Anatomy and Neuroscience - Research Publications

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    Uncovering genetic mechanisms of hypertension through multi-omic analysis of the kidney
    Eales, JM ; Jiang, X ; Xu, X ; Saluja, S ; Akbarov, A ; Cano-Gamez, E ; McNulty, MT ; Finan, C ; Guo, H ; Wystrychowski, W ; Szulinska, M ; Thomas, HB ; Pramanik, S ; Chopade, S ; Prestes, PR ; Wise, I ; Evangelou, E ; Salehi, M ; Shakanti, Y ; Ekholm, M ; Denniff, M ; Nazgiewicz, A ; Eichinger, F ; Godfrey, B ; Antczak, A ; Glyda, M ; Krol, R ; Eyre, S ; Brown, J ; Berzuini, C ; Bowes, J ; Caulfield, M ; Zukowska-Szczechowska, E ; Zywiec, J ; Bogdanski, P ; Kretzler, M ; Woolf, AS ; Talavera, D ; Keavney, B ; Maffia, P ; Guzik, TJ ; O'Keefe, RT ; Trynka, G ; Samani, NJ ; Hingorani, A ; Sampson, MG ; Morris, AP ; Charchar, FJ ; Tomaszewski, M (NATURE PORTFOLIO, 2021-05-06)
    The kidney is an organ of key relevance to blood pressure (BP) regulation, hypertension and antihypertensive treatment. However, genetically mediated renal mechanisms underlying susceptibility to hypertension remain poorly understood. We integrated genotype, gene expression, alternative splicing and DNA methylation profiles of up to 430 human kidneys to characterize the effects of BP index variants from genome-wide association studies (GWASs) on renal transcriptome and epigenome. We uncovered kidney targets for 479 (58.3%) BP-GWAS variants and paired 49 BP-GWAS kidney genes with 210 licensed drugs. Our colocalization and Mendelian randomization analyses identified 179 unique kidney genes with evidence of putatively causal effects on BP. Through Mendelian randomization, we also uncovered effects of BP on renal outcomes commonly affecting patients with hypertension. Collectively, our studies identified genetic variants, kidney genes, molecular mechanisms and biological pathways of key relevance to the genetic regulation of BP and inherited susceptibility to hypertension.
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    A Modified MTS Proliferation Assay for Suspended Cells to Avoid the Interference by Hydralazine and beta-Mercaptoethanol
    Wang, Y ; Nguyen, DT ; Yang, G ; Anesi, J ; Kelly, J ; Chai, Z ; Ahmady, F ; Charchar, F ; Golledge, J (MARY ANN LIEBERT, INC, 2021-01-20)
    The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay is one of the most commonly used tests of cell proliferation. Hydralazine has been reported to interfere with the performance of the MTS assay when used on adherent cells. This study aimed to investigate whether hydralazine interferes with the performance of the MTS assay on suspended cells. THP-1 (a monocytic leukemia cell line) cells were cultured in the presence or absence of hydralazine (0, 10, 50, 100, and 500 μM) for 2 or 24 h. Cell numbers were analyzed using the MTS, trypan blue exclusion, or microscopic assays. A modified version of the standard MTS assay was established by centrifuging the cells and replacing the test medium with fresh culture medium immediately before the addition of the MTS reagent. Culture of THP-1 cells with hydralazine at concentrations of 50, 100, and 500 μM for 2 h increased absorbance (p < 0.001) in the standard MTS assay, whereas both the trypan blue exclusion assay and microscopy suggested no change in cell numbers. Culture of THP-1 cells with 100 and 500 μm hydralazine for 24 h increased absorbance (p < 0.05) in the standard MTS assay; however, trypan blue exclusion and microscopy suggested a decrease in cell numbers. In a cell-free system, hydralazine (100 and 500 μM) increased absorbance in a time- and concentration-dependent manner. The modified MTS assay produced results consistent with trypan blue exclusion and microscopy using THP-1 cells. In addition, the modified MTS assay produced reliable results when K562 and Jurkat cells were incubated with hydralazine or β-mercaptoethanol (βME). In conclusion, a simple modification of the standard MTS assay overcame the interference of hydralazine and βME when assessing suspended cells.
