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Cellular hypoxia has been proposed as a major factor in the pathogenesis of chronic renal injury, yet to date there has been no direct evidence to support its importance. Therefore, we examined cortical hypoxia in an animal model of chronic renal injury (murine adriamycin nephrosis; AN) by assessing nuclear localization of the oxygen-dependent α-subunit of hypoxia-inducible factor-1 (HIF-1α) in animals 7, 14, and 28 days after adriamycin. Results were assessed in conjunction with quantitation of the cortical microvasculature (by CD34 immunostaining) and cortical expression of VEGF. Cortical apoptosis was also examined by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. A dramatic and significant increase in nuclear localization of HIF-1α was seen 28 days after adriamycin in the context of severe glomerular and tubulointerstitial damage. Areas of nuclear HIF-1α staining did not colocalize with areas of cellular apoptosis. AN was also associated with a significant attenuation of the peritubular capillaries that was significant at 14 and 28 days after adriamycin. Cortical VEGF expression fell in a stepwise manner from day 7 until day 28 after adriamycin. In conclusion, these data are consistent with a significant increase in cellular hypoxia occurring in the advanced stages of murine AN. Increased cortical hypoxia was preceded by significant reductions in both the number of peritubular capillaries (i.e., oxygen supply) and the angiogenic cytokine VEGF. Apart from providing the first direct evidence for cellular hypoxia in a model of chronic renal disease, these results suggest that a primary dysregulation of angiogenesis may be the cause of increased hypoxia in this model.
- capillary
- chronic
- tubular
cellular hypoxia in vitro has been shown to have effects potentially relevant to the pathogenesis of chronic renal injury. Hypoxia has profibrotic effects in vitro, invoking the transcription of matrix proteins (28, 29, 35), profibrotic growth factors (15, 28, 35), and endothelin-1 (ET-1) (23, 34). In addition, distinct hypoxia-dependent pathways may exist for the induction of apoptosis (6, 43). Since renal fibrosis and tubular atrophy are inevitable components of the tubulointerstitial disease that accompanies all progressive renal diseases, it has been suggested that cellular responses to hypoxia may represent a "final common pathway" for the development of these pathological changes (9, 10).
To date, supportive evidence for this theory has been indirect. Progressive renal diseases are associated with both a progressive loss of vascular structures (4, 21, 22, 26, 30) and an excess of vasoconstrictor substances such as ANG II and ET-1 (33, 52), both of which may decrease oxygen supply to the renal parenchyma. In conjunction with these changes, there are alterations in renal oxygen demand due to the processes of hypertrophy, atrophy, and loss of tissue components, all of which may be present in variable degrees. The net effect in terms of the timing and severity of tubular hypoxia, therefore, remains to be elucidated.
We previously reported a progressive reduction in binding of the hypoxia marker EF5 in murine adriamycin nephrosis, a model of human chronic renal disease (20a). Although this result could be interpreted as indicating less hypoxia in this model, we were unable to correct for alterations in delivery or binding of the EF5 marker occurring as a consequence of renal injury. To further assess hypoxia in this model, we examined a potentially more reliable marker of cellular hypoxia [nuclear localization of the α-subunit of hypoxia-inducible factor-1 (HIF-1α)]. HIF-1 is the prototypical factor involved in transcriptional responses to cellular hypoxia (46). While the β-subunit of HIF-1 is hypoxia independent, the α-subunit is stabilized in an oxygen-dependent manner (reviewed in Ref. 18). Stabilization of HIF-1α allows it to bind to the β-subunit and translocate to the nucleus, where it induces the transcription of multiple genes involved in homeostasis and disease pathogenesis. Detection of nuclear HIF-1α is therefore recognized as a marker of cellular hypoxia (20, 44, 50) that reflects local oxygen supply/demand ratios within the tissue.
Nuclear HIF-1α localization was examined at different time points in the course of murine adriamycin nephrosis by quantitative immunohistochemistry and Western blotting. The degree of nuclear HIF-1α accumulation was interpreted in the context of changes to the cortical microcirculation and measurement of the angiogenic growth factor VEGF.
Murine Adriamycin Nephrosis
BALB/c mice were cared for in the Department of Animal Care, Westmead Hospital, under the ethical guidelines outlined in the Code of Practice for the Care and Use of Animals for scientific purposes established by the Australian National Health and Medical Research Council (NHMRC). Six-week-old male inbred BALB/c mice, weighing 22–25 g, were kept under standard conditions with unrestricted access to food and water in a 12:12-h light-dark cycle. Murine adriamycin nephrosis (AN) (47) was established by a single injection of adriamycin (doxorubicin hydrochloride, Pharmacia and Upjohn, Perth, Australia) at a dose of 11 μg/g into the tail vein of 6-wk-old male BALB/c mice. Animals (6–8/time point) were killed at 7, 14, and 28 days after adriamycin injection. At death, samples of serum and bladder urine were collected. The kidneys were rapidly removed and transferred to a metal plate cooled on ice for sectioning. The kidney poles were snap frozen in liquid nitrogen and stored at −70°C. The remaining kidney was sectioned coronally. One-half of the kidneys were placed in cryomoulds, embedded in optimum cooling temperature compound (Tissue-Tek, Sakura Finetechnicals, Torrance, CA) by immersion in liquid nitrogen-cooled isopentane, and stored at −70°C. The other half was immersed in 10% neutral-buffered formalin for 24 h, dehydrated in graded alcohols, and embedded in paraffin. Creatinine was measured in serum and urine samples by the Jaffé reaction using a Hitachi 747 multianalyzer (Tokyo, Japan). The same machine was used to measure serum albumin by the bromocresyl green method and urine protein using the benzethonium chloride turbidimetric method.
Comparative Histology
Quantitative histological measurements were obtained from the outer cortex using a modification of previously published methods (40, 47). Five-micrometer paraffin-embedded sections were stained with periodic acid-Schiff (PAS). Thirty random cortical images were taken using an SV-microdigital camera (Sound Vision, Framingham, MA) fitted to an Axioskop microscope (Zeiss, Oberkochen, Germany). Blinded analysis was then made using image-analysis software (Optimas version 6.5, Media Cybernetics, Seattle, WA).
Twenty outer cortical glomeruli sectioned through the hilum were analyzed. The outline of the glomerular tuft was traced with a mouse for an estimate of tuft volume. As an index of glomerulosclerosis (GS%), a 24-bit color threshold was used to detect PAS-positive material within the glomerular tuft, and the proportion of PAS staining within the tuft was expressed as a percentage.
The volume fraction of the interstitium was estimated in 10 nonoverlapping fields using an 8 × 11 counting grid. The proportion of interstitial grid intersections was calculated and expressed as a percentage.
Tubular measurements were obtained from 50 randomly selected cortical tubules/animal. Using line morphometry, the tubular diameter was estimated as the shortest axis through the center of the imaged tubule. Tubular cell height was assessed using line morphometry by measuring the radial distance between the basement membrane and the lumen of a line drawn through the center of the largest nucleus visible in the tubular cross section of interest.
Immunostaining for HIF-1α
A well-characterized chicken polyclonal anti-HIF-1α antibody (5) was used as a primary antibody. Five-micrometer cryosections were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 10 min and incubated with a 1:50 concentration of primary antibody overnight at 4°C. A peroxidase-conjugated rabbit anti-chicken IgY antibody (1:100, Pierce) was used as a secondary antibody and peroxidase-conjugated goat anti-rabbit IgG (Dako, Carpinteria, CA) as a tertiary antibody. Antibody binding was localized using nickel-enhanced diaminobenzidine (Pierce). Kidney and liver tissue from mice exposed to in vivo hypoxia (10% O2, 6 h) was used as a positive control.
Double immunostaining was performed to look for evidence of HIF-1α nuclear staining within interstitial inflammatory cells. HIF-1α staining was performed as above with the substitution of a Texas red-conjugated goat anti-rabbit antibody (Molecular Probes) as the tertiary antibody. Following HIF-1α staining, sections were incubated with rat monoclonal antibodies against murine macrophages (clone F4/80, 1:100 dilution, BD Biosciences, San Diego, CA), murine CD4 cells (clone RM4–5, 1:100, BD Biosciences), and murine CD8 cells (clone 53–6.7, 1:100, BD Biosciences) for 1 h at room temperature. A fluorescein-conjugated goat Fab′ fragment to rat immunoglobulins (1:50, Serotec, Oxford, UK) was used as a secondary fluorescent reagent for the inflammatory cells.
