Podocyte Injury In Diabetic Nephropathy Research Articles

Tanshinone IIA suppresses ferroptosis to attenuate renal podocyte injury in diabetic nephropathy through the embryonic lethal abnormal visual-like protein 1 and acyl-coenzyme A synthetase long-chain family member 4 signaling pathway.

Tanshinone IIA (TIIA) is one of the main components of the root of the red-rooted Salvia miltiorrhiza Bunge. However, the molecular mechanisms underlying TIIA-mediated protective effects in diabetic nephropathy (DN) are still unclear. High glucose (HG)-induced mouse podocyte cell line (MPC5) cells were used as the in vitro model of DN and treated with TIIA. Cell viability, proliferation and apoptosis were detected using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide, 5-ethynyl-2'-deoxyuridine and flow cytometry assays. The protein levels were assessed using western blot assay. The levels of inflammatory factors were deleted by enzyme-linked immunoassay. Fe+ level, reactive oxygen species, malondialdehyde and glutathione products were detected using special assay kits. After ENCORI prediction, the interaction between embryonic lethal abnormal visual-like protein 1 (ELAVL1) and acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) was verified using co-immunoprecipitation assay and dual-luciferase reporter assays. ACSL4 messenger ribonucleic acid expression was measured using real-time quantitative polymerase chain reaction. TIIA repressed HG-induced MPC5 cell apoptosis, inflammatory response and ferroptosis. ACSL4 upregulation relieved the repression of TIIA on HG-mediated MPC5 cell injury and ferroptosis. ELAVL1 is bound with ACSL4 to positively regulate the stability of ACSL4 messenger ribonucleic acid. TIIA hindered HG-triggered MPC5 cell injury and ferroptosis by regulating the ELAVL1-ACSL4 pathway. TIIA blocked DN progression in in vivo research. TIIA treatment restrained HG-caused MPC5 cell injury and ferroptosis partly through targeting the ELAVL1-ACSL4 axis, providing a promising therapeutic target for DN treatment.

Dendrobium officinale polysaccharide decreases podocyte injury in diabetic nephropathy by regulating IRS-1/AKT signal and promoting mitophagy.

High glucose (HG) caused oxidative stress and mitochondrial dysfunction, resulting in insulin resistance in podocytes, a key mechanism of diabetic nephropathy. Dendrobium officinale polysaccharide (DOP) was able to improve insulin resistance and antioxidant capability. The purpose of this study is to explore the mechanism by which DOP decreases the podocyte injury induced by HG. MPC5 cells were treated with HG, DOP, and IRS-1/2 inhibitor NT157. Afterwards, glucose consumption, generations of ROS and MDA were measured using the detection kits. Mitophagy was monitored using both MtphagTracyker and LysoTracker. The mitochondrial membrane potential was evaluated by JC-1 staining. DOP was also used in a mouse model of diabetes, with the measurements of urine albumin, blood creatinine and blood urea nitrogen. Treatment with DOP suppressed the HG-induced reduction of glucose consumption, the phosphorylation of IRS-1 (phospho Y632), AKT (phospho Ser473 and Thr308) and Nephrin. In addition, HG-induced augment of ROS and MDA, formation of γ-H2A.X foci and translocation of AKT to nucleus were inhibited by DOP. DOP enhanced mitophagy, which was associated with decreased mitochondrial membrane potential and ROS production. DOP conferred protective effect on podocyte in the diabetic mouse by reducing the albumin/creatinine ratio and blood urea nitrogen, and restoring Nephrin expression in podocytes. DOP alleviates HG-induced podocyte injuryby regulating IRS-1/AKT signal and promoting mitophagy.

Sweroside attenuates podocyte injury and proteinuria in part by activating Akt/BAD signaling in mice.

