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DKD is the second leading cause of end-stage renal disease &#40;ESRD&#41; and accounts for approximately 16&#46;4&#37; of all cases of ESRD&#46;<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">5</span></a></p><p id="par0010" class="elsevierStylePara elsevierViewall">Early identification of patients with DKD who are at risk for progressive renal dysfunction enables early intervention and may lead to better clinical outcome&#46; Glomerular filtration rate &#40;GFR&#41; marker serum creatinine is the best marker of renal excretory function&#46; However&#44; an increased in serum creatinine level is only detectable when GFR declined significantly and thus is insensitive for early DKD diagnosis&#46; Currently&#44; moderately increased albuminuria&#44; which indicates widespread microvascular damage in kidney&#44; is the earliest and the most commonly used laboratory marker in evaluating DKD&#46; A constant increase in albuminuria can predict DKD progression in T2DM patients&#46;<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">6</span></a> However&#44; many factors may cause the fluctuation of albuminuria level&#44; including serum glucose&#44; blood pressure&#44; smoking&#44; pregnancy&#44; and urinary tract infection&#46; Evidence also shows that the incidence of normoalbumin diabetic kidney disease &#40;NA-DKD&#41; is increasing in T2DM patients&#46;<a class="elsevierStyleCrossRefs" href="#bib0190"><span class="elsevierStyleSup">7&#8211;9</span></a> Thus&#44; a more reliable marker is needed to identify T2DM patients with high risk for or has developed early DKD&#46;</p><p id="par0015" class="elsevierStylePara elsevierViewall">Urine proteome analysis in diabetic patients has provided insights into identification of novel diagnostic markers for DKD&#46;<a class="elsevierStyleCrossRefs" href="#bib0205"><span class="elsevierStyleSup">10&#44;11</span></a> Dihazi et al&#46;&#44;<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">12</span></a> using SELDI-TOF mass spectrometry and SAX2 protein arrays&#44; found a processed form of ubiquitin with m&#47;z 14766 called ubiquitin-52 amino acid fusion protein &#40;UbA52&#41; that was missing in the urine of patients with DM&#44; but existed in DKD patients&#46; Based on the finding&#44; they concluded UbA52 could be used as a diagnostic marker for DKD&#46; However&#44; the identification process for UbA52 based on mass spectrometry was complicated and expensive&#44; which may not be suitable for clinical use&#46; Thus&#44; other methods are needed to identify diabetic patients at high risk for DKD&#46; With early diagnosis of DKD&#44; there are opportunities for early medical intervention and preventing disease progression&#46; The objective of this study was to test urinary UbA52 using enzymatic ELISA approach in T2DM patients and healthy people&#44; and analyze its correlation with laboratory and pathological results&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Study design</span><p id="par0020" class="elsevierStylePara elsevierViewall">We recruited participants from the third hospital of Hebei Medical University &#40;Hebei&#44; 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examination of the optic fundus&#44; moderately increased albuminuria&#44; urinary albumin and urine creatinine ratio&#44; and urinary albumin excretion rate&#46; The groups included patients with DM with moderately increased albuminuria&#40;defined as the urinary albumin&#47;creatinine ratio &#40;UACR&#41; &#62;30<span class="elsevierStyleHsp" style=""></span>mg&#47;g&#44; or persistent microalbuminuria &#62;300<span class="elsevierStyleHsp" style=""></span>mg&#47;24<span class="elsevierStyleHsp" style=""></span>h&#44; or &#62;1&#46;0<span class="elsevierStyleHsp" style=""></span>g&#47;24<span class="elsevierStyleHsp" style=""></span>h for more than 3 months&#41;&#44; diabetic retinopathy&#44; excluding other renal diseases clinically were defined as diabetic kidney disease &#40;DM-NP&#59; <span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&#44; with DM without albuminuria or diabetic retinopathy&#44; some have chronic kidney disease due to hypertension&#44; gout&#44; nephrotoxic drugs and other reasons&#44; excluding diabetic kidney disease clinically &#40;DM-WNP&#59; <span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&#44; and healthy controls &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&#46; Three DM-NP patients had biopsy-proven DN&#46;</p><p id="par0030" class="elsevierStylePara elsevierViewall">The protocol was approved by the appropriate institutional review boards&#44; and written informed consent was obtained from all participants&#46; Clinical sample procurement and analysis as well as data management were approved by the local institutional ethical review committee of the third hospital of Hebei Medical University &#40;Hebei&#44; China&#41;&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Urine samples</span><p id="par0035" class="elsevierStylePara elsevierViewall">First morning midstream urine samples&#44; 10<span class="elsevierStyleHsp" style=""></span>ml&#44; were taken before breakfast&#46; Urine samples were centrifuged for 15<span class="elsevierStyleHsp" style=""></span>min at 3000<span class="elsevierStyleHsp" style=""></span>g and stored at &#8722;80<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The supernatant was divided into 1&#46;5-mL aliquots and immediately stored at &#8722;80<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; All samples were stored for 4&#8211;5 months and did not undergo any freeze-thaw cycles&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">UbA52 antibody testing</span><p id="par0040" class="elsevierStylePara elsevierViewall">In total&#44; 10<span class="elsevierStyleHsp" style=""></span>mg horseradish peroxidase &#40;HRP&#41;was added to 0&#46;2<span class="elsevierStyleHsp" style=""></span>ml of 1&#46;25&#37; glutaraldehyde solution and kept at room temperature overnight&#46; The reaction mixture was collected in a dialysis bag surrounded by normal saline&#46; The dialysis bag was stored for 24<span class="elsevierStyleHsp" style=""></span>h at 4<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; At the same time the normal saline was exchanged twice to remove unconjugated glutaraldehyde solution&#46; A few drops of normal saline were placed in the reaction mixture to ensure a total volume of 1<span class="elsevierStyleHsp" style=""></span>ml&#46; An amount of 5<span class="elsevierStyleHsp" style=""></span>mg goat anti-UbA52 polyclonal antibody &#40;Santa Cruz Biotechnology&#44; Santa Cruz&#44; CA&#44; US&#41; per 1<span class="elsevierStyleHsp" style=""></span>ml normal saline was added to the solution&#46; An amount of 0&#46;1<span class="elsevierStyleHsp" style=""></span>ml of 1<span class="elsevierStyleHsp" style=""></span>M carbonate buffer solution&#44; pH 9&#46;5&#44; was added&#59; the reaction mixture was vortex-mixed and kept for 24<span class="elsevierStyleHsp" style=""></span>h at 4<span class="elsevierStyleHsp" style=""></span>&#176;C effectively to form a crosslinked structure&#46; An amount of 0&#46;2<span class="elsevierStyleHsp" style=""></span>ml of 0&#46;2<span class="elsevierStyleHsp" style=""></span>M lysine was added to the reaction mixture with constant shaking&#46; The preparation was stored at room temperature for 2<span class="elsevierStyleHsp" style=""></span>h&#46; Then the same volume of saturated ammonium sulfate solution was added with constant stirring and incubated for 1<span class="elsevierStyleHsp" style=""></span>h at 4<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The mixture was centrifuged at 2862<span class="elsevierStyleHsp" style=""></span>g for 15<span class="elsevierStyleHsp" style=""></span>min&#46; The supernatant was discarded and the deposit was washed twice with half-saturated ammonium sulfate solution&#46; The final precipitation was suspended in 0&#46;15<span class="elsevierStyleHsp" style=""></span>M PBS&#44; pH 7&#46;4&#46; Then the mixture was collected in a dialysis bag surrounded by 0&#46;15<span class="elsevierStyleHsp" style=""></span>M PBS&#44; pH 7&#46;4&#44; to remove ammonium ion&#46; The mixture was centrifuged at 17886<span class="elsevierStyleHsp" style=""></span>g for 30<span class="elsevierStyleHsp" style=""></span>min&#46; The supernatant was the purified solution of HRP-conjugated goat anti-human UbA52 polyclonal antibody&#44; then repackaged into smaller packages to store at 4<span class="elsevierStyleHsp" style=""></span>&#176;C or &#8722;80<span class="elsevierStyleHsp" style=""></span>&#176;C&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">UbA52 measurement by double-antibody sandwich two-step ELISA</span><p id="par0045" class="elsevierStylePara elsevierViewall">An amount of 10<span class="elsevierStyleHsp" style=""></span>&#956;L goat anti-UbA52 polyclonal antibody was dissolved in coating buffer on 96- and 48-well ELISA plates&#46; 0&#46;15<span class="elsevierStyleHsp" style=""></span>ml of the mixture was putted in every well and allowed to incubate overnight at 4<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The concentration of the coating antibody was 2<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;ml&#46; The plate surface was then gently washed with washing buffer 4 times and dried with filter paper&#46; Eight wells were for standards&#46; Sample diluent solution was added to the standard solution and the concentrations of standard solution were 200&#44; 100&#44; 50&#44; 25&#44; 12&#46;5&#44; 6&#46;25&#44; 3&#46;12&#44; and 0<span class="elsevierStyleHsp" style=""></span>ng&#47;ml&#46; An amount of 100<span class="elsevierStyleHsp" style=""></span>&#956;L sample was added to 96- and 48-well ELISA plates and incubated for 90<span class="elsevierStyleHsp" style=""></span>min at 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The plate surface was then gently washed with washing buffer 5 times and dried with filter paper&#46; The HRP-conjugated antibody was diluted 5000 times with PBS&#46; Then 100<span class="elsevierStyleHsp" style=""></span>&#956;l was added to 96- and 48-well ELISA plates with constant shaking and incubated for 90<span class="elsevierStyleHsp" style=""></span>min at 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; Each well was washed 5 times in washing buffer and dried with filter paper&#46; After 100<span class="elsevierStyleHsp" style=""></span>&#956;L of TMB &#40;3&#44;3&#8242;&#44;5&#44;5&#8242;-tetramethylbenzidine hydrochloride&#41; solution was added&#44; the wells were incubated for 10<span class="elsevierStyleHsp" style=""></span>min at 37<span class="elsevierStyleHsp" style=""></span>&#176;C in the dark&#46; The reaction was stopped with 2<span class="elsevierStyleHsp" style=""></span>M of sulfuric acid&#46; The levels of each sample were extrapolated by referring to a standard curve &#40;4-parameter logistic curve fitting&#41; of optical density &#40;OD&#41; 450<span class="elsevierStyleHsp" style=""></span>nm&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Statistical analysis</span><p id="par0050" class="elsevierStylePara elsevierViewall">The data were analyzed by using SPSS 20 &#40;SPSS Inc&#46;&#44; Chicago&#44; IL&#41;&#46; For continuous variables&#44; data are presented as mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SD or median &#40;range&#41;&#44; and means were compared by one-way ANOVA&#46; Spearman&#39;s correlation analysis and multiple linear regression model were used to analyze the correlation between UbA52 and patients&#8217; laboratory indexes&#46; Receiver operating characteristic curve &#40;ROC&#41; was used to evaluate the value of UbA52 in T2DM mellitus and diabetic kidney disease diagnosis&#46; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 was considered statistically significant&#46;</p></span></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Results</span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Patient characteristics</span><p id="par0055" class="elsevierStylePara elsevierViewall">Of the 90 patients enrolled in the study&#44; 49 were male &#40;54&#46;4&#37;&#41;&#46; The mean age&#44; duration of diabetes&#44; gender distribution&#44; fasting blood glucose level&#44; body mass index and hemoglubulinAlc level did not differ between DM-NP and DM-WNP groups &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#46; The 24-h total