URINARY EXCRETION OF ALPHA-ACTININ-4 AND TIGHT JUNCTION PROTEIN 1 IN PATIENTS WITH TYPE 2 DIABETES AND DIFFERENT PATTERNS OF CHRONIC KIDNEY DISEASE.

Abnormalities of the cytoskeleton and the slit diaphragm of podocytes have been attributed to diabetic nephropathy. In this study, we assessed urinary excretion of alpha-actinin-4 (ACTN-4), a cytoskeleton protein and a component of the slit diaphragm, and tight junction protein 1 (TJP-1, or ZO-1), a peripheral membrane protein that forms molecular complexes with actin filaments, in patients with type 2 diabetes (T2D) and albuminuric or non-albuminuric chronic kidney disease (CKD). The study included 140 patients with long-term T2D (≥10 years) and 20 healthy subjects as control. Patterns of CKD were identified based on the estimated glomerular filtration rate (eGFR) and urinary albumin-to-creatinine ratio (UACR). Urinary ACTN-4 and TJP-1 were assessed by ELISA. Patients with T2D had increased urinary excretion of ACTN-4 (p=0.03) and TJP-1 (p=0.006). In logistic regression models, both ACTN-4 and TJP-1 demonstrated associations with albuminuric CKD (UACR ≥3.0 mg/mmol and eGFR <60 mL/min×1.73 m2) after adjusting to age, sex, diabetes duration, HbA1c, and smoking. In ROC-analysis, TJP-1 excretion ≥70 pg/mmol was associated with albuminuric CKD (OR 5.45, 95% CI 1.96-15.18, p=0.001). The results demonstrate that elevated urinary ACTN-4 and TJP-1 are associated specifically with albuminuric CKD, but not with non-albuminuric CKD, in T2D patients.

Study of Neutrophil Gelatinase Associated Lipocalin and Kidney Injury Molecule-1 in Patients with Type 2 Diabetes Mellitus Nephropathy.

BACKGROUND: There is a need for accurate and rapid biomarkers for the early diagnosis of diabetic nephropathy (DN). We aimed to study the accuracy of urinary neutrophil gelatinase-associated lipocalin (uNGAL), urinary kidney injury molecule-1 (uKIM-1), and blood NGAL (bNGAL) in type 2 diabetics as biomarkers for diagnosis of DN. METHODS: The study was a retrospective case-control study that included 30 control subjects, 40 diabetics with normo-albuminuria < 30 mg/g and eGFR > 60 mL/minute/1.73 m2, and 30 diabetics with albuminuria > 30mg/g and eGFR < 60mL/minute/1.73 m2. Blood and urine samples were obtained to determine levels of bNGAL, uNAGAL, and uKIM1. RESULTS: There was a significant increase in bNGAL, uNGAL, uKIM 1, uNGAL/creatinine and uKIM 1/creatinine among diabetics with albuminuria compared to diabetics with normoalbuminuria and normal control (p < 0.001 for all markers). For diagnosis of early DN, both bNGAL and uKIM 1 had sensitivity and specificity of 100% for each at cutoff values of 322.5 pg/mL and 74.25 ng/mL, respectively. uNGAL had a sensitivity of 97.5% and a spec-ificity of 100% at a cutoff point of 565 ng/mL. uKIM1/creatinine at a cutoff of 51.2 had a sensitivity of 100% and specificity of 100%. CONCLUSIONS: The present study highlights the accuracy of urinary KIM1 and NGAL and blood NGAL as biomarkers for the diagnosis of nephropathy in the early stage of diabetic nephropathy. There were positive correlations with kidney function tests creatinine, blood urea nitrogen, and the presence of albuminuria.

Soluble tumor necrosis factor receptor type 1 is an alternative marker of urinary albumin-creatinine ratio and estimated glomerular filtration rate for predicting the decline of renal function in subjects with type 2 diabetes mellitus.

