Diabetic Nephropathy Subjects Research Articles

Discovery of Free Glycated Amines and Glycated Urea in Diabetic Plasma: Potential Implications in Diabetes.

The role of protein glycation in the pathogenesis of diabetes has been well established. Akin to proteins, free amino acids and other small-molecule amines are also susceptible to glycation in hyperglycemic conditions and may have a role in the pathogenesis of the disease. However, information about glycation of free amino acids and other small-molecule amines is relatively obscure. In the quest to discover small-molecule glycated amines in the plasma, we have synthesized glycated amino acids, glycated creatine, and glycated urea, and by using a high-resolution accurate mass spectrometer, a mass spectral library was developed comprising the precursor and predominant fragment masses of glycated amines. Using this information, we report the discovery of the glycation of free lysine, arginine, and leucine/isoleucine from the plasma of diabetic patients. This has great physiological significance as glycation of these amino acids may create their deficiency and affect vital physiological processes such as protein synthesis, cell signaling, and insulin secretion. Also, these glycated amino acids could serve as potential markers of diabetes and its complications. While other amines, such as creatinine and urea, accumulate in the plasma and act as biomarkers of diabetic nephropathy. For the first time, we report the detection of glycated urea in diabetic plasma, which is confirmed by matching the precursor and fragment masses with the in vitro synthesized glycated urea by using 12C6 and 13C6-glucose. Further, we quantified glycated urea detected in two forms, monoglycated urea (MGU) and diglycated urea (DGU), by a targeted mass spectrometric approach in the plasma of healthy, diabetic, and diabetic nephropathy subjects. Both MGU and DGU showed a positive correlation with clinical parameters, such as blood glucose and HbA1c. Given that urea gets converted to glycated urea in hyperglycemic conditions, it is crucial to quantify MGU and DGU along with the urea for the diagnosis of diabetic nephropathy and study their physiological role in diabetes.

Angiotensin-Converting Enzyme Genetic Polymorphism rs4343 as Risk of Diabetic Nephropathy in Jambi-Malay Population

Background: Diabetic nephropathy (DN) is one of the frequent complications of type II diabetes mellitus (T2DM) in Jambi province. Controlling blood glucose and blood pressure does not guarantee DN prevention, since genetic factors may also contribute to this disease. Multi-ethnic studies showed that one of the strongest genetic factors associated with DN was single nucleotide polymorphism rs4343 of angiotensin-converting enzyme (ACE) gene. Study regarding phenotype-genotype association of ACE rs4343 and DN has not yet been performed in Jambi Province, which is dominated by Malay ethnicity. This study was conducted to reveal the association between ACE rs4343 and the risk of DN in the Jambi-Malay population.Materials and methods: This was a cross-sectional study involving 75 subjects (44 with DN and 31 without DN) who suffered from T2DM and hypertension. DN was defined as albumin to creatinine ratio (ACR) ≥30 mg/g. Genotyping was performed with one-step tetra amplification refractory mutation system-polymerase chain reaction (PCR) using specific primer for ACE rs4343. Bivariate and multivariate analyses were performed to analyze the genetic risk for DN.Results: The bivariate analysis showed the proportion of DN subjects was higher than non-DN within the AG genotype (11:1) than within the AA (33:30) genotype. This difference was statistically significant (p=0.012; OR (95% CI): 10.00 (1.22-82.15)). Multivariate analysis showed that AG genotype (p=0.047; OR (95% CI): 10.04 (1.03-97.31)) and uncontrolled blood pressure (p=0.001; OR (95% CI): 6.72 (2.08-21.71)) were the risk factors of DN in the Jambi-Malay population.Conclusion: Polymorphism of ACE rs4343 is a risk factor of DN in the Jambi-Malay Population.Keywords: rs4343, angiotensin-converting enzyme gene, diabetic nephropathy, Malay, Jambi

TRAIL and EGFR Pathways Targeting MicroRNAs are Predominantly Regulated in Human Diabetic Nephropathy.

Unbiased microRNA profiling of renal tissue and urinary extracellular vesicles (uEVs) from diabetic nephropathy (DN) subjects may unravel novel targets with diagnostic and therapeutic potential. Here we used the miRNA profile of uEVs and renal biopsies from DN subjects available on the GEO database. The miR expression profiles of kidney tissue (GSE51674) and urinary exosomes (GSE48318) from DN and control subjects were obtained by GEO2R tools from Gene Expression Omnibus (GEO) databases. Differentially expressed miRNAs in DN samples, relative to controls, were identified using a bioinformatic pipeline. Targets of miRs commonly regulated in both sample types were predicted by miRWalk, followed by functional gene enrichment analysis. Gene targets were identified by MiRTarBase, TargetScan and MiRDB. Eight miRs, including let-7c, miR-10a, miR-10b and miR-181c, were significantly regulated in kidney tissue and uEVs in DN subjects versus controls. The top 10 significant pathways targeted by these miRs included TRAIL, EGFR, Proteoglycan syndecan, VEGF and Integrin Pathway. Gene target analysis by miRwalk upon validation using ShinyGO 70 targets with significant miRNA-mRNA interaction. In silico analysis showed that miRs targeting TRAIL and EGFR signaling are predominately regulated in uEVs and renal tissue of DN subjects. After wet-lab validation, the identified miRs- target pairs may be explored for their diagnostic and/or therapeutic potential in diabetic nephropathy.

