Injury In Diabetic Nephropathy Research Articles

Thrombomodulin activation driven by LXR agonist attenuates renal injury in diabetic nephropathy.

Inflammation and thrombosis are recognized as interrelated biological processes. Both thrombomodulin (TM) and factor XIII-A (FXIII-A) are involved in inflammation and coagulation process. However, their role in the pathogenesis of diabetic nephropathy (DN) remains unclear. In vitro study, the liver X receptor (LXR) agonist T0901317 can up-regulate the expression of TM in glomerular endothelial cells. Now we evaluated the interaction between TM activation and FXIII-A and their effects against renal injury. We first evaluated the serum levels of FXIII-A and TM and the expression of TM, LXR-α and FXIII-A in renal tissues of patients with biopsy-proven DN. We then analyzed the expression of TM, LXR-α and FXIII-A in renal tissues of db/db DN mice after upregulating TM expression via T0901317 or downregulating its expression via transfection of TM shRNA-loaded adenovirus. We also investigated the serum levels of Tumor necrosis factor (TNF)-α, Interleukin (IL)-6, creatinine, and urinary microalbumin level in db/db mice. Our study showed that elevations in serum levels of FXIII-A positively correlated to the serum levels of TM and were also associated with end-stage kidney disease in patients with DN. The number of TM+ cells in the renal tissues of patients with DN negatively correlated with the number of FXIII-A+ cells and positively correlated with the number of LXR-α+ cells and estimated glomerular filtration rate (eGFR), whereas the number of FXIII-A+ cells negatively correlated with the eGFR. Thrombomodulin activation with T0901317 downregulated FXIII-A expression in the kidney tissue and alleviated renal injury in db/db mice.

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.

Identification of Ribonuclease 6 as an immunoinflammatory key gene associated with the glomerular injury in diabetic nephropathy

Diabetic nephropathy is one of the major causes of end-stage renal disease, and the pathogenesis of the disease has not been elucidated. While the immunoinflammatory response plays an essential role in the progression of diabetic nephropathy. Glomerular expression dataset in diabetic nephropathy was obtained from the GEO database. Differentially expressed genes were identified and functional enrichment analysis was performed to find genes associated with immunity and inflammation from them. The hub genes of immunoinflammatory were identified using MCODE after establishing the PPI network and gene expression was verified with diabetic nephropathy model rats. Xcell was used to assign immune cells to diabetic nephropathy glomerular samples to detect significant changes in immune cells and to analyze correlations with the hub gene. We found 120 DEGs associated with immunity and inflammation, Ribonuclease 6 was the Hub gene with the highest MCODE score. Xcell analysis revealed significant changes of immune cells in DN glomeruli, including upregulated Activated DCs, Conventional DCs, CD4+ Tem, Epithelial cells, Macrophages, Macrophages M1, and Memory B-cells. RNase6 expression showed the highest positive correlation with Macrophages M1, Activated DCs, and Conventional DCs. We verified through the Nephroseq v5 database that RNase6 expression was elevated in DN glomeruli and negatively correlated with glomerular filtration rate. Animal studies revealed that the kidney of DN model rats showed increased RNase6 expression together with inflammatory factor TNF-alpha and chemokine MCP-1. Our study identified RNase6 as a diagnostic and prognostic biomarker for diabetic nephropathy and found that it may play an essential role in the immunoinflammatory damage to the glomerulus.

KITLG Promotes Glomerular Endothelial Cell Injury in Diabetic Nephropathy by an Autocrine Effect.

