DN Patients Research Articles

MO609: Intrarenal TNF-α Expression in The Development of Diabetic Nephropathy

Abstract BACKGROUND AND AIMS Diabetic nephropathy (DN) develops in 40%–60% of people with type 2 diabetes mellitus (DM) and is characterized by a progressive and persistent decline in renal function, being the main cause of end-stage renal disease worldwide. TNF-α plays an important role in the development of inflammation, can lead to the destruction of connective tissue, release of lytic enzymes, has a direct effect on the processes of tissue regeneration, changing the expression of collagen types I, III and V. Also, TNF-α stimulates an increase in the synthesis of endothelin-1 by glomerular mesangial cells and has a direct cytotoxic effect on renal tissue, and increased cytokine production induces the processes of apoptosis and necrosis of glomerular cells. The aim of this work was to study the role of intrarenal TNF-α expression in the development of morphological changes in renal tissue in DN patients. METHOD We examined 50 patients with type 2 diabetes (mean age 66.58 ± 3.27 years). The duration of DM is 17.69 ± 0.35 years, and the duration of DN from the moment of detection of albuminuria to morphological examination of the renal tissue and diagnosis is 1.63 ± 0.34 years. Light and immunofluorescence microscopy of biopsy specimens of renal tissue of patients was carried out. Morphological changes were assessed in accordance with the Pathologic Classification of Diabetic Nephropathy (2010). According to light microscopy, 12 patients had class IIa (mild mesangial expansion), 13 patients had class IIb (severe mesangial expansion), 19 patients had class III (Kimmelstiel–Wilson nodular lesions) and 5 patients had class IV (extended diabetic glomerulosclerosis). In all patients, TNF-α expression in the glomerulus and in the interstitium was determined by immunofluorescence microscopy. RESULTS Correlation analysis showed the effect of TNF-α expression in the glomerulus in class IIa on the development of segmental sclerosis of the glomeruli (P = 0.04). A decrease in TNF-α expression in the glomerulus is accompanied by periglomerular sclerosis (P = 0.012), and also increases: thickening of the glomerular basement membrane (P = 0.03), expansion of the mesangium passes from soft to severe and affects more than 25% of the mesangial matrix (IIb stage) (P = 0.033), Kimmelstiel–Wilson nodular lesions are formed (P = 0.009). The interstitial expression of TNF-α remains high in the epithelium of the urinary tubules and in the interstitial cells of the kidney, regardless of the DN class and influences the development of tubulointerstitial fibrosis (P = 0.003). In order to clarify the prognostic significance of TNF-α and its role in the progression of DN, a linear regression analysis was carried out with the calculation of the coefficients of determination R2 (R Square) and using the Durban–Watson test and analysis of variance (ANOVA "Analysis of Variance") using the F criterion and calculating the standardized beta coefficient (ß) with a 95% confidence interval. It has been shown that glomerular expression of TNF-α affects the following: (i) progression of the expansion of the mesangial matrix (F = 4.817, P = 0.033; β = –0.311; CI –0.506 to –0.22, P = 0.033); (ii) the formation of Kimmelstiel–Wilson nodular lesions (F = 5.383, P = 0.025; β = –0.327; CI –1.375 to –0.97, P = 0.025). Interstitial expression of TNF-α contributes to the following: (i) the progression of arterial hyalinosis (F = 4.349, P = 0.043; β = 0.297; CI 0.009–0.497, P = 0.043); (ii) interstitial sclerosis (F = 9.541, P = 0.003; β = 0.422; CI 0.101–0.479, P = 0.003). CONCLUSION

MO624: The Relationship Between Renal Tubular Epithelial Cell Necroptosis and The Severity of Diabetic Nephropathy

Abstract BACKGROUND AND AIMS Diabetic nephropathy (DN), a common complication of diabetic patients, usually contributes to end-stage renal failure. Accidental cell death (ACD) and regulated cell death (RCD) cause renal cell loss and deteriorate renal function during the progression of DN. MLKL is the executor of necroptosis, a major form of RCD. The multimer of phosphorylated MLKL (p-MLKL) gather at the cell membrane and form the leaking pores, performing the last step of necroptosis. This study aims to study the relationship of p-MLKL expression and the disease severity in DN patients, providing promising treatment target. METHOD A total of 30 patients who diagnosed with DN clinical-pathologically in the Peking University First Hospital from January 1998 to August 2018 were enrolled (excluding DN with primary or secondary glomerular diseases, DN combined with thrombotic microangiopathies and small vasculitis). p-MLKL were stained on renal biopsy kidney sections by immunohistochemistry. Image-Pro Plus 6.0 software (IPP) was used to analyze the expression of p-MLKL on 10 randomly taken images for each patient. The relationship between p-MLKL expression and clinical-pathological parameters was explored. Clinical parameters of plasma creatinine, peak plasma creatinine during renal biopsy period, estimated glomerular filtration rate (eGFR) were used. DN pathological changes contained: (i) glomerular lesions, (ii) arteriole lesions (iii) tubular lesions and (iv) tubules chronic interstitial lesions. RESULTS Compared with normal control, the positive expression of p-MLKL could be detected in the renal tubular interstitium of patients with DN. However, no p-MLKL positive signal was seen in the glomeruli. Patients with p-MLKL positive signals accounted for 73.33% (22/30) of the enrolled patients. Among the 30 patients, the value of p-MLKL kidney tissue IOD (integrated optical density) was positively correlated with peak serum creatinine (r = 0.373, P = 0.042), the pathological score of spherical sclerosis (r = 0.364, P = 0.048) and tubular brush border (TBB) loss (r = 0.363, P = 0.049). CONCLUSION The expression of necroptosis marker p-MLKL was significantly correlated with the clinical-pathological changes of DN patients. Its role in the progression of DN deserves further exploration.