proteinuria level and serum creatinine level were higher for DM-NP than DM-WNP and controls &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; and did not differ between DM-WNP patients and controls &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">DM-NP patients showed increased urinary UbA52 content</span><p id="par0060" class="elsevierStylePara elsevierViewall">Three DM-NP patients had a diagnosis of diabetic kidney disease based on renal biopsy&#46; On ELISA&#44; the titers of UbA52 for these patients were 50&#46;4&#44; 58&#46;6&#44; 59&#46;3<span class="elsevierStyleHsp" style=""></span>ng&#47;ml&#46; The other patients in this group showed high moderately increased albuminuria without renal biopsy&#46; The urinary UbA52 content was higher for DM-NP than DN-WNP patients and controls &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; and did not differ between DM-WNP patients and controls &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46; The levels of urinary UbA52in DM-NP group was 1&#46;75 times and 2&#46;71 times of that in DN-WNP &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;004&#41; and normal control group &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41; respectively &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a> and <a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#41;&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Correlation analysis of urinary UbA52 level with clinical indicators</span><p id="par0065" class="elsevierStylePara elsevierViewall">The level of urinary UbA52 was significantly correlated with serum creatinine &#40;<span class="elsevierStyleItalic">r</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;468&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#44; GFR &#40;<span class="elsevierStyleItalic">r</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>&#8722;0&#46;300&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;004&#41; and proteinuria level &#40;<span class="elsevierStyleItalic">r</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;484&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#41;&#46; Multiple linear regression analysis &#40;Enter&#59; variables&#58; proteinuria level&#44; serum creatinine&#44; GFR&#44; FBS level&#44; HbAlc level&#44; BMI&#44; age&#44; and sex&#41; showed that only proteinuria level was independently associated with urinary UbA52 level &#40;<span class="elsevierStyleItalic">&#946;</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;833&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a>&#41;&#46; No evidence of influential collinearity was observed&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia><elsevierMultimedia ident="tbl0010"></elsevierMultimedia></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Diagnostic value of urinary UbA52 content</span><p id="par0070" class="elsevierStylePara elsevierViewall">The area under the receiver operating characteristic curve &#40;AUC&#41; of UbA52 content in diagnosing T2DM mellitus was 0&#46;751&#40;95&#37;CI 0&#46;684&#8211;0&#46;855&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#46; The sensitivity and specificity were 78&#46;3&#37; and 63&#46;3&#37;&#44; respectively&#44; and the cut off value was 4&#46;32&#46;</p><p id="par0075" class="elsevierStylePara elsevierViewall">The AUC of UbA52 content in diagnosing diabetic nephropathy was 0&#46;755&#40;95&#37; CI 0&#46;644&#8211;0&#46;866&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#46;The sensitivity and specificity were 63&#46;3&#37; and 85&#46;0&#37;&#44; respectively&#44; and the cut off value was 8&#46;97 &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a> and <a class="elsevierStyleCrossRefs" href="#tbl0015">Tables 3&#8211;5</a>&#41;&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia><elsevierMultimedia ident="tbl0015"></elsevierMultimedia><elsevierMultimedia ident="tbl0020"></elsevierMultimedia><elsevierMultimedia ident="tbl0025"></elsevierMultimedia></span></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Discussion</span><p id="par0080" class="elsevierStylePara elsevierViewall">DKD refers to kidney disease that is specific to diabetes&#46; In most cases&#44; careful screening instead of kidney biopsy is able to identify DKD patients&#46; The diagnosis of DKD is made based in part on the finding of elevated urinary albumin excretion and the presence of diabetic retinopathy&#46;<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">13</span></a> Diabetic nephropathy &#40;DN&#41; refers to specific pathological changes in kidney biopsy and functional changes in DM patients that result from detrimental effects of DM&#46;<a class="elsevierStyleCrossRefs" href="#bib0185"><span class="elsevierStyleSup">6&#44;7</span></a> Given that the diagnosis of DKD is based on clinical manifestations and laboratory findings&#44; however&#44; about 30&#37; DKD diagnosed by clinical criteria are biopsy-proven nondiabetic glomerular diseases combined with diabetes&#44; suggesting a urgent need for a sensitive yet specific marker for DKD&#46; SELDI-TOF&#47;MS found UbA52 can be considered as a reliable biomarker to identify patients with diabetic nephropathy among diabetic patients&#46; And more importantly&#44; to distinguish biopsy-proven diabetic nephropathy from nondiabetic-CKD in diabetic patients&#46;</p><p id="par0085" class="elsevierStylePara elsevierViewall">In current study&#44; we examined the value of urinary UbA52 as a diagnostic biomarker for DKD in patients with diabetes by two-step ELISA assay&#46; Multiple linear regression analysis showed that proteinuria level was independently associated with urinary UbA52 level &#40;<span class="elsevierStyleItalic">&#946;</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;833&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#46; The area under the receiver operating characteristic curve &#40;ROC&#41; of urinary UbA52 in diagnosing T2DM mellitus and diabetic nephropathy was 0&#46;751 and 0&#46;755&#44; respectively&#46; Based on our results&#44; urinary UbA52 measured by ELISA is expected to be a potential biomarker for the diagnosis of diabetic nephropathy&#46;</p><p id="par0090" class="elsevierStylePara elsevierViewall">DKD plays an important role in the development of ESRD&#46; Kidney biopsy&#44; the gold standard for diagnosis of glomerular disease&#44; represents the current method for a definite diagnosis of diabetic nephropathy&#44; but is only performed in diabetic patients with atypical presentations&#46; In 2010&#44; the Research Committee of the Renal Pathology Society first divide diabetic nephropathy into four hierarchical glomerular lesions with a separate evaluation for degrees of interstitial and vascular involvement&#46;<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">14</span></a> For the most part&#44; the diagnosis of DKD is based on the course of clinical manifestations and laboratory findings such as moderately increased albuminuria&#44; which is considered a prognostic predictor of the progression of DKD&#46; However&#44; moderately increased albuminuria has a limited predictive value because of its poor correlation with reduced glomerular filtration rate&#46;<a class="elsevierStyleCrossRefs" href="#bib0230"><span class="elsevierStyleSup">15&#44;16</span></a> Some patients with diabetic nephropathy proven by renal biopsy have normal serum creatinine level without moderately increased albuminuria&#44;<a class="elsevierStyleCrossRefs" href="#bib0240"><span class="elsevierStyleSup">17&#44;18</span></a> whereas others&#44; before moderately increased albuminuria is diagnosed&#44; may show progressive loss of kidney function&#46;<a class="elsevierStyleCrossRefs" href="#bib0250"><span class="elsevierStyleSup">19&#44;20</span></a> Recent studies suggested that the moderately increased albuminuria in several DKD patients can even return to normal values&#46;<a class="elsevierStyleCrossRefs" href="#bib0235"><span class="elsevierStyleSup">16&#44;21</span></a> While this phenomenon is more common in type 2 diabetes mellitus&#44; this situation is termed as normoalbuminuric diabetic kidney disease &#40;NADKD&#41; and is characterized by more obvious tubular interstitial lesions&#46;<a class="elsevierStyleCrossRefs" href="#bib0265"><span class="elsevierStyleSup">22&#44;23</span></a></p><p id="par0095" class="elsevierStylePara elsevierViewall">Therefore&#44; developing more sensitive and specific markers is urgently needed to identify patients with diabetes who are at high risk of DKD&#46; At the molecular level&#44; several proteins and their functions in DKD have been characterized&#44; whereas others remain to be discovered&#46; UbA52 is a ubiquitin fusion protein&#46; Sun et al&#46;<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">24</span></a> found that the expression of UbA52 mRNA in the kidney was proportional to blood glucose levels&#46; In situ hybridization and immunohistochemistry revealed that UbA52 exclusively localized to renal tubules&#44; and its expression was markedly increased in mice with DN&#46;<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">18</span></a> UbA52 can be detected in serum&#44; but at low concentrations which rule out its effect on urine concentration&#46;<a class="elsevierStyleCrossRef" href="#bib0280"><span class="elsevierStyleSup">25</span></a></p><p id="par0100" class="elsevierStylePara elsevierViewall">Our study shows the level of urinary UbA52 was significantly correlated with serum creatinine&#46; The expression of UbA52 is regulated by a variety of injuries&#44; such as oxidative and carbonyl stress&#44; which are important in the pathophysiology of DKD and apoptosis&#46;<a class="elsevierStyleCrossRefs" href="#bib0275"><span class="elsevierStyleSup">24&#44;26</span></a> High glucose in the cells of diabetic patients can lead to the formation of superoxide compounds and increased production of reactive oxygen species &#40;ROS&#41;&#46; Oxidative stress can increase the activity of ubiquitin activase &#40;E1&#41; and ubiquitin binding enzyme &#40;E2&#41; and then increase the expression of UbA52&#46;<a class="elsevierStyleCrossRefs" href="#bib0290"><span class="elsevierStyleSup">27&#44;28</span></a> Stimulation of cells with hydrogen peroxide&#44; which mimics oxidative stress&#44; can increase the expression of UbA52&#46;<a class="elsevierStyleCrossRef" href="#bib0300"><span class="elsevierStyleSup">29</span></a> The selective expression of UbA52 in renal tubules suggest that the ubiquitin proteasome proteolytic system is indeed operating in this compartment of the kidney&#46;<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">24</span></a></p><p id="par0105" class="elsevierStylePara elsevierViewall">We combined HRP-labeled antibody with ELISA to determine whether urine could be used for identifying a marker specific for DKD and evaluated the relationship between urinary UbA52 content and DKD&#46; Urinary UbA52 content was greater in DM-NP patients than in controls or DM-WNP patients&#46; Our results confirm the altered urinary UbA52 content in patients with DN&#46; UbA52 may be an important molecule relevant to the pathobiology of DKD&#46; With urinary UbA52 secretion&#44; we can estimate the degree of microangiopathy in the renal system of patients with diabetes&#46;</p><p id="par0110" class="elsevierStylePara elsevierViewall">The prevalence of nondiabetic renal disease in patients with T2DM mellitus &#40;T2DM&#41; varies from 27&#37; to 79&#37;&#46;<a class="elsevierStyleCrossRef" href="#bib0305"><span class="elsevierStyleSup">30</span></a> So a patient with T2DM with a high proteinuria could have a diagnosis of DKD or of T2DM with nondiabetic renal disease&#46; Because the proteinuria level was independently associated with urinary UbA52 level&#44; UbA52 may have a role in the pathogenesis of renal disease with proteinuria&#46; UBA52 was significantly increased in patients with focal segmental glomerular sclerosis &#40;FSGS&#41; and minimal change nephropathy &#40;MCD&#41;&#44;<a class="elsevierStyleCrossRef" href="#bib0310"><span class="elsevierStyleSup">31</span></a> which was much higher than that in patients with diabetic nephropathy in our study&#46; UBA52 may not be a specific marker&#44; but it may be helpful to identify these diseases with different cut off values&#46; Further studies with larger patient samples are needed to explore the function of UbA52 in DKD and nondiabetic renal diseases&#46;</p><p