Search

Objectives: Mesenchymal–epithelial interactions play a pivotal role in tubular morphogenesis and in maintaining the integrity of the kidney. During renal repair, similar mechanisms may regulate cellular reorganization and differentiation. We have hypothesized that soluble factors from proximal tubular epithelial cells (PTC) induce differentiation of adipose-derived adult mesenchymal stem cells (ASC). This hypothesis has been tested using cultured ASC and PTC. Material and methods: Conditioned medium was prepared from injured PTC and transferred to ASC cultures. ASC proliferation was analysed by a fluorometric and photometric assay. Signal transduction was analysed by phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2). Grade of ASC differentiation was assessed by morphological analysis and cell expression of characteristic markers. Results: Conditioned medium significantly induced proliferation and phosphorylation of ERK1/ERK2 of ASC. After 12 days of incubation, cell morphology changed to an epithelial-like monolayer. Expression of cytokeratin 18 was induced by conditioned medium, while α-smooth muscle actin, CD49a and CD90 expression decreased. These alterations strongly indicate onset of the differentiation process to the epithelial lineage. In summary, soluble factors from PTC induce signal transduction and differentiation of ASC. Conclusions: Our study shows that conditioned medium from renal tubular epithelial cells provides a convenient source of inductive signals to initiate differentiation of ASC towards epithelial lineage. We deduce that these interactions may play an important role during renal repair mechanisms. [ABSTRACT FROM AUTHOR]

Lymphocyte infiltration through vascular endothelium is one important step in the course of graft rejection. To investigate this process more exactly we established a monolayer invasion assay which enabled us to discriminate between adherent and penetrated cells. Detailed studies of adhesion and penetration kinetics of peripheral blood lymphocytes (PBL) acting on allogeneic human umbilical vein endothelial cells (HUVEC) were carried out by combined phase contrast and reflection interference contrast microscopy. Between 30 and 35% of all PBL attached to HUVEC after 4 hr. Out of these less than 10% penetrated. When HUVEC were prestimulated for 2 hr by interferon (IFN)-α, -β, -γ or interleukin (IL)-1, PBL adhesion in the early phase of cellular attachment to endothelial cells was accelerated. Overall adhesion however did not increase. Long-term pretreatment of HUVEC for 72 hr with IFN-γ or IL-1 also modified PBL-HUVEC interactions. However, a 72-hr pretreatment with IFN-α or -β did not influence lymphocyte binding behaviour. PBL penetration was not only accelerated but also enhanced by IFN-α, -β, -γ, irrespective of whether HUVEC were prestimulated for 2 hr or PBL and cytokines were added simultaneously to HUVEC. On the other hand IL-1 was not able to enhance the amount of penetrated cells but only accelerated the infiltration process. Up-regulation or de novo expression of the adhesion molecules ICAM-1 (intercellular adhesion molecule), ELAM-1 (endothelial leucocyte adhesion molecule) and VCAM-1 (vascular cell adhesion molecule) did not parallel PBL binding kinetics. Therefore an ICAM-, ELAM- and VCAM-independent modulation in the early phase of lymphocyte attachment to endothelium seems likely. The lymphocyte cytoskeleton may have a role in this process. [ABSTRACT FROM AUTHOR]