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    Serum antinuclear autoantibodies are associated with measures of oxidative stress and lifestyle factors - analysis of LIPIDOGRAM2015 and LIPIDOGEN2015 studies.
    Krzemień, P ; Kasperczyk, S ; Banach, M ; Kasperczyk, A ; Dobrakowski, M ; Tomasik, T ; Windak, A ; Mastej, M ; Catapano, A ; Ray, K ; Mikhailidis, D ; Toth, P ; Howard, G ; Lip, G ; Tomaszewski, M ; Charchar, F ; Sattar, N ; Williams, B ; MacDonald, T ; Penson, P ; Jóźwiak, J (Termedia Sp. z.o.o., 2021-07-03)
    Introduction Oxidative stress is one of many factors suspected to promote antinuclear autoantibody (ANA) formation. Reactive oxygen species can induce changes in the antigenic structure of macromolecules, causing the immune system to treat them as “neo-antigens” and start production of autoantibodies. This study was designed to evaluate the relationship between oxidative stress markers, lifestyle factors and the detection of ANA. Material and methods We examined measures of oxidative stress indices of free-radical damage to lipids and proteins, such as total oxidant status (TOS), concentration of protein thiol groups (PSH), and malondialdehyde (MDA), activity of superoxide dismutase (SOD) in in 1731 serum samples. The parameters of the non-enzymatic antioxidant system, such as total antioxidant status (TAS) and uric acid concentration (UA), were also measured and the oxidative stress index (OSI -index) was calculated. All samples were tested for the presence of ANA using an indirect immunofluorescence assay (IIFA). Results The presence of ANA in women was associated with lower physical activity (p=0.036), less frequent smoking (p=0.007) and drinking of alcohol (p=0.024) accompanied by significant changes in SOD isoenzymes activity (p<0.001) and a higher uric acid (UA) concentration (p<0.001). In ANA positive males we observed lower concentrations of PSH (p=0.046) and increased concentrations of MDA (p=0.047). Conclusions The results indicate that local oxidative stress may be associated with increased probability of ANA formation in a sex-specific manner.
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    Contributions of obesity to kidney health and disease - insights from mendelian randomisation and the human kidney transcriptomics.
    Xu, X ; Eales, JM ; Jiang, X ; Sanderson, E ; Drzal, M ; Saluja, S ; Scannali, D ; Williams, B ; Morris, AP ; Guzik, TJ ; Charchar, FJ ; Holmes, MV ; Tomaszewski, M ; Antoniades, C (Oxford University Press (OUP), 2021-12-10)
    AIMS: Obesity and kidney diseases are common complex disorders with an increasing clinical and economic impact on healthcare around the globe. Our objective was to examine if modifiable anthropometric obesity indices show putatively causal association with kidney health and disease and highlight biological mechanisms of potential relevance to the association between obesity and the kidney. METHODS AND RESULTS: We performed observational, one-sample, two-sample Mendelian randomisation (MR) and multivariable MR studies in approximately 300,000 participants of white-British ancestry from UK Biobank and participants of predominantly European ancestry from genome-wide association studies. The MR analyses revealed that increasing values of genetically predicted body mass index (BMI) and waist circumference (WC) were causally associated with biochemical indices of renal function, kidney health index (a composite renal outcome derived from blood biochemistry, urine analysis, and International Classification of Disease-based kidney disease diagnoses) and both acute and chronic kidney diseases of different aetiologies including hypertensive renal disease and diabetic nephropathy. Approximately 13-16% and 21-26% of the potentially causal effect of obesity indices on kidney health were mediated by blood pressure and type 2 diabetes, respectively. A total of 61 pathways mapping primarily onto transcriptional/translational regulation, innate and adaptive immunity, extracellular matrix and metabolism were associated with obesity measures in gene set enrichment analysis in up to 467 kidney transcriptomes. CONCLUSIONS: Our data show that a putatively causal association of obesity with renal health is largely independent of blood pressure and type 2 diabetes and uncover the signatures of obesity on the transcriptome of human kidney. TRANSLATIONAL PERSPECTIVE: These findings indicate that obesity is causally linked to indices of renal health and the risk of different kidney diseases. This evidence substantiates the value of weight loss as a strategy of preventing and/or counteracting a decline in kidney health as well as decreasing the risk of renal disease.