Diabetic nephropathy (DN) is an increasing threat to human health. The impact of hyperglycemia or its metabolites, advanced glycation end-products (AGEs), on glomerular endothelial cells (GECs) and their pathophysiologic mechanisms are not well explored. Our results reveal that AGEs increased the expression and secretion of the KIT ligand (KITLG) in GECs. Both AGEs and KITLG promoted endothelial-to-mesenchymal transition (EndoMT) in GECs and further increased the permeability of GECs through the AKT/extracellular-signal-regulated kinase pathway. Inhibition of KITLG’s effects by imatinib prevented AGE-medicated EndoMT in GECs, supporting the belief that KITLG is a critical factor for GEC injury. We found higher KITLG levels in the GECs and urine of db/db mice compared with db/m mice, and urinary KITLG levels were positively correlated with the urinary albumin-to-creatinine ratio (ACR). Furthermore, type 2 diabetic patients had higher urinary KITLG levels than normal individuals, as well as urinary KITLG levels that were positively correlated with urinary ACR and negatively correlated with the estimated glomerular filtration rate. KITLG plays a pathogenic role in GEC injury in DN and might act as a biomarker of DN progression.

TBHQ attenuates podocyte injury in diabetic nephropathy by inhibiting NADPH oxidase-derived ROS generation via the Nrf2/HO-1 signalling pathway

AimsOxidative stress plays a crucial role in podocyte injury in diabetic nephropathy (DN). tert-Butylhydroquinone (tBHQ) is an activator of Nrf2 that exerts protective effects in diabetic mice, but the underlying mechanism of tBHQ in the podocytes of DN is not fully understood. Materials and methodsA high glucose (HG)-induced HK2 cell model and streptozotocin-induced rat model of DN were established and treated with tBHQ or apocynin. The expression levels of Nrf2, HO-1, NOX2 and NOX4 were determined by Western blot or immunohistochemical staining. The level of oxidative stress in podocytes or kidney tissues was assessed using DCFH-DA or dihydroethidium (DHE) staining. Cell injury was assessed by F-actin staining and flow cytometry analysis. Key findingsWe showed that HG treatment increased the expressions of NOX2 and NOX4 and enhanced ROS production in podocytes. Inhibition of NADPH oxidase activity by apocynin dramatically attenuated HG-induced ROS production and further alleviated cell injury and apoptosis in podocytes. Moreover, we found that HG inhibited the Nrf2/HO-1 signalling pathway in podocytes; however, tBHQ treatment significantly activated the Nrf2 signalling pathway, inhibited NADPH oxidase activity, and attenuated ROS production and cell injury in HG-treated podocytes. Furthermore, we observed that tBHQ treatment partially attenuated renal injury, activated the Nrf2 signalling pathway, inhibited NADPH oxidase activity and reduced ROS generation in the kidneys of STZ-induced diabetic rats. SignificanceThese results suggest that tBHQ exerts a protective role in hyperglycaemia-induced podocyte injury, and that the potential protective mechanism of tBHQ involves inhibiting NADPH oxidase-derived ROS generation by activating the Nrf2/HO-1 signalling pathway.

Solasonine alleviates high glucose-induced podocyte injury through increasing Nrf2-medicated inhibition of NLRP3 activation.

The worldwide high prevalence of diabetic nephropathy is one of the common causes of renal failure in diabetic patients. Hyperglycemia-caused podocyte injury is considered as a major contributor to diabetic kidney disease, accompanied by a chronic inflammatory condition. Pyroptosis, a characterized inflammatory form of programmed cell death, is believed to be involved in the pathogenesis of diabetic nephropathy. Solasonine (SS) is a natural alkaloid and received attention as a potential anticancer agent. However, its protective effect against hyperglycemia-caused podocyte injury remains to be determined. Our study found that SS alleviates cell apoptosis, and reduces pyroptosis and oxidative damage in high glucose (HG)-treated MPC5 podocytes. Pro-inflammatory cytokines, including interleukin (IL)-1β and IL-18, and caspase-1 activity were markedly suppressed by SS in HG-treated MPC5 podocytes. SS also reduced HG-induced oxidative damage in MPC5 podocytes. Nrf2 expression was activated by SS in vitro under a HG condition. In addition, Nrf2 silencing attenuated the protective effect of SS against apoptosis, pro-inflammatory cytokines release, caspase-1 activity, and oxidative damage in MPC5 podocytes under a HG condition. Taken together, our findings revealed for the first time that SS alleviated high glucose-induced podocyte apoptosis, pyroptosis, and oxidative damage via regulating the Nrf2/NLRP3 signaling pathway. Our results indicate that SS has the potential as a therapeutic agent for podocyte injury in diabetic nephropathy.

