Biomedical subjects

Karl Schumacher

Publications and source records attributed to Karl Schumacher.

14 recordsLinked to original sources

PubMed2005

Temporal-spatial co-localisation of tissue transglutaminase (Tgase2) and matrix metalloproteinase-9 (MMP-9) with SBA-positive micro-fibres in the embryonic kidney cortex.

Growth of the kidney is a complex process piloted by the collecting duct (CD) ampullae. The dichotomous arborisation and consecutive elongation of this tubular element determines the exact site and time for the induction of nephrons in the overlaying mesenchymal cap condensates. The mechanism by which the CD ampullae find the correct orientation is currently unknown. Recently, we have demonstrated micro-fibres that originate from the basal aspect of the CD ampullae and extend through the mesenchyme to the organ capsule. The micro-fibres are assumed to be involved in the growth and arborisation process of the CD ampulla. Therefore, we have investigated the specific distribution of the micro-fibres during branching morphogenesis. We have also analysed whether the micro-fibres co-localise with extracellular matrix (ECM)-modulating enzymes and whether the co-localisation pattern changes during CD ampulla arborisation. Micro-fibres were detected in all stages of CD ampulla arborisation. Tissue transglutaminase (Tgase2) co-localised with soybean agglutinin (SBA)-positive micro-fibres, whose presence depended upon the degree of CD branching. Matrix metalloproteinase-9 (MMP-9) also co-localised with micro-fibres, but its expression pattern was different from that for Tgase2. Western blotting experiments demonstrated that Tgase2 and MMP-9 co-migrated with SBA-labelled proteins. Thus, the micro-fibres are developmentally modulated by enzymes of the ECM in embryonic kidney cortex. These findings illustrate the importance of micro-fibres in directing CD ampulla growth.

Animals↗

PubMed2005

Renal epithelia in long term gradient culture for biomaterial testing and tissue engineering.

In the organism epithelia perform perfect barrier functions. Strong rheological and mechanical influences constitute the normal environment of this tissue throughout life. Most epithelia are exposed to different fluids at the luminal and basal sides. To obtain realistic information about tissue development in modern biomaterial testing and tissue engineering it is necessary to mimick the natural environment of epithelia. Cultured cells are brought in contact with an artificial extracellular matrix to determine whether proper development into a functional epithelium occurs. As under natural conditions the cultures have to withstand mechanical and fluid stress over a prolonged period of time in close contact to a selected biomaterial. However, development of tissue-specific features such as polarization, tightness and transport under in vitro conditions will only occur, if the biomaterial and the culture conditions support tissue development. Leakage, edge damage and pressure differences during culture have to be avoided so that the natural functions of the growing epithelium can develop. Our aim is to generate functional epithelia derived from renal explants containing stem cells, which are microsurgically isolated and placed into specific O-ring carriers for optimal handling. The cells develop in combination with a collagenous matrix from an embryonic into a functional collecting duct (rCD) epithelium. To achieve optimal culture conditions the tissue is placed in a gradient culture container. A typical environment can be simulated by superfusing different culture media at the luminal and basal sides. Within days epithelia growing inside the gradient container build up a physiological barrier, which is maintained during the whole culture period. The described method allows to investigate the influence of new biomaterials over prolonged periods of time.

Animals↗

PubMed2004

Generation of renal tubules at the interface of an artificial interstitium.

During kidney development a multitude of tubular portions is formed. Little knowledge is available by which cellbiological mechanism a cluster of embryonic cells is able to generate the three-dimensional structure of a tubule. However, this know-how is most important in tissue engineering approaches such as the generation of an artificial kidney module or for the therapy of renal diseases using stem cells. To obtain cellbiological insights in parenchyme development we elaborate a new technique to generate under in vitro conditions renal tubules derived from the embryonic cortex of neonatal rabbits. The aim of the experiments is to establish a specific extracellular environment allowing optimal three-dimensional development of renal tubules under serum-free culture conditions. In the present paper we demonstrate features of the renal stem cell niche and show their isolation as intact microcompartments for advanced tissue culture. Perfusion culture in containers exhibiting a big dead fluid volume results in the development of a flat collecting duct (CD) epithelium at the surface of the tissue explant. In contrast, by fine-tuning the dead fluid volume within a perfusion culture container by an artificial interstitium made of a polyester fleece shows the generation of tubules. It is an up to date unknown morphogenetic information which tells the cells to form tubular structures.

Animals↗

PubMed2003

[From the renal stem cell niche to functional tubule].

In the present paper, a modular system to generate artificial tissues under optimal perfusion culture conditions is described. The microenvironment within the culture containers can be fine-tuned to meet the physiologic needs of individual tissues. An optimal physiologic environment is created by modulating a liquid phase as well as an artificial interstitium surrounding the growing construct. For example, the generation of tubular structures derived from renal stems cells under in vitro conditions is shown for the first time.