Impact of klotho on the expression of SRGAP2a in podocytes in diabetic nephropathy

BackgroundDiabetic nephropathy (DN) is the major cause of kidney failure, and glomerular podocytes play critical roles in the pathogenesis of DN by maintaining the glomerular structure and filtration barrier. Klotho and Slit-Robo GTP activating protein 2a (SRGAP2a) have been indicated to play protective roles in reducing kidney injury, but whether there is an internal relationship between these two factors is unclear.MethodsIn this study, we cultured differentiated rat podocytes in vitro and measured the SRGAP2a expressions by immunofluorescence staining, quantitative real-time PCR (qRT-PCR) and western blotting, after siRNA-mediated transforming growth factor β1 (TGF-β1) silencing, TGF-β1 overexpression and in the presence of a reactive oxygen species (ROS) inhibitor. And we detected the expressions of SRGAP2a, small mother against decapentaplegic (Smad)2/3, phosphorylated-Smad2/3 (p-Smad2/3), Smad7, and NAD(P)H oxidase 4 (NOX4), ROS levels and podocyte cytoskeletal remodelling under high glucose (HG) and exogenous klotho conditions. In addition, we performed haematoxylin–eosin (HE) staining and immunohistochemistry with diabetic rat models to confirm the in vitro results.ResultsThe results indicated that SRGAP2a expression was significantly upregulated under siRNA-mediated TGF-β1 silencing conditions or after adding a ROS inhibitor, but significantly downregulated with TGF-β1 overexpression, in the presence of HG. The supplementation of exogenous klotho under HG conditions significantly increased the SRGAP2a expression, remodelled the actin cytoskeleton and altered the expressions of Smad2/3, p-Smad2/3, Smad7 and NOX4 and reduced the ROS generation in podocytes. Moreover, klotho administration protected kidney injury in DN rats.ConclusionsThis study indicated that klotho may modulate the expression of SRGAP2a by regulating the ROS and TGF-β1 signalling pathways and provided theoretical support for klotho protein as a novel therapeutic strategy for treating DN patients.

Deranged Haematological Profile and Dyslipidaemiain Diabetes Induced Nephropathy

Background: Diabetic nephropathy caused by complication of diabetes mellitus and frequently increasing in the developed countries. It may be characterized by dyslipidaemia and deranged haematological indices that play a role in the development of late complications of diabetes. Therefore, there is an urgent need to develop strategies to diagnose the patients of diabetic nephropathy during its acute phase.
 Objective: To determine the haematological indices and the lipid profiles in diabetic nephropathy in comparison with diabetic controls
 Methodology: The present study was performed on total of 300 patients where 150 DN patients and 150 control with age range from 30-50 years. The haematological profile was done via ABX Micros 60 haematology analyser, analysis of lipids, fats and fatty substances lipid panel was also performed in both groups. Statistical analysis was done with SPSS through t-test where p≥ 0.05.
 Results: Current data indicated that the level of Haemoglobin A1c, Prothrombin time, White blood cells, platelet count, Low density lipoproteins and triglyceride is raised while significant reductions in red blood cell count and high-density lipoprotein levels have been noted in patients with diabetic nephropathy compared to diabetics.
 Conclusion: Our research indicates that both haematological and lipid profiles are highly disturbed in DN patients which may serve as a hallmark for early diagnosis of nephropathy in diabetics.

Computational Integration of Renal Histology and Urinary Proteomics using Neural Networks.

