Biomarker Profiling with Targeted Metabolomic Analysis of Plasma and Urine Samples in Patients with Type 2 Diabetes Mellitus and Early Diabetic Kidney Disease.

Background: Over the years, it was noticed that patients with diabetes have reached an alarming number worldwide. Diabetes presents many complications, including diabetic kidney disease (DKD), which can be considered the leading cause of end-stage renal disease. Current biomarkers such as serum creatinine and albuminuria have limitations for early detection of DKD. Methods: In our study, we used UHPLC-QTOF-ESI+-MS techniques to quantify previously analyzed metabolites. Based on one-way ANOVA and Fisher's LSD, untargeted analysis allowed the discrimination of six metabolites between subgroups P1 versus P2 and P3: tryptophan, kynurenic acid, taurine, l-acetylcarnitine, glycine, and tiglylglycine. Results: Our results showed several metabolites that exhibited significant differences among the patient groups and can be considered putative biomarkers in early DKD, including glycine and kynurenic acid in serum (p < 0.001) and tryptophan and tiglylglycine (p < 0.001) in urine. Conclusions: Although we identified metabolites as potential biomarkers in the present study, additional studies are needed to validate these results.

Mitochondrial DNA and Inflammation Are Associated with Cerebral Vessel Remodeling and Early Diabetic Kidney Disease in Patients with Type 2 Diabetes Mellitus.

Cerebrovascular disease accounts for major neurologic disabilities in patients with type 2 diabetes mellitus (DM). A potential association of mitochondrial DNA (mtDNA) and inflammation with cerebral vessel remodeling in patients with type 2 DM was evaluated. A cohort of 150 patients and 30 healthy controls were assessed concerning urinary albumin/creatinine ratio (UACR), synaptopodin, podocalyxin, kidney injury molecule-1 (KIM-1), N-acetyl-ß-(D)-glucosaminidase (NAG), interleukins IL-17A, IL-18, IL-10, tumor necrosis factor-alpha (TNFα), intercellular adhesion molecule-1 (ICAM-1). MtDNA-CN and nuclear DNA (nDNA) were quantified in peripheral blood and urine by qRT-PCR. Cytochrome b (CYTB) gene, subunit 2 of NADH dehydrogenase (ND2), and beta 2 microglobulin nuclear gene (B2M) were assessed by TaqMan assays. mtDNA-CN was defined as the ratio of the number of mtDNA/nDNA copies, through analysis of the CYTB/B2M and ND2/B2M ratio; cerebral Doppler ultrasound: intima-media thickness (IMT)-the common carotid arteries (CCAs), the pulsatility index (PI) and resistivity index (RI)- the internal carotid arteries (ICAs) and middle cerebral arteries (MCAs), the breath-holding index (BHI). The results showed direct correlations of CCAs-IMT, PI-ICAs, PI-MCAs, RI-ICAs, RI-MCAs with urinary mtDNA, IL-17A, IL-18, TNFα, ICAM-1, UACR, synaptopodin, podocalyxin, KIM-1, NAG, and indirect correlations with serum mtDNA, IL-10. BHI correlated directly with serum IL-10, and serum mtDNA, and negatively with serum IL-17A, serum ICAM-1, and NAG. In neurologically asymptomatic patients with type 2 DM cerebrovascular remodeling and impaired cerebrovascular reactivity may be associated with mtDNA variations and inflammation from the early stages of diabetic kidney disease.

Metabolites Potentially Derived from Gut Microbiota Associated with Podocyte, Proximal Tubule, and Renal and Cerebrovascular Endothelial Damage in Early Diabetic Kidney Disease in T2DM Patients.

Complications due to type 2 diabetes mellitus (T2DM) such as diabetic kidney disease (DKD) and cerebral small vessel disease (CSVD) have a powerful impact on mortality and morbidity. Our current diagnostic markers have become outdated as T2DM-related complications continue to develop. The aim of the investigation was to point out the relationship between previously selected metabolites which are potentially derived from gut microbiota and indicators of endothelial, proximal tubule (PT), and podocyte dysfunction, and neurosonological indices. The study participants were 20 healthy controls and 90 T2DM patients divided into three stages: normoalbuminuria, microalbuminuria, and macroalbuminuria. Serum and urine metabolites were determined by untargeted and targeted metabolomic techniques. The markers of endothelial, PT and podocyte dysfunction were assessed by ELISA technique, and the neurosonological indices were provided by an ultrasound device with high resolution (MYLAB 8-ESAOTE Italy). The descriptive statistical analysis was followed by univariable and multivariable linear regression analyses. In conclusion, in serum, arginine (sArg), butenoylcarnitine (sBCA), and indoxyl sulfate (sIS) expressed a biomarker potential in terms of renal endothelial dysfunction and carotid atherosclerosis, whereas sorbitol (sSorb) may be a potential biomarker of blood-brain barrier (BBB) dysfunction. In urine, BCA and IS were associated with markers of podocyte damage, whereas PCS correlated with markers of PT dysfunction.