Animals↗

PubMed2003

Differential regulation of renin and Cox-2 expression in the renal cortex of C57Bl/6 mice.

Based on the controversy about the relevance of cyclooxygenase-2 (Cox-2)-derived prostanoids from the macula densa for the control of the renin system, this study aimed to determine the interrelation between Cox-2 and renin expression in the mouse kidney. In control mice renin mRNA was readily detectable whilst renocortical Cox-2 mRNA abundance was at the detection limit of the RNase protection assay and no specific signals for Cox-2 were obtained by in situ hybridization or Western blot analysis. Experimental maneuvers such as low-salt diet, treatment with loop diuretics or angiotensin I converting enzyme inhibitors clearly increased renin mRNA abundance up to sevenfold, but under none of these conditions renocortical Cox-2 mRNA levels were significantly changed. Moreover, the strong stimulation of renin expression by angiotensin I-converting enzyme inhibition was not changed by the cyclooxygenase inhibitor ibuprofen, which in turn clearly lowered tissue prostanoid content. Our data suggest a marked divergence of renin and Cox-2 expression in the kidney cortex of C57Bl/6 mice with no clear evidence for a role of Cox-2-derived prostanoids from the macula densa in the regulation of renin expression.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2003

Urea restrains aldosterone-induced development of peanut agglutinin-binding on embryonic renal collecting duct epithelia.

Peanut agglutinin (PNA) represents a commonly used marker for beta-type intercalated (IC) cells and their distribution in the corticomedullary course of the collecting duct (CD) in the mature rabbit kidney. It has been shown that aldosterone is able to generate >90% of PNA-binding cells in an embryonic CD epithelium in vitro. In adult kidney, a maximum of only 25% PNA-positive cells is found in the cortical segment of the CD, and PNA-binding completely disappears in the inner-medullary CD. Molecules that regulate the gradual development of CD-specific cells during organ growth are unknown. In the present experiments, it was found that addition of physiologic concentrations of urea to the culture medium is able to restrain the action of aldosterone in embryonic CD epithelia. Urea antagonizes in a concentration-dependent manner the action of aldosterone finally leading to only 10% of PNA-binding cells. The data point to a urea-specific effect, because osmolytes such as NaCl and mannitol did not affect PNA binding. In addition, urea did not influence expression of principal-cell typical markers such as AQP2 and 3. The findings may explain that a higher number of PNA-positive cells is found in the cortical region of the kidney correlated with a low concentration of urea as compared with only few PNA-binding cells in the medullary CD, where a high concentration of urea occurs. Thus, an increasing concentration of urea may trigger the number of PNA-positive cells in the cortical-medullary course of the CD during organ development.

Aldosterone↗

PubMed2002

Detection of glycosylated sites in embryonic rabbit kidney by lectin chemistry.

The reciprocal cell biological interaction between mesenchymal and epithelial tissue plays a critical role during nephrogenesis. It is unknown to date whether the tissues interact during nephron induction by pure diffusion of substances or whether cellular contacts via gap junctions or focal adhesion molecules are involved. In neonatal rabbit kidney the interface between both tissues shows unique features. It consists of a distinct space, which is filled with specific extracellular matrix consisting of glycosylated proteins such as fibronectin, laminin, collagen, and proteoglycans. In the present experiments we tested by histochemistry whether it is possible to detect additional glycosylated proteins using Soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), Ulex europaeus I agglutinin (UEA I), and Peanut agglutinin (PNA) as molecular markers. All tested lectins showed distinct labeling patterns in embryonic renal tissue. Within the collecting duct ampulla, DBA and UEA I revealed intensive cellular reaction. In contrast, PNA and SBA reacted at the basal aspect of the collecting duct ampulla tip in addition to a cellular reaction. To identify the individual molecules labeled by the lectins, embryonic tissue was fractionated and separated by electrophoretic methods. For the first time, we were able to show by two-dimensional electrophoresis and subsequent western blot experiments that lectins bind to a series of individual protein spots, which have not been identified to date.

Animals↗

PubMed2002

Advanced technique for long term culture of epithelia in a continuous luminal-basal medium gradient.

