Soluble Klotho and Brain Atrophy in Alcoholism

Soluble Klotho and Brain Atrophy in Alcoholism Abstract Aim Fibroblast growth factor (FGF-23) and α-Klotho (Klotho) levels may be altered in inflammatory conditions, possibly as compensatory mechanisms. Klotho exerts a protective effect on neurodegeneration and improves learning and cognition. No data exist about the association of Klotho and FGF-23 levels with brain atrophy observed in alcoholics. The aim of this study is to explore these relationships. Short summary FGF-23 and Klotho levels are altered in inflammation, possibly as compensatory mechanisms. Klotho enhances learning, but its role in ethanol-mediated brain atrophy is unknown. We found higher FGF-23 and lower Klotho levels in 131 alcoholics compared with 41 controls. Among cirrhotics, Klotho was higher and inversely related to brain atrophy. Methods The study was performed on 131 alcoholic patients (54 cirrhotics) and 41 age- and sex-matched controls, in whom a brain computed tomography (CT) was performed and several indices were calculated. Results Marked brain atrophy was observed among patients when compared with controls. Patients also showed higher FGF-23 and lower Klotho values. However, among cirrhotics, Klotho values were higher. Klotho was inversely related to brain atrophy (for instance, ventricular index (ρ = −0.23, P = 0.008)), especially in cirrhotics. Klotho was also directly related to tumor necrosis factor (TNF) alpha (ρ = 0.22; P = 0.026) and inversely to transforming growth factor (TGF)-β (ρ = −0.34; P = 0.002), but not to C-reactive protein (CRP) or malondialdehyde levels. FGF-23 was also higher among cirrhotics but showed no association with CT indices. Conclusions Klotho showed higher values among cirrhotics, and was inversely related to brain atrophy. FGF-23, although high among patients, especially cirrhotics, did not show any association with brain atrophy. Some inflammatory markers or cytokines, such as CRP or TGF-β were related to brain atrophy. INTRODUCTION Fibroblast growth factor (FGF)-23 is a hormone produced mainly by osteocytes in response to high serum phosphate levels. Although initially it was thought that its action was restricted to inhibition of vitamin D synthesis and inhibition of the sodium phosphate transporters in the proximal tubule (Martin et al., 2012), the functions of this growth factor far beyond calcium/phosphate homeostasis are beginning to be disentangled (Kovesdy and Quarles, 2016). For instance, recent research has shown that bacterial lipopolysaccharide, tumor necrosis factor (TNF)-α and other proinflammatory cytokines upregulate the expression of FGF-23 and secretion by osteocytes (Ito et al., 2015). In acute inflammatory states, concomitant up-regulation of intact FGF-23 cleavage within the osteocyte may minimize changes in serum values of intact FGF-23. In chronic inflammation, the intra-osteocyte cleavage machinery becomes surpassed and serum FGF-23 levels rise (David et al., 2016). Therefore, a compensatory, anti-inflammatory role for FGF-23 has been suggested (Nakahara et al., 2016). In addition to increased osteocyte production, FGF-23 becomes expressed by macrophages in response to inflammation (Masuda et al., 2015). Alpha Klotho (Klotho) was first identified as an obligate co-receptor of FGF-23. This is true for membrane-bound Klotho. Alternative splicing of the Klotho gene and shedding of membrane-anchored Klotho by proteases of the ADAM (a disintegrin and metalloproteinase) group generate the soluble form of Klotho that can be detected in blood, cerebrospinal fluid and urine (Hu et al., 2013). Soluble Klotho is a pleiotropic hormone that exerts many FGF-23 independent functions, notably antioxidative (Yamamoto et al., 2005), anticell senescence (Kurosu et al., 2005), anti-apoptotic (Ikushima et al., 2006) and antibrogenic effects (Doi et al., 2011). As with FGF-23, some data suggest that Klotho exerts anti-inflammatory effects. Overexpression of Klotho counteracts increased expression of IL-6 after TNF-α stimulus, blunting the TNF proinflammatory effect (Xia et al., 2016). Some reports suggest that FGF-23 and Klotho values are altered in alcoholics. Reasons for this increase remain elusive. Given the proinflammatory nature of alcoholism, especially when it has led to liver cirrhosis (Bishehsari et al., 2017), it could be speculated that inflammation might play a role in these changes. One of the main features of alcoholic patients is brain atrophy (de la Monte and Kril, 2014). White matter atrophy, due to altered myelin synthesis, is the main target of ethanol (Navarro and Mandyam, 2015). Neurodegeneration of hippocampal neurons and circuits also occur (Qin and Crews, 2012). Although some effects are directly derived from ethanol and/or acetaldehyde, inflammation and oxidative damage may play a role (Qin et al., 2007; Umhau et al., 2014). On the other hand, excessively high FGF-23 serum levels may be associated with impaired learning and memory (Liu et al., 2011), especially in chronic kidney disease (Drew et al., 2014). The opposite is observed with Klotho (Haffner and Leifheit-Nestler, 2017). Indeed, there are Klotho dependent and Klotho independent signaling pathways in the hippocampal neurons. Klotho dependent pathways may enhance memory and learning, whereas Klotho independent pathways have the opposite effect (Hensel et al., 2016). In this study, we want to know whether changes in brain atrophy have any association with Klotho and FGF-23, and also whether serum levels of these molecules are related to systemic inflammation that occurs in alcoholism. Patients and methods We included 131 patients (7 women), consecutively admitted to our hospitalization unit, aged 57.60 ± 11.11 years and 41 controls (6 women) of similar age (54.49 ± 7.36 years, t = 1.60; NS) and sex (P = 0.08 by exact Fisher’s test), all of them workers in our hospital or their relatives, drinkers of <10 g ethanol/day. We did not include patients who also reported consumption of other drugs besides tobacco. Some biological data of the included patients are shown in Table 1. All the patients lived in the northern part of Tenerife (almost all of them in rural areas) and were heavy drinkers until admission or the day before admission (in the case of those 56 patients who were brought to the hospital because of major symptoms of withdrawal syndrome). Patients were not consumers of any other drug besides tobacco (94 patients) and were not receiving psychiatric medication. Table 1. Some epidemiological data and comorbid conditions of the patients included Ethanol consumption (g/day) 187 ± 154 150 (96–240) Years of consumption 33 ± 14 32 (25–40) Time since last drink (days)a 3–4 Antibodies anti-hepatitis C virus infection (yes/no) 8/123 Hypertension (yes/no) 52/78 Diabetes (yes/no) 23/108 Cirrhosis (yes/no) 54/77 Ascites (yes/no) 24/107 Encephalopathy (yes/no) 15/116 Obese (BMI over 30 kg/m2) 16/115 Tobacco (yes/no) 94/34 Packs/year 46 ± 36 38 (20–60) Admission due to Major withdrawal symptoms = 56 Decompensated ascites = 30 Acute alcoholic hepatitis = 11 Infection = 18 (including two spontaneous bacterial peritonitis) Other causes (mainly neoplasia, pancreatitis, heart failure) = 16 Ethanol consumption (g/day) 187 ± 154 150 (96–240) Years of consumption 33 ± 14 32 (25–40) Time since last drink (days)a 3–4 Antibodies anti-hepatitis C virus infection (yes/no) 8/123 Hypertension (yes/no) 52/78 Diabetes (yes/no) 23/108 Cirrhosis (yes/no) 54/77 Ascites (yes/no) 24/107 Encephalopathy (yes/no) 15/116 Obese (BMI over 30 kg/m2) 16/115 Tobacco (yes/no) 94/34 Packs/year 46 ± 36 38 (20–60) Admission due to Major withdrawal symptoms = 56 Decompensated ascites = 30 Acute alcoholic hepatitis = 11 Infection = 18 (including two spontaneous bacterial peritonitis) Other causes (mainly neoplasia, pancreatitis, heart failure) = 16 aDays of abstinence until blood extraction for FGF/Klotho determinations. Table 1. Some epidemiological data and comorbid conditions of the patients included Ethanol consumption (g/day) 187 ± 154 150 (96–240) Years of consumption 33 ± 14 32 (25–40) Time since last drink (days)a 3–4 Antibodies anti-hepatitis C virus infection (yes/no) 8/123 Hypertension (yes/no) 52/78 Diabetes (yes/no) 23/108 Cirrhosis (yes/no) 54/77 Ascites (yes/no) 24/107 Encephalopathy (yes/no) 15/116 Obese (BMI over 30 kg/m2) 16/115 Tobacco (yes/no) 94/34 Packs/year 46 ± 36 38 (20–60) Admission due to Major withdrawal symptoms = 56 Decompensated ascites = 30 Acute alcoholic hepatitis = 11 Infection = 18 (including two spontaneous bacterial peritonitis) Other causes (mainly neoplasia, pancreatitis, heart failure) = 16 Ethanol consumption (g/day) 187 ± 154 150 (96–240) Years of consumption 33 ± 14 32 (25–40) Time since last drink (days)a 3–4 Antibodies anti-hepatitis C virus infection (yes/no) 8/123 Hypertension (yes/no) 52/78 Diabetes (yes/no) 23/108 Cirrhosis (yes/no) 54/77 Ascites (yes/no) 24/107 Encephalopathy (yes/no) 15/116 Obese (BMI over 30 kg/m2) 16/115 Tobacco (yes/no) 94/34 Packs/year 46 ± 36 38 (20–60) Admission due to Major withdrawal symptoms = 56 Decompensated ascites = 30 Acute alcoholic hepatitis = 11 Infection = 18 (including two spontaneous bacterial peritonitis) Other causes (mainly neoplasia, pancreatitis, heart failure) = 16 aDays of abstinence until blood extraction for FGF/Klotho determinations. Fifty-one patients (out of 85 from whom reliable information was obtained) had suffered traumatic brain injury; 52 suffered hypertension; 33 dyslipemia, and 23, diabetes. Body mass index (BMI) was also recorded: 16 patients showed BMI over 30 kg/m2, 32 had BMI values between 25 and 30, and the remaining 83 patients a BMI below 25. All patients underwent an abdominal ultrasound (US) exploration and complete laboratory evaluation. Based on US data (liver with irregular shape, heterogeneous texture, and splenomegaly and/or portal dilatation) patients were classified as cirrhotics or non-cirrhotics. Methods Brain atrophy assessment All patients underwent, at admission, a brain CT examination. Gross atrophy was observed when CT images of alcoholics were compared with those of controls (Fig. 1a). From the CT images, we calculated the indices shown in Fig. 1b. In some cases, technical problems (moved images) precluded accurate calculation of these indices. We also evaluated the global diagnosis of the radiologist regarding the presence or not of cortical atrophy and/or cerebellar atrophy. Fig. 1. View largeDownload slide (a) A brain computerized tomography (CT) of a normal control (left) compared with that of an alcoholic patient (right). (b) Indices derived from computed tomography (CT). BICAUDATE = minimum width of lateral ventricles/skull width at the same level = B/E. BIFRONTAL = maximum width of frontal horns/skull width at the same level = A/D. EVANS = maximum width of frontal horns/skull width at the level of the III ventricle = A/F. CELLA = width of the III ventricle/skull width at the same level = C/F. VENTRICULAR = minimum width of lateral ventricles/maximum width of frontal horns = B/A. Fig. 1. View largeDownload slide (a) A brain computerized tomography (CT) of a normal control (left) compared with that of an alcoholic patient (right). (b) Indices derived from computed tomography (CT). BICAUDATE = minimum width of lateral ventricles/skull width at the same level = B/E. BIFRONTAL = maximum width of frontal