id="par0115" class="elsevierStylePara elsevierViewall">This study has several limitations&#46; First&#44; we had only 3 cases of diabetic nephropathy with a diagnosis by renal biopsy&#46; The other cases of DM-NP were defined by moderately increased albuminuria and diabetic retinopathy&#44; which are not accurate indictors and related to many factors&#46; Second&#44; we did not follow up patients to evaluate the prognosis&#44; which was whether relevant to the concentration of UbA52 in urine&#46; Third&#44; since we did not enroll patients with normoalbuminuric diabetic kidney disease &#40;NADKD&#41;&#44; the significance of UBA52 in NADKD needs further study&#46;</p><p id="par0120" class="elsevierStylePara elsevierViewall">In summary&#44; we found altered urinary UbA52 content in DM-NP patients&#46; The ubiquitin fusion protein UbA52 may be another important molecule relevant to the pathogenesis of DKD&#46; It is expected to facilitate identification of diabetic nephropathy among diabetic patients&#46; This observation gives an impetus to research on the function of UbA52&#44; which could further enhance our understanding of the pathogenesis of DKD&#46; The identification of this protease and longitudinal studies in larger patient groups will determine its usefulness as a marker for predicting the clinical course and the potential role of the protease in the pathophysiology of diabetic renal involvement&#46;</p></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0130">Funding</span><p id="par0140" class="elsevierStylePara elsevierViewall">This study was funded by the Science and Technology Program of Guangdong Province Grant 2016A090922005&#46;</p></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0135">Conflicts of interest</span><p id="par0125" class="elsevierStylePara elsevierViewall">The authors declare to have no conflicts of interest&#46;</p></span></span>"
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          "identificador" => "xres1573535"
          "titulo" => "Abstract"
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              "identificador" => "abst0005"
              "titulo" => "Background"
            ]
            1 => array:2 [
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              "titulo" => "Methods"
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              "identificador" => "abst0015"
              "titulo" => "Results"
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              "titulo" => "Conclusion"
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          "titulo" => "Keywords"
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          "titulo" => "Resumen"
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            0 => array:2 [
              "identificador" => "abst0025"
              "titulo" => "Antecedentes"
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            1 => array:2 [
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              "titulo" => "M&#233;todos"
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            2 => array:2 [
              "identificador" => "abst0035"
              "titulo" => "Resultados"
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              "titulo" => "Conclusi&#243;n"
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          "titulo" => "Palabras clave"
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          "titulo" => "Introduction"
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          "titulo" => "Materials and methods"
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              "identificador" => "sec0015"
              "titulo" => "Study design"
            ]
            1 => array:2 [
              "identificador" => "sec0020"
              "titulo" => "Urine samples"
            ]
            2 => array:2 [
              "identificador" => "sec0025"
              "titulo" => "UbA52 antibody testing"
            ]
            3 => array:2 [
              "identificador" => "sec0030"
              "titulo" => "UbA52 measurement by double-antibody sandwich two-step ELISA"
            ]
            4 => array:2 [
              "identificador" => "sec0035"
              "titulo" => "Statistical analysis"
            ]
          ]
        ]
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          "identificador" => "sec0040"
          "titulo" => "Results"
          "secciones" => array:4 [
            0 => array:2 [
              "identificador" => "sec0045"
              "titulo" => "Patient characteristics"
            ]
            1 => array:2 [
              "identificador" => "sec0050"
              "titulo" => "DM-NP patients showed increased urinary UbA52 content"
            ]
            2 => array:2 [
              "identificador" => "sec0055"
              "titulo" => "Correlation analysis of urinary UbA52 level with clinical indicators"
            ]
            3 => array:2 [
              "identificador" => "sec0060"
              "titulo" => "Diagnostic value of urinary UbA52 content"
            ]
          ]
        ]
        7 => array:2 [
          "identificador" => "sec0065"
          "titulo" => "Discussion"
        ]
        8 => array:2 [
          "identificador" => "sec0075"
          "titulo" => "Funding"
        ]
        9 => array:2 [
          "identificador" => "sec0070"
          "titulo" => "Conflicts of interest"
        ]
        10 => array:2 [
          "identificador" => "xack555571"
          "titulo" => "Acknowledgements"
        ]
        11 => array:1 [
          "titulo" => "References"
        ]
      ]
    ]
    "pdfFichero" => "main.pdf"
    "tienePdf" => true
    "fechaRecibido" => "2019-11-26"
    "fechaAceptado" => "2021-01-17"
    "PalabrasClave" => array:2 [
      "en" => array:1 [
        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Keywords"
          "identificador" => "xpalclavsec1417895"
          "palabras" => array:5 [
            0 => "Diabetic kidney disease"
            1 => "UbA52"
            2 => "Urine"
            3 => "Biomarkers"
            4 => "Diagnosis"
          ]
        ]
      ]
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          "titulo" => "Palabras clave"
          "identificador" => "xpalclavsec1417894"
          "palabras" => array:5 [
            0 => "Enfermedad renal diab&#233;tica"
            1 => "UbA52"
            2 => "Orina"
            3 => "Biomarcadores"
            4 => "Diagn&#243;stico"
          ]
        ]
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    "tieneResumen" => true
    "resumen" => array:2 [
      "en" => array:3 [
        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Background</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Ubiquitin-52 amino acid fusion protein &#40;UbA52&#41; is an important factor in the pathogenesis of diabetic kidney disease &#40;DKD&#41; and has been suggested a potential marker in the disease&#46; However&#44; whether upregulation of UbA52 marks early kidney injury in T2DM mellitus &#40;T2DM&#41; patients remains unclear&#46; In this study&#44; we examine the diagnostic value of UbA52 as a biomarker in predicting early diabetic kidney disease &#40;DKD&#41; in T2DM patients&#46;</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">We used two-step ELISA to test UbA52 level in urine of 3 defined patient groups&#46; Samples from T2DM patients without albuminuria or diabetic retinopathy &#40;DM-WNP&#59; <span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&#44; T2DM patients with albuminuria and diabetic retinopathy&#44; excluding other renal diseases clinically &#40;DM-NP&#59; <span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41; and healthy controls &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41; were analyzed&#46; Spearman&#39;s correlation analysis and multiple linear regression model were used to analyze the correlation of urinary UbA52 level with laboratory results regarding kidney function&#46; Receiver operating characteristic curve &#40;ROC&#41; was used to evaluate the diagnostic value of UbA52 in predicting T2DM and early DKD&#46;</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Urinary UbA52 level in DM-NP group was 1&#46;75 times and 2&#46;71 times higher than in DN-WNP &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;004&#41; and normal control group &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#44; respectively&#46; The level of urinary UbA52 correlated significantly with serum creatinine &#40;<span class="elsevierStyleItalic">r</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;468&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#44; GFR &#40;<span class="elsevierStyleItalic">r</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>&#8722;0&#46;300&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;004&#41; and proteinuria &#40;<span class="elsevierStyleItalic">r</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;484&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#46; Multiple linear regression analysis showed that proteinuria level was independently associated with urinary UbA52 level &#40;<span class="elsevierStyleItalic">&#946;</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;833&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#46; The area under the ROC of urinary UbA52 in diagnosing T2DM and DKD was 0&#46;751 and 0&#46;755&#44; respectively&#46;</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusion</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">The level of urinary UbA52 increased significantly in T2DM patients with DKD&#46; The level of proteinuria is independently associated with urinary UbA52 level&#46; Urinary UbA52 could serve as an early marker in the diagnosis of DKD&#46;</p><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">ClinicalTrials&#46;gov Identifier&#58; <span class="elsevierStyleInterRef" id="intr0005" href="ctgov:NCT02204280">NCT02204280</span>&#46;</p></span>"
        "secciones" => array:4 [
          0 => array:2 [
            "identificador" => "abst0005"
            "titulo" => "Background"
          ]
          1 => array:2 [
            "identificador" => "abst0010"
            "titulo" => "Methods"
          ]
          2 => array:2 [
            "identificador" => "abst0015"
            "titulo" => "Results"
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          3 => array:2 [
            "identificador" => "abst0020"
            "titulo" => "Conclusion"
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      "es" => array:3 [
        "titulo" => "Resumen"
        "resumen" => "<span id="abst0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Antecedentes</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">La prote&#237;na de fusi&#243;n de amino&#225;cidos ubiquitina-52 &#40;UbA52&#41; es un factor importante en la patog&#233;nesis de la enfermedad renal diab&#233;tica &#40;ERD&#41;&#44; y se ha sugerido como marcador potencial en la enfermedad&#46; Sin embargo&#44; a&#250;n no est&#225; claro si la regulaci&#243;n al alza de UbA52 indica una lesi&#243;n renal temprana en pacientes con diabetes mellitus de tipo 2 &#40;DMT2&#41;&#46; En este estudio&#44; analizamos el valor diagn&#243;stico de UbA52 como biomarcador para predecir la ERD temprana en pacientes con DMT2&#46;</p></span> <span id="abst0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">M&#233;todos</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Utilizamos un ELISA de 2 pasos para analizar el nivel de UbA52 en la orina de 3 grupos de pacientes definidos&#46; Se analizaron muestras de pacientes con DMT2 sin albuminuria o retinopat&#237;a diab&#233;tica &#40;DM-WNP&#59; n<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&#44; pacientes con DMT2 y con albuminuria y retinopat&#237;a diab&#233;tica&#44; excluyendo cl&#237;nicamente otras enfermedades renales &#40;DM-NP&#59; n<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41; y controles sanos &#40;n<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&#46; Se utiliz&#243; el an&#225;lisis de correlaci&#243;n de Spearman y el modelo de regresi&#243;n lineal m&#250;ltiple para analizar la correlaci&#243;n del nivel de UbA52 en orina con los resultados de laboratorio relativos a la funci&#243;n renal&#46; Se utiliz&#243; la curva de caracter&#237;sticas operativas del receptor &#40;ROC&#41; para evaluar el valor diagn&#243;stico de UbA52 en la predicci&#243;n de la DMT2 y de la