Subtotal nephrectomy impairs ischemia-induced angiogenesis and hindlimb re-perfusion in rats.

Kidney disease is associated with increased cardiovascular morbidity, but underlying mechanisms are poorly understood. We tested the hypothesis that chronic renal insufficiency impairs angioadaptation in a rat model of hindlimb ischemia. Twenty male Sprague-Dawley rats (8 weeks old) underwent subtotal nephrectomy (5/6SNX) or sham surgery (each n=10). Ten weeks later, unilateral hindlimb ischemia was induced in all animals. Hindlimb perfusion was assessed by laser Doppler perfusion imaging and fluorescent microsphere injection studies 2 weeks after surgery. Ischemia-induced angiogenesis was measured by analyzing capillary density using CD31 immunofluorescence. Expression of vascular endothelial growth factor (VEGF), its receptors (VEGFRs) and inducible as well as endothelial nitric oxide (NO) synthase was measured by real-time reverse transcription-polymerase chain reaction. Laser Doppler hindpaw perfusion was significantly reduced in 5/6SNX compared to sham-operated animals. Impaired hindlimb re-perfusion in 5/6SNX vs control rats was confirmed by fluorescent microsphere injection studies (relative perfusion of ischemic vs non-ischemic limb: 68.9+/-6.4 vs 92.4+/-3.6%, P=0.005). Ischemic skeletal muscle neovascularization increased to a greater extent in sham-operated compared to 5/6SNX rats (69+/-8 vs 29+/-7%, P<0.05). VEGF and VEGFR-1/2 mRNA expression increased in ischemic hindlimbs of control rats, whereas no change or a decrease was observed in 5/6SNX. In contrast, inducible and endothelial NO synthase expression did not significantly differ between sham and 5/6SNX rats. Chronic renal insufficiency impairs angiogenesis and limb perfusion in a rat hindlimb ischemia model. Impaired angioadaptation may contribute to the poor prognosis of patients with renal failure suffering from peripheral arterial disease.

Fibrillin-1 regulates mesangial cell attachment, spreading, migration and proliferation.

The microfibrillar protein fibrillin-1 is present in many organs, including the vasculature, eye, and dermis, and is thought to convey structural anchorage and elastic strength. Fibrillin-1 is also a component of the mesangial matrix. To assess the functional relevance of fibrillin-1 for cell-matrix interactions in the glomerulus, we studied the attachment, spreading, migration and proliferation of mesangial cells on fibrillin-1 and the regulation of fibrillin-1 in experimental anti-Thy1.1 nephritis displaying mesangial cell migration and proliferation in vivo. During the acute phase of experimental Thy1.1 nephritis, glomerular fibrillin-1 messenger ribonucleic acid expression and protein immunoreactivity were significantly induced as compared to controls. In a hexosaminidase-based adhesion assay, mesangial cells showed concentration-dependent attachment to fibrillin-1, similar to what was observed for fibronectin. The cell attachment was Arg-Gly-Asp dependent. Further, fibrillin-1 significantly promoted spreading and focal contact formation detected by immunostaining for vinculin. Mesangial cell migration, assessed by a transmigration assay, and proliferation, measured by a 5-bromo-2'-deoxy-uridine incorporation assay, were augmented by fibrillin-1. In diabetic mice underexpressing fibrillin-1, glomerular cell proliferation, determined by counting proliferating cell nuclear antigen-positive cells in renal sections, was significantly lower than in diabetic control mice. We conclude that fibrillin-1 promotes mesangial cell attachment, spreading, migration, and proliferation. We speculate that fibrillin-1 may thus contribute to mesangial hypercellularity during glomerular disease.

Angiotensin II type 1 receptor blockade prevents lethal malignant hypertension: relation to kidney inflammation.