Identification of Hub Genes Involved in Tubulointerstitial Injury in Diabetic Nephropathy by Bioinformatics Analysis and Experiment Verification.

Diabetic nephropathy (DN) is the most important cause of end-stage renal disease with a poorer prognosis and high economic burdens of medical treatments. It is of great research value and clinical significance to explore potential gene targets of renal tubulointerstitial lesions in DN. To properly identify key genes associated with tubulointerstitial injury of DN, we initially performed a weighted gene coexpression network analysis of the dataset to screen out two nonconserved gene modules (dark orange and dark red). The regulation of oxidative stress-induced intrinsic apoptotic signaling pathway, PI3K-Akt signaling pathway, p38MAPK cascade, and Th1 and Th2 cell differentiation were primarily included in Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of these two modules. Next, 199 differentially expressed genes (DEGs) were identified via the limma package. Then, the GO annotation and KEGG pathways of the DEGs were primarily enriched in extracellular matrix (ECM) organization, epithelial cell migration, cell adhesion molecules (CAMs), NF-kappa B signaling pathway, and ECM-receptor interaction. Gene set enrichment analysis showed that in the DN group, the interaction of ECM-receptor, CAMs, the interaction of cytokine-cytokine receptor, and complement and coagulation cascade pathways were significantly activated. Eleven key genes, including ALB, ANXA1, ANXA2, C3, CCL2, CLU, EGF, FOS, PLG, TIMP1, and VCAM1, were selected by constructing a protein-protein interaction network, and expression validation, ECM-related pathways, and glomerular filtration rate correlation analysis were performed in the validated dataset. The upregulated expression of hub genes ANXA2 and FOS was verified by real-time quantitative PCR in HK-2 cells treated with high glucose. This study revealed potential regulatory mechanisms of renal tubulointerstitial damage and highlighted the crucial role of extracellular matrix in DN, which may promote the identification of new biomarkers and therapeutic targets.

The role of mitochondrial fission factor in podocyte injury in diabetic nephropathy

Diabetic nephropathy (DN) is one of the most important complications of diabetes mellitus (DM) and has become the second cause of end-stage renal disease (ESRD). This study intends to investigate the molecular mechanism of increased mitochondrial fission in podocytes under the effect of high glucose (HG), and to preliminarily study the role of mitochondrial fission factor (MFF)-mediated mitochondrial fission in podocyte injury of DN. In vitro studies, we found that HG induced increased mitochondrial fission and podocyte damage. At the same time MFF mRNA and protein levels was increased, suggesting that MFF was transcriptional upregulated under HG conditions. Consistent with this, in vivo studies found that mitochondrial fission was also significantly increased in podocytes of diabetic nephropathy mice, and MFF expression was up-regulated. Therefore, our study proves that mitochondrial fission increases in podocytes under DM both in vitro and in vivo, and the up-regulation of MFF expression may be one of the reasons for the increase of mitochondrial fission. After inhibiting the expression of MFF, the survival rate of podocytes was significantly decreased under HG conditions, suggesting that MFF may play a protective role in podocyte injury in DN.

WTAP-mediated N6-methyladenosine modification of NLRP3 mRNA in kidney injury of diabetic nephropathy