Cortical nuclear staining of HIF-1α was analyzed semiquantitatively using image-analysis software (Optimas version 6.5). Ten random cortical images (each measuring 257 × 214 μm) were taken from each animal. Using the software, positively stained nuclei within the positive control tissue (hypoxic mouse liver) were used to set a 24-bit color "threshold" that recognized positively stained nuclei within the positive control image. When a suitable threshold was selected, it was then applied to the experimental slides to enable an unbiased comparison of the number of positive nuclei within the samples.
Western Blotting for HIF-1α
Nuclear proteins were extracted from snap-frozen samples of renal cortex as previously described (40). Aliquots (25 μg) of nuclear proteins were resolved on an 8% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The chicken IgY anti-HIF-1α (1:100) was used as a primary antibody and peroxidase-conjugated rabbit anti-chicken IgY antibody (1:1,000, Pierce) as the secondary antibody. Sites of antibody binding were revealed by immersion in an enhanced chemiluminescent substrate (ECL, Amersham) and exposure to autoradiographic film (Amersham). Comparative densitometry was performed using a Molecular Dynamics personal densitometer SI (Amersham) and ImageQuant image-analysis software (Amersham).
Colocalization of HIF-1α and Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling Staining
Apoptosis within tissue samples was examined by terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) staining. HIF-1α staining was performed as above using a Texas red-conjugated anti-rabbit antibody (Molecular Probes) as the tertiary antibody. TUNEL staining was then performed using a commercially available kit (in situ cell death detection kit, Roche Diagnostics, Mannheim, Germany). Fluorescent microscopy was performed using a Leica DM-LB fluorescent microscope (Wetzlar, Germany) fitted with a digital camera (Spot RT, Diagnostic Instruments, Sterling Heights, MI).
Assessment of Microvasculature
Immunostaining for murine CD34.
The distribution of the endothelial cell marker CD34 (7, 12, 39) within the cortex was examined by immunohistochemistry and image analysis. Five-micrometer paraffin-embedded sections were dewaxed. Antigen retrieval was performed for 20 min at 95–99°C using a commercial target retrieval buffer (Dako). A rat monoclonal antibody to mouse CD34 (1:400, MEC 14.7, Cedarlane Laboratories) was used for 1 h at room temperature. Biotin-conjugated rabbit anti-rat immunoglobulin (Dako) was used as a secondary antibody. Sites of secondary antibody binding were demonstrated using a streptavidin-peroxidase conjugate (Dako) with diaminobenzidine as the enzyme substrate.
Peritubular capillary density was estimated using an adaptation of previously published methods (21). Ten random outer cortical fields (measuring 257 × 214 μm) were obtained from each animal. The capillary rarefaction index (CRI) was then measured using image-analysis software (Optimas version 6.5). Using a 10 × 12 counting grid, the proportion of grid squares that did not contain CD34-staining capillaries was calculated and expressed as a percentage.
Western blotting for CD34.
Aliquots (50 μg) of cytoplasmic proteins were resolved on an 8% SDS-polyacrylamide gel and transferred to a nitrocellulose membranes. The MEC14.7 antibody was used at a concentration of 1:2,000, and biotinylated rabbit anti-rat IgG (1:2,000, Dako) was used as the secondary antibody. Sites of antibody binding were revealed by immersion in streptavidin-HRP (1:3,000, Dako) before chemiluminescent development. Comparative densitometry was performed as described above.
Assessment of Cortical VEGF
Immunostaining for VEGF.
Four-micrometer paraffin-embedded sections were incubated with a 1:50 dilution of goat polyclonal antibody against VEGF (P20, Santa Cruz Biotechnology, Santa Cruz, CA). Preliminary staining of acetone-fixed cryosections showed VEGF staining in the glomerular mesangium, cortical tubules, and vascular smooth muscle. The degree of glomerular VEGF staining was less prominent in paraffin-embedded sections but was restored if antigen retrieval was performed. Antigen retrieval was not performed for routine staining due to the minimal contribution of glomerular VEGF to the total.
Peroxidase-conjugated rabbit anti-goat antibody (1:200, Dako) was used as the secondary antibody and peroxidase-conjugated goat anti-rabbit antibody (1:100, Dako) as the tertiary antibody. Cortical VEGF staining was analyzed semiquantitatively using image-analysis software (Optimas version 6.5) using an adaptation of published methods (21). Ten random images (measuring 257 × 214 μm) were obtained from the outer cortex of each animal. Using the software, a 24-bit color threshold was used to identify positively stained areas in each image. The area of each image represented by positively stained areas was then calculated by the software and expressed as a percentage.
Western blotting for VEGF.
Aliquots (50 μg) of cytoplasmic proteins were resolved on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. Staining was performed using a 1:100 dilution of the primary antibody and peroxidase-conjugated rabbit anti-goat Ig (Dako, 1:2,000) as the secondary antibody. Comparative densitometry was performed as described above.
Statistical Comparisons
Statistical comparisons between animal groups were performed using a computer-based statistical package (SPSS version 8, SPSS, Chicago, IL). Statistical significance between groups was tested using one-way ANOVA and the least squares method of post hoc analysis. A P value of 0.05 was considered to represent statistical significance.
Functional and Pathological Effects of Murine AN
As previously described (47), these animals developed progressive renal disease characterized by proteinuria, hypoalbuminemia, and impaired renal function (Table 1). Three mice died after adriamycin treatment (2 in the 28-day group and 1 in the 14-day group). Measured values for serum creatinine at 7 days after adriamycin (5.8 ± 5.5 μM) were outside the linear range of the analyzer used (stated as 9–2,000 μM) and were excluded from analysis. Serum creatinine levels were statistically greater than control at 28 days after adriamycin. AN animals developed proteinuria, as evidenced by a stepwise increase in the urine protein/creatinine ratio that was significantly greater than control after 7 days.
Pathologically, this model was associated with progressive glomerular, tubular, and interstitial injury, all of which were severe at 28 days after adriamycin (Table 1). Glomerular changes observed included progressive glomerular enlargement and an increase in glomerulosclerosis. Progressive tubular atrophy was reflected by loss of the luminal brush border and a stepwise reduction in tubular cell height. The interstitium was expanded with a mononuclear cell infiltrate and increased PAS-positive matrix.
Immunostaining and Western Blotting for HIF-α
Strong nuclear staining of HIF-1α was seen in the kidney and liver tissue from animals exposed to 10% oxygen (Fig. 1A). In the normal kidney, there was faint staining in cortical nuclei, predominantly in distal tubules (Fig. 1B). Glomeruli demonstrated no nuclear staining, and proximal tubules showed faint nuclear staining only. No nuclear staining was seen in the medulla or papilla.
The pattern of nuclear staining of HIF-1α was significantly altered in AN. Although HIF-1α staining was not obviously different from control at days 7 and 14 after adriamycin, a dramatic increase in nuclear HIF-1α staining was seen in the renal cortex at 28 days after adriamycin (Fig. 1C). Nuclear HIF-1α staining was most apparent in areas of extensive tubular dilatation and interstitial inflammation. In these areas, nuclear HIF-1α staining was seen in both tubular and interstitial cells. Double fluorescent staining for HIF-1α and inflammatory cell-surface markers showed nuclear HIF-1α staining within macrophages, CD4 lymphocytes, and CD8 lymphocytes in cortical samples from animals obtained 28 days after adriamycin (Fig. 2).
Semiquantitative analysis of the number of positive nuclei per unit of cortical area reflected these observations (Fig. 1D). There was a dramatic increase in the number of positively stained nuclei at the 28-day time point (191 ± 115 vs. 9 ± 17 nuclei/unit area, P < 0.01). An identical pattern was seen when nuclear protein extracts were probed for HIF-1α (Fig. 1E).