ERD temprana&#46;</p></span> <span id="abst0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Resultados</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">El nivel de UbA52 en orina en el grupo DM-NP fue 1&#44;75 y 2&#44;71 veces mayor que en el grupo DN-WNP &#40;p<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#44;004&#41;&#44; y en el grupo de control normal &#40;p<span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#44; respectivamente&#46; El nivel de UbA52 en orina se correlacion&#243; significativamente con la creatinina s&#233;rica &#40;r<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#44;468&#59; p<span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#44; la TFG &#40;r<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>&#8722;0&#44;300&#59; p<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#44;004&#41; y la proteinuria &#40;r<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#44;484&#59; p<span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#46; El an&#225;lisis de regresi&#243;n lineal m&#250;ltiple mostr&#243; que el nivel de proteinuria se asociaba de forma independiente al nivel de UbA52 en orina &#40;&#946;<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#44;833&#59; p<span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#46; El &#225;rea bajo las ROC de UbA52 en orina en el diagn&#243;stico de la DMT2 y de la ERD fue de 0&#44;751 y 0&#44;755&#44; respectivamente&#46;</p></span> <span id="abst0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Conclusi&#243;n</span><p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">El nivel de UbA52 en orina aument&#243; significativamente en pacientes con DMT2 y ERD&#46; El nivel de proteinuria se asocia independientemente al nivel de UbA52 en orina&#46; UbA52 en orina podr&#237;a servir como marcador temprano en el diagn&#243;stico de la ERD&#46;</p></span>"
        "secciones" => array:4 [
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            "identificador" => "abst0025"
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          2 => array:2 [
            "identificador" => "abst0035"
            "titulo" => "Resultados"
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            "identificador" => "abst0040"
            "titulo" => "Conclusi&#243;n"
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          "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">Concentration of UbA52 in urine of patients&#46; Horizontal bars are median&#44; box edges are the range of 25&#8211;75&#37;&#44; whiskers are minimum and maximum&#46; The numbers above the boxes are outlier samples&#46;</p>"
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          "en" => "<p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">The correlation of the concentration of UbA52 with serum creatinine&#44; and proteinuria level in patients&#46;</p>"
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        "etiqueta" => "Fig&#46; 3"
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          "en" => "<p id="spar0060" class="elsevierStyleSimplePara elsevierViewall">The ROC curve for Uba52 content as a diagnostic marker of T2DM and diabetic nephropathy&#46; &#40;A&#41; ROC curve of Uba52 content in diagnosis of T2DM mellitus&#59; Patients were divided into normal control group &#40;control&#41; and T2DM group &#40;DM-NP<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>DM-NP&#41;&#46; &#40;B&#41; ROC curve of Uba52 content in diagnosis of diabetic nephropathy&#59; Patients were divided into groups proteinuric group &#40;DM-NP&#41; and non-proteinuric group &#40;DM-WNP<span class="elsevierStyleHsp" style=""></span>&#43;<span class="elsevierStyleHsp" style=""></span>control&#41;&#46;</p>"
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          "leyenda" => "<p id="spar0070" class="elsevierStyleSimplePara elsevierViewall">FBS&#44; fasting blood glucose&#59; HbA1c&#44; hemoglobulinA1c&#59; BMI&#44; body mass index&#59; DM-WNP&#44; T2DM without nephropathy and without moderately increased albuminuria&#59; DM-NP&#44; DM with moderately or severely increased albuminuria and diabetic retinopathy&#59; Data are mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SD unless indicated&#46;</p>"
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Characteristics&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">DM-NP &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">Age &#40;year&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">58<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>13&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">60<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>9&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t">43<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>11&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">Male&#47;female&#44; no&#46;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t">20&#47;10&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t">13&#47;17&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">16&#47;14&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">Duaration of diabetes &#40;year&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t">11&#46;1<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>5&#46;42&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">8&#46;98<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>5&#46;34&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t">FBS level &#40;mmol&#47;L&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">8&#46;71<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;69&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">5&#46;01<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;44&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t">HbAlc level &#40;&#37;&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">7&#46;78<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;70&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">7&#46;98<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;92&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8211;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Proteinuria level &#40;mg&#47;d&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t">450&#46;00 &#40;54&#44;11000&#41;<a class="elsevierStyleCrossRef" href="#tblfn0005">&#42;</a>&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">8&#46;52 &#40;3&#46;23&#44;22&#46;5&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t">&#8211;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">26&#46;53<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;15&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">23&#46;72<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;30&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Serum creatinine &#40;mmol&#47;L&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">70 &#40;29&#46;2&#44;102&#46;95&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">64&#46;50 &#40;45&#44;85&#46;1&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">GFR &#40;ml&#47;min&#47;1&#46;73<span class="elsevierStyleHsp" style=""></span>m<span class="elsevierStyleSup">2</span>&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">87&#46;00 &#40;23&#44;150&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">82&#46;50 &#40;56&#44;140&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">104&#46;50 &#40;78&#44; 138&#41;<a class="elsevierStyleCrossRef" href="#tblfn0010">&#42;&#42;</a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">UbA52 level &#40;ng&#47;ml&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t">10&#46;95 &#40;1&#46;07&#44;59&#46;25&#41;<a class="elsevierStyleCrossRef" href="#tblfn0015"><span class="elsevierStyleSup">a</span></a>&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t">6&#46;24 &#40;0&#46;37&#44;30&#46;87&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">4&#46;04 &#40;0&#46;59&#44;14&#46;99&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\tvoid\n
                  \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td-with-role" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t" scope="col">Standardized coefficients&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="" valign="\n
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">B</span>&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Std&#46; error&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Beta&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">T</span>&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Sig&#46;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Tolerance&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">VIF&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t">&#40;Constant&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">7&#46;313&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;767&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">9&#46;538&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;000&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Proteinuria level&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;006&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;000&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;833&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">14&#46;119&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;000&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;401&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">2&#46;492&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Serum creatinine&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t">GFR&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t">FBS level&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">HbAlc Level&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">BMI&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">Age&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t">Sex&nbsp;\t\t\t\t\t\t\n
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                  """
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        "tipo" => "MULTIMEDIATABLA"
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          "leyenda" => "<p id="spar0090" class="elsevierStyleSimplePara elsevierViewall">PPV&#44; positive predictive value&#59; NPV&#44; negative predictive value&#46;</p>"
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            0 => array:2 [
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                0 => """
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                  \t\t\t\t\tvoid\n
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                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Prediction evaluation&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Sensitivity&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Specific&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">PPV&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">NPV&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Youden&#39;s index&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t">T2DM&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
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                  \t\t\t\t">78&#46;3&#37;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t\ttop\n
                  \t\t\t\t">63&#46;3&#37;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t">Diabetic nephropathy&nbsp;\t\t\t\t\t\t\n
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                          "etal" => true
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                            0 => "L&#46; Wang"
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        "texto" => "<p id="par0130" class="elsevierStylePara elsevierViewall">We would like to express our gratitude to all the patients who have participated&#44; as well as to all the staff of the Division of Nephrology of the Third Affiliated Hospital of Hebei Medical University for their collaboration and enthusiasm in this study&#46;</p>"
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Original article
Clinical significance of UbA52 level in the urine of patients with type 2 diabetes mellitus and diabetic kidney disease
Importancia clínica del nivel de UbA52 en la orina de pacientes con diabetes mellitus tipo 2 y enfermedad renal diabética
Xiaochang Xua, Yejing Donga, Maodong Liub, Ying Lib, Yimin Zhanga,
Autor para correspondencia
zhangyim@mail.sysu.edu.cn

Corresponding author.