BackgroundDiabetic nephropathy (DN) is prevalent in patients with diabetes. N6-methyladenosine (m6A) methylation has been found to cause modification of nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing (NLRP) 3, which is involved in cell pyroptosis and inflammation. WTAP is a key gene in modulating NLRP3 m6A.MethodsIn this study, WTAP was silenced or overexpressed in high glucose (HG)-treated HK-2 cells to determine its influence on pyroptosis, NLRP3 inflammasome-related proteins, and the release of pro-inflammatory cytokines. NLRP3 expression and m6A levels were assessed in the presence of WTAP shRNA (shWTAP). WTAP expression in HK-2 cells was examined with the introduction of C646, a histone acetyltransferase p300 inhibitor.ResultsWe found that WTAP expression was enhanced in patients with DN and in HG-treated HK-2 cells. Knockdown of WTAP attenuated HG-induced cell pyroptosis and NLRP3-related pro-inflammatory cytokines in both HK-2 cells and db/db mice, whereas WTAP overexpression promoted these cellular processes in HK-2 cells. WTAP mediated the m6A of NLRP3 mRNA that was stabilized by insulin-like growth factor 2 mRNA binding protein 1. Histone acetyltransferase p300 regulated WTAP expression. WTAP mRNA levels were positively correlated with NLRP3 inflammasome components and pro-inflammatory cytokines.ConclusionTaken together, WTAP promotes the m6A methylation of NLRP3 mRNA to upregulate NLRP3 inflammasome activation, which further induces cell pyroptosis and inflammation.

The Role of the NLRP3 Inflammasome in Mediating Glomerular and Tubular Injury in Diabetic Nephropathy.

The NOD-like receptor protein 3 (NLRP3) inflammasome is a multi-protein signalling complex integral to the chronic inflammatory response, activated in response to sterile and non-sterile cellular damage. The assembly and activation of the NLRP3 inflammasome comprise a two-step process involving nuclear factor kappa B (NFkB)-mediated priming, followed by canonical, non-canonical or alternative signalling pathways. These result in the maturation and release of inflammatory cytokines interleukin 1 beta (IL1ß) and interleukin-18 (IL18), which are associated with chronic inflammatory conditions including diabetic kidney disease. Diabetic nephropathy is a condition affecting ∼40% of people with diabetes, the key underlying pathology of which is tubulointerstitial inflammation and fibrosis. There is growing evidence to suggest the involvement of the NLRP3 inflammasome in this chronic inflammation. Early deterioration of kidney function begins in the glomerulus, with tubular inflammation dictating the progression of late-stage disease. Priming and activation of the NLRP3 inflammasome have been linked to several clinical markers of nephropathy including proteinuria and albuminuria, in addition to morphological changes including mesangial expansion. Treatment options for diabetic nephropathy are limited, and research that examines the impact of directly targeting the NLRP3 inflammasome, or associated downstream components are beginning to gain favour, with several agents currently in clinical trials. This review will explore a role for NLRP3 inflammasome activation and signalling in mediating inflammation in diabetic nephropathy, specifically in the glomerulus and proximal tubule, before briefly describing the current position of therapeutic research in this field.

Identification and Verification of Diagnostic Biomarkers for Glomerular Injury in Diabetic Nephropathy Based on Machine Learning Algorithms.

Diabetic nephropathy (DN) is regarded as the leading cause of end-stage renal disease worldwide and lacks novel therapeutic targets. To screen and verify special biomarkers for glomerular injury in patients with DN, fifteen datasets were retrieved from the Gene Expression Omnibus (GEO) database, correspondingly divided into training and testing cohorts and then merged. Using the limma package, 140 differentially expressed genes (DEGs) were screened out between 81 glomerular DN samples and 41 normal ones from the training cohort. With the help of the ConsensusClusterPlus and WGCNA packages, the 81 glomerular DN samples were distinctly divided into two subclusters, and two highly associated modules were identified. By using machine learning algorithms (LASSO, RF, and SVM-RFE) and the Venn diagram, two overlapping genes (PRKAR2B and TGFBI) were finally determined as potential biomarkers, which were further validated in external testing datasets and the HFD/STZ-induced mouse models. Based on the biomarkers, the diagnostic model was developed with reliable predictive ability for diabetic glomerular injury. Enrichment analyses indicated the apparent abnormal immune status in patients with DN, and the two biomarkers played an important role in the immune microenvironment. The identified biomarkers demonstrated a meaningful correlation between the immune cells’ infiltration and renal function. In conclusion, two robust genes were identified as diagnostic biomarkers and may serve as potential targets for therapeutics of DN, which were closely associated with multiple immune cells.