Histological image data and molecular profiles provide context into renal condition. Often, a biopsy is drawn to diagnose or monitor a suspected kidney problem. However, molecular profiles can go beyond a pathologist's ability to see and diagnose. Using AI, we computationally incorporated urinary proteomic profiles with microstructural morphology from renal biopsy to investigate new and existing molecular links to image phenotypes. We studied whole slide images of periodic acid-Schiff stained renal biopsies from 56 DN patients matched with 2,038 proteins measured from each patient's urine. Using Seurat, we identified differentially expressed proteins in patients that developed end-stage renal disease within 2 years of biopsy. Glomeruli, globally sclerotic glomeruli, and tubules were segmented from WSI using our previously published HAIL pipeline. For each glomerulus, 315 handcrafted digital image features were measured, and for tubules, 207 features. We trained fully connected networks to predict urinary protein measurements that were differentially expressed between patients who did/ did not progress to ESRD within 2 years of biopsy. The input to this network was either glomerular or tubular histomorphological features in biopsy. Trained network weights were used as a proxy to rank which morphological features correlated most highly with specific urinary proteins. We identified significant image feature-protein pairs by ranking network weights by magnitude. We also looked at which features on average were most significant in predicting proteins. For both glomeruli and tubules, RGB color values and variance in PAS+ areas (specifically basement membrane for tubules) were, on average, more predictive of molecular profiles than other features. There is a strong connection between molecular profile and image phenotype, which can be elucidated through computational methods. These discovered links can provide insight to disease pathways, and discover new factors contributing to incidence and progression.

SETD8 cooperates with MZF1 to participate in hyperglycemia-induced endothelial inflammation via elevation of WNT5A levels in diabetic nephropathy

ObjectiveDiabetic nephropathy (DN) is regarded as the main vascular complication of diabetes mellitus, directly affecting the outcome of diabetic patients. Inflammatory factors were reported to participate in the progress of DN. Wingless-type family member 5 (WNT5A), myeloid zinc finger 1 (MZF1), and lysine methyltransferase 8 (SETD8) have also been reported to elevate inflammatory factor levels and activate the nuclear factor kappa B (NF-κB) pathway to induce endothelial dysfunction. In the current study, it was assumed that MZF1 associates with SETD8 to regulate WNT5A transcription, thus resulting in hyperglycemia-induced glomerular endothelial inflammation in DN.MethodsThe present study recruited 25 diagnosed DN patients (type 2 diabetes) and 25 control participants (nondiabetic renal cancer patients with normal renal function, stage I–II) consecutively. Moreover, a DN rat and cellular model was constructed in the present study. Immunohistochemistry, Western blot, and quantitative polymerase chain reaction (qPCR) were implemented to determine protein and messenger RNA (mRNA) levels. Coimmunoprecipitation (CoIP) and immunofluorescence were implemented in human glomerular endothelial cells (HGECs). Chromatin immunoprecipitation assays and dual luciferase assays were implemented to determine transcriptional activity.ResultsThe results of this study indicated that levels of WNT5A expression, p65 phosphorylation (p-p65), and inflammatory factors were all elevated in DN patients and rats. In vitro, levels of p-p65 and inflammatory factors increased along with the increase of WNT5A expression in hyperglycemic HGECs. Moreover, high glucose increased MZF1 expression and decreased SETD8 expression. MZF1 and SETD8 inhibit each other under the stimulus of high glucose, but cooperate to regulate WNT5A expression, thus influencing p-p65 and endothelial inflammatory factors levels. Overexpression of MZF1 and silencing of SETD8 induced endothelial p-p65 and inflammatory factors levels, which can be reversed by si-WNT5A. Mechanistic research indicated that MZF1, SETD8, and its downstream target histone H4 lysine 20 methylation (H4K20me1) all occupied the WNT5A promoter region. sh-SETD8 expanded the enrichment of MZF1 on WNT5A promoter. Our in vivo study proved that SETD8 overexpression inhibited levels of WNT5A, p-p65 expression, and inflammatory factors in DN rats.ConclusionsMZF1 links with SETD8 to regulate WNT5A expression in HGECs, thus elevating levels of hyperglycemia-mediated inflammatory factors in glomerular endothelium of DN patients and rats.Trial registration ChiCTR, ChiCTR2000029425. 2020/1/31, http://www.chictr.org.cn/showproj.aspx?proj=48548

The axis of long non-coding RNA MALAT1/miR-1-3p/CXCR4 is dysregulated in patients with diabetic neuropathy

BackgroundDiabetic neuropathy (DN) is a prevalent complication of diabetes mellitus characterized by pain and inflammation. Long non-coding RNAs (lncRNAs) have been associated with DN. This study aimed to investigate transcript levels of Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), microRNA (miR)-1-3p, and C-X-C motif chemokine receptor 4 (CXCR4) in the DN patients and type 2 diabetes mellitus (T2DM) cases without neuropathy. MethodsHere, 20 cases with DN and 20 T2DM subjects without neuropathy (as the control group) were included. Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of all participants. The expression levels of targets were evaluated by Real-time-PCR. ResultsResults showed that MALAT1 (Fold change = 2.47, P = 0.03) and CXCR4 (Fold change = 1.65, P = 0.023) were significantly upregulated, while miR-1-3p was downregulated (Fold change = 0.9, P = 0.028) in whole blood samples from DN patients compared to the control group. A significant correlation was found between transcript levels of MALAT1 and CXCR4 (rho = 0.84; P < 0.0001). ConclusionsThis study suggests a possible involvement of the MALAT1/miR-1-3p/CXCR4 axis in the pathogenesis of DN.