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    Relationship Between Anti-DFS70 Autoantibodies and Oxidative Stress.
    Krzemień, P ; Kasperczyk, S ; Banach, M ; Kasperczyk, A ; Dobrakowski, M ; Tomasik, T ; Windak, A ; Mastej, M ; Catapano, A ; Ray, KK ; Mikhailidis, DP ; Toth, PP ; Howard, G ; Lip, GY ; Tomaszewski, M ; Charchar, FJ ; Sattar, N ; Williams, B ; MacDonald, TM ; Penson, PE ; Jóźwiak, JJ (SAGE Publications, 2022)
    BACKGROUND: The anti-DFS70 autoantibodies are one of the most commonly and widely described agent of unknown clinical significance, frequently detected in healthy individuals. It is not known whether the DFS70 autoantibodies are protective or pathogenic. One of the factors suspected of inducing the formation of anti-DFS70 antibodies is increased oxidative stress. We evaluated the coexistence of anti-DFS70 antibodies with selected markers of oxidative stress and investigated whether these antibodies could be considered as indirect markers of oxidative stress. METHODS: The intensity of oxidative stress was measured in all samples via indices of free-radical damage to lipids and proteins such as total oxidant status (TOS), concentrations of lipid hydroperoxides (LPH), lipofuscin (LPS), and malondialdehyde (MDA). The parameters of the non-enzymatic antioxidant system, such as total antioxidant status (TAS) and uric acid concentration (UA), were also measured, as well as the activity of superoxide dismutase (SOD). Based on TOS and TAS values, the oxidative stress index (OSI) was calculated. All samples were also tested with indirect immunofluorescence assay (IFA) and 357 samples were selected for direct monospecific anti DFS70 enzyme-linked immunosorbent assay (ELISA) testing. RESULTS: The anti-DFS70 antibodies were confirmed by ELISA test in 21.29% of samples. Compared with anti-DFS70 negative samples we observed 23% lower concentration of LPH (P = .038) and 11% lower concentration of UA (P = .005). TOS was 20% lower (P = .014). The activity of SOD was up to 5% higher (P = .037). The Pearson correlation showed weak negative correlation for LPH, UA, and TOS and a weak positive correlation for SOD activity. CONCLUSION: In samples positive for the anti-DFS70 antibody a decreased level of oxidative stress was observed, especially in the case of samples with a high antibody titer. Anti-DFS70 antibodies can be considered as an indirect marker of reduced oxidative stress or a marker indicating the recent intensification of antioxidant processes.
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    The Differences in the Prevalence of Cardiovascular Disease, Its Risk Factors, and Achievement of Therapeutic Goals among Urban and Rural Primary Care Patients in Poland: Results from the LIPIDOGRAM 2015 Study
    Studzinski, K ; Tomasik, T ; Windak, A ; Banach, M ; Wojtowicz, E ; Mastej, M ; Tomaszewski, M ; Mikhailidis, DP ; Toth, PP ; Catapano, A ; Ray, KK ; Howard, G ; Lip, GYH ; Charchar, FJ ; Sattar, N ; Williams, B ; MacDonald, TM ; Penson, PE ; Jozwiak, JJ (MDPI, 2021-12-01)
    A nationwide cross-sectional study, LIPIDOGRAM2015, was carried out in Poland in the years 2015 and 2016. A total of 438 primary care physicians enrolled 13,724 adult patients that sought medical care in primary health care practices. The prevalence of hypertension, diabetes mellitus, dyslipidaemia, and CVD were similar in urban and rural areas (49.5 vs. 49.4%; 13.7 vs. 13.1%; 84.2 vs. 85.2%; 14.4 vs. 14.2%, respectively). The prevalence of obesity (32.3 vs. 37.5%, p < 0.01) and excessive waist circumference (77.5 vs. 80.7%, p < 0.01), as well as abdominal obesity (43.2 vs. 46.4%, p < 0.01), were higher in rural areas in both genders. Mean levels of LDL-C (128 vs. 130 mg/dL, p = 0.04) and non-HDL-C (147 vs. 148 mg/dL, p = 0.03) were slightly higher in rural populations. Altogether, 14.3% of patients with CVD from urban areas and 11.3% from rural areas reached LDL <70 mg/dL (p = 0.04). There were no important differences in the prevalence of hypertension, diabetes, dyslipidaemia, and CVD, or in mean levels of blood pressure, cholesterol fractions, glucose, and HbA1c between Polish urban and rural primary care patient populations. A high proportion of patients in cities and an even-higher proportion in rural areas did not reach the recommended targets for blood pressure, LDL-C, and HbA1c, indicating the need for novel CVD-prevention programs.
