Wilms tumor protein-dependent transcription of VEGF receptor 2 and hypoxia regulate expression of the testis-promoting gene Sox9 in murine embryonic gonads.

Wilms tumor protein 1 (WT1) has been implicated in the control of several genes in sexual development, but its function in gonad formation is still unclear. Here, we report that WT1 stimulates expression of Kdr, the gene encoding VEGF receptor 2, in murine embryonic gonads. We found that WT1 and KDR are co-expressed in Sertoli cells of the testes and somatic cells of embryonic ovaries. Vivo-morpholino-mediated WT1 knockdown decreased Kdr transcripts in cultured embryonic gonads at multiple developmental stages. Furthermore, WT1 bound to the Kdr promoter in the chromatin of embryonic testes and ovaries. Forced expression of the WT1(-KTS) isoform, which functions as a transcription factor, increased KDR mRNA levels, whereas the WT1(+KTS) isoform, which acts presumably on the post-transcriptional level, did not. ChIP indicated that WT1(-KTS), but not WT1(+KTS), binds to the KDR promoter. Treatment with the KDR tyrosine kinase inhibitor SU1498 or the KDR ligand VEGFA revealed that KDR signaling represses the testis-promoting gene Sox9 in embryonic XX gonads. WT1 knockdown abrogated the stimulatory effect of SU1498-mediated KDR inhibition on Sox9 expression. Exposure to 1% O2 to mimic the low-oxygen conditions in the embryo increased Vegfa expression but did not affect Sox9 mRNA levels in gonadal explants. However, incubation in 1% O2 in the presence of SU1498 significantly reduced Sox9 transcripts in cultured testes and increased Sox9 levels in ovaries. These findings demonstrate that both the local oxygen environment and WT1, which enhances KDR expression, contribute to sex-specific Sox9 expression in developing murine gonads.

Characterization of polymer membranes by MALDI mass-spectrometric imaging techniques.

For physical and chemical characterization of polymers, a wide range of analytical methods is available. Techniques like NMR and X-ray are often combined for a detailed characterization of polymers used in medical applications. Over the past few years, MALDI mass-spectrometry has been developed as a powerful tool for space-resolved analysis, not least because of its mass accuracy and high sensitivity. MALDI imaging techniques combine the potential of mass-spectrometric analysis with imaging as additional spatial information. MALDI imaging enables the visualization of localization and distribution of biomolecules, chemical compounds, and other molecules on different surfaces. In this study, surfaces of polymeric dialyzer membranes, consisting of polysulfone (PS) and polyvinylpyrrolidone (PVP), were investigated, regarding chemical structure and the compound's distribution. Flat membranes as well as hollow fiber membranes were analyzed by MALDI imaging. First, analysis parameters like laser intensity and laser raster step size (spatial resolution in resulting image) were established in accordance with polymer's characteristics. According to the manufacturing process, luminal and abluminal membrane surfaces are characterized by differences in chemical composition and physical characteristics. The MALDI imaging demonstrated that the abluminal membrane surface consists more of polysulfone than polyvinylpyrrolidone, and the luminal membrane surface displayed more PVP than PS. The addition of PVP as hydrophilic modifier to polysulfone-based membranes increases the biocompatibility of the dialysis membranes. The analysis of polymer distribution is a relevant feature for characterization of dialysis membranes. In conclusion, MALDI imaging is a powerful technique for polymer membrane analysis, regarding not only detection and identification of polymers but also localization and distribution in membrane surfaces.

Calcium-dependent expression of transient receptor potential canonical type 3 channels in patients with chronic kidney disease.

