V. Fanos

Overlapping between CYP3A4 and CYP3A7 expression in the fetal human liver during development

Abstract Objective: The cytochrome P450 (CYP450) superfamily is implicated in important life processes, including metabolism of many molecules. CYP3A account for the largest portion of CYP450 proteins in human, including CYP3A4, CYP3A5 and CYP3A7. The purpose of this study was to investigate the immunohistochemical expression of CYP3A4 and CYP3A7 in human liver at different post-conceptional (PC) ages. Methods: Human liver samples from 30 fetuses and newborns were, clustered according with the PC age, routinely processed for immunohistochemical analysis of CYP3A4 and CYP3A7. Results: CYP3A4 was positive in all but two cases, CYP3A7 was positive in all but one case, which was negative also for CYP3A4. Conclusions: Our data on immunohistochemical detection of CYP3A4 and CYP3A7 during development show that CYP3A4 expression is not restricted to the post-natal age, being the immunostaining for both CYP3A4 and CYP3A7 identical after 25 weeks of PC age, thus the relationship between these CYP450 isoforms should be considered much more complex than previous thought. A high interindividual variability was observed among subjects at all gestational age. The variable CYP3A expression suggests the existence of a marked interindividual variability in drug metabolism during the intrauterine life and in perinatal period.

Reduced brachial flow-mediated vasodilation in young adult ex extremely low birth weight preterm: a condition predictive of increased cardiovascular risk?

Background. Sporadic data present in literature report how preterm birth and low birth weight constitute the risk factors for the development of cardiovascular diseases in later life. Aim. To assess the presence of potential alterations to endothelial function in young adults born preterm at extremely low birth weight (<1000 g; ex ELBW). Method. Thirty-two ex-ELBW subjects (10 males [M] and 22 females [F], aged 17–28 years, mean [±DS] 20.1 ± 2.5 years) were compared with 32 healthy, age-matched subjects born at term (C, 9 M and 23 F). Exclusion criteria: 1) pathological conditions known to affect endothelial function; 2) administration of drugs known to affect endothelial function. Endothelial function was assessed by non-invasive finger plethysmography, previously validated by the US Food and Drug Administration (Endopath; Itamar Medical Ltd., Cesarea, Israel). Results. Endothelial function was significantly reduced in ex-ELBW subjects compared to C (1.94 ± 0.37 vs 2.68 ± 0.41, p < 0.0001). Moreover, this function correlated significantly with gestational age (r = 0.56, p < 0.0009) and birth weight (r = 0.63, p < 0.0001). Conclusions. The results obtained reveal a significant decrease in endothelial function of ex-ELBW subjects compared to controls, underlining a probable correlation with preterm birth and low birth weight. Taken together, these results suggest that an ELBW may underlie the onset of early circulatory dysfunction predictive of increased cardiovascular risk.

Perinatal nutrient restriction reduces nephron endowment increasing renal morbidity in adulthood: A review

Perinatal malnutrition has been included among the causes of renal disease in adulthood. Here, we consider the relationships between early supply of specific nutrients (such as protein, fat, vitamins and electrolytes) and renal endowment. Prenatal and postnatal nutrition mismatch is also discussed. In addition, this article presents the role of nutrition of both mothers and pre-term infants on nephron endowment, with final practical considerations.

Cytochrome P450 genetic polymorphism in neonatal drug metabolism: role and practical consequences towards a new drug culture in neonatology.

The cytochrome P450 superfamily (CYP450) in humans is formed by 57 functional monooxygenases critical for the metabolism of numerous endogenous and exogenous compounds. The superfamily is organized into 18 families and 44 subfamilies. CYP nomenclature is based on the identity of amino acids. The most important functions of the CYP450 are related to metabolism of endogenous compounds, detoxification of exogenous xenobiotics and decomposition of the vast majority of currently used drugs. The expression of CYP450 enzymes in the human body is characterized by a marked substrate and tissue specificity, the most important being localized in the liver, but also present in kidney, lung, brain, breast, prostate and in the small intestine. The human cytochrome P450 3A gene family (CYP3A) accounts for the largest portion of CYP450 proteins in human liver and includes 4 genes: CYP3A4, CYP3A5, CYP3A7, CYP3A43. Multiple and complex genetic variations, marked interindividual, interethnic and gender variability have been reported regarding CYP3A isoform expression and activity. Multiple factors may affect CYP3A expression and activity, such as inducers like rifampicin, phenobarbital, 3-methylcholantrene, beta-naphtoflavone, and dexamethasone. The maturation of organ systems, paralleled by ontogeny of drug-metabolizing enzymes during fetal life and in the first months of postnatal life, surely exerts profound effects on drug disposition, probably being the predominant factor accounting for age-associated changes in drug clearance. In fact, drug dosage in the perinatal period represents a continuous challenge for neonatologists. The purpose of this article is to provide a brief review of the pharmacokinetic differences between neonates and adults, showing the peculiarities of liver CYP450-related drug metabolism in the perinatal period and at birth, and to report the toxic mechanisms of liver injury in neonates, due to the most frequently utilized drugs in NICU centers.