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    Analysis of the impact of sex and age on the variation in the prevalence of antinuclear autoantibodies in Polish population: a nationwide observational, cross-sectional study.
    Krzemień, P ; Kasperczyk, S ; Banach, M ; Kasperczyk, A ; Dobrakowski, M ; Tomasik, T ; Windak, A ; Mastej, M ; Catapano, A ; Ray, KK ; Mikhailidis, DP ; Toth, PP ; Howard, G ; Lip, GYH ; Tomaszewski, M ; Charchar, FJ ; Sattar, N ; Williams, B ; MacDonald, TM ; Penson, PE ; Jóźwiak, JJ ; LIPIDOGRAM2015 Investigators, (Springer Science and Business Media LLC, 2022-02)
    The detection of antinuclear autoantibody (ANA) is dependent on many factors and varies between the populations. The aim of the study was first to assess the prevalence of ANA in the Polish adult population depending on age, sex and the cutoff threshold used for the results obtained. Second, we estimated the occurrence of individual types of ANA-staining patterns. We tested 1731 patient samples using commercially available IIFA using two cutoff thresholds of 1:100 and 1:160. We found ANA in 260 participants (15.0%), but the percentage of positive results strongly depended on the cutoff level. For a cutoff threshold 1:100, the positive population was 19.5% and for the 1:160 cutoff threshold, it was 11.7%. The most prevalent ANA-staining pattern was AC-2 Dense Fine speckled (50%), followed by AC-21 Reticular/AMA (14.38%) ANA more common in women (72%); 64% of ANA-positive patients were over 50 years of age. ANA prevalence in the Polish population is at a level observed in other highly developed countries and is more prevalent in women and elderly individuals. To reduce the number of positive results released, we suggest that Polish laboratories should set 1:160 as the cutoff threshold.
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    Secondary Stroke Prevention in Polish Adults: Results from the LIPIDOGRAM2015 Study
    Labuz-Roszak, B ; Banach, M ; Skrzypek, M ; Windak, A ; Tomasik, T ; Mastej, M ; Tomaszewski, M ; Mikhailidis, DP ; Toth, PP ; Catapano, A ; Ray, KK ; Howard, G ; Lip, GYH ; Charchar, FJ ; Sattar, N ; Williams, B ; MacDonald, TM ; Penson, P ; Jozwiak, JJ (MDPI, 2021-10-01)
    BACKGROUND: The purpose of the study was to evaluate secondary stroke prevention in Poland and its association with sociodemographic factors, place of residence, and concomitant cardiovascular risk factors. MATERIAL AND METHODS: From all patients in LIPIDOGRAM2015 Study (n = 13,724), 268 subjects had a history of ischaemic stroke and were included. RESULTS: 165 subjects (61.6%) used at least one preventive medication. Oral antiplatelet and anticoagulation agents were used by 116 (43.3%) and 70 (26.1%) patients, respectively. Only 157 (58.6%) participants used lipid-lowering drugs, and 205 (76.5%) were treated with antihypertensive drugs. Coronary heart disease (CHD) and dyslipidaemia were associated with antiplatelet treatment (p = 0.047 and p = 0.012, respectively). A history of atrial fibrillation, CHD, and previous myocardial infarction correlated with anticoagulant treatment (p = 0.001, p = 0.011, and p < 0.0001, respectively). Age, gender, time from stroke onset, place of residence, and level of education were not associated with antiplatelet or anticoagulant treatment. Only 31.7% of patients were engaged in regular physical activity, 62% used appropriate diet, and 13.6% were current smokers. CONCLUSIONS: In Poland drugs and lifestyle modification for secondary stroke prevention are not commonly adhered to. Educational programmes for physicians and patients should be developed to improve application of effective secondary prevention of stroke.
