Thymoquinone protects renal tubular cells against tubular injury

In this work the effect of angiotensin II (AT II) on proximal tubular epithelial cells (pTECs) in vitro was studied. AT II was found to activate the nuclear factor kappaB (NF-kappaB) and its controlled genes, for example, interleukin 6 (IL-6) of pTECs in a time-dependent manner. Two points with maximum NF-kappaB activation were found, the first after 12 h and the second after 3.5 days. The first point may be due to activation of NF-kappaB in pTECs in response to AT II while the second may be due to activation of the advanced glycation end product (AGE)/receptor of the AGE (RAGE) system. Thymoquinone (TQ) was found to decrease NF-kappaB activation in a dose-dependant manner with maximum inhibitory effect at a concentration of 500 nM. Also, pre-incubation of pTECs with TQ leads to disappearance of the second peak of NF-kappaB. These data are consistent with results obtained from IL-6 enzyme-linked immunosorbent assay (ELISA) and transient transfection experiments. The results explain the therapeutic value of TQ which can be used to delay end stage renal diseases in diabetics.     

Salmonella-induced tubular networks

Salmonella virulence relies on its capacity to replicate inside various cell types in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). A unique feature of Salmonella-infected cells is the presence of tubular structures originating from and connected to the SCV, which often extend throughout the cell cytoplasm. These tubules include the well-studied Salmonella-induced filaments (SIFs), enriched in lysosomal membrane proteins. However, recent studies revealed that the Salmonella-induced tubular network is more extensive than previously thought and includes three types of tubules distinct from SIFs: sorting nexin tubules, Salmonella-induced secretory carrier membrane protein 3 (SCAMP3) tubules and lysosome-associated membrane protein 1 (LAMP1)-negative tubules. In this review, we examine the molecular mechanisms involved in the formation of Salmonella-induced tubular networks and discuss the importance of the tubules for Salmonella virulence and establishment of a Salmonella intracellular replicative niche.     

Engineering tubular bone constructs

Cell-sheet techniques have been proven effective in various soft tissue engineering applications. In this experiment, we investigated the feasibility of bone tissue engineering using a hybrid of mesenchymal stem cell (MSC) sheets and PLGA meshes. Porcine MSCs were cultured to a thin layer of cell sheets via osteogenic induction. Tube-like long bones were constructed by wrapping the cell sheet on to PLGA meshes resulting in constructs which could be cultured in spinner flasks, prior to implantation in nude rats. Our results showed that the sheets were composed of viable cells and dense matrix with a thickness of about 80-120 microm, mineral deposition was also observed in the sheet. In vitro cultures demonstrated calcified cartilage-like tissue formation and most PLGA meshes were absorbed during the 8-week culture period. In vivo experiments revealed that dense mineralized tissue was formed in subcutaneous sites and the 8-week plants shared similar micro-CT characteristics with native bone. The neo tissue demonstrated histological markers for both bone and cartilage, indicating that the bone formation pathway in constructs was akin to endochondral ossification, with the residues of PLGA having an effect on the neo tissue organization and formation. These results indicate that cell-sheet approaches in combination with custom-shaped scaffolds have potential in producing bone tissue.     

Scale-up of tubular photobioreactors

The effect of the light/dark cycle frequency on theproductivity of algal culture at differentday-averaged irradiance conditions was evaluated forPhaeodactylum tricornutum grown in outdoortubular photobioreactors. The photobioreactor scale-upproblem was analyzed by establishing the frequency oflight–dark cycling of cells and ensuring that thecycle frequency remained unchanged on scale-up. Thehydrodynamics and geometry related factors wereidentified for assuring an unchanged light/dark cycle.The light/dark cycle time in two different tubularphotobioreactors was shown to be identical when thelinear culture velocity in the large scale device(U _LL) and that in the small scale unit (>U _LS)were related as follows:U_LL = \frac f ^9/7 \alpha^8/7 U_LS.Here f is the scale factor (i.e., the ratio oflarge-to-small tube diameters), is afunction of the illuminated volumes in the tworeactors, and `dark' refers to any zone of the reactorwhere the light intensity is less than the saturationvalue. The above equation was tested in continuouscultures of P. tricornutum in reactors with 0.03 mand 0.06 m diameter tubes, and over the workableculture velocity range of 0.23 to 0.50 m s^-1. Thepredicted maximum realistic photobioreactor tubediameter was about 0.10 m for assuring a cultureperformance identical to that in reactors with smaller tubes.     