The role of immunohistochemistry in the study of the newborn kidney.

The identification of the different cell types involved in human nephrogenesis, when solely based on morphology, may lead to errors in its interpretation, given the complexity of the histological picture of the fetal and of the newborn kidney. In this study, the most recent works utilizing immunhistochemistry for the identification of the multiple cell types involved in human nephrogenesis are reviewed. The role of WT1, MUC1, Thymosin beta 10, Thymosin beta 4, CD10 and CD44 in the different phases of glomerulogenesis and of tubulogenesis is here described, with particular emphasis on their expression in the early phases of nephrogenesis. On the basis of our data, immunohistochemistry appears to be a useful tool in the study of human nephrogenesis, giving new data on the different steps of the differentiation of metanephric mesenchyme towards the multiple cell types characterizing the mature human kidney. Moreover, allowing a better knowledge of the protein products involved in the generation of new nephrons, immunohistochemistry could open new perspectives in the field of renal regenerating medicine, evidencing the factors able to prolong nephrogenesis after birth, helping us to reach our goal: allowing newborn kidneys to restore their nephron endowment, escaping susceptibility to hypertension and renal disease in adulthood.

CD10 in the developing human kidney: immunoreactivity and possible role in renal embryogenesis

CD10 was first identified in tumor cells of acute lymphoblastic leukemia. Most studies on CD10 expression have dealt with tumor pathology. Since no data are available for specific role in the fetal kidney, this study aimed at investigating CD10 expression during the different phases of renal embryogenesis. To this end, the expression of CD10 was evaluated in the kidney of two human fetus and in three newborns. In both fetuses, immunostaining for CD10 was compartmentalized and mainly concentrated in the mid-deep cortex. Reactivity for CD10 was stronger in the glomerular epithelium, in proximal tubules and in metanephric mesenchymal cells. At 25 weeks of gestation, CD10 was also detected in the subcapsular regions, including some pretubular aggregates of cap mesenchymal cells and renal vesicles. At 34 weeks of gestation, we observed an increased immunoreactivity for CD10 in visceral and parietal glomerular epithelium. At 39 weeks of gestation, CD10 was also expressed in the collecting tubules and in the Henle loops. Our data show a strong expression of CD10 in all stage of human kidney development, characterized by dynamic changes and support the hypothesis that CD10 plays a relevant role in renal embryogenesis.

The role of novel biomarkers in early diagnosis and prognosis of acute kidney injury in newborns.

Acute kidney injury (AKI) refers to the rapid loss of renal function. In clinical practice, AKI is common among hospitalized patients of all age groups including neonates and remains an important cause of morbidity and mortality due to its late diagnosis and therefore delayed therapeutic intervention. Although the precise incidence of AKI in newborn is unknown, several studies have reported that 8 to 24% of all critically ill newborns in neonatal intensive care units may develop the condition. We aim at reviewing the existing literature on novel serum and urinary biomarkers and discuss their role in the early diagnosis and prognosis of AKI in newborns. Specifically, this review will focus on cystatin C (CysC), neutrophil gelatinase-associated lipocalin (NGAL) and interleukin-18 (IL-18) in serum and on CysC, NGAL, kidney injury molecule-1, and IL-18 in urine.