Quercetin attenuates the proliferation, inflammation, and oxidative stress of high glucose-induced human mesangial cells by regulating the miR-485-5p/YAP1 pathway.

BackgroundDiabetic nephropathy (DN) is a kidney damage caused by diabetes and the main cause of end-stage renal disease. However, the current treatment of DN has many limitations. Quercetin is a bioflavonoid compound with therapeutic benefits in metabolic diseases. This study aims to determine the therapeutic potentials and underlying mechanism of quercetin on DN.MethodsWe collected blood samples from DN patients and healthy controls and treated human mesangial cells (HMCs) with high glucose (HG) to establish an in vitro model of DN. Then we assessed the expression difference of miR-485-5p as well as YAP1 in serum of DN patients and healthy controls and between HG-induced HMCs and control cells. qRT-PCR and western blot were performed to assess miR-485-5p and YAP1 expression levels; CCK-8 and ELISAs were used to examine cell proliferation, inflammation, and oxidative stress. Dual luciferase reporter assay was implemented to detect the binding of miR-485-5p and YAP1 mRNA sequence.ResultsQuercetin suppressed proliferation, inflammation, and oxidative stress of HMCs induced by HG. As for mechanism, miR-485-5p directly bound to YAP1 and inhibited YAP1 expression. The downregulation of miR-485-5p and upregulation of YAP1 were also observed in the serum of DN patients. Quercetin modulated miR-485-5p/YAP1 axis to regulate HG-induced inflammation and oxidative stress.Conclusion:Quercetin inhibits the proliferation, inflammation, and oxidative stress of HMCs induced by HG through miR-485-5p/YAP1 axis, which might provide a novel treatment strategy for DN.

Circular RNA_0037128 aggravates high glucose-induced damage in HK-2 cells via regulation of microRNA-497-5p/nuclear factor of activated T cells 5 axis

ABSTRACT Circular RNAs (CircRNAs) were reported to play vital roles in the progression of DN. Herein, the action of circular RNA_0037128 (circ_0037128) was investigated in DN. The level of circ_0037128, microRNA-497-5p (miR-497-5p) and nuclear factor of activated T cells 5 (NFAT5) was determined using quantitative real-time polymerase chain reaction (qRT-PCR). The feature of circ_0037128 was tested by RNase R and Actinomycin D treatment assays. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2ʹ-deoxyuridine (EdU) staining assays were conducted to evaluate the proliferation ability. The relative protein expression was determined via Western blot analysis. Levels of the inflammatory cytokines, like tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), were assessed by enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) production, lactate dehydrogenase (LDH) and superoxide dismutase (SOD) activity were determined by the matched kits. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were conducted for evaluating the correlation between miR-497-5p and circ_0037128 or NFAT5. Circ_0037128 and NFAT5 were enhanced, while miR-497-5p was weakened in kidney tissues of DN patients and high glucose (HG)-cultured HK-2 cells. Circ_0037128 inhibition bated HG-caused inhibition effect on cell proliferation and promotion effects on oxidative stress, inflammation and fibrosis in HK-2 cells. Moreover, circ_0037128 knockdown alleviated HG-caused cell damage via regulating miR-497-5p. In addition, NFAT5 overexpression could reverse the influence of miR-497-5p on HG-induced injury in HK-2 cells. Mechanically, circ_0037128 sponged miR-497-5p to modulate NFAT5. Circ_0037128 downregulation could mitigate HG-stimulated cell damage via regulating the miR-497-5p/NFAT5 axis in HK-2 cells in vitro, providing a possible therapy target for DN.

CDKN2B-AS1 participates in high glucose-induced apoptosis and fibrosis via NOTCH2 through functioning as a miR-98-5p decoy in human podocytes and renal tubular cells

BackgroundDiabetic nephropathy (DN) is the most common causes of end-stage renal disease. Long non-coding RNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) is connected with the development of DN, but the role of CDKN2B-AS1 in DN has not been entirely elucidated.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was carried out to measure CDKN2B-AS1 and miR-98-5p levels. Cell viability, proliferation, and apoptosis were analyzed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) or flow cytometry assays. Protein levels were measured by western blotting. The relationship between CDKN2B-AS1 or notch homolog 2 (NOTCH2) and miR-98-5p was verified via dual-luciferase reporter assay.ResultsCDKN2B-AS1 and NOTCH2 were upregulated in the serum of DN patients and high glucose-disposed human podocytes (HPCs) and human renal tubular cells (HK-2), whereas miR-98-5p was downregulated. High glucose repressed viability and accelerated apoptosis of HPCs and HK-2 cells. CDKN2B-AS1 knockdown impaired high glucose-induced apoptosis and fibrosis of HPCs and HK-2 cells. Mechanistically, CDKN2B-AS1 sponged miR-98-5p to regulate NOTCH2 expression. Also, CDKN2B-AS1 inhibition-mediated effects on apoptosis and fibrosis of high glucose-disposed HPCs and HK-2 cells were weakened by miR-98-5p inhibitor. Also, NOTCH2 knockdown partly reversed miR-98-5p inhibitor-mediated impacts on apoptosis and fibrosis of high glucose-disposed HPCs and HK-2 cells.ConclusionHigh glucose-induced CDKN2B-AS1 promoted apoptosis and fibrosis via the TGF-β1 signaling mediated by the miR-98-5p/NOTCH2 axis in HPCs and HK-2 cells.