In this study, we investigated the effects of sweroside on podocyte injury in diabetic nephropathy (DN) mice and elucidated its molecular mechanisms. We conducted in vivo experiments using a C57BL/6 mice model of DN to explore the effects of sweroside on proteinuria and podocyte injury in DN mice. In in vitro experiments, conditionally immortalized mouse podocytes were treated with high glucose and sweroside, and the protective effects of sweroside on podocyte injury were analyzed. In vitro, Akt/BAD pathways were detected using gene siRNA silencing assays and found to be involved in the protective roles of sweroside in high glucose-mediated podocyte injury. In vivo, sweroside significantly decreased albuminuria in DN mice (p < 0.01). periodic acid-Schiff staining showed that sweroside alleviated the glomerular volume and mesangium expansion in DN mice. Consistently, western blot and reverse transcription-polymerase chain reaction analyses showed that the profibrotic molecule expression in the glomeruli declined in sweroside-treated DN mice. Immunofluorescent results showed that sweroside preserved nephrin and podocin expression, and transmission electron microscopy showed that sweroside attenuated podocyte injury. In DN mice, sweroside decreased podocyte apoptosis, and increased nephrin, podocin expression and decreased desmin and HIF1α expression. These results confirmed that sweroside ameliorated albuminuria, glomerulomegaly, and glomerulosclerosis in these mice. Experiments in vitro revealed that sweroside improved HG-induced podocyte injury and apoptosis. Sweroside stimulated activation of the Akt/BAD pathway and upregulated Bcl-2-associated death promoter (BAD) and p-Akt. Overall, sweroside protected podocytes from injury and prevented the progression of DN, providing a novel strategy for the treatment of DN.

Integrated Network Pharmacology and Cellular Assay to Explore the Mechanisms of Selenized Tripterine Phytosomes (Se@Tri-PTs) Alleviating Podocyte Injury in Diabetic Nephropathy.

This work aimed to elucidate the mechanisms of Se@Tri-PTs in alleviating podocyte injury via network pharmacology and in vitro cellular assay. Selenized tripterine phytosomes (Se@Tri-PTs) have been confirmed to undertake synergistic and sensitized effects on inflammation, which may be curatively promising for diabetic nephropathy (DN). However, the mechanisms of Se@Tri-PTs in alleviating podocyte injury, a major contributor to DN, still remain unclear. The objective of the study was to find out the underlying mechanisms of Se@Tri-PTs in alleviating podocyte injury in diabetic nephropathy. The key components and targets of Tripterygium wilfordii (TW) significant for DN as well as the signaling pathways involved have been identified. A high glucose-induced podocyte injury model was established and verified by western blot. The protective concentration of Se@Tri-PTs was screened by CCK-8 assay. Podocytes cultured with high glucose were treated with Se@Tri-PTs under protective levels. The expression of key protective proteins, nephrin and desmin, in podocytes, was assayed by western blot. Further, autophagy- related proteins and factors, like NLRP3, Beclin-1, LC3II/LC3, P62, and SIRT1, were analyzed, which was followed by apoptosis detection. Network pharmacology revealed that several monomeric components of TW, especially Tri, act on DN through multiple targets and pathways, including the NLRP3-mediated inflammatory pathway. Se@Tri-PTs improved the viability of podocytes and alleviated their injury induced by high glucose at 5 μg/L or above. High-glucose induction promoted the expression of NLRP3 in podocytes, while a low concentration of Se@Tri-PTs suppressed the expression. A long-term exposure of high glucose significantly inhibited the autophagic activity of podocytes, as manifested by decreased Beclin-1 level, lower ratio of LC3 II/LC3 I, and up- regulation of P62. This abnormality was efficiently reversed by Se@Tri-PTs. Importantly, the expression of SIRT1 was up-regulated and podocyte apoptosis was reduced. Se@Tri-PTs can alleviate podocyte injury associated with DN by modulating NLRP3 expression through the pathway of SIRT1-mediated autophagy.