Background: For biotechnological use of cells in tissue engineered applications, such as biohybrid renal devices, optimal culture conditions are required. Oxygen delivery is one of the most important cell determined system criterion for ex vivo applications. It is involved in the maintenance of highly oxygen-dependent renal tubular epithelial cells, affecting metabolic state, differentiation, and desired transport functions. The purpose of this study was to examine respiratory patterns such as basal oxygen consumption, solute transport-related oxygen demand, and oxygen concentration-dependent oxygen uptake of renal tubular epithelial cells in vitro., Methods: Respiratory patterns of highly purified human primary renal proximal (hPTC) and early distal tubular cells (hTALDC) were analyzed by perfusion respirometry. Spontaneous oxygen consumptions and maximum respirations after carbonyl cyanide m-chlorophenyl hydrazone (CCCP) uncoupling were measured. Respiration fractions contributing to basolateral Na(+) /K(+) -ATPase transport activities were assessed via ouabain inhibition and Na(+) -free medium. Furthermore, we determined oxygen uptake in dependency of oxygen concentration and morphology in various culture conditions (shaken, static)., Results: Respiration of solely hPTC strongly depended on oxygen concentration in a Michaelis-Menten pattern at noncritical oxygen concentrations. Respiration of both cell types was significantly increased by CCCP, whereas average Na(+) /K(+) -ATPase-based oxygen uptake fractions differ significantly between the two cell types. Nevertheless, no significant differences were found in spontaneous respiration between hPTC and hTALDC., Conclusions: Our results clearly indicate that cell-specific oxygen consumption parameters have to be considered in the design of biotechnological devices intended to support kidney function by cell-supported renal replacement therapy., (Copyright © 2011 American Institute of Chemical Engineers (AIChE).)

The molecular mechanisms underlying the aging process are still unclear, but the clonal accumulation of mitochondrial deletion mutants is one of the prime candidates. An important question for the mitochondrial theory of aging is to discover how defective organelles might be selected at the expense of wild-type mitochondria. We propose that mitochondrial fission and fusion events are of critical importance for resolving this apparent contradiction. We show that the occurrence of fusions removes the problems associated with the idea that smaller DNA molecules accumulate because they replicate in a shorter time--the survival of the tiny (SOT) hypothesis. Furthermore, stochastic simulations of mitochondrial replication, mutation and degradation show that two important experimental findings, namely the overall low mosaic pattern of oxidative phosphorylation (OXPHOS) impaired cells in old organisms and the distribution of deletion sizes, can be reproduced and explained by this hypothesis. Finally, we make predictions that can be tested experimentally to further verify our explanation for the age-related accumulation of mitochondrial deletion mutants.

The fluorescent dyes DASPMI and rhodamine 6 GO specifically stain mitochondria in living cells. Dye concentrations from 10(-8) to 5 X 10(-6) mole l-1 can be used. Excitation and emission spectra, and quantum efficiency of DASPMI depend on solvent characteristics. Spectra of mitochondria in living cells correspond to those in phospholipids (excitation around 470 nm, emission 560-570 nm). Fluorescence intensity of DASPMI is a measure for the energization of mitochondria, as revealed by in vitro studies. In living cells uptake of the dye is strongly influenced by inhibitors of oxidative phosphorylation (i.e. by oligomycin, FCCP). Distribution of fluorescence intensity indicates an intracellular gradient in energy load of endothelial cells. Single mitochondria exhibit oscillations in fluorescence. Mitochondria loaded with DASPMI release the dye suddenly into the cytoplasm on treatment with FCCP. Cyanide and antimycin however, do not diminish fluorescence in vivo under optimal nutritional conditions, while they do so in mitochondrial suspension, indicating different mitochondrial behaviour in vivo and in suspension.

In perfused rat hearts alterations of aortic flow and mitochondrial membrane potential resulting from uncoupling of oxidative phosphorylation, hypoxia and treatment with a cardioprotective drug (2-mercaptopropionylglycine (MPG) have been studied. Mitochondrial membrane potential was followed by surface fluorimetry on DASPMI stained hearts. This fluorochrome specifically stains mitochondria in living cells; fluorescence intensity is related to the electrochemical gradient. Aortic flow turned out to be a much more sensitive indicator of heart function than ventricular pressure or mitochondrial membrane potential. No direct relationship exists between mitochondrial membrane potential and ATP production under the different metabolic conditions. Two phases of hypoxic mitochondrial damage have been deduced: the first results in derangement of ATP synthases while membrane potential is maintained, the second in irreversible damage of mitochondrial membranes with loss of membrane potential.