Cancer before and after the start of hemodialysis and association with mortality - an Eastern-European multicenter study.

INTRODUCTION: East-European data on cancer in patients undergoing hemodialysis (HD) are scarce. This study aimed to assess the pattern of cancer and related mortality in patients with end-stage kidney disease (ESKD) undergoing HD. METHODS: Retrospectively analyzing data from 7 HD centers, this study examined 1377 incident HD patients divided into three groups: no-cancers (NoC), cancers that occurred prior to HD initiation (CPI) and de novo cancer developed after HD initiation (DNC). Mortality risk and survival trends within groups were analyzed using Cox regression and Kaplan-Meier methods. RESULTS: In the cohort, 89.46% of the patients had no cancer (NoC group), 3.63% had cancer before (CPI group), and 6.89% had cancer after HD initiation (DNC group). The mean time from HD initiation to DNC diagnosis was 1 [2.75] years. Older age was associated with a higher risk of developing DNC (p < 0.001). Chronic tubulointerstitial nephritis (CTIN) is more prevalent in cancer patients. The most common cancer sites among DNC patients were the digestive (29.47%) and urinary tracts (18.95%), while those in CPI subjects were hematologic (22%) and digestive (20%). Cancer was an independent predictor of mortality risk (HR = 6.9, 95% [CI]:4.5-10.6, p < 0.001). CONCLUSIONS: East-European ESKD patients undergoing HD have a high incidence of de novo cancers whose primary cancer sites are the digestive and urinary tracts. Almost half of the HD patients with CPI have hematologic and digestive tract cancers. Age and CTIN were associated with cancer risk. Cancer is an independent risk factor for all-cause mortality in patients undergoing hemodialysis (HD).

Quantitative, Targeted Analysis of Gut Microbiota Derived Metabolites Provides Novel Biomarkers of Early Diabetic Kidney Disease in Type 2 Diabetes Mellitus Patients.

Diabetic kidney disease (DKD) is one of the most debilitating complications of type 2 diabetes mellitus (T2DM), as it progresses silently to end-stage renal disease (ESRD). The discovery of novel biomarkers of early DKD becomes acute, as its incidence is reaching catastrophic proportions. Our study aimed to quantify previously identified metabolites from serum and urine through untargeted ultra-high-performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry (UHPLC-QTOF-ESI+-MS) techniques, such as the following: arginine, dimethylarginine, hippuric acid, indoxyl sulfate, p-cresyl sulfate, L-acetylcarnitine, butenoylcarnitine and sorbitol. The study concept was based on the targeted analysis of selected metabolites, using the serum and urine of 20 healthy subjects and 90 T2DM patients with DKD in different stages (normoalbuminuria-uACR < 30 mg/g; microalbuminuria-uACR 30-300 mg/g; macroalbuminuria-uACR > 300 mg/g). The quantitative evaluation of metabolites was performed with pure standards, followed by the validation methods such as the limit of detection (LOD) and the limit of quantification (LOQ). The following metabolites from this study resulted as possible biomarkers of early DKD: in serum-arginine, dimethylarginine, hippuric acid, indoxyl sulfate, butenoylcarnitine and sorbitol and in urine-p-cresyl sulfate.