Alterations in the gut virome are associated with type 2 diabetes and diabetic nephropathy

ABSTRACT Although changes in gut microbiome have been associated with the development of T2D and its complications, the role of the gut virome remains largely unknown. Here, we characterized the gut virome alterations in T2D and its complications diabetic nephropathy (DN) by metagenomic sequencing of fecal viral-like particles. Compared with controls, T2D subjects, especially those with DN, had significantly lower viral richness and diversity. 81 viral species were identified to be significantly altered in T2D subjects, including a decrease in some phages (e.g. Flavobacterium phage and Cellulophaga phaga). DN subjects were depleted of 12 viral species, including Bacteroides phage, Anoxybacillus virus and Brevibacillus phage, and enriched in 2 phages (Shigella phage and Xylella phage). Multiple viral functions, particularly those of phage lysing host bacteria, were markedly reduced in T2D and DN. Strong viral-bacterial interactions in healthy controls were disrupted in both T2D and DN. Moreover, the combined use of gut viral and bacterial markers achieved a powerful diagnostic performance for T2D and DN, with AUC of 99.03% and 98.19%, respectively. Our results suggest that T2D and its complication DN are characterized by a significant decrease in gut viral diversity, changes in specific virus species, loss of multiple viral functions, and disruption of viral-bacterial correlations. The combined gut viral and bacterial markers have diagnostic potential for T2D and DN.

#2969 IS DIABETES MELLITUS ASSOCIATED WITH WORSE KIDNEY OUTCOME IN PATIENTS WITH GLOMERULOPATHIES?

Abstract Background and Aims Diabetes mellitus (DM) is the leading cause of end stage kidney disease and a major risk factor for cardio-vascular disease and death. While it is known that subjects with diabetic nephropathy (DN) have a worse kidney outcome compared to other non-diabetic glomerulopathies, it is not certain whether DM independently influences the kidney outcome in subjects diagnosed with glomerulopathies (GP) other than diabetic nephropathy. Method This retrospective study included 1200 adults [50 (95%CI 48 to 51) years, 56% males, eGFR 48.5 (95%CI 45.9 to 51.3) mL/min], with kidney biopsy (KB) proven GP between 1st Jan. 2008 and 31st Dec. 2017. Subjects were followed for a mean of 89 (95%CI 85.5 to 92.4) months until 31st May 2018. The primary endpoints were the initiation of chronic renal replacement therapy (RRT) or death. Subjects with inappropriate biopsy sample, insufficient data and RRT prior to KB were excluded. Demographic, clinical and laboratory data at the time of biopsy were retrieved from medical records. Kidney survival was evaluated by Kaplan-Meier method and a competitive risk to event analysis was used to estimate the risk of RRT, considering death as a competing event. Variables related to kidney outcome were evaluated by the subdistribution hazard function using Fine-Gray model. According to the presence or absence of DM and the types of GP, subjects were divided in three groups: GP without DM (n = 987 pts.), GP with DM (n = 65 pts.), and DN (n = 148 pts.). Results GP with DM subjects were older (60 (95%CI 56 to 64) years vs. 55.5 (95%CI 54 to 59) years in DN group vs. 47 (95%CI 45 to 48) years in GP without DM; p<0.001). Males were predominant in all groups, with higher frequency in DN group (66.9% vs. 54.9% - GP without DM vs. 61.5% - GP with DM; p = 0.01). DN group had higher Charlson comorbidity index [5 (95%CI 4 to 5) vs. 2 (95%CI 2 to 2) in GP without DM vs. 3 (95%CI 3 to 4) in GP with DM; p<0.001], lower eGFR (31.3 (95%CI 25.4 to 36.7)mL/min vs. 51.9 (95%CI 48.9 to 55.8)mL/min in GP without DM vs. 43.2 (95%CI 35.4 to 51.3) mL/min in GP with DM; p<0.001) and higher proteinuria (4.9 (95%CI 3.8 to 6) g/g vs. 2.6 (95%CI 2.35 to 2.9) g/g in GP without DM vs. 4.3 (95%CI 2.7 to 6.7) g/g in GP with DM); p<0.001]. During the follow-up period, 24.3% needed RRT, while 11.4% died. The highest RRT initiation and death frequency was in DN group (40.5% vs. 21.7% in GP without DM vs. 27.7% in GP with DM; p<0.001, and 18.2% vs. 10% in GP without DM vs. 19.9% in GP with DM; p = 0.004). In the univariate time-dependent analysis, subjects with DN had the worse kidney outcome (log rank p<0.001) (Fig. 1), however the kidney survival was similar between GP with DM and GP without DM subjects (log rank p = 0.1). In the competitive risk time to event analysis where death is considered the competing event, diabetic nephropathy subjects had worse kidney survival than GP with DM subjects [CIF 2.3 vs. 1.3, p<0.001], while GP with DM had similar survival with GP without DM (CIF 1,3; p = 0.2) (Fig. 2). After adjusting for the risk factors for CKD progression, DM was not associated with an increased risk of RRT (HR = 0.98, p = 0.9). Predictors for RRT were diabetic nephropathy on KB, along with younger age, lower serum albumin, and lower baseline eGFR. Conclusion In this large cohort of subjects with glomerulopathies, diabetes mellitus complicated with diabetic nephropathy seem to have a worse kidney outcome, while DM in subjects with glomerulopathies other than DN doesn't seem to influence the progression to RRT.