Each niche has an actor: multiple stem cell niches in the preterm kidney

The preterm kidney cannot be simply considered as a kidney small in size: as compared to the adult kidney, the developing organ of the preterm infant is characterized by marked differences regarding the architecture and cell components. At macroscopy, fine linear demarcations indenting the renal surface characterize the fetal and preterm kidney. At microscopy, multiple major architectural changes differentiate the developing kidney from the adult one: a large capsule with a high cellularity; the branching ureteric bud, extending from the hilum towards the renal capsule; striking morphological differences among superficial (just born) and deep (more mature) glomeruli; persistence of remnants of the metanephric mesenchyme in the hylum; incomplete differentiation of developing proximal and distal tubules. At cellular level, kidneys of preterm infants are characterized by huge amounts of stem/precursor cells showing different degrees of differentiation, admixed with mature cell types. The most striking difference between the preterm and adult kidney is represented by the abundance of stem/progenitor cells in the former. Multiple stem cell niches may be identified in the preterm kidney, including the capsule, the sub-capsular nephrogenic zone, the cap mesenchyme embracing the ureteric bud tips, the cortical and medullary interstitium, and the hilar zone in proximity of the ureteric origin. The sub-capsular area represents the major stem cell niche in the prenatal kidney. It has been defined “blue strip”, due to the scarcity of cytoplasm of the undifferentiated stem/progenitors, which appear as small cells arranged in a solid pattern. All these data taken together, the morphological approach to the analysis of the preterm kidney appears completely different from that typically utilized in kidney biopsies from adult subjects. Such a different structure should be taken into account when evaluating renal function in a preterm infant in clinical practice. Moreover, a better knowledge of molecular biology of the blue strip stem/progenitor cells could be at the basis of a new “endogenous” regenerative medicine, finalized to maintain and protect the nephrogenic potential of preterm infants till the 36th week of post-conceptional age, allowing them to escape oligonephronia and chronic kidney disease later in life.

Advanced intrauterine growth restriction is associated with reduced excretion of asymmetric dimethylarginine

BACKGROUND High blood levels of asymmetric dimethylarginine (ADMA) are associated with future development of adverse cardiovascular events. The ADMA/symmetric dimethylarginine (SDMA) ratio is a marker of ADMA catabolism, with a high ADMA/SDMA ratio being suggestive of reduced ADMA excretion. AIMS This study aimed a) to verify the presence of a statistically significant difference between ADMA/SDMA ratio levels in a group of young adult subjects who were born preterm with an extremely low birth weight (ex-ELBW) and a group of healthy adults born at term and b) to seek correlations between ADMA/SDMA ratio levels in ex-ELBW and anthropometric and clinical parameters (gender, chronological age, gestational age, birth weight, and length of stay in the Neonatal Intensive Care Unit). SUBJECTS, STUDY DESIGN, OUTCOME MEASURES Thirty-seven ex-ELBW subjects (11 males [M] and 26 females [F], aged 17-28 years, mean age: 22.2 ± 1.8 years) were compared with 37 controls (11 M and 26 F). ADMA/SDMA ratio levels were assessed for each patient included in the study. RESULTS ADMA/SDMA ratio in ex-ELBW subjects was higher compared to controls (1.42 ± 0.31 vs 0.95 ± 0.14, p<0.002) and inversely correlated with birth weight (r=-0.68, p<0.0001) and gestational age (r=-0.54, p<0.0005). CONCLUSIONS ADMA catabolism is significantly decreased in ex-ELBW subjects compared to controls, underlining a probable correlation with restriction of intrauterine growth. These results suggest the onset of early circulatory dysfunction predictive of increased cardiovascular risk in ex-ELBW.

WT1 expression in the human fetus during development

Wilms’ Tumor 1 (WT1) is a transcription factor involved in the development of the urogenital system. The purpose of this study was to analyze the immunoreactivity for WT1 protein in different tissues and organs in human fetuses in early phases of gestation. To this end, samples from multiple organs were obtained from 4 human fetuses, ranging from 7 up to 12 weeks of gestation. Each sample was formalin-fixed, paraffin embedded and immunostained for WT1. Our data show that WT1 is involved in development of multiple human organs in a more vast series of cells types than previously reported. Immunostaining for WT1 was characterized by a predominant cytoplasmic reactivity in the vast majority of cell types. Mesenchimal progenitors in the fetal lung, ductal plate progenitors in fetal liver, cap mesenchimal cells in the developing kidney, fetal zone cells in adrenal glands, atrial and ventricular cardiomyocytes in the fetal heart, radial glial cells in the fetal cerebral cortex and skeletal muscle cell precursors showed the highest levels of WT1 immunoreactivity. Future studies will be needed to detect differences in the expression of WT1 in various organs at different gestational ages, in order to better evaluate the role of WT1 in cell proliferation and differentiation during intrauterine human development.