Colocalization of HIF-1α and TUNEL Staining
TUNEL staining revealed apoptotic nuclei within the middle epithelial layers of the positive control tissue (murine esophagus) consistent with the normal process of keratinocyte maturation (Fig. 3A). Double staining revealed occasional apoptotic nuclei within the epithelial layers of the esophagus that also stained positive for HIF-1α, although the majority of apoptotic nuclei were negative (Fig. 3A). No apoptosis was visible in the normal kidney (not shown). Apoptotic nuclei were detectable after adriamycin. There were scanty apoptotic nuclei at day 7 after adriamycin, with apoptotic nuclei at days 14 and 28 after adriamycin largely confined to occasional epithelial cells lining atrophic tubules and sloughed nuclei present within the tubular lumen (Fig. 3, B and C). Double staining of the AN samples did not demonstrate areas of overlap of the HIF-1α or apoptotic stains (Fig. 3, B and C).
Assessment of Cortical Microvasculature
CD34 immunostaining of the normal renal cortex revealed a dense capillary network that in some areas appeared to completely encircle individual tubules (Fig. 4A). The glomerular capillary network also exhibited positive staining. A dense intertubular capillary network was also noted in the medulla and papilla (not shown). AN was associated with significant changes in the density of the cortical microvasculature. There was a significant reduction in visible capillaries from day 14 that was most prominent 28 days after adriamycin (Fig. 4B). The reduction in capillaries was particularly prominent in areas of interstitial expansion and tubular atrophy. In keeping with these observations, there was a significant increase in the capillary rarefaction index after adriamycin (Fig. 4C). Microvascular density was reduced threefold at 14 days after adriamycin (P < 0.01) and fivefold at 28 days (P < 0.01). Western blotting of protein extracts (Fig. 4D) showed a similar stepwise reduction in cortical CD34 expression that was significant at 14 and 28 days after adriamycin.
Immunostaining and Western Blotting for VEGF
Extensive tubular staining for VEGF was seen in the normal kidney, with accentuation of distal tubules (Fig. 5A). There was a reduction in tubular VEGF staining following adriamycin, particularly in areas of tubular damage or atrophy (Fig. 5B). Semiquantitative analysis of cortical VEGF expression reflected these changes. There was a significant reduction in cortical VEGF detectable at 7 days after adriamycin (Fig. 5C), with further stepwise reductions at later time points. Western blotting of cortical proteins also showed a similar decremental pattern of VEGF expression (Fig. 5D).
The results presented in this paper describe in detail the time course of cortical HIF-1α expression in murine AN, a robust model of human focal glomerulosclerosis. Increased expression and nuclear localization of HIF-1α were seen in the late stages of this model and were localized to tubular and interstitial cells in areas of extensive parenchymal injury. This finding represents the first direct evidence of hypoxia in an animal model of chronic renal disease. Increased HIF-1α expression in these experiments followed significant reductions in both peritubular capillary density (a surrogate measure of oxygen supply) and reduced cortical expression of the angiogenic growth factor VEGF. Areas of cellular hypoxia did not colocalize with areas of tubular apoptosis in this model.
Two separate methods were used to compare nuclear localization of HIF-1α in the current experiments (immunostaining and Western blotting). Consistent with previous reports, we found minimal nuclear staining of HIF-1α in the normal kidney (44, 45, 51). Cortical nuclear HIF-1α was significantly increased by whole animal hypoxia as well as in the later stages of AN. This pattern of nuclear localization is consistent with increased stabilization of HIF-1α in the advanced stages of this model. The cellular fate of HIF-1α is determined by the activity of a redox-sensitive pathway (reviewed in Ref. 18). In the presence of oxygen, the α-subunit undergoes prolyl hydroxylation and is targeted for proteasomic degradation. In the absence of oxygen, this hydroxylation does not occur, and after translocation to the nucleus, HIF-1α is free to bind to the β-subunit (the aryl hydrocarbon nuclear receptor translocator) to form the HIF-1 complex that, in turn, interacts with hypoxia-responsive elements in the promoter region of multiple genes. Other than hypoxia, a number of other stimuli may potentially affect the stabilization of HIF-1α in this model, including the proinflammatory molecules TNF-α, IL-1β (2, 17), and nitric oxide (NO) (19, 37). It is noteworthy, however, that the effects of TNF-α, IL-1β, and NO on HIF-1α stabilization are based on in vitro experiments. A consistent difficulty with the interpretation of these experimental systems is that these molecules may well exert their in vitro effects by influencing oxygen consumption, weakening the case for a direct effect of these stimuli on HIF-1α stabilization. Furthermore, extrapolation of the results obtained with the use of NO donors in vitro is made more complex by the fact that the production of NO in vivo is an oxygen-dependent process (16), and exogenous NO donors may themselves disrupt the redox-dependent process of HIF-1α stabilization in a way that is not relevant to NO produced in vivo (19, 37). The effects of endogenous TNF-α, IL-1β, and NO on HIF-1α stabilization in the current experiments are therefore uncertain.
The presence of increased nuclear HIF-1α in the later stages of this model is strongly suggestive of a significant increase in cellular hypoxia in advanced disease. Before these data were gathered, experimental evidence to support the presence of "chronic renal hypoxia" has been based largely on indirect findings, including the observed reduction in peritubular capillaries (4, 21, 22, 26, 30) and extrapolation of the profibrotic effects of hypoxia in vitro (28, 29, 35). The principal advantage of a HIF-1α-based method of hypoxia detection is that it gives direct evidence of transcriptionally relevant hypoxia at a cellular level. The HIF-1 pathway is activated in vivo when oxygen demand outweighs supply (18). This delicate balance cannot be inferred from microelectrode measurements that have previously been regarded as the "gold standard" for direct hypoxia measurement.
The finding of increased HIF-1α stabilization in AN warrants a consideration of whether HIF-1-dependent stimuli may contribute to the pathogenesis of renal injury in this model. Although hypoxia in vitro induces a wide range of profibrotic effects, including the stimulation of profibrotic growth factors (15, 28, 35), matrix proteins (28, 29, 35), and matrix stabilizers, it was notable in the current experiments that nuclear HIF-1α was increased only in the later stages of this model, at a time when interstitial fibrosis was already present. This temporal association makes it unlikely that hypoxia contributes significantly to the development of interstitial fibrosis in this model. However, hypoxia could contribute to pathological changes occurring in the late stages of AN. Our finding that apoptosis was more extensive in the later stages of this model in conjunction with the possible existence of HIF-1-dependent pathways for apoptosis (6) prompted us to examine for evidence of colocalization of HIF-1α and a nuclear marker of apoptosis (TUNEL staining). Using a previously described fluorescent technique (49), we found evidence of colocalization of HIF-1α and TUNEL staining in nuclei of the esophageal epithelium, a tissue in which both cellular hypoxia (24, 38) and apoptosis (48) occur under normoxic conditions. In the kidney sections, apoptotic nuclei were most prominent in the later stages of AN, with TUNEL labeling of occasional tubular epithelial cells and intraluminal cellular casts. Although HIF-1α staining was detectable in the same sections, there was no colocalization of HIF-1α or apoptosis in the kidney samples. This result suggests that HIF-1-dependent pathways are unlikely to contribute significantly to tubular apoptosis in this model but may instead represent an attempted homeostatic response to relative oxygen deficiency within the tubular cells in advanced AN.
To investigate the potential causes of hypoxia in this model, we measured peritubular capillary density as a surrogate measure of oxygen supply. AN was associated with a significant reduction in CD34-labeled peritubular capillaries, consistent with similar findings in other chronic renal diseases (4, 21, 22, 26, 30). This reduction in capillary density preceded the detection of increased cortical hypoxia and would suggest that a reduction in oxygen supply plays a role in the development of hypoxia. Consistent with this hypothesis was the finding that the areas of greatest capillary rarefaction at day 28 after adriamycin were those with the greatest degree of tubulointerstitial injury and correlated morphologically with the areas within which HIF-1α nuclear staining was seen. Two methods (CD34 immunostaining and Western blotting) were used to assess the cortical microvasculature in this model. Western blotting was performed because the method used for calculation of the capillary rarefaction index assumes that the volume of tissue assessed in the normal kidney is unchanged in AN. This model is associated with tubular dilatation, tubular loss, and interstitial expansion, all of which affect the volume of tissue to a variable amount. The reduction of CD34 protein in AN by Western blotting (which is not subject to the same limitations) was further evidence that the cortical microvasculature is reduced in this model.