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    Plasma Proteomics of Renal Function: A Transethnic Meta-Analysis and Mendelian Randomization Study
    Matias-Garcia, PR ; Wilson, R ; Guo, Q ; Zaghlool, SB ; Eales, JM ; Xu, X ; Charchar, FJ ; Dormer, J ; Maalmi, H ; Schlosser, P ; Elhadad, MA ; Nano, J ; Sharma, S ; Peters, A ; Fornoni, A ; Mook-Kanamori, DO ; Winkelmann, J ; Danesh, J ; Di Angelantonio, E ; Ouwehand, WH ; Watkins, NA ; Roberts, DJ ; Petrera, A ; Graumann, J ; Koenig, W ; Hveem, K ; Jonasson, C ; Koettgen, A ; Butterworth, A ; Prunotto, M ; Hauck, S ; Herder, C ; Suhre, K ; Gieger, C ; Tomaszewski, M ; Teumer, A ; Waldenberger, M (AMER SOC NEPHROLOGY, 2021-07-01)
    BACKGROUND: Studies on the relationship between renal function and the human plasma proteome have identified several potential biomarkers. However, investigations have been conducted largely in European populations, and causality of the associations between plasma proteins and kidney function has never been addressed. METHODS: A cross-sectional study of 993 plasma proteins among 2,882 participants in four studies of European and admixed ancestries (KORA, INTERVAL, HUNT, QMDiab) identified trans-ethnic associations between eGFR/CKD and proteomic biomarkers. For the replicated associations, two-sample bidirectional Mendelian randomization (MR) was used to investigate potential causal relationships. Publicly available datasets and transcriptomic data from independent studies were used to examine the association between gene expression in kidney tissue and eGFR . RESULTS: Fifty-seven plasma proteins were associated with eGFR, including one novel protein. Twenty-three of these were additionally associated with CKD. The strongest inferred causal effect was the positive effect of eGFR on testican-2, in line with the known biological role of this protein and the expression of its protein-coding gene (SPOCK2) in renal tissue. We also observed suggestive evidence of an effect of melanoma inhibitory activity (MIA), carbonic anhydrase III, and cystatin-M on eGFR. CONCLUSIONS: In a discovery-replication setting, we identified 57 proteins trans-ethnically associated with eGFR. The revealed causal relationships are an important stepping-stone in establishing testican-2 as a clinically relevant physiological marker of kidney disease progression, and point to additional proteins warranting further investigation.
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    Establishment of sex difference in circulating uric acid is associated with higher testosterone and lower sex hormone-binding globulin in adolescent boys.
    Wang, Y ; Charchar, FJ (Springer Science and Business Media LLC, 2021-08-30)
    Men have higher circulating levels of uric acid than women. This sex difference is suspected to be a result of suppressive effects of estradiol on uric acid. If so, estradiol would be inversely associated with circulating uric acid. This study aimed to test this hypothesis. This cross-sectional study included 9472 participants (weighted sample size of 184,342,210) aged 12-80 years from the 2013 to 2016 US National Health and Nutrition Examination Survey. Associations of sex hormones with uric acid were analyzed using weighted least squares regression, adjusting for demographic characteristics, lifestyle risk factors, and comorbidities. Neither free nor bioavailable estradiol was inversely associated with circulating uric acid in adolescent boys or girls, or adult men or women, or perimenopausal women after full adjustment. The sex difference in uric acid was established during adolescence as a result of a dramatic increase in uric acid in adolescent boys. During adolescence, the increase in estradiol in girls over time was accompanied by a relatively unchanged level of uric acid. All three fractions of estradiol (free, bioavailable, and total) were positively associated with uric acid in adolescent boys and girls after full adjustment. In adolescent boys, all three fractions of testosterone were positively associated with serum uric acid, and sex hormone-binding globulin was inversely associated with uric acid after full adjustment. These results suggest that estradiol is not inversely associated with circulating uric acid in adolescents and the establishment of sex difference in circulating uric acid during adolescence is associated with higher testosterone and lower sex hormone-binding globulin in adolescent boys.