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S Kloth

Publications and source records attributed to S Kloth.

33 records · Page 2Linked to original sources
PubMed1994

Interrelationship of renal vascular development and nephrogenesis.

Within the cortex region of the neonatal rabbit kidney the developing microvasculature was investigated by means of two endothelium-detecting antibodies (EnPo 1 and EC1). Rows of antibody-labelled cells were found within tissue regions that had previously been described as avascular. We conclude that these vessel-like structures detected by EnPo 1 and EC1 are capillary precursors without lumina. Furthermore, beneath the fibrous capsule within the morphologically homogeneous mesenchyme two cell populations can be discriminated by use of differential antigen expression. The EnPo 1 antigen, which is abundant on endothelial cells and podocytes at different developmental stages, was detected on a subpopulation of mesenchymal cells. These cells were exclusively detected surrounding the tip of the collecting duct ampulla. Due to the unique specificity of EC1 and EnPo 1 the process of microvascular development can be readily followed on serial optical sections gained by laser scan microscopy. (1) Adjacent to EnPo 1-positive mesenchymal cell islets vessel-like structures are found that are in contact with the differentiated vasculature. (2) The renal vesicle is enclosed by a network of vessel-like structures establishing contact with differentiated vessels. (3) No guidance of invading capillary sprouts toward the developing glomerulus and nephron is required, since vascular elements already accompany the earliest detectable nephron stage.

Animals↗
PubMed1994

Detection of intracellular interleukin 2: evidence for novel immunologically related forms of the lymphokine.

At present, few data are available on intracellular interleukin 2 (IL-2) and its posttranscriptional regulation. Unlike other lymphokines, IL-2 does not accumulate within the cell, but is rapidly secreted following its production. The process of detection and biochemical characterization of intracellular IL-2 involved using a high producer subclone of the Jurkat T-lymphoma line as a source for IL-2, in combination with a two-step separation protocol and a sensitive detection method. Following phytohemagglutinin (PHA)/4 beta-phorbol 12-myristate 13 acetate (TPA) stimulation, a 14 kDa molecule could be visualized in Western blots by means of two monoclonal anti-IL-2 antibodies possessing different epitope specificities. This molecule exhibited biological activity of IL-2 as determined by a murine cytotoxic T-cell proliferation assay. In addition to this biologically active form of the lymphokine, a strongly immunoreactive protein with a molecular weight of 54 kDa (P54) was found in Jurkat cell lysates. Further biochemical characterization of this intracellular variant revealed an isoelectric point similar to that of secreted forms of IL-2. All attempts to split the 54 kDa molecule into smaller subunits failed, and no biological IL-2 activity could be measured in response to P54. However, the appearance of this high molecular weight variant followed clear-cut time kinetics. The highest concentration of P54 was found to occur after 2 h of stimulation. Thereafter its concentration decreased continuously, while the amount of the biologically active 14 kDa variant increased under ongoing stimulation. One possible explanation for these results is that P54 may represent an immature form of IL-2 that is tightly linked to a carrier molecule.

Extracellular Space↗
PubMed1994

Maturation of renal collecting duct cells in vivo and under perifusion culture.

The embryonic collecting duct epithelium of neonatal kidney undergoes profound functional changes during maturation. In its initial state as inductor epithelium it appears homogeneous, but differentiates into a heterogeneously composed collecting duct epithelium consisting of principal and intercalated cells. The mechanism of this terminal differentiation process is unknown. We used morphological and immunohistochemical methods to investigate the maturation of the collecting duct system in neonatal rabbit kidney and under organotypic culture conditions. The new perifusion culturing method allowed us to follow the differentiation of the ampullary collecting duct epithelium under conditions as close as possible to the situation within the organ. With this technique we were able to induce a differentiation process similar to that in the in situ situation. This process led to the appearance of a mixed cell population consisting of principal and intercalated-like cells, respectively. A continuous perifusion of the medium made it possible to stabilize the microenvironment under culture conditions and thus to maintain the heterogeneous composed collecting duct epithelium in a differentiated status over long periods of time.