Metabolomic Investigation of Blood and Urinary Amino Acids and Derivatives in Patients with Type 2 Diabetes Mellitus and Early Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease; however, few biomarkers of its early identification are available. The aim of the study was to assess new biomarkers in the early stages of DKD in type 2 diabetes mellitus (DM) patients. This cross-sectional pilot study performed an integrated metabolomic profiling of blood and urine in 90 patients with type 2 DM, classified into three subgroups according to albuminuria stage from P1 to P3 (30 normo-, 30 micro-, and 30 macroalbuminuric) and 20 healthy controls using high-performance liquid chromatography and mass spectrometry (UPLC-QTOF-ESI* MS). From a large cohort of separated and identified molecules, 33 and 39 amino acids and derivatives from serum and urine, respectively, were selected for statistical analysis using Metaboanalyst 5.0. online software. The multivariate and univariate algorithms confirmed the relevance of some amino acids and derivatives as biomarkers that are responsible for the discrimination between healthy controls and DKD patients. Serum molecules such as tiglylglycine, methoxytryptophan, serotonin sulfate, 5-hydroxy lysine, taurine, kynurenic acid, and tyrosine were found to be more significant in the discrimination between group C and subgroups P1-P2-P3. In urine, o-phosphothreonine, aspartic acid, 5-hydroxy lysine, uric acid, methoxytryptophan, were among the most relevant metabolites in the discrimination between group C and DKD group, as well between subgroups P1-P2-P3. The identification of these potential biomarkers may indicate their involvement in the early DKD and 2DM progression, reflecting kidney injury at specific sites along the nephron, even in the early stages of DKD.

Mitochondrial DNA Changes in Blood and Urine Display a Specific Signature in Relation to Inflammation in Normoalbuminuric Diabetic Kidney Disease in Type 2 Diabetes Mellitus Patients.

Mitochondrial dysfunction is an important mechanism contributing to the development and progression of diabetic kidney disease (DKD). Mitochondrial DNA (mtDNA) levels in blood and urine were evaluated in relation to podocyte injury and proximal tubule (PT) dysfunction, as well as to a specific inflammatory response in normoalbuminuric DKD. A total of 150 type 2 diabetes mellitus (DM) patients (52 normoalbuminuric, 48 microalbuminuric, and 50 macroalbuminuric ones, respectively) and 30 healthy controls were assessed concerning the urinary albumin/creatinine ratio (UACR), biomarkers of podocyte damage (synaptopodin and podocalyxin), PT dysfunction (kidney injury molecule-1 (KIM-1) and N-acetyl-ß-(D)-glucosaminidase (NAG)), and inflammation (serum and urinary interleukins (IL-17A, IL-18, and IL-10)). MtDNA-CN and nuclear DNA (nDNA) were quantified in peripheral blood and urine via qRT-PCR. MtDNA-CN was defined as the ratio of the number of mtDNA/nDNA copies via analysis of the CYTB/B2M and ND2/B2M ratio. Multivariable regression analysis provided models in which serum mtDNA directly correlated with IL-10 and indirectly correlated with UACR, IL-17A, and KIM-1 (R2 = 0.626; p < 0.0001). Urinary mtDNA directly correlated with UACR, podocalyxin, IL-18, and NAG, and negatively correlated with eGFR and IL-10 (R2 = 0.631; p < 0.0001). Mitochondrial DNA changes in serum and urine display a specific signature in relation to inflammation both at the podocyte and tubular levels in normoalbuminuric type 2 DM patients.

Untargeted Metabolomics by Ultra-High-Performance Liquid Chromatography Coupled with Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometry Analysis Identifies a Specific Metabolomic Profile in Patients with Early Chronic Kidney Disease.

Chronic kidney disease (CKD) has emerged as one of the most progressive diseases with increased mortality and morbidity. Metabolomics offers new insights into CKD pathogenesis and the discovery of new biomarkers for the early diagnosis of CKD. The aim of this cross-sectional study was to assess metabolomic profiling of serum and urine samples obtained from CKD patients. Untargeted metabolomics followed by multivariate and univariate analysis of blood and urine samples from 88 patients with CKD, staged by estimated glomerular filtration rate (eGFR), and 20 healthy control subjects was performed using ultra-high-performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry. Serum levels of Oleoyl glycine, alpha-lipoic acid, Propylthiouracil, and L-cysteine correlated directly with eGFR. Negative correlations were observed between serum 5-Hydroxyindoleacetic acid, Phenylalanine, Pyridoxamine, Cysteinyl glycine, Propenoylcarnitine, Uridine, and All-trans retinoic acid levels and eGFR. In urine samples, the majority of molecules were increased in patients with advanced CKD as compared with early CKD patients and controls. Amino acids, antioxidants, uremic toxins, acylcarnitines, and tryptophane metabolites were found in all CKD stages. Their dual variations in serum and urine may explain their impact on both glomerular and tubular structures, even in the early stages of CKD. Patients with CKD display a specific metabolomic profile. Since this paper represents a pilot study, future research is needed to confirm our findings that metabolites can serve as indicators of early CKD.