Effects of valsartan combined with \u03b1-lipoic acid on renal function in patients with diabetic nephropathy: a systematic review and meta-analysis

BackgroundDiabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes, valsartan and α-lipoic acid alone or in combination has been used for the treatment of patients with DN. However, some results in these clinical reports were still controversial. The purpose of this study was to evaluate the efficacy of valsartan combined with α-lipoic acid on renal function in patients with DN.MethodsWe searched the electronic databases including PubMed, Sciencedirect, EMBASE, Cochrane library, Chinese national knowledge infrastructure (CNKI) and Wanfang databases, and the publication deadline was limited to January 2020. Randomized controlled trials (RCTs) evaluating the effects of valsartan combined with α-lipoic acid in DN patients were included. Pooled estimates were conducted using a fixed or random effect model. The outcomes included urinary albumin excretion rate (UAER), and the level of urinary albumin, β2-microglobulin (β2-MG), hypersensitive C-reactive protein (hs-CRP) and oxidative stress.Results11 studies with 1294 participants were included in this study. The pooled analysis indicated that α-lipoic acid combined with valsartan could remarkably reduce UAER (P < 0.00001, SMD = -1.95, 95%CI = -2.55 to − 1.20; P = 0.03, SMD = -0.85, 95%CI = -1.59 to − 0.1) and the level of urinary albumin (P = 0.001, SMD = -1.48, 95%CI = − 2.38 to − 0.58; P = 0.01, SMD = -1.67, 95%CI = -3.00 to − 0.33), β2-MG (P < 0.001,SMD = − 2.59, 95%CI = -3.78 to − 1.40; P = 0.03, SMD = -0.48, 95%CI = -0.93 to − 0.04) when compared with valsartan or lipoic acid monotherapy in patients with DN. However, there was no significant difference in the level of hs-CRP among the three therapies (P = 0.06, SMD = -2.80, 95%CI = -5.67 to 0.07; P = 0.10, SMD = -0.42, 95%CI = − 0.92 to 0.08). In addition, α-lipoic acid combined with valsartan markedly increased the level of SOD (P = 0.03, SMD = 1.24, 95%CI = 0.32 to 1.03; P = 0.0002, SMD = 0.68, 95%CI = 0.32 to 1.03) and T-AOC (P < 0.00001, SMD = 0.89, 95%CI = 0.62 to 1.16; P = 0.02, SMD = 0.58, 95%CI = 0.10 to1.07), and reduced the level of MDA(P = 0.0002, SMD = -1.99, 95%CI = -3.02 to − 0.96; P = 0.0001, SMD = -0.69, 95%CI = -1.04 to − 0.34).Conclusionsα-lipoic acid combined with valsartan could significantly reduce the level of urinary albumin and oxidative stress, increase antioxidant capacity and alleviate renal function damage in patients with DN, and this will provide a reference for the selection of treatment drugs for DN.

Urinary small extracellular vesicles derived CCL21 mRNA as biomarker linked with pathogenesis for diabetic nephropathy

BackgroundDiabetic nephropathy (DN) is a leading cause of renal failure, whereas the effective and early diagnostic biomarkers are still lacking.MethodsFourteen cytokines and chemokines mRNA were detected in urinary extracellular vesicles (EVs) from the screening cohort including 4 healthy controls (HC), 4 diabetes mellitus (DM) and 4 biopsy-proven DN patients, and was validated in another 16 HC and 15 DM and 28 DN patients. Correlation analysis was performed between the candidate biomarkers and clinic parameters as well as kidney histological changes. The findings were also confirmed in DN rat model with single injection of STZ.ResultsThe number of small EVs secreted in urine was increased in DN patients compared to DM patients and healthy controls, with expression of AQP1 (a marker of proximal tubules) and AQP2 (a marker of distal/collecting tubules). Small EVs derived CCL21 mRNA increased significantly in DN patients and correlated with level of proteinuria and eGFR. Interestingly, elevated CCL21 mRNA from urine small EVs was observed in DN patients with normal renal function and could discriminate early DN patients from DM more efficiently compared to eGFR and proteinuria. CCL21 also showed an accurate diagnostic ability in distinguishing incipient from overt DN. Histologically, CCL21 mRNA expression increased progressively with the deterioration of tubulointerstitial inflammation and showed the highest level in nodular sclerosis group (class III) in DN patients. Remarkable infiltration of CD3 positive T cells including both CD4 and CD8 positive T cell population were observed in DN patients with high-CCL21 expression. Besides, accumulation of CD3 positive T cells correlated with level of urinary small EVs derived CCL21 and co-localized with CCL21 in the tubulointerstitium in DN patients. Finally, the correlation of CCL21 expression in renal cortex and urinary small EVs was confirmed in STZ-induced DN rat model.ConclusionsUrinary small EVs derived CCL21 mRNA may serve as early biomarker for identifying DN linked with pathogenesis. CCL21 mRNA mediated T cell infiltration may constitute the key mechanism of chronic inflammation in DN.