Animals↗
PubMed1994

Developing renal microvasculature can be maintained under perfusion culture conditions.

The cortex corticis of the neonatal rabbit kidney consists of developing nephrons, vessels, collecting duct ampullae and the nephrogenic mesenchyme. Inductive interactions between embryonic mesenchyme and collecting duct ampullae lead to the coordinated development of the nephrons and the collecting duct system. The factors regulating nephrogenesis and vascular development within this tissue region are unknown. In order to analyze the hormonal regulation of vascular development an organotypic culture system was established. Cortex explants from neonatal rabbit kidneys were prepared, mounted in a set of holding rings and cultured under serum-free conditions for 14 days in conventional culture plates or under permanent medium perfusion in a newly developed culture container. The detection of endothelial cells was carried out by means of two monoclonal antibodies. Within the renal cortex corticis EnPo 1 detected developing vasculature as well as podocytes and a subset of mesenchymal cells. EC1 displayed exclusive specificity for endothelial cells. The antibody did not discriminate between arteries and veins. Endothelial cells of different developmental stages were labeled with the same intensity. A combination of both antibodies allowed the discrimination between developing endothelial cells and podocytes. Following 14 days of culture under permanent medium exchange, excellent tissue preservation as well as endothelial cell proliferation was observed in cortex explants. In contrast, tissue kept in stationary culture revealed a high degree of disintegration. Endothelial antigen expression was also severely disturbed. Tissue maintenance under stationary conditions was improved by the application of a hormone mixture consisting of aldosterone and 1,25-hydroxyvitamin D3. However, the high degree of spatial organization shown by developing endothelial cells in vivo was maintained exclusively in explants cultured in the presence of hormone under permanent perfusion.

Aldosterone↗
PubMed1993

Histochemical markers reveal an unexpected heterogeneous composition of the renal embryonic collecting duct epithelium.

The ampullary collecting duct epithelium acts as an inductor in the embryonic and neonatal kidney. It induces the formation of all nephron generations and thus determines the whole architecture of the kidney. As the organ matures, the collecting duct epithelium itself transdifferentiates. The ampullary inductor epithelium, which appears homogeneous as revealed by light microscopy, develops into the well-known heterogeneous epithelium of the mature collecting duct consisting of light principal and dark intercalated cells. Up to now the mechanisms initiating and regulating this transdifferentiation step are unknown. Only very few data are available concerning functional characteristics of the ampullary epithelial cells of neonatal rabbit kidney. Therefore, a characterization of the collecting duct ampullary cells was carried out by means of immunohistological techniques using a set of different monoclonal antibodies and the lectin peanut agglutinin. All epithelial cells within the ampullary tip and neck were positive for cytokeratin 19, an intermediate filament protein. On the other hand, the monoclonal antibody CD 7 revealed a clear cut boundary between the ampullary neck and the ampullary tip region. Furthermore, after incubation with the monoclonal antibody BO-7 specifically reacting with intercalated cells of the mature collecting duct, both labeled and unlabeled cells were observed within the whole ampullary epithelium. These results were confirmed by scanning electron microscopical investigations which revealed two distinct epithelial cell populations. Thus, an unexpected heterogeneity of the ampullary epithelium could be demonstrated.

Amino Acid Sequence↗
PubMed1993

Aldosterone modulates PNA binding cell isoforms within renal collecting duct epithelium.

