Normand Brière

Growth regulatory properties of endothelins

Endothelins are produced by endothelial and epithelial cells, macrophages, fibroblasts, and many other types of cells. Their receptors are present in numerous cells, including smooth muscle cells, myocytes, and fibroblasts. Evidence now suggests that the three isoforms of endothelins (ET-1 and the other two related isopeptides, ET-2 and ET-3) regulate growth in several of these cells. Endothelin-1 influences DNA synthesis, the expression of protooncogenes, cell proliferation, and hypertrophy. The participation of ET in mitogenesis involves activation of multiple transduction pathways, such as the production of second messengers, the release of intracellular pools of calcium, and influx of extracellular calcium. Moreover, ET-1 acts in synergism with various factors, such as EGF, PDGF, bFGF, TGFs, insulin, etc., to potentiate cellular transformation or replication. Several of these factors may in turn stimulate the synthesis and/or the release of endothelins. The production and release of endothelins are also increased in acute and chronic pathological processes, e.g., atherosclerosis, postangioplastic restenosis, hypertension, and carcinogenesis. It is postulated that endothelins act in a paracrine/autocrine manner in growth regulation and play an important role mediating vascular remodeling in some cardiovascular diseases. The present review analyses the implication of endothelins (ET-1, -2, and -3) in physiopathology related to their growth regulatory properties.

Peroxisomes in human foetal kidney: variations in size and number during development

SummaryThe kidneys of 15 human foetuses (10–18 weeks of age) were used for morphometric studies on peroxisomes during gestational development and in organ culture. The catalase positive organelles revealed by DAB were found to ovoid with a granular matrix delimited by a membrane occasionally deformed by marginal plates. Generally, the distribution was uniform in cells of proximal tubules. In the same cell, size and density varied. The number fluctuated from cell to cell. No significant difference in the mean diameter was observed from the 10th to 18th weeks of gestation, although the mean value (0.36±0.1 μm) was significantly less than the adult figure. These results indicate that size modifications might occur later on in gestation or after birth to reach the adult value. During the studied period, the mean number of peroxisomes per 100 μm2 of surface area did not differ significantly from that of the 10–12 week group (10.5±1.97). No important changes of peroxisome morphology in kidney explants cultured for 7 days were noticed on day 3–4. Thereafter, the shape of many peroxisomes became elongated or irregular; marginal plates were frequent. A decrease in the diameter of peroxisomes began at day 4, became significant on day 5 and more accentuated on day 7. In addition, as the culture matured, there was a progressive reduction in catalase activity, revealed by a diminished density of the peroxisomal matrix. The number of DAB positive organelles per surface area decreased steadily with culture age, and significantly on day 2 (p0.01) to become drastically low on day 5 and negligible on day 7. The present experiments provided for the first time basic quantitative data on peroxisomes of the foetal human nephron during gestational development and maturation in organ culture.

Fetal mouse kidney maturation in vitro: coordinated influences of epidermal growth factor, transferrin and hydrocortisone.

SummaryWe have investigated the individual and combined actions of epidermal growth factor (EGF), transferrin and hydrocortisone on the maturation of whole fetal mouse metanephrol maintained in serum-free conditions for up to 5 days. The presence of EGF (100 ng/ml) resulted in elevated levels of [3H]-thymidine incorporation when compared to controls; autoradiograms showed that the proliferation of mesenchymal cells in the nephrogenic zone is particularly enhanced as verified by cell counting. Brush border hydrolase activities (alkaline phosphatase and γ-glutamyltransferase), on the other hand, were significantly diminished. Transferrin (5 μg/ml) slightly stimulated DNA synthesis and potentiated EGF mitogenic action. The activation of DNA replication by the growth factor seems to be mediated through the protein kinase C pathway. When added alone, hydrocortisone (10−6 M) strongly inhibited DNA synthesis, stimulated hydrolase activities and exerted a positive effect on brush border differentiation. When combined with EGF or to EGF + transferrin, hydrocortisone counteracted the effects of these latter peptides on DNA synthesis and enzyme activities. Considering the earlier observation of a reciprocal relation between proliferation and differentiation during the neotubulogenic phase of kidney development, the results described in the current study suggest that synergistic and synarchic actions of these heterologous factors are involved in the regulation of tubulogenesis.

