrations in the maternal plasma of smokers were lower than this value in non-smokers. The study has demonstrated that maternal smoking was associated with toxic metals (Cd and Pb) levels, urate concentration and alteration of miRNA expression. Given that the effects of maternal smoking on miRNA expression are inadequate, all compartments of the feto-placental unit should be analyzed to obtain a complete picture. This paper is the first to report on the results of expression of cellular and circulating miRNAs simultaneously in maternal and fetal compartments and in the placenta.

Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs.

INTRODUCTION: The assessment of circulating miRNAs is challenging and still limited due to their low concentrations, small size and lack of reference values in human biological samples. Pre-amplification of complementary DNAs may facilitate reliable miRNA quantification. The aim of our study was to evaluate the efficacy of pre-amplification as a step to increase the sensitivity of qPCR analysis for five candidate circulating miRNAs presumably related to toxic metals and cigarette smoke exposure: miR-1537, miR-190b, miR-16, miR-21, and miR-146a. MATERIALS AND METHODS: Candidate miRNAs expression was analysed in plasma samples of 19 mother-newborn pairs. For isolation, transcription, pre-amplification and qPCR quantification kits and protocols by Qiagen (Hilden, Germany) were used. Paired t-test or Wilcoxon rank test were used to compare miRNAs expression levels with and without a pre-amplification step prior to qPCR, separately in maternal and cord plasma. Intraclass correlation (ICC) was calculated as an agreement measure between procedures for each miRNA. RESULTS: Pre-amplification facilitated the detection of all assayed miRNAs with an overall cycle threshold (CT) improvement of 6.6 ± 0.89 (P < 0.05). Excellent ICCs (> 0.90) were found between data for preamplified and not preamplified miR-16, miR-21 and miR-146a. However, these correlations for low expressed miR-190b were moderate (0.79 in maternal; 0.61 in cord plasma) and poor for miR-1537 (0.49 in maternal; no correlation in cord plasma). CONCLUSION: Pre-amplification is a useful, necessary step in the analysis of miR-1537 and miR-190b as a reliable procedure facilitating extracellular miRNA expression detection in human plasma by real-time PCR quantification.

Cigarette Smoking during Pregnancy: Effects on Antioxidant Enzymes, Metallothionein and Trace Elements in Mother-Newborn Pairs.

The effect of maternal smoking as a source of exposure to toxic metals Cd and Pb on superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity, metallothionein (MT), Cd, Pb, Cu, Fe, Mn, Se and Zn concentrations were assessed in maternal and umbilical cord blood and placenta in 74 healthy mother-newborn pairs after term delivery. Sparse discriminant analysis (SDA) was used to identify elements with the strongest impact on the SOD, GPx and MT in the measured compartments, which was then quantified by multiple regression analysis. SOD activity was lower in maternal and cord plasma, and higher in the placenta of smokers compared to non-smokers, whereas GPx activity and MT concentration did not differ between the groups. Although active smoking during pregnancy contributed to higher maternal Cd and Pb concentrations, its contribution to the variability of SOD, GPx or MT after control for other elements identified by SDA was not significant. However, an impaired balance in the antioxidant defence observed in the conditions of relatively low-to-moderate exposure levels to Cd and Pb could contribute to an increased susceptibility of offspring to oxidative stress and risk of disease development later in life. Further study on a larger number of subjects will help to better understand complex interactions between exposure to toxic elements and oxidative stress related to maternal cigarette smoking.

[Analysis of patients with acute renal failure hospitalized at department of nephrology and dialysis, Rijeka University Hospital, during the five-year period]. / Analiza akutnog bubreznog zatajenja tijekom petogodisnjeg razdoblja u zavodu za nefrologiju i dijalizu klinickog bolnickog centra Rijeka.

INTRODUCTION: Acute renal failure (ARF) is a serious complication that occurs in 5%-18% of hospitalized patients and in up to 30% of patients admitted to Intensive Care Unit. The hospital mortality rate of patients with ARF is between 28% and 90%. The incidence of ARF is proportional to patient age. Therefore, despite all improvements in modern medicine, the annual incidence of ARF has not changed over the two past decades. The aim of our study was to analyze the incidence and causes of ARF in our Center during the five-year period, to analyze the characteristics of patients and their comorbid conditions, variations in laboratory parameters during hospitalization, and therapy administered. Also, we analyzed the outcome and length of hospitalization. SUBJECTS AND METHODS: During the five-year period (from January 2008 till December 2012), we analyzed 316 patients treated for ARF at Department of Nephrology and Dialysis, Rijeka University Hospital Center. Data were obtained by searching medical records. ARF was defined according to the KDIGO recommendations: increase in serum creatinine (sCR) > 26 µmol/L within 48 h, or increase in sCR by 1.5 times compared to the reference values, which is known or assumed to have appeared within a week of hospitalization, or diuresis < 0.5 mL/kg/h for ≥ 6 hours. RESULTS: Out of 316 ARF patients analyzed, 57 were hospitalized at our Department in 2008 (50.9% of men and 49.1% of women), 56 in 2009 (39.3% of men and 60.7% of women), 66 in 2010 (55.3% of men and 44.7% of women) and 76 in 2011 (55.3% of men and 44.7% of women). In 2012, we analyzed 61 ARF patients (42.6% of men and 53.4% of women). There were no statistically significant age and gender differences, although we noticed an increasing tendency in the number of elderly patients hospitalized for ARF. Furthermore, analyzing the frequency of patient arrival from home, nursing home or transfer from other departments we recorded an increase in the arrival of patients from nursing homes during the study period. Analysis of the proportion of patients hospitalized for ARF in our Department in relation to the total number of hospitalized patients revealed that ARF was the cause of hospitalization in 8.2%-9.9% of all patients. There was no significant change in the number of patients hospitalized for ARF during the period observed. Analyzing the frequency of hospitalization due to ARF by months, we noticed that the largest number of patients were hospitalized during summer months (from June to September). The most common form of ARF was prerenal (56.1%-67.9%). The largest number of patients were treated by parenteral rehydration and antibiotics (52.6%-71.4%). Renal replacement therapy was performed in 12.5%- 21.1% of all patients. The mortality rate throughout the period of observation ranged from 21.2%-30.4%. Furthermore, complete recovery of renal function was achieved in 30.5%-40.4% of all patients. The mean length of hospital stay ranged from 11.8 to 15.1 days. CONCLUSION: Acute renal failure is a significant cause of hospitalization, especially in elderly patients. Therefore, early identification along with appropriate and early treatment of patients with ARF is needed to improve survival and recovery of renal function in these patients.