Annalisa Locci

Marked interindividual variability in renal maturation of preterm infants: lessons from autopsy

The kidney of low birthweight preterm infants is characterized by a reduced number of mature nephrons at birth. The aim of the present study was to determine whether, in preterms, active glomerulogenesis occurs in the postnatal period and whether it may compensate the reduced number of nephrons developed during the intrauterine life. Kidney samples were obtained at autopsy from 8 human fetuses, 12 premature infants, and 3 term newborns. Glomerulogenesis, as measured by radial glomerular count (RGC), was markedly decreased in all preterm infants as compared with term newborns. A marked interindividual variability was detected in the level of glomerulogenesis, which, in the vast majority of cases, did neither correlate with the gestational age at birth nor with birthweight. Active glomerulogenesis, as demonstrated by the presence of S-shaped bodies in the subcapsular region, was present in all preterm infants in the perinatal period, but it ceased in a preterm surviving for 3 months. Our data show that active glomerulogenesis continues even after birth for a short period, although it is not able to compensate a marked oligonephronia at birth. As a consequence, the incomplete nephrogenesis typical of all extremely low birthweight preterm infants possibly results in a persistent oligonephronia which should likelihood represent a major risk factors of progressive renal disease in adulthood.

Expression of WT1 during normal human kidney development

Wilms Tumor 1 (WT1) is a zinc finger protein, expressed by human podocytes in the adult kidney, which plays a relevant role in different phases of nephrogenesis in experimental animals. Since no data are available for specific role in the human fetal kidney, this study aimed at investigating the expression of WT1 during the different phases of nephrogenesis. To this end, the expression of WT1 was evaluated in the kidneys, from four human fetuses and two newborns. WT1 immunoreactivity was detected in all the examined kidneys, but not in the kidneys of the newborn at term. Immunostaining for WT1 was observed in podocytes of the glomeruli and in the subcapsular regions, in areas of active glomerulogenesis. The extent and the intensity of immunoreactivity for WT1 changed from one case to the next according to the different gestational age. This study confirms in human kidney the relevant role played by WT1 during nephrogenesis. Its expression pattern suggests a main role in the regulation of the process of Mesenchimal-Epithelial-Transition and in the development and maturation of podocytes. Further studies are needed to verify the correlation between the expression pattern of WT1 and that of other genes products involved in nephrogenesis, in order to better understand their relationship at protein level.

Expression pattern of thymosin beta 4 in the adult human liver

Thymosin beta-4 (Tβ4) is a member of beta-thymosins, a family of small peptides involved in polymerization of G-actin, and in many critical biological processes including apoptosis, cell migration, angiogenesis, and fibrosis. Previous studies in the newborn liver did not reveal any significant reactivity for Tβ4 during the intrauterine life. The aim of the present study was to investigate by immunohistochemistry Tβ4 expression in the adult normal liver. Thirty-five human liver samples, including 11 needle liver biopsies and 24 liver specimens obtained at autopsy, in which no pathological change was detected at the histological examination, were immunostained utilizing an anti-Tβ4 commercial antibody. Tβ4 was detected in the hepatocytes of all adult normal livers examined. A zonation of Tβ4 expression was evident in the vast majority of cases. Immunostaining was preferentially detected in zone 3, while a minor degree of reactivity was detected in periportal hepatocytes (zone 1). At higher power, Tβ4-reactive granules appeared mainly localized at the biliary pole of hepatocytes. In cases with a strong immunostaining, even perinuclear areas and the sinusoidal pole of hepatocytes appeared interested by immunoreactivity for Tβ4. The current work first evidences a strong diffuse expression of Tβ4 in the adult human liver, and adds hepatocytes to the list of human cells able to synthesize large amounts of Tβ4 in adulthood. Moreover, Tβ4 should be added to the liver proteins characterized by a zonate expression pattern, in a descending gradient from the terminal vein to the periportal areas of the liver acinus. Identifying the intimate role played by this peptide intracellularly and extracellularly, in physiology and in different liver diseases, is a major challenge for future research focusing on Tβ4.