Human foetal kidney explant in serum-free organ culture

SummaryThe purpose of the work was to develop an in vitro model for the study of human kidney development. Human metanephric explants from foetuses 10–18 weeks of gestational age were cultured in serum-free Leibovitz L-15 medium without hormones. Under the current minimal conditions for growth, the system permitted to maintain the renal tissues in culture for periods up to 9 days, although no evident sign of morphological differentiation was observed. However, during the studied period the overall architecture of the explants was preserved as well as the ultrastructural features of cytoplasmic organelles. The incorporation of 3H-thymidine and 3H-leucine indicated that DNA and protein synthesis was maintained or increased. Glycoprotein synthesis evaluated by 3H-glucosamine incorporation and radioautography continued in mesangium as well as in glomerular and tubular basement membranes. Alkaline phosphatase, γ-glutamyltranspeptidase (brush border) and catalase (peroxisomes) activities remained histochemically active. The proposed organ culture system appears as a reliable and promising model that will provide basic data on the morphology and functional characteristics of the developing kidney. Since it is achieved in a completely controlled environment, it will permit to study the role of growth factors and hormones in proliferation and differentiation of the cell populations during development of the human foetal kidney.

Alkaline phosphatase activity at unusual sites of human foetal kidney

SummaryFrozen sections of human foetal kidneys were treated by the lead citrate method in order to demonstrate alkaline phosphatase (ALP) activity. Unexpectedly, ALP activity was observed over the parietal layer of Bowmans capsule, in addition to usual localization in proximal tubules and blood vessels. The enzyme was confined to the membrane of microvilli belonging to tall columnar cells. Normally, in the mature nephron, these high cells are absent from the parietal layer that is instead entirely composed of squamous epithelium.ALP activity was also revealed at another unusual site in the kidney medulla. A collar of mesenchymal cells encapsulating groups of tubules and calyces gave an intense enzymatic reaction. The activity was present over the membrane of long cytoplasmic processes. The cytoplasm showed a well developed rough endoplasmic reticulum, an indication of a high rate of protein synthesis. The nearby presence of numerous collagenous fibrils is concordant with this assumption. Moreover, these ALP-positive cells might represent an intermediary stage through which cells have to pass before differentiating.

Comparison between mouse kidneys of pre- and postnatal ages maturing in vivo and in serum-free organ culture.

1. To evaluate the influence of age, DNA synthesis and brush border hydrolase activities were determined in mouse kidneys maturing in vivo and in serum-free organ culture. 2. DNA synthesis decreased with advancing age. 3. The protein content and leucylnaphthylamidase, maltase, trehalase, alkaline phosphatase and gamma-glutamyltransferase activities increased with aging. 4. The differences due to age were reproduced in kidneys maturing in culture. 5. These results show that age has a significant effect on the parameters determined, but apparently has no influence on the viability of the kidney explants in culture.

Rat prostate explants in serum-free organ culture: A comparison of two media and gas mixtures

Urologists are looking for a way to easily discriminate between aggressive and very slow‐growing prostate tumors. A sound way to appreciate such developing activities would be to identify an appropriate cell marker in prostate explants maintained in a defined culture system.