A further result from these experiments is that cortical expression of the angiogenic cytokine VEGF was significantly reduced as the disease progressed, consistent with results obtained in other models of chronic renal injury (8, 21, 31). This result may seem contradictory to the finding of increased hypoxia in this model, particularly since VEGF is both induced by hypoxia and transcriptionally activated by HIF-1 (11, 25, 27). There are several explanations for this apparent disparity. First, HIF-1 is not the only known transcriptional activator of VEGF, with experimental evidence suggesting a role for SP-1 (1, 41) as well as the oncogene ras (3, 32). VEGF transcription is also inhibited by the anti-oncogenes p53 (36) and p73 (42). Also relevant are previous experiments showing that VEGF expression is reduced in cutaneous models of inflammation at a time when HIF-1α expression is increased (2) and the finding that VEGF production in response to hypoxia was reduced in the presence of macrophage-derived cytokines in vitro (21). These latter findings are potentially relevant to the reduction of VEGF observed in the current experiments, since AN is associated with a prominent mononuclear infiltrate (47). It is therefore likely that the finding of reduced VEGF in AN (as well as in other renal disease models) is multifactorial in origin.
Whatever the cause, the finding that VEGF is reduced in AN has potential relevance to the increased HIF-1α expression that occurs in the late stages of this model. If cellular hypoxia is caused by a reduction in oxygen supply engendered by microvascular loss in this model, it is also possible, in turn, that the reduction in peritubular capillaries may in fact be secondary to reduced local production of VEGF, since VEGF is an important survival factor for endothelial cells (13, 14). In support of this, the reductions in cortical VEGF seen in AN were observed before a detectable reduction in peritubular capillaries, which, in turn, preceded a detectable increase in cellular hypoxia.
In summary, we showed a significant increase in nuclear HIF-1α localization in murine AN, consistent with the presence of increased cellular hypoxia in the later stages of this model. HIF-1-dependent mechanisms do not appear to be the predominant cause of apoptosis in this model, and the timing of the increase in nuclear HIF-1α would argue against a significant role for hypoxia in either parenchymal injury or interstitial fibrosis in this model. The observed reductions in both the density of peritubular capillaries and expression of the principal angiogenic growth factor VEGF suggest that the increase in nuclear HIF-1α in AN is secondary to a disruption of mechanisms involved in the maintenance of the normally extensive capillary network within the kidney.
This work was supported by grants from the National Health and Medical Research Council of Australia and the Swiss National Science Foundation.
In vivo hypoxia experiments to provide positive controls for HIF-1α staining were performed in collaboration with Dr. Scott Geller and Prof. Jonathon Stone from the Department of Retinal Biology, the University of Sydney.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2005 the American Physiological Society


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Does Sleeping On Back Cause Flat Spot On Head
What could cause a quarter sized tingling spot on back of head? Could it be related to the fact that i'm sleep deprived?
Superficial nerve: This sounds like merely an irritable, superficial nerve in your scalp and probably is not related to being sleep deprived. If symptoms worsen or headaches develop, have a physician check it. ...Read more
Sleep (Definition)
Sleep is a topic that involves concepts such as how long it takes to fall asleep, how many times one wakes up at night, how many hours one sleeps at night, sleep stages, ...Read more
Do babies get flat spots on their heads if they sleep on their backs?
Yes: Some babies develop a flattened head (positional plagiocephaly) due to spending a lot of time on their backs. The soft bones of the scalp facilitate brain growth during the first year and can be molded during this time. Make sure to rotate sleeping positions and give your baby supervised tummy time while awake. ...Read moreSee 2 more doctor answers
When in tired and I try to lay down and sleep sometime I hear thesse pops in my head followed by like a warm sensation at the spot where it happens. What could this be?
Sinus disease: This sounds like you may have fluid in your sinuses that drains when you change positions. This is a guess but I am not aware of any other things this may be. Does not sound life threatening. ...Read more
Pain on left side of head when bending, changing sleep positions, touchingthat spot. Lately weird drooling feeling on left eye and some pain on left leg
Head and other pain: You need to go see a doctor to evaluate these symptoms. ...Read more
Baby sleeps on back with head turned, can this cause any problems?
Back is good: Sleeping on the back is good. Most babies turn their head to one side or the other. As long as the head is not always turned to the same side to the point of having trouble turning it the opposite direction, or causing flat spots, then it is ok. ...Read more
What does it mean if I'm alwase tired but can't sleep no energy light headed an dulled hearing sometimes sounds like pulse an sometimes see spots?
For : tiredness check hemoglobin, thyroid and vitamin D levels. Dull hearing could be middle ear issues ( effusions). Spots may have many causes. FM in differential. ...Read more
Baby has flat spot right back side and in front left side. Will her head shape be fine later or will it be same. Worried. Birth time she was fine. ?
"back to sleep": When it was established that putting an infant tummy side down increased the risk of SIDS, parents were told to put infants on their backs to sleep. Since an infant's skull is very malleable, it can cause flattening of the back of the skull. Give your baby plenty of tummy time while awake. As her brain grows and her sleep position varies, this flattening may get less apparent with time. ...Read moreSee 1 more doctor answer
Should i worry about my son's flat spot? Our pediatrician recently told us that our son had a flat spot on the back of his head. She said we could leave it or have a pediatric plastic surgeon take a look at it to see if a helmet is needed. While she act
How much ??: A statement that the kid has a flat spot doesn't need to send you to a orthotic person for a helmet or anything else.Explore how much of an issue your son has and the doc's recommendations.Most babies that back sleep as recommended will develop a flat spot. Most will disappear naturally as the kid assumes other positions for sleep or play. If the shape worsens over time a consult is reasonable. ...Read moreSee 2 more doctor answers
I have a tender spot on the back of my head. What could be the cause?
Probably nothing: You have probably bumped or irritated the "spot" in some manner. Have someone look to see if there is anything visable. If not, give it a while. Remember, in medicine, something bad usually does not go away and gets worse and becomes apparent, if it goes away, it was nothing to start with. ...Read moreSee 1 more doctor answer
I have a flat tender spot on the back of my head like where the swirl is what can this be?
Swelling: If you didn't injure the spot where the head is tender (bumped your head) then it is most likely some skin irritation. You could have an allergic response to some shampoo or other hair treatment. As long as you are not an infant, the skull is closed and it is only the scalp you are feeling. There are rare situations when the skull is not closed from birth, but you would have known about that. ...Read more
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Dynamic projection mapping onto deforming non-rigid surface
We realize dynamic projection mapping onto deforming non-rigid surface based on two original technologies. The first technology is a high-speed projector "DynaFlash" that can project 8-bit images up to 1,000 fps with 3 ms delay. The second technology is a high-speed non-rigid surface tracking at 1,000 fps. Since the projection and sensing are operated at a speed of 1,000 fps, a human cannot perceive any misalignment between the dynamically-deforming target and the projected images. Especially, focusing on new paradigms in the field of user interface and fashion, we have demonstrated dynamic projection mapping onto a deformed sheet of paper and T-shirt. Also we show that projection to multiple targets can be controlled flexibly by using our recognition technique. http://www.k2.t.u-tokyo.ac.jp/vision/DPM/
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Large Balance Commercial Collector
Seeking large balance, commercial collector. Srictly business to business collections with no auto-dialer. Hrly wage, commission, + other benefits.
Looking to hire reliable hard working individuals for our small framing company. Must have transportation. We will provide a signing bonus. Competi...
Mortage/Title Closer- St. Paul area, MN - Direct Hire
Excellent position for a Closer at a well known company in St. Paul, MN. This is a direct hire position paying $35to$45K + commission and benefits.
Mortgage Loan Processor - Minneapolis/St. Paul, MN - $18-21/hr +commission
Staffing Partners' client is seeking experienced Mortgage Loan Processors. These are temp to hire positions. Pay is $18-$21/hr plus commission.
Training and Development Specialist
We have an immediate opening for a Training & Development Specialist with our growing client! Great opportunity to develop new training programs!
Human Resources Generalist-Minneapolis, MN
Staffing Partners has an immediate opening for a Human Resources Generalist to join our growing client's team in Minneapolis, MN!
Doherty Top Talent's Walk in Wednesday Job Fair!
Come down to Doherty's Roseville office for a job fair! We have many employers looking for candidates just like you!!