Therapeutic potential of urine exosomes derived from rats with diabetic kidney disease.

Kidney disease is prevalent in diabetes. Urinary exosomes (uE) from animal models and patients with Diabetic nephropathy (DN) showed increased levels of miRs with reno-protective potential. We examined whether urinary loss of such miRs is associated with their reduced renal levels in DN patients. We also tested whether injecting uE can leverage kidney disease in rats. In this study (study-1) we performed microarray profiling of miRNA in uE and renal tissues in DN patients and subjects with diabetes without DN (controls). In study-2, diabetes was induced in Wistar rats by Streptozotocin (i.p. 50 mg/kg of body weight). Urinary exosomes were collected at 6th, 7th and 8th weeks, and injected back into the rats (100ug/biweekly, uE-treated n=7) via tail vein on weeks 9 and 10. Equal volume of vehicle was injected in controls (vehicle, n=7). uE from the human and rat showed the presence of exosome-specific proteins by immunoblotting. Microarray profiling revealed a set of 15 miRs having high levels in the uE, while lower in renal biopsies, from DN, compared to controls (n=5-9/group). Bioinformatic analysis also confirmed the Renoprotective potential of these miRs. Taqman qPCR confirmed the opposite regulation of miR-200c-3p and miR-24-3p in paired uE and renal biopsy samples from DN patients (n=15), relative to non-DN controls. A rise in 28 miRs levels, including miR-200c-3p, miR-24-3p, miR-30a-3p and miR-23a-3p were observed in the uE of DN rats, collected between 6th-8th weeks, relative to baseline (before diabetes induction). uE- treated DN rats had significantly reduced urine albumin-to-creatinine ratio, attenuated renal pathology, and lower miR-24-3p target fibrotic/inflammatory genes (TGF-beta, and Collagen IV), relative to vehicle treated DN rats. In uE treated rats, the renal expression of miR-24-3p, miR-30a-3p, let-7a-5p and miR-23a-3p was increased, relative to vehicle control. Patients with diabetic nephropathy had reduced renal levels, while higher uE abundance of miRs with reno-protective potential. Reverting the urinary loss of miRs by injecting uE attenuated renal pathology in diabetic rats.

Role of serum free fatty acids in determining severity of diabetic nephropathy: A case control study

Introduction and Aim: The Diabetic Nephropathy (DN) is a result of impaired renal function in Type 2 diabetes. At the onset of diabetes, microvascular complication increases due to accumulation of free fatty acids (FFA) causing renal damage. A study was conducted to estimate the concentration of serum FFA causing severity of diabetic nephropathy.
 
 Materials and Methods: 90 Type 2 diabetic subjects and 30 study controls (age group 35 to 65 years) were selected from the medicine OPD of S N Medical College and HSK Hospital, Bagalkot. Based on the presence of microalbuminuria, the 90 Type 2 diabetic patients were equally divided in to 3 groups, named as stage I to stage III. The serum FFA was estimated by ELISA method in these three groups and control subjects. The statistical analysis was done using SPSS software version 19 utilizing unpaired “t” test for quantitative data and Pearson’s correlation tests.
 