Metabolite Profiling of the Gut-Renal-Cerebral Axis Reveals a Particular Pattern in Early Diabetic Kidney Disease in T2DM Patients.

Type 2 diabetes mellitus (T2DM) represents an important microvascular disease concerning the kidney and the brain. Gut dysbiosis and microbiota-derived metabolites may be in relation with early pathophysiological changes in diabetic kidney disease (DKD). The aim of the study was to find new potential gut-derived biomarkers involved in the pathogenesis of early DKD, with a focus on the complex interconnection of these biomarkers with podocyte injury, proximal tubule dysfunction, renal and cerebrovascular endothelial dysfunction. The study design consisted of metabolite profiling of serum and urine of 90 T2DM patients (subgroups P1-normoalbuminuria, P2-microalbuminuria, P3-macroalbuminuria) and 20 healthy controls (group C), based on ultra-high-performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry analysis (UHPLC-QTOF-ESI+-MS). By multivariate and univariate analyses of serum and urine, which included Partial Least Squares Discriminant Analysis (PLSDA), Variable Importance Plots (VIP), Random Forest scores, One Way ANOVA and Biomarker analysis, there were discovered metabolites belonging to nitrogen metabolic pathway and retinoic acid signaling pathway which differentiate P1 group from P2, P3, C groups. Tyrosine, phenylalanine, indoxyl sulfate, serotonin sulfate, and all-trans retinoic acid express the metabolic fingerprint of P1 group vs. P2, P3, C groups, revealing a particular pattern in early DKD in T2DM patients.

Acute Kidney Injury after Endoscopic Retrograde Cholangiopancreatography-A Hospital-Based Prospective Observational Study.

Background: Endoscopic retrograde cholangiopancreatography (ERCP) represents a major pivotal point in gastrointestinal endoscopy. Little is known about acute kidney injury (AKI) post-ERCP. This study analyses the incidence, risk factors, and prognosis of post-ERCP AKI. Methods: A total of 396 patients were prospectively studied. AKI was defined by an increase in serum creatinine (SCr) ≥ 0.3 mg/dL or by an increase in SCr ≥ 50% in the first 48 h post-ERCP. Logistic regression analysis was used to identify the predictors of AKI and in-hospital mortality. A two-tailed p value < 0.05 was considered significant. Results: One hundred and three patients (26%) developed post-ERCP AKI. Estimated glomerular filtration rate (adjusted odds ratio (aOR) = 0.95, 95% confidence interval (CI): 0.94−0.96, p < 0.001), nonrenal Charlson Comorbidity Index (Aor = 1.19, 95% CI: 1.05−1.35, p = 0.006), choledocholithiasis (aOR = 4.05, 95% CI: 1.98−8.29, p < 0.001), and bilirubin (aOR = 1.1, 95% CI: 1.05−1.15, p < 0.001) were associated with post-ERCP AKI. Post-ERCP AKI was associated with longer hospital stay (p < 0.001) and with increased in-hospital mortality (7.76% versus 0.36%, p < 0.001). Moderate-to-severe (stage 2 and 3) AKI was independently associated with in-hospital mortality (aOR = 6.43, 95% CI: 1.48−27.88, p < 0.013). Conclusions: Post-ERCP AKI represented an important complication associated with longer hospital stay. Moderate-to-severe post-ERCP AKI was an independent risk factor for in-hospital mortality.

Long noncoding RNAs may impact podocytes and proximal tubule function through modulating miRNAs expression in Early Diabetic Kidney Disease of Type 2 Diabetes Mellitus patients.