horns/skull width at the same level = A/D. EVANS = maximum width of frontal horns/skull width at the level of the III ventricle = A/F. CELLA = width of the III ventricle/skull width at the same level = C/F. VENTRICULAR = minimum width of lateral ventricles/maximum width of frontal horns = B/A. Laboratory evaluation Within the first 72–96 h after admission, blood samples were taken at 8.00 a.m. in fasting conditions and were immediately frozen at −20°C. In addition to routine laboratory evaluation, including C-reactive protein (CRP), serum α Klotho, FGF-23, TNF-α and transforming growth factor (TGF)-β were determined by the following procedures: FGF-23 (plasma levels) was measured using a second-generation ELISA (Immutopics, Inc. San Clemente, California). The sensitivity of this assay as determined by the 95% confidence limit on 20 duplicate determinations of the 0 RU/ml standard is 1.5 RU/ml. The inter-assay and intra-assay coefficients of variation are <5%. The percentage of recovery for this assay ranged from 91% to 116%. Serum α Klotho levels were determined by ELISA (Immuno-Biological Laboratories, Fujioka, Japan). Inter-assay and intra-assay variation coefficients range from 2.9 to 11.4% and 2.7 to 3.5%, respectively. The percentage of recovery ranged 84.7–97.5%. Sensitivity is 6.15 pg/ml, and cross-reactivity with other molecules such as osteopontin, platelet-derived growth factor or vascular endothelial growth factor is <0.1%. Serum TNF-α was determined by immunometric chemiluminiscent assay (intra-assay variation coefficient ranging 4–6.5%, inter-assay variation coefficient ranging 2.6–3.6%, recovery 92–112%, Diagnostic Products Corporation, Los Angeles, CA, USA); TGF-β levels were determined by quantitative ELISA (Quantikine ELISA kit), with a sensitivity of 15.4 pg/ml, recovery 86–115%; intra-assay variation coefficient 2.5–2.9; inter-assay variation coefficient 6.4–9.1% (R&D Systems, Abindong Science Park, Abindong, UK). Lipid peroxidation products Serum malondialdehyde (MDA) levels were measured as thiobarbituric acid-reactive substance (TBARS) using the method reported by Kikugawa et al. (1992). In this method, 0.2 ml of plasma was added to 0.2 ml of H3PO4 (0.2 M). In order to obtain a color reaction, 25 μl of 0.11 M TBA solution was added. The samples were then heated at 90°C for 45 min. The MDA-TBA condensation produces a pink color species (the intensity of the color indicates the degree of lipid peroxidation). The samples were then cooled and the TBARS were extracted with 0.4 ml of n-butanol. The butanol phase was separated by centrifugation at 6000×g for 10 min. The aliquots of this phase were then placed in a 96-well plate and read at 535 nm in a microlate spectrophotometer reader (Benchmark Plus, Bio-Rad, Hercules, CA, USA). The calibration curve was prepared with authentic MDA standards ranging from 0 to 20 μM. The intra- and inter-assay variation coefficients were 1.82 and 4.01, respectively. Statistics The Kolmogorov–Smirnov test was used to test for normal distribution, a condition not fulfilled by several variables. Therefore, non-parametric tests, such as Mann–Whitney’s U-test and Kruskall–Wallis were used to analyze differences in these parameters between groups. Student’s t-test, variance analysis and Pearson’s correlation analysis were used for the variables with a normal distribution, whereas Spearman’s rho (instead of Pearson’s correlation) was utilized in the case of non-parametric variables. All of these analyses were performed using SPSS software (Chicago, Ill., USA). Multivariate analyses, including multiple linear correlation analysis and logistic regression analysis (after transformation of linear variables into qualitative ones according to median values), were also performed when necessary. The study protocol was approved by the local ethical committee of our Hospital (number 2017/50) and conforms to the ethical guidelines of the 1975 Declaration of Helsinki. All patients and controls gave their written informed consent. Results Despite similar creatinine levels among patients and controls (Table 2), patients showed significantly higher FGF-23 values than controls (Z = 3.63; P < 0.001), but lower plasma Klotho values (Z = 2.12; P = 0.034). No relationship was observed between alpha Klotho and FGF-23 levels (ρ = 0.14; P = 0.14 (NS) for the entire population; ρ= 0.08 among cirrhotics and ρ = 0.05 among non-cirrhotics). Logistic regression analysis also showed that differences between patients and controls were not attributable to differences in creatinine. Table 2. Differences in some biological variables between patients and controls, including differences in brain computerized tomography-derived indices Patients (n = 131) Controls (n = 41) FGF-23 (RU/ml) 318.02 ± 867.45 94.97 (55.15–252.53) 67.02 ± 37.05 59.36 (51.44–73.25) Z = 3.63; P < 0.001 Klotho (pg/ml) 540.12 ± 371.70 459.00 (291.40–690.30) 594.83 ± 247.45 525.60 (427.50–782.70) Z = 2.12; P = 0.034 Serum calcium (mg/dl) 8.99 ± 0.80 Normal range = 9.0–10.4 Serum phosphate (mg/dl)a 3.10 ± 0.84 Normal range = 2.5–7.0 Serum magnesium (mg/dl)b 1.82 ± 0.41 Normal range = 1.6–2.6 Parathyroid hormone (mg/dl)c 41.17 ± 56.70 38.46 ± 22.09 T= 054; P = 0.60 (NS) Vitamin D (25 OH) (mg/dl)d 17.81 ± 11.27 82.51 ± 27.55 T = 11.75; P < 0.001 TNF-α (pg/ml)e 15.22 ± 96.50 5.00(1.59–5.00) 5.49 ± 2.53 5.00 (1.96–5.02) Z = 2.54; P = 0.014 TGF-β (pg/ml)f 19,785 ± 12017 15,896 ± 9967 T = 1.13; P = 0.26 (NS) Malondialdehyde (μmol/l)g 3.04 ± 2.84 2.30(1.30-3.64) 1.60 ± 0.26 1.62 (1.43–1.74) Z = 3.05; P = 0.002 Creatinine (mg/dl) 0.9471 ± 0.6545 0.9690 ± 0.1435 T = 0.52; P = 0.60 (NS) Ventricular index 0.4834 ± 0.0860 0.4114 ± 0.0781 T = 4.66; P < 0.001 Bicaudate index 0.1612 ± 0.0362 0.1235 ± 0.0247 T = 7.34; P < 0.001 Bifrontal index 0.3506 ± 0.0422 0.3213 ± 0.0333 T = 4.02; P < 0.001 Evans index 0.2999 ± 0.0366 0.2993 ± 0.0363 T= 3.19; P = 0.002 Cella index 0.0638 ± 0.0199 0.0443 ± 0.0134 T = 6.60; P < 0.001 Patients (n = 131) Controls (n = 41) FGF-23 (RU/ml) 318.02 ± 867.45 94.97 (55.15–252.53) 67.02 ± 37.05 59.36 (51.44–73.25) Z = 3.63; P < 0.001 Klotho (pg/ml) 540.12 ± 371.70 459.00 (291.40–690.30) 594.83 ± 247.45 525.60 (427.50–782.70) Z = 2.12; P = 0.034 Serum calcium (mg/dl) 8.99 ± 0.80 Normal range = 9.0–10.4 Serum phosphate (mg/dl)a 3.10 ± 0.84 Normal range = 2.5–7.0 Serum magnesium (mg/dl)b 1.82 ± 0.41 Normal range = 1.6–2.6 Parathyroid hormone (mg/dl)c 41.17 ± 56.70 38.46 ± 22.09 T= 054; P = 0.60 (NS) Vitamin D (25 OH) (mg/dl)d 17.81 ± 11.27 82.51 ± 27.55 T = 11.75; P < 0.001 TNF-α (pg/ml)e 15.22 ± 96.50 5.00(1.59–5.00) 5.49 ± 2.53 5.00 (1.96–5.02) Z = 2.54; P = 0.014 TGF-β (pg/ml)f 19,785 ± 12017 15,896 ± 9967 T = 1.13; P = 0.26 (NS) Malondialdehyde (μmol/l)g 3.04 ± 2.84 2.30(1.30-3.64) 1.60 ± 0.26 1.62 (1.43–1.74) Z = 3.05; P = 0.002 Creatinine (mg/dl) 0.9471 ± 0.6545 0.9690 ± 0.1435 T = 0.52; P = 0.60 (NS) Ventricular index 0.4834 ± 0.0860 0.4114 ± 0.0781 T = 4.66; P < 0.001 Bicaudate index 0.1612 ± 0.0362 0.1235 ± 0.0247 T = 7.34; P < 0.001 Bifrontal index 0.3506 ± 0.0422 0.3213 ± 0.0333 T = 4.02; P < 0.001 Evans index 0.2999 ± 0.0366 0.2993 ± 0.0363 T= 3.19; P = 0.002 Cella index 0.0638 ± 0.0199 0.0443 ± 0.0134 T = 6.60; P < 0.001 a67 Patients. b119 Patients. c25 Controls and 94 patients. d26 Controls and 113 patients. e102 Patients and 20 controls. f77 Patients and 19 controls. g126 Patients and 28 controls. FGF, fibroblast growth factor; TNF, tumor necrosis factor; TGF, transforming growth factor. Table 2. Differences in some biological variables between patients and controls, including differences in brain computerized tomography-derived indices Patients (n = 131) Controls (n = 41) FGF-23 (RU/ml) 318.02 ± 867.45 94.97 (55.15–252.53) 67.02 ± 37.05 59.36 (51.44–73.25) Z = 3.63; P < 0.001 Klotho (pg/ml) 540.12 ± 371.70 459.00 (291.40–690.30) 594.83 ± 247.45 525.60 (427.50–782.70) Z = 2.12; P = 0.034 Serum calcium (mg/dl) 8.99 ± 0.80 Normal range = 9.0–10.4 Serum phosphate (mg/dl)a 3.10 ± 0.84 Normal range = 2.5–7.0 Serum magnesium (mg/dl)b 1.82 ± 0.41 Normal range = 1.6–2.6 Parathyroid hormone (mg/dl)c 41.17 ± 56.70 38.46 ± 22.09 T= 054; P = 0.60 (NS) Vitamin D (25 OH) (mg/dl)d 17.81 ± 11.27 82.51 ± 27.55 T = 11.75; P < 0.001 TNF-α (pg/ml)e 15.22 ± 96.50 5.00(1.59–5.00) 5.49 ± 2.53 5.00 (1.96–5.02) Z = 2.54; P = 0.014 TGF-β (pg/ml)f 19,785 ± 12017 15,896 ± 9967 T = 1.13; P = 0.26 (NS) Malondialdehyde (μmol/l)g 3.04 ± 2.84 2.30(1.30-3.64) 1.60 ± 0.26 1.62 (1.43–1.74) Z = 3.05; P = 0.002 Creatinine (mg/dl) 0.9471 ± 0.6545 0.9690 ± 0.1435 T = 0.52; P = 0.60 (NS) Ventricular index 0.4834 ± 0.0860 0.4114 ± 0.0781 T = 4.66; P < 0.001 Bicaudate index 0.1612 ± 0.0362 0.1235 ± 0.0247 T = 7.34; P < 0.001 Bifrontal index 0.3506 ± 0.0422 0.3213 ± 0.0333 T = 4.02; P < 0.001 Evans index 0.2999 ± 0.0366 0.2993 ± 0.0363 T= 3.19; P = 0.002 Cella index 0.0638 ± 0.0199 0.0443 ± 0.0134 T = 6.60; P < 0.001 Patients (n = 131) Controls (n = 41) FGF-23 (RU/ml) 318.02 ± 867.45 94.97 (55.15–252.53) 67.02 ± 37.05 59.36 (51.44–73.25) Z = 3.63; P < 0.001 Klotho (pg/ml) 540.12 ± 371.70 459.00 (291.40–690.30) 594.83 ± 247.45 525.60 (427.50–782.70) Z = 2.12; P = 0.034 Serum calcium (mg/dl) 8.99 ± 0.80 Normal range = 9.0–10.4 Serum phosphate (mg/dl)a 3.10 ± 0.84 Normal range = 2.5–7.0 Serum magnesium (mg/dl)b 1.82 ± 0.41 Normal range = 1.6–2.6 Parathyroid hormone (mg/dl)c 41.17 ± 56.70 38.46 ± 22.09 T= 054; P = 0.60 (NS) Vitamin D (25 OH) (mg/dl)d 17.81 ± 11.27 82.51 ± 27.55 T = 11.75; P < 0.001 TNF-α (pg/ml)e 15.22 ± 96.50 5.00(1.59–5.00) 5.49 ± 2.53 5.00 (1.96–5.02) Z = 2.54; P = 0.014 TGF-β (pg/ml)f 19,785 ± 12017 15,896 ± 9967 T = 1.13; P = 0.26 (NS) Malondialdehyde (μmol/l)g 3.04 ± 2.84 2.30(1.30-3.64) 1.60 ± 0.26 1.62 (1.43–1.74) Z = 3.05; P = 0.002 Creatinine (mg/dl) 0.9471 ± 0.6545 0.9690 ± 0.1435 T = 0.52; P = 0.60 (NS) Ventricular index 0.4834 ± 0.0860 0.4114 ± 0.0781 T = 4.66; P < 0.001 Bicaudate index 0.1612 ± 0.0362 0.1235 ± 0.0247 T = 7.34; P < 0.001 Bifrontal index 0.3506 ± 0.0422 0.3213 ± 0.0333 T = 4.02; P < 0.001 Evans index 0.2999 ± 0.0366 0.2993 ± 0.0363 T= 3.19; P = 0.002 Cella index 0.0638 ± 0.0199 0.0443 ± 0.0134 T = 6.60; P < 0.001 a67 Patients. b119 Patients. c25 Controls and 94 patients. d26 Controls and 113 patients. e102 Patients and 20 controls. f77 Patients and 19 controls. g126 Patients and 28 controls. FGF, fibroblast growth factor; TNF, tumor necrosis factor; TGF, transforming growth factor. When the sample was classified in cirrhotics and non-cirrhotics (Table 3), we found that Klotho levels were significantly ‘higher’ among cirrhotics (Z = 4.69; P < 0.001), a result that was independent of creatinine (odds ratio (OR) for Klotho over the median = 0.20, 95% confidence interval (CI) = 0.09–0.424; B = −1.61; P < 0.001). FGF-23 levels were also significantly higher (Z = 2.25; P = 0.024) among cirrhotics when compared with non-cirrhotics, but when a logistic regression between cirrhosis and median values of creatinine and FGF-23 was performed, none of the variables showed an independent relationship with the presence or not of cirrhosis. No differences were observed between cirrhotics and controls by SNK test regarding Klotho values, but cirrhotics showed markedly higher FGF-23 values than controls (Z = 4.89; P < 0.001). Table 3. Differences in some biological variables between cirrhotics and non-cirrhotics, including differences in brain computerized