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.

MUC1 in mesenchymal-to-epithelial transition during human nephrogenesis: changing the fate of renal progenitor/stem cells?

Background: The development of the human kidney is a complex process requiring interactions between epithelial and mesenchymal cells. The condensed cap mesenchyme is hypothesized to generate a population of stem/progenitor cells that undergo mesenchymal-epithelial transition (MET) originating nephrons. Few immunohistochemical markers are available for detecting cap mesenchymal cells in the early phases of MET. Methods: The expression of MUC1 was evaluated in the kidneys, of 4 human foetuses and 2 newborns. Results: MUC1 immunoreactivity was detected in all the examined kidneys in the cap mesenchyme and in the renal vesicles. Immunostaining for MUC1 in cap mesenchymal cells changed from one nodule to the next: some mesenchymal nodules were negative, some showed MUC1 reactivity in scattered cells, whereas in others, positive cells revealed the presence of a roundish developing epithelial structure. Conclusions: Our data clearly indicates, for the first time to the best of our knowledge, immunohistochemical evidence of MUC1 expression during human kidney development. We focused on MUC1 reactivity in the cap mesenchyme. On the basis of these preliminary data, we speculate that MUC1 may be involved in human nephrogenesis and may play a relevant role in MET from the cap mesenchyme to the renal vesicle, changing the fate of renal stem/progenitor cells.

MUC1 marks collecting tubules, renal vesicles, comma- and S-shaped bodies in human developing kidney

MUC1 is a transmembrane glycoprotein, apically expressed in most epithelial cells, used in the differential diagnosis of carcinomas and for discrimination of tumors of non-epithelial origin showing epithelioid features. Little attention has been paid so far though, on its possible significance in embryonic tissues. A preliminary study from our group revealed MUC1 expression in the cap mesenchymal cells during human nephrogenesis, suggesting a role for MUC1 in the process of mesenchymal-to-epithelial transition. This study aimed at investigating the expression pattern of MUC1 in various developing structures of human fetal kidney. Expression of MUC1 was examined in kidneys of 5 human fetuses. MUC1 immunoreactivity was detected in ureteric bud tips, in collecting tubules, in cap mesenchymal cells undergoing the initial phases of mesenchymal-to-epithelial transition, in renal vesicles, comma-bodies, and S-shaped bodies. Our previous preliminary report suggested a role for MUC1 in the initial phases of the process of mesenchymal-to-epithelial transition. The present data suggest that MUC1 expression characterizes multiple structures during human nephrogenesis, from the ureteric bud, to the initial phases of mesenchymal-to-epithelial transition and that MUC1 should be added to the genes activated during the process of mesenchymal-to-epithelial transition in the cap mesenchyme of human kidney.

Hypoxia/reoxygenation-induced myocardial lesions in newborn piglets are related to interindividual variability and not to oxygen concentration

OBJECTIVE: Evaluation of myocardial histological changes in an experimental animal model of neonatal hypoxia-reoxygenation. METHODS: Normocapnic hypoxia was induced in 40 male Landrace/Large White piglets. Reoxygenation was initiated when the animals developed bradycardia (HR <60 beats/min) or severe hypotension (MAP <15 mmHg). The animals were divided into four groups based on the oxygen (O2) concentration used for reoxygenation; groups 1, 2, 3, and 4 received 18%, 21%, 40%, and 100% O2, respectively. The animals were further classified into five groups based on the time required for reoxygenation: A: fast recovery (<15 min); B: medium recovery (15-45 min); C: slow recovery (45-90 min); D: very slow recovery (>90 min), and E: nine deceased piglets. RESULTS: Histology revealed changes in all heart specimens. Interstitial edema, a wavy arrangement, hypereosinophilia and coagulative necrosis of cardiomyocytes were observed frequently. No differences in the incidence of changes were observed among groups 1-4, whereas marked differences regarding the frequency and the degree of changes were found among groups A-E. Coagulative necrosis was correlated with increased recovery time: this condition was detected post-asphyxia in 14%, 57%, and 100% of piglets with fast, medium, and slow or very slow recovery rates, respectively. CONCLUSIONS: The significant myocardial histological changes observed suggest that this experimental model might be a reliable model for investigating human neonatal cardiac hypoxia-related injury. No correlation was observed between the severity of histological changes and the fiO2 used during reoxygenation. Severe myocardial changes correlated strictly with recovery time, suggesting an unreported individual susceptibility of myocardiocytes to hypoxia, possibly leading to death after the typical time-sequence of events.