Executive Assistant
Doherty needs an Executive Assistant for a Fortune 400 food distribution company in Edina. Contact us today for more details or to apply. 952-832-8363
Recruiting Specialist-Minneapolis, MN
Staffing Partners is seeking a motivated recruiting specialist for a great contract to hire opportunity with our client in Minneapolis!
Doherty Top Talent's Walk in Wednesday Job Fair!
Come down to Doherty's Roseville office for a job fair! We have many employers looking for candidates just like you!!


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You are going to Watch Rosario + Vampire Episode 5 English dubbed online free episodes with HQ / high quality. Stream Anime Rosario + Vampire Episode 5 Online English dub Episode Description : School Swimsuits and a Vampire. The students learn that they are to choose a school activity and Tsukune is having a hard time choosing when he comes across the swimming club with Moka, who is a bit reluctant to investigate because water has a purifying effect on vampires and drains their powers. As Kurumu and Yukari try to garner the most attention through events, Tamao Ichinose, the head of the swim club, tries to isolate Tsukune for herself. After she teaches Tamao and the mermaids the error of their ways, Inner Moka reprimands Tsukune for not understanding Outer Moka.
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Meals: Breakfast & Dinner
Arrive Delhi by Air or Train. Arrive at bus stand, Travel over night in Volvo bus from Delhi to Manali.
Manali Local sightseeing, Morning arrive at Manali and transfer to hotel After fresh up at Manali hotel, depart for half day local sightseeing tour covering places like Hidimba Devi temple, Manu temple, Vashisht Bath, Van Vihar and evening shopping at the local market ( Mall ). Overnight stay & dinner at Manali hotel
Meals: Breakfast & Dinner
Rohtang pass(up to possible) which is located at height of 13050 Feet from sea level, Generally Rohtang pass open in mid May and closes in mid November, depending up on snowfall, If Rohtang pass is not open sight seeing will be done for available snow point like Gulaba , Kothi or Marhi or Solang valley. Solang valley is heaven for adventure lovers, and Paragliding . zorbing, skiing can be done, return back to hotel & overnight stay at Manali hotel.
Meals: Breakfast & Dinner
After Breakfast in the Morning excursion to Kullu valley, enjoy Rafting in the Beas River, enroute visit Kullu market, Kullu Shawl factories and sale outlets, Angora Farm, Vaishno Mata Temple and Parvati River. Kasol Valley, Manikaran Shiva Temple, Gurudwara Sahib and Hot water springs, return back to hotel & overnight stay at Manali hotel.
Meals: Breakfast
In the morning, you would check out from the hotel. You can explore the Shopping malls of Manali on your own. In the evening board the Volvo AC bus to Delhi for overnight journey.
Mr. Vinod Singh Gill, Ms. Sunaina Sharma
Set No. 2, Bhawani Bhwan, near May villa, Summer Hill, Shimla, Himachal Pradesh, India - 171005
+91-9816078074, 8091579000, 8091479000
Kerala Short Tour - 5 D / 4 N
Munnar - Thekkady - Alleppey - Cochin
Kullu - Manali Special Tour Package - 5 D / 4 N
Kullu - Manali
Hills Queen Summer Special Tour Package - 3 D / 2 N
Shimla - Kufri - Chail
Shimla - Kullu - Manali Tour Package - 6 D / 5 N
Shimla - Kullu - Manali
Hill Queen Special Honeymoon Tour Package - 5 D / 4 N
Shimla - Kullu - Manali Special Honeymoon Tour Package - 5 D / 4 N
Shimla - Kullu - Manali
Kinner Kailash Package Tour Program - 7 D / 6 N
Shimla - Sangla - Kalpa
Mystic Himachal Tour - 13 D / 12 N
Chandigarh - Shimla - Manali - Kullu
Himachal Excursion & Golden Temple Package - 10 D / 9 N
Shimla - Manali - Dharamsala - Dalhousie - Amritsar
Shimla Volvo Package - 3 D / 2 N


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5 Tips to Help Businesses Return from a Blogging Hiatus
September 17, 2016
Sometimes you have to take a break from blogging. For small businesses especially, where priorities extend away from blogging and social media towards more pressing matters that require all hands on deck, this break can spiral into an indefinite hiatus, and the longer it lasts, the more it feels...
Get More Traffic to Your Blog with this One Simple (But Powerful) Strategy
August 21, 2015
The real power of the web for businesses lies in its social nature. The web is built for sharing, collaborating, including. So when starting - or growing - a blog is on your to-do list, social strategies are your best best. Things like SEO, bold headlines, and good visual appeal are great, too. But the biggest boosts in blog traffic and engagement I've ever had didn't come from independent things like keywords. They came from including others in my work.
How to Use Popups on Your Blog to Get More Fans & Followers
July 03, 2015
Your blog has more traffic than all of your social profiles combined. Not to mention your visitors have made an informed decision to read your blog - as opposed to aimlessly clicking on a social profile of yours while they're on the phone. This article will cover just one of the many strategies that can turn your blog into a goldmine of fans and followers: website scroll popups. I'll show you exactly how they work and give you four essential elements of setting them up right.
How to Fix a High Bounce Rate
May 14, 2015
Let's go over the 9 main reasons for high bounce rates and, more importantly, what you can do to make people keep scrolling downward and clicking through more pages.
7 Ways to Create a Personality for Your Corporate Blog
May 13, 2015
This first impression is made up of a fairly complex set of factors - many of which are subtle and hard to measure. Things such as writing style, blog layout, architecture of your site, choice of imagery etc. Even if you were to ask your readers what it is they are looking for, you may get a blank stare or vague response. What they feel and inevitably how they perceive your brand is predominantly subconscious. But all of these factors in some way add up to a visitors perception of your brand and their likelihood to return to your site.
14 Awesome Free Resources to Drive More Traffic to Your Blog
April 21, 2015
We've collected 14 awesome free resources to help you grow your blog traffic. We've tried and tested them for ourselves, and we've only included the tools that have really made a difference to our blog traffic.
Demystifying the Technical Aspects of Building a Successful Blog
March 25, 2015
In order for a blog to be considered a success, it has to abide not only by the strategy implemented to reach its goals, but also the foundation that supports it. A content strategy that brings fruit to a content marketing and social media marketing strategy is important in generating blog traffic, which you can turn into leads or customers. But how the blog is built and set up is just a crucial, if not even more so.
5 Ways to Boost Your B2B Business Blog
March 25, 2015
70% of B2B marketers plan on creating more content in 2015 than they did the previous year, according to the 2015 Content Marketing Report. It's a great goal but it does suggest a new problem - how to create all that new content? As content manager for a B2B business blog it is something that I can certainly resonate with. Here I'll address some of the ways to solve that challenge.
Why Bother With a Small Business Blog?
March 10, 2015
Why it is so important to have a business blog and not just a social media presence? Creating a blog will give you access to a cohort of people online.
The Real Reason Your Posts Aren't Converting Like They Should
January 15, 2015
Blogging is an art form and it should be treated as such. To truly master blogging you're going to need to spend a lot of time perfecting your writing style and getting to know your customers. The deeper you know your customers and the better you can serve them with clarity the more likely your posts are to resonate and inspire action.


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Explore these ideas and much more!
Kollabora.com is a community of makers sharing DIY projects. Discover the latest fashion-forward projects in sewing, jewelry, knitting, crocheting, quilting, and more. Learn from a large technique database and shop the marketplace for the best craft supplies.
Since the start of the new series of Sewing Bee, we can't get enough of sewing our own wardrobes! First stop for summer is always dresses, with sunshine on it's way, and what's better than dresses you've sewn yourself?
We've fallen in love with this elegant style! #NewLook #pattern 6447 can be made with a fitted and flared skirt and the length can be tailored to perfectly suit you.
And favorite fabrics: | 17 Useful Wedding Cheat Sheets For Any Bride-To-Be


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9 Celebs Sued For Catching Fade
Celebrities have to defend themselves against crazy fans, paparazzi, and other folks, but some catch fade when it isn't necessary —and that's when they have to give up some of those coins. Here are 9 celebs sued for catching fade.
The Game
Struggle rapper 40 Glocc won a $4.5 million judgment against The Game for whooping his azz on camera.
Drake was sued along with Chris Breezy for catching fade in a bottle bustin' brawl over Rihanna at a night club for $16 million. The judge ruled that they did not have to pay up.