The majority of epithelia in our organism perform barrier functions on being exposed to different fluids at the luminal and basal sides. To simulate this natural situation under in vitro conditions for biomaterial testing and tissue engineering the epithelia have to withstand mechanical and fluid stress over a prolonged period of time. Leakage, edge damage and pressure differences in the culture system have to be avoided so that the epithelial barrier function is maintained. Besides, the environmental influences on important cell biological features such as, sealing or transport functions, have to remain upregulated and a loss of characteristics by dedifferentiation is prevented. Our aim is to expose embryonic renal collecting duct (CD) epithelia as model tissue for 14 days to fluid gradients and to monitor the development of tissue-specific features. For these experiments, cultured embryonic epithelia are placed in tissue carriers and in gradient containers, where different media are superfused at the luminal and basal sides. Epithelia growing on the tissue carriers act as a physiological barrier during the whole culture period. To avoid mechanical damage of the tissue and to suppress fluid pressure differences between the luminal and basal compartments improved transport of the medium and an elimination of unilaterally accumulated gas bubbles in the gradient container compartments by newly developed gas expander modules is introduced. By the application of these tools the yield of embryonic renal collecting duct epithelia with intact barrier function on a fragile natural support material could be increased significantly as compared to earlier experiments. Epithelia treated with a luminal NaCl load ranging from 3 to 24 mmol l were analyzed by immunohistochemical methods to determine the degree of differentiation. The tissue showed an upregulation of individual CD cell features as compared to embryonic epithelia in the neonatal kidney.

Animals↗

PubMed2002

Proliferating cells versus differentiated cells in tissue engineering.

The efficiency of cell or tissue cultures is usually judged by how quickly confluence is reached within a Petri dish or on a scaffold. Growth factors and fetal bovine serum are employed to drive cultured cells from one mitosis to the next as quickly as possible. The tissue specific interphase is extremely short under these conditions, so that the degree of differentiation desired in tissue engineering cannot be achieved. To reach the goal of functional differentiation in vitro mitosis and interphase must be separated experimentally and tailored to the specific requirements of the cell-type used. This could be achieved by a three step concept for tissue-engineering in vitro as we present here. The expansion phase is followed by a phase in which tissue differentiation is initiated. The final phase serves to express and maintain histotypical differentiation of the generated tissue.

Animals↗

PubMed2002

Low tonicity mediates a downregulation of cyclooxygenase-1 expression by furosemide in the rat renal papilla.

It is well known that loop diuretics enhance the renal excretion of prostanoids; therefore, this study aimed to characterize the influence of loop diuretics on the intrarenal expression of cyclooxygenases, which are the key enzymes for prostanoid formation. Male Sprague-Dawley rats were infused with furosemide (12 mg/kg per d) for 6 d, and the expression of cyclooxygenase-1 and -2 (Cox-1 and Cox-2) was analyzed in the different kidney zones. Furosemide increased Cox-2 mRNA expression approximately twofold in the cortex, but it left Cox-1 mRNA expression unaltered there. In the outer medulla, furosemide changed neither Cox-1 nor Cox-2 mRNA expression. In the inner medulla, however, furosemide decreased Cox-1 and Cox-2 mRNA levels to approximately 30% and 60% of their control levels, respectively. The downregulation of mRNA was paralleled by a decrease of Cox protein in the collecting ducts and interstitial cells. Moreover, tissue prostaglandin E(2) (PGE(2)) concentrations in the papilla were markedly decreased by furosemide to about 30% of the control level. Furosemide lowered urine osmolality from 1550 mosmol/kg to 480 mosmol/kg; therefore, further consideration was given to the influence of tonicity as a possible mediator of the effects of furosemide on the Cox expression. Water loading was therefore used to reduce the medullary tonicity by a second maneuver. Water loading led to a similar reduction in papillary Cox mRNA expression and PGE(2) content like furosemide. To investigate the influence of the osmolarity on the expression of Cox and the production of PGE(2) under defined in vitro conditions, inner medullary collecting duct cells were incubated with culture medium containing graded amounts of NaCl ranging from 200 mmol/L to 600 mmol/L, and Cox-1 and Cox-2 mRNA abundance were determined after 24 h an 48 h. Cox-1 and Cox-2 mRNA abundance changed in parallel with the osmolarity. The data suggest that loop diuretics decrease the expression of cyclooxygenases and consequently tissue PGE(2) concentrations in the kidney inner medulla. This effect could be related to the breakdown of the papillary osmotic gradient induced by loop diuretics.

Actins↗

PubMed2002

SBA-positive fibers between the CD ampulla, mesenchyme, and renal capsule.

During kidney development, the CD shows two peculiarities. First, the tip of the CD ampulla is always found at a specific distance from the organ capsule. Second, the CD growth occurs as a perfectly straight elongation. It is unknown whether the CD-specific growth is dependent on hormonal action or on structural elements. Histochemical experiments on neonatal rabbit kidney yielded new insight into the interface of the CD ampulla and the surrounding nephrogenic mesenchyme. Incubation of tissue sections with soybean lectin (SBA) showed the existence of fibers extending in a radial course from the ampullar tip through the mesenchyme toward the organ capsule. SBA labeling did not colocalize with collagen type I, III, IV, V, and VI, laminin, fibronectin, and tenascin. It is assumed that while the kidney increases in volume the structural fixation of the ampullar tip by the SBA-positive fibers causes CD ampullae to maintain a constant distance from the organ capsule. The connection would explain the linear extension of the CD in relation to the organ capsule. In addition, the presented data suggest that the SBA-positive fibers between ampullar tip and organ capsule create a structural microcompartmentation of the nephrogenic zone.