It is unknown whether extracellular calcium may regulate the expression of transient receptor potential canonical type 3 (TRPC3) channels in patients with chronic kidney disease. Using quantitative in-cell Western assay we compared the expression of TRPC3 channel protein in monocytes from 20 patients with chronic kidney disease and 19 age- and sex-matched healthy control subjects. TRPC3 channels were identified by immunoblotting using specific antibodies and TRPC3 protein was further confirmed by mass spectrometry. We observed a significant increase of TRPC3 channel protein expression in patients with chronic kidney disease compared to healthy control subjects (normalized expression, 0.42±0.06 vs. 0.19±0.03; p<0.01). Expression of TRPC3 was significantly inversely correlated with estimated glomerular filtration rates (Spearman r=-0.41) or serum calcium concentration (Spearman r=-0.34). During a hemodialysis session serum calcium concentrations significantly increased, whereas the expression of TRPC3 channels and calcium influx significantly decreased. In vitro studies confirmed that higher calcium concentrations but not magnesium, barium nor sodium concentrations significantly decreased TRPC3 expression in human monocytes. This study indicates that reduced extracellular calcium concentrations up-regulate TRPC3 channel protein expression in patients with chronic kidney disease.

Superoxide dismutase type 1 in monocytes of chronic kidney disease patients.

We analyzed proteomic profiles in monocytes of chronic kidney disease (CKD) patients and healthy control subjects. Two-dimensional electrophoresis (2-DE) and silver staining indicated differences in protein pattern. Among the analyzed proteins, superoxide dismutase type 1 (SOD1), which was identified both by MS/MS mass-spectrometry and immunoblotting, was reduced in kidney disease. We characterized SOD1 protein amount, using quantitative in-cell Western assay and immunostaining of 2-DE gel blots, and SOD1 gene expression, using quantitative real-time polymerase chain reaction (PCR), in 98 chronic hemodialysis (HD) and 211 CKD patients, and 34 control subjects. Furthermore, we showed that different SOD1 protein species exist in human monocytes. SOD1 protein amount was significantly lower in HD (normalized SOD1 protein, 27.2 ± 2.8) compared to CKD patients (34.3 ± 2.8), or control subjects (48.0 ± 8.6; mean ± SEM; P < 0.05). Analysis of SOD1 immunostaining showed significantly more SOD1 protein in control subjects compared to patients with CKD or HD (P < 0.0001, analysis of main immunoreactive protein spot). SOD1 gene expression was significantly higher in HD (normalized SOD1 gene expression, 17.8 ± 2.3) compared to CKD patients (9.0 ± 0.7), or control subjects (5.5 ± 1.0; P < 0.0001). An increased SOD1 gene expression may indicate increased protein degradation in patients with CKD and compensatory increase of SOD1 gene expression. Taken together, we show reduced SOD1 protein amount in monocytes of CKD, most pronounced in HD patients, accompanied by increased SOD1 gene expression.

Single-institution cross-sectional study to evaluate need for information and need for referral to psychooncology care in association with depression in brain tumor patients and their family caregivers.

BACKGROUND: The prognosis of patients with brain tumors is widely varying. Psychooncologic need and depression are high among these patients and their family caregivers. However, the need for counselling and need for referral to psychooncology care is often underestimated. METHODS: We performed a single-institution cross-sectional study to evaluate psychooncologic need, depression and information need in both patients and their family caregivers. The Hornheider Screening Instrument (HSI) and the Patient Health Questionnaire (PHQ-9) were used to evaluate psychooncologic need and depression, and a study-specific questionnaire was developed to evaluate information need. Multivariable analyses were performed to detect correlations. RESULTS: A total of 444 patients and their family caregivers were approached to participate, with a survey completion rate of 35.4%. More than half of the patients and family caregivers were in need for referral to psychooncology care and 31.9% of patients suffered from clinically relevant depression. In multivariable analysis, psychooncologic need were positively associated with mild (odds ratio, OR, 7.077; 95% confidence interval, CI, 2.263-22.137; p = 0.001) or moderate to severe (OR 149.27, 95% CI 26.690-737.20; p <  0.001) depression. Patient information need was associated with depression (OR 3.007, 95% CI 1.175-7.695; p = 0.022). CONCLUSIONS: Unmet counselling need in brain tumor patients and their family caregivers associate to high psychooncologic need and depression. Adequate information may decrease the need for referral to psychooncology care and treatment of depression in these patients. Future studies should further explore these relations to promote development of supportive structures.