Kanye West
Kanye was sued for putting them paws on a photographer at LAX and in the recent incident involving Yezzy and a rude teen who hurled a racial slur at Kimmy Cakes, he settled with her heckler for $250K.
Chris Brown
Recently, Breezy was sued by man who he allegedly fought on the basketball court for $250k. Chris denies the allegations.
Lil Wayne
Lil Wayne and his goons were sued for allegedly hitting a fan over the head with a skateboard for trying to take a pic with him.
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Selita Banks
Selita was sued for allegedly breaking a woman's nose after she kissed then boo thang Terrence J.
Patti LaBelle
Patti was sued after yelling at a toddler for running around in an apartment building lobby. She became so irritated that she began to throw water and tried to punch the kid's mom.
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Discover a selection of top forums gamearena order on the internet. Come to share your passion on forums gamearena order and meet other fans of gamearena order. Then, why don't you create your own forum of gamearena order ?
Liaw and order is not dead, it has only changed.
Free forum: A public place for digimon believers and any passing members to hang out and talk about all things digimon.
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Greyknight fan site for table top gameing This order of light is dedicated to the way of the emperor and to stamp chaos in to dust
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The Order is a Herosmash clan made up of the most skilled PvPers. We're dedicated to staying on top, while being team players and a good example to others. Check the Joining topic if you want to apply.
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Welcome Comrades!
This is the forum for The New Sith Order. It is a place for people to speak their minds, and come together in a community of like minded individuals.
Marauders Era Alternate Universe RPG, Enter a Universe you have not yet explored. Attend Hogwarts or our very own Unique school, WolfClaw Academy of Magic, Join as an Original Character or Canon Character. Attend Classes, get a job and start working
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A Star Wars: TOR guild
I have looked for places to debate this topic online and found nothing easily done. The goal of this is to argue points of both sides in order to truly understand.
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Forum for the H.O.O.D. alliance of Ogame's Izar Universe.
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The home of the JEDI (Jedi Enclave of the Dawning Illumination) a combined Role Play and Jedi Philosophy group.
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In order to survive, packs must be formed, however, a nuclear war has left the world in a state of disarray. Mutated, alone, lost... Can you make it?
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This forum was created to connect those in the CG/l scene, in order for them to express themselves, to connect and to find local resources.
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Free forum : This is a text based roleplay where you create a character then live and fight within the jedi order.
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The official forums of the CyberNations alliance The Sovereign Order
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Your online resort for force teaching from both Ashla and Bogan We are the keepers of the flame and the mystery of the dark. keeping the force in balance.
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Imperial Order Alliance - Clash of Kings
Unable to remain idle as his former apprentice destroys what they'd spent years defending, Obi-Wan Kenobi has come out of exile to secretly form a new Jedi Order.
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Holy Storm Order is a Illyriad miltary alliance.
This is a traditional, original Yu-Gi-Oh-based roleplay site, whom utilizes the external Duelist Network in order to create a multipurpose, enjoyable, experience.
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Where all the international alliances of Orbis comes together.
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Create your own Fursona and interact in post apocalyptic role play. Humans and anthros have to get along in order to survive the apocalypse.
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The world is rotting, our mother, the goddess, mother earth, with wars and religions, it kills her, and the cause oF it all is the Gods.
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A more accurate accounting of history, by and for the Information Age.
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This Forum was made to create a Poll system in order to figure out what were going to play.
The communications office for the Order of Tenebris. We have a dream that one day, the cyberverse will burn to ashes, and in its place we will build a better world.
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This Guild Spans Many Worlds (And games) (Sometimes known as The imperial Crimson Knights, or The imperial Cobalt Knights)
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To you or me, pictures of them might be perfectly innocent, but police and courts might take different view. She is not letting anyone stop her from making those beautiful parts of her body visible to everyone. Available for children up to the age of 15 (16 for disabled children), Childcare Vouchers is a government backed scheme to reduce registered childcare costs.
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Free WiFi! WiFi is available in all areas and is free of charge.
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Hotels Vicchio
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Is Flaherty Dr John incorrect or missing key business information? Update your Hotfrog listing today.
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Hotfrog Ireland provides information regarding Flaherty Dr John in Loughrea Galway. Flaherty Dr John is located at The Sycamores, Main St, Loughrea Galway and provides health services. Contact them on (091) 842 144 or by visiting them on their website http://www.mainstreetclinicloughrea.ie.
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Ultrastructure of trout spermatozoa: Changes after dilution and deep-freezing
- Accepted:
- Cite this article as:
- Billard, R. Cell Tissue Res. (1983) 228: 205. doi:10.1007/BF00204873
Trout spermatozoa taken from the testis, vas deferens and ejaculate are described at the ultrastructural level. The morphology of the spermatozoa head, changes in the middle-piece structure, and the relationship between the centrioles and the flagellum were studied under consideration of their role in the reproduction of this species. Morphological changes observed after dilution of the spermatozoa in freshwater or saline and subsequent freezing deserve attention in connection with certain manipulations used in fish farms and laboratories.
Key wordsSpermatozoaUltrastructureDeep-freezingSperm manipulationTrout
Copyright information
© Springer-Verlag GmbH & Co. KG 1983


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Whoa!! What was going on betweeen 5:08-5:11am 2/22? It lloked like the guy walked in front of the camera and all of a sudden the camera blurred and awful roaring sounds floowed for a few minutes. Right now its much more quiet. anyone knows what's happened?
Are the rest of you hearing this?1
2 posts / 0 new
Thu, 22/02/2007 - 5:14am#1
Are the rest of you hearing this?1
Thu, 22/02/2007 - 5:46am#2
Oh My Jane,
Please join in the watering hole of the day & there you can see blow by blow descriptions of what is going on by some very experienced cammers.
I believe "the guy' you saw was the auto focus changing from infra red to normal. At the smame time there was awesome baboon calls.
I hope that you recover from your scare


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22 Blacksmith Court Jimboomba QLD 4280
Email Agent
Large quality built home with shed on 2 fenced acres22 Blacksmith Court, Jimboomba
For the family that are after ample living space this quality built home features open plan kitchen/family/ dining room, living room and rumpus/games area.
The study also could be utilized for the business person that works from home.
This home also has our Queensland climate under control with the zoned, ducted ,reverse cycle air conditioning.
Added features are:
* Modern kitchen with stone benchtops and gas cooking,dishwasher and walk in pantry
* Master bedroom featuring walk in wardrobe and ensuite
* 3rd toilet next to the laundry at the opposite end of the bedroom areas
* Quality fittings and fixtures
* Fans
* Concrete Drive way
* Large covered alfresco area
* Double lock up garage with remote access
* 2 remote vehicle accommodation under roof
* Fans
* Security screens
* New carpets throughout.
* Pets on Application (SORRY NO HORSES)
An inspection will not disappoint, please contact our office today to arrange a time to view.
Inspection Times
- No inspections scheduled
- Contact Agent
Key Features
- Air Conditioning
- Built-in Wardrobes
- Dishwasher
- Ducted Cooling
- Ducted Heating
- Remote Garage
- Study
- Heating:
- Other...
- Hot Water:
- Gas
- Land
- 8000m²
- Ensuites
- 1
Other FeaturesClose to schools, Dining Room, Established Gardens, Family Room, Formal Lounge, Fully Fenced Yard, G
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LJ Hooker Jimboomba
Jimboomba QLD, 4280
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Doctor insights on:
What Is Cause Of Num And Dull Pain From My Thigh To My Feet
Would could cause me to have numbness in my right thigh after being on my feet for a while?
Numbness: Probably a pinched nerve - see your neurologist for assistance ...Read more
I have a dull pain and cramp on my left leg from the thigh to my feet.?
See your doctor.: Without the benefit of an exam it is impossible to say. Could be a pulled muscle, inflamed or pinched nerve, painful varicose vein or a more serious condition such as a blood clot. If worse with walking could be peripheral arterial disease involving a blocked artery. See your doctor asap! ...Read more
From past 1 week I am feeling numbness in my left thigh if i sit for half an hour in 1 position. Prior to this my feet are also paining, feeling heavy
Pinched nerve: With such a positional relationship, you may have a nerve entrapment when you're in the sitting position. This wouldn't necessarily explain the symptoms in both feet, however. An evaluation by a neurologist would be recommended if these symptoms persist. ...Read more
Can sciatica cause numbness in right thigh and cold right foot?