tomography-derived indices Cirrhotics (n = 54) Non-cirrhotics (n = 77) Age 59.39 ± 9.81 57.03 ± 12.42 T= 1.24; P = 0.25 (NS) FGF-23 (RU/ml) 294.99 ± 363.87 333.04 ± 1078.97 81.63 (38.97–221.01) Z = 2.25; P = 0.024 127.76 (68.73–429.17) Klotho (pg/ml) 697.80 ± 412.65 429.54 ± 295.85 360.50 (241.25–521.10) Z = 4.69; P < 0.001 666.15 (422.55–828.60) Creatinine (mg/dl) 1.099 ± 0.901 0.841 ± 0.370 T = 1.99; P = 0.05 Glomerular filtration rate (ml/min) 104.89 ± 50.80 113.38 ± 41.29 T= 1.05; P = 0.30 (NS) Daily ethanol (g) 185 ± 113 190 ± 157 T = 0.20; P = 0.84 (NS) Years of addiction 33 ± 13 32 ± 14 T= 0.26; P = 0.79 (NS) TNF-α (pg/ml)a 26.77 ± 150.18 4.06 (1.59–5.00) 7.36 ± 11.23 5.00 (1.59–5.19) Z = 1.37; P = 0.17 (NS) TGF-β (pg/ml)b 16,951 ± 11095 21,347 ± 12573 T= 1.55; P = 0.13 (NS) Malondialdehyde (μmol/l)c 3.78 ± 3.56 2.90 (1.82–4.31) 2.53 ± 2.09 1.88 (1.24–3.32) Z = 2.85; P = 0.004 C-reactive protein (mg/l) 27.78 ± 40.65 13.95 (4.53–32.43) 24.78 ± 43.04 10.15 (3.53–28.50) Z = 0.77; P = 0.44 (NS) Albumin (g/dl) 3.46 ± 0.72 3.72 ± 0.59 T = 2.32; P = 0.022 MCV (fl) 100.59 ± 10.73 99.54 ± 6.77 T= 0.64; P = 0.49 (NS) Prothrombin activity (%) 68.76 ± 21.37 68.00 (55.75-86.25) 87.77 ± 14.18 90.00 (77.50-100.00) Z = 5.21; P < 0.001 Serum bilirubin (mg/dl) 3.52 ± 4.54 1.65 (1.00-3.43) 1.36 ± 1.58 1.00 (1.00-1.00) Z = 5.19; P < 0.001 Serum GGT (U/l) 391.57 ± 662.21 203.50 (81.25–388.50) 188.92 ± 255.04 96.00 (45.00–228.75) Z = 2.28; P = 0.005 Serum ASAT (U/l) 85.55 ± 84.86 45.00 (28.50–109.75) 58.25 ± 85.75 31.00 (21.00–71.50) Z = 2.27; P = 0.007 Serum ALAT (U/l) 55.65 ± 66.31 36.50 (18.50–67.00) 47.70 ± 60.13 31.00 (17.00–49.00) Z = 1.15; P = 0.25 (NS) Bicaudate indexd 0.1641 ± 0.0385 0.1591 ± 0.0347 T = 0.76; P = 0.45 (NS) Bifrontal indexd 0.3528 ± 0.0465 0.3490 ± 0.0391 T = 0.50; P = 0.62 (NS) Evans’ indexd 0.3020 ± 0.0405 0.2984 ± 0.0337 T = 0.54; P = 0.59 (NS) Cella indexd 0.0646 ± 0.0215 0.0632 ± 0.0188 T= 0.40; P = 0.69 (NS) Ventricular indexd 0.4881 ± 0.0914 0.4800 ± 0.0791 T= 0.53; P = 0.60 (NS) Cirrhotics (n = 54) Non-cirrhotics (n = 77) Age 59.39 ± 9.81 57.03 ± 12.42 T= 1.24; P = 0.25 (NS) FGF-23 (RU/ml) 294.99 ± 363.87 333.04 ± 1078.97 81.63 (38.97–221.01) Z = 2.25; P = 0.024 127.76 (68.73–429.17) Klotho (pg/ml) 697.80 ± 412.65 429.54 ± 295.85 360.50 (241.25–521.10) Z = 4.69; P < 0.001 666.15 (422.55–828.60) Creatinine (mg/dl) 1.099 ± 0.901 0.841 ± 0.370 T = 1.99; P = 0.05 Glomerular filtration rate (ml/min) 104.89 ± 50.80 113.38 ± 41.29 T= 1.05; P = 0.30 (NS) Daily ethanol (g) 185 ± 113 190 ± 157 T = 0.20; P = 0.84 (NS) Years of addiction 33 ± 13 32 ± 14 T= 0.26; P = 0.79 (NS) TNF-α (pg/ml)a 26.77 ± 150.18 4.06 (1.59–5.00) 7.36 ± 11.23 5.00 (1.59–5.19) Z = 1.37; P = 0.17 (NS) TGF-β (pg/ml)b 16,951 ± 11095 21,347 ± 12573 T= 1.55; P = 0.13 (NS) Malondialdehyde (μmol/l)c 3.78 ± 3.56 2.90 (1.82–4.31) 2.53 ± 2.09 1.88 (1.24–3.32) Z = 2.85; P = 0.004 C-reactive protein (mg/l) 27.78 ± 40.65 13.95 (4.53–32.43) 24.78 ± 43.04 10.15 (3.53–28.50) Z = 0.77; P = 0.44 (NS) Albumin (g/dl) 3.46 ± 0.72 3.72 ± 0.59 T = 2.32; P = 0.022 MCV (fl) 100.59 ± 10.73 99.54 ± 6.77 T= 0.64; P = 0.49 (NS) Prothrombin activity (%) 68.76 ± 21.37 68.00 (55.75-86.25) 87.77 ± 14.18 90.00 (77.50-100.00) Z = 5.21; P < 0.001 Serum bilirubin (mg/dl) 3.52 ± 4.54 1.65 (1.00-3.43) 1.36 ± 1.58 1.00 (1.00-1.00) Z = 5.19; P < 0.001 Serum GGT (U/l) 391.57 ± 662.21 203.50 (81.25–388.50) 188.92 ± 255.04 96.00 (45.00–228.75) Z = 2.28; P = 0.005 Serum ASAT (U/l) 85.55 ± 84.86 45.00 (28.50–109.75) 58.25 ± 85.75 31.00 (21.00–71.50) Z = 2.27; P = 0.007 Serum ALAT (U/l) 55.65 ± 66.31 36.50 (18.50–67.00) 47.70 ± 60.13 31.00 (17.00–49.00) Z = 1.15; P = 0.25 (NS) Bicaudate indexd 0.1641 ± 0.0385 0.1591 ± 0.0347 T = 0.76; P = 0.45 (NS) Bifrontal indexd 0.3528 ± 0.0465 0.3490 ± 0.0391 T = 0.50; P = 0.62 (NS) Evans’ indexd 0.3020 ± 0.0405 0.2984 ± 0.0337 T = 0.54; P = 0.59 (NS) Cella indexd 0.0646 ± 0.0215 0.0632 ± 0.0188 T= 0.40; P = 0.69 (NS) Ventricular indexd 0.4881 ± 0.0914 0.4800 ± 0.0791 T= 0.53; P = 0.60 (NS) a44 Cirrhotics and 58 non-cirrhotics. b29 Cirrhotics and 48 non-cirrhotics. c51 Cirrhotics and 75 non-cirrhotics. d52 Cirrhotics and 74 non-cirrhotics. FGF, fibroblast growth factor; TNF, tumor necrosis factor; TGF, transforming growth factor; MCV, mean corpuscular volume; GGT, gamma-glutamyl transferase; ASAT, aspartate aminotransferase; ALAT, alanine aminotransferase; NS, non-significant. Table 3. Differences in some biological variables between cirrhotics and non-cirrhotics, including differences in brain computerized tomography-derived indices Cirrhotics (n = 54) Non-cirrhotics (n = 77) Age 59.39 ± 9.81 57.03 ± 12.42 T= 1.24; P = 0.25 (NS) FGF-23 (RU/ml) 294.99 ± 363.87 333.04 ± 1078.97 81.63 (38.97–221.01) Z = 2.25; P = 0.024 127.76 (68.73–429.17) Klotho (pg/ml) 697.80 ± 412.65 429.54 ± 295.85 360.50 (241.25–521.10) Z = 4.69; P < 0.001 666.15 (422.55–828.60) Creatinine (mg/dl) 1.099 ± 0.901 0.841 ± 0.370 T = 1.99; P = 0.05 Glomerular filtration rate (ml/min) 104.89 ± 50.80 113.38 ± 41.29 T= 1.05; P = 0.30 (NS) Daily ethanol (g) 185 ± 113 190 ± 157 T = 0.20; P = 0.84 (NS) Years of addiction 33 ± 13 32 ± 14 T= 0.26; P = 0.79 (NS) TNF-α (pg/ml)a 26.77 ± 150.18 4.06 (1.59–5.00) 7.36 ± 11.23 5.00 (1.59–5.19) Z = 1.37; P = 0.17 (NS) TGF-β (pg/ml)b 16,951 ± 11095 21,347 ± 12573 T= 1.55; P = 0.13 (NS) Malondialdehyde (μmol/l)c 3.78 ± 3.56 2.90 (1.82–4.31) 2.53 ± 2.09 1.88 (1.24–3.32) Z = 2.85; P = 0.004 C-reactive protein (mg/l) 27.78 ± 40.65 13.95 (4.53–32.43) 24.78 ± 43.04 10.15 (3.53–28.50) Z = 0.77; P = 0.44 (NS) Albumin (g/dl) 3.46 ± 0.72 3.72 ± 0.59 T = 2.32; P = 0.022 MCV (fl) 100.59 ± 10.73 99.54 ± 6.77 T= 0.64; P = 0.49 (NS) Prothrombin activity (%) 68.76 ± 21.37 68.00 (55.75-86.25) 87.77 ± 14.18 90.00 (77.50-100.00) Z = 5.21; P < 0.001 Serum bilirubin (mg/dl) 3.52 ± 4.54 1.65 (1.00-3.43) 1.36 ± 1.58 1.00 (1.00-1.00) Z = 5.19; P < 0.001 Serum GGT (U/l) 391.57 ± 662.21 203.50 (81.25–388.50) 188.92 ± 255.04 96.00 (45.00–228.75) Z = 2.28; P = 0.005 Serum ASAT (U/l) 85.55 ± 84.86 45.00 (28.50–109.75) 58.25 ± 85.75 31.00 (21.00–71.50) Z = 2.27; P = 0.007 Serum ALAT (U/l) 55.65 ± 66.31 36.50 (18.50–67.00) 47.70 ± 60.13 31.00 (17.00–49.00) Z = 1.15; P = 0.25 (NS) Bicaudate indexd 0.1641 ± 0.0385 0.1591 ± 0.0347 T = 0.76; P = 0.45 (NS) Bifrontal indexd 0.3528 ± 0.0465 0.3490 ± 0.0391 T = 0.50; P = 0.62 (NS) Evans’ indexd 0.3020 ± 0.0405 0.2984 ± 0.0337 T = 0.54; P = 0.59 (NS) Cella indexd 0.0646 ± 0.0215 0.0632 ± 0.0188 T= 0.40; P = 0.69 (NS) Ventricular indexd 0.4881 ± 0.0914 0.4800 ± 0.0791 T= 0.53; P = 0.60 (NS) Cirrhotics (n = 54) Non-cirrhotics (n = 77) Age 59.39 ± 9.81 57.03 ± 12.42 T= 1.24; P = 0.25 (NS) FGF-23 (RU/ml) 294.99 ± 363.87 333.04 ± 1078.97 81.63 (38.97–221.01) Z = 2.25; P = 0.024 127.76 (68.73–429.17) Klotho (pg/ml) 697.80 ± 412.65 429.54 ± 295.85 360.50 (241.25–521.10) Z = 4.69; P < 0.001 666.15 (422.55–828.60) Creatinine (mg/dl) 1.099 ± 0.901 0.841 ± 0.370 T = 1.99; P = 0.05 Glomerular filtration rate (ml/min) 104.89 ± 50.80 113.38 ± 41.29 T= 1.05; P = 0.30 (NS) Daily ethanol (g) 185 ± 113 190 ± 157 T = 0.20; P = 0.84 (NS) Years of addiction 33 ± 13 32 ± 14 T= 0.26; P = 0.79 (NS) TNF-α (pg/ml)a 26.77 ± 150.18 4.06 (1.59–5.00) 7.36 ± 11.23 5.00 (1.59–5.19) Z = 1.37; P = 0.17 (NS) TGF-β (pg/ml)b 16,951 ± 11095 21,347 ± 12573 T= 1.55; P = 0.13 (NS) Malondialdehyde (μmol/l)c 3.78 ± 3.56 2.90 (1.82–4.31) 2.53 ± 2.09 1.88 (1.24–3.32) Z = 2.85; P = 0.004 C-reactive protein (mg/l) 27.78 ± 40.65 13.95 (4.53–32.43) 24.78 ± 43.04 10.15 (3.53–28.50) Z = 0.77; P = 0.44 (NS) Albumin (g/dl) 3.46 ± 0.72 3.72 ± 0.59 T = 2.32; P = 0.022 MCV (fl) 100.59 ± 10.73 99.54 ± 6.77 T= 0.64; P = 0.49 (NS) Prothrombin activity (%) 68.76 ± 21.37 68.00 (55.75-86.25) 87.77 ± 14.18 90.00 (77.50-100.00) Z = 5.21; P < 0.001 Serum bilirubin (mg/dl) 3.52 ± 4.54 1.65 (1.00-3.43) 1.36 ± 1.58 1.00 (1.00-1.00) Z = 5.19; P < 0.001 Serum GGT (U/l) 391.57 ± 662.21 203.50 (81.25–388.50) 188.92 ± 255.04 96.00 (45.00–228.75) Z = 2.28; P = 0.005 Serum ASAT (U/l) 85.55 ± 84.86 45.00 (28.50–109.75) 58.25 ± 85.75 31.00 (21.00–71.50) Z = 2.27; P = 0.007 Serum ALAT (U/l) 55.65 ± 66.31 36.50 (18.50–67.00) 47.70 ± 60.13 31.00 (17.00–49.00) Z = 1.15; P = 0.25 (NS) Bicaudate indexd 0.1641 ± 0.0385 0.1591 ± 0.0347 T = 0.76; P = 0.45 (NS) Bifrontal indexd 0.3528 ± 0.0465 0.3490 ± 0.0391 T = 0.50; P = 0.62 (NS) Evans’ indexd 0.3020 ± 0.0405 0.2984 ± 0.0337 T = 0.54; P = 0.59 (NS) Cella indexd 0.0646 ± 0.0215 0.0632 ± 0.0188 T= 0.40; P = 0.69 (NS) Ventricular indexd 0.4881 ± 0.0914 0.4800 ± 0.0791 T= 0.53; P = 0.60 (NS) a44 Cirrhotics and 58 non-cirrhotics. b29 Cirrhotics and 48 non-cirrhotics. c51 Cirrhotics and 75 non-cirrhotics. d52 Cirrhotics and 74 non-cirrhotics. FGF, fibroblast growth factor; TNF, tumor necrosis factor; TGF, transforming growth factor; MCV, mean corpuscular volume; GGT, gamma-glutamyl transferase; ASAT, aspartate aminotransferase; ALAT, alanine aminotransferase; NS, non-significant. Klotho was related to liver function derangement (albumin, ρ= −0.30; P < 0.001; prothrombin activity ρ = −0.38; P < 0.001; bilirubin ρ = 0.35; P < 0.001). These relationships were independent of creatinine. Also, higher Klotho values were observed among patients with ascites (Z = 4.78; P < 0.001) or encephalopathy (Z = 2.16; P = 0.031), also independent of age and creatinine (OR for Klotho over the median = 0.0.57, 95 CI = 0.013–0.257; B = −2.87; P < 0.001 for ascites and 0.30, 95 CI = 0.09–1.01; B = −1.21; P = 0.052 for encephalopathy). This was not the case for FGF-23, which did not show any association with biochemical or clinical variables related to liver function impairment. No relationships were observed between Klotho and vitamin D (ρ = −0.034; P = 0.7), parathyroid hormone (PTH; ρ = −0.06; P = 0.6); serum phosphate (ρ = −0.010; P = 0.9) or magnesium (ρ = −0.076; P = 0.4), but a significant inverse correlation with serum calcium (ρ = −0.19; P = 0.032) did exist. Serum FGF levels were not related to any of these last five variables (P > 0.40 in all the cases). As shown in Table 2, marked differences were observed in CT indices among patients and controls, but no differences among cirrhotics and non-cirrhotics were found (Table 3). Klotho levels were inversely related to the ventricular index (ρ= −0.23, P = 0.008), a result that was also found among non-cirrhotic patients (ρ= −0.328; P = 0.004; Table 4). Multiple correlation analyses, also including the variables age and creatinine, revealed that age (P = 0.016), Klotho (P = 0.032) and creatinine (P = 0.033), in this order, were related to the ventricular index. Table 4. Correlations between CT indices and Klotho and FGF-23 values. Whereas indices followed a normal distribution, FGF-23 always showed a non-parametric distribution. Klotho was normally distributed among cirrhotics (KS = 1.15; P = 0.14), although not in the whole sample (KS = 1.41; P = 0.038) All the patients Cirrhotics only Non-cirrhotics only Klotho FGF-23 Klotho FGF-23 Klotho FGF-23 Bicaudate index ρ = −0.13; P = 0.14 ρ = −0.072; P = 0.45 r = −0.28; P = 0.047 ρ = −0.10 P = 0.51 ρ = −0.19; P = 0.11 ρ = −0.045; P = 0.72 Bifrontal index ρ = 0.015; P = 0.87 ρ = −0.17; P = 0.06 r = −0.31; P = 0.026 ρ = −0.35; P = 0.021 ρ = 0.11; P = 0.35 ρ = −0.19; P = 0.11 Evans index ρ = 0.061; P = 0.50 ρ = −0.14; P = 0.13 r = −0.33; P = 0.017 ρ = −0.20; P = 0.16 ρ = 0.19; P = 0.11 ρ = −0.20; P = 0.10 Cella index ρ = −0.10; P = 0.26 ρ = −0.023; P = 0.81 r = −0.27; P = 0.058 ρ = −0.023; P = 0.81 ρ = −0.10; P = 0.39 ρ = −0.026; P = 0.84 Ventricular index ρ = −0.23; P = 0.008 ρ = −0.014; P = 0.89 r = −0.12; P = 0.39 ρ = −0.014; P = 0.89 ρ = −0.33; P = 0.004 ρ = 0.098; P = 0.43 All the patients Cirrhotics only Non-cirrhotics only Klotho FGF-23 Klotho FGF-23 Klotho FGF-23 Bicaudate index ρ = −0.13; P = 0.14 ρ = −0.072; P = 0.45 r = −0.28; P = 0.047 ρ = −0.10 P = 0.51 ρ = −0.19; P = 0.11 ρ = −0.045; P = 0.72 Bifrontal index ρ = 0.015; P = 0.87 ρ = −0.17; P = 0.06 r = −0.31; P = 0.026 ρ = −0.35; P = 0.021 ρ = 0.11; P = 0.35 ρ = −0.19; P = 0.11 Evans index ρ = 0.061; P = 0.50 ρ = −0.14; P = 0.13 r = −0.33; P = 0.017 ρ = −0.20; P = 0.16 ρ = 0.19; P = 0.11 ρ = −0.20; P = 0.10 Cella index ρ = −0.10; P = 0.26 ρ = −0.023; P = 0.81 r = −0.27; P = 0.058 ρ = −0.023; P = 0.81 ρ = −0.10; P = 0.39 ρ = −0.026; P = 0.84 Ventricular index ρ = −0.23; P = 0.008 ρ = −0.014; P = 0.89 r = −0.12; P = 0.39 ρ = −0.014; P = 0.89 ρ = −0.33; P = 0.004 ρ = 0.098; P = 0.43 Table 4. Correlations between CT indices and Klotho and FGF-23 values. Whereas indices followed a normal distribution, FGF-23 always showed a non-parametric distribution. Klotho was normally distributed among cirrhotics (KS = 1.15; P = 0.14), although not in the whole sample (KS = 1.41; P = 0.038) All the patients Cirrhotics only Non-cirrhotics only Klotho FGF-23 Klotho FGF-23 Klotho FGF-23 Bicaudate index ρ = −0.13; P = 0.14 ρ = −0.072; P = 0.45 r = −0.28; P = 0.047 ρ = −0.10 P = 0.51 ρ = −0.19; P = 0.11 ρ = −0.045; P = 0.72 Bifrontal index ρ = 0.015; P = 0.87 ρ = −0.17; P = 0.06 r = −0.31; P = 0.026 ρ = −0.35; P = 0.021 ρ = 0.11; P = 0.35 ρ = −0.19; P = 0.11 Evans index ρ = 0.061; P = 0.50 ρ = −0.14; P = 0.13 r = −0.33; P = 0.017 ρ = −0.20; P = 0.16 ρ = 0.19; P = 0.11 ρ = −0.20; P = 0.10 Cella index ρ = −0.10; P = 0.26 ρ = −0.023; P = 0.81 r = −0.27; P = 0.058 ρ = −0.023; P = 0.81 ρ = −0.10; P = 0.39 ρ = −0.026; P = 0.84 Ventricular index ρ = −0.23; P = 0.008 ρ = −0.014; P = 0.89 r = −0.12; P = 0.39 ρ = −0.014; P = 0.89 ρ = −0.33; P = 0.004 ρ = 0.098; P = 0.43 All the patients Cirrhotics only Non-cirrhotics only Klotho FGF-23 Klotho FGF-23 Klotho FGF-23 Bicaudate index ρ = −0.13; P = 0.14 ρ = −0.072; P = 0.45 r = −0.28; P = 0.047 ρ = −0.10 P = 0.51 ρ = −0.19; P = 0.11 ρ = −0.045; P = 0.72 Bifrontal index ρ = 0.015; P = 0.87 ρ = −0.17; P = 0.06 r = −0.31; P = 0.026 ρ = −0.35; P = 0.021 ρ = 0.11; P = 0.35 ρ = −0.19; P = 0.11 Evans index ρ = 0.061; P = 0.50 ρ = −0.14; P = 0.13 r = −0.33; P = 0.017 ρ = −0.20; P = 0.16 ρ = 0.19; P = 0.11 ρ = −0.20; P = 0.10 Cella index ρ = −0.10; P = 0.26 ρ = −0.023; P = 0.81 r = −0.27; P = 0.058 ρ = −0.023; P = 0.81 ρ = −0.10; P = 0.39 ρ = −0.026; P = 0.84 Ventricular index ρ = −0.23; P = 0.008 ρ = −0.014; P = 0.89 r = −0.12; P = 0.39 ρ = −0.014; P = 0.89 ρ = −0.33; P = 0.004 ρ = 0.098; P = 0.43 Analyzing cirrhotics and non-cirrhotics as separate groups, we observed: Klotho levels were inversely related to several CT indices: bicaudate (r= −0.28; P = 0.047); bifrontal (r = −0.31; P = 0.02), Evans (r = −0.33; P = 0.017) and a trend with cella index (r = −0.27; P = 0.058), so that the higher the Klotho values, the less brain atrophy (Table 4). The relationships between Klotho and bifrontal index and between Klotho and Evans index were fully independent of age and creatinine; whereas creatinine was also selected (after Klotho) in the relationship with bicaudate index. FGF-23 levels were inversely related to the bifrontal index (ρ= −0.35; P = 0.021), a relationship that was independent of age and creatinine. Among non-cirrhotics, no relationships were observed between Klotho or FGF-23 and CT indices, besides the already mentioned correlation between Klotho and ventricular index. We found a relationship between atrophy and inflammation: CRP was related to bicaudate index (ρ = 0.30), bifrontal (ρ= 0.299) and Evans index (ρ = 0.30; P < 0.001 in all the cases), and also to cella index (ρ = 0.27; P = 0.002); and TGF-β (determined to 77 patients) also showed direct relationships with bifrontal index (ρ = 0.27; P = 0.020), cella index (ρ = 0.25; P = 0.03), and ventricular index (ρ = 0.24; P = 0.025). Klotho was also directly related to TNF-α (ρ = 0.22; P = 0.026) and inversely to TGF-β (ρ = −0.34; P = 0.002), but not to CRP. FGF-23 did not show any significant relationships with the aforementioned variables. Serum MDA levels were higher among patients than among controls (Table 2), and higher in cirrhotics than in non-cirrhotics (Table 3), but did not show any relationships with FGF-23 or Klotho levels. DISCUSSION We found significantly lower Klotho levels among non-cirrhotic alcoholics than among controls, but significantly higher ones among cirrhotics than among non-cirrhotics. Klotho levels showed a relationship with liver function derangement that was independent of creatinine. Among cirrhotics higher Klotho levels were associated with less brain atrophy. Therefore, two main questions should be addressed, the first one relative to the higher values of Klotho among cirrhotics, and the second one, about the relationship between Klotho and brain atrophy. Few studies have analyzed the behavior of Klotho in cirrhotics. In a preliminary report on 97 alcoholics (most of them also included in this study), searching for the association between vascular changes and FGF-23/Klotho levels, Klotho levels were higher among patients, especially if they were cirrhotics (Quintero-Platt et al., 2017). Prystupa et al. (2016) failed to find differences among 54 cirrhotics and 18 controls, but a clear trend to higher values was observed in Child C patients, in accordance with the results obtained in this study. Previously, in a study on hepatoma cells belonging to 52 patients, 22 of them cirrhotics, Chen et al. (2013) found that immunohistochemical Klotho staining was significantly higher among cirrhotics, and, more importantly, it was related to mortality, that was significantly higher among those with more intense Klotho expression. In these three studies, Klotho was higher among cirrhotics, and, in an opposite fashion to what is expected for an anti-aging substance, it was related to liver function impairment, and even, to higher mortality. The biological role of α Klotho has been studied mainly in patients with chronic kidney disease and in several experimental models (Hu et al., 2012), and the current knowledge strongly supports the role of Klotho as a cell protective, anti-aging factor, with antioxidant, anti-vascular calcification, antifibrotic and antisenescence properties. The results reported here (and perhaps those reported by Prystupa et al., 2016) are the best explainable as an expression of a compensatory rise of Klotho in an attempt to protect the cell from aggression. In this sense, Dounousi et al. (2016), in septic patients, found that Klotho levels were increased in the peak of the infection, returning to normal values at the end of the infection. As in the present study, in which a significant relationship was obtained between TNFα and Klotho, these data suggest that Klotho may increase in acute inflammation, perhaps as a compensatory mechanism. In patients with cystic fibrosis, Krick et al. (2017) showed that TGF-β leads to an increase in Klotho expression in the bronchial epithelium. Increased Klotho levels provoked a decrease in IL-8 levels in these patients, in accordance with its anti-inflammatory action. It was also reported that Klotho inhibits TGF β signaling, acting as an antifibrogenic factor (Doi et al., 2011). Therefore, Klotho tends to blunt the secretion of some cytokines that, in turn, have a stimulatory effect on Klotho secretion. In alcoholics, increased TGF β (Gu et al., 2013) is involved in liver fibrogenesis (Ceni et al., 2014). Hypothetically, as it happens in bronchial epithelium, increased TGF β in cirrhotic liver might trigger increased secretion of Klotho, a speculation that could explain the increased levels of Klotho found in cirrhotics in the present study. Indeed, we also found an inverse relationship between Klotho and TGF-β, in accordance with the aforementioned inhibitory effect of Klotho on TGF- β. Cirrhotics also showed higher MDA and a trend to higher TNF-α levels (cause and consequence of oxidative damage). Klotho counteracts oxidative damage, leading to up-regulation of antioxidant enzymes (Kops et al., 2002), so it could be speculated that the increased Klotho levels observed in cirrhotics would obey to a protective homeostatic mechanism, although this interpretation is hypothetical, given the observational nature of this study. FGF-23 levels were higher among alcoholics, but were not related to liver function derangement. Prié et al. (2013) found higher values among cirrhotics, and an association with mortality. They also showed that diseased liver (mice treated with diethylnitrosamine) produced and secreted FGF-23. The increase in FGF-23 in inflammation is well-established: both endotoxemia and circulating TNF-α (both observed in alcoholics and cirrhotics) lead to an amplified expression of FGF-23 in osteocytes (Ito et al., 2015). This effect could explain the increased FGF-23 levels reported in this study, but the lack of correlation between TNF α and FGF-23 does not support this possibility. In contrast with the poor relationships observed between FGF-23 and brain alterations, Klotho levels and brain atrophy showed an inverse relationship, in accordance with the protective effects of this hormone, and with the observations reported by other authors. In the Inchianti study, increased plasma Klotho was associated with a lower cognitive decline in older adults (Shardell et al., 2016) and lower probability of frailty (Shardell et al., 2017). In general, higher Klotho levels are associated with better cognitive performance (Cararo-Lopes et al., 2017). Nephrectomized rats show cognitive impairment that is associated with low Klotho levels (and increased TNF-α, Degaspari et al., 2015). The enhancing effect of Klotho in oligodendrocyte maturation and differentiation (Chen et al., 2013) and the protection of neurons from oxidative damage and excitotoxicity (Zeldich et al., 2014) may account for the inverse relationship of Klotho and brain atrophy. Ethanol-mediated oligodendrocyte damage (Navarro and Mandyam, 2015), oxidative stress (Miller et al., 2013), increased inflammation (Umhau et al., 2014) and excitotoxicity (Zhou and Crews, 2005) play outstanding roles in brain alterations observed in alcoholics, so the potential protective effect of high Klotho on brain atrophy is logical. Interestingly, we also found a relationship between atrophy and inflammation, in accordance with the well stablished effect of proinflammatory cytokines on neurodegeneration (Qin and Crews, 2012). Therefore, we conclude that soluble α Klotho levels are increased among alcoholic cirrhotics compared with non-cirrhotic alcoholics, in a fashion independent of serum creatinine. This elevation is related to liver function impairment. FGF-23 levels are also increased in alcoholics compared with controls, but differences among cirrhotics and non-cirrhotics are subtle. 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Medical Council on Alcohol and Oxford University Press. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Alcohol and Alcoholism Oxford University Press