Formononetin Attenuates Renal Tubular Injury and Mitochondrial Damage in Diabetic Nephropathy Partly via Regulating Sirt1/PGC-1α Pathway.

Mitochondrial abnormality is one of the main factors of tubular injury in diabetic nephropathy (DN). Formononetin (FMN), a novel isoflavonoid isolated from Astragalus membranaceus, has diverse pharmacological activities. However, the beneficial effects of FMN on renal tubular impairment and mitochondrial dysfunction in DN have yet to be studied. In this study, we performed in vivo tests in Streptozotocin (STZ) -induced diabetic rats to explore the therapeutic effects of FMN on DN. We demonstrated that FMN could ameliorate albuminuria and renal histopathology. FMN attenuated renal tubular cells apoptosis, mitochondrial fragmentation and restored expression of mitochondrial dynamics-associated proteins, such as Drp1, Fis1 and Mfn2, as well as apoptosis-related proteins, such as Bax, Bcl-2 and cleaved-caspase-3. Moreover, FMN upregulated the protein expression of Sirt1 and PGC-1α in diabetic kidneys. In vitro studies further demonstrated that FMN could inhibit high glucose-induced apoptosis of HK-2 cells. FMN also reduced the production of mitochondrial superoxide and alleviated mitochondrial membrane potential (MMP) loss. Furthermore, FMN partially restored the protein expression of Drp1, Fis1 and Mfn2, Bax, Bcl-2, cleaved-caspase-3, Sirt1 and PGC-1α in HK-2 cells exposure to high glucose. In conclusion, FMN could attenuate renal tubular injury and mitochondrial damage in DN partly by regulating Sirt1/PGC-1α pathway.

Integrated Analysis of Multiple Microarray Studies to Identify Core Gene-Expression Signatures Involved in Tubulointerstitial Injury in Diabetic Nephropathy.

Diabetic nephropathy is a leading cause of end-stage renal disease in both developed and developing countries. It is lack of specific diagnosis, and the pathogenesis remains unclarified in diabetic nephropathy, following the unsatisfactory effects of existing treatments. Therefore, it is very meaningful to find biomarkers with high specificity and potential targets. Two datasets, GSE30529 and GSE47184 from GEO based on diabetic nephropathy tubular samples, were downloaded and merged after batch effect removal. A total of 545 different expression genes screened with log2FC > 0.5 were weighted gene coexpression correlation network analysis, and green module and blue module were identified. The results of KEGG analyses both in green module and GSEA analysis showed the same two enriched pathway, focal adhesion and viral myocarditis. Based on the intersection among WGCNA focal adhesion/Viral myocarditis, GSEA focal adhesion/viral myocarditis, and PPI network, 17 core genes, ACTN1, CAV1, PRKCB, PDGFRA, COL1A2, COL6A3, RHOA, VWF, FN1, HLA-F, HLA-DPB1, ITGB2, HLA-DRA, HLA-DMA, HLA-DPA1, HLA-B, and HLA-DMB, were identified as potential biomarkers in diabetic tubulointerstitial injury and were further validated externally for expression at GSE99325 and GSE104954 and clinical feature at nephroseq V5 online platform. CMap analysis suggested that two compounds, LY-294002 and bufexamac, may be new insights for therapeutics of diabetic tubulointerstitial injury. Conclusively, it was raised that a series of core genes may be as potential biomarkers for diagnosis and two prospective compounds.

Src Activation Aggravates Podocyte Injury in Diabetic Nephropathy via Suppression of FUNDC1-Mediated Mitophagy.