a Division of Nephrology, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
b Division of Nephrology, The Third Affiliated Hospital, Hebei Medical University, Shijiazhuang, China
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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">The incidence of T2DM as well as life expectancy of T2DM patients has been increasing worldwide&#44; resulting in the soaring of patients with diabetic nephropathy&#46;<a class="elsevierStyleCrossRefs" href="#bib0160"><span class="elsevierStyleSup">1&#44;2</span></a> In developed countries&#44; diabetes has become the leading cause of kidney disease&#46; It is estimated about 1 in 4 adults with diabetes has kidney disease in the United States&#46;<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">3</span></a> In developing countries&#44; T2DM is rapidly replacing infectious diseases to be the leading cause of kidney failure&#46;<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">4</span></a> In China&#44; DKD is the second leading cause of end-stage renal disease &#40;ESRD&#41; and accounts for approximately 16&#46;4&#37; of all cases of ESRD&#46;<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">5</span></a></p><p id="par0010" class="elsevierStylePara elsevierViewall">Early identification of patients with DKD who are at risk for progressive renal dysfunction enables early intervention and may lead to better clinical outcome&#46; Glomerular filtration rate &#40;GFR&#41; marker serum creatinine is the best marker of renal excretory function&#46; However&#44; an increased in serum creatinine level is only detectable when GFR declined significantly and thus is insensitive for early DKD diagnosis&#46; Currently&#44; moderately increased albuminuria&#44; which indicates widespread microvascular damage in kidney&#44; is the earliest and the most commonly used laboratory marker in evaluating DKD&#46; A constant increase in albuminuria can predict DKD progression in T2DM patients&#46;<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">6</span></a> However&#44; many factors may cause the fluctuation of albuminuria level&#44; including serum glucose&#44; blood pressure&#44; smoking&#44; pregnancy&#44; and urinary tract infection&#46; Evidence also shows that the incidence of normoalbumin diabetic kidney disease &#40;NA-DKD&#41; is increasing in T2DM patients&#46;<a class="elsevierStyleCrossRefs" href="#bib0190"><span class="elsevierStyleSup">7&#8211;9</span></a> Thus&#44; a more reliable marker is needed to identify T2DM patients with high risk for or has developed early DKD&#46;</p><p id="par0015" class="elsevierStylePara elsevierViewall">Urine proteome analysis in diabetic patients has provided insights into identification of novel diagnostic markers for DKD&#46;<a class="elsevierStyleCrossRefs" href="#bib0205"><span class="elsevierStyleSup">10&#44;11</span></a> Dihazi et al&#46;&#44;<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">12</span></a> using SELDI-TOF mass spectrometry and SAX2 protein arrays&#44; found a processed form of ubiquitin with m&#47;z 14766 called ubiquitin-52 amino acid fusion protein &#40;UbA52&#41; that was missing in the urine of patients with DM&#44; but existed in DKD patients&#46; Based on the finding&#44; they concluded UbA52 could be used as a diagnostic marker for DKD&#46; However&#44; the identification process for UbA52 based on mass spectrometry was complicated and expensive&#44; which may not be suitable for clinical use&#46; Thus&#44; other methods are needed to identify diabetic patients at high risk for DKD&#46; With early diagnosis of DKD&#44; there are opportunities for early medical intervention and preventing disease progression&#46; The objective of this study was to test urinary UbA52 using enzymatic ELISA approach in T2DM patients and healthy people&#44; and analyze its correlation with laboratory and pathological results&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Study design</span><p id="par0020" class="elsevierStylePara elsevierViewall">We recruited participants from the third hospital of Hebei Medical University &#40;Hebei&#44; China&#41;&#46; Patients with T2DM were eligible if they were &#60;80 years old and &#62;18years&#44; and diagnosed with T2DM&#46; T2DM was defined according to the following criteria&#58; &#40;i&#41; HbAlc<span class="elsevierStyleHsp" style=""></span>&#8805;<span class="elsevierStyleHsp" style=""></span>6&#46;5&#37;&#59; &#40;ii&#41; fasting blood glucose<span class="elsevierStyleHsp" style=""></span>&#8805;<span class="elsevierStyleHsp" style=""></span>7&#46;0<span class="elsevierStyleHsp" style=""></span>mmol&#47;L&#59; &#40;iii&#41; 2-h plasma blood glucose<span class="elsevierStyleHsp" style=""></span>&#8805;<span class="elsevierStyleHsp" style=""></span>11&#46;1<span class="elsevierStyleHsp" style=""></span>mmol&#47;L during an oral glucose tolerance test &#40;OGTT&#41;&#59; &#40;iv&#41; with classic symptoms of hyperglycemic crisis&#44; a random blood glucose<span class="elsevierStyleHsp" style=""></span>&#8805;<span class="elsevierStyleHsp" style=""></span>11&#46;1<span class="elsevierStyleHsp" style=""></span>mmol&#47;L and had not received renal replacement therapy&#46; Patients with T2DM were excluded if they had acute complications of diabetes&#44; such as ketoacidosis or hyperosmolar coma&#59; malignancy&#44; particularly tumors affecting hepatic and renal function&#44; and history of radiotherapy and&#47;or chemotherapy&#59; heart failure&#44; particularly New York Heart Association grade<span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>III&#59; incomplete information that could affect the experimental results&#59; out-patients&#59; pregnant or breastfeeding&#59; severe hepatic insufficiency&#44; with aminotransferase level 2 times higher than normal&#59; history of respiratory infections or other serious illnesses&#59; or unwilling to cooperate in the study&#46; Another 30 healthy subjects were recruited from the Center of Health Examination of the Third Affiliated Hospital&#44; Hebei Medical University &#40;Hebei&#44; China&#41;&#46;</p><p id="par0025" class="elsevierStylePara elsevierViewall">Three patient groups were defined on the basis of clinical course&#44; examination of the optic fundus&#44; moderately increased albuminuria&#44; urinary albumin and urine creatinine ratio&#44; and urinary albumin excretion rate&#46; The groups included patients with DM with moderately increased albuminuria&#40;defined as the urinary albumin&#47;creatinine ratio &#40;UACR&#41; &#62;30<span class="elsevierStyleHsp" style=""></span>mg&#47;g&#44; or persistent microalbuminuria &#62;300<span class="elsevierStyleHsp" style=""></span>mg&#47;24<span class="elsevierStyleHsp" style=""></span>h&#44; or &#62;1&#46;0<span class="elsevierStyleHsp" style=""></span>g&#47;24<span class="elsevierStyleHsp" style=""></span>h for more than 3 months&#41;&#44; diabetic retinopathy&#44; excluding other renal diseases clinically were defined as diabetic kidney disease &#40;DM-NP&#59; <span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&#44; with DM without albuminuria or diabetic retinopathy&#44; some have chronic kidney disease due to hypertension&#44; gout&#44; nephrotoxic drugs and other reasons&#44; excluding diabetic kidney disease clinically &#40;DM-WNP&#59; <span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&#44; and healthy controls &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&#46; Three DM-NP patients had biopsy-proven DN&#46;</p><p id="par0030" class="elsevierStylePara elsevierViewall">The protocol was approved by the appropriate institutional review boards&#44; and written informed consent was obtained from all participants&#46; Clinical sample procurement and analysis as well as data management were approved by the local institutional ethical review committee of the third hospital of Hebei Medical University &#40;Hebei&#44; China&#41;&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Urine samples</span><p id="par0035" class="elsevierStylePara elsevierViewall">First morning midstream urine samples&#44; 10<span class="elsevierStyleHsp" style=""></span>ml&#44; were taken before breakfast&#46; Urine samples were centrifuged for 15<span class="elsevierStyleHsp" style=""></span>min at 3000<span class="elsevierStyleHsp" style=""></span>g and stored at &#8722;80<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The supernatant was divided into 1&#46;5-mL aliquots and immediately stored at &#8722;80<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; All samples were stored for 4&#8211;5 months and did not undergo any freeze-thaw cycles&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">UbA52 antibody testing</span><p id="par0040" class="elsevierStylePara elsevierViewall">In total&#44; 10<span class="elsevierStyleHsp" style=""></span>mg horseradish peroxidase &#40;HRP&#41;was added to 0&#46;2<span class="elsevierStyleHsp" style=""></span>ml of 1&#46;25&#37; glutaraldehyde solution and kept at room temperature overnight&#46; The reaction mixture was collected in a dialysis bag surrounded by normal saline&#46; The dialysis bag was stored for 24<span class="elsevierStyleHsp" style=""></span>h at 4<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; At the same time the normal saline was exchanged twice to remove unconjugated glutaraldehyde solution&#46; A few drops of normal saline were placed in the reaction mixture to ensure a total volume of 1<span class="elsevierStyleHsp" style=""></span>ml&#46; An amount of 5<span class="elsevierStyleHsp" style=""></span>mg goat anti-UbA52 polyclonal antibody &#40;Santa Cruz Biotechnology&#44; Santa Cruz&#44; CA&#44; US&#41; per 1<span class="elsevierStyleHsp" style=""></span>ml normal saline was added to the solution&#46; An amount of 0&#46;1<span class="elsevierStyleHsp" style=""></span>ml of 1<span class="elsevierStyleHsp" style=""></span>M carbonate buffer solution&#44; pH 9&#46;5&#44; was added&#59; the reaction mixture was vortex-mixed and kept for 24<span class="elsevierStyleHsp" style=""></span>h at 4<span class="elsevierStyleHsp" style=""></span>&#176;C effectively to form a crosslinked structure&#46; An amount of 0&#46;2<span class="elsevierStyleHsp" style=""></span>ml of 0&#46;2<span class="elsevierStyleHsp" style=""></span>M lysine was added to the reaction mixture with constant shaking&#46; The preparation was stored at room temperature for 2<span class="elsevierStyleHsp" style=""></span>h&#46; Then the same volume of saturated ammonium sulfate solution was added with constant stirring and incubated for 1<span class="elsevierStyleHsp" style=""></span>h at 4<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The mixture was centrifuged at 2862<span class="elsevierStyleHsp" style=""></span>g for 15<span class="elsevierStyleHsp" style=""></span>min&#46; The supernatant was discarded and the deposit was washed twice with half-saturated ammonium sulfate solution&#46; The final precipitation was suspended in 0&#46;15<span class="elsevierStyleHsp" style=""></span>M PBS&#44; pH 7&#46;4&#46; Then the mixture was collected in a dialysis bag surrounded by 0&#46;15<span class="elsevierStyleHsp" style=""></span>M PBS&#44; pH 7&#46;4&#44; to remove ammonium ion&#46; The mixture was centrifuged at 17886<span class="elsevierStyleHsp" style=""></span>g for 30<span class="elsevierStyleHsp" style=""></span>min&#46; The supernatant was the purified solution of HRP-conjugated goat anti-human UbA52 polyclonal antibody&#44; then repackaged into smaller packages to store at 4<span class="elsevierStyleHsp" style=""></span>&#176;C or &#8722;80<span class="elsevierStyleHsp" style=""></span>&#176;C&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">UbA52 measurement by double-antibody sandwich two-step ELISA</span><p id="par0045" class="elsevierStylePara elsevierViewall">An amount of 10<span class="elsevierStyleHsp" style=""></span>&#956;L goat anti-UbA52 polyclonal antibody was dissolved in coating buffer on 96- and 48-well ELISA plates&#46; 0&#46;15<span class="elsevierStyleHsp" style=""></span>ml of the mixture was putted in every well and allowed to incubate overnight at 4<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The concentration of the coating antibody was 2<span class="elsevierStyleHsp" style=""></span>&#956;g&#47;ml&#46; The plate surface was then gently washed with washing buffer 4 times and dried with filter paper&#46; Eight wells were for standards&#46; Sample diluent solution was added to the standard solution and the concentrations of standard solution were 200&#44; 100&#44; 50&#44; 25&#44; 12&#46;5&#44; 6&#46;25&#44; 3&#46;12&#44; and 0<span class="elsevierStyleHsp" style=""></span>ng&#47;ml&#46; An amount of 100<span class="elsevierStyleHsp" style=""></span>&#956;L sample was added to 96- and 48-well ELISA plates and incubated for 90<span class="elsevierStyleHsp" style=""></span>min at 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The plate surface was then gently washed with washing buffer 5 times and dried with filter paper&#46; The HRP-conjugated antibody was diluted 5000 times with PBS&#46; Then 100<span class="elsevierStyleHsp" style=""></span>&#956;l was added to 96- and 48-well ELISA plates with constant shaking and incubated for 90<span