WT1 and ACE mRNAs of blood extracellular vesicle as biomarkers of diabetic nephropathy

BackgroundDiabetic nephropathy (DN) has an increasing global prevalence with excessive health expenditure and burden. Exosomal mRNAs regulate intercellular communications and participate in the pathogenesis of various disorders like DN. This study aimed to assess the expression levels of ACE, ELMO1, and WT1 mRNAs in the blood extracellular vesicles (EVs) of DN patients and diabetic patients without nephropathy (DM group) in comparison to healthy controls and investigate their correlations with the severity of DN.MethodsThe performed investigation is a cross-sectional study of 256 participants including 103 DN patients, 100 DM patients, and 53 healthy controls. The quantification of WT1, ACE, and ELMO1 mRNAs in the blood EVs were executed using qRT-PCR. The ROC analysis was performed to determine the diagnostic accuracy of mRNAs.ResultsDN patients had significantly higher expressed WT1 mRNA (1.70-fold change) and lower expressed ACE mRNA (0.55-fold change) in the blood EVs compared to DM patients and controls. ELMO1 mRNA was not expressed in EVs of any groups. A positive correlation between WT1 mRNA level and urine Alb/Cr ratio (r = 0.602, p < 0.001) and a negative correlation between ACE mRNA expression and urine Alb/Cr ratio within DN patients (r = − 0.474, p < 0.001) was identified. The accuracy of WT1 mRNA and 1/ACE mRNA for predicting incipient DN was 0.63 (95% CI 0.55, 0.72) and 0.62 (95% CI 0.54, 0.71), and for predicting overt DN was 0.83 (95% CI 0.74, 0.92) and 0.75 (95% CI 0.66, 0.83), respectively.ConclusionsWT1 and ACE mRNAs level in blood EVs were predictors for early diagnosis of DN therefore their quantifications might be used to determine the severity of albuminuria and glomerular injuries.

Ets1 associates with KMT5A to participate in high glucose-mediated EndMT via upregulation of PFN2 expression in diabetic nephropathy

BackgroundDiabetic nephropathy (DN) is currently the leading cause of end-stage renal disease globally. The endothelial-to-mesenchymal transition (EndMT) of glomerular endothelial cells has been reported to play a crucial role in DN. As a specific form of epithelial-to-mesenchymal transition, EndMT and epithelial-to-mesenchymal transition may exhibit mutual modulators. Profilin 2 (PFN2) has been reported to participate in epithelial-to-mesenchymal transition. Moreover, ETS proto-oncogene 1 (ets1) and lysine methyltransferase 5A (KMT5A) have been reported to contribute to high glucose-mediated endothelial injury and epithelial-to-mesenchymal transition. In this study, we hypothesize ets1 associates with KMT5A to modulate PFN2 transcription, thus participating in high glucose-mediated EndMT in glomerular endothelial cells.MethodsImmunohistochemistry (IHC) was performed to detect protein levels in the kidney tissues and/or aorta tissues of human subjects and rats. Western blot, qPCR and immunofluorescence were performed using human umbilical vein endothelial cells (HUVECs). Chromatin immunoprecipitation (ChIP) assays and dual luciferase assays were performed to assess transcriptional activity. The difference between the groups was compared by two-tailed unpaired t-tests or one-way ANOVAs.ResultsOur data indicated that vimentin, αSMA, S100A4 and PFN2 levels were increased, and CD31 levels were reduced in glomerular endothelial cells of DN patients and rats. Our cell experiments showed that high glucose induced EndMT by augmenting PFN2 expression in HUVECs. Moreover, high glucose increased ets1 expression. si-ets1 suppressed high glucose-induced PFN2 levels and EndMT. ets1 overexpression-mediated EndMT was reversed by si-PFN2. Furthermore, ets1 was determined to associate with KMT5A. High glucose attenuated KMT5A levels and histone H4 lysine 20 methylation (H4K20me1), one of the downstream targets of KMT5A. KMT5A upregulation suppressed high glucose-induced PFN2 levels and EndMT. sh-KMT5A-mediated EndMT was counteracted by si-PFN2. Furthermore, H4K20me1 and ets1 occupied the PFN2 promoter region. sh-KMT5A cooperated with ets1 overexpression to activate PFN2 promoter activity. Our in vivo study demonstrated that KMT5A was reduced, while ets1 was augmented, in glomerular endothelial cells of DN patients and rats.ConclusionsThe present study indicated that ets1 cooperated with KMT5A to transcribe PFN2, thus contributing to hyperglycemia-induced EndMT in the glomerular endothelial cells of DN patients and rats.Trial registration ChiCTR, ChiCTR2000029425. 2020/1/31, http://www.chictr.org.cn/showproj.aspx?proj=48548