Deletion of an intronic HIF-2α binding site suppresses hypoxia-induced WT1 expression.

Hypoxia-inducible factors (HIFs) play a key role in the adaptation to low oxygen by interacting with hypoxia response elements (HREs) in the genome. Cellular levels of the HIF-2α transcription factor subunit influence the histopathology and clinical outcome of neuroblastoma, a malignant childhood tumor of the sympathetic ganglia. Expression of the Wilms tumor gene, WT1, marks a group of high-risk neuroblastoma. Here, we identify WT1 as a downstream target of HIF-2α in Kelly neuroblastoma cells. In chromatin immunoprecipitation assays, HIF-2α bound to a HRE in intron 3 of the WT1 gene, but not to another predicted HIF binding site (HBS) in the first intron. The identified element conferred oxygen sensitivity to otherwise hypoxia-resistant WT1 and SV40 promoter constructs. Deletion of the HBS in the intronic HRE by genome editing abolished WT1 expression in hypoxic neuroblastoma cells. Physical interaction between the HRE and the WT1 promoter in normoxic and hypoxic Kelly cells was shown by chromosome conformation capture assays. These findings demonstrate that binding of HIF-2α to an oxygen-sensitive enhancer in intron 3 stimulates transcription of the WT1 gene in neuroblastoma cells by hypoxia-independent chromatin looping. This novel regulatory mechanism may have implications for the biology and prognosis of neuroblastoma.

The Wilms tumor protein WT1 stimulates transcription of the gene encoding insulin-like growth factor binding protein 5 (IGFBP5).

Insulin-like growth factor (IGF) binding proteins (IGFBPs) constitute a family of six secreted proteins that regulate the signaling of insulin-like growth factors (IGFs). IGFBP5 is the most conserved family member in vertebrates and the major IGF binding protein in bone. IGFBP5 is required for normal development of the musculoskeletal system, and various types of cancer frequently express high levels of IGFP5. Here we identify the gene encoding IGFBP5 as a novel downstream target of the Wilms tumor protein WT1. IGFBP5 and WT1 are expressed in an overlapping pattern in the condensing metanephric mesenchyme of embryonic murine kidneys. Down-regulation of WT1 by transfection with antisense vivo-morpholino significantly decreased Igfbp5 transcripts in murine embryonic kidney explants. Likewise, silencing of Wt1 in a mouse mesonephros-derived cell line reduced Igfbp5 mRNA levels by approximately 80%. Conversely, induction of the WT1(-KTS) isoform, whose role as transcriptional regulator has been firmly established, significantly increased IGFBP5 mRNA and protein levels in osteosarcoma cells. IGFBP5 expression was not significantly changed by WT1(+KTS) protein, which exhibits lower DNA binding affinity than the WT1(-KTS) isoform and has a presumed role in post-transcriptional gene regulation. Luciferase reporter constructs harboring 0.8 and 1.6 kilobases of the murine Igfbp5 promoter, respectively, were stimulated approximately 5-fold by co-transfection of WT1(-KTS). The WT1(+KTS) variant had no significant effect on IGFBP5 promoter activity. Binding of WT1(-KTS), but not of WT1(+KTS) protein, to the IGFBP5 promoter in human osteosarcoma cells was proven by chromatin immunoprecipitation (ChIP) and confirmed by electrophoretic mobility shift assay. These findings demonstrate that WT1 activates transcription of the IGFBP5 gene with possible implications for kidney development and bone (patho)physiology.

Transient receptor potential channels in essential hypertension.