BACKGROUND: Urinary albumin-creatinine ratio (UACR) and estimated glomerular filtration rates (eGFR) help predict worsening diabetic kidney disease (DKD) but have their limitations. Soluble tumor necrosis factor receptor type 1 (sTNFR1) is a biomarker of DKD. The predictive abilities of sTNFR1 and UACR plus eGFR have not been compared. METHODS: This prospective cohort study included patients with type 2 diabetes (T2D) to identify the risk factors of worsening DKD. Renal events were defined as > 30 % loss in eGFR based on consecutive tests after 6 months. The associations of sTNFR1, UACR, and eGFR levels and the risks of renal events were tested using a Cox regression model and the area under the curve (AUC) was compared between sTNFR1 levels and UACR plus eGFR using receiver-operating characteristic (ROC) analysis. The accuracy of stratification was evaluated using Kaplan-Meier analysis. RESULTS: Levels of sTNFR1 and UACR were associated with risks of > 30 % decline in eGFR after adjusting for relevant factors. The association between sTNFR1 levels and renal outcomes was independent of UACR and eGFR at baseline. The AUC of sTNFR1 level was comparable with that of combined UACR and eGFR (0.73 vs. 0.71, respectively, p = 0.72) and the results persisted for quartile groups of sTNFR1 and risk categories of Kidney Disease: Improving Global Outcomes (KDIGO) (0.70 vs. 0.71, respectively, p = 0.84). Both stratifications by sTNFR1 levels and KDIGO were accurate. CONCLUSION: sTNFR1 could be an alternative marker for identifying patients with diabetes at risk of declining renal function.

Screening candidate diagnostic biomarkers for diabetic kidney disease.

BACKGROUND: There are big differences in treatments and prognosis between diabetic kidney disease (DKD) and non-diabetic renal disease (NDRD). However, DKD patients couldn't be diagnosed early due to lack of special biomarkers. Urine is an ideal non-invasive sample for screening DKD biomarkers. This study aims to explore DKD special biomarkers by urinary proteomics. MATERIALS AND METHODS: According to the result of renal biopsy, 142 type 2 diabetes mellitus (T2DM) patients were divided into 2 groups: DKD (n = 83) and NDRD (n = 59). Ten patients were selected from each group to define urinary protein profiles by label-free quantitative proteomics. The candidate proteins were further verifyied by parallel reaction monitoring (PRM) methods (n = 40). Proteins which perform the same trend both in PRM and proteomics were verified by enzyme-linked immunosorbent assays (ELISA) with expanding the sample size (n = 82). The area under the receiver operating characteristic curve (AUC) was used to evaluate the accuracy of diagnostic biomarkers. RESULTS: We identified 417 peptides in urinary proteins showing significant difference between DKD and NDRD. PRM verification identified C7, SERPINA4, IGHG1, SEMG2, PGLS, GGT1, CDH2, CDH1 was consistent with the proteomic results and p < 0.05. Three potential biomarkers for DKD, C7, SERPINA4, and gGT1, were verified by ELISA. The combinatied SERPINA4/Ucr and gGT1/Ucr (AUC = 0.758, p = 0.001) displayed higher diagnostic efficiency than C7/Ucr (AUC = 0.632, p = 0.048), SERPINA4/Ucr (AUC = 0.661, p = 0.032), and gGT1/Ucr (AUC = 0.661, p = 0.029) respectively. CONCLUSIONS: The combined index SERPINA4/Ucr and gGT1/Ucr can be considered as candidate biomarkers for diabetic nephropathy after adjusting by urine creatinine.

RISK FACTORS FOR DIABETIC NEPHROPATHY IN DIABETES MELLITUS TYPE 1.

OBJECTIVE: The aim: To find the risk factors of microalbuminuria and estimated Glomerular Filtration Rate (eGFR) in patients with type 1 diabetes mellitus. PATIENTS AND METHODS: Materials and methods: One hundred ten patients of type 1 diabetes mellitus in this cross-sectional study at diabetic and endocrinology center in Al-Najaf during the period from September 2021 to March 2022. All patients were asked about sociodemographic characteristics (age, gender, smoking, duration of DM type1, family history of DM type1), measured (body mass index BMI, blood pressure) and laboratory investigations done to all patients (G.U.E, s. creatinine, lipid profile, HBA1C, calculated estimated Glomerular Filtration Rate (eGFR) and Spot Urine Albumin-Creatinine Ratio (ACR). RESULTS: Results: Out of 110 patients, 62 male and 48 female, the mean age was (22±12). The patients with microalbuminuria (ACR ≥ 30 mg/g) show statistically significant with increase HBA1C, duration of DM type 1, total cholesterol (T.C), low density lipoprotein (LDL), triglycerides (TG) and family history of DM type 1, while there were not statistically significant with age, gender, smoking, BMI, eGFR, high density lipoprotein (HDL) and hypertension. Patients with eGFR<90mL/min/1.73m2 show statistically significant with increase HBA1C, duration of DM type1, LDL, TG, T.C, while significantly decrease in HDL and there were not statistically significant with age, gender, smoking, family history of DM type 1, BMI and hypertension. CONCLUSION: Conclusions: The degree of glycemic control, duration of type1 (DM) and dyslipidemia were associated with increased microalbuminuria and reduced eGFR (nephropathy). Family history of DM type1 was risk factor for microalbuminuria.

