MO778ASSOCIATION OF ADVANCED GLYCATION END PRODUCTS (AGE) AND INDOXYL SULPHATE WITH ARTERIO-VENOUS FISTULA NON-MATURATION

Background and Aims The main hindrance in adequate utilization of Arteriovenous fistula (AVF) is its high rates of primary failure. Its etiology is multi factorial and several uremic toxins are under investigation. Here we explore the potential influence of Advanced Glycation End Products (AGE) and Human Indoxyl Sulphate (HIS) on AVF non maturation. Method This one year prospective observational study included single stage AVF in consecutive CKD 4 and 5 patients. At baseline, demographic, biochemical (including AGE and HIS levels) and doppler ultrasound of upper limb were done followed by AVF construction. At 12 weeks, AVF was assessed for clinical maturation (usage of fistula with 2 needles for 75% of dialysis sessions during 15 day period); accordingly divided in to matured or non-matured groups. Demographic and biochemical parameters between both groups were compared; ROC and correlation of AGE and HIS were analyzed. Results Of 129 AVF, 67.4% were matured and 32.5% non-matured. Overall mean age was 40.9 year, male predominance (77.5%) and more non-matured AVF among diabetes. The mean value of AGE and IS was 0.18 µg/ml and 0.18µg/ml respectively; but difference was non significant between two group. AGE levels were further showing positive correlation with CRP (p 0.01, r 0.21) and venous diameter (p 0.02, r -0.22). There were significant positive correlation between AGE and HIS (p<0.00, r 0.5) but not with other parameters. AUC of AGE and HIS were 0.48 and 0.5 respectively and association could not be demonstrated between all studied parameters and AVF outcome. Conclusion Study cohort showed high incidence of primary failure without any association of AGE and HIS with clinical AVF non maturation.

Endothelial glycocalyx damage in kidney disease correlates with uraemic toxins and endothelial dysfunction

Background Damage to the endothelial glycocalyx is an early indicator of vascular damage and a potential marker of endothelial dysfunction. This study aimed to assess the relationship between markers of glycocalyx damage, endothelial dysfunction, and uraemic toxins in patients with chronic kidney disease. Methods Healthy controls, CKD patients, dialysis patients, and kidney transplant recipients had biochemical markers of glycocalyx damage (syndecan-1 and hyaluronan), endothelial dysfunction (von Willebrand factor; vWF and vascular cell adhesion molecule; VCAM-1), and uraemic toxins (indoxyl sulphate and p-cresyl sulphate) measured. In addition, Sidestream Darkfield imaging was performed using the novel GlycoCheck™ device to measure glycocalyx width by the perfused boundary region (PBR) in the sublingual microcirculation. Results Serum markers of glycocalyx damage were highest in the dialysis group (n = 33), followed by CKD patients (n = 32) and kidney transplant recipients (n = 30) compared to controls (n = 30): hyaluronan: 137 (16-1414), 79 (11–257), 57 (14–218) and 23 (8-116) ng/mL, respectively, p < 0.0001; syndecan-1: 81 (40–529), 46 (21–134), 39 (23–72), and 30 (12–138) ng/mL, respectively, p < 0.0001. Markers of endothelial dysfunction followed a similar pattern. No difference in the width of the PBR was detected between these groups (2.01 ± 0.35, 2.07 ± 0.27, 2.06 ± 0.28, and 2.05 ± 0.3 µm, respectively, p = 0.89). Glycocalyx damage correlated with markers of endothelial dysfunction (log-hyaluronan and log-VCAM-1: r = 0.64, p < 0.001) and levels of uraemic toxins (log-hyaluronan and log-indoxyl sulphate: r = 0.48, p < 0.001). Conclusions Levels of biochemical markers of glycocalyx and endothelial cell damage are highest in patients receiving dialysis. Glycocalyx and endothelial damage markers correlated with each other, and with uraemic toxins. Although we could not demonstrate a change in PBR, the biochemical markers suggest that glycocalyx damage is most marked in patients with higher levels of uraemic toxins.