To investigate the differentiation of the ampullary collecting duct cells into adult principal and intercalated cells, the embryonic cortex of newborn New Zealand rabbit kidney was isolated and brought in culture. With this culture technique the ampullary cells formed a polarized collecting duct epithelium which was kept under permanent exchange of medium and in the presence of aldosterone, arginine vasopressin and/or insulin. After 14 days of perfusion culture the epithelia showed light and dark cells resembling the principal and intercalated cells of the adult collecting duct. The differentiation from embryonic into adult collecting duct cells was controlled by applying the monoclonal antibody CD 7. Independent of the hormonal treatment all of the epithelial cells matured in culture and expressed the CD 7 antigen. This corresponded with the situation found within the adult kidney, where the CD 7 antigen was localized in all principal and intercalated (IC) cells, whereas the embryonic ampullary epithelium in the neonatal kidney remained negative. A differentiation feature of the beta-type intercalated cell was investigated by labeling the cultured epithelia with peanut agglutinin (PNA). In contrast to the CD 7 antigen the development of PNA binding was highly dependent of time and individual hormone administration. While in control epithelia only 8% of PNA positive cells were found, aldosterone induced epithelia revealed 72% PNA labeled cells. The combination of aldosterone and insulin increased the number of PNA-positive cells to 90%. By scanning electron microscopy it could further be shown that several isoforms of cells were reactive with PNA. Thus, in culture the PNA label is not restricted to the typical beta-type IC cells.

Aldosterone↗
PubMed1992

Characterization of an endothelial protein in the developing rabbit kidney.

A new protein (EnPo 1 antigen) abundant on endothelial cells and glomerular podocytes has been characterized by means of the mouse monoclonal antibody EnPo 1. Following electrophoretical separation of rabbit kidney homogenates EnPo 1 recognized a protein with a molecular weight of 110 kDa and an isoelectric point of 5.9 in Western blots. Using immunohistological techniques, the EnPo 1 antigen has been localized in high concentrations on glomerular podocytes of different developmental stages. Furthermore, the EnPo 1 antigen was expressed on endothelial cells of all adult rabbit organs tested so far. Detailed analysis of neonatal rabbit kidney revealed the abundance of EnPo 1 antigen on both differentiated vessels as well as on immature endothelial cells and endothelium of the microvasculature. Thus, for the first time a marker for in situ investigations of angiogenic processes within the mammalian kidney is available. Analysis of kidney cryosections by confocal laser scan microscopy revealed a direct connection between mature and differentiating vessels in the outer kidney cortex. Furthermore, two differentially organized cell populations discriminated by their EnPo 1 binding pattern were localized in the embryonic renal cortex. Morphologically, these cells were not distinguishable from other mesenchymal cells.

Animals↗
PubMed1992

Construction of an apparatus for perfusion cell cultures which enables in vitro experiments under organotypic conditions.

The value of cultured cells in cell biological, pharmaceutical or biotechnological research depends on the degree of terminal cell differentiation. In conventional Petri dishes or tissue culture plates it is often difficult to achieve culture conditions which resemble the in situ situation of intact tissue, as regards optimal cell adhesion, exchange of nutrients and metabolic products. These limitations prompted us to develop simple laboratory tools which optimize the environment of cultured cells. A perfusion apparatus with various culture containers and compatible cell holder sets was constructed which allows the simulation of organotypic conditions. (i) The cells can be kept on individual and interchangeable support materials for an optimal cell attachment. (ii) Culture medium can be perfused during the whole culture period. (iii) One type of the new culture container can be perfused with different media at the apical and basal side of the cells, thus mimicking the organotypic environment that applies for epithelial monolayers. Cell culture experiments with renal collecting duct epithelia exhibited an excellent morphological appearance showing typical features of principal and intercalated cells.

Animals↗
PubMed1991

Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67.