[Association study of serum LncRNA MALAT1 and SAA with type 2 diabetic kidney disease].

To investigate the correlation of serum long noncoding RNA-metastasis associated lung adenocarcinoma transcript 1(LncRNA MALAT1) and serum amyloid A(SAA) with diabetic kidney disease. Retrospective research was used, and 40 patients with type 2 diabetes and 80 patients with type 2 diabetic kidney disease patients who were treated in Tianjin Medical University Chu Hsien-I Memorial Hospital from August 2021 to February 2022 were selected, and 40 healthy subjects were selected during the same period. Reverse transcription-polymerase chain reaction(RT-PCR) was used to detect serum LncRNA MALAT1. SAA were detected with enzyme linked immunosorbent assay (ELISA). Automatic biochemistry analyzer was used to detect serum creatinine (CREA) and low-density lipoprotein cholesterol(LDL-C),automatic blood glucose analyzer to detect serum fasting plasma glucose (FPG), automatic glycated hemoglobin analyzer to detect hemoglobin A1C (HbA1c), and automatic immunoassay analyzer to detect urinary albumin to creatinine ratio(UACR). Differences between groups were compared by t test and analysis of variance. Pearson analysis was used to analyze the correlation between MALAT1, SAA and other indicators. Receiver operating characteristic curve(ROC) was used to evaluate the auxiliary diagnostic value of MALAT1 and SAA for diabetic kidney disease. The results showed that MALAT1 and SAA in the diabetic kidney disease with mass albuminuria group were higher than those in the type 2 diabetes mellitus group (q=8.57, P<0.01; q=11.09, P<0.01) and the diabetic kidney disease with microalbuminuria group (q=3.96, P<0.05; q=7.85, P<0.01). MALAT1 had a high correlation with UACR, CREA, SAA, HbA1c and FPG (r value was 0.706, 0.643, 0.578, 0.553, and 0.524, all P<0.01), and SAA had a high correlation with UACR, HbA1c and FPG (r value was 0.664, 0.617, and 0.595, all P<0.01). ROC curve analysis of the diagnostic value of LncRNA MALAT1 and protein SAA for diabetic kidney disease showed that the areas under curve (AUC) were 0.741 and 0.744, respectively. The combined diagnostic value of the two was the greatest (AUC=0.801). In summary, MALAT1 and SAA were elevated in the serum of patients with type 2 diabetes. Their concentrations in the serum of group with diabetic kidney disease were higher than that in the type 2 diabetes group, and the serum concentrations of MALAT1 and SAA in group with mass albuminuria are higher than the group with microalbuminuria. MALAT1 and SAA were both closely related to UACR and HbA1c, and there is a correlation between them. Both of them may have ancillary diagnostic value for diabetic kidney disease.

Serum Trimethylamine N-oxide and the Diversity of the Intestinal Microbial Flora in Type 2 Diabetes Complicated by Diabetic Kidney Disease.

BACKGROUND: Trimethylamine N-oxide (TMAO) serves as a metabolite of intestinal bacteria as well as a urotoxin influencing the prognosis of chronic kidney disease (CKD), which has become a research hotspot in the field of kidney disease. This study preliminarily explored the alternations of the microbial flora and serum TMAO in patients with type 2 diabetes mellitus (T2DM) complicated with diabetic kidney disease (DKD). METHODS: Seventeen T2DM patients at the Affiliated Hospital of Zunyi Medical University between September 2018 and February 2019 were included. Among these patients, 8 patients had T2DM complicated with DKD. Eight healthy volunteers constituted the control group. Fresh stool was collected for Illumina sequencing. Based on the sequencing outcomes, the flora diversity and species differences were analyzed. Serum TMAO, cystatin C, urinary albumin/urine creatinine ratios (ACRs), and routine biochemical outcomes were also compared. RESULTS: The DKD group exhibited a significantly higher TMAO level than the remaining groups. The high-TMAO group had a significantly increased ACR level compared with the low-TMAO group. TMAO positively correlated with the ACR. Compared with the control group, the DKD group exhibited a decreased flora diversity. At the genus level, both the T2DM group and the DKD group showed decreased numbers of Alloprevotella and Megasphaera compared with the control group. The difference in Megasphaera between the DKD group and the control group was significant. CONCLUSIONS: The alternation of the intestinal microbial flora may participate in the development of DKD, and TMAO and chronic inflammation might be important factors for DKD development.