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Abstract

Abstract Aim Fibroblast growth factor (FGF-23) and α-Klotho (Klotho) levels may be altered in inflammatory conditions, possibly as compensatory mechanisms. Klotho exerts a protective effect on neurodegeneration and improves learning and cognition. No data exist about the association of Klotho and FGF-23 levels with brain atrophy observed in alcoholics. The aim of this study is to explore these relationships. Short summary FGF-23 and Klotho levels are altered in inflammation, possibly as compensatory mechanisms. Klotho enhances learning, but its role in ethanol-mediated brain atrophy is unknown. We found higher FGF-23 and lower Klotho levels in 131 alcoholics compared with 41 controls. Among cirrhotics, Klotho was higher and inversely related to brain atrophy. Methods The study was performed on 131 alcoholic patients (54 cirrhotics) and 41 age- and sex-matched controls, in whom a brain computed tomography (CT) was performed and several indices were calculated. Results Marked brain atrophy was observed among patients when compared with controls. Patients also showed higher FGF-23 and lower Klotho values. However, among cirrhotics, Klotho values were higher. Klotho was inversely related to brain atrophy (for instance, ventricular index (ρ = −0.23, P = 0.008)), especially in cirrhotics. Klotho was also directly related to tumor necrosis factor (TNF) alpha (ρ = 0.22; P = 0.026) and inversely to transforming growth factor (TGF)-β (ρ = −0.34; P = 0.002), but not to C-reactive protein (CRP) or malondialdehyde levels. FGF-23 was also higher among cirrhotics but showed no association with CT indices. Conclusions Klotho showed higher values among cirrhotics, and was inversely related to brain atrophy. FGF-23, although high among patients, especially cirrhotics, did not show any association with brain atrophy. Some inflammatory markers or cytokines, such as CRP or TGF-β were related to brain atrophy. INTRODUCTION Fibroblast growth factor (FGF)-23 is a hormone produced mainly by osteocytes in response to high serum phosphate levels. Although initially it was thought that its action was restricted to inhibition of vitamin D synthesis and inhibition of the sodium phosphate transporters in the proximal tubule (Martin et al., 2012), the functions of this growth factor far beyond calcium/phosphate homeostasis are beginning to be disentangled (Kovesdy and Quarles, 2016). For instance, recent research has shown that bacterial lipopolysaccharide, tumor necrosis factor (TNF)-α and other proinflammatory cytokines upregulate the expression of FGF-23 and secretion by osteocytes (Ito et al., 2015). In acute inflammatory states, concomitant up-regulation of intact FGF-23 cleavage within the osteocyte may minimize changes in serum values of intact FGF-23. In chronic inflammation, the intra-osteocyte cleavage machinery becomes surpassed and serum FGF-23 levels rise (David et al., 2016). Therefore, a compensatory, anti-inflammatory role for FGF-23 has been suggested (Nakahara et al., 2016). In addition to increased osteocyte production, FGF-23 becomes expressed by macrophages in response to inflammation (Masuda et al., 2015). Alpha Klotho (Klotho) was first identified as an obligate co-receptor of FGF-23. This is true for membrane-bound Klotho. Alternative splicing of the Klotho gene and shedding of membrane-anchored Klotho by proteases of the ADAM (a disintegrin and metalloproteinase) group generate the soluble form of Klotho that can be detected in blood, cerebrospinal fluid and urine (Hu et al., 2013). Soluble Klotho is a pleiotropic hormone that exerts many FGF-23 independent functions, notably antioxidative (Yamamoto et al., 2005), anticell senescence (Kurosu et al., 2005), anti-apoptotic (Ikushima et al., 2006) and antibrogenic effects (Doi et al., 2011). As with FGF-23, some data suggest that Klotho exerts anti-inflammatory effects. Overexpression of Klotho counteracts increased expression of IL-6 after TNF-α stimulus, blunting the TNF proinflammatory effect (Xia et al., 2016). Some reports suggest that FGF-23 and Klotho values are altered in alcoholics. Reasons for this increase remain elusive. Given the proinflammatory nature of alcoholism, especially when it has led to liver cirrhosis (Bishehsari et al., 2017), it could be speculated that inflammation might play a role in these changes. One of the main features of alcoholic patients is brain atrophy (de la Monte and Kril, 2014). White matter atrophy, due to altered myelin synthesis, is the main target of ethanol (Navarro and Mandyam, 2015). Neurodegeneration of hippocampal neurons and circuits also occur (Qin and Crews, 2012). Although some effects are directly derived from ethanol and/or acetaldehyde, inflammation and oxidative damage may play a role (Qin et al., 2007; Umhau et al., 2014). On the other hand, excessively high FGF-23 serum levels may be associated with impaired learning and memory (Liu et al., 2011), especially in chronic kidney disease (Drew et al., 2014). The opposite is observed with Klotho (Haffner and Leifheit-Nestler, 2017). Indeed, there are Klotho dependent and Klotho independent signaling pathways in the hippocampal neurons. Klotho dependent pathways may enhance memory and learning, whereas Klotho independent pathways have the opposite effect (Hensel et al., 2016). In this study, we want to know whether changes in brain atrophy have any association with Klotho and FGF-23, and also whether serum levels of these molecules are related to systemic inflammation that occurs in alcoholism. Patients and methods We included 131 patients (7 women), consecutively admitted to our hospitalization unit, aged 57.60 ± 11.11 years and 41 controls (6 women) of similar age (54.49 ± 7.36 years, t = 1.60; NS) and sex (P = 0.08 by exact Fisher’s test), all of them workers in our hospital or their relatives, drinkers of <10 g ethanol/day. We did not include patients who also reported consumption of other drugs besides tobacco. Some biological data of the included patients are shown in Table 1. All the patients lived in the northern part of Tenerife (almost all of them in rural areas) and were heavy drinkers until admission or the day before admission (in the case of those 56 patients who were brought to the hospital because of major symptoms of withdrawal syndrome). Patients were not consumers of any other drug besides tobacco (94 patients) and were not receiving psychiatric medication. Table 1. Some epidemiological data and comorbid conditions of the patients included Ethanol consumption (g/day) 187 ± 154 150 (96–240) Years of consumption 33 ± 14 32 (25–40) Time since last drink (days)a 3–4 Antibodies anti-hepatitis C virus infection (yes/no) 8/123 Hypertension (yes/no) 52/78 Diabetes (yes/no) 23/108 Cirrhosis (yes/no) 54/77 Ascites (yes/no) 24/107 Encephalopathy (yes/no) 15/116 Obese (BMI over 30 kg/m2) 16/115 Tobacco (yes/no) 94/34 Packs/year 46 ± 36 38 (20–60) Admission due to Major withdrawal symptoms = 56 Decompensated ascites = 30 Acute alcoholic hepatitis = 11 Infection = 18 (including two spontaneous bacterial peritonitis) Other causes (mainly neoplasia, pancreatitis, heart failure) = 16 Ethanol consumption (g/day) 187 ± 154 150 (96–240) Years of consumption 33 ± 14 32 (25–40) Time since last drink (days)a 3–4 Antibodies anti-hepatitis C virus infection (yes/no) 8/123 Hypertension (yes/no) 52/78 Diabetes (yes/no) 23/108 Cirrhosis (yes/no) 54/77 Ascites (yes/no) 24/107 Encephalopathy (yes/no) 15/116 Obese (BMI over 30 kg/m2) 16/115 Tobacco (yes/no) 94/34 Packs/year 46 ± 36 38 (20–60) Admission due to Major withdrawal symptoms = 56 Decompensated ascites = 30 Acute alcoholic hepatitis = 11 Infection = 18 (including two spontaneous bacterial peritonitis) Other causes (mainly neoplasia, pancreatitis, heart failure) = 16 aDays of abstinence until blood extraction for FGF/Klotho determinations. Table 1. Some epidemiological data and comorbid conditions of the patients included Ethanol consumption (g/day) 187 ± 154 150 (96–240) Years of consumption 33 ± 14 32 (25–40) Time since last drink (days)a 3–4 Antibodies anti-hepatitis C virus infection (yes/no) 8/123 Hypertension (yes/no) 52/78 Diabetes (yes/no) 23/108 Cirrhosis (yes/no) 54/77 Ascites (yes/no) 24/107 Encephalopathy (yes/no) 15/116 Obese (BMI over 30 kg/m2) 16/115 Tobacco (yes/no) 94/34 Packs/year 46 ± 36 38 (20–60) Admission due to Major withdrawal symptoms = 56 Decompensated ascites = 30 Acute alcoholic hepatitis = 11 Infection = 18 (including two spontaneous bacterial peritonitis) Other causes (mainly neoplasia, pancreatitis, heart failure) = 16 Ethanol consumption (g/day) 187 ± 154 150 (96–240) Years of consumption 33 ± 14 32 (25–40) Time since last drink (days)a 3–4 Antibodies anti-hepatitis C virus infection (yes/no) 8/123 Hypertension (yes/no) 52/78 Diabetes (yes/no) 23/108 Cirrhosis (yes/no) 54/77 Ascites (yes/no) 24/107 Encephalopathy (yes/no) 15/116 Obese (BMI over 30 kg/m2) 16/115 Tobacco (yes/no) 94/34 Packs/year 46 ± 36 38 (20–60) Admission due to Major withdrawal symptoms = 56 Decompensated ascites = 30 Acute alcoholic hepatitis = 11 Infection = 18 (including two spontaneous bacterial peritonitis) Other causes (mainly neoplasia, pancreatitis, heart failure) = 16 aDays of abstinence until blood extraction for FGF/Klotho determinations. Fifty-one patients (out of 85 from whom reliable information was obtained) had suffered traumatic brain injury; 52 suffered hypertension; 33 dyslipemia, and 23, diabetes. Body mass index (BMI) was also recorded: 16 patients showed BMI over 30 kg/m2, 32 had BMI values between 25 and 30, and the remaining 83 patients a BMI below 25. All patients underwent an abdominal ultrasound (US) exploration and complete laboratory evaluation. Based on US data (liver with irregular shape, heterogeneous texture, and splenomegaly and/or portal dilatation) patients were classified as cirrhotics or non-cirrhotics. Methods Brain atrophy assessment All patients underwent, at admission, a brain CT examination. Gross atrophy was observed when CT images of alcoholics were compared with those of controls (Fig. 1a). From the CT images, we calculated the indices shown in Fig. 1b. In some cases, technical problems (moved images) precluded accurate calculation of these indices. We also evaluated the global diagnosis of the radiologist regarding the presence or not of cortical atrophy and/or cerebellar atrophy. Fig. 1. View largeDownload slide (a) A brain computerized tomography (CT) of a normal control (left) compared with that of an alcoholic patient (right). (b) Indices derived from computed tomography (CT). BICAUDATE = minimum width of lateral ventricles/skull width at the same level = B/E. BIFRONTAL = maximum width of frontal horns/skull width at the same level = A/D. EVANS = maximum width of frontal horns/skull width at the level of the III ventricle = A/F. CELLA = width of the III ventricle/skull width at the same level = C/F. VENTRICULAR = minimum width of lateral ventricles/maximum width of frontal horns = B/A. Fig. 1. View largeDownload slide (a) A brain computerized tomography (CT) of a normal control (left) compared with that of an alcoholic patient (right). (b) Indices derived from computed tomography (CT). BICAUDATE = minimum width of lateral ventricles/skull width at the same level = B/E. BIFRONTAL = maximum width of frontal horns/skull width at the same level = A/D. EVANS = maximum