class="elsevierStyleHsp" style=""></span>min at 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; Each well was washed 5 times in washing buffer and dried with filter paper&#46; After 100<span class="elsevierStyleHsp" style=""></span>&#956;L of TMB &#40;3&#44;3&#8242;&#44;5&#44;5&#8242;-tetramethylbenzidine hydrochloride&#41; solution was added&#44; the wells were incubated for 10<span class="elsevierStyleHsp" style=""></span>min at 37<span class="elsevierStyleHsp" style=""></span>&#176;C in the dark&#46; The reaction was stopped with 2<span class="elsevierStyleHsp" style=""></span>M of sulfuric acid&#46; The levels of each sample were extrapolated by referring to a standard curve &#40;4-parameter logistic curve fitting&#41; of optical density &#40;OD&#41; 450<span class="elsevierStyleHsp" style=""></span>nm&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Statistical analysis</span><p id="par0050" class="elsevierStylePara elsevierViewall">The data were analyzed by using SPSS 20 &#40;SPSS Inc&#46;&#44; Chicago&#44; IL&#41;&#46; For continuous variables&#44; data are presented as mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SD or median &#40;range&#41;&#44; and means were compared by one-way ANOVA&#46; Spearman&#39;s correlation analysis and multiple linear regression model were used to analyze the correlation between UbA52 and patients&#8217; laboratory indexes&#46; Receiver operating characteristic curve &#40;ROC&#41; was used to evaluate the value of UbA52 in T2DM mellitus and diabetic kidney disease diagnosis&#46; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05 was considered statistically significant&#46;</p></span></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Results</span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Patient characteristics</span><p id="par0055" class="elsevierStylePara elsevierViewall">Of the 90 patients enrolled in the study&#44; 49 were male &#40;54&#46;4&#37;&#41;&#46; The mean age&#44; duration of diabetes&#44; gender distribution&#44; fasting blood glucose level&#44; body mass index and hemoglubulinAlc level did not differ between DM-NP and DM-WNP groups &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#46; The 24-h total proteinuria level and serum creatinine level were higher for DM-NP than DM-WNP and controls &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; and did not differ between DM-WNP patients and controls &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">DM-NP patients showed increased urinary UbA52 content</span><p id="par0060" class="elsevierStylePara elsevierViewall">Three DM-NP patients had a diagnosis of diabetic kidney disease based on renal biopsy&#46; On ELISA&#44; the titers of UbA52 for these patients were 50&#46;4&#44; 58&#46;6&#44; 59&#46;3<span class="elsevierStyleHsp" style=""></span>ng&#47;ml&#46; The other patients in this group showed high moderately increased albuminuria without renal biopsy&#46; The urinary UbA52 content was higher for DM-NP than DN-WNP patients and controls &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; and did not differ between DM-WNP patients and controls &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#62;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46; The levels of urinary UbA52in DM-NP group was 1&#46;75 times and 2&#46;71 times of that in DN-WNP &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;004&#41; and normal control group &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41; respectively &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a> and <a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#41;&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Correlation analysis of urinary UbA52 level with clinical indicators</span><p id="par0065" class="elsevierStylePara elsevierViewall">The level of urinary UbA52 was significantly correlated with serum creatinine &#40;<span class="elsevierStyleItalic">r</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;468&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#44; GFR &#40;<span class="elsevierStyleItalic">r</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>&#8722;0&#46;300&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;004&#41; and proteinuria level &#40;<span class="elsevierStyleItalic">r</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;484&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#41;&#46; Multiple linear regression analysis &#40;Enter&#59; variables&#58; proteinuria level&#44; serum creatinine&#44; GFR&#44; FBS level&#44; HbAlc level&#44; BMI&#44; age&#44; and sex&#41; showed that only proteinuria level was independently associated with urinary UbA52 level &#40;<span class="elsevierStyleItalic">&#946;</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;833&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a>&#41;&#46; No evidence of influential collinearity was observed&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia><elsevierMultimedia ident="tbl0010"></elsevierMultimedia></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Diagnostic value of urinary UbA52 content</span><p id="par0070" class="elsevierStylePara elsevierViewall">The area under the receiver operating characteristic curve &#40;AUC&#41; of UbA52 content in diagnosing T2DM mellitus was 0&#46;751&#40;95&#37;CI 0&#46;684&#8211;0&#46;855&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#46; The sensitivity and specificity were 78&#46;3&#37; and 63&#46;3&#37;&#44; respectively&#44; and the cut off value was 4&#46;32&#46;</p><p id="par0075" class="elsevierStylePara elsevierViewall">The AUC of UbA52 content in diagnosing diabetic nephropathy was 0&#46;755&#40;95&#37; CI 0&#46;644&#8211;0&#46;866&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#46;The sensitivity and specificity were 63&#46;3&#37; and 85&#46;0&#37;&#44; respectively&#44; and the cut off value was 8&#46;97 &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a> and <a class="elsevierStyleCrossRefs" href="#tbl0015">Tables 3&#8211;5</a>&#41;&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia><elsevierMultimedia ident="tbl0015"></elsevierMultimedia><elsevierMultimedia ident="tbl0020"></elsevierMultimedia><elsevierMultimedia ident="tbl0025"></elsevierMultimedia></span></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Discussion</span><p id="par0080" class="elsevierStylePara elsevierViewall">DKD refers to kidney disease that is specific to diabetes&#46; In most cases&#44; careful screening instead of kidney biopsy is able to identify DKD patients&#46; The diagnosis of DKD is made based in part on the finding of elevated urinary albumin excretion and the presence of diabetic retinopathy&#46;<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">13</span></a> Diabetic nephropathy &#40;DN&#41; refers to specific pathological changes in kidney biopsy and functional changes in DM patients that result from detrimental effects of DM&#46;<a class="elsevierStyleCrossRefs" href="#bib0185"><span class="elsevierStyleSup">6&#44;7</span></a> Given that the diagnosis of DKD is based on clinical manifestations and laboratory findings&#44; however&#44; about 30&#37; DKD diagnosed by clinical criteria are biopsy-proven nondiabetic glomerular diseases combined with diabetes&#44; suggesting a urgent need for a sensitive yet specific marker for DKD&#46; SELDI-TOF&#47;MS found UbA52 can be considered as a reliable biomarker to identify patients with diabetic nephropathy among diabetic patients&#46; And more importantly&#44; to distinguish biopsy-proven diabetic nephropathy from nondiabetic-CKD in diabetic patients&#46;</p><p id="par0085" class="elsevierStylePara elsevierViewall">In current study&#44; we examined the value of urinary UbA52 as a diagnostic biomarker for DKD in patients with diabetes by two-step ELISA assay&#46; Multiple linear regression analysis showed that proteinuria level was independently associated with urinary UbA52 level &#40;<span class="elsevierStyleItalic">&#946;</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;833&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#46; The area under the receiver operating characteristic curve &#40;ROC&#41; of urinary UbA52 in diagnosing T2DM mellitus and diabetic nephropathy was 0&#46;751 and 0&#46;755&#44; respectively&#46; Based on our results&#44; urinary UbA52 measured by ELISA is expected to be a potential biomarker for the diagnosis of diabetic nephropathy&#46;</p><p id="par0090" class="elsevierStylePara elsevierViewall">DKD plays an important role in the development of ESRD&#46; Kidney biopsy&#44; the gold standard for diagnosis of glomerular disease&#44; represents the current method for a definite diagnosis of diabetic nephropathy&#44; but is only performed in diabetic patients with atypical presentations&#46; In 2010&#44; the Research Committee of the Renal Pathology Society first divide diabetic nephropathy into four hierarchical glomerular lesions with a separate evaluation for degrees of interstitial and vascular involvement&#46;<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">14</span></a> For the most part&#44; the diagnosis of DKD is based on the course of clinical manifestations and laboratory findings such as moderately increased albuminuria&#44; which is considered a prognostic predictor of the progression of DKD&#46; However&#44; moderately increased albuminuria has a limited predictive value because of its poor correlation with reduced glomerular filtration rate&#46;<a class="elsevierStyleCrossRefs" href="#bib0230"><span class="elsevierStyleSup">15&#44;16</span></a> Some patients with diabetic nephropathy proven by renal biopsy have normal serum creatinine level without moderately increased albuminuria&#44;<a class="elsevierStyleCrossRefs" href="#bib0240"><span class="elsevierStyleSup">17&#44;18</span></a> whereas others&#44; before moderately increased albuminuria is diagnosed&#44; may show progressive loss of kidney function&#46;<a class="elsevierStyleCrossRefs" href="#bib0250"><span class="elsevierStyleSup">19&#44;20</span></a> Recent studies suggested that the moderately increased albuminuria in several DKD patients can even return to normal values&#46;<a class="elsevierStyleCrossRefs" href="#bib0235"><span class="elsevierStyleSup">16&#44;21</span></a> While this phenomenon is more common in type 2 diabetes mellitus&#44; this situation is termed as normoalbuminuric diabetic kidney disease &#40;NADKD&#41; and is characterized by more obvious tubular interstitial lesions&#46;<a class="elsevierStyleCrossRefs" href="#bib0265"><span class="elsevierStyleSup">22&#44;23</span></a></p><p id="par0095" class="elsevierStylePara elsevierViewall">Therefore&#44; developing more sensitive and specific markers is urgently needed to identify patients with diabetes who are at high risk of DKD&#46; At the molecular level&#44; several proteins and their functions in DKD have been characterized&#44; whereas others remain to be discovered&#46; UbA52 is a ubiquitin fusion protein&#46; Sun et al&#46;<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">24</span></a> found that the expression of UbA52 mRNA in the kidney was proportional to blood glucose levels&#46; In situ hybridization and immunohistochemistry revealed that UbA52 exclusively localized to renal tubules&#44; and its expression was markedly increased in mice with DN&#46;<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">18</span></a> UbA52 can be detected in serum&#44; but at low concentrations which rule out its effect on urine concentration&#46;<a class="elsevierStyleCrossRef" href="#bib0280"><span class="elsevierStyleSup">25</span></a></p><p id="par0100" class="elsevierStylePara elsevierViewall">Our study shows the level of urinary UbA52 was significantly correlated with serum creatinine&#46; The expression of UbA52 is regulated by a variety of injuries&#44; such as oxidative and carbonyl stress&#44; which are important in the pathophysiology of DKD and apoptosis&#46;<a class="elsevierStyleCrossRefs" href="#bib0275"><span class="elsevierStyleSup">24&#44;26</span></a> High glucose in the cells of diabetic patients can lead to the formation of superoxide compounds and increased production of reactive oxygen species &#40;ROS&#41;&#46; Oxidative stress can increase the activity of ubiquitin activase &#40;E1&#41; and ubiquitin binding enzyme &#40;E2&#41; and then increase the expression of UbA52&#46;<a class="elsevierStyleCrossRefs" href="#bib0290"><span class="elsevierStyleSup">27&#44;28</span></a> Stimulation of cells with hydrogen peroxide&#44; which mimics oxidative stress&#44; can increase the expression of UbA52&#46;<a class="elsevierStyleCrossRef" href="#bib0300"><span class="elsevierStyleSup">29</span></a> The selective expression of UbA52 in renal tubules suggest that the ubiquitin proteasome proteolytic system is indeed operating in this compartment of the kidney&#46;<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">24</span></a></p><p id="par0105" class="elsevierStylePara elsevierViewall">We combined HRP-labeled antibody with ELISA to determine whether urine could be used for identifying a marker specific for DKD and evaluated the relationship between urinary UbA52 content and DKD&#46; Urinary UbA52 content was greater in DM-NP patients than in controls or DM-WNP