Circ_0015756 modulates high glucose-induced podocyte injury through miR-136 in diabetic nephropathy

This study was to discuss the impact of Circ_0015756 on podocyte injury in diabetic nephropathy and its mechanism. After transfection of podocytes with si-NC and si-Circ_0015756, they were then exposed to normal glucose and high glucose (HG). Cell invasion and migration ability were detected by Transwell and scratch test, while cell proliferation and apoptosis were analyzed by flow cytometry, and apoptosis and anti-apoptotic protein expression were analyzed by Western blotting. Bioinformatics software and gene assay were employed to detect and verify the relationship between miR-136 and Circ_0015756. Finally, the activation state of NF-κB pathway was analyzed by Western blot. HG treatment induced podocytes injury and suppressed invasion and migration activity, accompanied by increased Circ_0015756 expression. And silencing of Circ_0015756 alleviated cell injury induced by HG treatment, while miR-136 knock down abolished Circ_0015756 silencing’ protective effect. Further, silencing of Circ_0015756 inhibited NF-κB pathways by regulating miR-136. HG environment induced upregulation of Circ_0015756 expression, while silencing of Circ_0015756 regulated the NF-κB pathway by upregulating miR-136 expression, thus alleviating podocytes injury induced by HG treatment.

LINC00355 mediates CTNNBIP1 promoter methylation and promotes ER stress-induced podocyte injury in diabetic nephropathy.

Endoplasmic reticulum stress (ER stress) plays an important role in podocyte injury in diabetic nephropathy. Wnt/β-catenin signaling modulates ER stress, yet the epigenetic regulation of β-catenin in ER stress and podocyte injury remains largely unknown. Herein, we tested the hypothesis that LINC00355 recruits EZH1 to the promoter region of CTNNBIP1 and trimethylates H3K4 to regulate ER-stress induced podocyte injury in DN. LINC00355 is upregulated in podocytes and correlates with renal function decline in DN patients. LINC00355 localizes in the nucleus and exerts biological functions by directly binding EZH1, which epigenetically targets CTNNBIP1 through repressive trimethylation of H3K4 and activates Wnt/β-catenin signaling and ER stress. Further, we provide mechanistic evidences that LINC00355 recruits EZH1 to the promoter region of CTNNBIP1 and regulates ER-stress induced podocyte injury in DN. Our data reveal a major role of LINC00355/EZH1/CTNNBIP1 network in triggering podocyte injury, providing new evidences for understanding the role of ER stress in DN.

Triptolide protects against podocyte injury in diabetic nephropathy by activating the Nrf2/HO-1 pathway and inhibiting the NLRP3 inflammasome pathway

Objectives Diabetic nephropathy (DN) is the most common microvascular complication of diabetes mellitus. This study investigated the mechanism of triptolide (TP) in podocyte injury in DN. Methods DN mouse models were established by feeding with a high-fat diet and injecting with streptozocin and MPC5 podocyte injury models were induced by high-glucose (HG), followed by TP treatment. Fasting blood glucose and renal function indicators, such as 24 h urine albumin (UAlb), serum creatinine (SCr), blood urea nitrogen (BUN), and kidney/body weight ratio of mice were examined. H&E and TUNEL staining were performed for evaluating pathological changes and apoptosis in renal tissue. The podocyte markers, reactive oxygen species (ROS), oxidative stress (OS), serum inflammatory cytokines, nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway-related proteins, and pyroptosis were detected by Western blotting and corresponding kits. MPC5 cell viability and pyroptosis were evaluated by MTT and Hoechst 33342/PI double-fluorescence staining. Nrf2 inhibitor ML385 was used to verify the regulation of TP on Nrf2. Results TP improved renal function and histopathological injury of DN mice, alleviated podocytes injury, reduced OS and ROS by activating the Nrf2/heme oxygenase-1 (HO-1) pathway, and weakened pyroptosis by inhibiting the nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome pathway. In vitro experiments further verified the inhibition of TP on OS and pyroptosis by mediating the Nrf2/HO-1 and NLRP3 inflammasome pathways. Inhibition of Nrf2 reversed the protective effect of TP on MPC5 cells. Conclusions Overall, TP alleviated podocyte injury in DN by inhibiting OS and pyroptosis via Nrf2/ROS/NLRP3 axis.

Niaoduqing alleviates podocyte injury in high glucose model via regulating multiple targets and AGE/RAGE pathway: Network pharmacology and experimental validation.