The monoclonal antibody Ki-67 detects a human nuclear antigen that is present in proliferating cells, but absent in quiescent cells. The aim of this study was to characterize the Ki-67 antigen by means of immunobiochemical and molecular biology techniques. Enzymatic digestion experiments showed that this antigen is highly susceptible to protease treatment, and the antigen cannot be extracted by 0.1 normal HCl, indicating that Ki-67 antigen is a nonhistone protein. Immunoblot analysis of cell lysates with Ki-67 showed a double band with apparent molecular weights of 395 kd and 345 kd, regardless of whether the gels were run under reducing or nonreducing conditions. It is noteworthy that these bands were exclusively detectable in lysates prepared from proliferating cells, whereas they were absent in lysates obtained from quiescent cells. These immunobiochemical data are further substantiated by our molecular cloning approaches. By means of immunocloning with Ki-67, the authors isolated and sequenced several cDNA fragments from lambda gt11 libraries. A 1095-bp fragment gave a strong hybridization signal at 7.5 to 9.5 kb in Northern blot analysis with RNA prepared from proliferating cells, whereas it was negative with RNA prepared from quiescent cells. This cDNA fragment could be bacterially expressed, and in subsequent immunoblot analysis Ki-67 reacted exclusively with those fusion proteins that were derived from bacteria containing the insert in the right reading frame.

Antibodies, Monoclonal↗
PubMedDate not supplied

Modulation of cell differentiation in perfusion culture.

An in vitro model was used to investigate the terminal differentiation mechanisms leading from embryonic to adult renal tissue. For these experiments the capsula fibrosa with adherent embryonic tissue was isolated from neonatal rabbit kidneys. These explants were mounted onto special tissue carriers and cultured in medium containing serum for 24 h. During that time collecting duct (CD) cells grew out and formed a monolayered epithelium covering the whole surface of the explant. The carriers were then transferred to perfusion culture containers to obtain an optimal degree of differentiation. A special type of container allowed us to continuously superfuse the epithelia with individual media on the luminal and basal sides. Using this method it became possible to culture embryonic CD epithelia in a fluid gradient for weeks. The epithelia were superfused with standard Iscove's modified Dulbecco's medium (IMDM) on the basal side, while IMDM containing additional NaCl was used on the luminal side. In controls IMDM was superfused on both the luminal and basal sides. It was found that the degree of differentiation in the CD epithelia is dependent on the influence of fluid gradient exposure. Perfusion culture under isotonic conditions revealed that less than 5% of cells were immunopositive for principal and intercalated cell features, while epithelia cultured in a luminal-basal gradient showed more than 80% positive cells. Immunoreactivity for characteristic markers started to develop after an unexpectedly long latent period of 3-6 days, then increased continuously during the following 5 days and reached a maximum on day 14. After switching back from the gradient to isotonic culture conditions the immunoreactivity for some markers decreased within 5 days, while other characteristic features remained stable. Thus, differentiation was not only under the control of growth factors but was also regulated by the electrolyte environment.

Animals↗
PubMedDate not supplied

Electrolyte environment modulates differentiation in embryonic renal collecting duct epithelia.

The influence of electrolytes on the development of renal principal and intercalated collecting duct cells is unknown. Consequently embryonic collecting duct epithelia were exposed to different electrolyte concentrations, and their degree of differentiation was registered by immunohistochemical methods. Embryonic collecting duct epithelia were isolated from neonatal rabbit kidneys and placed on tissue carriers. The apical urine and the basal serum compartments were simulated in a gradient culture container. The two sides of the epithelium were each constantly superfused with medium for 13 days. In controls the medium on both apical and basal side was standard Iscove's modified Dulbecco's Medium (IMDM) with 112 mmol/l Na+ and 85 mmol/l Cl-. In experimental series the NaCl concentration at the basal side of the epithelium was increased up to 137 mmol/l Na+ and 99 mmol/l Cl- as found in the serum of neonatal rabbits. Light microscopy revealed morphologically faultless epithelia following gradient perfusion culture in standard and NaCl-adapted IMDM. The development of principal and intercalated cell features was monitored with the monoclonal antibodies 703, 503, PCD9, and peanut lectin. Cells immunopositive for monoclonal antibody 703, for example, increased from less than 10% in controls to more than 80% in NaCl-adapted IMDM. It is a new finding that the development of collecting duct cell features is influenced by the extracellular electrolyte environment.

Animals↗