Aims: Long noncoding RNAs (lncRNAs) play key roles in the pathophysiology of DKD involving actions of microRNAs (miRNAs). The aims of the study were to establish the involvement of selected lncRNAs in the epigenetic mechanisms of podocyte damage and tubular injury in DKD of type 2 diabetes mellitus (DM) patients in relation to a particular miRNAs profile. Methods: A total of 136 patients with type 2 DM and 25 healthy subjects were assessed in a cross-sectional study concerning urinary albumin: creatinine ratio (UACR), eGFR, biomarkers of podocyte damage (synaptopodin, podocalyxin) and of proximal tubule (PT) dysfunction (Kidney injury molecule-1-KIM-1, N-acetyl-D-glucosaminidase-NAG), urinary lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), nuclear-enriched abundant transcript 1 (NEAT1), myocardial infarction-associated transcript (MIAT), taurine-upregulated gene 1 (TUG1), urinary miRNA21, 124, 93, 29a. Results: Multivariable regression analysis showed that urinary lncMALAT1 correlated directly with urinary synaptopodin, podocalyxin, KIM-1, NAG, miRNA21, 124, UACR, and negatively with eGFR, miRNA93, 29a (p<0.0001; R2=0.727); urinary lncNEAT1 correlated directly with synaptopodin, KIM-1, NAG, miRNA21, 124, and negatively with eGFR, miRNA93, 29a (p<0.0001; R2=0.702); urinary lncMIAT correlated directly with miRNA93 and 29a, eGFR (p<0.0001; R2=0.671) and negatively with synaptopodin, KIM-1, NAG, UACR, miRNA21, 124 (p<0.0001; R2=0.654); urinary lncTUG1 correlated directly with eGFR, miRNA93, 29a, and negatively with synaptopodin, podocalyxin, NAG, miRNA21, 124 (p<0.0001; R2=0.748). Conclusions: In patients with type 2 DM lncRNAs exert either deleterious or protective functions within glomeruli and PT. LncRNAs may contribute to DKD through modulating miRNAs expression and activities. This observation holds true independently of albuminuria and DKD stage.

Urinary proteins detected using modern proteomics intervene in early type 2 diabetic kidney disease - a pilot study.

Aim: An advanced proteomics platform for protein biomarker discovery in diabetic chronic kidney disease (DKD) was developed, validated and implemented. Materials & methods: Three Type 2 diabetes mellitus patients and three control subjects were enrolled. Urinary peptides were extracted, samples were analyzed on a hybrid LTQ-Orbitrap Velos Pro instrument. Raw data were searched using the SEQUEST algorithm and integrated into Proteome Discoverer platform. Results & discussion: Unique peptide sequences, resulted sequence coverage, scoring of peptide spectrum matches were reported to albuminuria and databases. Five proteins that can be associated with early DKD were found: apolipoprotein AI, neutrophil gelatinase-associated lipocalin, cytidine deaminase, S100-A8 and hemoglobin subunit delta. Conclusion: Urinary proteome analysis could be used to evaluate mechanisms of pathogenesis of DKD.

MiRNA Expression is Associated with Clinical Variables Related to Vascular Remodeling in the Kidney and the Brain in Type 2 Diabetes Mellitus Patients.

Background: The association of vascular remodeling in the kidney and the brain with a particular microRNAs (miRNA) profile is not well studied.Methods: Seventy-six patients with Type 2 diabetes and 11 healthy subjects were assessed concerning urine albumin: creatinine ratio (UACR), biomarkers of podocyte injury and of proximal tubule (PT) dysfunction. MiRNA were quantified in blood and urine by a real-time PCR System. Cerebrovascular ultrasound measurements were performed in the carotid and middle cerebral arteries.Results: MiRNA21 and miRNA124 correlated positively with nephrin, podocalyxin, synaptopodin, urinary N-acetyl-D-glucosaminidase (NAG), urinary kidney-injury molecule-1 (KIM-1), UACR, and negatively with eGFR; miRNA125a, 126, 146a, 192 correlated negatively with nephrin, podocalyxin, synaptopodin, urinary NAG, urinary KIM-1, UACR, and directly with eGFR. Plasma miRNA-21 and miRNA192 correlated directly with cerebral hemodynamics parameters of atherosclerosis and arteriosclerosis. MiRNA-124, 125a, 126, 146a showed negative correlations with the same parameters.Conclusions: In Type 2 diabetes patients there is an association of vascular remodeling in the brain and the kidney with a specific miRNAs pattern. Cerebrovascular changes occur even in normoalbuminuric patients, with 'high-to-normal' levels of podocyte injury and PT dysfunction biomarkers. These phenomena may be explained by the variability of miRNA expression within the two organs in early DKD.

Pro-inflammatory cytokines are associated with podocyte damage and proximal tubular dysfunction in the early stage of diabetic kidney disease in type 2 diabetes mellitus patients.