OBJECTIVE: The role of nonselective cation channels of the transient receptor potential channel (TRPC) family in essential hypertension has not yet been investigated. METHODS: We studied TRPCs in 51 patients with essential hypertension and 51 age-matched and sex-matched normotensive control subjects. Calcium and gadolinium influx into human monocytes was determined using the fluorescent dye technique. TRPC expression was measured using reverse transcriptase-polymerase chain reaction and in-cell western assay. Gene silencing by small interfering RNA for specific TRPC knockdown was also performed. RESULTS: We observed an increased gadolinium/calcium-influx ratio through TRPC in essential hypertensive patients compared with normotensive control subjects [cation influx ratio (mean +/- SEM), 125 +/- 14 versus 80 +/- 7%; each n = 51; P < 0.01], due to an increase of gadolinium influx in hypertensive patients compared with normotensive control subjects (48 +/- 4 versus 36 +/- 3%; each n = 51; P < 0.05). We observed a significant increase of TRPC3 and TRPC5 protein expression in essential hypertensive patients compared with normotensive control subjects (normalized TRPC3 expression, 3.21 +/- 0.59 versus 1.36 +/- 0.07; each n = 20; P < 0.01; normalized TRPC5 expression, 2.10 +/- 0.28 versus 1.40 +/- 0.52; each n = 12; P < 0.05). We used small interfering RNA for knockdown of TRPC5. The thereby reduced channel expression caused a significant attenuation of calcium and gadolinium influx. CONCLUSION: This study points to an important role of TRPCs in essential hypertension.

Lower Superoxide Dismutase 2 (SOD2) Protein Content in Mononuclear Cells Is Associated with Better Survival in Patients with Hemodialysis Therapy.

Mitochondrial superoxide dismutase 2 (SOD2) converts superoxide anions to hydrogen peroxide and oxygen. Human data on SOD2 protein content in chronic kidney disease (CKD) are sparse and mortality data are lacking. We investigated SOD2 protein content in monocytes from patients with hemodialysis therapy (n = 81), CKD stage 1-5 (n = 120), and healthy controls (n = 13) using in-cell Western assays. SOD2 protein decreased from CKD stage 1 until stage 4 whereas it increased again in stage 5 with and without hemodialysis. SOD2 gene expression, analyzed by quantitative real-time PCR, was not significantly different between the groups. Elevating cellular superoxide production reduced SOD2 protein content. This effect was abolished by the superoxide dismutase mimetic Tempol. Using gelelectrophoresis and Western blot we did not detect nitrotyrosine modifications of SOD2 in CKD. Finally, in patients with CKD stage 5 with hemodialysis therapy higher than median SOD2 protein content was associated with higher all-cause mortality. In conclusion, SOD2 protein content declined in CKD until stage 4 while SOD2 gene expression did not. Increased cellular superoxide anion production might affect SOD2 protein content. In advanced CKD (stage 5) SOD2 protein content increased again, but higher than median SOD2 protein content in these patients did not confer a survival benefit.

Surface characterization of dialyzer polymer membranes by imaging ToF-SIMS and quantitative XPS line scans.

The surfaces of polymeric dialyzer membranes consisting of polysulfone and polyvinylpyrrolidone were investigated regarding the lateral distribution and quantitative surface composition using time-of-flight secondary-ion-mass-spectrometry and x-ray photoelectron spectroscopy. Knowledge of the distribution and composition on the outer surface region is of utmost importance for understanding the biocompatibility of such dialyzer membranes. Both flat membranes and hollow fiber membranes were studied.

Proteomics of vitreous humor of patients with exudative age-related macular degeneration.