Yes: yes it can. Especially, with activity.however, I would see a neurologist to make sure that you do not have a nerve disease, I eat underwear apathy whether the sciatica. The floor more is more likely to cause these symptoms. I meant former. ...Read more
Hi .. What can cause ;leg numbness, just the leg part, not knee not calf not thigh not foot... Anything to do with nf2?
Could be: One of the superficial cutaneous nerves in the leg may be affected by a neurofibroma. ...Read more
Im Post-op L foot bunionextomy 7mo. Still Have numbness/stiff/sore in toe area. & L leg a constant pain/ache right above knee on inside thigh area?
Ask if Normal!: Recovering from a bunionectomy is based on many factors that can vary from patient to patient and even surgeon to surgeon. The important point to remember is that as the postoperative weeks go by, you should be enjoying improvement in your wound condition. Less swelling, less pain, less stiffness and less numbness not more. Prolonged symptoms can cause a change in mechanics causing new symptoms. ...Read more
Low prob for clots, ddimer neg, dr thinks numbness from knee into foot, slight ache in inner thigh is pinched nerve. Is ddimer secure test for clots?
D dimer - screening: D dimer is only a screening test . Confirmation of a blood clot should be done through a doppler ultrasound. Discuss with your doctor if he would consider ordering a doppler ultrasound of the lower extremity that is affected. ...Read more
What is the cause of cramp and dull pain from my left buttocks down to my feet always?
Many possibilities: There are many possible causes for the pattern of pain you're describing. A careful history and thorough physical exam would be helpful in determining the cause. I would suggest you see your doc about this. The possible causes could be muscular (a fascial distortion or a pulled/strained muscle), neurological (a slipped disc in the low back, sciatica) or some combination of the two. ...Read moreSee 1 more doctor answer
What would cause severe hip and thigh pain after being on my feet for hours?
Could be your back: Problems with the lower back can often be felt in the hip and back of the thigh....Standing in one place for hours can often bring on pain and stiffness in the lumbar spine....See your doctor and have your back examined. ...Read more
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- Got a lump on the middle of my penis and there is no pain what could it be
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- I have a dull pain and cramp on my left leg from the thigh to my feet


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- Professionally Installed French Doors in Laurel, MT
- Dependable Laurel French Door Professionals
- Free Quotes on French Doors in Laurel, MT
French Doors in Laurel, MT
The French doors Laurel, MT has to offer might be the perfect addition to your house, so you will certainly want to take the time to look into this option before you make a final decision. These doors are considered to be very stylish right now and an increasing number of homeowners are getting them installed. French doors offer an absolutely lovely aesthetic and they are very easy to open and close, so you won't have to worry about their functionality at all.
Avg Costs for French Doors
Min Cost
Max Cost
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$833.46 - $1,205.30
Find the Best Quality French Doors - Laurel, MT
If you have been looking for a way to enhance the aesthetic of your home's interior, you might find that Laurel French doors are exactly what you need. Many homeowners have them because they are such a nice touch aesthetically. These doors are great for beach houses as well as those in the country, so you won't be limited at all when it comes to whether or not they are going to work inside your house. Because French doors are so versatile in terms of the way they look, you will almost certainly be able to get them installed and have them look excellent in your home.
French Doors Companies
Home Base Renovation
514 N 29th St
Billings, Mt 59101
Billings Builders Exchange Inc
2050 Broadwater Ave Ste A
Billings, MT 59102
Stroebel Welding, Inc.
1520 Foothill Dr
Billings, MT 59105
Tiensvold Fire Protection
2101 Columbine Dr
Billings, MT 59105
Accent Windows Doors & Siding Of Montana
960 S 24th St West Suite D
Billings, MT 59101
Alpha Omega Disaster Restoration
204 E Main St
Laurel, Mt 59044


text:
Robber threatens woman with 'HIV syringe'
The Local · 31 Aug 2015, 16:31
Published: 31 Aug 2015 16:31 GMT+02:00
- Police arrest teen over Berlin knife attack (25 Aug 15)
- East Germany sees half of racist violent crime (18 Aug 15)
- Thieves open 1,000 beers, don't drink any (18 Aug 15)
The 59-year-old was in the middle of parking her car when a man in his late 30s climbed into the passenger seat, police wrote in an incident report.
He brandished a syringe full of yellow liquid in his left hand as he demanded cash and warned the woman that he could inject her with the virus.
His horrified target reached into her purse and gave the man a small amount of cash she had on her, prompting him to jump out of the car and flee on foot towards a nearby U-Bahn station.
The woman immediately called the police, who were unable to find the robber despite sending ten patrol cars to search for him.
For more news from Germany, join us on Facebook and Twitter.


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Sharp Humor works for Derby
Sharp Humor, winner of the Swale Stakes and second in the Florida Derby, tuned up for the 132nd Kentucky Derby with a swift one-mile workout Thursday morning at Churchill Downs. Sharp Humor, trained by Dale Romans, was clocked in 1:38 over a fast track.
Sharp Humor is one of many front-running types expected for the Derby, for which the final major prep, the $325,000 Coolmore Lexington Stakes, will be run Saturday at Keeneland. A field of 10, headed by Showing Up and Like Now, was entered Thursday for the 1 1/16-mile Lexington.


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Bank of Baroda Recruitment 2016 Notification and Online Application Form download link has made available to apply for the total 250 vacancies of BOB Specialist Officer Project at www.bankofbaroda.co.in:
Now one of the most reputed banks in India, Bank of Baroda has come up with fresh employment news for appointing eligible candidates to various MMGS-II and MMGS-III Grade/ Scale Posts. As per a notification released by the department, there are 250 BOB Specialist Officer vacancies going to fill up in varied disciplines such as Finance/ Credit, Information Technology, Planning, Risk Management, Human Resources Management, IT Specialist, Data Analyst, Economist, and Law. It's a right opportunity for those all appropriate and career-oriented individuals looking to grab latest bank jobs after clearing their graduation degree. Interested job-seekers can apply to this Bank of Baroda Recruitment 2016 of 250 SO Posts by submitting their application form via online mode and that before the closing date 13th of April 2016. Here, we kindly suggest each and every willing candidate read out this article without wasting time for being sure about their eligibility.
ABOUT BANK OF BARODA: It is an Indian state-owned banking and financial services company, established on 20th July 1908 in Gujarat state. The main headquarters of this company is located in Vadodara city but its corporate office is in Mumbai, Maharashtra. At present, it is the second largest bank in India only next to State Bank Of India. The Maharaja Sayajirao Gaekwad III has founded this bank in the Princely State of Baroda. On 19th July 1969, this organization along with other 13 major commercial banks of India was nationalized by the Indian Government (GOI). Mr. P S Jayakumar (CEO & MO) and Bharat Dangar (Director) are the key people of this public sector company. The tagline of BOB is "India's International Bank". It has more than 104 branches in 24 countries which also include 61 branches of the bank, 38 branches of its 08 subsidiaries and 1 representative office in Thailand. It also has a joint venture in Zambia with 18 branches/ offices. Today, BOB is the FIRST choice for every job-hunter because it provides a range of opportunities and also allows them to gain rich banking exposure by serving a variety of customers.
Check: Latest RBI Recruitment 2016 Notification
Right now, it has invites applications only from well-qualified people for the appointment of mentioned 250 Middle Management Grade/ Scale II & Scale III Officers in various disciplines. All intrigued candidates must note that the Bank of Board Apply Online Link for registration of online applications will be open from 30.03.2016 to 13.04.2016 at its official website www.bankofbaroda.co.in. After the due date, the registration forms of remaining contestants will not be accepted so type to apply as early as possible within the deadline. Willing individuals need to have a qualification of graduation degree from any recognized university for participating in BOB Recruitment 2016 of Specialist Officer Posts. According to age criteria, contestant's age should be above 25 years and under 40 years. The board members will announce a date for hosting written test and downloading admit card/ call letter soon after completion of registration process.