BACKGROUND: Angiotensin II is elevated in malignant hypertension. We tested the hypothesis that angiotensin II type 1 receptor blockade can prevent the development of malignant hypertension even in the absence of a blood pressure-lowering effect. METHODS AND RESULTS: Two-kidney, 1-clip rats were followed up for 28 days; blood pressure was measured by tail-cuff plethysmography and intra-arterially. After a 2-week run-in phase, rats received valsartan at a dose of 0.3 (n=14) or 3 (n=12) mg. kg(-1). d(-1) or solvent (n=27). Only the higher dose of valsartan, but not the lower dose, decreased blood pressure. Both doses of valsartan prevented the development of lethal malignant hypertension. Twenty of 27 solvent-treated renovascular hypertensive rats died, but only 3 of 14 rats treated with the low dose and 1 of 12 rats treated with the high dose of valsartan died. Histological signs of malignant nephrosclerosis were found in all rats examined that had died throughout the study and in 6 of 7 surviving solvent-treated renovascular hypertensive animals. Increased expression of monocyte chemoattractant protein-1 and prominent interstitial influx of macrophages occurred in the nonclipped kidneys exposed to high pressure in solvent-treated rats. These alterations were prevented by valsartan at both doses, irrespective of blood pressure effects. CONCLUSIONS: Angiotensin II type 1 receptor blockade by valsartan prevents lethal malignant hypertension independently of blood pressure. The results suggest that reduction of angiotensin-induced inflammation in the kidney may contribute to the protective effects of valsartan.

Inducible nitric oxide synthase and glomerular hemodynamics in rats with liver cirrhosis.

This study was designed to test the hypothesis that glomerular de novo expression of inducible nitric oxide synthase (iNOS) contributes to renal hemodynamic abnormalities in liver cirrhosis developed 3 wk after common bile duct ligature (CBDL). De novo expression of iNOS mRNA was detected by RT-PCR in RNA extracts from isolated CBDL rat glomeruli whereas no iNOS mRNA was found in control rat glomerular RNA. Immunohistochemical staining for iNOS was negative in control animals whereas, in CBDL rats, positive iNOS staining was detected in an apparently mesangial pattern in all glomeruli. Western blots of protein extracts from isolated glomeruli of CBDL rats, but not control animals, showed a prominent iNOS band of 130 kDa. Mean arterial pressure (MAP), renal plasma flow (RPF; p-aminohippurate clearance), and glomerular filtration rate (GFR; inulin clearance) were unaltered in CBDL rats, but the application of 4 mg/kg L-N(6)-(1-iminoethyl)lysine, a specific inhibitor of iNOS, reduced GFR and RPF significantly in CBDL rats, whereas control animals were not affected. Similar results were obtained with lipopolysaccharide (LPS)-pretreated animals, which were studied as a positive control for iNOS expression and as a model for recent iNOS induction. We conclude that de novo expression of iNOS occurs in glomeruli of rats with liver cirrhosis and that nitric oxide, generated by iNOS, contributes to the maintenance of glomerular filtration in the early state of this disease.

Glomerular osteopontin expression and macrophage infiltration in glomerulosclerosis of DOCA-salt rats.

Expression of the chemoattractant osteopontin (OPN) may contribute to macrophage infiltration in many types of tubulointerstitial kidney disease, but the role of OPN in chronic glomerulosclerosis is unknown. We hypothesized that glomerular OPN expression and macrophage infiltration occur in deoxycorticosterone acetate (DOCA)-salt glomerulosclerosis in rats. Uninephrectomized rats receiving DOCA pellets and 1% saline were compared with control rats. OPN mRNA was determined by Northern blot, and OPN protein was determined by Western blot. The localization of OPN was studied by in situ hybridization and double immunohistochemistry with glomerular cell markers. Macrophage infiltration was quantified by counting ED-1-positive cells, and semiquantitative glomerulosclerosis scores were obtained. In DOCA-salt rats, OPN mRNA in the kidney was increased 2-fold over control after 9 days and 3 weeks and 20-fold after 6 weeks. Tubulointerstitial OPN staining was apparent after 21 days of DOCA treatment. Glomerular OPN mRNA and protein was detected after 42 days in parietal and visceral epithelial cells, activated myofibroblasts, and occasionally mesangial cells. Progressive glomerular macrophage infiltration occurred during the development of DOCA hypertension, paralleling the degree of glomerulosclerosis. Glomeruli staining positive for osteopontin contained more macrophages (18.4 +/- 3.4 per cross-section) than osteopontin-negative glomeruli (3.6 +/- 0.5; P < 0.05). Glomerular OPN expression occurs in chronic hypertensive glomerulosclerosis and is associated with macrophage infiltration. The data suggest a role for OPN as a chemoattractant in hypertensive glomerulosclerosis.

Photosynthetic performance of the Atlantic brown macroalgae, Cystoseira abies-marina, Dictyota dichotoma and Sargassum vulgare, measured in Gran Canaria on site.