 Results: The estimated serum FFA levels in stage I to III was found to be higher and highly significant as compared to control (p=0.001). We find the best cut off value of serum FFA was 4.75 mmol/L, causing severity of diabetic nephropathy. The area under the curve (AUC) is 0.92 with the specificity of 86%, sensitivity 89% and the diagnostic accuracy was found to be of 87%.
 
 Conclusion: Serum free fatty acid levels were higher in diabetic nephropathy subjects, which could be used as diagnostic marker for the severity of renal damage with cut off value of 4.75 mmol/L.

Expression of pro-inflammatory cytokines (IL-6 & IL-18) exacerbate the risk of diabetic nephropathy in the Pakistani population.

Diabetic nephropathy (DN) is a micro-chronic diabetic consequence induced by metabolic and hemodynamic abnormalities. Free radicals react with other critical cellular components, causing progression ofaberrant renal function. This case control study was aimed to determine the role of IL-6 and IL-18in diabetic nephropathy in Pakistani population. The study's subjects (n = 180 from Lahore, Gujranwala, and Karachi) were divided into control, diabetes mellitus(DM) and diabetic nephropathy (DN) groups. The serum concentration of IL-6 & IL-18 were determined by enzyme-linked immunosorbent assay (ELISA). The expression analysis of IL-6 & IL-18 were performed by Real Time PCR. The significant increase in serum levels of IL-6 were observed among the control, DM and DN groups (15.3 ± 24.1pg/ml, 34.7 ± 24.0pg/ml, 52.6 ± 33.2pg/ml) whereas no significant difference was observed in serum levels of IL-18. The expression analysis of IL-6 was increased by more than forty three fold in DN group (n-fold = ~43.6) as compared to DM & control whereas the expression profile of IL-18 decreased in DN group (n-fold = ~0.89). In DN group the correlation analysis revealed direct association of GFR with serum IL-6(r = 0.1114) & inverse relationship with serum IL-18(r = -0.097). In multiple regression analysis using GFR as the dependent variable, BMI and expression of IL-18 were determinants in DM subjects, but only uric acid in DN subjects. The present study implicates that increased expression of IL-6 and decreased of IL-18 was associated with development of DN in Pakistani population.

MiRNA-200b level in peripheral blood predicts renal interstitial injury in patients with diabetic nephropathy.

To uncover the diagnostic potential of peripheral blood microRNA-200b (miRNA-200b) in renal interstitial injury in diabetic nephropathy (DN) patients. A total of 50 diabetes subjects, 50 mild DN subjects, 50 moderate-severe DN subjects and 50 healthy subjects were included. Peripheral blood level of miRNA-200b in every subject was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Serum levels of renal function indicators were determined by enzyme-linked immunosorbent assay (ELISA). Meanwhile, relative levels of fibrosis damage indicators were examined by chemiluminescent immunoassay. Diagnostic potentials of miRNA200b in diabetes, mild DN and moderate-severe DN were assessed by depicting receiver operating characteristic (ROC) curves. Peripheral blood level of miRNA-200b was higher in DN subjects than diabetes subjects without vascular complications, especially moderate-severe DN patients. Peripheral blood level of miRNA-200b in DN subjects was negatively correlated to relative levels of serum creatinine, urinary nitrogen, cystatin, TGF-b, CIV and PCIII. ROC curves demonstrated diagnostic potentials of miRNA-200b in mild and moderate-severe DN. Peripheral blood level of miRNA-200b is closely linked to the degree of renal interstitial injury in DN patients. MiRNA-200b may be a vital indicator in predicting the development of DN.

Biomarkers for Detecting Kidney Dysfunction in Type-2 Diabetics and Diabetic Nephropathy Subjects: A Case-Control Study to Identify Potential Biomarkers of DN to Stratify Risk of Progression in T2D Patients

IntroductionCurrently there are no biomarkers that are predictive of when patients with type-2 diabetes (T2D) will progress to more serious kidney disease i.e., diabetic nephropathy (DN). Biomarkers that could identify patients at risk of progression would allow earlier, more aggressive treatment intervention and management, reducing patient morbidity and mortality.Materials and MethodsStudy participants (N=88; control n=26; T2D n=32; DN n=30) were recruited from the renal unit at Antrim Area Hospital, Antrim, UK; Whiteabbey Hospital Diabetic Clinic, Newtownabbey, UK; Ulster University (UU), Belfast, UK; and the University of the Third Age (U3A), Belfast, UK; between 2019 and 2020. Venous blood and urine were collected with a detailed clinical history for each study participant.ResultsIn total, 13/25 (52.0%) biomarkers measured in urine and 25/34 (73.5%) biomarkers measured in serum were identified as significantly different between control, T2D and DN participants. DN patients, were older, smoked more, had higher systolic blood pressure and higher serum creatinine levels and lower eGFR function. Serum biomarkers significantly inversely correlated with eGFR.ConclusionThis pilot-study identified several serum biomarkers that could be used to predict progression of T2D to more serious kidney disease: namely, midkine, sTNFR1 and 2, H-FABP and Cystatin C. Our results warrant confirmation in a longitudinal study using a larger patient cohort.