Urinary interleukin-9 in youth with type 1 diabetes mellitus.

AIMS: Interleukin-9 (IL-9) attenuates podocyte injury in experimental kidney disease, but its role in diabetic nephropathy is unknown. We sought to relate urinary IL-9 levels to the release of podocyte-derived extracellular vesicles (EVs) in youth with type 1 diabetes. We related urinary IL-9 levels to clinical variables and studied interactions between urinary IL-9, vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) on urinary albumin/creatinine ratio (ACR) a functional measure of podocyte injury. METHODS: We performed an analysis of urine samples and clinical data from a cohort of youth with type 1 diabetes (n = 53). Cytokines were measured using a Luminex platform (Eve Technologies), and nanoscale flow cytometry was employed to quantify urinary podocyte-derived EVs. All urinary measures were normalized to urinary creatinine. RESULTS: Mean age was 14.7 ± 1.6 years, and the mean time from diagnosis was 6.7 ± 2.9 years. Mean HbA1c was 70.3 ± 13.9 mmol/mol, mean ACR was 1.3 ± 1.9 mg/mmol, and mean eGFR was 140.3 ± 32.6 ml/min/1.73 m2. IL-9 was inversely related to podocyte EVs (r = - 0.56, p = 0.003). IL-9 was also inversely related to blood glucose, HbA1C and eGFR (r = - 0.44, p = 0.002; r = - 0.41, p = 0.003; r = - 0.49, p < 0.001, respectively) and positively correlated with systolic BP (r = 0.30, p = 0.04). There was a significant interaction between IL-9, EVs and ACR (p = 0.0143), and the relationship between IL-9 and ACR depended on VEGF (p = 0.0083), TNFα (p = 0.0231) and IL-6 levels (p = 0.0178). CONCLUSIONS: IL-9 is associated with podocyte injury in early type 1 diabetes, and there are complex interactions between urinary IL-9, inflammatory cytokines and ACR.

d-allulose protects against diabetic nephropathy progression in Otsuka Long-Evans Tokushima Fatty rats with type 2 diabetes.

d-allulose is a rare sugar that has been reported to possess anti-hyperglycemic effects. In the present study, we hypothesized that d-allulose is effective in attenuating the progression of diabetic nephropathy in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat model of type 2 diabetes mellitus. Drinking water with or without 3% d-allulose was administered to OLETF rats for 13 weeks. Long-Evans Tokushima Otsuka rats that received drinking water without d-allulose were used as non-diabetic control rats. d-allulose significantly attenuated the increase in blood glucose levels and progressive mesangial expansion in the glomerulus, which is regarded as a characteristic of diabetic nephropathy, in OLETF rats. d-allulose also attenuated the significant increases in renal IL-6 and tumor necrosis factor-α mRNA levels in OLETF rats, which is a proinflammatory parameter. Additionally, we showed that d-allulose suppresses mesangial matrix expansion, but its correlation with suppressing renal inflammation in OLETF rats should be investigated further. Collectively, our results support the hypothesis that d-allulose can prevent diabetic nephropathy in rats.

Urinary mRNA Signatures as Predictors of Renal Function Decline in Patients With Biopsy-Proven Diabetic Kidney Disease.

The clinical manifestations of diabetic kidney disease (DKD) are more heterogeneous than those previously reported, and these observations mandate the need for the recruitment of patients with biopsy-proven DKD in biomarker research. In this study, using the public gene expression omnibus (GEO) repository, we aimed to identify urinary mRNA biomarkers that can predict histological severity and disease progression in patients with DKD in whom the diagnosis and histologic grade has been confirmed by kidney biopsy. We identified 30 DKD-specific mRNA candidates based on the analysis of the GEO datasets. Among these, there were significant alterations in the urinary levels of 17 mRNAs in patients with DKD, compared with healthy controls. Four urinary mRNAs-LYZ, C3, FKBP5, and G6PC-reflected tubulointerstitial inflammation and fibrosis in kidney biopsy and could predict rapid progression to end-stage kidney disease independently of the baseline eGFR (tertile 1 vs. tertile 3; adjusted hazard ratio of 9.68 and 95% confidence interval of 2.85-32.87, p < 0.001). In conclusion, we demonstrated that urinary mRNA signatures have a potential to indicate the pathologic status and predict adverse renal outcomes in patients with DKD.