Photosynthetic performance was determined in three common Atlantic brown macroalgae, Cystoseira abies-marina, Dictyota dichotoma and Sargassum vulgare, in Gran Canaria, Canary Islands, on site. The photosynthetic quantum yield was measured with both a portable PAM instrument on site and a diving PAM under water in the habitat. In parallel, solar radiation was measured continuously above and under water by means of two three-channel dosimeters, ELDONET (Real Time Computer, Möhrendorf, Germany), in three wavelength ranges, UV-A, UV-B and PAR. The effective photosynthetic quantum yield decreased in all species in response to exposure to 15 min of solar radiation but recovered in the subsequent shade conditions within several hours. A 30-min exposure caused an even more profound photoinhibition from which the algae recovered only partially. Most of the effect was due to visible radiation, however, the UV wavelength range, and especially UV-B, considerably enhanced the decrease in photosynthetic quantum yield. In all species except Sargassum a significant photoinhibition was detected at their growth sites at high solar angles in the water column, measured with the diving PAM.

Monocyte chemoattractant protein-1 and macrophage infiltration in hypertensive kidney injury.

BACKGROUND: We investigated whether monocyte chemoattractant protein-1 (MCP-1) is expressed in hypertensive nephrosclerosis, and tested the effect of angiotensin II type 1 receptor blockade on MCP-1 expression and macrophage (MPhi) infiltration. METHODS: Rats with two-kidney, one-clip (2K1C) hypertension with and without treatment with the angiotensin II type 1 receptor antagonist valsartan (3 mg/kg/day) were studied. In these animals as well as in spontaneously hypertensive rats (SHR), stroke-prone SHR (SHR-SP), hypertensive mRen-2 transgenic rats (TGR), and respective control strains, MCP-1 expression in the kidney was investigated by Northern and Western blots and by immunohistochemistry. Glomerular and interstitial MPhis were counted. RESULTS: In the nonclipped kidney of 2K1C rats, MCP-1 expression was elevated at 14 and 28 days when significant MPhi infiltration was present. MCP-1 was localized to glomerular endothelial and epithelial cells, interstitial and tubular cells, MPhis, and vascular smooth muscle cells. A similar pattern of MCP-1 staining was present in TGR kidneys, whereas MCP-1 expression was not increased in SHR and SHR-SP. Valsartan reduced but did not normalize blood pressure, blocked the induction of MCP-1 protein in 2K1C kidneys, and decreased interstitial MPhi infiltration significantly. CONCLUSION: MCP-1 expression is increased in angiotensin II-dependent models of hypertensive nephrosclerosis and is temporally and spatially related to MPhi infiltration. The angiotensin II type 1 receptor mediates the induction of MCP-1.

On site photosynthetic performance of Atlantic green algae.

Photosynthetic performance was measured on site in four common Atlantic green algae, Asparagopsis taxiforme, Valonia utricularia, Caulerpa racemosa and Codium taylori, in Gran Canaria, Canary Islands. The photosynthetic quantum yield was determined with a portable PAM instrument and with a diving PAM in the water column. Solar radiation was measured continuously above and in the water column by means of two three-channel dosimeters, ELDONET (Real Time Computer, Möhrendorf, Germany), in the UV-A, UV-B and PAR ranges. The effective photosynthetic quantum yield was found to be affected by exposure to solar radiation in as short as 15 min but recovered in the shade in most species within several hours. Only A. taxiforme failed to recover completely, and a 30-min exposure caused severe photoinhibition from which the algae recovered only partially. While most of the effect was due to the PAR wavelength range, the UV, and especially the UV-B, range considerably enhanced photoinhibition. In all four species, a significant inhibition was found even at their growth sites in the water column, measured with the diving PAM, at high solar angles.

Physical characterization of gravitaxis in Euglena gracilis.

Gravitaxis in unicellular microorganisms like Euglena gracilis has been known for more than 100 years. The current model explains this phenomenon on the basis of a specific density difference between cell body and surrounding medium. In order to test the feasibility of the current model in terms of physical considerations the specific density of different Euglena gracilis cultures was determined. Depending on the culture conditions the specific density was in a range between 1.046 g mL-1 and 1.054 g mL-1. Size and gravitaxis measurements were performed in parallel, which allowed to relate the force applied to the lower membrane to the kinetic properties of gravitactic reorientation. A linear relationship between force and gravitaxis kinetics was found. A comparison between estimated activation energy of the proposed stretch-sensitive ion channels and energy supplied by the displacement of the lower membrane by the sedimentation of the cell body revealed that a focusing, an amplification and/or an integration period over time must be involved in the gravitactic signal transduction chain. Analysis of stimulus-response curves revealed an integration period of about 5 seconds before a gravitactic reorientation starts. The kinetics of gravitaxis at 1 x gn, and 0.12 x gn, was found to be similar. A hypothesis is presented that explains this finding on the basis of a combination of an integration period and an all-or-none reaction during gravitactic reorientation.