Quality of life in patients with diabetic nephropathy: findings from the KNOW-CKD (Korean Cohort Study for Outcomes in Patients with Chronic Kidney Disease) cohort.

BackgroundDiabetic nephropathy (DN) can affect quality of life (QoL) because it requires arduous lifelong management. This study analyzed QoL differences between DN patients and patients with other chronic kidney diseases (CKDs).MethodsThe analysis included subjects (n = 1,766) from the KNOW-CKD (Korean Cohort Study for Outcomes in Patients with Chronic Kidney Disease) cohort who completed the Kidney Disease Quality of Life Short Form questionnaire. After implementing propensity score matching (PSM) using factors that affect the QoL of DN patients, QoL differences between DN and non-DN participants were examined.ResultsAmong all DN patients (n = 390), higher QoL scores were found for taller subjects, and lower scores were found for those who were unemployed or unmarried, received Medical Aid, had lower economic status, had higher platelet counts or alkaline phosphatase levels, or used clopidogrel or insulin. After PSM, the 239 matched DN subjects reported significantly lower patient satisfaction (59.9 vs. 64.5, p = 0.02) and general health (35.3 vs. 39.1, p = 0.04) than the 239 non-DN subjects. Scores decreased in both groups during the 5-year follow-up, and the scores in the work status, sexual function, and role-physical domains were lower among DN patients than non-DN patients, though those differences were not statistically significant.ConclusionSocioeconomic factors of DN were strong risk factors for impaired QoL, as were high platelet, alkaline phosphatase, and clopidogrel and insulin use. Clinicians should keep in mind that the QoL of DN patients might decrease in some domains compared with non-DN CKDs.

Association of Fetuin-A with Thr256Ser exon polymorphism of α2-Heremans Schmid Glycoprotein (AHSG) gene in type 2 diabetic patients with overt nephropathy

BackgroundCirculatory Fetuin-A has been well reported to elevate the risk for Diabetic Nephropathy (DN) and is associated with many vascular complications. Compelling reports have well documented that the circulatory levels of Fetuin-A directly have an impact on its AHSG (α2- Heremans- Schmid Glycoprotein) gene single nucleotide polymorphisms (SNP). Thus, in this study among the South Indian T2DM population, we aim to explore the association of AHSG Thr256Ser (rs4918) SNP in subjects with DN and correlate with the circulatory levels of Fetuin-A at various stages of DN patients. MethodsA total of 975 subjects were recruited, such as Group-I, consisting of Controls (n = 300), Group-II, with normoalbuminuria (n = 300), Group-IIIa, with incipient microalbuminuria (n = 195), Group-IIIb, with persistent macroalbuminuria (n = 180)] and were subjected for genotyping using PCR-Restriction Fragment Length Polymorphism (RFLP). Circulatory Fetuin-A was measured using sandwich enzyme-linked immunosorbent assay (ELISA). ResultsThe ‘G’ allele of AHSG exon-7 (C/G) SNP is significantly concomitant and conferred significant risk for normoalbuminuria subjects. In the DN subjects, the ‘G' allele showed the risk for persistent macroalbuminuria. A noticeable stepwise decrease was evidenced in the circulatory Fetuin-A among persistent macroalbuminuria over incipient microalbuminuria from normoalbuminuria. Further, the circulatory Fetuin-A was lowered in DN subjects with mutant GG genotype than the wild CC. ConclusionAHSG Thr256Ser (rs4918) SNP was associated with renal complications among South Indian T2DM subjects.

POS-332 QUALITY OF LIFE IN PATIENTS WITH DIABETIC NEPHROPATHY: FINDINGS FROM THE KNOW-CKD (KOREAN COHORT STUDY FOR OUTCOME IN PATIENTS WITH CHRONIC KIDNEY DISEASE) COHORT