Purpose: The aim of present study was to explore the pharmacological mechanisms of Niaoduqing granules on the treatment of podocyte injury in diabetic nephropathy (DN) via network pharmacology and experimental validation. Methods: Active ingredients and related targets of Niaoduqing, as well as related genes of podocyte injury, proteinuria and DN, were obtained from public databases. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) network analysis were performed to investigate the potential mechanisms. High glucose (HG) -induced MPC5 cell injury model was treated with the major core active ingredients of Niaoduqing and used to validate the predicted targets and signaling pathways. Results: Totally, 16 potential therapeutic targets were identified by intersecting the targets of Niaoduqing and disease, in which 7 of them were considered as the core targets via PPI network analysis. KEGG enrichment analysis showed that AGE-RAGE signaling pathway was identified as the most crucial signaling pathway. The results of in vitro experiments revealed that the treatment of Niaoduqing active ingredients significantly protected MPC5 cells from HG-induced apoptosis. Moreover, Niaoduqing could significantly attenuate the HG-induced activation of AGE-RAGE signaling pathway, whereas inhibited the over-expression of VEGF-A, ICAM-1, PTGS-2 and ACE in HG-induced MPC5 cells. Conclusion: Niaoduqing might protect against podocyte injury in DN through regulating the activity of AGE/RAGE pathway and expression of multiple genes. Further clinical and animal experimental studies are necessary to confirm present findings.

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.

Histone modification in podocyte injury of diabetic nephropathy.

Diabetic nephropathy (DN), an important complication of diabetic microvascular disease, is one of the leading causes of end-stage renal disease (ESRD), which brings heavy burdens to the whole society. Podocytes are terminally differentiated glomerular cells, which act as a pivotal component of glomerular filtration barrier. When podocytes are injured, glomerular filtration barrier is damaged, and proteinuria would occur. Dysfunction of podocytes contributes to DN. And degrees of podocyte injury influence prognosis of DN. Growing evidences have shown that epigenetics does a lot in the evolvement of podocyte injury. Epigenetics includes DNA methylation, histone modification, and non-coding RNA. Among them, histone modification plays an indelible role. Histone modification includes histone methylation, histone acetylation, and other modifications such as histone phosphorylation, histone ubiquitination, histone ADP-ribosylation, histone crotonylation, and histone β-hydroxybutyrylation. It can affect chromatin structure and regulate gene transcription to exert its function. This review is to summarize documents about pathogenesis of podocyte injury, most importantly, histone modification of podocyte injury in DN recently to provide new ideas for further molecular research, diagnosis, and treatment.

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.

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.

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.

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.

POS-404 LINC00355 MEDIATES CTNNBIP1 PROMOTOR METHYLATION AND PROMOTES ER STRESS-INDUCED PODOCYTE INJURY IN DIABETIC NEPHROPATHY

Podocyte injury is a crucial feature of many glomerular disease,including diabetic nephropathy (DN) which is one of the most common microvascular complications of diabetes mellitus and the main cause of death and disability in diabetic patients. Recently,a series of studies have shed lighti on the role of epigenetic changes such as histone protein modifications played in podocyte injury of glomerular disease.Endoplasmic reticulum stress (ER stress) plays an important role in podocyte injury in diabetic nephropathy.

Asparaginyl endopeptidase protects against podocyte injury in diabetic nephropathy through cleaving cofilin-1