Circadian rhythm of gravitaxis in Euglena gracilis.

Euglena gracilis, a unicellular, photosynthetic flagellate is a model system for environmentally controlled behavioral reactions. One pronounced reaction is the orientation with respect to gravity. In synchronized cultures with no cell growth a distinct circadian rhythm of negative gravitactic orientation could be observed. The main maximum of sensitivity was detected 5 h after the beginning of the subjective day, the main minimum 5 h before the beginning of the subjective day. Transferring synchronized cultures to continuous light resulted in an almost instantaneous loss of rhythmicity. In contrast, after transfer to permanent darkness cells exhibited a circadian rhythm with a progressive shortening of the period for more than 5 days. These findings are in contrast to the circadian rhythm of phototaxis in Euglena, where a free-running period of 24 h was observed. Parallel measurements of negative gravitactic orientation, velocity, cell shape as well as cAMP concentration in synchronized cultures revealed a circadian rhythm of all reactions. The results are discussed with regard to the possible role of cell shape and cAMP in gravitactic orientation.

Long-term cultivation of the flagellate Euglena gracilis.

Euglena gracilis, a unicellular photosynthetic flagellate, serves as a model system in signal transduction research. To further study its complex gravitaxis, experiments under microgravity are desirable. In preparation for long-term experiments on a space station, an autonomous cultivation unit has been developed and the culture conditions and surveillance methods have been established. The running time of more than 600 d under closed conditions with light as the only source of energy confirmed the stability of the Euglena population and gave new insights into its behavior. Physicochemical parameters such as oxygen concentration, temperature and pH as well as physiological parameters including cell density, motility, gravitactic orientation and pigmentation were recorded on a frequent basis. The suitability of the botanical bioreaction to serve as an oxygen supplier for animals in a closed system was demonstrated.

A rapid, simple method for the isolation and characterization of the photoreceptor of Dictyostelium discoideum amoebae.

A membrane-bound 45.5 kDa protein has been isolated from Dictyostelium discoideum amoebae. It shows an absorption spectrum, which closely resembles the action spectrum for amoebal phototaxis, leading to the conclusion that this protein might play an important role in the photoreception of Dictyostelium amoebae. For further characterization we employed phase partition in an aqueous polymer two-phase system, which was developed by Widell and Larsson for the separation of plasma membrane proteins of higher plants. This method clearly shows that the 45.5 kDa protein is a plasma membrane protein and not an intracellular protein. Furthermore, by using phase systems with increasing polymer concentrations, this simple and rapid purification of plasma membrane proteins allowed us to isolate the putative photoreceptor in one single step. Compared to standard biochemical methods phase partition provides an enormous facilitation of the isolation of D. discoideum membrane proteins.

Blue Light Induces Phosphorylation at Seryl Residues on a Pea (Pisum sativum L.) Plasma Membrane Protein.

We have partially characterized the blue-light-stimulated in vitro phosphorylation of a membrane protein from etiolated Pisum sativum L. stems. Properties of the response have implicated its involvement in signal transduction of phototropic stimuli (T.W. Short, W.R. Briggs [1990] Plant Physiol 92: 179-185; P. Reymond, T.W. Short, W.R. Briggs [1992] Proc Natl Acad Sci USA 89: 4718- 4721). Analysis of proteolysis products and phosphoamino acidanalysis indicate that the substrate protein is phosphorylated on multiple seryl residues. Kinetics of the in vitro reaction show phosphorylation to be complete within 2 to 5 min at 30[deg]C in either light-exposed or dark-control plasma membrane preparations, regardless of whether the membranes were first solubilized in Triton X-100. Nucleotide competition assays show the kinase to be ATP specific. The pH optimum covers a broad range with a maximum near 7.5. A wide array of salts inhibits the phosphorylation at high concentrations, but millimolar concentrations of Mg2+ are required to form Mg.ATP complexes for maximal activity, whereas excess free Mg2+ or Ca2+ are not required for the reaction.