Background and purpose: Mitophagy plays a significant role in the progression of diabetic nephropathy (DN), although the regulatory mechanisms remain unclear. Recently, accumulating evidence demonstrated that impaired mitochondrial function and mitophagy are involved in DN. Here, we are aimed to explore the role of c-Src (Src) and FUNDC1-related mitophagy in the development of DN. Methods: The db/db mice were used to establish a DN mice model. The mice accepted PP2 (Src inhibitor) treatment to study the role of Src in DN. Kidney function was measured via biochemical testing. Renal histopathology and morphometric analysis were conducted via hematoxylin-eosin (HE), periodic acid-Schiff (PAS), Masson’s staining, and transmission electron microscopy (TEM). We measured degree of apoptosis in kidney by TUNEL assay. Indices of mitophagy (LC3 and p62) were evaluated by Western blotting and immunofluorescence. Complementary in vitro assays were conducted using human podocytes subjected to high glucose in combination with PP2 treatment or FUNDC1 small interfering RNAs (siRNAs). Flow cytometry was used to detect the apoptotic cells. Mitochondrial function was evaluated by JC-1 staining. Double immunofluorescence labeling of LC3 and TOMM20 used to assess the degree of mitophagy. Results: Increased Src activation was detected in the kidneys of db/db mice, and its expression was positively correlated with mitochondrial damage, podocyte apoptosis, and renal dysfunction. Inhibition of Src activation with PP2 protected against mitochondrial damage and podocyte apoptosis. In vitro experiments in podocytes established that high glucose increased Src activation, promoting FUNDC1 phosphorylation and inhibiting mitophagy. Consistent with the mouse model, inhibiting Src activity protected podocytes against mitochondrial damage. FUNDC1 silencing negated the actions of PP2, indicating that FUNDC1-mediated mitophagy is downstream pathway of Src. Conclusion: In summary, our data indicated that Src is a culprit factor in diabetic renal damage via suppression of FUNDC1-mediated mitophagy, promoting the development of DN.

Upregulation of BRD7 protects podocytes against high glucose-induced apoptosis by enhancing Nrf2 in a GSK-3β-dependent manner

Bromodomain-containing protein 7 (BRD7) is linked to a variety of pathophysiological conditions. However, it is still unclear whether BRD7 is connected with diabetic nephropathy. This research explored the relevance of BRD7 in diabetic nephropathy using high glucose (HG)-stimulated podocytes in vitro. BRD7 expression in podocytes was decreased after HG stimulation. Podocytes with forced BRD7 expression were protected from HG-induced apoptosis, oxidative stress and inflammation. Further data revealed that forced expression of BRD7 led to enhanced nuclear factor erythroid-2-related factor 2 (Nrf2) activation in HG-stimulated podocytes, associated with the upregulation of glycogen synthase kinase-3β (GSK-3β) phosphorylation. Reactivation of GSK-3β diminished BRD7-elicited Nrf2 activation. In addition, restraining of Nrf2 diminished the BRD7 overexpression-induced beneficial effects on HG-induced podocyte damage. Taken together, these data document that BRD7 defends against HG-induced podocyte damage by enhancing Nrf2 via regulation of GSK-3β. Our work indicates that the BRD7/GSK-3β/Nrf2 axis may play a key role in mediating podocyte injury in diabetic nephropathy.

Activation of Complement Pathways in Kidney Tissue May Mediate Tubulointerstitial Injury in Diabetic Nephropathy.