Podocyte injury and loss are critical events in diabetic nephropathy (DN); however, the underlying molecular mechanisms remain unclear. Here, we demonstrate that asparaginyl endopeptidase (AEP) protects against podocyte injury through modulating the dynamics of the cytoskeleton. AEP was highly upregulated in diabetic glomeruli and hyperglycemic stimuli treated-podocytes; however, AEP gene knockout and its compound inhibitor treatment accelerated DN in streptozotocin-induced diabetic mice, whereas specific induction of AEP in glomerular cells attenuated podocyte injury and renal function deterioration. In vitro, elevated AEP was involved in actin cytoskeleton maintenance and anti-apoptosis effects. Mechanistically, we found that AEP directly cleaved the actin-binding protein cofilin-1 after the asparagine 138 (N138) site. The protein levels of endogenous cofilin-1 1-138 fragments were upregulated in diabetic podocytes, consistent with the changes in AEP levels. Importantly, we found that cofilin-1 1-138 fragments were remarkably unphosphorylated than full-length cofilin-1, indicating the enhanced cytoskeleton maintenance activity of cofilin-1 1-138. Then we validated cofilin-1 1-138 could rescue podocytes from cytoskeleton disarrangement and injury in diabetic conditions. Taken together, our data suggest a protective role of elevated AEP in podocyte injury during DN progression through cleaving cofilin-1 to maintain podocyte cytoskeleton dynamics and defend damage.

CAMP-response element binding protein mediates podocyte injury in diabetic nephropathy by targeting lncRNA DLX6-AS1

BackgroundProgressive proteinuria is one of the earliest clinical features of diabetic nephropathy (DN). In our previous study, lncRNA DLX6-AS1 (DLX6-AS1, Dlx6os1 in the mouse) was found to be associated with the extent of albuminuria in DN patients. Furthermore, the lack of Dlx6os1 was pivotal in switching off the inflammatory response in db/db mouse model. However, the regulatory factors responsible for elevated DLX6-AS1 in DN remains unknown. MethodsTo identify potential regulatory factors for DLX6-AS1, JASPAR database and DNA pull down combined subsequent liquid chromatography-tandem mass spectrometry were used. Dual-luciferase reporter assay and chromatin immunoprecipitation were then performed to confirm binding sites. We also investigated the effects of the regulatory factors on DN progression in db/db mouse model and cultured human podocytes. ResultsOur analyses demonstrated that cAMP-response element binding protein (CREB) was highly expressed and closely associated with DLX6-AS1 in DN. In db/db mouse and in cultured podocytes, CREB silencing significantly reduced the level of DLX6-AS1 or Dlx6os1 and attenuated renal damage. Mechanistically, CREB overexpression aggravated renal inflammation and destroyed the structure of podocytes by targeting DLX6-AS1. The damaging role of CREB in podocyte injury was also inhibited by 666–15, a selective inhibitor, in a dose-dependent manner. In vivo, the inhibition of CREB by 666–15 significantly attenuated albuminuria and ameliorated inflammatory infiltration in podocytes. ConclusionsOur findings indicated that CREB is a key mediator of podocyte injury and acts by regulating DLX6-AS1. Thus, CREB may be an effective and potential therapeutic target for the treatment of DN.

METTL3-mediated m6A modification of TIMP2 mRNA promotes podocyte injury in diabetic nephropathy

Epigenetic changes are present in many physiological and pathological processes. The N6-methyladenosine (m6A) modification is the most common modification in eukaryotic mRNA. However, the role of m6A modification in diabetic nephropathy (DN) remains elusive. Here, we found that m6A modification was significantly upregulated in the kidney of type 1 and type 2 diabetic mice, which was caused by elevated levels of METTL3. Moreover, METTL3 is increased in podocyte of renal biopsy from patients with DN, which is related to renal damage. METTL3 knockout significantly reduced the inflammation and apoptosis in high glucose (HG)-stimulated podocytes, while its overexpression significantly aggravated these responses invitro. Podocyte-conditional knockout METTL3 significantly alleviated podocyte injury and albuminuria in streptozotocin (STZ)-induced diabetic mice. Therapeutically, silencing METTL3 with adeno-associated virus serotype-9 (AAV9)-shMETTL3 invivo mitigated albuminuria and histopathological injury in STZ-induced diabetic mice and db/db mice. Mechanistically, METTL3 modulated Notch signaling via the m6A modification of TIMP2 in an insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2)-dependent manner and exerted pro-inflammatory and pro-apoptotic effects. In summary, this study suggested that METTL3-mediated m6A modification is an important mechanism of podocyte injury in DN. Targeting m6A through the writer enzyme METTL3 is a potential approach for the treatment of DN.