Check: SBI Clerk Recruitment 2016 of 17168 vacancies
Before proceeding further for filling the form, applicants should ensure that he/she fulfills every eligibility criteria and other described conditions in Bank of Baroda SO Notification 2016. Here in this page, we have tried our best to mention complete details about vacancies such as age limit, upper age limit for reserved categories, education qualifications post wise, emoluments, pay scale, grade pay, intimation charge (fee), selection process, important dates and "Procedure to Submit On-line Registration Form". Those Contestants who regularly looking for latest govt job updates, they can follow our website on a daily basis.
Bank of Baroda Recruitment 2016 Details:-
Kind of Job: Central Government Jobs
Job Category: Banking Jobs
Job Placement: Anywhere in India
Registration Mode: On-line
Total No of Vacancies: 250 posts
Post Name: Specialist Officers (SO)
Vacancy Details (Code):
- Finance/ Credit (01) MMGS-II: 95 seats
- Information Technology (02) MMGS-II: 35 seats
- Planning (03) MMGS-II: 06 seats
- Risk Management (04) MMGS-II: 10 seats
- Human Resources Management (05) MMGS-II: 15 seats
- Economist (06) MMGS-II: 04 seats
- Law (07) MMGS-II: 20 seats
- Finance/ Credit (08) MMGS-III: 50 seats
- Information Technology (09) MMGS-III: 15 seats
Scale of Pay (per month):
- Middle Management Grade/ Scale-II: 31705 x 1145 (1) – 32850 x 1310(10) – 45950
- Middle Management Grade/ Scale-III: 42020 x 1310 (5) – 48570 x 1460 (2) – 51490
- The selected candidates will get initial monthly emoluments of Middle Management Grade/ Scale II & Scale III Officers, including DA, Special Allowance, HRA & CCA about Rs. 49,585/- and Rs. 65,434/- per month, respectively in a Metropolitan Centre.
- NOTE: Allowances may vary depending upon the place of posting of applicants.
Application Fee & Intimation Charge:
- General (GEN) and OBC Quota – Rs. 600/- (rupees six hundred only)
- SC/ ST/ Persons with Disability (PWD) Aspirants – Rs. 100/- (rupees one hundred only)
Mode of Payment:
- Every contestant has to make the payment of requisite fee via ON-LINE mode only.
BOB Specialist Officer Project 2015-16 Eligibility Criteria:-
Educational Qualifications:
- Interested applicants should have at least cleared their Graduation/ Bachelors Degree in relevant discipline from a Govt approved University or Institution.
- For more brief qualification details post wise, we request aspirants to check out official notification using a link below.
Age Limitation (13.04.16):
- Applicants age must be mini 25 years and maxi 40 years
- Upper Age Limit will be given to Reserved Categories as under:-
- SC/ ST – 05 (five) years
- OBC – 03 (three) years
- Persons with Disabilities (PWD) – 10 (ten) years
- Ex-Servicemen – 05 (five) years
- Persons affected by 1984 riots: 05 (five) years
Selection Methodology:
Only most eligible and suitable candidates will be appointed based on their performance in following exams:
- On-line Written Test
- Interview
- Psychometric Test/ Group Discussion (GD)
How to fill BOB Specialist Officer Online Application Form 2016?
- First, open an authorized web page.
- Navigate to "Careers" section of the homepage.
- Now, press on a link written as "Advertise for Recruitment of Specialist Officers in Bank of Baroda – Project 2015-16".
- Read the official advertisement before going further.
- Fill up online application form with correct information.
- Pay fees using online sources and don't forget to download e-receipt.
- Attach scanned copy of passport size photo, digital signature, and supporting documents.
- Verify filled up details and then submit it.
- Save the submitted copy and also, print a copy for examination purpose.
Useful Dates for the Candidates:-
- Starting Date for Online Registration and Payment of Application Fees: 30th March 2016
- Closing Date for Submission of Online Application and Fee Payment: 13th April 2016
- Date of On-line Written Examination: Upgrade Soon
Important Links:-
Download BOB Specialist Officer Notification Pdf
Bank of Baroda Recruitment 2016 Apply Online
Official website: www.bankofbaroda.co.in
Bookmarks this page for further updated news of Bank of Baroda SO Syllabus, Exam Pattern, Admit Card, Modal Papers, Study Materials, Key answers, Cut off Marks, Results, and Merit List. Please share your suggestions or feedbacks with us regarding this article by writing it in a comment box below. Our techufo.in web team will try to reply every reader as quickly as they can, THANK YOU.


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67, Milton, WAI have a sparkle in my eyes, a pretty smile and a warm and trusting heart. I am a mother, grandmother, sister and friend. I take very good care of myself mentally, physically ...
20, Federal Way, WAI would like to talk to a person who is honest, respect to other. I like outgoing with people I trust. When I have free time I like hang out with my family and friends. I usua...
60, Puyallup, WAI am taking a new career path and recently taken a new job in the insurance business, an employee benefit group and just love the people and it is a branch of what I already k...
25, Federal Way, WAI am originally from California and moved to Washington a year ago in hopes of pursuing my teaching career. I am a High School English Teacher and I absolutely love what I do....
61, Puyallup, WAI am passionate about my life. I love my children/grandchildren, I like my profession, but am looking forward to retirement in a few years! I enjoy adventures, am open to try...
60, Federal Way, WAI was divorced from my 2 exes, both were not spirituals, but instead they both love to humiliates n condemns about my faith as a Catholic. Looking for someone who has the same...
68, Puyallup, WAI'm very passionate about family, staying active and healthy, and my faith. I'm very close to my only son and his family. We travel and enjoy special times together. God fi...
55, Puyallup, WAI am passionate about day to day tasks, (do every little thing with thankfulness and gratitude). as well as special events, sports, family and special occasions, too numerous ...
73, Tacoma, WAHi there, I am passionate about my Catholic faith and helping a team of volunteers provide winter overnight shelter for people without homes during the coldest winter months....
45, Tacoma, WAI am a mom of two who values family. I work full time at a benefits consulting firm as a paralegal, accounting, consultant and office manager - "Jill of all Trades". My idea...
40, Federal Way, WAI have my own home, car, and dog. I am very close to my family and love spending time with my nieces I am looking for someone to do things with a take it slow. I would like to...
34, Puyallup, WAHi, I live my life with love and passion. I would like to find a good person who can appreciate the wonderful woman I am. I'm tired of being used and hurt. I want to be happ...
Catholic Dating By Major City:
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- Renton,
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It's time to re-imagine backup and recovery. Protect your business in the cloud or on premise with powerful backup software that assures recovery of your virtual, physical and cloud data, systems and applications. Experience the difference. Start your free trial today.Sponsored Listing
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Beasley is a 7 week old female. She is a tad shy with new people but warms up quickly. Beasley adores rubs on her belly and under her chin. She will cuddle up to the crook of your arm to melt into you while she purrs away. Beasley loves her toys, playing chase and climbing the scratch post to look out the window. Beadley has had her shots, tested negative for FIV and Feline Leukemia and had her Vet wellness exam. Please contact LDDR to meet her today.
Last Day Dog Rescue now microchips all dogs/cats before adoption. Microchips are implanted between the dog's/cat's shoulder blades and are a permanent form of identification. The microchips are registered to Last Day Dog Rescue with the adopter's info added once adopted. If the dog/cat is ever lost and slips his/her collar/tags the microchip allows the finder to know who to contact if scanned for a microchip. Dogs and cats MUST still have a collar with tags on at all times. Per our contract, we take all our dogs and cats back if for any reason you can't keep your adopted dog or cat.
The petfinder.com system requires that we choose a predominant breed or breed mix for our dogs and cats. Visual breed identification in dogs is unreliable so for most of the dogs we are only guessing at predominant breed or breed mix. We get to know each dog as an individual and will do our best to describe each of our dogs based on personality, not by breed label.
Thank You!
Your message has been sent to Last Day Dog Rescue.
You'll receive a copy, too, at to help you keep track of which pets you've inquired about, and which shelters and rescues you've emailed.
NOTE: Some shelters have physical locations you can visit; some of these shelters may only have pets for a limited time, so please do not wait for a reply—just go visit the shelter! Other organizations are rescue groups run by busy volunteers who may take a while to reply. You can find information about the shelter or rescue group caring for this pet, and their adoption procedures, on the pet's details page on Adopt-a-Pet.com.


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SOFA : Chicago
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Team Work | Polymer Art Archive
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