BACKGROUND: There is absence of specific biomarkers and an incomplete understanding of the pathophysiology of exudative age-related macular degeneration (AMD). METHODS AND FINDINGS: Eighty-eight vitreous samples (73 from patients with treatment naïve AMD and 15 control samples from patients with idiopathic floaters) were analyzed with capillary electrophoresis coupled to mass spectrometry in this retrospective case series to define potential candidate protein markers of AMD. Nineteen proteins were found to be upregulated in vitreous of AMD patients. Most of the proteins were plasma derived and involved in biological (ion) transport, acute phase inflammatory reaction, and blood coagulation. A number of proteins have not been previously associated to AMD including alpha-1-antitrypsin, fibrinogen alpha chain and prostaglandin H2-D isomerase. Alpha-1-antitrypsin was validated in vitreous of an independent set of AMD patients using Western blot analysis. Further systems biology analysis of the data indicated that the observed proteomic changes may reflect upregulation of immune response and complement activity. CONCLUSIONS: Proteome analysis of vitreous samples from patients with AMD, which underwent an intravitreal combination therapy including a core vitrectomy, steroids and bevacizumab, revealed apparent AMD-specific proteomic changes. The identified AMD-associated proteins provide some insight into the pathophysiological changes associated with AMD.

Do cysteine residues regulate transient receptor potential canonical type 6 channel protein expression?

The regulation of calcium influx through transient receptor potential canonical type 6 (TRPC6) channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine (HC) or acetylcysteine (ACC) affect TRPC6 expression in human monocytes. We observed that patients with chronic renal failure had significantly elevated HC levels and TRPC6 mRNA expression levels in monocytes compared with control subjects. We further observed that administration of HC or ACC significantly increased TRPC6 channel protein expression compared with control conditions. We, therefore, hypothesize that cysteine residues increase TRPC6 channel protein expression in humans.

Pulsatile atheroprone shear stress affects the expression of transient receptor potential channels in human endothelial cells.

The goal of the study was to assess whether pulsatile atheroprone shear stress modulates the expression of transient receptor potential (TRP) channels, TRPC3, TRPC6, TRPM7, and TRPV1 mRNA, in human umbilical vascular endothelial cells. Exposure of cultured vascular endothelial cells to defined shear stress, producing a constant laminar flow (generating a shear stress of 6 dyne/cm(2)), laminar pulsatile atheroprotective flow (with a mean shear stress of 20 dyne/cm(2)), or laminar atheroprone bidirectional flow (with a mean shear stress of 0 dyne/cm(2)) differentially induced TRPC6 and TRPV1 mRNA as measured by quantitative real-time RT-PCR and normalized to GAPDH expression. Thereby, TRPC6 and TRPV1 mRNA expressions were significantly increased after 24 hours of exposure to an atheroprone flow profile compared with an atheroprotective flow profile. Furthermore, the expression of transcription factors GATA1 and GATA4 was significantly correlated with the expression of TRPC6 mRNA. In contrast, after 24 hours of constant laminar flow, the expression of TRPC6 and TRPV1 mRNA was unchanged, whereas the expression of TRPC3 and TRPM7 was significantly higher in endothelial cells exposed to shear stress in comparison with endothelial cells grown under static conditions. There was a significant association between the expression of TRPC6 and tumor necrosis factor-α mRNA in human vascular tissue. No-flow and atheroprone flow conditions are equally characterized by an increase in the expression of tumor necrosis factor-α; however, inflammation-associated endothelial cell reactions may be further aggravated at atheroprone flow conditions by the increase of TRPV1 and TRPC6, as observed in our study.

High glucose-induced oxidative stress increases transient receptor potential channel expression in human monocytes.