IntroductionKey genes involved in tubulointerstitial injury may influence the development and progression of diabetic nephropathy (DN). We investigated whether complement-related genes are linked to the mechanism underlying tubulointerstitial injury in DN.MethodsWe analyzed the microarray data of 17 tubulointerstitial tissue samples from DN patients and 21 normal controls from the Gene Expression Omnibus. A gene co-expression network was constructed, and genes were divided into modules by weighted gene co-expression network analysis (WGCNA). We also investigated the association of C3 and C1q deposits in kidney tissues with a composite outcome of end-stage renal disease or a 50% reduction in the estimated glomerular filtration rate (eGFR) in DN patients. Finally, we performed immunohistochemical analyses of C3, C1q, C5b-9, mannose-binding lectin (MBL), and factor B in kidney tissues.ResultsNine co-expression modules were constructed using 12,075 genes from the 38 human tubulointerstitial tissue samples. Black module with more genes was positively correlated with tubulointerstitial injury in DN. C3, one of the top 10 genes in tubulointerstitial injury, was verified in an independent dataset; C3 was significantly overexpressed in tubulointerstitial tissue from patients with DN compared to the normal controls. The mRNA level of C3 in renal tubulointerstitium was negatively correlated with eGFR in DN patients (r = −0.75; p = 0.001). Analysis of the follow-up data of 54 DN patients demonstrated that codeposits of C3 and C1q in kidney tissues were independently associated with the renal outcome in DN (hazard ratio, 2.3, 95% confidence interval, 1.01–5.2, p < 0.05). Immunohistochemical analysis showed that patients with higher C1q, C3, C5b-9, MBL, or factor B expression in renal tubulointerstitium were more likely to progress to kidney failure.ConclusionLocal complement activation of the classical, lectin and alternative pathways appears linked to tubulointerstitial injury and disease progression in DN.

NAD+ Anabolism Disturbance Causes Glomerular Mesangial Cell Injury in Diabetic Nephropathy.

The homeostasis of NAD+ anabolism is indispensable for maintaining the NAD+ pool. In mammals, the mainly synthetic pathway of NAD+ is the salvage synthesis, a reaction catalyzed by nicotinamide mononucleotide adenylyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase (NMNATs) successively, converting nicotinamide (NAM) to nicotinamide mononucleotide (NMN) and NMN to NAD+, respectively. However, the relationship between NAD+ anabolism disturbance and diabetic nephropathy (DN) remains elusive. Here our study found that the disruption of NAD+ anabolism homeostasis caused an elevation in both oxidative stress and fibronectin expression, along with a decrease in Sirt1 and an increase in both NF-κB P65 expression and acetylation, culminating in extracellular matrix deposition and globular fibrosis in DN. More importantly, through constitutively overexpressing NMNAT1 or NAMPT in human mesangial cells, we revealed NAD+ levels altered inversely with NMN levels in the context of DN and, further, their changes affect Sirt1/NF-κB P65, thus playing a crucial role in the pathogenesis of DN. Accordingly, FK866, a NAMPT inhibitor, and quercetin, a Sirt1 agonist, have favorable effects on the maintenance of NAD+ homeostasis and renal function in db/db mice. Collectively, our findings suggest that NMN accumulation may provide a causal link between NAD+ anabolism disturbance and diabetic nephropathy (DN) as well as a promising therapeutic target for DN treatment.

Penehyclidine hydrochloride suppresses inflammation response and reduces podocyte injury in diabetic nephropathy by targeting fibrinogen-like protein 2

BackgroundDiabetic nephropathy (DN) is one of the main complications of diabetes. Penehyclidine hydrochloride (PHC) has anti-inflammatory, anti-apoptotic and anti-oxidative stress effects. Nevertheless, whether PHC can be used to prevent podocyte injury has not been reported. ObjectivesThis present study aimed to identify the functional role of PHC in DN as well as its underlying mechanism. MethodsThe high-glucose (HG)-induced podocyte damage in vitro model was established. The proliferation, apoptotic rate, inflammatory factors, and gene/protein expressions of HG-induced MPC5 cells were determined using CCK-8 assay, flow cytometry, ELISA, real-time PCR, and Western blot upon PHC treatment. Co-immunoprecipitation experiments and pull-down assay were performed to verify the interactions between fibrinogen-like protein 2 (Fgl2) and toll-like receptor 4 (TLR4) as well as TLR4 and NLRP3. A rat in vivo model was used to confirm the effect of PHC treatment. ResultsPHC treatment reduced Fgl2 expression and inhibited HG-induced podocyte injury and DN-induced kidney damage. Flg2 was associated with TLR4 and NLRP3. It was further proved that PHC treatment suppressed the TLR4-NF-кB and NLRP3-Caspase-1 pathways through Fgl2, which eventually inhibited inflammatory cytokines and prevented HG-induced podocyte injury both in vitro and in vivo. ConclusionPHC treatment possibly ameliorates DN by preventing podocyte injury via inactivating the TLR4-NF-кB and NLRP3-Caspase-1 signaling pathways by Flg2.