Thymosin β4 expression reveals intriguing similarities between fetal and cancer cells

Thymosin β4 (Tβ4) is highly expressed in saliva of human newborns but not in adults. Here preliminary immunohistochemical analyses on different human tissues are reported. Immunoreactivity for Tβ4 in human salivary glands show high quantities of Tβ4 before birth, followed by downregulation of expression in adulthood. In contrast, Tβ4 is detected in tumors of salivary glands, suggesting that tumor cells might utilize fetal programs, including Tβ4 synthesis. Immunohistochemical analyses in the gastrointestinal tract showed strong reactivity for Tβ4 in enterocytes during development, but weak immunostaining in mature enterocytes. In colorectal cancer, the association of a high expression of Tβ4 with epithelial–mesenchymal transition was observed. On the basis of these data, the process of epithelial–mesenchymal transition could represent the unifying process that explains the role of Tβ4 during fetal development and in cancer progression.

Thymosin beta-10 expression in developing human kidney

Thymosin beta-10 (Tβ10) is a member of beta-thymosins (Tβs), a family of low molecular mass peptides, which play essential roles in many cellular functions, including apoptosis, cell proliferation, cell migration, and endocytosis. The report that the Tβ10 gene is expressed at high levels in embryonic human brain as well in human kidney induced us to study Tβ10 reactivity in the preterm kidney in order to verify, at tissue level, the expression of this peptide during renal embryogenesis. To this end, we analyzed, using immunocytochemistry, the expression of Tβ10 in samples of human kidney obtained, at autopsy, from 8 fetuses, 12 preterm infants, ranging from 25 to 36 weeks of gestation and 3 at term newborns. Tβ10 immunoreactivity was detected in 20 out of 22 kidneys examined, and was mainly localized in proximal and distal tubular structures, in the cytoplasm and occasionally in the nuclei of ductal cells. In 11 cases, we also detected a focal and mild reactivity for the peptide in glomeruli. In 13 kidneys, we also observed immunostaining for Tβ10 inside the “comma-shaped bodies” and the “S-shaped bodies” during active glomerulogenesis. Our data show, for the first time, the expression of Tβ10 in the human kidney during the initial phases of its physiological development, mainly restricted in the proximal and the distal tubuli. Further studies are needed in order to better characterize the role of Tβ10 in kidney embryogenesis.

Toward nephrogenesis in the pig kidney: the composite tubulo-glomerular nodule

Background: Significant differences regarding nephrogenesis and its completion among different animal species have been reported. Since many informations on clinical conditions (i.e. asphyxia, drugs) are extrapolated from piglets, this study aimed at analyzing nephrogenesis in piglets, in order to compare it with existing data on nephrogenesis in humans. Methods: Six male newborn piglets were subjected to euthanasia and their kidneys were harvested. Necropsy revealed no injury and no underlying pathology in any of the animals used in the experiment. Results: The analysis of the renal cortex evidenced in all the animals studied the presence of active nephrogenesis. The sequence of events identified during porcine nephrogenesis was characterized by the appearance, in the metanephric mesenchyme, of nodules undergoing mesenchymal-epithelial transition originating a specific picture that we named the tubulo-glomerular nodule. This peculiar developmental structure gives rise to the precursor of tubular and glomerular structures, till the extrusion of developing glomeruli that progressively migrate toward the mid and deep cortex. Conclusions: Nephrogenesis in pig is characterized by a peculiar morphological event, with marked differences compared with humans. These informations should be taken into account when the experimental data in piglets are extrapolated to humans, especially for clinical purposes.