patients&#46; Our results confirm the altered urinary UbA52 content in patients with DN&#46; UbA52 may be an important molecule relevant to the pathobiology of DKD&#46; With urinary UbA52 secretion&#44; we can estimate the degree of microangiopathy in the renal system of patients with diabetes&#46;</p><p id="par0110" class="elsevierStylePara elsevierViewall">The prevalence of nondiabetic renal disease in patients with T2DM mellitus &#40;T2DM&#41; varies from 27&#37; to 79&#37;&#46;<a class="elsevierStyleCrossRef" href="#bib0305"><span class="elsevierStyleSup">30</span></a> So a patient with T2DM with a high proteinuria could have a diagnosis of DKD or of T2DM with nondiabetic renal disease&#46; Because the proteinuria level was independently associated with urinary UbA52 level&#44; UbA52 may have a role in the pathogenesis of renal disease with proteinuria&#46; UBA52 was significantly increased in patients with focal segmental glomerular sclerosis &#40;FSGS&#41; and minimal change nephropathy &#40;MCD&#41;&#44;<a class="elsevierStyleCrossRef" href="#bib0310"><span class="elsevierStyleSup">31</span></a> which was much higher than that in patients with diabetic nephropathy in our study&#46; UBA52 may not be a specific marker&#44; but it may be helpful to identify these diseases with different cut off values&#46; Further studies with larger patient samples are needed to explore the function of UbA52 in DKD and nondiabetic renal diseases&#46;</p><p id="par0115" class="elsevierStylePara elsevierViewall">This study has several limitations&#46; First&#44; we had only 3 cases of diabetic nephropathy with a diagnosis by renal biopsy&#46; The other cases of DM-NP were defined by moderately increased albuminuria and diabetic retinopathy&#44; which are not accurate indictors and related to many factors&#46; Second&#44; we did not follow up patients to evaluate the prognosis&#44; which was whether relevant to the concentration of UbA52 in urine&#46; Third&#44; since we did not enroll patients with normoalbuminuric diabetic kidney disease &#40;NADKD&#41;&#44; the significance of UBA52 in NADKD needs further study&#46;</p><p id="par0120" class="elsevierStylePara elsevierViewall">In summary&#44; we found altered urinary UbA52 content in DM-NP patients&#46; The ubiquitin fusion protein UbA52 may be another important molecule relevant to the pathogenesis of DKD&#46; It is expected to facilitate identification of diabetic nephropathy among diabetic patients&#46; This observation gives an impetus to research on the function of UbA52&#44; which could further enhance our understanding of the pathogenesis of DKD&#46; The identification of this protease and longitudinal studies in larger patient groups will determine its usefulness as a marker for predicting the clinical course and the potential role of the protease in the pathophysiology of diabetic renal involvement&#46;</p></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0130">Funding</span><p id="par0140" class="elsevierStylePara elsevierViewall">This study was funded by the Science and Technology Program of Guangdong Province Grant 2016A090922005&#46;</p></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0135">Conflicts of interest</span><p id="par0125" class="elsevierStylePara elsevierViewall">The authors declare to have no conflicts of interest&#46;</p></span></span>"
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              "titulo" => "Antecedentes"
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              "titulo" => "Urine samples"
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              "titulo" => "UbA52 antibody testing"
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              "identificador" => "sec0030"
              "titulo" => "UbA52 measurement by double-antibody sandwich two-step ELISA"
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              "titulo" => "Patient characteristics"
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              "identificador" => "sec0050"
              "titulo" => "DM-NP patients showed increased urinary UbA52 content"
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              "identificador" => "sec0055"
              "titulo" => "Correlation analysis of urinary UbA52 level with clinical indicators"
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              "identificador" => "sec0060"
              "titulo" => "Diagnostic value of urinary UbA52 content"
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    "fechaRecibido" => "2019-11-26"
    "fechaAceptado" => "2021-01-17"
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            1 => "UbA52"
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        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Background</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Ubiquitin-52 amino acid fusion protein &#40;UbA52&#41; is an important factor in the pathogenesis of diabetic kidney disease &#40;DKD&#41; and has been suggested a potential marker in the disease&#46; However&#44; whether upregulation of UbA52 marks early kidney injury in T2DM mellitus &#40;T2DM&#41; patients remains unclear&#46; In this study&#44; we examine the diagnostic value of UbA52 as a biomarker in predicting early diabetic kidney disease &#40;DKD&#41; in T2DM patients&#46;</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">We used two-step ELISA to test UbA52 level in urine of 3 defined patient groups&#46; Samples from T2DM patients without albuminuria or diabetic retinopathy &#40;DM-WNP&#59; <span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&#44; T2DM patients with albuminuria and diabetic retinopathy&#44; excluding other renal diseases clinically &#40;DM-NP&#59; <span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41; and healthy controls &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41; were analyzed&#46; Spearman&#39;s correlation analysis and multiple linear regression model were used to analyze the correlation of urinary UbA52 level with laboratory results regarding kidney function&#46; Receiver operating characteristic curve &#40;ROC&#41; was used to evaluate the diagnostic value of UbA52 in predicting T2DM and early DKD&#46;</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Urinary UbA52 level in DM-NP group was 1&#46;75 times and 2&#46;71 times higher than in DN-WNP &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;004&#41; and normal control group &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#44; respectively&#46; The level of urinary UbA52 correlated significantly with serum creatinine &#40;<span class="elsevierStyleItalic">r</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;468&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#44; GFR &#40;<span class="elsevierStyleItalic">r</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>&#8722;0&#46;300&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;004&#41; and proteinuria &#40;<span class="elsevierStyleItalic">r</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;484&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#46; Multiple linear regression analysis showed that proteinuria level was independently associated with urinary UbA52 level &#40;<span class="elsevierStyleItalic">&#946;</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;833&#44; <span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#46; The area under the ROC of urinary UbA52 in diagnosing T2DM and DKD was 0&#46;751 and 0&#46;755&#44; respectively&#46;</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusion</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">The level of urinary UbA52 increased significantly in T2DM patients with DKD&#46; The level of proteinuria is independently associated with urinary UbA52 level&#46; Urinary UbA52 could serve as an early marker in the diagnosis of DKD&#46;</p><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">ClinicalTrials&#46;gov Identifier&#58; <span class="elsevierStyleInterRef" id="intr0005" href="ctgov:NCT02204280">NCT02204280</span>&#46;</p></span>"
        "secciones" => array:4 [
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            "titulo" => "Background"
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          1 => array:2 [
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            "titulo" => "Methods"
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          2 => array:2 [
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        "resumen" => "<span id="abst0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Antecedentes</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">La prote&#237;na de fusi&#243;n de amino&#225;cidos ubiquitina-52 &#40;UbA52&#41; es un factor importante en la patog&#233;nesis de la enfermedad renal diab&#233;tica &#40;ERD&#41;&#44; y se ha sugerido como marcador potencial en la enfermedad&#46; Sin embargo&#44; a&#250;n no est&#225; claro si la regulaci&#243;n al alza de UbA52 indica una lesi&#243;n renal temprana en pacientes con diabetes mellitus de tipo 2 &#40;DMT2&#41;&#46; En este estudio&#44; analizamos el valor diagn&#243;stico de UbA52 como biomarcador para predecir la ERD temprana en pacientes con DMT2&#46;</p></span> <span id="abst0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">M&#233;todos</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Utilizamos un ELISA de 2 pasos para analizar el nivel de UbA52 en la orina de 3 grupos de pacientes definidos&#46; Se analizaron muestras de pacientes con DMT2 sin albuminuria o retinopat&#237;a diab&#233;tica &#40;DM-WNP&#59; n<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&#44; pacientes con DMT2 y con albuminuria y retinopat&#237;a diab&#233;tica&#44; excluyendo cl&#237;nicamente otras enfermedades renales &#40;DM-NP&#59; n<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41; y controles sanos &#40;n<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&#46; Se utiliz&#243; el an&#225;lisis de correlaci&#243;n de Spearman y el modelo de regresi&#243;n lineal m&#250;ltiple para analizar la correlaci&#243;n del nivel de UbA52 en orina con los resultados de laboratorio relativos a la funci&#243;n renal&#46; Se utiliz&#243; la curva de caracter&#237;sticas operativas del receptor &#40;ROC&#41; para evaluar el valor diagn&#243;stico de UbA52 en la predicci&#243;n de la DMT2 y de la ERD temprana&#46;</p></span> <span id="abst0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Resultados</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">El nivel de UbA52 en orina en el grupo DM-NP fue 1&#44;75 y 2&#44;71 veces mayor que en el grupo DN-WNP &#40;p<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#44;004&#41;&#44; y en el grupo de control normal &#40;p<span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#44; respectivamente&#46; El nivel de UbA52 en orina se correlacion&#243; significativamente con la creatinina s&#233;rica &#40;r<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#44;468&#59; p<span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#44; la TFG &#40;r<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>&#8722;0&#44;300&#59; p<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#44;004&#41; y la proteinuria &#40;r<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#44;484&#59; p<span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#46; El an&#225;lisis de regresi&#243;n lineal m&#250;ltiple mostr&#243; que el nivel de proteinuria se asociaba de forma independiente al nivel de UbA52 en orina &#40;&#946;<span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#44;833&#59; p<span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#44;001&#41;&#46; El &#225;rea bajo las ROC de UbA52 en orina en el diagn&#243;stico de la DMT2 y de la ERD fue de 0&#44;751 y 0&#44;755&#44; respectivamente&#46;</p></span> <span id="abst0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Conclusi&#243;n</span><p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">El nivel de UbA52 en orina aument&#243; significativamente en pacientes con DMT2 y ERD&#46; El nivel de proteinuria se asocia independientemente al nivel de UbA52 en orina&#46; UbA52 en orina podr&#237;a servir como marcador temprano en el diagn&#243;stico de la ERD&#46;</p></span>"
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          "leyenda" => "<p id="spar0070" class="elsevierStyleSimplePara elsevierViewall">FBS&#44; fasting blood glucose&#59; HbA1c&#44; hemoglobulinA1c&#59; BMI&#44; body mass index&#59; DM-WNP&#44; T2DM without nephropathy and without moderately increased albuminuria&#59; DM-NP&#44; DM with moderately or severely increased albuminuria and diabetic retinopathy&#59; Data are mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>SD unless indicated&#46;</p>"
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                  <table border="0" frame="\n
                  \t\t\t\t\tvoid\n