Hsa_circ_0037128 aggravates high glucose-induced podocytes injury in diabetic nephropathy through mediating miR-31-5p/KLF9

Background Circular RNA is a key regulator involved in the progression of many human diseases including diabetic nephropathy (DN). However, the role and mechanism of hsa_circ_0037128 in the occurrence and development of DN remains to be explored. Methods High glucose (HG)-induced podocytes were used to construct in vitro DN models. The expression of hsa_circ_0037128, microRNA (miR)-31-5p, and Kruppel-like factor 9 (KLF9) was determined using quantitative real-time polymerase chain reaction. The viability and apoptosis of podocytes was measured using cell counting kit 8 assay and flow cytometry. Western blot analysis was performed to examine the protein levels of apoptosis markers and KLF9 in podocytes. Inflammation factors were detected by ELISA assay, and oxidative stress markers were assessed by corresponding Assay Kits. In addition, the interaction between miR-31-5p and hsa_circ_0037128 or KLF9 was verified using dual-luciferase reporter assay and RIP assay. Results Our data suggested that hsa_circ_0037128 was highly expressed in DN patients and HG-induced podocytes. In HG-induced podocytes, hsa_circ_0037128 knockdown could alleviate HG-induced podocytes injury. In the term of mechanism, hsa_circ_0037128 could sponge miR-31-5p to upregulate KLF9. MiR-31-5p inhibitor could reverse the negative regulation of hsa_circ_0037128 silencing on HG-induced podocytes injury. Also, miR-31-5p relieved HG-induced podocytes injury, and this effect also could be reversed by KLF9 overexpression. Conclusion In summary, our data showed that hsa_circ_0037128 could promote HG-induced podocytes injury via regulating miR-31-5p/KLF9 axis, showing that hsa_circ_0037128 might be a target for DN treatment.

Microencapsulated islet transplantation alleviates podocyte injury in diabetic nephropathy via inhibiting Notch-1 signaling

ObjectivePodocyte injury has a critical role in the pathogenesis of diabetic nephropathy (DN). Microencapsulated islet transplantation (MIT) is identified as an effective method for improving the clinical condition of DN. This study aimed to explore the role and mechanism of MIT in alleviating podocyte injury in DN. MethodsA mouse model of DN was constructed using streptozotocin (STZ). Mice were divided into 3 groups: the untreated diabetic nephropathy group (DN group), the microencapsulated islet transplantation-treated group (MIT group) and the control group. The mice were raised for 6 weeks posterior to islet transplantation to identify the role of MIT. Renal function and structure of glomerular filtration barrier were assessed by urine analysis, histopathological examination, and transmission electron microscopy. The expression levels of several proteins including Caspase-3, Bcl2/Bax, β-galactosidase, Ki-67, synaptopodin, WT-1, Jagged-1, Notch-1, and Hes-1 in renal tissues were identified via immunohistochemistry (IHC), immunofluorescence (IF), and western blotting techniques. ResultsCompared with the DN group, the MIT group presented decreased levels of blood glucose, urinary albumin/creatinine, urea nitrogen, and serum creatinine while their body weight gradually increased. Glomerular injury in the MIT group was significantly better than that in the DN group. The MIT group indicated significantly decreased expression of Caspase-3, β-galactosidase, Bax/Bcl-2, and Ki-67 when compared with DN group, while the proportion of synaptopodin- and WT-1-positive cells was significantly increased (P < 0.05). The protein expression of Jagged-1, Notch-1, and Hes-1 in the glomerulus of the MIT group was significantly lower than that in the DN group (P < 0.05). ConclusionMIT alleviates podocyte injury induced by DN by inhibiting Notch-1 signaling. The identification of signaling pathways influencing podocyte restoration can help evaluate personalized medicine efficacy for patients treated with islet transplantation.