Animals↗

PubMed2002

Role of prostanoids in regulation of the renin-angiotensin-aldosterone system by salt intake.

We investigated the effect of cyclooxygenase (COX) activity on the regulation of the renin-angiotensin-aldosterone system by salt intake. Therefore, Sprague-Dawley rats were subjected to different salt diets [0.02, 0.6, and 8% NaCl (wt/wt)] and treated with the selective COX-2 inhibitor rofecoxib (10 mg x kg body wt(-1) x day(-1)) or with ketorolac at a dose selective for COX-1 inhibition (2 mg x kg body wt(-1) x day(-1)) for 3, 7, 14, and 21 days. Rofecoxib and ketorolac caused a similar reduction of renocortical PGE2 formation with a low-salt diet. Rofecoxib did not change plasma renin activity or renocortical renin mRNA abundance with any of the diets but clearly lowered plasma aldosterone concentration. In contrast, ketorolac delayed the increase in plasma renin activity and of renin mRNA in response to low salt intake but did not change plasma aldosterone concentration. Prolonged treatment with rofecoxib but not with ketorolac caused an upregulation of COX-2 expression while COX-1 mRNA abundance remained unchanged. These findings suggest that COX-1-derived, but not COX-2-derived, prostanoids are of relevance for the regulation of the renin system by salt intake.

Animals↗

PubMed2002

Cyclooxygenases in the collecting duct of neonatal rabbit kidney.

COX 1 and 2 expression during the terminal phase of kidney development is poorly understood. To obtain information about this process we followed the primary appearance of cyclooxygenases in collecting duct (CD) epithelia of the neonatal rabbit kidney with immunohistochemical and two dimensional electrophoretical methods. In the fully embryonic cortical zone immunohistochemical expression of COX 1 is seen in all cells of the CD ampulla, while COX 2 is lacking within the nephron inducer. Within the matured cortical collecting duct (CCD) COX 1 and 2 immunoreactivity could not be detected. In contrast, a heterogeneous expression profile for COX 1 and 2 is found in the outer medullary CD (OMCD), since not all cells showed immunohistochemical labeling. Within the inner medullary CD (IMCD) nearly all cells express both cyclooxygenases. As revealed by western blot experiments generated embryonic CD epithelia in perfusion culture demonstrate high COX 1 presence at the begin of culture, while COX 2 is found to a minor degree. From day 3 until day 14 continuous levels of COX 1 and 2 expression are detected. Administration of 1 x 10(-7) mmol/l aldosterone does not influence COX expression, while application of 100 mmol/l NaCl increases COX 2 fourfold. The upregulation of COX 2 by a chronic NaCl load in embryonic epithelia suggests in part a constitutive and in part a facultative expression during CD cell differentiation.

Animals↗

PubMed2001

Role of renocortical cyclooxygenase-2 for renal vascular resistance and macula densa control of renin secretion.

This study aimed to assess the role of cyclooxygenase-2 (COX-2)-derived prostanoids for the macula densa control of renal afferent arteriolar resistance and for renin secretion. For this purpose, studied were the effects of blocking macula densa salt transport by the loop diuretic bumetanide (100 microM) on renal perfusate flow and on renin secretion in isolated perfused rats, in which renocortical COX-2 expression was prestimulated in vivo by treatment with the angiotensin-converting enzyme inhibitor ramipril, with low-salt diet, or with a combination of both. These maneuvers stimulated COX-2 expression in an order of ramipril + low salt>> low salt > ramipril > controls. Flow rates through isolated kidneys at a constant pressure of 100 mmHg were dependent on the pretreatment regimen, in the way that they went in parallel with COX-2 expression. The COX-2 inhibitor NS-398 (10 microM) lowered flow rates depending on the COX-2 expression level and was most pronounced therefore after pretreatment with low salt + ramipril. NS-398 did not change the increase of flow in response to bumetanide but attenuated the stimulation of renin secretion in response to bumetanide in a manner depending on the expression level of COX-2. These findings suggest that in states of increased renocortical expression of COX-2, overall renal vascular resistance and the macula densa control of renin secretion become dependent on COX-2-derived prostanoids.

Angiotensin-Converting Enzyme Inhibitors↗