                  \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Characteristics&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">DM-NP &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">DM-WNP &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Controls &#40;<span class="elsevierStyleItalic">n</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>30&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Age &#40;year&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">58<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>13&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">60<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>9&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">43<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>11&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Male&#47;female&#44; no&#46;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">20&#47;10&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">13&#47;17&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">16&#47;14&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Duaration of diabetes &#40;year&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">11&#46;1<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>5&#46;42&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">8&#46;98<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>5&#46;34&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">FBS level &#40;mmol&#47;L&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">9&#46;29<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>4&#46;15&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">8&#46;71<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;69&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">5&#46;01<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>0&#46;44&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">HbAlc level &#40;&#37;&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">7&#46;78<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;70&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">7&#46;98<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>1&#46;92&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Proteinuria level &#40;mg&#47;d&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">450&#46;00 &#40;54&#44;11000&#41;<a class="elsevierStyleCrossRef" href="#tblfn0005">&#42;</a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">8&#46;52 &#40;3&#46;23&#44;22&#46;5&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">BMI &#40;kg&#47;m<span class="elsevierStyleSup">2</span>&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">27&#46;24<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;43&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">26&#46;53<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;15&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">23&#46;72<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>3&#46;30&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Serum creatinine &#40;mmol&#47;L&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">73&#46;2 &#40;50&#46;4&#44;201&#41;<a class="elsevierStyleCrossRef" href="#tblfn0015"><span class="elsevierStyleSup">a</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">70 &#40;29&#46;2&#44;102&#46;95&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">64&#46;50 &#40;45&#44;85&#46;1&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">GFR &#40;ml&#47;min&#47;1&#46;73<span class="elsevierStyleHsp" style=""></span>m<span class="elsevierStyleSup">2</span>&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">87&#46;00 &#40;23&#44;150&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">82&#46;50 &#40;56&#44;140&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">104&#46;50 &#40;78&#44; 138&#41;<a class="elsevierStyleCrossRef" href="#tblfn0010">&#42;&#42;</a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">UbA52 level &#40;ng&#47;ml&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="left" valign="\n
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                  \t\t\t\t">10&#46;95 &#40;1&#46;07&#44;59&#46;25&#41;<a class="elsevierStyleCrossRef" href="#tblfn0015"><span class="elsevierStyleSup">a</span></a>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t">6&#46;24 &#40;0&#46;37&#44;30&#46;87&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t">4&#46;04 &#40;0&#46;59&#44;14&#46;99&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Collinearity statistics</th></tr><tr title="table-row"><th class="td" title="\n
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                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Std&#46; error&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Beta&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">T</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Sig&#46;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Tolerance&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">VIF&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#40;Constant&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">7&#46;313&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;767&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">9&#46;538&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;000&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Proteinuria level&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;006&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;000&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;833&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">14&#46;119&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;000&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;401&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">2&#46;492&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Serum creatinine&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8722;0&#46;051&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;095&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8722;0&#46;098&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8722;0&#46;540&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;592&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;249&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">4&#46;734&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">GFR&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;010&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;092&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;018&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;109&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;914&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;290&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">4&#46;260&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">FBS level&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8722;0&#46;406&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;343&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8722;0&#46;100&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8722;1&#46;183&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;242&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;686&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">1&#46;458&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">HbAlc Level&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;212&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;645&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;027&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;329&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;744&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;749&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">1&#46;334&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">BMI&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8722;0&#46;055&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;336&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8722;0&#46;013&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8722;0&#46;163&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;871&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;836&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">1&#46;196&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Age&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">4&#46;530&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">2&#46;170&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;158&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">2&#46;088&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;052&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">0&#46;857&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">1&#46;167&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">Sex&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&#8722;0&#46;118&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
                  \t\t\t\t\ttop\n
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                  \t\t\t\t">&#8722;0&#46;088&nbsp;\t\t\t\t\t\t\n
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                    0 => array:2 [
                      "titulo" => "Prevalence and ethnic pattern of diabetes and prediabetes in China in 2013"
                      "autores" => array:1 [
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                          "etal" => true
                          "autores" => array:6 [
                            0 => "L&#46; Wang"
                            1 => "P&#46; Gao"
                            2 => "M&#46; Zhang"
                            3 => "Z&#46; Huang"
                            4 => "D&#46; Zhang"
                            5 => "Q&#46; Deng"
                          ]
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                    0 => array:2 [
                      "doi" => "10.1001/jama.2017.7596"
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                        "volumen" => "317"
                        "paginaInicial" => "2515"
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                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/28655017"
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                          ]
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                0 => array:2 [
                  "contribucion" => array:1 [
                    0 => array:2 [
                      "titulo" => "IDF Diabetes Atlas&#58; global estimates of diabetes prevalence for 2017 and projections for 2045"
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                        0 => array:2 [
                          "etal" => true
                          "autores" => array:6 [
                            0 => "N&#46;H&#46; Cho"
                            1 => "J&#46;E&#46; Shaw"
                            2 => "S&#46; Karuranga"
                            3 => "Y&#46; Huang"
                            4 => "J&#46;D&#46; Da Rocha Fernandes"
                            5 => "A&#46;W&#46; Ohlrogge"
                          ]
                        ]
                      ]
                    ]
                  ]
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                    0 => array:1 [
                      "Revista" => array:6 [
                        "tituloSerie" => "Diabetes Res Clin Pract"
                        "fecha" => "2018"
                        "volumen" => "138"
                        "paginaInicial" => "271"
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                        "itemHostRev" => array:3 [
                          "pii" => "S0735109719337970"
                          "estado" => "S300"
                          "issn" => "07351097"
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              ]
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                0 => array:2 [
                  "contribucion" => array:1 [
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                      "titulo" => "Clinical manifestations of kidney disease among US adults with diabetes&#44; 1988&#8211;2014"
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                          "etal" => true
                          "autores" => array:6 [
                            0 => "M&#46; Afkarian"
                            1 => "L&#46;R&#46; Zelnick"
                            2 => "Y&#46;N&#46; Hall"
                            3 => "P&#46;J&#46; Heagerty"
                            4 => "K&#46; Tuttle"
                            5 => "N&#46;S&#46; Weiss"
                          ]
                        ]
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                    ]
                  ]
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                    0 => array:2 [
                      "doi" => "10.1001/jama.2016.10924"
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                            "url" => "https://www.ncbi.nlm.nih.gov/pubmed/27532915"
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                      "titulo" => "The global epidemiology of diabetes and kidney disease"
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        "texto" => "<p id="par0130" class="elsevierStylePara elsevierViewall">We would like to express our gratitude to all the patients who have participated&#44; as well as to all the staff of the Division of Nephrology of the Third Affiliated Hospital of Hebei Medical University for their collaboration and enthusiasm in this study&#46;</p>"
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