Differential staining of preneoplastic foci in rat liver parenchyma during azo dye carcinogenesis

SummaryIn preneoplastic liver of rats fed the azo dye 4-dimethylaminoazobenzene, certain cellular populations are characterized by cytologic changes typical of tumor cells and appear as the sites of neoplastic transformation. With a basic dye such as toluidine blue, cytoplasmic RNA in preneoplastic foci and hepatomas stains more intensely than in surrounding tissue. In the present study, it was found that when a basic dye (hematoxylin) was combined with an acid dye (tartrazine), these areas stained differentially from the surrounding liver parenchyma. RNAse hydrolysis has shown that such staining properties might be due to the increased proportion of cytoplasmic RNA to components stainable with tartrazine in hyperbasophilic cells, while the surrounding parenchymal cells are probably distinguished by the opposite ratio.It is suggested that the increased basophilia in preneoplastic areas and hepatomas results from the presence of excess RNA and a corresponding decrease in cellular components stained with tartrazine. However, the present method does not permit us to determine whether hyperbasophilia is due to a normal type or an altered form of RNA present in excess.

Positive influence of tetracycline on human fetal kidney in serum-free organ culture

SummaryHuman fetal kidney explants can be maintained during 5 days in Leibovitz’s L15, a basic serum-free medium. Because culture conditions are minimal for growth and differentiation, DNA synthesis drastically decreases during the first 48 h, but stabilizes thereafter. The addition of insulin plus transferrin significantly restores this important cellular function in kidneys of fetuses younger than 16 wk. However, renal explants from older fetuses are more difficult to culture: they respond less to growth factors and are more prone to necrosis. The objective of this study was to verify the influence of tetracycline, an antibiotic with anti-collagenase potential, on cultured kidney explants aged 17 to 20 wk. The addition of 20µg/ml tetracycline did not influence DNA synthesis nor the effectiveness of insulin plus transferrin on cell proliferation. Nor did it change the activities of alkaline phosphatase and γ-glutamyltransferase, two enzymic markers of brush border differentiation. After 5 days in L15 alone, explants often showed necrosis and an important reduction in both weight and volume. Insulin plus transferrin significantly restored these parameters to control values observed at Day 0, but evidence of necrosis was still present. Tetracycline alone markedly reduced explant necrosis resulting in a significant increase in weight and volume. The effectiveness of insulin plus transferrin on explant morphometry was not improved when tetracycline was added as third factor. These results indicate that insulin plus transferrin restores explant mass through cell proliferation, whereas tetracycline does so possibly through a reduction in extracellular matrix degradation. The two effects are not additive in cultured mid-term fetal kidneys.

EFFECTS OF HYDROCORTISONE ON HUMAN FOETAL KIDNEY IN SERUM-FREE ORGAN CULTURE

The influence of hydrocortisone (HC) on the in vitro maturation of human foetal kidney was studied. Following legal therapeutic abortions, explants (2 × 2 mm) of renal cortex from foetuses aged 12-18 weeks, were placed on a lens paper covering a stainless steel grid lying over the central well of a Falcon culture dish. The explants were cultured for 2 and 5 days in serum-free Leibovitz L-15 medium at 37°C in a mixture of 95% air-5% CO2, without hormones (controls) or with HC at concentrations of 12.5, 25 or 50 ng/ml. During the studied period, the overall architecture of the renal structures was preserved without evident signs of nephrogenesis. DNA synthesis was measured by incorporation of 3H-thymidine and was increased on day 5 by 80% with the addition of HC at 12.5 ng/ml and by 131% with 50 ng/ml. The sites of 3H-TdR incorporation were the same after HC addition. The activity of some brush border hydrolases was modified by HC. Gamma-glutamyltranspeptidase activity was reduced by 68% on day 2 and by 42% on day 5 with 12.5 ng of HC. Maltase activity was increased by 19% on day 2 with 50 ng of the hormone. Trehalase activity was reduced by 61% with 12.5 ng and by 50% with 50 ng of HC, on day 5. Alkaline phosphatase activity was decreased on day 2, by 50% with 12.5 ng and by 39% with 25 and 50 ng of HC. This study provides for the first time, basic data on the direct effects of hydrocortisone on proliferation and brush border enzyme activities in the human foetal kidney maintained in organ culture. (MRC of Canada)