Dapagliflozin Reduces Urinary Albumin Excretion by Downregulating the Expression of cAMP, MAPK, and cGMP-PKG Signaling Pathways Associated Genes.

Objective: Diabetic nephropathy (DN), the most severe complication of diabetes mellitus, is characterized by albuminuria and progressive loss of kidney function. Dapagliflozin (DAP), a sodium-glucose cotransporter inhibitor, is an oral medication that improves blood glucose control in diabetic patients. However, the effects and mechanisms of DAP on DN remain unclear. Materials and Methods: The effect of DAP was based on a retrospective cohort study of patients who underwent 2-year surveillance, and the concentration of urine albumin-to-creatinine ratio, glomerular filtration rate, and serum creatinine were collected after treatment with DAP. To investigate the underlying mechanisms through which DAP reduces urinary albumin excretion, we used RNA-sequencing (RNA-seq) to analyze gene expression in human kidney 2 (HK-2) cells treated with DAP. Results: The retrospective cohort analysis indicated that DAP could reduce the excretion rate of urinary albumin in patients with type 2 diabetes and renal impairment. The results of the RNA-seq experiments showed 349 differentially expressed genes between DAP-treated HK-2 cells and control cells. Gene ontology annotation enrichment analysis showed that DAP mainly affected the expression of integral component of membrane- and cell junction-related genes, while the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that DAP primarily downregulated the expression of gene clusters associated with cyclic adenosine monophosphate, mitogen-activated protein kinase, and cyclic guanosine monophosphate-protein kinase G signaling pathways, which play critical roles in the progression of DN. Conclusion: Our results shed light on the mechanism by which DAP controls DN progression and provide a theoretical basis for the clinical treatment of DN.

Untargeted serum metabolomics and tryptophan metabolism profiling in type 2 diabetic patients with diabetic glomerulopathy.

Diabetic glomerulopathy (DG) remains the prevalent microvascular complication and leading cause of shortened lifespan in type-2 diabetes mellitus (T2DM) despite improvement in hyperglycemia control. Considering the pivotal role of kidney in metabolism, using untargeted metabolomic techniques to globally delineate the serum metabolite profiles will help advance understanding pathogenetic underpinnings of renal biopsy-confirmed DG from the perspective of metabolism specifically. Fourteen pathologically diagnosed DG patients secondary to T2DM and 14 age- and gender-matched healthy controls (HCs) were recruited for study. We employed mass spectrometry-based untargeted metabolomic methods to reveal the metabolite profiles of serum samples collected from all included subjects. We identified a total of 334 and 397 metabolites in positive and negative ion mode respectively. One hundred and eighty-two important differential metabolites whose variable importance in projection (VIP) > 1 and p value <0.05 were selected and annotated to metabolic pathways. KEGG pathway enrichment analysis revealed tryptophan metabolism enriched most significantly. Among the tryptophan derivatives, L-tryptophan (L-Trp) and serotonin were relatively accumulated in DGs compared with HCs, while 5-hydroxyindoleacetic acid (5-HIAA) and indole-3-acetamide were depleted. Correlation analysis showed serotonin and L-Trp are negatively correlated with 24 h urine protein and glycosylated hemoglobin (Ghb). To exclude the interference of preexisting T2DM on DG exacerbation, we selected 5-HIAA and 3-(3-hydroxyphenyl) propionic acid (3-OHPPA) which are not correlated with Ghb and analyzed their correlation relationship with crucial renal indices. We found 3-OHPPA is positively correlated with urine total protein and creatinine ratio (T/Cr) and 24 h urine protein, 5-HIAA is positively correlated with serum creatinine and urea.

Efficacy and safety of esaxerenone (CS-3150) in Japanese patients with type 2 diabetes and macroalbuminuria: a multicenter, single-arm, open-label phase III study.