Cytotoxicity of alkylating agents in isolated rat kidney proximal tubular and distal tubular cells

Patterns of chemical-induced cytotoxicity in different regions of the nephron were studied with freshly isolated proximal tubular and distal tubular cells from rat kidney. Three model alkylating agents, methyl vinyl ketone, allyl alcohol, and N-dimethylnitrosamine, were used as test chemicals. Methyl vinyl ketone and a metabolite of allyl alcohol, acrolein, are Michael acceptors that bind to cellular protein sulfhydryl groups and GSH. N-Dimethylnitrosamine binds to cellular protein and DNA. Lactate dehydrogenase leakage was used to assess irreversible cellular injury. Distal tubular cells were more susceptible than proximal tubular cells to injury produced by methyl vinyl ketone or allyl alcohol while the two cell populations were equally susceptible to injury produced by N-dimethylnitrosamine. Preincubation of both proximal tubular and distal tubular cells with GSH protected them from methyl vinyl ketone- and allyl alcohol-induced cytotoxicity but had no effect on N-dimethylnitrosamine-induced cytotoxicity. Similarly, incubation of cells with methyl vinyl ketone or allyl alcohol, but not N-dimethylnitrosamine, altered cellular GSH status. As with GSH status, incubation of cells with methyl vinyl ketone or allyl alcohol, but not N-dimethylnitrosamine, caused pronounced inhibitory effects on mitochondrial function, as evidenced by ATP depletion and inhibition of cellular oxygen consumption. These results demonstrate that alkylating agents are cytotoxic to both proximal tubular and distal tubular cells, and that interaction with cellular GSH is a factor determining nephron cell type specificity of injury.     

Design of scraped tubular fermentors

Conventional stirred-tank fermentors are inefficient in carrying out certain fermentation processes because of one or more of the following constraints: media backmix–flow, solids wall–deposits, microbial growth–disruption. Two series of novel design of aerated scraped tubular fermentors have been developed to over come the deleterious effects of these constraints. One design is based on a horizontal tube fitted with an internal mechanical wall-scraper that also promotes media segregation; the other design is based on a vertical array of vessels interconnected by small gas-jetting orifices that promote media segregation and clean-surface operation. Tests with cultures of Trichdorma viride (for single-cell protein production) and Candida lipolytica (for lipase production) have been carried out. It is shown that these novel fermentors can minimize the effects of catabolite repression inherent in both cultures and of wall growth in the former.     

Solution flow in tubular semi-permeable membranes

Summary A recent suggestion that the term volume flow should be used for the flow along sieve tubes when the pressure gradient involved is very small is considered to be unwarranted. It involves no new concepts and does not constitute any mechanism that is not implicit in the Münch theory.     

Renal tubular actions of ANF.