OBJECTIVE: Transient receptor potential (TRP) channel-induced cation influx activates human monocytes, which play an important role in the pathogenesis of atherosclerosis. In the present study, we investigated the effects of high glucose-induced oxidative stress on TRP channel expression in human monocytes. RESEARCH DESIGN AND METHODS: Human monocytes were exposed to control conditions (5.6 mmol/l d-glucose), high glucose (30 mmol/l d-glucose or l-glucose), 100 micromol/l peroxynitrite, or high glucose in the presence of the superoxide dismutase mimetic tempol (100 micromol/l). TRP mRNA and TRP protein expression was measured using quantitative real-time RT-PCR and quantitative in-cell Western assay, respectively. Calcium influx and intracellular reactive oxygen species were measured using fluorescent dyes. RESULTS: Administration of high d-glucose significantly increased reactive oxygen species. High d-glucose or peroxynitrite significantly increased the expression of TRP canonical type 1 (TRPC1), TRPC3, TRPC5, TRPC6, TRP melastatin type 6 (TRPM6), and TRPM7 mRNA and TRPC3 and TRPC6 proteins. High d-glucose plus tempol or high l-glucose did not affect TRP expression. Increased oxidative stress by lipopolysaccharide or tumor necrosis factor-alpha increased TRP mRNA expression, whereas the reduction of superoxide radicals using diphenylene iodonium significantly reduced TRP mRNA expression. Increased TRPC3 and TRPC6 protein expression was accompanied by increased 1-oleoyl-2-acetyl-sn-glycerol-induced calcium influx, which was blocked by the TRPC inhibitor 2-aminoethoxydiphenylborane. TRPC6 mRNA was significantly higher in monocytes from 18 patients with type 2 diabetes compared with 28 control subjects (P < 0.05). CONCLUSIONS: High d-glucose-induced oxidative stress increases TRP expression and calcium influx in human monocytes, pointing to a novel pathway for increased activation of monocytes and hence atherosclerosis in patients with diabetes.

Low expression of thiosulfate sulfurtransferase (rhodanese) predicts mortality in hemodialysis patients.

OBJECTIVES: To test the hypothesis that impaired expression of the thiosulfate sulfurtransferase rhodanese is associated with oxidative stress and may predict mortality in hemodialysis patients. DESIGN AND METHODS: Sixty-two hemodialysis patients were investigated to determine protein and mRNA expression of rhodanese in monocytes. Whole cell reactive oxygen species and mitochondrial superoxide production were measured by fluorescence spectrophotometry. RESULTS: Compared to healthy subjects, hemodialysis patients showed significantly lower rhodanese mRNA and protein expression and significantly increased reactive oxygen species. Lower rhodanese protein expression was significantly associated with higher mitochondrial superoxide production. The hazard ratio for mortality in hemodialysis patients with rhodanese mRNA below compared to patients above the median was 2.22. Survival was shorter with rhodanese mRNA below compared to patients above the median. CONCLUSION: Impaired rhodanese expression is associated with increased whole cell reactive oxygen species as well as higher mitochondrial superoxide production and predicts mortality in hemodialysis patients.

Transient receptor potential canonical type 3 channels and blood pressure in humans.

OBJECTIVE: There is evidence that transient receptor potential canonical type 3 (TRPC3) cation channels are involved in the regulation of blood pressure, but this has not been studied using human renal tissue. We tested the hypothesis that the expression of TRPC3 in human renal tissue is associated with blood pressure in patients. MATERIAL AND METHODS: TRPC3 was detected in cultured human endothelial cells and in vascular endothelium cells from human renal tissue by immunoblotting, immunohistochemistry, and quantitative real-time reverse transcriptase-PCR. The changes of TRPC3 and vascular endothelial growth factor receptor type 2 expression in cultured human endothelial cells were measured after administration of vascular endothelial growth factor isoform 121. RESULTS: In cultured human endothelial cells, vascular endothelial growth factor isoform 121 significantly reduced TRPC3 expression by 57% and vascular endothelial growth factor receptor type 2 by 70%. This reduction was partly blocked by phosphatidylinositol 3-kinase inhibitors, wortmannin, or LY294002. Downregulation of TRPC3 channel expression was associated with reduced calcium influx. The changes of calcium influx could be abolished by the inhibitor of TRPC channels, 2-aminoethoxydiphenylborane, pointing to their functional importance. TRPC3 expression was significantly higher in patients with SBP more than 140 mmHg compared with patients with SBP of 140 mmHg or less (0.00181 +/- 0.00059 versus 0.00037 +/- 0.00012 arbitrary units; P < 0.01). CONCLUSION: The data support the hypothesis that TRPC3 expression in human renal tissue including vascular endothelium is associated with blood pressure regulation in humans.