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, and can affect quality of life (QoL) because it requires arduous lifelong management. This study analyzed QoL differences at baseline and after 5 years between DN anDiabetic nephropathy (DN) is a major cause of end-stage renal disease, and can affect quality of life (QoL) because it requires arduous lifelong management. This study analyzed QoL differences at baseline and after 5 years between DN and non-DN patients with other chronic kidney disease (CKD).d non-DN patients with other chronic kidney disease (CKD). The analysis included subjects (n=1766) from the KNOW-CKD (KoreaN cohort study for Outcome in patients With Chronic Kidney Disease) cohort who completed the Kidney Disease Quality of Life Short Form (KDQOL-SF). The factors that influenced the QoL of participants with DN (n=390) were first analyzed, and differences in QoL between DN and non-DN participants was examined. To maintain homogeneity, most factors that influenced the QoL of participants with DN were controlled by propensity score-matched pair sampling using the greedy matching technique. In total, 239 DN and 239 non-DN subjects were finally selected, and differences in the mean KDQOL-SF scores between the 2 groups were then analyzed. In the multivariate linear regression model, higher QoL scores were found for taller DN subjects and lower QoL scores were found for those who were unemployed or unmarried, received Medical Aid, had lower economic status, had higher platelet counts and alkaline phosphatase levels, and used clopidogrel or insulin. Patient satisfaction (59.9 vs. 64.5, P=0.022) and general health (35.3 vs. 39.1, P=0.041) were significantly lower in the DN group than in the non-DN group. Scores generally decreased in both groups during the 5-year follow-up, and the scores in the work status, sexual function, and role-physical domains were lower among patients with DN than among non-DN patients, but the differences were not statistically significant. In conclusion, among the DN subjects, socioeconomic factors were found to be strong risk factors for impaired QoL, as well as high platelet counts, high alkaline phosphatase levels, and clopidogrel and insulin use. The DN subjects showed lower QoL than the non-DN subjects in the domains of patient satisfaction and general health. In conclusion, we confirmed that DN itself affected QoL more strongly than other types of CKD.

Clinical, histopathologic and molecular features of idiopathic and diabetic nodular mesangial sclerosis in humans.

Idiopathic nodular mesangial sclerosis, also called idiopathic nodular glomerulosclerosis (ING), is a rare clinical entity with an unclear pathogenesis. The hallmark of this disease is the presence of nodular mesangial sclerosis on histology without clinical evidence of diabetes mellitus or other predisposing diagnoses. To achieve insights into its pathogenesis, we queried the clinical, histopathologic and transcriptomic features of ING and nodular diabetic nephropathy (DN). All renal biopsy reports accessioned at Indiana University Health from 2001 to 2016 were reviewed to identify 48 ING cases. Clinical and histopathologic features were compared between individuals with ING and DN (n = 751). Glomeruli of ING (n = 5), DN (n = 18) and reference (REF) nephrectomy (n = 9) samples were isolated by laser microdissection and RNA was sequenced. Immunohistochemistry of proline-rich 36 (PRR36) protein was performed. ING subjects were frequently hypertensive (95.8%) with a smoking history (66.7%). ING subjects were older, had lower proteinuria and had less hyaline arteriolosclerosis than DN subjects. Butanoate metabolism was an enriched pathway in ING samples compared with either REF or DN samples. The top differentially expressed gene, PRR36, had increased expression in glomeruli 248-fold [false discovery rate (FDR) P = 5.93 × 10-6] compared with the REF and increased 109-fold (FDR P = 1.85 × 10-6) compared with DN samples. Immunohistochemistry revealed a reduced proportion of cells with perinuclear reaction in ING samples as compared to DN. Despite similar clinical and histopathologic characteristics in ING and DN, the uncovered transcriptomic signature suggests that ING has distinct molecular features from nodular DN. Further study is warranted to understand these relationships.

Association between serum bilirubin and albuminuria in type 2 diabetes mellitus and diabetic nephropathy

Introduction: Diabetic nephropathy develops due to oxidative stress and inflammation resulting from chronic hyperglycemia. Bilirubin, a product of heme catabolism is found to have antioxidant and anti-inflammatory properties. Though previous studies have examined the relationship between total bilirubin and diabetic nephropathy, very few studies have focused on indirect and direct bilirubin levels. Hence, the present study aimed to compare serum bilirubin (total, indirect and direct) levels between non-diabetics, type 2 diabetics and diabetic nephropathy subjects and also to correlate albuminuria with serum bilirubin in type 2 diabetics and diabetic nephropathy subjects. Materials and Methods: 50 non-diabetics, 50 type 2 diabetics and 50 diabetic nephropathy subjects were included in the study. Fasting blood glucose, HbA1C, serum bilirubin (total, indirect and direct), serum creatinine, urine microalbumin and urine creatinine were measured. Estimated glomerular filtration rate (eGFR) and urine albumin creatinine ratio (ACR) was calculated. Results: Total bilirubin, direct and indirect bilirubin were significantly decreased in type 2 diabetics and diabetic nephropathy subjects compared to non-diabetics. Total bilirubin and indirect bilirubin were also significantly decreased in diabetic nephropathy subjects compared to type 2 diabetics. Total bilirubin, direct and indirect bilirubin showed significant negative correlation with albuminuria (Urine ACR) in type 2 diabetics and diabetic nephropathy subjects. Conclusion: Our study suggests that low bilirubin levels might be a risk factor for the development of type 2 diabetes and diabetic nephropathy. Bilirubin can be used as a marker to predict the risk of developing type 2 diabetes among general population and diabetic nephropathy among type 2 diabetes patients. Keywords: Albuminuria, Antioxidant, Bilirubin, Diabetes mellitus, Diabetic nephropathy

Crosstalk between endoplasmic reticulum stress and oxidative stress in the progression of diabetic nephropathy.