Spectrum of non-diabetic renal disease in patients with diabetes mellitus in a tertiary care hospital

Aim and Objectives: To analyse frequency and spectrum of non diabetic renal disease (NDRD) in type 2 diabetes patients and also determine the clinical markers associated with NDRD. Methodology: Single centre based prospective observational study carried out during the period from M a y 2016 to September 2019. Type 2 DM patients with atypical clinical renal disease who underwent renal biopsy to rule out NDRD at King George Hospital, Visakhapatnam were included in the study. Results: In the present study, the overall prevalence of NDRD with or without underlying DN in our study was 71.7%. The prevalence rates of isolated NDRD, NDRD with DN and isolated DN were 51.2%, 20.5%, and 28.2% respectively. Shorter duration of diabetes, absence of retinopathy, presence of active urine sediment are markers associated with NDRD in type 2 diabetics and are strong indicators for biopsy. Isolated DN group patients suffer significantly with hypertension compared to NDRD group. Isolated NDRD group patients had low HbA1c (

MiRNA-483-5p Targets HDCA4 to Regulate Renal Tubular Damage in Diabetic Nephropathy.

This study was designed to evaluate the diagnostic value of miR-483-5p in diabetic nephropathy (DN), and its effect and mechanism on apoptosis and inflammation of human proximal renal tubular cells (HK2) induced by high glucose (HG). Thirty healthy controls, 30 types 2 diabetes mellitus (T2DM) patients, and 28 DN patients were enrolled. miR-483-5p mRNA levels in serum were analyzed by RT-qPCR assays. The receiver operating characteristic curve (ROC) was used to analyze the diagnostic value of miR-483-5p in DN. HK2 cells were induced by HG to establish an in vitro study model. CCK-8 and flow cytometry was used to detect cell viability, apoptosis, and reactive oxygen species (ROS) generation. Inflammation levels were measured by ELISA. Luciferase reporter assay was used to detect target genes of miR-483-5p. miR-483-5p was decreased in DN patients. The decreased level of miR-483-5p was positively correlated with estimated glomerular filtration rate (eGFR) and negatively correlated with proteinuria. miR-483-5p can significantly distinguish DN patients from healthy controls and T2DM and has a high diagnostic value. miR-483-5p decreased in HK2 cells induced by HG, and overexpression of miR-483-5p reversed HG-induced decreased cell activity, increased apoptosis, ROS production, and inflammation. Histone deacetylase 4 (HDCA4) was markedly increased in DN patients and HG-induced HK2 cells. miR-483-5p directly targeted HDCA4, and increasing miR-483-5p inhibited HDCA4 increased in HG-induced HK2. In conclusion, the results indicate that reduction of miR-483-5p has a high diagnostic value in DN, and overexpression of miR-483-5p has a certain protective effect on HK2 cells induced by HG by targeting HDCA4.

MO161DIABETIC NEPHROPATHY AS AN ADDITIONAL FACTOR CAUSES DISTURBANCES IN T CELL IMMUNITY IN CHRONIC KIDNEY DISEASE PATIENTS

Abstract Background and Aims Chronic kidney disease (CKD) and Diabetes Mellitus (DM), are chronic inflammatory conditions, and may both affect T-cell immunity. However, our knowledge about alterations of specific T cell subpopulations, as well as the further modifications occurring after dialysis initiation, is limited. The present study aimed to investigate the changes of T lymphocyte subtypes in CKD patients due to Diabetic Nephropathy (DN-CKD) and compare to nonDN-CKD patients. Furthermore, the effect of dialysis, was also estimated in both groups. Method In 30 DN-CKD, 30 nonDN-CKD patients and 25 healthy individuals, cytometric analysis of T cell subpopulations was performed, including CD4+, CD8+, CD4+CD28null, CD8+CD28null, Natural Killer cells and Tregs. Measurements were performed at the day started (T0) on either hemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD) and repeated six months later (T6). Results At time T0, CD4+, NK cells and Tregs were significantly reduced (p=0.001, p=0.01, p&amp;lt;0.0001, respectively), while CD28null cells were increased (p=0.005) in CKD patients compared to controls. CD4CD28null and CD8CD28null (%) cells were increased in DN-CKD compared to nonDN-CKD (p=0.03, p=0.02, respectively). At time T6, a significant increase in total CD28null cells were noticed in DN patients (p=0.05) but not in nonDN patients. More interestingly, DN-CKD patients who started on HD showed a significant increase in CD4CD28null cells [from 30(14-100) to 52.7(15-203), p=0.02], CD8CD28null cells [from 130(47-400) to 212(25-1192), p=0.02] and NK cells [from 137(56-275) to 266(103-456), p=0.01], while those DN-CKD started on CAPD showed a significant reduction in the percentage of CD4CD28null cells [from 9.8(2.9-19.8) to 6.9(1.3-+18), p=0.05]. Conclusion Patients with CKD due to DN are more likely to have impaired T cell immunity, mainly regarding CD28null cells, which further deteriorates after initiation of HD. Instead, CAPD method seems to ameliorate those changes.