width of frontal horns/skull width at the level of the III ventricle = A/F. CELLA = width of the III ventricle/skull width at the same level = C/F. VENTRICULAR = minimum width of lateral ventricles/maximum width of frontal horns = B/A. Laboratory evaluation Within the first 72–96 h after admission, blood samples were taken at 8.00 a.m. in fasting conditions and were immediately frozen at −20°C. In addition to routine laboratory evaluation, including C-reactive protein (CRP), serum α Klotho, FGF-23, TNF-α and transforming growth factor (TGF)-β were determined by the following procedures: FGF-23 (plasma levels) was measured using a second-generation ELISA (Immutopics, Inc. San Clemente, California). The sensitivity of this assay as determined by the 95% confidence limit on 20 duplicate determinations of the 0 RU/ml standard is 1.5 RU/ml. The inter-assay and intra-assay coefficients of variation are <5%. The percentage of recovery for this assay ranged from 91% to 116%. Serum α Klotho levels were determined by ELISA (Immuno-Biological Laboratories, Fujioka, Japan). Inter-assay and intra-assay variation coefficients range from 2.9 to 11.4% and 2.7 to 3.5%, respectively. The percentage of recovery ranged 84.7–97.5%. Sensitivity is 6.15 pg/ml, and cross-reactivity with other molecules such as osteopontin, platelet-derived growth factor or vascular endothelial growth factor is <0.1%. Serum TNF-α was determined by immunometric chemiluminiscent assay (intra-assay variation coefficient ranging 4–6.5%, inter-assay variation coefficient ranging 2.6–3.6%, recovery 92–112%, Diagnostic Products Corporation, Los Angeles, CA, USA); TGF-β levels were determined by quantitative ELISA (Quantikine ELISA kit), with a sensitivity of 15.4 pg/ml, recovery 86–115%; intra-assay variation coefficient 2.5–2.9; inter-assay variation coefficient 6.4–9.1% (R&D Systems, Abindong Science Park, Abindong, UK). Lipid peroxidation products Serum malondialdehyde (MDA) levels were measured as thiobarbituric acid-reactive substance (TBARS) using the method reported by Kikugawa et al. (1992). In this method, 0.2 ml of plasma was added to 0.2 ml of H3PO4 (0.2 M). In order to obtain a color reaction, 25 μl of 0.11 M TBA solution was added. The samples were then heated at 90°C for 45 min. The MDA-TBA condensation produces a pink color species (the intensity of the color indicates the degree of lipid peroxidation). The samples were then cooled and the TBARS were extracted with 0.4 ml of n-butanol. The butanol phase was separated by centrifugation at 6000×g for 10 min. The aliquots of this phase were then placed in a 96-well plate and read at 535 nm in a microlate spectrophotometer reader (Benchmark Plus, Bio-Rad, Hercules, CA, USA). The calibration curve was prepared with authentic MDA standards ranging from 0 to 20 μM. The intra- and inter-assay variation coefficients were 1.82 and 4.01, respectively. Statistics The Kolmogorov–Smirnov test was used to test for normal distribution, a condition not fulfilled by several variables. Therefore, non-parametric tests, such as Mann–Whitney’s U-test and Kruskall–Wallis were used to analyze differences in these parameters between groups. Student’s t-test, variance analysis and Pearson’s correlation analysis were used for the variables with a normal distribution, whereas Spearman’s rho (instead of Pearson’s correlation) was utilized in the case of non-parametric variables. All of these analyses were performed using SPSS software (Chicago, Ill., USA). Multivariate analyses, including multiple linear correlation analysis and logistic regression analysis (after transformation of linear variables into qualitative ones according to median values), were also performed when necessary. The study protocol was approved by the local ethical committee of our Hospital (number 2017/50) and conforms to the ethical guidelines of the 1975 Declaration of Helsinki. All patients and controls gave their written informed consent. Results Despite similar creatinine levels among patients and controls (Table 2), patients showed significantly higher FGF-23 values than controls (Z = 3.63; P < 0.001), but lower plasma Klotho values (Z = 2.12; P = 0.034). No relationship was observed between alpha Klotho and FGF-23 levels (ρ = 0.14; P = 0.14 (NS) for the entire population; ρ= 0.08 among cirrhotics and ρ = 0.05 among non-cirrhotics). Logistic regression analysis also showed that differences between patients and controls were not attributable to differences in creatinine. Table 2. Differences in some biological variables between patients and controls, including differences in brain computerized tomography-derived indices Patients (n = 131) Controls (n = 41) FGF-23 (RU/ml) 318.02 ± 867.45 94.97 (55.15–252.53) 67.02 ± 37.05 59.36 (51.44–73.25) Z = 3.63; P < 0.001 Klotho (pg/ml) 540.12 ± 371.70 459.00 (291.40–690.30) 594.83 ± 247.45 525.60 (427.50–782.70) Z = 2.12; P = 0.034 Serum calcium (mg/dl) 8.99 ± 0.80 Normal range = 9.0–10.4 Serum phosphate (mg/dl)a 3.10 ± 0.84 Normal range = 2.5–7.0 Serum magnesium (mg/dl)b 1.82 ± 0.41 Normal range = 1.6–2.6 Parathyroid hormone (mg/dl)c 41.17 ± 56.70 38.46 ± 22.09 T= 054; P = 0.60 (NS) Vitamin D (25 OH) (mg/dl)d 17.81 ± 11.27 82.51 ± 27.55 T = 11.75; P < 0.001 TNF-α (pg/ml)e 15.22 ± 96.50 5.00(1.59–5.00) 5.49 ± 2.53 5.00 (1.96–5.02) Z = 2.54; P = 0.014 TGF-β (pg/ml)f 19,785 ± 12017 15,896 ± 9967 T = 1.13; P = 0.26 (NS) Malondialdehyde (μmol/l)g 3.04 ± 2.84 2.30(1.30-3.64) 1.60 ± 0.26 1.62 (1.43–1.74) Z = 3.05; P = 0.002 Creatinine (mg/dl) 0.9471 ± 0.6545 0.9690 ± 0.1435 T = 0.52; P = 0.60 (NS) Ventricular index 0.4834 ± 0.0860 0.4114 ± 0.0781 T = 4.66; P < 0.001 Bicaudate index 0.1612 ± 0.0362 0.1235 ± 0.0247 T = 7.34; P < 0.001 Bifrontal index 0.3506 ± 0.0422 0.3213 ± 0.0333 T = 4.02; P < 0.001 Evans index 0.2999 ± 0.0366 0.2993 ± 0.0363 T= 3.19; P = 0.002 Cella index 0.0638 ± 0.0199 0.0443 ± 0.0134 T = 6.60; P < 0.001 Patients (n = 131) Controls (n = 41) FGF-23 (RU/ml) 318.02 ± 867.45 94.97 (55.15–252.53) 67.02 ± 37.05 59.36 (51.44–73.25) Z = 3.63; P < 0.001 Klotho (pg/ml) 540.12 ± 371.70 459.00 (291.40–690.30) 594.83 ± 247.45 525.60 (427.50–782.70) Z = 2.12; P = 0.034 Serum calcium (mg/dl) 8.99 ± 0.80 Normal range = 9.0–10.4 Serum phosphate (mg/dl)a 3.10 ± 0.84 Normal range = 2.5–7.0 Serum magnesium (mg/dl)b 1.82 ± 0.41 Normal range = 1.6–2.6 Parathyroid hormone (mg/dl)c 41.17 ± 56.70 38.46 ± 22.09 T= 054; P = 0.60 (NS) Vitamin D (25 OH) (mg/dl)d 17.81 ± 11.27 82.51 ± 27.55 T = 11.75; P < 0.001 TNF-α (pg/ml)e 15.22 ± 96.50 5.00(1.59–5.00) 5.49 ± 2.53 5.00 (1.96–5.02) Z = 2.54; P = 0.014 TGF-β (pg/ml)f 19,785 ± 12017 15,896 ± 9967 T = 1.13; P = 0.26 (NS) Malondialdehyde (μmol/l)g 3.04 ± 2.84 2.30(1.30-3.64) 1.60 ± 0.26 1.62 (1.43–1.74) Z = 3.05; P = 0.002 Creatinine (mg/dl) 0.9471 ± 0.6545 0.9690 ± 0.1435 T = 0.52; P = 0.60 (NS) Ventricular index 0.4834 ± 0.0860 0.4114 ± 0.0781 T = 4.66; P < 0.001 Bicaudate index 0.1612 ± 0.0362 0.1235 ± 0.0247 T = 7.34; P < 0.001 Bifrontal index 0.3506 ± 0.0422 0.3213 ± 0.0333 T = 4.02; P < 0.001 Evans index 0.2999 ± 0.0366 0.2993 ± 0.0363 T= 3.19; P = 0.002 Cella index 0.0638 ± 0.0199 0.0443 ± 0.0134 T = 6.60; P < 0.001 a67 Patients. b119 Patients. c25 Controls and 94 patients. d26 Controls and 113 patients. e102 Patients and 20 controls. f77 Patients and 19 controls. g126 Patients and 28 controls. FGF, fibroblast growth factor; TNF, tumor necrosis factor; TGF, transforming growth factor. Table 2. Differences in some biological variables between patients and controls, including differences in brain computerized tomography-derived indices Patients (n = 131) Controls (n = 41) FGF-23 (RU/ml) 318.02 ± 867.45 94.97 (55.15–252.53) 67.02 ± 37.05 59.36 (51.44–73.25) Z = 3.63; P < 0.001 Klotho (pg/ml) 540.12 ± 371.70 459.00 (291.40–690.30) 594.83 ± 247.45 525.60 (427.50–782.70) Z = 2.12; P = 0.034 Serum calcium (mg/dl) 8.99 ± 0.80 Normal range = 9.0–10.4 Serum phosphate (mg/dl)a 3.10 ± 0.84 Normal range = 2.5–7.0 Serum magnesium (mg/dl)b 1.82 ± 0.41 Normal range = 1.6–2.6 Parathyroid hormone (mg/dl)c 41.17 ± 56.70 38.46 ± 22.09 T= 054; P = 0.60 (NS) Vitamin D (25 OH) (mg/dl)d 17.81 ± 11.27 82.51 ± 27.55 T = 11.75; P < 0.001 TNF-α (pg/ml)e 15.22 ± 96.50 5.00(1.59–5.00) 5.49 ± 2.53 5.00 (1.96–5.02) Z = 2.54; P = 0.014 TGF-β (pg/ml)f 19,785 ± 12017 15,896 ± 9967 T = 1.13; P = 0.26 (NS) Malondialdehyde (μmol/l)g 3.04 ± 2.84 2.30(1.30-3.64) 1.60 ± 0.26 1.62 (1.43–1.74) Z = 3.05; P = 0.002 Creatinine (mg/dl) 0.9471 ± 0.6545 0.9690 ± 0.1435 T = 0.52; P = 0.60 (NS) Ventricular index 0.4834 ± 0.0860 0.4114 ± 0.0781 T = 4.66; P < 0.001 Bicaudate index 0.1612 ± 0.0362 0.1235 ± 0.0247 T = 7.34; P < 0.001 Bifrontal index 0.3506 ± 0.0422 0.3213 ± 0.0333 T = 4.02; P < 0.001 Evans index 0.2999 ± 0.0366 0.2993 ± 0.0363 T= 3.19; P = 0.002 Cella index 0.0638 ± 0.0199 0.0443 ± 0.0134 T = 6.60; P < 0.001 Patients (n = 131) Controls (n = 41) FGF-23 (RU/ml) 318.02 ± 867.45 94.97 (55.15–252.53) 67.02 ± 37.05 59.36 (51.44–73.25) Z = 3.63; P < 0.001 Klotho (pg/ml) 540.12 ± 371.70 459.00 (291.40–690.30) 594.83 ± 247.45 525.60 (427.50–782.70) Z = 2.12; P = 0.034 Serum calcium (mg/dl) 8.99 ± 0.80 Normal range = 9.0–10.4 Serum phosphate (mg/dl)a 3.10 ± 0.84 Normal range = 2.5–7.0 Serum magnesium (mg/dl)b 1.82 ± 0.41 Normal range = 1.6–2.6 Parathyroid hormone (mg/dl)c 41.17 ± 56.70 38.46 ± 22.09 T= 054; P = 0.60 (NS) Vitamin D (25 OH) (mg/dl)d 17.81 ± 11.27 82.51 ± 27.55 T = 11.75; P < 0.001 TNF-α (pg/ml)e 15.22 ± 96.50 5.00(1.59–5.00) 5.49 ± 2.53 5.00 (1.96–5.02) Z = 2.54; P = 0.014 TGF-β (pg/ml)f 19,785 ± 12017 15,896 ± 9967 T = 1.13; P = 0.26 (NS) Malondialdehyde (μmol/l)g 3.04 ± 2.84 2.30(1.30-3.64) 1.60 ± 0.26 1.62 (1.43–1.74) Z = 3.05; P = 0.002 Creatinine (mg/dl) 0.9471 ± 0.6545 0.9690 ± 0.1435 T = 0.52; P = 0.60 (NS) Ventricular index 0.4834 ± 0.0860 0.4114 ± 0.0781 T = 4.66; P < 0.001 Bicaudate index 0.1612 ± 0.0362 0.1235 ± 0.0247 T = 7.34; P < 0.001 Bifrontal index 0.3506 ± 0.0422 0.3213 ± 0.0333 T = 4.02; P < 0.001 Evans index 0.2999 ± 0.0366 0.2993 ± 0.0363 T= 3.19; P = 0.002 Cella index 0.0638 ± 0.0199 0.0443 ± 0.0134 T = 6.60; P < 0.001 a67 Patients. b119 Patients. c25 Controls and 94 patients. d26 Controls and 113 patients. e102 Patients and 20 controls. f77 Patients and 19 controls. g126 Patients and 28 controls. FGF, fibroblast growth factor; TNF, tumor necrosis factor; TGF, transforming growth factor. When the sample was classified in cirrhotics and non-cirrhotics (Table 3), we found that Klotho levels were significantly ‘higher’ among cirrhotics (Z = 4.69; P < 0.001), a result that was independent of creatinine (odds ratio (OR) for Klotho over the median = 0.20, 95% confidence interval (CI) = 0.09–0.424; B = −1.61; P < 0.001). FGF-23 levels were also significantly higher (Z = 2.25; P = 0.024) among cirrhotics when compared with non-cirrhotics, but when a logistic regression between cirrhosis and median values of creatinine and FGF-23 was performed, none of the variables showed an independent relationship with the presence or not of cirrhosis. No differences were observed between cirrhotics and controls by SNK test regarding Klotho values, but cirrhotics showed markedly higher FGF-23 values than controls (Z = 4.89; P < 0.001). Table 3. Differences in some biological variables between cirrhotics and non-cirrhotics, including differences in brain computerized tomography-derived indices Cirrhotics (n = 54) Non-cirrhotics (n = 77) Age 59.39 ± 9.81 57.03 ± 12.42 T= 1.24; P = 0.25 (NS) FGF-23 (RU/ml) 294.99 ± 363.87 333.04 ± 1078.97 81.63 (38.97–221.01) Z = 2.25; P = 0.024 127.76 (68.73–429.17) Klotho (pg/ml) 697.80 ± 412.65 429.54 ± 295.85 360.50 (241.25–521.10) Z = 4.69; P < 0.001 666.15 (422.55–828.60) Creatinine (mg/dl) 1.099 ± 0.901 0.841 ± 0.370 T = 1.99; P = 0.05 Glomerular filtration rate (ml/min) 104.89 ± 50.80 113.38 ± 41.29 T= 1.05; P = 0.30 (NS) Daily ethanol (g) 185 ± 113 190 ± 157 T = 0.20; P = 0.84 (NS) Years of addiction 33 ± 13 32 ± 14 T= 0.26; P = 0.79 (NS) TNF-α (pg/ml)a 26.77 ± 150.18 4.06 (1.59–5.00) 7.36 ± 11.23 5.00 (1.59–5.19) Z = 1.37; P = 0.17 (NS) TGF-β (pg/ml)b 16,951 ± 11095 21,347 ± 12573 T= 1.55; P = 0.13 (NS) Malondialdehyde (μmol/l)c 3.78 ± 3.56 2.90 (1.82–4.31) 2.53 ± 2.09 1.88 (1.24–3.32) Z = 2.85; P = 0.004 C-reactive protein (mg/l) 27.78 ± 40.65 13.95 (4.53–32.43) 24.78 ± 43.04 10.15 (3.53–28.50) Z = 0.77; P = 0.44 (NS) Albumin (g/dl) 3.46 ± 0.72 3.72 ± 0.59 T = 2.32; P = 0.022 MCV (fl) 100.59 ± 10.73 99.54 ± 6.77 T= 0.64; P = 0.49 (NS) Prothrombin activity (%) 68.76 ± 21.37 68.00 (55.75-86.25) 87.77 ± 14.18 90.00 (77.50-100.00) Z = 5.21; P < 0.001 Serum bilirubin (mg/dl) 3.52 ± 4.54 1.65 (1.00-3.43) 1.36 ± 1.58 1.00 (1.00-1.00) Z = 5.19; P < 0.001 Serum GGT (U/l) 391.57 ± 662.21 203.50 (81.25–388.50) 188.92 ± 255.04 96.00 (45.00–228.75) Z = 2.28; P = 0.005 Serum ASAT (U/l) 85.55 ± 84.86 45.00 (28.50–109.75) 58.25 ± 85.75 31.00 (21.00–71.50) Z = 2.27; P = 0.007 Serum ALAT (U/l) 55.65 ± 66.31 36.50 (18.50–67.00) 47.70 ± 60.13 31.00 (17.00–49.00) Z = 1.15; P = 0.25 (NS) Bicaudate indexd 0.1641 ± 0.0385 0.1591 ± 0.0347 T = 0.76; P = 0.45 (NS) Bifrontal indexd 0.3528 ± 0.0465 0.3490 ± 0.0391 T = 0.50; P = 0.62 (NS) Evans’ indexd 0.3020 ± 0.0405 0.2984 ± 0.0337 T = 0.54; P = 0.59 (NS) Cella indexd 0.0646 ± 0.0215 0.0632 ± 0.0188 T= 0.40; P = 0.69 (NS) Ventricular indexd 0.4881 ± 0.0914 0.4800 ± 0.0791 T= 0.53; P = 0.60 (NS) Cirrhotics (n = 54) Non-cirrhotics (n = 77) Age 59.39 ± 9.81 57.03 ± 12.42 T= 1.24; P = 0.25 (NS) FGF-23 (RU/ml) 294.99 ± 363.87 333.04 ± 1078.97 81.63 (38.97–221.01) Z = 2.25; P = 0.024 127.76 (68.73–429.17) Klotho (pg/ml) 697.80 ± 412.65 429.54 ± 295.85 360.50 (241.25–521.10) Z = 4.69; P < 0.001 666.15 (422.55–828.60) Creatinine (mg/dl) 1.099 ± 0.901 0.841 ± 0.370 T = 1.99; P = 0.05 Glomerular filtration rate (ml/min) 104.89 ± 50.80 113.38 ± 41.29 T= 1.05; P = 0.30 (NS) Daily ethanol (g) 185 ± 113 190 ± 157 T = 0.20; P = 0.84 (NS) Years of addiction 33 ± 13 32 ± 14 T= 0.26; P = 0.79 (NS) TNF-α (pg/ml)a 26.77 ± 150.18 4.06 (1.59–5.00) 7.36 ± 11.23 5.00 (1.59–5.19) Z = 1.37; P = 0.17 (NS) TGF-β (pg/ml)b 16,951 ± 11095 21,347 ± 12573 T= 1.55; P = 0.13 (NS) Malondialdehyde (μmol/l)c 3.78 ± 3.56 2.90 (1.82–4.31) 2.53 ± 2.09 1.88 (1.24–3.32) Z = 2.85; P = 0.004 C-reactive protein (mg/l) 27.78 ± 40.65 13.95 (4.53–32.43) 24.78 ± 43.04 10.15 (3.53–28.50) Z = 0.77; P = 0.44 (NS) Albumin (g/dl) 3.46 ± 0.72 3.72 ± 0.59 T = 2.32; P = 0.022 MCV (fl) 100.59 ± 10.73 99.54 ± 6.77 T= 0.64; P = 0.49 (NS) Prothrombin activity (%) 68.76 ± 21.37 68.00 (55.75-86.25) 87.77 ± 14.18 90.00 (77.50-100.00) Z = 5.21; P < 0.001 Serum bilirubin (mg/dl) 3.52 ± 4.54 1.65 (1.00-3.43) 1.36 ± 1.58 1.00 (1.00-1.00) Z = 5.19; P < 0.001 Serum GGT (U/l) 391.57 ± 662.21 203.50 (81.25–388.50) 188.92 ± 255.04 96.00 (45.00–228.75) Z = 2.28; P = 0.005 Serum ASAT (U/l) 85.55 ± 84.86 45.00 (28.50–109.75) 58.25 ± 85.75 31.00 (21.00–71.50) Z = 2.27; P = 0.007 Serum ALAT (U/l) 55.65 ± 66.31 36.50 (18.50–67.00) 47.70 ± 60.13 31.00 (17.00–49.00) Z = 1.15; P = 0.25 (NS) Bicaudate indexd 0.1641 ± 0.0385 0.1591 ± 0.0347 T = 0.76; P = 0.45 (NS) Bifrontal indexd 0.3528 ± 0.0465 0.3490 ± 0.0391 T = 0.50; P = 0.62 (NS) Evans’ indexd 0.3020 ± 0.0405 0.2984 ± 0.0337 T = 0.54; P = 0.59 (NS) Cella indexd 0.0646 ± 0.0215 0.0632 ± 0.0188 T= 0.40; P = 0.69 (NS) Ventricular indexd 0.4881 ± 0.0914 0.4800 ± 0.0791 T= 0.53; P = 0.60 (NS) a44 Cirrhotics and 58 non-cirrhotics. b29 Cirrhotics and 48 non-cirrhotics. c51 Cirrhotics and 75 non-cirrhotics. d52 Cirrhotics and 74 non-cirrhotics. FGF, fibroblast growth factor; TNF, tumor necrosis factor; TGF, transforming growth factor; MCV, mean corpuscular volume; GGT, gamma-glutamyl transferase; ASAT, aspartate aminotransferase; ALAT, alanine aminotransferase; NS, non-significant. Table 3. Differences in some biological variables between cirrhotics and non-cirrhotics, including differences in brain computerized tomography-derived indices Cirrhotics (n = 54) Non-cirrhotics (n = 77) Age 59.39 ± 9.81 57.03 ± 12.42 T= 1.24; P = 0.25 (NS) FGF-23 (RU/ml) 294.99 ± 363.87 333.04 ± 1078.97 81.63 (38.97–221.01) Z = 2.25; P = 0.024 127.76 (68.73–429.17) Klotho (pg/ml) 697.80 ± 412.65 429.54 ± 295.85 360.50 (241.25–521.10) Z = 4.69; P < 0.001 666.15 (422.55–828.60) Creatinine (mg/dl) 1.099 ± 0.901 0.841 ± 0.370 T = 1.99; P = 0.05 Glomerular filtration rate (ml/min) 104.89 ± 50.80 113.38 ± 41.29 T= 1.05; P = 0.30 (NS) Daily ethanol (g) 185 ± 113 190 ± 157 T = 0.20; P = 0.84 (NS) Years of addiction 33 ± 13 32 ± 14 T= 0.26; P = 0.79 (NS) TNF-α (pg/ml)a 26.77 ± 150.18 4.06 (1.59–5.00) 7.36 ± 11.23 5.00 (1.59–5.19) Z = 1.37; P = 0.17 (NS) TGF-β (pg/ml)b 16,951 ± 11095 21,347 ± 12573 T= 1.55; P = 0.13 (NS) Malondialdehyde (μmol/l)c 3.78 ± 3.56 2.90 (1.82–4.31) 2.53 ± 2.09 1.88 (1.24–3.32) Z = 2.85; P = 0.004 C-reactive protein (mg/l) 27.78 ± 40.65 13.95 (4.53–32.43) 24.78 ± 43.04 10.15 (3.53–28.50) Z = 0.77; P = 0.44 (NS) Albumin (g/dl) 3.46 ± 0.72 3.72 ± 0.59 T = 2.32; P = 0.022 MCV (fl) 100.59 ± 10.73 99.54 ± 6.77 T= 0.64; P = 0.49 (NS) Prothrombin activity (%) 68.76 ± 21.37 68.00 (55.75-86.25) 87.77 ± 14.18 90.00 (77.50-100.00) Z = 5.21; P < 0.001 Serum bilirubin (mg/dl) 3.52 ± 4.54 1.65 (1.00-3.43) 1.36 ± 1.58 1.00 (1.00-1.00) Z = 5.19; P < 0.001 Serum GGT (U/l) 391.57 ± 662.21 203.50 (81.25–388.50) 188.92 ± 255.04 96.00 (45.00–228.75) Z = 2.28; P = 0.005 Serum ASAT (U/l) 85.55 ± 84.86 45.00 (28.50–109.75) 58.25 ± 85.75 31.00 (21.00–71.50) Z = 2.27; P = 0.007 Serum ALAT (U/l) 55.65 ± 66.31 36.50 (18.50–67.00) 47.70 ± 60.13 31.00 (17.00–49.00) Z = 1.15; P = 0.25 (NS) Bicaudate indexd 0.1641 ± 0.0385 0.1591 ± 0.0347 T = 0.76; P = 0.45 (NS) Bifrontal indexd 0.3528 ± 0.0465 0.3490 ± 0.0391 T = 0.50; P = 0.62 (NS) Evans’ indexd 0.3020 ± 0.0405 0.2984 ± 0.0337 T = 0.54; P = 0.59 (NS) Cella indexd 0.0646 ± 0.0215 0.0632 ± 0.0188 T= 0.40; P = 0.69 (NS) Ventricular indexd 0.4881 ± 0.0914 0.4800 ± 0.0791 T= 0.53; P = 0.60 (NS) Cirrhotics (n = 54) Non-cirrhotics (n = 77) Age 59.39 ± 9.81 57.03 ± 12.42 T= 1.24; P = 0.25 (NS) FGF-23 (RU/ml) 294.99 ± 363.87 333.04 ± 1078.97 81.63 (38.97–221.01) Z = 2.25; P = 0.024 127.76 (68.73–429.17) Klotho (pg/ml) 697.80 ± 412.65 429.54 ± 295.85 360.50 (241.25–521.10) Z = 4.69; P < 0.001 666.15 (422.55–828.60) Creatinine (mg/dl) 1.099 ± 0.901 0.841 ± 0.370 T = 1.99; P = 0.05 Glomerular filtration rate (ml/min) 104.89 ± 50.80 113.38 ± 41.29 T= 1.05; P = 0.30 (NS) Daily ethanol (g) 185 ± 113 190 ± 157 T = 0.20; P = 0.84 (NS) Years of addiction 33 ± 13 32 ± 14 T= 0.26; P = 0.79 (NS) TNF-α (pg/ml)a 26.77 ± 150.18 4.06 (1.59–5.00) 7.36 ± 11.23 5.00 (1.59–5.19) Z = 1.37; P = 0.17 (NS) TGF-β (pg/ml)b 16,951 ± 11095 21,347 ± 12573 T= 1.55; P = 0.13 (NS) Malondialdehyde (μmol/l)c 3.78 ± 3.56 2.90 (1.82–4.31) 2.53 ± 2.09 1.88 (1.24–3.32) Z = 2.85; P = 0.004 C-reactive protein (mg/l) 27.78 ± 40.65 13.95 (4.53–32.43) 24.78 ± 43.04 10.15 (3.53–28.50) Z = 0.77; P = 0.44 (NS) Albumin (g/dl) 3.46 ± 0.72 3.72 ± 0.59 T = 2.32; P = 0.022 MCV (fl) 100.59 ± 10.73 99.54 ± 6.77 T= 0.64; P = 0.49 (NS) Prothrombin activity (%) 68.76 ± 21.37 68.00 (55.75-86.25) 87.77 ± 14.18 90.00 (77.50-100.00) Z = 5.21; P < 0.001 Serum bilirubin (mg/dl) 3.52 ± 4.54 1.65 (1.00-3.43) 1.36 ± 1.58 1.00 (1.00-1.00) Z = 5.19; P < 0.001 Serum GGT (U/l) 391.57 ± 662.21 203.50 (81.25–388.50) 188.92 ± 255.04 96.00 (45.00–228.75) Z = 2.28; P = 0.005 Serum ASAT (U/l) 85.55 ± 84.86 45.00 (28.50–109.75) 58.25 ± 85.75 31.00 (21.00–71.50) Z = 2.27; P = 0.007 Serum ALAT (U/l) 55.65 ± 66.31 36.50 (18.50–67.00) 47.70 ± 60.13 31.00 (17.00–49.00) Z = 1.15; P = 0.25 (NS) Bicaudate indexd 0.1641 ± 0.0385 0.1591 ± 0.0347 T = 0.76; P = 0.45 (NS) Bifrontal indexd 0.3528 ± 0.0465 0.3490 ± 0.0391 T = 0.50; P = 0.62 (NS) Evans’ indexd 0.3020 ± 0.0405 0.2984 ± 0.0337 T = 0.54; P = 0.59 (NS) Cella indexd 0.0646 ± 0.0215 0.0632 ± 0.0188 T= 0.40; P = 0.69 (NS) Ventricular indexd 0.4881 ± 0.0914 0.4800 ± 0.0791 T= 0.53; P = 0.60 (NS) a44 Cirrhotics and 58 non-cirrhotics. b29 Cirrhotics and 48 non-cirrhotics. c51 Cirrhotics and 75 non-cirrhotics. d52 Cirrhotics and 74 non-cirrhotics. FGF, fibroblast growth factor; TNF, tumor necrosis factor; TGF, transforming growth factor; MCV, mean corpuscular volume; GGT, gamma-glutamyl transferase; ASAT, aspartate aminotransferase; ALAT, alanine aminotransferase; NS, non-significant. Klotho was related to liver function derangement (albumin, ρ= −0.30; P < 0.001; prothrombin activity ρ = −0.38; P < 0.001; bilirubin ρ = 0.35; P < 0.001). These relationships were independent of creatinine. Also, higher Klotho values were observed among patients with ascites (Z = 4.78; P < 0.001) or encephalopathy (Z = 2.16; P = 0.031), also independent of age and creatinine (OR for Klotho over the median = 0.0.57, 95 CI = 0.013–0.257; B = −2.87; P < 0.001 for ascites and 0.30, 95 CI = 0.09–1.01; B = −1.21; P = 0.052 for encephalopathy). This was not the case for FGF-23, which did not show any association with biochemical or clinical variables related to liver function impairment. No relationships were observed between Klotho and vitamin D (ρ = −0.034; P = 0.7), parathyroid hormone (PTH; ρ = −0.06; P = 0.6); serum phosphate (ρ = −0.010; P = 0.9) or magnesium (ρ = −0.076; P = 0.4), but a significant inverse correlation with serum calcium (ρ = −0.19; P = 0.032) did exist. Serum FGF levels were not related to any of these last five variables (P > 0.40 in all the cases). As shown in Table 2, marked differences were observed in CT indices among patients and controls, but no differences among cirrhotics and non-cirrhotics were found (Table 3). Klotho levels were inversely related to the ventricular index (ρ= −0.23, P = 0.008), a result that was also found among non-cirrhotic patients (ρ= −0.328; P = 0.004; Table 4). Multiple correlation analyses, also including the variables age and creatinine, revealed that age (P = 0.016), Klotho (P = 0.032) and creatinine (P = 0.033), in this order, were related to the ventricular index. Table 4. Correlations between CT indices and Klotho and FGF-23 values. Whereas indices followed a normal distribution, FGF-23 always showed a non-parametric distribution. Klotho was normally distributed among cirrhotics (KS = 1.15; P = 0.14), although not in the whole sample (KS = 1.41; P = 0.038) All the patients Cirrhotics only Non-cirrhotics only Klotho FGF-23 Klotho FGF-23 Klotho FGF-23 Bicaudate index ρ = −0.13; P = 0.14 ρ = −0.072; P = 0.45 r = −0.28; P = 0.047 ρ = −0.10 P = 0.51 ρ = −0.19; P = 0.11 ρ = −0.045; P = 0.72 Bifrontal index ρ = 0.015; P = 0.87 ρ = −0.17; P = 0.06 r = −0.31; P = 0.026 ρ = −0.35; P = 0.021 ρ = 0.11; P = 0.35 ρ = −0.19; P = 0.11 Evans index ρ = 0.061; P = 0.50 ρ = −0.14; P = 0.13 r = −0.33; P = 0.017 ρ = −0.20; P = 0.16 ρ = 0.19; P = 0.11 ρ = −0.20; P = 0.10 Cella index ρ = −0.10; P = 0.26 ρ = −0.023; P = 0.81 r = −0.27; P = 0.058 