Increasing evidence in substantiating the roles of endoplasmic reticulum stress, oxidative stress, and inflammatory responses and their interplay is evident in various diseases. However, an in-depth mechanistic understanding of the crosstalk between the intracellular stress signaling pathways and inflammatory responses and their participation in disease progression has not yet been explored. Progress has been made in our understanding of the cross talk and integrated stress signaling network between endoplasmic reticulum stress and oxidative stress towards the pathogenesis of diabetic nephropathy. In this present study, we studied the crosstalk between the endoplasmic reticulum stress and oxidative stress by understanding the role of protein disulfide isomerase and endoplasmic reticulum oxidase 1α, a key player in redox protein folding in the endoplasmic reticulum. We had recruited a total of 90 subjects and divided into three groups (control (n = 30), type 2 diabetes mellitus (n = 30), and diabetic nephropathy (n = 30)). We found that endoplasmic reticulum stress markers, activating transcription factor 6, inositol-requiring enzyme 1α, protein kinase RNA-like endoplasmic reticulum kinase, C/EBP homologous protein, and glucose-regulated protein-78; oxidative stress markers, thioredoxin-interacting protein and cytochrome b-245 light chain; and the crosstalk markers, protein disulfide isomerase and endoplasmic reticulum oxidase-1α, were progressively elevated in type 2 diabetes mellitus and diabetic nephropathy subjects. The association between the crosstalk markers showed a positive correlation with endoplasmic reticulum stress and oxidative stress markers. Further, the interplay between endoplasmic reticulum stress and oxidative stress was investigated in vitro using a human leukemic monocytic cell line under a hyperglycemic environment and examined the expression of protein disulfide isomerase and endoplasmic reticulum oxidase-1α. DCFH-DA assay and flow cytometry were performed to detect the production of free radicals. Further, phosphorylation of eIF2α in high glucose-exposed cells was studied using western blot. In conclusion, our results shed light on the crosstalk between endoplasmic reticulum stress and oxidative stress and significantly contribute to the onset and progression of diabetic nephropathy and therefore represent the major therapeutic targets for alleviating micro- and macrovascular complications associated with this metabolic disturbance. Graphical abstract.

CORRELATION BETWEEN OXIDATIVE STRESS AND ANTIOXIDANT IN DIABETIC NEPHROPATHY

Background: Diabetic Nephropathy is consider as one of the major micro-vascular problems of diabetes mellitus and has become the most general single factor of end stage of kidney disease. It is defined traditionally by kidney morphological and modification like: glomerular hyper filtration, glomerular and kidney hypertrophy, increased urinary albumin excretion (> 300 mg/24 hours), increased GBM (Glomerular Basement Membrane) thickness and mesangial expansion and also accumulation of extracellular proteins comprising laminin, collagens and fibronectin worldwide. Oxidative stress (OS) has been characterized as the imbalance between reactive oxygen species (ROS) yielding and the possessive antioxidant defense system.
 Objective of the study: Correlation between oxidative stress and antioxidant in diabetic nephropathy.
 Materials and methods: The investigation was conducted on 100 DN subjects of both sex and aged 20 or more and 100 age and sex matched healthy control subjects. MDA, SOD and Catalase of each subject was measured.
 Results: the present investigation shows that the MDA was elevated significantly and SOD and Catalase level was found to be significantly low in DN individuals as compared to controls. Conclusion: This study concluded that the MDA could be better marker for early recognition of DN.
 Keywords: Diabetic nephropathy (DN), MDA, SOD, Catalase, Kidney disease

Link between ACE I/D Gene Polymorphism and Dyslipidemia in Diabetic Nephropathy: A Case-control Study from Hyderabad, India.