BACKGROUND: Esaxerenone has potential renoprotective effects and reduces the urinary albumin-to-creatinine ratio (UACR) in patients with diabetic kidney disease and overt nephropathy. We investigated the efficacy and safety of esaxerenone in Japanese patients with type 2 diabetes (T2D) and macroalbuminuria (UACR ≥ 300 mg/g creatinine). METHODS: We conducted a multicenter, single-arm, open-label phase III study in 56 patients with T2D and UACR ≥ 300 mg/g creatinine with estimated glomerular filtration rate (eGFR) ≥ 30 mL/min/1.73 m2 and treated with a renin-angiotensin system inhibitor. Patients received esaxerenone for 28 weeks at 1.25 mg/day initially with titration to 2.5 mg/day based on serum potassium (K+) monitoring. Efficacy was evaluated as the change in UACR from baseline to week 28. Safety endpoints included adverse events (AEs), incidence of serum K+ increase, and change in eGFR from baseline. RESULTS: UACR decreased by 54.6% (95% CI 46.9%, 61.3%) on average from baseline (544.1 mg/g creatinine) to the end of treatment (246.8 mg/g creatinine); 51.8% of patients showed improvement to early nephropathy. AE incidence was 69.6%. Three patients (5.4%) had serum K+ levels ≥ 6.0 mEq/L or ≥ 5.5 mEq/L on two consecutive occasions. Hyperkalemia in two patients was transient and resolved during the treatment period. One patient discontinued following two consecutive serum K+ values ≥ 5.5 mEq/L. The maximum change from baseline in eGFR was - 8.3 mL/min/1.73 m2 at week 24. CONCLUSIONS: Esaxerenone reduced UACR in Japanese patients with T2D and UACR ≥ 300 mg/g creatinine; more than half experienced a transition from UACR ≥ 300 mg/g creatinine to UACR < 300 mg/g creatinine. CLINICAL TRIAL REGISTRATION: JapicCTI-173696.

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.

What can urinary exosomes tell us?

Exosomes are involved in a wide variety of biochemical processes in human body homeostasis. Exosomes also provide important information regarding communications among several organ systems. Additionally, they can serve as molecular vehicles to deliver drugs. Therefore, exosomes have received wide attention in current biomedical research for unraveling pathogenic mechanisms of diseases, searching for novel biomarkers, and discovering new drugs. This paper reviews and discusses the significance of urinary exosomes for a better understanding of human disease pathophysiology and their potential use as therapeutic targets. Isolation methods of exosomes and the latest technological advances are also discussed. Furthermore, novel urinary exosomal biomarkers are highlighted with special emphasis on their clinical applicability (particularly sensitivity, specificity, reliability, and other aspects). Finally, future trends for this field are analyzed and our perspectives are provided.

Significant association of urinary alpha-1-microglobulin compared to urinary neutrophil gelatinase-associated lipocalin with renal insufficiency in patients with type 2 diabetes.

AIM: Various studies have reported that urinary neutrophil gelatinase-associated lipocalin (NGAL), an indicator of tubular damage, may be an effective biomarker of renal impairment in patients with diabetes. This study aimed to compare the ability of urinary alpha-1-microglobulin (a traditional tubular damage marker) with NGAL for evaluating renal insufficiency in patients with type-2 diabetes. METHODS: Urinary albumin-to-creatinine ratio (ACR) and estimated glomerular filtration rate (eGFR) were used to determine whether 513 participants with type-2 diabetes had renal dysfunction. Urinary alpha-1-microglobulin-to-creatinine ratio (A1MCR) and NGAL-to-creatinine ratio (NCR) were calculated. RESULTS: Although both A1MCR and NCR were significantly higher among participants with renal insufficiency than among participants without renal damage, the difference in A1MCR values between participants with and without renal insufficiency was relatively greater than the difference in NCR values, especially among the male subjects. The correlation of ACR or eGFR with A1MCR was stronger than that of ACR or eGFR with NCR. A1MCR showed a good capability for detecting renal dysfunction (area under the curve = 0.80), its cut-off value was 14.82 mg/g, corresponding to 71.4% sensitivity and 73.1% specificity. The diagnostic efficiency of A1MCR was significantly higher than that of NCR. CONCLUSION: The results indicated that the traditional tubular damage marker A1MCR was more significantly associated with renal insufficiency defined by ACR and/or eGFR and may have a higher diagnostic efficiency compared with the efficiency of NCR in patients with type-2 diabetes.

Urinary Extracellular Vesicles as a Source of NGAL for Diabetic Kidney Disease Evaluation in Children and Adolescents With Type 1 Diabetes Mellitus.