ρ = −0.023; P = 0.81 ρ = −0.10; P = 0.39 ρ = −0.026; P = 0.84 Ventricular index ρ = −0.23; P = 0.008 ρ = −0.014; P = 0.89 r = −0.12; P = 0.39 ρ = −0.014; P = 0.89 ρ = −0.33; P = 0.004 ρ = 0.098; P = 0.43 All the patients Cirrhotics only Non-cirrhotics only Klotho FGF-23 Klotho FGF-23 Klotho FGF-23 Bicaudate index ρ = −0.13; P = 0.14 ρ = −0.072; P = 0.45 r = −0.28; P = 0.047 ρ = −0.10 P = 0.51 ρ = −0.19; P = 0.11 ρ = −0.045; P = 0.72 Bifrontal index ρ = 0.015; P = 0.87 ρ = −0.17; P = 0.06 r = −0.31; P = 0.026 ρ = −0.35; P = 0.021 ρ = 0.11; P = 0.35 ρ = −0.19; P = 0.11 Evans index ρ = 0.061; P = 0.50 ρ = −0.14; P = 0.13 r = −0.33; P = 0.017 ρ = −0.20; P = 0.16 ρ = 0.19; P = 0.11 ρ = −0.20; P = 0.10 Cella index ρ = −0.10; P = 0.26 ρ = −0.023; P = 0.81 r = −0.27; P = 0.058 ρ = −0.023; P = 0.81 ρ = −0.10; P = 0.39 ρ = −0.026; P = 0.84 Ventricular index ρ = −0.23; P = 0.008 ρ = −0.014; P = 0.89 r = −0.12; P = 0.39 ρ = −0.014; P = 0.89 ρ = −0.33; P = 0.004 ρ = 0.098; P = 0.43 Table 4. Correlations between CT indices and Klotho and FGF-23 values. Whereas indices followed a normal distribution, FGF-23 always showed a non-parametric distribution. Klotho was normally distributed among cirrhotics (KS = 1.15; P = 0.14), although not in the whole sample (KS = 1.41; P = 0.038) All the patients Cirrhotics only Non-cirrhotics only Klotho FGF-23 Klotho FGF-23 Klotho FGF-23 Bicaudate index ρ = −0.13; P = 0.14 ρ = −0.072; P = 0.45 r = −0.28; P = 0.047 ρ = −0.10 P = 0.51 ρ = −0.19; P = 0.11 ρ = −0.045; P = 0.72 Bifrontal index ρ = 0.015; P = 0.87 ρ = −0.17; P = 0.06 r = −0.31; P = 0.026 ρ = −0.35; P = 0.021 ρ = 0.11; P = 0.35 ρ = −0.19; P = 0.11 Evans index ρ = 0.061; P = 0.50 ρ = −0.14; P = 0.13 r = −0.33; P = 0.017 ρ = −0.20; P = 0.16 ρ = 0.19; P = 0.11 ρ = −0.20; P = 0.10 Cella index ρ = −0.10; P = 0.26 ρ = −0.023; P = 0.81 r = −0.27; P = 0.058 ρ = −0.023; P = 0.81 ρ = −0.10; P = 0.39 ρ = −0.026; P = 0.84 Ventricular index ρ = −0.23; P = 0.008 ρ = −0.014; P = 0.89 r = −0.12; P = 0.39 ρ = −0.014; P = 0.89 ρ = −0.33; P = 0.004 ρ = 0.098; P = 0.43 All the patients Cirrhotics only Non-cirrhotics only Klotho FGF-23 Klotho FGF-23 Klotho FGF-23 Bicaudate index ρ = −0.13; P = 0.14 ρ = −0.072; P = 0.45 r = −0.28; P = 0.047 ρ = −0.10 P = 0.51 ρ = −0.19; P = 0.11 ρ = −0.045; P = 0.72 Bifrontal index ρ = 0.015; P = 0.87 ρ = −0.17; P = 0.06 r = −0.31; P = 0.026 ρ = −0.35; P = 0.021 ρ = 0.11; P = 0.35 ρ = −0.19; P = 0.11 Evans index ρ = 0.061; P = 0.50 ρ = −0.14; P = 0.13 r = −0.33; P = 0.017 ρ = −0.20; P = 0.16 ρ = 0.19; P = 0.11 ρ = −0.20; P = 0.10 Cella index ρ = −0.10; P = 0.26 ρ = −0.023; P = 0.81 r = −0.27; P = 0.058 ρ = −0.023; P = 0.81 ρ = −0.10; P = 0.39 ρ = −0.026; P = 0.84 Ventricular index ρ = −0.23; P = 0.008 ρ = −0.014; P = 0.89 r = −0.12; P = 0.39 ρ = −0.014; P = 0.89 ρ = −0.33; P = 0.004 ρ = 0.098; P = 0.43 Analyzing cirrhotics and non-cirrhotics as separate groups, we observed: Klotho levels were inversely related to several CT indices: bicaudate (r= −0.28; P = 0.047); bifrontal (r = −0.31; P = 0.02), Evans (r = −0.33; P = 0.017) and a trend with cella index (r = −0.27; P = 0.058), so that the higher the Klotho values, the less brain atrophy (Table 4). The relationships between Klotho and bifrontal index and between Klotho and Evans index were fully independent of age and creatinine; whereas creatinine was also selected (after Klotho) in the relationship with bicaudate index. FGF-23 levels were inversely related to the bifrontal index (ρ= −0.35; P = 0.021), a relationship that was independent of age and creatinine. Among non-cirrhotics, no relationships were observed between Klotho or FGF-23 and CT indices, besides the already mentioned correlation between Klotho and ventricular index. We found a relationship between atrophy and inflammation: CRP was related to bicaudate index (ρ = 0.30), bifrontal (ρ= 0.299) and Evans index (ρ = 0.30; P < 0.001 in all the cases), and also to cella index (ρ = 0.27; P = 0.002); and TGF-β (determined to 77 patients) also showed direct relationships with bifrontal index (ρ = 0.27; P = 0.020), cella index (ρ = 0.25; P = 0.03), and ventricular index (ρ = 0.24; P = 0.025). Klotho was also directly related to TNF-α (ρ = 0.22; P = 0.026) and inversely to TGF-β (ρ = −0.34; P = 0.002), but not to CRP. FGF-23 did not show any significant relationships with the aforementioned variables. Serum MDA levels were higher among patients than among controls (Table 2), and higher in cirrhotics than in non-cirrhotics (Table 3), but did not show any relationships with FGF-23 or Klotho levels. DISCUSSION We found significantly lower Klotho levels among non-cirrhotic alcoholics than among controls, but significantly higher ones among cirrhotics than among non-cirrhotics. Klotho levels showed a relationship with liver function derangement that was independent of creatinine. Among cirrhotics higher Klotho levels were associated with less brain atrophy. Therefore, two main questions should be addressed, the first one relative to the higher values of Klotho among cirrhotics, and the second one, about the relationship between Klotho and brain atrophy. Few studies have analyzed the behavior of Klotho in cirrhotics. In a preliminary report on 97 alcoholics (most of them also included in this study), searching for the association between vascular changes and FGF-23/Klotho levels, Klotho levels were higher among patients, especially if they were cirrhotics (Quintero-Platt et al., 2017). Prystupa et al. (2016) failed to find differences among 54 cirrhotics and 18 controls, but a clear trend to higher values was observed in Child C patients, in accordance with the results obtained in this study. Previously, in a study on hepatoma cells belonging to 52 patients, 22 of them cirrhotics, Chen et al. (2013) found that immunohistochemical Klotho staining was significantly higher among cirrhotics, and, more importantly, it was related to mortality, that was significantly higher among those with more intense Klotho expression. In these three studies, Klotho was higher among cirrhotics, and, in an opposite fashion to what is expected for an anti-aging substance, it was related to liver function impairment, and even, to higher mortality. The biological role of α Klotho has been studied mainly in patients with chronic kidney disease and in several experimental models (Hu et al., 2012), and the current knowledge strongly supports the role of Klotho as a cell protective, anti-aging factor, with antioxidant, anti-vascular calcification, antifibrotic and antisenescence properties. The results reported here (and perhaps those reported by Prystupa et al., 2016) are the best explainable as an expression of a compensatory rise of Klotho in an attempt to protect the cell from aggression. In this sense, Dounousi et al. (2016), in septic patients, found that Klotho levels were increased in the peak of the infection, returning to normal values at the end of the infection. As in the present study, in which a significant relationship was obtained between TNFα and Klotho, these data suggest that Klotho may increase in acute inflammation, perhaps as a compensatory mechanism. In patients with cystic fibrosis, Krick et al. (2017) showed that TGF-β leads to an increase in Klotho expression in the bronchial epithelium. Increased Klotho levels provoked a decrease in IL-8 levels in these patients, in accordance with its anti-inflammatory action. It was also reported that Klotho inhibits TGF β signaling, acting as an antifibrogenic factor (Doi et al., 2011). Therefore, Klotho tends to blunt the secretion of some cytokines that, in turn, have a stimulatory effect on Klotho secretion. In alcoholics, increased TGF β (Gu et al., 2013) is involved in liver fibrogenesis (Ceni et al., 2014). Hypothetically, as it happens in bronchial epithelium, increased TGF β in cirrhotic liver might trigger increased secretion of Klotho, a speculation that could explain the increased levels of Klotho found in cirrhotics in the present study. Indeed, we also found an inverse relationship between Klotho and TGF-β, in accordance with the aforementioned inhibitory effect of Klotho on TGF- β. Cirrhotics also showed higher MDA and a trend to higher TNF-α levels (cause and consequence of oxidative damage). Klotho counteracts oxidative damage, leading to up-regulation of antioxidant enzymes (Kops et al., 2002), so it could be speculated that the increased Klotho levels observed in cirrhotics would obey to a protective homeostatic mechanism, although this interpretation is hypothetical, given the observational nature of this study. FGF-23 levels were higher among alcoholics, but were not related to liver function derangement. Prié et al. (2013) found higher values among cirrhotics, and an association with mortality. They also showed that diseased liver (mice treated with diethylnitrosamine) produced and secreted FGF-23. The increase in FGF-23 in inflammation is well-established: both endotoxemia and circulating TNF-α (both observed in alcoholics and cirrhotics) lead to an amplified expression of FGF-23 in osteocytes (Ito et al., 2015). This effect could explain the increased FGF-23 levels reported in this study, but the lack of correlation between TNF α and FGF-23 does not support this possibility. In contrast with the poor relationships observed between FGF-23 and brain alterations, Klotho levels and brain atrophy showed an inverse relationship, in accordance with the protective effects of this hormone, and with the observations reported by other authors. In the Inchianti study, increased plasma Klotho was associated with a lower cognitive decline in older adults (Shardell et al., 2016) and lower probability of frailty (Shardell et al., 2017). In general, higher Klotho levels are associated with better cognitive performance (Cararo-Lopes et al., 2017). Nephrectomized rats show cognitive impairment that is associated with low Klotho levels (and increased TNF-α, Degaspari et al., 2015). The enhancing effect of Klotho in oligodendrocyte maturation and differentiation (Chen et al., 2013) and the protection of neurons from oxidative damage and excitotoxicity (Zeldich et al., 2014) may account for the inverse relationship of Klotho and brain atrophy. Ethanol-mediated oligodendrocyte damage (Navarro and Mandyam, 2015), oxidative stress (Miller et al., 2013), increased inflammation (Umhau et al., 2014) and excitotoxicity (Zhou and Crews, 2005) play outstanding roles in brain alterations observed in alcoholics, so the potential protective effect of high Klotho on brain atrophy is logical. Interestingly, we also found a relationship between atrophy and inflammation, in accordance with the well stablished effect of proinflammatory cytokines on neurodegeneration (Qin and Crews, 2012). Therefore, we conclude that soluble α Klotho levels are increased among alcoholic cirrhotics compared with non-cirrhotic alcoholics, in a fashion independent of serum creatinine. This elevation is related to liver function impairment. FGF-23 levels are also increased in alcoholics compared with controls, but differences among cirrhotics and non-cirrhotics are subtle. 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Medical Council on Alcohol and Oxford University Press. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Alcohol and AlcoholismOxford University Press

Published: May 26, 2018

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