AIMS: To evaluate if there is a link between inflammation (expressed by inflammatory cytokines) and the early stage of diabetic kidney disease (DKD), as shown by markers of podocyte damage and proximal tubular (PT) dysfunction. METHODS: In this study were enrolled 117 type 2 DM patients (36-normoalbuminuria, 42-microalbuminuria, 39- macroalbuminuria), and 11 healthy subjects. Serum and urinary IL-1 alpha, IL-8, IL-18, urinary albumin:creatinine ratio (UACR), eGFR, biomarkers of podocyte damage (podocalyxin, synaptopodin, nephrin) and of PT dysfunction (KIM-1, NAG) were assessed. RESULTS: In multivariable regression urinary Il-1 alpha correlated positively with podocalyxin and NAG (p < 0.0001, R2= 0.57); urinary IL-8 correlated directly with synaptopodin, NAG, nephrin, and KIM-1 (p < 0.0001, R2 = 0.67); urinary IL-18 correlated directly with synaptopodin, NAG, and nephrin (p < 0.0001, R2 = 0.59). Serum IL-1 alpha correlated positively with nephrin, synaptopodin, NAG (P < 0.0001, R2 = 0.68); serum IL-8 correlated directly with synaptopodin and NAG (p < 0.0001, R2 = 0.66); serum IL-18 correlated directly with NAG, KIM-1, and podocalyxin (p < 0.0001, R2=0.647). CONCLUSIONS: Pro-inflammatory interleukins are associated with podocyte injury and PT dysfunction in early DKD. These could exert a key role in the pathogenesis of early DKD, before the development of albuminuria.

Interleukins and miRNAs intervene in the early stages of diabetic kidney disease in Type 2 diabetes mellitus patients.

Aim: The involvement of proinflammatory interleukins (IL) in diabetic kidney disease of Type 2 diabetes mellitus (DM) patients was studied in relation to a particular miRNA profile. Materials & methods: A total of 117 patients with Type 2 DM and 11 controls were enrolled in a case series study. Serum and urinary ILs and miRNAs were assessed. Results: IL-1α correlated with miRNA21, 124, estimated glomerular filtration rate (eGFR) and negatively with miRNA125a and 192; IL-8 with miRNA21, 124, eGFR and negatively with miRNA125a, 126 and 146a; IL-18 with miRNA21, 124 and negatively with miRNA146a, 192, eGFR. Conclusion: There is an association between specific serum and urinary ILs and serum and urinary miRNAs profiles in the inflammatory response in Type 2 DM patients with diabetic kidney disease.

Deregulated profiles of urinary microRNAs may explain podocyte injury and proximal tubule dysfunction in normoalbuminuric patients with type 2 diabetes mellitus.

MicroRNAs (miRNAs) are short non-coding RNA species that are important post-transcriptional regulators of gene expression. The aim of the study was to establish a potential explanation of podocyte damage and proximal tubule (PT) dysfunction induced by deregulated miRNAs expression in the course of type 2 diabetes mellitus (DM). A total of 68 patients with type 2 DM and 11 healthy subjects were enrolled in a cross-sectional study and assessed concerning urinary albumin:creatinine ratio (UACR), urinary N-acetyl-ß-D-glucosamininidase (NAG), urinary kidney injury molecule-1, urinary nephrin, podocalyxin, synaptopodin, estimated glomerular filtration rate (eGFR), urinary miRNA21, miRNA124, and miRNA192. In univariable regression analysis, miRNA21, miRNA124, and miRNA192 correlated with urinary nephrin, synaptopodin, podocalyxin, NAG, KIM-1, UACR, and eGFR. Multivariable regression analysis yielded models in which miRNA192 correlated with synaptopodin, uNAG, and eGFR (R2=0.902; P<0.0001), miRNA124 correlated with synaptopodin, uNAG, UACR, and eGFR (R2=0.881; P<0.0001), whereas miRNA21 correlated with podocalyxin, uNAG, UACR, and eGFR (R2=0.882; P<0.0001). Urinary miRNA192 expression was downregulated, while urinary miRNA21 and miRNA124 expressions were upregulated. In patients with type 2 DM, there is an association between podocyte injury and PT dysfunction, and miRNA excretion, even in the normoalbuminuria stage. This observation documents a potential role of the urinary profiles of miRNA21, miRNA124, and miRNA192 in early DN. Despite their variability across the segments of the nephron, urinary miRNAs may be considered as a reliable tool for the identification of novel biomarkers in order to characterize the genetic pattern of podocyte damage and PT dysfunction in early DN of type 2 DM.