Background: Tubular damage has a role in Diabetic Kidney Disease (DKD). We evaluated the early tubulointerstitial damage biomarkers in type-1 Diabetes Mellitus (T1DM) pediatric participants and studied the correlation with classical DKD parameters. Methods: Thirty-four T1DM and fifteen healthy participants were enrolled. Clinical and biochemical parameters [Glomerular filtration Rate (GFR), microalbuminuria (MAU), albumin/creatinine ratio (ACR), and glycated hemoglobin A1c (HbA1c)] were evaluated. Neutrophil gelatinase-associated lipocalin (NGAL), Hypoxia-inducible Factor-1α (HIF-1α), and Nuclear Factor of Activated T-cells-5 (NFAT5) levels were studied in the supernatant (S) and the exosome-like extracellular vesicles (E) fraction from urine samples. Results: In the T1DM, 12% had MAU >20 mg/L, 6% ACR >30 mg/g, and 88% had eGFR >140 ml/min/1.72 m2. NGAL in the S (NGAL-S) or E (NGAL-E) fraction was not detectable in the control. The NGAL-E was more frequent (p = 0.040) and higher (p = 0.002) than NGAL-S in T1DM. The T1DM participants with positive NGAL had higher age (p = 0.03), T1DM evolution (p = 0.03), and serum creatinine (p = 0.003) than negative NGAL. The NGAL-E correlated positively with tanner stage (p = 0.0036), the median levels of HbA1c before enrollment (p = 0.045) and was independent of ACR, MAU, and HbA1c at the enrollment. NFAT5 and HIF-1α levels were not detectable in T1DM or control. Conclusion: Urinary exosome-like extracellular vesicles could be a new source of early detection of tubular injury biomarkers of DKD in T1DM patients.

Mechanism-based modeling of the effect of a novel inhibitor of vascular adhesion protein-1 on albuminuria and renal function markers in patients with diabetic kidney disease.

The vascular adhesion protein-1 (VAP-1) inhibitor ASP8232 reduces albuminuria in patients with type 2 diabetes and chronic kidney disease. A mechanism-based model was developed to quantify the effects of ASP8232 on renal markers from a placebo-controlled Phase 2 study in diabetic kidney disease with 12 weeks of ASP8232 treatment. The model incorporated the available pharmacokinetic, pharmacodynamic (plasma VAP-1 concentration and activity), serum and urine creatinine, serum cystatin C, albumin excretion rate, urinary albumin-to-creatinine ratio, and urine volume information in an integrated manner. Drug-independent time-varying changes and different drug effects could be quantified for these markers using the model. Through simulations, this model provided the opportunity to dissect the relationship and longitudinal association between the estimated glomerular filtration rate and albuminuria and to quantify the pharmacological effects of ASP8232. The developed drug-independent model may be useful as a starting point for other compounds affecting the same biomarkers in a similar time scale.

Serum myeloid-related protein 8/14 is a potential predictor of diabetic kidney disease.

BACKGROUND: Myeloid-related protein 8/14 (MRP8/14) is secreted by macrophages and formed by MRP8 and MRP14, which is closely related to vascular inflammation. Chronic vascular inflammation plays a significant role in the development and progression of diabetic kidney disease (DKD). This study aims to investigate the relationship between MRP8/14 and DKD. METHODS: A total of 80 individuals with type 2 diabetes were divided into four groups, according to the baseline urinary albumin/creatinine ratio (ACR) levels Serum concentrations of MRP8/14 were measured by ELISA. The clinical variables were obtained through physical examination, illness history, or laboratory evidence. RESULTS: As DKD worsened, the level of serum MRP8/14 increased gradually, and MRP8/14 has a significantly positive correlation with ACR (r = 0.349, P = 0.002), body mass index (BMI) (r = 0.288, P = 0.009), serum creatinine (Cre) (r = 0.392, P < 0.001), blood urine nitrogen (BUN) (r = 0.333, P = 0.003), systolic blood pressure (SBP) (r = 0.301, P = 0.007), and a negative correlation with the estimated glomerular filtration rate (eGFR) (r = -0.478, P < 0.001). Logistic regression analysis showed that age, Cre, eGFR, ACR, and MRP8/14 were associated with the progression of DKD (P < 0.05). CONCLUSIONS: The serum MRP8/14 is correlated significantly with the progression of DKD, suggesting that MRP8/14 may be an independent predictor of the progression of DKD.

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.