Many of the earliest investigations of the renal effects of atrial natriuretic factor (ANF) pointed to the glomerulus as a major site of the peptide's action. More recently, there have been many reports showing various effects of ANF on renal tubular epithelia, including collecting ducts, thick ascending limbs of Henle's loop, thin limbs of Henle's loops, and proximal tubules. The purpose of this review is to summarize the evidence for renal tubular actions of ANF and analyze it from the perspective of the specialized functions of the individual nephron segments, addressing the question: can renal tubule effects of ANF play a significant role in the precise day-to-day regulation of renal NaCl and water excretion? Based on these considerations, we propose that long-term renal tubular action of ANF may be distinct from its short-term natriuretic effect. The short-term action of ANF to accelerate salt and water excretion may play a role in the overall response to acute volume overload. This action of ANF appears to be largely due to an ANF-mediated increase in glomerular filtration rate accompanied by a blunting of the tubuloglomerular feedback mechanism, perhaps with some contribution from ANF-mediated inhibition of fluid absorption in the proximal tubule. In contrast, contributions of ANF to the precise day-to-day regulation of salt and water excretion are likely to be chiefly due to ANF-mediated inhibition of NaCl and water absorption in collecting ducts, but may also involve actions of ANF on the loop of Henle.     

Transcytosis in cultured proximal tubular cells

Summary Studies were designed to examine fluid-phase pinocytosis in proximal tubular cells. Canine proximal tubules were obtained from the band IV of Percoll^® gradient centrifugation of the dispersed renal cortex, and were seeded on collagen-coated polycarbonate membranes. Integrity of monolayers was confirmed by electrophysiologic measurements, and by scanning electron microscopy. At confluence cell monolayers were studied in Ussing chambers. The rate of transfer of a marker of fluidphase pinocytosis, Lucifer Yellow CH, from the luminal to the basolateral bath was three times higher than that occurring in the opposite direction. Fluorescence microscopy demonstrated that Lucifer Yellow was trapped exclusively in the vesicular compartment. Electron microscopy of the monolayers incubated with cationized ferritin added to the luminal or to the basolateral bath revealed that endocytic vesicles were formed only at the luminal surface. Luminal-to-basolateral transfer of Lucifer Yellow was almost completely blocked at 0°C, and was significantly diminished by K⁺ depletion. Transcytosis of Lucifer Yellow was stimulated twofold by 1-oleoyl-2-acetyl-glycerol. Transfer of quin-2 acetoxymethylester across the monolayer was used as a marker of the paracellular pathway, demonstrating the lack of directional selectivity of this transport route. In summary, vectorial fluid-phase pinocytosis in proximal tubular cells represents an additional mechanism contributing to fluid transport in this segment of the nephron.     

Solution flow in tubular semipermeable membranes

Summary Solution flow in tubular semipermeable membranes was studied as a model for assimilate transport in sieve tubes. A mass flow of solution was demonstrated both in closed turgid tubes and in open tubes without turgor pressure. These results can be explained in terms of hydrostatic and osmotic pressure differences across the semipermeable membrane without consideration of a decrease in hydrostatic pressure along the direction of solution flow. A theoretical model based on nonequilibrium thermodynamics is developed that is in fairly good quantitative agreement with the experimental results. Münch's original experiment demonstrating solution flow is analyzed and shown not to depend on a gradient of hydrostatic pressure but rather to depend on the same driving forces operative in these experiments. On the basis of these findings a volume-flow mechanism of phloem transport is proposed.Supported by the Deutsche Forschungsgemeinschaft and the U.S. National Science Foundation with research grants to the first two authors, and by a U.S. Public Health Service Research Career Development Award (K4-GM-21, 171) from the institute of General Medical Sciences to the third author.     

Presynaptic tubular structures in photoreceptor cells

Summary After the application of fixatives including phosphotungstic acid or a mixture of osmium tetroxide and zinc iodide, complex tubular structures are evident in the presynaptic side of the synapses between photoreceptor and bipolar cells of the rat's retina. In the first case only the limiting membranes are visualized, while in the second only the content of the tubules is stained. These tubules seem to be related, on a morphological ground, with the formation of synaptic vesicles. These tubular structures are not observed when fixation is done with osmium tetroxide or glutaraldehyde-osmium tetroxide.This work has been supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina, and from National Institutes of Health, U.S.A., (5 RO1 NS 06953-05 NEUA).We want to express our gratitude to Mrs. Haydée Agoff de Zimman and Mr. Alberto Saénz for their skillful technical assistance.