Urinary podocyte-associated mRNA levels correlate with proximal tubule dysfunction in early diabetic nephropathy of type 2 diabetes mellitus.

AIM: The study assessed mRNA expression of podocyte-associated molecules in urinary sediments of patients with type 2 diabetes mellitus (DM) in relation to urinary podocytes, biomarkers of podocyte injury and of proximal tubule (PT) dysfunction. METHODS: A total of 76 patients with type 2 DM and 20 healthy subjects were enrolled in a cross-sectional study, and assessed concerning urinary podocytes, urinary mRNA of podocyte-associated genes, urinary biomarkers of podocyte damage and of PT dysfunction. RESULTS: We found significant differences between urinary mRNA of podocyte-associated molecules in relation with albuminuria stage. In multivariable regression analysis, urinary mRNA of nephrin, podocin, alpha-actinin-4, CD2-associated protein, glomerular epithelial protein 1 (GLEPP1), ADAM 10, and NFκB correlated directly with urinary podocytes, albuminuria, urinary alpha1-microglobulin, urinary kidney-injury molecule-1, nephrinuria, urinary vascular endothelial growth factor, urinary advanced glycation end-products (AGE), and indirectly with eGFR (p < 0.0001, R2 = 0.808; p < 0.0001, R2 = 0.825; p < 0.0001, R2 = 0.805; p < 0.0001, R2 = 0.663; p < 0.0001, R2 = 0.726; p < 0.0001, R2 = 0.720; p < 0.0001, R2 = 0.724). CONCLUSIONS: In patients with type 2 DM there is an association between urinary mRNA of podocyte-associated molecules, biomarkers of podocyte damage, and of PT dysfunction. GLEPP1, ADAM10, and NFκB may be considered additional candidate molecules indicative of early diabetic nephropathy. AGE could be involved in this association.

Glycated peptides are associated with proximal tubule dysfunction in type 2 diabetes mellitus.

BACKGROUND: Advanced glycation end-products have been involved in the pathogenesis of proximal tubule dysfunction which characterizes diabetic tubulopathy. METHODS: A total of 76 Type 2 diabetes mellitus patients and 28 healthy controls were evaluated concerning a potential association of glycated peptides with proximal tubule dysfunction by assessing urine albumin:creatinine ratio, urinary alpha1-microglobulin, urinary neutrophil gelatinase-associated lipocalin, plasma and urinary advanced glycation end-products, plasma asymmetric dimethyl-arginine, serum cystatin C. Fully automated chip-nanoelectrospray ionization and high-capacity ion trap multistage mass spectrometry characterized the urinary proteomic profile. RESULTS: The urinary glycated proteins displayed a molecular weight of 15,121.4 Da in normoalbuminuric patients and of 30,180.4 Da in microalbuminuric patients. Urinary alpha1-microglobulin and neutrophil gelatinase-associated lipocalin correlated with urinary advanced glycation end-products (R(2)=0.586; R(2)=0.415), urine albumin: creatinine ratio (R(2)=0.292; R(2)=0.116), estimated glomerular filtration rate (R(2)=0.172; R(2)=0.135), serum cystatin C (R(2)=0.146; R(2)=0.129), but not with asymmetric dimethyl-arginine. In multivariable regression analysis models, the correlations for urinary alpha1-microglobulin and neutrophil gelatinase-associated lipocalin remained significant with urine albumin: creatinine ratio, urinary advanced glycation end-products, estimated glomerular filtration rate (P<0.0001, R(2)=0.674; P<0.0001, R(2)=0.551; P<0.0001, R(2)=0.482). CONCLUSIONS: In patients with Type 2 diabetes mellitus urinary glycated peptides are associated with proximal tubule dysfunction. The proteomic patterns of urinary glycated peptides could differentiate normo- from microalbuminuric patients and may explain a potential relation between the size and the glycation status of glycated peptides, and the extent of proximal tubule dysfunction. The lack of correlation between parameters of endothelial dysfunction and proximal tubule dysfunction cannot exclude glomerular involvement in early diabetic nephropathy.

Proximal tubule dysfunction is associated with podocyte damage biomarkers nephrin and vascular endothelial growth factor in type 2 diabetes mellitus patients: a cross-sectional study.