ACE ID gene polymorphism contributes to chronic kidney disease progression but not NOS3 gene among Type 2 diabetes with nephropathy patients

AimDiabetic nephropathy (DN) is one of the major life-threatening complications of diabetes and it leads to end-stage renal disease. Altered angiotension converting enzyme and nitric oxide synthase are probably the cause of initiation and the progression of diabetic nephropathy. The present study aims to investigate the effect of ACE ID and NOS3 VNTR gene polymorphisms on the progression of chronic kidney disease among diabetic nephropathy. Methods253 DN patients and 104 controls were genotyped for ACE ID and NOS3 VNTR polymorphisms by following PCR-RFLP method. The diabetic nephropathy cases were divided into two groups based on CKD stages: 138 DN cases were at early stage (CKD1 to CKD3) and 115 DN cases were at advanced stage (CKD4 and CKD5). Association χ2 and univariate analysis were performed. ResultsA significant difference was found in genotype frequencies of ACE ID and NOS3 VNTR polymorphisms between the DN patients and the controls. On univariate analysis, the DD genotype of ACE gene was found to have a significant association with the advancement of CKD in DN (OR=0.37; 95 % CI=0.14–0.94; p=0.033). ConclusionsThe results suggest the association of ACE ID and NOS3 VNTR polymorphism with diabetic nephropathy in South Indian population. Furthermore, the present study evidences the association between DD genotype of ACE gene and advancement of CKD progression in DN.

LncRNA ANRIL play an important role in renal injury induced by diabetes

IntroductionThe aim of this study was to discuss the effects and mechanisms of lncRNA in renal injury induced by diabetes by vivo and vitro study.Material and methodsCollecting healthy and DN patients, and measuring lncRNA ANRIL expression by RT-qPCR assay in serum. Measuring the ANRIL expression in normal, 10d and 20d model rats in kidney. Measuring LDH, MDA, SOD, IL-6 and TNF-α concentration by Elisa assay, evaluating the kidney pathology by HE staining, cell apoptosis by TUNEL assay, relative proteins expression by IHC assay and relative mRNA expression by RT-qPCR assay. In the cell experiment, Measuring LDH, MDA, SOD, IL-6 and TNF-α concentration by Elisa assay, measuring the cell proliferation by CCK-8, cell apoptosis by flow cytometry, relative mRNA and proteins expression by RT-qPCR and WB assay. The NF-κB(p65) nuclear volume of difference groups were evaluated by cell immunofluorescence.ResultsCompared with healthy people, lncRNA ANRIL was significantly increased in DN patients. Compared with sham group, the ANRIL expression, LDH, MDA, IL-6 and TNF-α concentration was significantly increased, SOD concentrations and cell apoptosis rate were significantly depressed with day increasing. TLR4, MyD88 and NF-κB(p65) mRNA and proteins expressions were significantly up-regulation.ConclusionslncRNA ANRIL knockdown had effects to improve renal injury induced by diabetes via regulation TLR4/NF-κB(p65).

Evidence of Resilience in Kidney Donors: A New York Statewide Cohort Analysis

IntroductionDonor nephrectomy (DN) is a procedure performed to provide recipients with a kidney to treat end-stage renal disease. The following analysis evaluated depression diagnosis in DN patients compared to controls. MethodsDN patients and matched controls were identified between 2000 and 2009 from the Statewide Planning and Research Cooperative System database. Cohorts were tracked for depression incidence. Multivariable logistic regression was used to determine independent predictors of a postoperative depression diagnosis. ResultsThe total study cohort included 2108 DN cases and 2108 controls. In both donors and controls, the baseline rate of depression was 0.95% (n = 20). The 5-year incidence of depression diagnosis after exposure increased in both cohorts (donors: 2.5%, n = 53; controls: 7.2%, n = 152; P < .001). The 5-year relative risk for developing depression was 2.65 (CI 1.59-4.42, P = .0002) in donors and 7.60 (CI 4.79-12.07, P < .001) in controls. On multivariable regression, being a donor was associated with reduced risk of developing postoperative depression (OR = 0.322, CI 0.233-0.445, P < .001), and the greatest risk factor for postoperative depression was a prior depressive diagnosis (OR = 7.811, CI 3.814-15.997, P < .001). ConclusionOur analysis shows that the strongest risk factor for depression was a prior diagnosis of depression. However, willingness to undergo donor nephrectomy is associated with less subsequent depression than the control population, suggesting that kidney donors may be a more resilient cohort.