P0744CLINICAL IMPACT OF SERUM INDOXYL SULPHATE MEASURED BY NEW ENZYMATIC METHOD IN PATIENTS WITH CHRONIC KIDNEY DISEASE

Background and Aims Uremic toxins have been highlighted as serious risk factors for deterioration of renal function and onset/progression of cardiovascular diseases (CVD). Serum level of indoxyl sulphate (IS), a major uremic toxin, was demonstrated its significant association with vascular disease and mortality in a cohort of chronic kidney disease (CKD) patients, however, IS has not been available in clinical setting due to time consuming and expensive measurement cost. Recently epoch-making IS measurement method applicable for general auto analyzer has been developed, which could explore new therapeutic avenue in CKD from the view point of uremic toxin management. In this study, clinical utility of new enzymatic IS measurement method was analyzed in association with renal function and CVD among CKD patients. Method Subjects were consecutive 150 CKD patients in Nagoya University Hospital whose serum samples were collected between 2009 and 2014. Serum IS levels were measured by “NIPRO” reagent and analyzed with eGFR, CVD events and renal outcomes defined by 30% decrease in eGFR. Results Characteristics of patients were 69 ± 10 years old, 29% female, eGFR: 44 ± 20 mL/min/1.73m2 (∼G3a: 43％, G3b: 29％, G4: 24％, Ｇ5: 4％), proteinuria 2.8 ± 3. 5ｇ/ｇCr (A1: 29％, A2: 29％, A3: 42％), HTN: 83％, and DM: 39％. Serum IS levels (μmol/L) were 10.5 ± 7.5 (∼G3a: 1.8 ± 0.6, G3b: 2.1 ± 0.6, G4: 15.8 ± 8.1, Ｇ5: 22.9 ± 13.5), and strongly correlated with eGFR (r =0.518, P&lt;0.001). Among IS low (&lt;6), middle and high (≥12) tertiles, significantly different factors were age, eGFR, Hb, iPTH and LDL-C. In multiple logistic regression analysis, only eGFR was significant associating factor with IS tertiles. IS levels in 2 patients prescribed AST-120 (Kremezin) were 8.1 and 10.3, which seems to be very low in comparison to their eGFR of 13.8 and 13.7. During observation period of 5.1 ± 1.0 years, 59 renal outcomes and 9 CVD events were observed. Kaplan-Meier survival curve analysis free from renal outcomes revealed better tendency in IS low group (p= 0.0617 in log-rank test). According to IS levels adjusted by eGFR, only 1 out of 9 CVD events occurred in low IS/eGFR tertile group. Conclusion Serum IS levels could be measured in new enzymatic method. Strong correlation between IS and eGFR was demonstrated, and AST-120 might be effective to improve IS. In renal and CVD outcome analysis, more sample size is needed for further study.

P0876THE ROLE OF TRPM7 IN VASCULAR CALCIFICATION: COMPARISION BETWEEN PHOSPHATE AND UREMIC TOXIN

Background and Aims Vascular calcification is a common complication in patients with chronic kidney disease (CKD) and associated with increased morbidity and mortality. Pathogenesis of vascular calcification in CKD is multiple and phenotypic transformation of vascular smooth muscle cell (VSMC) is of critical importance. TRPM7 is a magnesium channel that regulate magnesium transport. The role of TRPM7 in VSMC transformation during vascular calcification is not clear. We aim to investigate the effects of phosphate and indoxyl sulphate on the expression of TRPM7 in VSMC and 2-APB, the TRPM7 blocker was used to evaluate the alteration of calcification-related molecules. Method Human aortic smooth muscle cells (HASMC) were used and treated either with phosphate (3.3mM) or indoxyl sulphate (500uM) for 7 days. 2-APB, a channel blocker of TRPM7 was added simultaneously in blocking experiment. Cells were then examined grossly and alizarin red solution was employed for calcification staining assessment. For calcification process study, cells were harvested for gene expression and protein abundance analysis. Results Gross examination did not find evidence of cell death during experiment on both treatments. Alizarin staining revealed apparent calcification in phosphate-treated HASMC but it was unremarkable in indoxyl sulphate. Phosphate treatment induced significant increase in RUNX2(151±35.8% of control), BMP2(124±10.1%), BMP7(155±9.3%), vitamin D receptor (VDR, 138±21%), calcium sensing receptor (CaSR, 185± 39%) and TRPM7(172±33.7%, all p&lt; 0.05), but DKK1 and sclerostin were not changed. The addition of 2-APB prevented the increase of RUNX2(107±31.2%), BMP2(80±4.9%), BMP7(72±32.9%), VDR (94±43.2%), CaSR (83±17%) and TRPM7(118±23.9%). Indoxyl sulphate treatment was associated with decrease in TRPM7 (45±22%, p&lt;0.05), but increase in BMP2(136±10.4%), VDR (154±53.5%) were noted. There were no significant alterations in CaSR (108±43.7%) and sclerostin (110±27.1%). Co-administration with 2-APB partially reduced the increase in RUNX2(158±55.1%), VDR (149±35.9%). The abundance of BMP2/7were not changed. TRPM7 was decreased significantly (33±23.1%). Conclusion We concluded that both phosphate and indoxyl sulphate induced calcification in VSMC but it was less in indoxyl sulphate. TRPM7 was upregulated by phosphate but decreased in indoxyl sulphate treatment. Blocking TRPM7 with 2-APB can prevent phosphate-induced calcification but partially attenuated calcification during indoxyl sulphate administration.