BACKGROUND: There is an ongoing debate as to whether early diabetic nephropathy in Type 2 diabetes mellitus may be attributed to the glomerulus or to the proximal tubule. Urinary excretion of nephrin and vascular endothelial growth factor may increase even in the normoalbuminuria stage. In the course of diabetic nephropathy, the proximal tubule may be involved in the uptake of urinary nephrin and vascular endothelial growth factor. MATERIALS AND METHODS: Two groups of consecutive Type 2 diabetes mellitus outpatients (38 normo-, 32 microalbuminuric) and 21 healthy subjects were enrolled in a cross-sectional study and evaluated concerning the relation of proximal tubule dysfunction with the podocyte biomarkers excretion, assessed by ELISA methods. The impact of advanced glycation end-products on this relation was also queried. RESULTS: Urinary alpha1-microglobulin and kidney injury molecule-1 correlated with urinary albumin:creatinine ratio (R2 = 0.269; p < 0.001; R2 = 0.125; p < 0.001), nephrinuria (R2 = 0.529; p<0.001; R2 = 0.203; p < 0.001), urinary vascular endothelial growth factor (R2 = 0.709; p < 0.001; R2 = 0.360; p < 0.001), urinary advanced glycation end-products (R2 = 0.578; p < 0.001; R2 = 0.405; p < 0.001), serum cystatin C (R2 = 0.130; p < 0.001; R2 = 0.128; p<0.001), and glomerular filtration rate (R2 = 0.167; p < 0.001; R2 = 0.166; p < 0.001); nephrinuria and urinary vascular endothelial growth factor correlated with urinary albumin:creatinine ratio (R2 = 0.498; p < 0.001; R2 = 0.227; p<0.001), urinary advanced glycation end-products (R2 = 0.251; p < 0.001; R2 = 0.308; p < 0.001), serum cystatin C (R2 = 0.157; p < 0.001; R2 = 0.226; p < 0.001), and glomerular filtration rate (R2 = 0.087; p = 0.007; R2 = 0.218; p < 0.001). CONCLUSIONS: In Type 2 diabetes mellitus there is an association of proximal tubule dysfunction with podocyte damage biomarkers, even in the normoalbuminuria stage. This observation suggests a potential role of the proximal tubule in urinary nephrin and urinary vascular endothelial growth factor processing in early diabetic nephropathy, a fact which could be related to advanced glycation end-products intervention. Podocyte damage and proximal tubule dysfunction biomarkers could be validated as a practical approach to the diagnosis of early diabetic nephropathy by further studies on larger cohorts.

Pioglitazone delays proximal tubule dysfunction and improves cerebral vessel endothelial dysfunction in normoalbuminuric people with type 2 diabetes mellitus.

AIM: The renal and cerebral protective effects of pioglitazone were assessed in normoalbuminuric patients with type 2 diabetes mellitus (DM). METHODS: A total of 68 normoalbuminuric type 2 DM patients were enrolled in a one-year open-label randomized controlled trial: 34 patients (pioglitazone-metformin) vs. 34 patients (glimepiride-metformin). All patients were assessed concerning urinary albumin: creatinine ratio (UACR), urinary alpha1-microglobulin, urinary beta2-microglobulin, plasma asymmetric dymethyl-arginine (ADMA), GFR, hsC-reactive protein, fibrinogen, HbA1c; pulsatility index, resistance index in the internal carotid artery and middle cerebral artery, intima-media thickness in the common carotid artery; cerebrovascular reactivity was evaluated through the breath-holding test. RESULTS: At 1 year there were differences between groups regarding ADMA, urinary beta2-microglobulin, urinary alpha1-microglobulin, parameters of inflammation, serum creatinine, GFR, UACR, the cerebral haemodynamic indices. Significant correlations were found between alpha 1-microglobulin-UACR (R(2)=0.143; P=0.001) and GFR (R(2)=0.081; P=0.01); beta2-microglobulin-UACR (R(2)=0.241; P=0.0001) and GFR (R(2)=0.064; P=0.036); ADMA-GFR (R(2)=0.338; P=0.0001), parameters of inflammation, HbA1c, duration of DM, cerebral indices. There were no correlations between ADMA-UACR, urinary alpha1-microglobulin and beta2-microglobulin. CONCLUSION: Proximal tubule (PT) dysfunction precedes albuminuria and is dissociated from endothelial dysfunction in patients with type 2 DM. Pioglitazone delays PT dysfunction and improves cerebral vessels endothelial dysfunction in normoalbuminuric patients with type 2 DM.