Introduction:Diabetic nephropathy (DN) is the commonest single cause of end-stage renal failure, and dyslipidemia is a critical risk factor in the occurrence of DN. In the light of recent reports emphasizing the importance of angiotensin I-converting enzyme (ACE) in the modulation of plasma lipids, we sought to evaluate the influence of ACE I/D gene polymorphism with dyslipidemia status among type 2 diabetic (T2D) patients with and without nephropathy in the genetic predisposition and the progression to DN.Method:This study comprised of 600 subjects, which include patients with DN, T2D, and healthy controls (HC). Polymerase chain reaction based genotyping of ACE I/D polymorphism was performed and appropriate statistical analysis was done.Results:Out of the 600 subjects, 20 (10%) of the HC, 73 (36.5%) of the T2D group, and 125 (62.5%) of the DN subjects had dyslipidemia. The D allele (0.62) and DD (42.5) genotype frequencies were higher in the DN group in comparison with T2D and HC (P < 0.05). The genotypes also varied among patients with dyslipidemia (χ2 5.04; P < 0.05) but not in the non-dyslipidemia group. Under the co-dominant model, DD genotype conferred a risk of 1.26 (P < 0.001) toward DN, whereas the ID genotype offered protection from DN among the dyslipidemic subjects (OR = 0.05; P < 0.01). In addition, genotype-dependent difference was seen in the plasma lipid levels among study groups. A multiple logistic regression analysis revealed male gender, BMI, HbA1c, TG, HDL, and ACE DD genotype as independent risk factors for the development of DN.Conclusion:The study showed a significant predisposing association of ACE DD genotype with DN and protective effect of ID genotype on DN in the dyslipidemia subgroup.

Complement C5a inhibition moderates lipid metabolism and reduces tubulointerstitial fibrosis in diabetic nephropathy.

Complement C5 mediates pro-inflammatory responses in many immune-related renal diseases. Given that the C5a level is elevated in diabetes, we investigated whether activation of C5a/C5aR signalling plays a pathogenic role in diabetic nephropathy (DN) and the therapeutic potential of C5a inhibition for renal fibrosis. Human renal biopsies from patients with DN and control subjects were used for immunohistochemical staining of complement C5 components. Renal function and tubulointerstitial injury were compared between db/m mice, vehicle-treated mice and C5a inhibitor-treated db/db mice. A cell culture model of tubule epithelial cells (HK-2) was used to demonstrate the effect of C5a on the renal fibrotic pathway. Increased levels of C5a, but not of its receptor C5aR, were detected in renal tubules from patients with DN. The intensity of C5a staining was positively correlated with the progression of the disease. In db/db mice, administration of a novel C5a inhibitor, NOX-D21, reduced the serum triglyceride level and attenuated the upregulation of diacylglycerolacyltransferase-1 and sterol-regulatory element binding protein-1 expression and lipid accumulation in diabetic kidney. NOX-D21-treated diabetic mice also had reduced serum blood urea nitrogen and creatinine levels with less glomerular and tubulointerstitial damage. Renal transforming growth factor beta 1 (TGF-β1), fibronectin and collagen type I expressions were reduced by NOX-D21. In HK-2 cells, C5a stimulated TGF-β production through the activation of the PI3K/Akt signalling pathway. Blockade of C5a signalling by NOX-D21 moderates altered lipid metabolism in diabetes and improved tubulointerstitial fibrosis by reduction of lipid accumulation and TGF-β-driven fibrosis in diabetic kidney.

Increased levels of circulating (TNF-α) is associated with (-308G/A) promoter polymorphism of TNF-α gene in Diabetic Nephropathy

The crucial role of Tumor Necrosis Factor-α (TNF-α) on renal function in patients with Diabetic Nephropathy (DN) has been well documented. The present study was designed to investigate the association of TNF-α [-308G/A, (rs1800629)] single nucleotide polymorphism (SNP) on the susceptibility to DN subjects and to correlate it with the plasma levels of TNF-α along with circulatory TNF-α receptor super family cytokines (sTNFR-1 and sTNFR-2). A total of 756 subjects, were recruited and divided into groups [Group-I, Control (n=218), Group-II, Normoalbuminuria (n=196), Group-IIIa, Microalbuminuria (n=178), Group-IIIb, Macroalbuminuria (n=164)] and were genotyped by PCR-restriction fragment length polymorphism (RFLP). Circulatory levels of TNF-α and sTNFR-1 & sTNFR-2 were measured using multiplex bead based assay. The ‘A’ allele of TNF-α (-308 G/A) SNP was associated with a significant risk for macroalbuminuria subjects (OR: 2.1; 95% CI: 0.8–3.7; P<0.001). A marked stepwise increase was observed in the levels of circulatory biomarkers such as TNF-α, sTNF-R1 and sTNF-R2 from normo to macroalbuminuria subjects. In DN subjects, the TNF-α level was higher in individuals who had mutant AA, than the wild GG genotype of TNF-α gene. Our results conclude that rs1800629 polymorphism in TNF-α gene is associated with renal complications in T2DM subjects.