Probiotic versus Symbiotic in cardiac protection against indoxyl sulphate and urea toxins in experimental model of chronic kidney disease in rats

Background The occurrence of reno-cardiac syndrome is associated with poor clinical outcome. Uremic toxins in chronic kidney diseases disrupt intestinal equilibrium and increase aerobic bacteria/anerobic bacteria ratio. As a result indoxyl sulphate level in blood increases and contributes to pathogenesis of reno-cardiac syndrome. Probiotic and symbiotic are anerobic bacteria that correct the intestinal equilibrium. Aim of work We aimed to study the pathogenesis of reno-cardiac syndrome and compare the possible protective effects of probiotics and symbiotic. Design 48 white albino rats were assigned into 6 groups: Sham group, 5/6 nephrectomy, early and late probiotic treated nephrectomy rats, early and late symbiotic treated nephrectomy rats. Methods Heart was exposed to 30 min ischemia followed by 30 min reperfusion. PT, TPT, HRT, HR and CBF were measured at 5, 15 and 30 min of reperfusion. We measured serum levels of creatinine, urea, indoxyl sulphate, and heart tissue level of TGF-β and NADPH. Results uremic toxins, TGF-β and NADPH levels were significantly increased in all groups compared to sham group. In treated groups, uremic toxins significantly decreased while TGF-β and NADPH levels increased compared to nephrectomy group. PT, TPT, HRT, HR and CBF of the heart at 30 min reperfusion were decreased in nephrectomy group compared to sham one, while these functions were improved in treated groups compared to nephrectomy one. Conclusion Use of probiotic and symbiotic in reno-cardiac syndrome can protect the heart by improving the intestinal barrier, antioxidant effects and decreasing indoxyl sulphate level. Symbiotic had better outcomes than probiotic.

Specific Urinary Metabolites in Malignant Melanoma

Background and objectives: Melanin, which has a confirmed role in melanoma cell behaviour, is formed in the process of melanogenesis and is synthesized from tryptophan, L-tyrosine and their metabolites. All these metabolites are easily detectable by chromatography in urine. Materials and Methods: Urine samples of 133 individuals (82 malignant melanoma patients and 51 healthy controls) were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC). The diagnosis of malignant melanoma was confirmed histologically. Results: Chromatograms of melanoma patients showed increased levels of 5,6-dihydroxyindole-2-carboxylic acid, vanilmandelic acid, homovanilic acid, tryptophan, 5-hydroxyindole-3-acetic acid, and indoxyl sulphate compared to healthy controls. Concentration of indoxyl sulphate, homovanilic acid and tryptophan were significantly increased even in the low clinical stage 0 of the disease (indoxyl sulphate, homovanilic acid and tryptophan in patients with clinical stage 0 vs. controls expressed as medium/ interquartile range in µmol/mmol creatinine: 28.37/15.30 vs. 5.00/6.91; 47.97/33.08 vs. 7.33/21.25; and 16.38/15.98 vs. 3.46/6.22, respectively). Conclusions: HPLC detection of metabolites of L-tyrosine and tryptophan in the urine of melanoma patients may play a significant role in diagnostics as well as a therapeutic strategy of melanoma cancer.